# Src promotes tumor cell invasion by hijacking the translation machineries

Benjamin Bonnard, Anouk Chatefau, Cyril Dourthe, Sylvaine Di Tommaso, Jean-William Dupuy, Isabelle Mahouche, Jacobo Solorzano, Morgane Le Bras, Anne-Aurélie Raymond, Violaine Moreau, et al.

► To cite this version:

Benjamin Bonnard, Anouk Chatefau, Cyril Dourthe, Sylvaine Di Tommaso, Jean-William Dupuy, et al.. Src promotes tumor cell invasion by hijacking the translation machineries. Cell Reports, 2026, 45 (3), pp.116989. 10.1016/j.celrep.2026.116989. hal-05534504

HAL Id: hal-05534504

https://hal.science/hal-05534504v1

Submitted on 3 Mar 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
**Cell Reports**

# Src promotes tumor cell invasion by hijacking the translation machineries

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Benjamin Bonnard, Anouk Chatefau, Cyril Dourthe, ..., Anne Blangy, Yvan Martineau, Frédéric Saltel

## Correspondence

benjamin.bonnard@inserm.fr (B.B.), frederic.saltel@inserm.fr (F.S.)

## In brief

Bonnard et al. demonstrate the determinant role of the Src oncogene in protein synthesis regulation through canonical and non-canonical pathways. The specific Src-regulated targets are essential for invadosome formation leading to tumor cell invasion.

## Highlights

* Src activity regulates protein synthesis and specific eIF3 subunit expression

* Src-regulated eIF3 subunits control invadosome formation

* Src activity enhances both canonical and non-canonical translation initiation

* Src-regulated eIF3 subunits are enriched in liver cancer

<figure><img src="image_3.png" /></figure>
Article

# Src promotes tumor cell invasion by hijacking the translation machineries

Benjamin Bonnard,<fnref n="1" />,<fnref n="5" />,* Anouk Chatefau,<fnref n="1" />,<fnref n="5" /> Cyril Dourthe,<fnref n="1" />,<fnref n="2" /> Sylvaine Di Tommaso,<fnref n="2" /> Jean-William Dupuy,<fnref n="2" />
Isabelle Mahouche,<fnref n="1" /> Jacobo Solorzano,<fnref n="3" /> Morgane Le Bras,<fnref n="3" /> Anne-Aurélie Raymond,<fnref n="1" />,<fnref n="2" /> Violaine Moreau,<fnref n="1" />
Arnaud Jabouille,<fnref n="1" /> Anne Blangy,<fnref n="4" /> Yvan Martineau,<fnref n="3" /> and Frédéric Saltel<fnref n="1" />,<fnref n="2" />,<fnref n="6" />,*

## SUMMARY

The Src oncogene controls cancer cell invasiveness by promoting invadosome formation and extracellular matrix (ECM) degradation. Invadosomes are translational hotspots enriched in the eukaryotic translation initiation factor 3 (eIF3) complex that is mandatory for their maintenance. Here, we determined that Src regulates mRNA translation and controls the expression of eIF3 subunits, including eIF3h, eIF3e, and eIF3d. These subunits are crucial for invadosome formation and ECM degradation. Src also modulates both canonical eIF4E-dependent translation via the activation of the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway and the non-canonical translation mechanism through eIF3d overexpression, both of which are necessary for invadosome function. Moreover, overexpression of Src and eIF3h/e/d correlates with poor prognosis in patients with hepatocellular carcinoma (HCC), promoting ECM degradation and tumor invasiveness of HCC cells. This study identifies Src as a major regulator of translation initiation, modulating invadosome formation, ECM degradation, and tumor cell invasion.

## INTRODUCTION

The degradation of the extracellular matrix (ECM) is a determinant step for cancer cell migration and invasion leading to tumor progression and metastasis development.<fnref n="1" /> Invadosomes are dynamic F-actin-based structures mainly involved in ECM remodeling in normal and cancer cells.<fnref n="2" />,<fnref n="3" /> Beyond their cytoskeletal architecture, invadosomes have emerged as sites of localized mRNA translation,<fnref n="4" />,<fnref n="5" /> as observed for cell migration,<fnref n="6" />,⁷ ensuring spatially restricted synthesis of proteins required for cell adhesion and invasion. We previously demonstrated that invadosomes from Rous sarcoma virus-transformed NIH 3T3 cells (NIH 3T3 Src) are enriched in translational components, including ribosomes and β-actin mRNA, and display local translation activity.⁸,⁹ Furthermore, we identified several translation initiation factors enriched in invadosomes as compared to the whole cell. Mass spectrometry revealed the presence of several eukaryotic initiation factors (eIFs), notably 8 out of the 13 subunits of the eukaryotic translation initiation factor 3 (eIF3) complex, suggesting that specialized eIF3-dependent translation machinery could operate within invadosomes. Aberrant expression of individual eIF3 subunits at both proteins and mRNAs has been reported in several cancers, including liver, lung, breast, and colorectal cancers.¹⁰ Functional studies have shown that

depletion of specific eIF3 subunits (eIF3a, eIF3b, eIF3c, eIF3e, eIF3g, and eIF3i) reduces global protein synthesis and impairs cell proliferation,¹¹ highlighting their essential role in ribosome biogenesis and the MAPK pathway regulation.¹² Some eIF3 subunits have also been involved in tumor invasion and metastasis: eIF3h depletion reduces the invasive capacity of hepatocellular carcinoma (HCC) cells,¹³ while eIF3d promotes breast cancer invasion and metastasis through a non-canonical cap-binding activity involving eIF4G2, a distinct variant of eIF4G.¹⁴ However, whether the eIF3 complex directly participates in invadosome formation and the localized translation processes driving cell invasion remains unknown.

The eIF3 complex plays a major role in translation initiation, mediating ribosome recruitment to mRNAs through coordinated interactions with the eIF4F complex (composed of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the RNA-helicase eIF4A). eIF3 promotes the assembly of the 43S preinitiation complex, comprising the 40S ribosomal subunit and numerous other eIFs (including eIF1, eIF1A, and eIF2), and strengthens mRNA binding to the eIF4F complex and scanning for the start codon.¹⁰ Cap-dependent mRNA translation is controlled by the phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway,¹⁵ which can be activated by the tyrosine kinase (PTK) Src.¹⁶ Src kinase activity has been shown to drive

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Bordeaux Institute of Oncology, BRIC U1312, INSERM, Université de Bordeaux, 33076 Bordeaux, France</footnote>

<footnote n="2">²Oncoprot Platform, TBM-Core US 005, 33076 Bordeaux, France</footnote>

<footnote n="3">³Centre de Recherche en Cancérologie de Toulouse (CRCT), INSERM U1037, Université Toulouse III Paul Sabatier, ERL5294 CNRS, 31037 Toulouse, France</footnote>

<footnote n="4">⁴Montpellier Cell Biology Research Center, CRBM, Université de Montpellier, CNRS, 34293 Montpellier, France</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>

<footnote n="6">⁶Lead contact</footnote>

<footnote>*Correspondence: benjamin.bonnard@inserm.fr (B.B.), frederic.saltel@inserm.fr (F.S.)
https://doi.org/10.1016/j.celrep.2026.116989</footnote>
the translation of polycomb complex components in breast cancer tumors, leading to their overexpression.¹⁷ In addition, Src signaling has been described to regulate various cellular functions, including proliferation, angiogenesis, and cytoskeleton organization,¹⁸ and may also promote CAP-dependent mRNA translation.¹⁶ Nevertheless, the direct link between Src activity and translation regulation remains unclear.

In this study, we demonstrate that Src activity regulates protein synthesis through activation of both canonical and non-canonical mRNA translation pathways and selective regulation of eIF3 subunits, identifying individual eIF3 subunits that are essential for invadosome formation and ECM degradation. Using next-generation sequencing (NGS) of laser-captured invadosomes, we identify eIF3-dependent target mRNAs involved in invadosome formation and function, including the RNA-binding protein IGF2BP2 (insulin-like growth factor 2 mRNA-binding protein 2) and transketolase (TKT), an enzyme involved in the pentose-phosphate pathway. Finally, we provide evidence that Src and eIF3 cooperate in HCC invasion, supporting a model in which Src-driven reprogramming of translation initiations sustains invasive cell behavior.

## RESULTS

### Src kinase activity regulates protein synthesis and specific eIF3 subunit expression

We reported previously that invadosome NIH 3T3 Src cells contain actively translating ribosomes and that the inhibition of mRNA translation with cycloheximide (CHX) reduced invadosome stability,⁸ as illustrated in Figures S1A and S1B. Here, we found that NIH 3T3 Src cells expressing the constitutively active form of Src (NIH 3T3 Src) have an overall translation level higher than parental NIH 3T3 wild-type (WT) cells, as assessed by surface sensing of translation (SUnSET) assays (Figure 1A). In NIH 3T3 Src cells, Src kinase inhibitors (PP2 or SU6656) reduced Src activity (Figures 1B and S1C), decreasing not only invadosome formation (Figures 1C and S1D) but also protein synthesis levels (Figures 1D and S1E), both of which are reversible (Figures 1D and S1E). Furthermore, small interfering RNA (siRNA) specifically targeting Src expression reduced translation activity in NIH 3T3 Src cells (Figure S1F). In addition, we also found that Src phosphorylation, assessed by the level of Y419 phosphorylation, positively correlates with the translation level in NIH 3T3 Src cells, as assessed by SUnSET (dot graph in Figure 1D). Finally, we found, using polysome profiling, that PP2 treatment caused a loss of heavy polysomes and a corresponding increase of 60S and 80S fractions (Figure S1G). Taken together, these observations suggest that the Src activation that controls invadosome formation also affects translation.

To further the way Src activation modulates protein expression, we compared the proteomes of NIH 3T3 WT cells and NIH 3T3 Src cells, treated or not with PP2. We found that 135 proteins were upregulated in NIH 3T3 Src cells as compared to NIH 3T3 WT cells, while 15 proteins were downregulated (volcano plot of Figure 1E). Conversely, PP2 treatment in NIH 3T3 Src cells downregulated 106 proteins and upregulated 38 (Figure S1H). We compared proteins upregulated in NIH 3T3 Src cells with NIH 3T3 WT cells with proteins previously found

enriched in invadosomes.⁹ We highlighted 10 proteins regulating mRNA translation. These included six subunits (eIF3c, eIF3e, eIF3f, eIF3g, eIF3h, and eIF3l) of the eIF3 complex, the largest translation initiation factor with a determinant role in protein synthesis¹⁰ (Venn diagram in Figure 1E; Table S1). In agreement with the proteomic data, immunoblot experiments showed that eIF3e and eIF3h are expressed at higher levels in NIH 3T3 Src cells as compared to NIH 3T3 WT cells (Figures 1F and S1I). Moreover, Src kinase inhibitors PP2 and SU6656 reduced expression of eIF3c, eIF3e, eIF3h, and eIF3l (Figures 1F, S1J, and S1K), whereas Src activity did not regulate the expression of eIF3i (Figures 1G and S1L). This suggests that Src activity controls the level of a subset of eIF3 subunits.

Altogether, the above results show that Src is a determining driver of protein synthesis, regulating important elements of the translational machinery, including a subset of eIF3 subunits.

To rigorously address the possibility that eIF3e/h/d subunit expression might be regulated specifically by the Src oncogenic pathway, we modulated the activity of several well-established oncogenes (β-catenin, KRAS, and cMyc) in distinct cancer cell models using a pharmacological approach. We therefore overexpressed active murine forms of the oncogenes Src (Src Y527F), β-catenin (β-catenin S33Y), and cMyc (cMyc T58A-His) in WT NIH 3T3 cells (Figure S2A). These fibroblast-derived cells do not form invadosomes or degrade the ECM under basal conditions. Indeed, the cytoskeleton of NIH 3T3 WT cells displays stress fibers (Figure S2B). As expected, overexpression of the Src Y527F construct induced invadosome formation and degradation of the ECM. A clear colocalization between the active form of Src and invadosomes was observed (Figure S2B). In contrast, in cells overexpressing β-catenin or cMyc, no changes were observed in cytoskeletal organization: there was no formation of invadosomes and no ECM degradation (Figure S2B). Under the same experimental conditions, when we labeled different subunits of the eIF3 complex (namely eIF3e, eIF3h, and eIF3d), we observed a specific enrichment of these eIF3 subunits only in the active Src condition (Figure S3).

Collectively, these results consistently demonstrate that the regulation of eIF3e/h/d expression is largely specific to Src activation, with minimal or no contribution from the oncogenic pathways tested.

### Src-regulated eIF3 subunits control invadosome formation and ECM degradation

Among the 13 subunits of the eIF3 complex, 8 were found to be enriched in invadosomes⁵ (Figure 2A), and 6 of these had their expression level regulated by Src, as demonstrated above. We confirmed by immunofluorescence that eIF3h and eIF3e localize to invadosomes (Figure 2B). To examine their role in invadosome formation, we treated NIH 3T3 Src cells with siRNAs targeting eIF3h or eIF3e. We found that the individual depletion of either subunit reduced invadosome formation, ECM degradation, and cell invasiveness (Figures 2C, 2D, S4A, and S4B). These results demonstrate that both eIF3h and eIF3e are mandatory for invadosome formation and function. As eIF3h and eIF3e contribute to the recruitment of eIF3k, eIF3l, and eIF3d subunits on the eIF3 complex,¹⁹ we assessed whether their depletion affected the level of other eIF3 subunits.
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Src kinase activity regulates protein synthesis and specific eIF3 subunit expression</figcaption></figure>

(A) The surface sensing of translation (SUnSET) assay was conducted on transfected NIH 3T3 WT (WT) and NIH 3T3 Src (Src) cells. The bar graph depicts the quantification of puromycin incorporation.

(B–E) Src cells were treated with Src inhibitor for 1 h (PP2 1H) followed by a recovery period in fresh medium (PP2 1H + R6H). DMSO treatment served as the control group (Ctrl).

(B) Src phosphorylation was evaluated by western blot.

(C) The top image displays representative images of invadosomes stained with phalloidin, with the accompanying bar graph indicating the number of invadosomes per nucleus. Scale bar: 40 µm. Error bars (mean ± SEM, *n* = 20 fields, four independent experiments, *p* < 0.05, a paired *t* test was used for statistical analysis).

(D) The SUnSET assay was performed, and cycloheximide (CHX) treatment was used as a negative control (left). The middle image illustrates the quantification of puromycin incorporation, while the right image displays the Pearson correlation between Src phosphorylation and puromycin incorporation in Src cells.

(E) The volcano plot displays downregulated (blue) and upregulated (red) proteins in Src cells compared to WT cells evaluated by mass spectrometry. Protein expression of eIF3c/e/h/i/l subunits is indicated in purple. The Venn diagram illustrates the overlap between deregulated translational proteins resulting from Src activation and those enriched into invadosomes compared to whole cells.

(F and G) Protein expression levels of eIF3h, eIF3e, and eIF3i in WT and Src cells treated or untreated with PP2 during 24 h. Protein expression was normalized with total protein content using stain-free (SF) technology. Bar graphs for western blot quantification are presented with error bars (mean ± SEM, four independent experiments, *p* < 0.05, a paired *t* test was used for statistical analysis). Protein expression was normalized with total protein content using SF technology.

The depletion of eIF3h reduced the levels of eIF3e, eIF3d, and eIF3k, whereas the depletion of eIF3e reduced expression of eIF3d and eIF3k but not eIF3h. The expression of eIF3a, eIF3b, and eIF3c core subunits of the complex was not affected (Figures S4C and S4D).

As they are regulated by eIF3h and eIF3e, we examined whether eIF3d and eIF3k also participate in invadosome formation and degradation activity using specific siRNAs. We demonstrated that eIF3d is required for invadosome formation and function (Figures 2E and S4E), as are eIF3h and eIF3e.
A

eIF3 enrichment
(invadosome vs whole cells)

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

D

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

E

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

F

<figure><img src="image_14.png" /></figure>

(legend on next page)
In contrast, eIF3k appears dispensable (Figure S4F). Of note, the depletion of eIF3d or eIF3k did not affect the expression of eIF3h, eIF3e, and eIF3l, nor the expression of the core subunits, except for a slight increase in eIF3c levels upon eIF3d knockdown (Figures S4G and S4H). These results demonstrate that eIF3h and eIF3e play a crucial role in NIH 3T3 Src cell invasiveness by regulating invadosome formation and ECM degradation, as does eIF3d, whose expression level is controlled by those of eIF3h and eIF3e.

As the eIF3 complex is a master regulator of mRNA translation initiation, we assessed the overall protein synthesis upon siRNA-mediated depletion of individual eIF3 subunits in NIH 3T3 Src cells. SUnSET experiments revealed that depletion of eIF3h, eIF3e, or eIF3d strongly depressed translation levels, whereas eIF3k depletion had no effect (Figure 2F). The above results show that eIF3 subunits regulated by Src activation control invadosome formation and function by controlling the level of individual eIF3 subunits.

## Specific eIF3-dependent mRNAs and product proteins colocalize in invadosomes and control ECM degradation and invasion

As eIF3 localizes to invadosomes that correspond to an active translation site, we hypothesized that eIF3-dependent mRNAs essential for invadosome formation and function could also localize into these structures. We established the transcriptome of invadosomes by combining the laser capture microdissection of invadosomes and NGS (Figure 3A). Thereby, we identified 2,984 mRNAs enriched in invadosomes as compared to whole cells (Figure 3B). Among the most significantly enriched mRNAs in invadosomes, many encode for proteins of the cytoskeleton (Figure S5A) that play a key role in invadosomes,²⁰⁻²² such as ACTA1/2, ACTG2, TUBB2A/B, TUBB3A/B, and TUBB5 (Table S2). We noted a 21% overlap, corresponding to 37 mRNAs + proteins, between the proteome⁸ and the transcriptome of invadosomes (Figure 3C; Table S3), which may correspond to mRNAs translated in invadosomes. Second, using the proteomic approach, we identified the proteins affected by eIF3e depletion (Table S4). Among the 37 mRNAs, we found 3 common hits: eIF3e itself, as expected, as well as IGF2BP2 and TKT. IGF2BP2 is a well-described mRNA-binding protein known to participate in cell motility and invadosome formation.²³ TKT is a key enzyme involved in the non-oxidative pentose phosphate pathway and is deregulated in several cancers, including liver, pancreatic, and colorectal cancers.²⁴ TKT was shown to promote the proliferation, migration, and invasion of colorectal

cancer cell lines,²⁵ but its role in invadosomes was unknown so far. We confirmed that both are present at the protein level in invadosomes (Figures 3D and S5B). Moreover, their depletion using siRNA impacts invadosome formation and ECM degradation activity (Figures 3E, S5C, and S5D), as well as cell invasiveness (Figure S5F).

However, using fluorescence *in situ* hybridization (FISH), we were only able to localize and quantify IGF2BP2 mRNA at the invadosome level; this was not the case for TKT (Figures 3F and S5E).

These results demonstrate that eIF3 controls the expression of specific mRNAs in NIH 3T3 cells that are essential for invadosome formation and function. Some of these mRNAs localize with their product protein in invadosomes and are essential for their function, such as IGF2BP2.

## Src kinase activity enhances canonical “eIF4E-dependent” and non-canonical “eIF3d-dependent” eukaryotic translation initiation leading to invadosome formation

To uncover how Src activity could regulate eIF3-dependent translation, we performed a comparative Ingenuity Pathway Analysis on the proteome of NIH 3T3 Src cells compared to NIH 3T3 parental cells. This revealed that Src activation mostly triggers the mTOR, the ribosomal protein S6 kinase B1 (S6K1), and the eIF2 signaling pathways (Figure 4A, blue dots). Interestingly, we also found that mTOR, S6K1, and eIF2 signaling pathways were enriched in the invadosome proteome⁵ (Figure 4A, yellow dots). To identify the kinases whose activity is regulated downstream of Src, we performed a wide serine/threonine (STK) and PTK activity profiling comparing NIH 3T3 WT cells and NIH 3T3 Src cells, treated or not with PP2 (Tables S5 and S6). Thereby, we highlighted 8 PTKs and 7 STKs, including S6K1, modulated by Src activity (Tables S5 and S6). A STRING protein network analysis showed that 11 out of these 15 kinases are linked to mTOR (Figure S6A), suggesting that Src could be involved in the regulation of mTOR in NIH 3T3 Src cells. As the PI3K/AKT axis is the major upstream activator of mTOR signaling,¹⁵ known to be activated by Src,²⁶ we analyzed the phosphorylation status of PI3K/mTOR downstream effectors. We confirmed that the activating phosphorylation of AKT (S473 and T308), S6K1 (T389), and 4E-BP1 (T37/46) was higher in NIH 3T3 Src cells as compared to NIH 3T3 WT cells and lower upon PP2 or SU6656 treatment (Figure S6B). These results show that Src kinase activity activates the mTOR signaling pathway in NIH 3T3 Src cells.

## Figure 2. Key role of Src-regulated eIF3 subunits in invadosome formation and ECM degradation

(A) Schematic representation of eukaryotic translation initiation factor 3 (eIF3) complex showing the individual enrichment of eIF3 subunit into invadosomes vs. whole cells from Ezzoukhry et al.⁸ The colored bar represents the enrichment ratio (invadosome vs. whole cells) from <2 (white) to >10 (red).

(B) Confocal microscopy images of F-actin (red) and eIF3h or eIF3e (green) in NIH 3T3 Src cells. Scale bar: 40 µm.

(C-E) NIH 3T3 Src cells were transfected with siRNA control (si Ctrl) or with two independent siRNAs targeting eIF3h (C), eIF3e (D), or eIF3d (E). Transfected NIH 3T3 Src cells were seeded on a fluorescent gelatin matrix. The top image shows representative images of invadosomes stained with phalloidin; the bar graph shows the number of invadosomes per nucleus. The bottom image shows representative images of the degradation area (black); the bar graph shows the quantification of the matrix degradation area per nucleus after 24 h. Scale bar: 40 µm. Error bars (mean ± SEM, n = 20 fields, three independent experiments, *p* < 0.05, a paired t test was used for statistical analysis).

(F) SUnSET assay performed on transfected NIH 3T3 Src cells. Protein expression was normalized with total protein content using stain-free (SF) technology. The bar graphs show the quantification of puromycin incorporation. Error bars (mean ± SEM, three independent experiments, *p* < 0.05, a paired t test was used for statistical analysis).
<figure><img src="image_15.png" /></figure>

The PI3K/mTOR pathway plays a crucial role in canonical translation initiation by phosphorylating 4E-BP1 and enhancing eIF4E/eIF4G interaction, leading to the recruitment of the eIF3 complex and formation of the 48S preinitiation complex.¹⁵ In agreement with the above activation of the mTOR signaling pathway by Src, we found that the Src inhibitor PP2 reduced eIF4E/eIF4G interaction in NIH 3T3 Src cells. This was assessed by eIF4E immunoprecipitation from NIH 3T3 Src cells (Figure 4B) and confirmed by cap-column (Figure S6C). PP2-mediated disruption of eIF4G/eIF4E was similar to that observed after treatment with the PI3K inhibitor LY294002, the mTOR inhibitor rapamycin, or a 4EGi-1 inhibitor (Figure 4B). This demonstrated that canonical eIF4E-dependent translation initiation was abol-

<caption>Figure 3. Specific mRNAs and product proteins eIF3-dependently colocalize in invadosomes and control ECM degradation and invasion</caption>

(A) Schematic workflow of invadosome isolation for transcriptomic analysis.

(B) Volcano plot displaying enriched mRNAs in invadosomes compared to whole cells, with downregulated mRNAs (blue) and upregulated mRNAs (red). *IGF2BP2* and *TKT* expression is highlighted in purple.

(C) Venn diagram demonstrating the overlap of enriched mRNAs (red circle) and enriched proteins (gray circle) in invadosomes.

(D) Confocal microscopy images of F-actin (red) and IGFBP2 (green) in NIH 3T3 Src cells. Scale bar: 20 µm.

(E) NIH 3T3 Src cells were transfected with either siRNA control (si Ctrl) or two independent siRNAs targeting IGFBP2 and then seeded on a fluorescent gelatin matrix. The top image presents representative images of invadosomes stained with phalloidin, with the bar graph indicating the number of invadosomes per nucleus. The bottom image displays representative images of the degradation area (depicted in black), with the bar graph quantifying the matrix degradation area per nucleus after 24 h. Scale bar: 40 µm. Error bars (mean ± SEM, n = 20 fields, four independent experiments, *p* < 0.05, a paired *t* test was used for statistical analysis).

(F) Single-molecule fluorescence *in situ* hybridization was used to localize IGF2BP2 mRNAs in NIH 3T3 Src cells. Z-projections of 2 out of 13 slices of the z stacks encompassing the whole cell height. Scale bar: 10 µm. The right image shows pixel quantification of IGF2BP2 mRNAs in invadosomes and whole cells (n = 12 cells with associated invadosomes were considered, *p* < 0.05, a paired *t* test was used for statistical analysis).

ished by PP2, leading to a reduction in invadosome formation in NIH 3T3 Src cells (Figure 4C). Interestingly, the Src inhibitor displays a stronger effect on protein synthesis compared to other compounds (Figure 4D), suggesting that Src exerts

an additional control on protein synthesis in NIH 3T3 Src cells. We hypothesized that eIF3d was involved in this process, as it has been shown to have a direct cap-binding capacity²⁷ and to mediate translation initiation upon mTOR inhibition.²⁸,²⁹ Furthermore, we identified the overexpression of eIF3d in NIH 3T3 Src cells when compared to NIH 3T3 cells, which is reversed by PP2 or SU6656 treatment (Figure S6D). Indeed, the combination of the mTOR inhibitor rapamycin with eIF3d silencing resulted in a similar inhibition of protein synthesis (Figure 4E) and invadosome disruption (Figure 4F) to PP2 treatment. Altogether, these results demonstrate Src involvement in both canonical eIF4E-dependent and non-canonical eIF3d-dependent mRNA translation initiation, through the activation of mTOR signaling and the
<figure><img src="image_16.png" /><figcaption>**Figure 4.** Src kinase activity enhances canonical and non-canonical eukaryotic translation initiation leading to invadosome formation</figcaption></figure>

(A) The bubble plot illustrates the gene set enrichment analysis conducted on the Ingenuity Pathways Analysis database based on the Src-dependent proteome (depicted in blue) and identified proteome enrichment in invadosomes (depicted in yellow). The color and size of the circles indicate the significance level and number of proteins, respectively, from each enriched pathway.

(B-D) NIH 3T3 Src cells underwent treatment with various inhibitors—Src inhibitor (PP2), PI3K inhibitor (LY294002), mTOR inhibitor (rapamycin), and inhibitor of eIF4E/eIF4G interaction (4EGi-1)—for 1 h. DMSO treatment served as the control group (Ctrl).

(B) The left image presents immunoblot results of immunoprecipitated (IPed) and input fractions following eIF4E pull-down. The right image displays the quantification of the eIF4E/eIF4G ratio in the IPed fraction, normalized with respective expression in the input fraction. Error bars (mean ± SEM, four independent experiments, \*p < 0.05, a paired t test was used for statistical analysis).

(C) The left image exhibits representative images of invadosomes stained with phalloidin, while the bar graph illustrates the number of invadosomes per nucleus. Scale bar: 40 µm. Error bars (mean ± SEM, n = 20 fields, four independent experiments, \*p < 0.05, a paired t test was used for statistical analysis).

(D) SUnSET assay performed on treated NIH 3T3 Src cells. The bar graphs show the quantification of puromycin incorporation. As a negative control, NIH 3T3 Src cells were incubated with cycloheximide (CHX).

*(legend continued on next page)*
<figure><img src="image_17.png" /></figure>

overexpression of eIF3d, which both contribute to invadosome
formation in NIH 3T3 Src cells.

The Src-eIF3h/3e/3d axis is enriched in patients with
HCC and enhances the invasiveness of the HCC cell line

In order to explore whether the Src-eIF3 pathway could
contribute to cancer invasion, we explored the correlative
expression of Src and eIF3 subunit genes in human cancers us-
ing the Human Protein Atlas database. We found that high
expression of Src, eIF3h, eIF3e, and eIF3d negatively correlated
with patient survival in 4 types of cancers: liver, lung, pancreatic,
and renal (Figure S7A). Based on GEO databases (GEO:
GSE25097), we found a positive correlation between Src,
eIF3h, eIF3e, and eIF3d gene expression only in patients with
liver cancer (Figures 5A and S7B). We also extracted information
from open data on patients with HCC, the most common type of
liver cancer. Here, we found that Src, eIF3h, eIF3e, and eIF3d
mRNA levels were increased in tumor (T) as compared to non-tu-
moral (NT) tissue (Figure 5B). Immunohistochemistry on liver bi-
opsies from patients with HCC revealed that eIF3h, eIF3e, and
eIF3d protein staining is higher in the tumor as compared to
the adjacent tissue (Figure 5C), while total Src staining was
similar. This suggests that eIF3 is upregulated in tumor sections
(or at least specific eIF3 subunits), and this could have a role in
the progression of HCC.

In order to examine whether the Src/eIF3 axis could promote
invasiveness in HCC, we used the Huh7 HCC cell line. Huh7 cells
seeded on type I collagen matrix and treated with TGF-β1 are
known to acquire an invasive phenotype due to the formation
of ECM-degrading linear invadosomes (Figures 5D and 5E).³⁰
This is accompanied by increased Src phosphorylation
(Figure 5F) and higher expression of eIF3h and eIF3e as well as
a slight increase in eIF3d (Figure 5F). We found that the downre-
gulation of eIF3h, eIF3e, or eIF3d with siRNAs prevented the
TGF-β1-induced ECM degradation by Huh7 cells and reduced
their invasive capacity (Figures 5G–5I and S7C–S7E).

Altogether, these results show that eIF3h, eIF3e, and eIF3d
subunits could participate in tumor progression in patients with
HCC by contributing to the regulation of ECM degradation.

DISCUSSION

Basement membrane crossing and ECM remodeling are two
critical steps that enable invasive cancer cells to migrate and
metastasize. Our study revealed a molecular mechanism down-
stream of Src kinase leading to the modulation of protein synthe-
sis, both canonically through the AKT/mTOR pathway and non-
canonically through eIF3d-dependent translation initiation
(Figure 6). To support the notion of active mRNA translation at
the invadosome, we defined the enrichment of several eIF3 sub-

units within this structure, as well as eIF3-dependent mRNAs,
and encoded protein products, such as IGF2BP2, which are
functionally involved in invadosome formation and ECM degra-
dation. Finally, we demonstrated that the Src/eIF3 signaling
pathway is critical for HCC cell invasiveness.

Oncogenes, such as Myc or Ras, favor the expression of the
general translation machinery, including ribosome biogenesis
and translation initiation factors.³¹ Although the underlying
mechanisms remain elusive, this observation corresponds to a
cellular response to the need to increase cell body mass (protein
content) in order to cope with intensive cell division and duplica-
tion of cell organelles.³¹ Similarly, Src appears to control protein
synthesis requirements through multiple mechanisms. First, the
stimulation of one of the major axes of translational control, the
PI3K/AKT/mTOR pathway, is achieved through sequential phos-
phorylation controlled by Src. Activation of mTOR is observed
upregulated up to 80% of breast, ovarian, and colorectal can-
cers as a result of upstream mutations (EGFR, PI3K, etc.) or de-
letions (PTEN, LKB1, etc.) and favors phosphorylation of 4E-BP1
and the association of cap-bound eIF4E with eIF4G. Second, Src
upregulates several eIF3 subunits. Several eIF3 subunits have
oncogenic properties, including eIF3a, eIF3b or eIF3h,³² and
eIF3e.³³ Finally, Src induces the overexpression of eIF3d. This
subunit is able to drive an eIF4E-independent translation initia-
tion by binding to the cap structure. Taken together, our study
highlights the ability of Src to modulate mRNA translation
through multiple mechanisms, and this is essential for invado-
some formation and cell-invasive properties.

Interestingly, Shin et al. identified that inhibition of the mTOR/
eIF4E axis promotes cell phenotype switching from a prolifera-
tive to a migratory status through the activation of non-canonical
eIF3d-dependent translation.²⁸ In addition, another study
demonstrated that the formation of the eIF4G2 (DAP5)/eIF3d
dimer can promote cancer cell EMT, invasiveness, and metas-
tasis.¹⁴ Whereas we identify the role of eIF3d in global protein
synthesis and invadosome formation, eIF3d is not specifically
enriched in invadosomes, in contrast to eIF3e and eIF3h. This
suggests that these other subunits may have a more specific
function driving local mRNA translation at the invadosome.

Locally enriched translation machinery plays a crucial role in
stabilizing cellular protrusion.⁷,⁸,³⁴ Src has been shown to regu-
late the spatial translation of β-actin mRNA through phosphory-
lation of IGF2BP1 in neuronal outgrowth,⁴ supporting the poten-
tial role of local translation into invadosomes. In search of the
underlying molecular mechanism, we explored the BioGRID
database and uncovered the specific interaction of eIF3h with
components of the invadosome actin structure, such as ARP3
(ACTR3) and cortactin (CTTN).³⁵ Importantly, other eIF3 subunits
did not show such enrichment, although each subunit was found
to interact with the rest of the eIF3 complex. Future experiments

<footnote>(E and F) NIH 3T3 Src cells were transfected with siRNA control (si Ctrl) or with two independent siRNAs targeting eIF3d and then treated with rapamycin or PP2 or with DMSO as a control for 1 h.</footnote>

<footnote>(E) The SUrSET assay was performed on treated NIH 3T3 Src cells. The bar graphs show the quantification of puromycin incorporation.</footnote>

<footnote>(F) The bar graph illustrates the number of invadosomes per nucleus. Scale bar: 20 µm. Error bars (mean ± SEM, n = 20 fields, four independent experiments, \*p < 0.05, a paired t test was used for statistical analysis). Protein expression was normalized with total protein content using stain-free (SF) technology. Bar graphs for western blot quantification were presented with error bars (mean ± SEM, four to five independent experiments, \*p < 0.05, a paired t test was used for statistical analysis).</footnote>
A

<figure><img src="image_18.png" /></figure>

B

<figure><img src="image_19.png" /></figure>

C

<figure><img src="image_20.png" /></figure>

D

Huh7 seeded on Collagen I + TGF-β1

<figure><img src="image_21.png" /></figure>

E

<figure><img src="image_22.png" /></figure>

F

<figure><img src="image_23.png" /></figure>

G

<figure><img src="image_24.png" /></figure>

H

<figure><img src="image_25.png" /></figure>

I

<figure><img src="image_26.png" /></figure>

(legend on next page)
should aim to define whether specific interactions of eIF3h with invadosome components favor the sustained assembly of this actin structure by maintaining locally specific mRNAs or the translation machinery per se. We also showed that Src-induced PI3K/mTOR activation promotes interaction between the eIF4F subunits eIF4E and eIF4G, which are necessary for invadosome formation and ECM degradation. This supports the determining role of cap-dependent translation in cell invasiveness. Since eIF4E/eIF4G interaction is required for cap-dependent translation initiation¹⁵ and the eIF3 complex directly interacts with eIF4G through the eIF3e subunit,³⁶ we demonstrated that impairment of eIF4E/eIF4G interaction and deregulation of eIF3e expression are required for invadosome formation. Interestingly, the collagen-induced β1 integrin activation increased Src and eIF4G activity in human lung fibroblasts.³⁷

We described the differential role of eIF3 subunits in invado-
some formation. While depletion of eIF3k has no major effect
on invadosomes, depletion of eIF3h, eIF3e, and eIF3d strongly
impacts both invadosome formation and ECM degradation
and is associated with a lower translation level. In our study,
we suggest that eIF3h and eIF3d drive canonical and non-
canonical translation, respectively, while eIF3e acts at the
interconnection of both modes of translation according to its
own role in the dynamics of eIF3 complex formation. ¹²
This explains the huge effects of eIF3e depletion on protein
synthesis in NIH 3T3 Src cells. Another study confirmed our
interest in studying the eIF3 complex in invadosomes, as 11
of the 13 eIF3 subunits were found to interact with p190Rho-
GAP,³⁸ a key driver of invadosome formation.³⁹ Interestingly,
a recent phosphoproteomic-based study confirmed that
Src phosphorylation activates translational initiation factor
eIF4A1,⁴⁰ supporting the idea that Src activation promotes
translation. However, we found that eIF4A1 and eIF4A3 were
enriched in NIH 3T3 Src cells compared to NIH 3T3 WT cells,
while Src inhibition reduced the expression of eIF4G1. None
of the eIF4F subunits was found to be enriched in invado-
somes compared to whole cells. Instead of eIF4F, the eIF3
complex appears to be a key driver in the formation of
invadosomes.

Local translation was first identified in neurons,⁴¹ where
mRNA localization and translation at a specific site are involved
in various processes, such as neuron development, synapto-
genesis, and experience-dependent plasticity and survival in
dendrites and axons. Local translation of mRNAs is also
required for cellular protrusion stabilization.³⁴,⁴² Many studies
reviewed and deciphered the regulation of mRNA trafficking
to their translation; nevertheless, the exact molecular mecha-
nisms remain unclear.⁴³ In order to decipher the translatome
in invadosomes, we used an innovative method combining
transcriptomic and proteomic analysis on isolated invado-
somes from the cell body via laser capture microdissection.⁸
We were able to highlight 37 mRNAs and their respective
coding proteins enriched into invadosomes, suggesting that
they could be translated locally. Among these, we identified
IGF2BP2 and TKT, whose mRNA translation depends on
eIF3, and they are both required for invadosome formation
and ECM degradation. IFG2BP2 was also shown to be involved
in dot-like invadosome formation in the triple-negative breast
cancer cell line MDA-MB-231.²³ IFG2BP2 is known to enhance
mRNA stability and translation, including eIF3-target mRNAs
such as c-Myc.⁴⁴ Furthermore, a study demonstrated that
IFG2BP2 is one of the most enriched proteins in the β-actin
mRNA interactome. This suggests that IFG2BP2 could promote
translation of β-actin⁴⁵ and stabilize cellular protrusions,
including invadosomes. Interestingly, TKT, an enzyme involved
in the non-oxidative pentose phosphate pathway, is highly ex-
pressed in several types of cancer, and its expression is asso-
ciated with poor prognosis and increased proliferation and
metastasis.²⁴ Moreover, TKT depletion reduced cell invasive-
ness in colorectal cancer cells,²⁵ which is consistent with our
finding that TKT is essential for invadosome formation. Howev-
er, among the 37 targets that overlap between invadosome
proteome and transcriptome, some of them have a determinant
role in cancer (including TRPM1 or MYOF). Indeed, TRPM1 is
an acting-binding protein and is involved in epithelial-mesen-
chymal plasticity leading to metastasis development and
chemotherapy resistance.⁴⁶ MYOF mediates breast cancer in-
vasion and epithelial-to-mesenchymal transition,⁴⁷,⁴⁸ and

<caption>Figure 5. The Src-eIF3h/3e/3d axis is enriched in patients with HCC and enhances the invasiveness of the HCC cell line</caption>

(A) The heatmap illustrates the correlation between the mRNA expression of Src and eIF3 subunits across four distinct cancer types (liver, lung, pancreatic, and renal).

(B) Comparative transcriptomic analysis ([http://lifeome.net/database/hccdb/home.html](http://lifeome.net/database/hccdb/home.html)) conducted on tumor (T) vs. non-tumor (NT) sections obtained from liver biopsies.

(C) Liver biopsy samples from patients with hepatocellular carcinoma (HCC) were stained with hematoxylin and eosin, as well as stained for Src, eIF3h, eIF3e, and eIF3d proteins. Bar graphs show a positive staining area normalized with total area. Scale bar: 400 µm (n = 4, *p* < 0.05, a paired t test was used for statistical analysis).

(D) Huh7 cells were treated with transforming growth factor β1 (TGF-β1) for 48 h and then seeded on fluorescent collagen type I matrix. TKS5 has been used to reveal linear invadosomes. The arrowheads indicate the colocalization of TKS5 with actin and collagen I fibers showing linear invadosomes. Scale bar: 20 µm.

(E) Huh7 cells were cultured on a fluorescent gelatin matrix coated with collagen type I and treated or not with TGF-β1. The left image presents representative images of the degradation area (depicted in black), with the accompanying bar graph quantifying the matrix degradation area per nucleus after 24 h. Scale bar: 20 µm. Error bars (mean ± SEM, n = 20 fields, four independent experiments, *p* < 0.05, a paired t test was used for statistical analysis).

(F) Bar graphs illustrate the phosphorylation status of Src and the protein expression levels of eIF3h, eIF3e, and eIF3d. Protein expression was normalized with total protein content using stain-free (SF) technology. Bar graphs for western blot quantification were presented with error bars (mean ± SEM, four to five independent experiments, *p* < 0.05, a paired *t* test was used for statistical analysis).

(G–I) Huh7 cells were transfected with siRNA control (si Ctrl) or two independent siRNAs targeting eIF3h (G), eIF3e (H), or eIF3d (I) and then cultured on a fluorescent gelatin matrix coated with collagen type I and treated with TGF-β1. The left image presents representative images of the degradation area (depicted in black), with the accompanying bar graph quantifying the matrix degradation area per nucleus after 24 h. Scale bar: 40 µm. Error bars (mean ± SEM, n = 20 fields, four independent experiments, *p* < 0.05, a paired *t* test was used for statistical analysis).
<figure><img src="image_27.png" /><figcaption>Figure 6. The Src activation regulates translation leading to invadosome formation</figcaption></figure>

Src-mediated phosphorylation drives protein translation through two distinct pathways: the canonical PI3K/mTOR signaling cascade and a non-canonical mechanism involving the regulation of specific eIF3 subunits—namely eIF3h, eIF3e, and eIF3d. These processes collectively facilitate the formation of mature invadosomes. The significance of these findings is further supported by both pharmacological inhibition and targeted genetic approaches, which confirm the critical role of Src-induced translational signaling.

It remains unclear how Src modulates transcriptional factors to promote or reduce gene expression of identified targets. Further experiments to investigate the localized translation in subcellular fraction could provide an additional information related to Src-dependent translation. In addition, comparing the translatome between various types of invadosomes could highlight the specific downstream mechanism of these subcellular structures. Finally, targeting the identified Src-regulated mRNAs should be investi-

gated in an *in vivo* model of HCC to evaluate the potential therapeutic translatability of our findings.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Frederic Saltel (frederic.saltel@inserm.fr).

### Materials availability

This study did not generate new unique reagents.

### Data and code availability

Proteomic data have been deposited to the ProteomeXchange Consortium, and bulk RNA sequencing (RNA-seq) data have been deposited to the Science Data Bank and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Furthermore, the Excel table is also available as Table S7. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. This work does not contain any custom software for the data analysis.

## ACKNOWLEDGMENTS

We thank Prof. Paulette Bioulacl-Sage for the expertise in histological analysis. We also thank the Bordeaux Imaging Center (confocal and live-cell imaging), Vect'UB ( lentivirus production), and Neurocentre Magendie (laser microdissection and transcriptomic facilities) for their support of this study. This research has been supported and funded by the Agence Nationale de la Recherche (ANR SubRnaAct, Projet-ANR-23-CE13-0033), La Ligue Contre le Cancer (équipe labélisée 2023), and Fondation ARC 2023 et L'Institut National du Cancer.

specific MYOF inhibitors showed an anti-tumor effect on breast cancer.⁴⁹

While the protein form of Src was similar between tumor and non-tumor regions in biopsies from patients with HCC, the activated form of Src (pY416) was positively correlated with tumor grade and associated with poor patient survival in HCC.⁵⁰ Furthermore, the activation of pY416-Src remains an important driver for α₂-macroglobin-mediated invasion and metastasis in HCC cell lines.⁵¹ In our study, we demonstrated that inhibition of Src phosphorylation by PP2 reduced TGF-β1-induced Huh7 invasiveness. More recently, Yang et al. demonstrated that the depletion of Src also reduced both tumor growth and invasion in Huh7 and HepG2 cells.⁵² Similarly to Src, the overexpression of several individual eIF3 subunits has been detected in several types of cancer, including HCC.⁵³ We and others demonstrated an increase of eIF3h mRNA and protein expression in liver cancer, including HCC.¹³,⁵⁴ Interestingly, eIF3h overexpression was also associated with microvascular invasion and advanced tumor-node-metastasis T stage.¹³ Our results showed that depletion of eIF3h or eIF3e reduced HCC cell line invasiveness in accordance with the literature.¹³ Overall, our data demonstrate that specific eIF3 subunits regulated by Src activity represent a signaling pathway involved in the invasive process and could represent a potential emerging therapeutic target in HCC.

## Limitations of the study

Our current study focused on investing the role of the Src oncogene that regulates mRNA translation and controls the expression of specific eIF3 subunits leading to invadosome formation.
## AUTHOR CONTRIBUTIONS

B.B. designed and performed the experiments. A.C. contributed to the experiment realization. S.D.T., J.-W.D., and A.-A.R. performed and interpreted the mass spectrometry analysis. M.L.B. and J.S. performed polysome profiling and the associated bioinformatic analysis. I.M. helped with IHC staining. A.B. and C.D. contributed to RNA-seq and bioinformatic analyses, respectively. B.B., A.J., A.B., Y.M., and F.S. designed the study, and F.S. led the study. B.B. wrote the manuscript.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS**

* Cell culture

* Patient samples

* **METHOD DETAILS**

* Transfection

* Western Blot

* eIF4E immunoprecipitation and eIF4G blot analysis

* Sunset assay

* Degradation assay

* Invasion assay

* Polysome profiling and bioinformatical analyses

* Immunofluorescence and imaging

* Co-staining β-actin mRNA and coding protein

* Single Molecule Fluorescence In Situ Hybridization

* Laser microdissection

* Label-free quantitative proteomics

* RNA extraction

* Libraries synthesized

* Human database

* Bioinformatics analysis

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2026.116989.

Received: August 10, 2024

Revised: November 5, 2025

Accepted: January 20, 2026

## REFERENCES

1. Kai, F., Drain, A.P., and Weaver, V.M. (2019). The Extracellular Matrix Modulates the Metastatic Journey. Dev. Cell 49, 332-346. https://doi.org/10.1016/j.devcel.2019.03.026.

2. Linder, S., Cervero, P., Eddy, R., and Condeelis, J. (2023). Mechanisms and roles of podosomes and invadopodia. Nat. Rev. Mol. Cell Biol. 24, 86-106. https://doi.org/10.1038/s41580-022-00530-6.

3. Chen, W.T., and Wang, J.Y. (1999). Specialized surface protrusions of invasive cells, invadopodia and lamellipodia, have differential MT1-MMP, MMP-2, and TIMP-2 localization. Ann. N. Y. Acad. Sci. 878, 361-371. https://doi.org/10.1111/j.1749-6632.1999.tb07695.x.

4. Hüttelmaier, S., Zenklusen, D., Lederer, M., Dictenberg, J., Lorenz, M., Meng, X., Bassell, G.J., Condeelis, J., and Singer, R.H. (2005). Spatial

regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 512-515. https://doi.org/10.1038/nature04115.

5. Dermit, M., Dodel, M., Lee, F.C.Y., Azman, M.S., Schwenzer, H., Jones, J.L., Blagden, S.P., Ule, J., and Mardakheh, F.K. (2020). Subcellular mRNA Localization Regulates Ribosome Biogenesis in Migrating Cells. Dev. Cell 55, 298-313.e10. https://doi.org/10.1016/j.devcel.2020.10.006.

6. Moriarty, R.A., Mili, S., and Stroka, K.M. (2022). RNA localization in confined cells depends on cellular mechanical activity and contributes to confined migration. iScience 25, 103845. https://doi.org/10.1016/j.isci.2022.103845.

7. Moissoglu, K., Yasuda, K., Wang, T., Chrisafis, G., and Mili, S. (2019). Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport. eLife 8, e44752. https://doi.org/10.7554/eLife.44752.

8. Ezzoukhry, Z., Henriet, E., Cordelières, F.P., Dupuy, J.-W., Maître, M., Gay, N., Di-Tommaso, S., Mercier, L., Goetz, J.G., Peter, M., et al. (2018). Combining laser capture microdissection and proteomics reveals an active translation machinery controlling invadosome formation. Nat. Commun. 9, 2031. https://doi.org/10.1038/s41467-018-04461-9.

9. Normand, L., Bonnard, B., Sala, M., Di Tommaso, S., Dourthe, C., Raymond, A.-A., Dupuy, J.-W., Mercier, L., Goetz, J.G., Moreau, V., et al. (2025). Tks5 interactome reveals endoplasmic-reticulum-associated translation machinery in invadosomes. FEBS J. 292, 6400-6419. https://doi.org/10.1111/febs.70196.

10. Gomes-Duarte, A., Lacerda, R., Menezes, J., and Romão, L. (2018). eIF3: a factor for human health and disease. RNA Biol. 15, 26-34. https://doi.org/10.1080/15476286.2017.1391437.

11. Wagner, S., Herrmannová, A., Sikrová, D., and Valášek, L.S. (2016). Human eIF3b and eIF3a serve as the nucleation core for the assembly of eIF3 into two interconnected modules: the yeast-like core and the octamer. Nucleic Acids Res. 44, 10772-10788. https://doi.org/10.1093/nar/gkw972.

12. Herrmannová, A., Jelínek, J., Pospíšilová, K., Kerényi, F., Vomastek, T., Watt, K., Brábek, J., Mohammad, M.P., Wagner, S., Topisirovic, I., and Valášek, L.S. (2024). Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathways. eLife 13, RP95846. https://doi.org/10.7554/eLife.95846.

13. Zhu, Q., Qiao, G.-L., Zeng, X.-C., Li, Y., Yan, J.-J., Duan, R., and Du, Z.-Y. (2016). Elevated expression of eukaryotic translation initiation factor 3H is associated with proliferation, invasion and tumorigenicity in human hepatocellular carcinoma. Oncotarget 7, 49888-49901. https://doi.org/10.18632/oncotarget.10222.

14. Alard, A., Katsara, O., Rios-Fuller, T., Parra, C.d.l., Ozerdem, U., Ernlund, A., and Schneider, R.J. (2023). Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation. Cell Rep. 42, 112646. https://doi.org/10.1016/j.celrep.2023.112646.

15. Yang, M., Lu, Y., Piao, W., and Jin, H. (2022). The Translational Regulation in mTOR Pathway. Biomolecules 12, 802. https://doi.org/10.3390/biom12060802.

16. Karni, R., Gus, Y., Dor, Y., Meyuhas, O., and Levitzki, A. (2005). Active Src elevates the expression of beta-catenin by enhancement of cap-dependent translation. Mol. Cell Biol. 25, 5031-5039. https://doi.org/10.1128/MCB.25.12.5031-5039.2005.

17. Smith, H.W., Hirukawa, A., Sanguin-Gendreau, V., Nandi, I., Dufour, C.R., Zuo, D., Tandoc, K., Leibovitch, M., Singh, S., Rennhack, J.P., et al. (2019). An ErbB2/c-Src axis links bioenergetics with PRC2 translation to drive epigenetic reprogramming and mammary tumorigenesis. Nat. Commun. 10, 2901. https://doi.org/10.1038/s41467-019-10681-4.

18. Parkin, A., Man, J., Timpson, P., and Pajic, M. (2019). Targeting the complexity of Src signalling in the tumour microenvironment of pancreatic cancer: from mechanism to therapy. FEBS J. 286, 3510-3539. https://doi.org/10.1111/febs.15011.
19. Smith, M.D., Arake-Tacca, L., Nitido, A., Montabana, E., Park, A., and Cate, J.H. (2016). Assembly of eIF3 Mediated by Mutually Dependent Subunit Insertion. Structure **24**, 886–896. https://doi.org/10.1016/j.str.2016.02.024.

20. Guérit, D., Marie, P., Morel, A., Maurin, J., Verollet, C., Raynaud-Messina, B., Urbach, S., and Blangy, A. (2020). Primary myeloid cell proteomics and transcriptomics: importance of β-tubulin isotypes for osteoclast function. J. Cell Sci. **133**, jcs239772. https://doi.org/10.1242/jcs.239772.

21. Maurin, J., Blangy, A., and Bompard, G. (2020). Regulation of invadosomes by microtubules: Not only a matter of railways. Eur. J. Cell Biol. **99**, 151109. https://doi.org/10.1016/j.ejcb.2020.151109.

22. Peláez, R., Pariente, A., Pérez-Sala, Á., and Larrayoz, I.M. (2019). Integrins: Moonlighting Proteins in Invadosome Formation. Cancers (Basel) **11**, 615. https://doi.org/10.3390/cancers11050615.

23. Kim, H.-Y., Ha Thi, H.T., and Hong, S. (2018). IMP2 and IMP3 cooperate to promote the metastasis of triple-negative breast cancer through destabilization of progesterone receptor. Cancer Lett. **415**, 30–39. https://doi.org/10.1016/j.canlet.2017.11.039.

24. Hao, S., Meng, Q., Sun, H., Li, Y., Li, Y., Gu, L., Liu, B., Zhang, Y., Zhou, H., Xu, Z., and Wang, Y. (2022). The role of transketolase in human cancer progression and therapy. Biomed. Pharmacother. **154**, 113607. https://doi.org/10.1016/j.biopha.2022.113607.

25. Li, M., Zhao, X., Yong, H., Xu, J., Qu, P., Qiao, S., Hou, P., Li, Z., Chu, S., Zheng, J., and Bai, J. (2022). Transketolase promotes colorectal cancer metastasis through regulating AKT phosphorylation. Cell Death Dis. **13**, 99. https://doi.org/10.1038/s41419-022-04575-5.

26. Dai, X., Wang, L.-J., Wu, J., Shi, Y.-X., Li, G.-P., and Yang, X.-Q. (2018). Src kinase inhibitor PP2 regulates the biological characteristics of A549 cells via the PI3K/Akt signaling pathway. Oncol. Lett. **16**, 5059–5065. https://doi.org/10.3892/ol.2018.9282.

27. Lee, A.S., Kranzusch, P.J., Doudna, J.A., and Cate, J.H.D. (2016). eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation. Nature **536**, 96–99. https://doi.org/10.1038/nature18954.

28. Shin, S., Han, M.-J., Jedrychowski, M.P., Zhang, Z., Shokat, K.M., Plas, D.R., Dephoure, N., and Yoon, S.-O. (2023). mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching. Cell Rep. **42**, 112868. https://doi.org/10.1016/j.celrep.2023.112868.

29. Roiuk, M., Neff, M., and Teleman, A.A. (2024). eIF4E-independent translation is largely eIF3d-dependent. Nat. Commun. **15**, 6692. https://doi.org/10.1038/s41467-024-51027-z.

30. Ezzoukhry, Z., Henriet, E., Piquet, L., Boyé, K., Bioulac-Sage, P., Balabaud, C., Couchy, G., Zucman-Rossi, J., Moreau, V., and Saltel, F. (2016). TGF-β1 promotes linear invadosome formation in hepatocellular carcinoma cells, through DDR1 up-regulation and collagen I cross-linking. Eur. J. Cell Biol. **95**, 503–512. https://doi.org/10.1016/j.ejcb.2016.09.003.

31. Fabbri, L., Chakraborty, A., Robert, C., and Vagner, S. (2021). The plasticity of mRNA translation during cancer progression and therapy resistance. Nat. Rev. Cancer **21**, 558–577. https://doi.org/10.1038/s41568-021-00380-y.

32. Zhang, L., Pan, X., and Hershey, J.W.B. (2007). Individual overexpression of five subunits of human translation initiation factor eIF3 promotes malignant transformation of immortal fibroblast cells. J. Biol. Chem. **282**, 5790–5800. https://doi.org/10.1074/jbc.M606284200.

33. Grzmil, M., Rzymski, T., Milani, M., Harris, A.L., Capper, R.G., Saunders, N.J., Salhan, A., Ragoussis, J., and Norbury, C.J. (2010). An oncogenic role of eIF3e/INT6 in human breast cancer. Oncogene **29**, 4080–4089. https://doi.org/10.1038/onc.2010.152.

34. Mardakheh, F.K., Paul, A., Kümper, S., Sadok, A., Paterson, H., McCarthy, A., Yuan, Y., and Marshall, C.J. (2015). Global Analysis of mRNA Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions. Dev. Cell **35**, 344–357. https://doi.org/10.1016/j.devcel.2015.10.005.

35. Zhou, Z., Zhou, H., Ponzoni, L., Luo, A., Zhu, R., He, M., Huang, Y., Guan, K.-L., Bahar, I., Liu, Z., and Wan, Y. (2020). EIF3H Orchestrates Hippo Pathway-Mediated Oncogenesis via Catalytic Control of YAP Stability. Cancer Res. **80**, 2550–2563. https://doi.org/10.1158/0008-5472.CAN-19-3718.

36. LeFebvre, A.K., Korneeva, N.L., Trutschl, M., Cvek, U., Duzan, R.D., Bradley, C.A., Hershey, J.W.B., and Rhoads, R.E. (2006). Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit. J. Biol. Chem. **281**, 22917–22932. https://doi.org/10.1074/jbc.M605418200.

37. Nho, R.S., and Peterson, M. (2011). Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (4EBP-1) Function Is Suppressed by Src and Protein Phosphatase 2A (PP2A) on Extracellular Matrix. J. Biol. Chem. **286**, 31953–31965. https://doi.org/10.1074/jbc.M111.222299.

38. Parasuraman, P., Mulligan, P., Walker, J.A., Li, B., Boukhali, M., Haas, W., and Bernards, A. (2017). Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis. J. Biol. Chem. **292**, 26797–2689. https://doi.org/10.1074/jbc.M116.769216.

39. Héraud, C., Pinault, M., Neaud, V., Saltel, F., Lagrée, V., and Moreau, V. (2022). Identification of an inhibitory domain in GTPase-activating protein p190RhoGAP responsible for masking its functional GAP domain. J. Biol. Chem. **299**, 102792. https://doi.org/10.1016/j.jbc.2022.102792.

40. Ferrando, I.M., Chaerkady, R., Zhong, J., Molina, H., Jacob, H.K.C., Herbst-Robinson, K., Dancy, B.M., Katju, V., Bose, R., Zhang, J., et al. (2012). Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics. Mol. Cell. Proteomics **11**, 355–369. https://doi.org/10.1074/mcp.M111.015750.

41. Holt, C.E., Martin, K.C., and Schuman, E.M. (2019). Local translation in neurons: visualization and function. Nat. Struct. Mol. Biol. **26**, 557–566. https://doi.org/10.1038/s41594-019-0263-5.

42. Costa, D.S., Kenny-Ganzert, I.W., Chi, Q., Park, K., Kelley, L.C., Garde, A., Matus, D.Q., Park, J., Yogev, S., Goldstein, B., et al. (2023). The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane. Development **150**, dev201570. https://doi.org/10.1242/dev.201570.

43. Gasparski, A.N., Mason, D.E., Moissoglu, K., and Mili, S. (2022). Regulation and outcomes of localized RNA translation. Wiley Interdiscip. Rev. RNA **13**, e1721. https://doi.org/10.1002/wrna.1721.

44. Huang, H., Weng, H., Sun, W., Qin, X., Shi, H., Wu, H., Zhao, B.S., Mesquita, A., Liu, C., Yuan, C.L., et al. (2018). Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat. Cell Biol. **20**, 285–295. https://doi.org/10.1038/s41556-018-0045-z.

45. Mukherjee, J., Hermesh, O., Eliscovich, C., Nalpas, N., Franz-Wachtel, M., Maćek, B., and Jansen, R.-P. (2019). β-Actin mRNA interactome mapping by proximity biotinylation. Proc. Natl. Acad. Sci. USA **116**, 12863–12872. https://doi.org/10.1073/pnas.1820737116.

46. Xu, T., Verhagen, M.P., Teeuwssen, M., Sun, W., Joosten, R., Sacchetti, A., Ewing-Graham, P.C., Jansen, M.P.H.M., Boere, I.A., Bryce, N.S., et al. (2024). Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer. Cell Death Differ. **31**, 360–377. https://doi.org/10.1038/s41418-024-01267-9.

47. Li, R., Ackerman, W.E., Mihai, C., Volakis, L.I., Ghadiali, S., and Kniss, D.A. (2012). Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion. PLoS One **7**, e39766. https://doi.org/10.1371/journal.pone.0039766.

48. Volakis, L.I., Li, R., Ackerman, W.E., Mihai, C., Bechel, M., Summerfield, T.L., Ahn, C.S., Powell, H.M., Zielinski, R., Rosol, T.J., et al. (2014). Loss of myoferlin redirects breast cancer cell motility towards collective migration. PLoS One **9**, e86110. https://doi.org/10.1371/journal.pone.0086110.

49. Zhang, T., Li, J., He, Y., Yang, F., Hao, Y., Jin, W., Wu, J., Sun, Z., Li, Y., Chen, Y., et al. (2018). A small molecule targeting myoferlin exerts promoting anti-tumor effects on breast cancer. Nat. Commun. **9**, 3726. https://doi.org/10.1038/s41467-018-06179-0.
50. Chen, M.-L., Chai, C.-Y., Yeh, K.-T., Wang, S.-N., Tsai, C.-J., Yeh, Y.-T., and Yang, S.-F. (2011). Crosstalk between activated and inactivated c-Src in hepatocellular carcinoma. Dis. Markers 30, 325-333. https://doi.org/10.3233/DMA-2011-0792.

51. Zhao, S., Li, H., Wang, Q., Su, C., Wang, G., Song, H., Zhao, L., Luan, Z., and Su, R. (2015). The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M. BMC Cancer 15, 389. https://doi.org/10.1186/s12885-015-1401-z.

52. Yang, J., Zhang, X., Liu, L., Yang, X., Qian, Q., and Du, B. (2021). c-Src promotes the growth and tumorigenesis of hepatocellular carcinoma via the Hippo signaling pathway. Life Sci. 264, 118711. https://doi.org/10.1016/j.lfs.2020.118711.

53. Hershey, J.W.B. (2015). The role of eIF3 and its individual subunits in cancer. Biochim. Biophys. Acta 1849, 792-800. https://doi.org/10.1016/j.bbagrm.2014.10.005.

54. Okamoto, H., Yasui, K., Zhao, C., Arii, S., and Inazawa, J. (2003). PTK2 and EIF3S3 genes may be amplification targets at 8q23-q24 and are associated with large hepatocellular carcinomas. Hepatology 38, 1242-1249. https://doi.org/10.1053/jhep.2003.50457.

55. Liang, S., Bellato, H.M., Lorent, J., Lupinacci, F.C.S., Oertlin, C., van Hoef, V., Andrade, V.P., Roffé, M., Masvidal, L., Hajj, G.N.M., and Larsson, O. (2018). Polysome-profiling in small tissue samples. Nucleic Acids Res. 46, e3. https://doi.org/10.1093/nar/gkx940.

56. Oertlin, C., Lorent, J., Murie, C., Furic, L., Topisirovic, I., and Larsson, O. (2019). Generally applicable transcriptome-wide analysis of translation using anota2seq. Nucleic Acids Res. 47, e70. https://doi.org/10.1093/nar/gkz223.

57. Wright, G.W., and Simon, R.M. (2003). A random variance model for detection of differential gene expression in small microarray experiments. Bioinformatics 19, 2448-2455. https://doi.org/10.1093/bioinformatics/btg345.

58. Larsson, O., Sonenberg, N., and Nadon, R. (2010). Identification of differential translation in genome wide studies. Proc. Natl. Acad. Sci. USA 107, 21487-21492. https://doi.org/10.1073/pnas.1006821107.

59. Yoon, Y.J., Wu, B., Buxbaum, A.R., Das, S., Tsai, A., English, B.P., Grimm, J.B., Lavis, L.D., and Singer, R.H. (2016). Glutamate-induced RNA localization and translation in neurons. Proc. Natl. Acad. Sci. USA 113, E6877-E6886. https://doi.org/10.1073/pnas.1614267113.

60. Tsanov, N., Samacoits, A., Chouaib, R., Traboulsi, A.-M., Gostan, T., Weber, C., Zimmer, C., Zibara, K., Walter, T., Peter, M., et al. (2016). smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability. Nucleic Acids Res. 44, e165. https://doi.org/10.1093/nar/gkw784.

61. Bouyssié, D., Hesse, A.-M., Mouton-Barbosa, E., Rompais, M., Macron, C., Carapito, C., Gonzalez de Peredo, A., Couté, Y., Dupierris, V., Burel, A., et al. (2020). Proline: an efficient and user-friendly software suite for large-scale proteomics. Bioinformatics 36, 3148-3155. https://doi.org/10.1093/bioinformatics/btaa118.

62. Henriet, E., Abou Hammoud, A., Dupuy, J.-W., Dartigues, B., Ezoukry, Z., Dugot-Senant, N., Leste-Lasserre, T., Pallares-Lupon, N., Nikolski, M., Le Bail, B., et al. (2017). Argininosuccinate synthase 1 (ASS1): A marker of unclassified hepatocellular adenoma and high bleeding risk. Hepatology 66, 2016-2028. https://doi.org/10.1002/hep.29336.

63. Deutsch, E.W., Bandeira, N., Perez-Riverol, Y., Sharma, V., Carver, J.J., Mendoza, L., Kundu, D.J., Wang, S., Bandla, C., Kamatchinathan, S., et al. (2023). The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. 51, D1539-D1548. https://doi.org/10.1093/nar/gkac1040.

64. Szklarczyk, D., Franceschini, A., Wyder, S., Forslund, K., Heller, D., Huerta-Cepas, J., Simonovic, M., Roth, A., Santos, A., Tsafou, K.P., et al. (2015). STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 43, D447-D452. https://doi.org/10.1093/nar/gku1003.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>IgG1 Isotype Control from murine myeloma</td><td>Sigma</td><td>M5284</td></tr><tr><td>Mouse monoclonal anti-DDK</td><td>ThermoFisher</td><td>TA50011</td></tr><tr><td>Mouse monoclonal anti-eIF4E</td><td>Santa Cruz</td><td>sc-9976</td></tr><tr><td>Mouse monoclonal anti-GFP</td><td>Santa Cruz</td><td>sc-9996</td></tr><tr><td>Mouse monoclonal anti-Igf2bp2</td><td>Santa Cruz</td><td>sc-377014</td></tr><tr><td>Mouse monoclonal anti-Puromycin</td><td>Millipore</td><td>MABE343</td></tr><tr><td>Rabbit monoclonal anti-eIF3H</td><td>Cell signaling</td><td>3413S</td></tr><tr><td>Rabbit monoclonal anti-eIF4E</td><td>Cell signaling</td><td>2067</td></tr><tr><td>Rabbit monoclonal anti-eIF4G</td><td>Cell signaling</td><td>8701</td></tr><tr><td>Rabbit monoclonal anti-phospho-AKT(T308)</td><td>Cell signaling</td><td>2965</td></tr><tr><td>Rabbit monoclonal anti-phospho-S6K1(T389)</td><td>Cell signaling</td><td>9234</td></tr><tr><td>Rabbit monoclonal anti-Src</td><td>Cell signaling</td><td>2109</td></tr><tr><td>Rabbit polyclonal anti-4E-BP1</td><td>Cell signaling</td><td>9452</td></tr><tr><td>Rabbit polyclonal anti-AKT</td><td>Cell signaling</td><td>9272</td></tr><tr><td>Rabbit polyclonal anti-eIF3A</td><td>Cell signaling</td><td>2538</td></tr><tr><td>Rabbit polyclonal anti-eIF3B</td><td>Novus Biologicals</td><td>NBP238597</td></tr><tr><td>Rabbit polyclonal anti-eIF3C</td><td>AB Clonal</td><td>a7022</td></tr><tr><td>Rabbit polyclonal anti-eIF3D</td><td>AB Clonal</td><td>a5947</td></tr><tr><td>Rabbit polyclonal anti-eIF3E</td><td>Fortis Life Science</td><td>A302-985A</td></tr><tr><td>Rabbit polyclonal anti-eIF3H</td><td>Novus Biologicals</td><td>NBP184870</td></tr><tr><td>Rabbit polyclonal anti-eIF3I</td><td>Sigma</td><td>HPA029940</td></tr><tr><td>Rabbit polyclonal anti-eIF3K</td><td>Abcam</td><td>ab85968</td></tr><tr><td>Rabbit polyclonal anti-eIF3L</td><td>AB Clonal</td><td>A9972</td></tr><tr><td>Rabbit polyclonal anti-phospho-4E-BP1(T37/46)</td><td>Cell signaling</td><td>9459</td></tr><tr><td>Rabbit polyclonal anti-phospho-AKT(S473)</td><td>Cell signaling</td><td>9271</td></tr><tr><td>Rabbit polyclonal anti-phospho-Src (T419)</td><td>Cell signaling</td><td>2101</td></tr><tr><td>Rabbit polyclonal anti-S6K1</td><td>Cell signaling</td><td>9202</td></tr><tr><td>Rabbit polyclonal anti-Src</td><td>Cell signaling</td><td>2108</td></tr><tr><td>Rabbit polyclonal anti-TKT</td><td>Sigma</td><td>HPA029480</td></tr><tr><td>Rabbit polyclonal anti-TKS5</td><td>Santa Cruz</td><td>sc-30122</td></tr><tr><td>Mouse monoclonal anti-β-Catenin</td><td>BD Transduction lab</td><td>61054</td></tr><tr><td>Rabbit polyclonal anti-His</td><td>Cell Signaling</td><td>2365</td></tr><tr><td colspan="3">Biological samples</td></tr><tr><td>Patients with HCC</td><td>CHU Bordeaux</td><td>Bordeaux Liver Biobank BB- 0033- 00036</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>PP2</td><td>Sigma</td><td>529576</td></tr><tr><td>SU6656</td><td>Sigma</td><td>S9692</td></tr><tr><td>LY294002</td><td>Cell Signaling</td><td>9901</td></tr><tr><td>Rapamycin</td><td>Sigma</td><td>553211</td></tr><tr><td>4EGi-1</td><td>Sigma</td><td>324517</td></tr><tr><td>TGF-b1</td><td>Invitrogen</td><td>AF-100-21C</td></tr><tr><td>Protein G Dynabeads</td><td>Life Technologies</td><td>10009D</td></tr><tr><td>RIPA lysis buffer</td><td>Life Technologies</td><td>89900</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Puromycin</td><td>Sigma</td><td>P9620</td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C7698</td></tr><tr><td>Oregon Green 488-conjugated Gelatin</td><td>Invitrogen</td><td>G13186</td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>49629</td></tr><tr><td>Type 1 collagen Corning</td><td>Sigma</td><td>CLS354236</td></tr><tr><td>Matrigel Corning</td><td>Sigma</td><td>CLS354277</td></tr><tr><td>Mitomycin</td><td>Sigma</td><td>M4287</td></tr><tr><td>Paraformaldehyde solution</td><td>Electron microscope Science</td><td>15700</td></tr><tr><td>Crystal Violet</td><td>Sigma</td><td>C0775</td></tr><tr><td>Dulbecco's modified Eagle's medium with 4.5 g/L glucose Glutamax-I</td><td>Invitrogen</td><td>10564011</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778100</td></tr><tr><td>JetPRIME</td><td>Polyplus</td><td>201508</td></tr><tr><td>Phospho Stop</td><td>Roche</td><td>4906845001</td></tr><tr><td>Pierce BCA Protein Assay kit</td><td>Invitrogen</td><td>23225</td></tr><tr><td>TGX Stain-Free FastCast gels</td><td>Biorad</td><td>1610183</td></tr><tr><td>Acrylamide 10%</td><td></td><td></td></tr><tr><td>Trizol-LS</td><td>Invitrogen</td><td>15596026</td></tr><tr><td>Halo-ligand JF549</td><td>Promega</td><td>GA1110</td></tr><tr><td>Citifluor Mountant solution</td><td>Electron microscope Science</td><td>50-192-7398</td></tr><tr><td>Dapi</td><td>Sigma Aldrich</td><td>D9542</td></tr><tr><td>Phalloidin</td><td>Interchim</td><td>FP-BZ-9630</td></tr><tr><td>Complete</td><td>Roche</td><td>04693116001</td></tr><tr><td>Maxima Reverse Transcriptase</td><td>Thermofisher</td><td>10869200</td></tr><tr><td>PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa</td><td>Thermo Scientific</td><td>26620</td></tr><tr><td>FBS</td><td>Eurobio</td><td>CVFSVF00-01</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190144</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td></tr><tr><td>OptiMEM</td><td>Gibco</td><td>11058021</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284</td></tr><tr><td>BSA</td><td>Sigma</td><td>05470</td></tr><tr><td>Torin</td><td>Sigma</td><td>475991</td></tr><tr><td>PP242</td><td>Sigma</td><td>P0037</td></tr><tr><td>Fluoromount-G mounting media</td><td>SouthernBiotech</td><td>0100-01</td></tr><tr><td>TBS 10X</td><td>Euromedex</td><td>ET220</td></tr><tr><td>Tween</td><td>Sigma Aldrich</td><td>P7949</td></tr><tr><td>SYBR® Green SuperMix for iQTM</td><td>Quanta Biosciences, Inc</td><td>95053-500</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>Proteomic (Src vs. WT) and (Src + PP2 vs. Src)</td><td>This paper</td><td>https://www.ebi.ac.uk/pride/archive/projects/PXD071367<br>Accession numbers PXD071367</td></tr><tr><td>Bulk RNAseq (invadosomes vs. whole cells)</td><td>This paper</td><td>Benjamin Bonnard, Anouk Chatefau, Cyril Dourthe et al. Bulk RNAseq on invadosomes vs. whole cells [DS/OL]. V1. Science DataBank, 2025[2025-12-17]. https://cstr.cn/31253.11.sciencedb.33451. CSTR:31253.11.sciencedb.33451.</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Experimental models: Cell lines</td></tr><tr><td>NIH-3T3</td><td>Sara A. Courtneidge</td><td>Burnham Institute for Medical Research, LaJolla, CA.</td></tr><tr><td>NIH-3T3 Src</td><td>Sara A. Courtneidge</td><td>Burnham Institute for Medical Research, LaJolla, CA.</td></tr><tr><td>NIH-3T3-Src Lifeact-mRuby</td><td>Sara A. Courtneidge</td><td>Burnham Institute for Medical Research, LaJolla, CA.</td></tr><tr><td>NIH-3T3-Src Lifeact-GFP</td><td>Sara A. Courtneidge</td><td>Burnham Institute for Medical Research, LaJolla, CA.</td></tr><tr><td>HuH7</td><td>ABM</td><td>T8973</td></tr></tbody></table>

Oligonucleotides

<table><tr><td>Mm-lgf2bp2-01</td><td>CTGTGGGGCGACTCCCTGAGGGTATCT TACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-02</td><td>CCTGATCTTGCGTTGTGCAGTCTGGCTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-03</td><td>GGCACGATAACTTCTGCACTTGTCAAGT TCTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-04</td><td>CCAGCGGTCGACGAGGGGACTCGGATTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-05</td><td>ACATCTGGACCTTCTGCTGGAGCAATTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-06</td><td>CTTACAGTCTCCTGCTCTGGGTAGGAGTTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-07</td><td>TCTCTCGGGGTTATAAATGCTCAAATCCTG TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-08</td><td>AGAATCATGCGGCATGCTTCAGATGTCCTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-09</td><td>GATGTTCTTTATGGTCAAGCCCTCCTTTCTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-10</td><td>CTTGTTCCCGGGCACGATGAGGGGGTTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-11</td><td>AGGAGAGCTCACCTCTTCATCGGGGATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-12</td><td>GTTTCTGTATCTGTGTTGACTTGCTCCACG TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-13</td><td>GAATCTGGATTCTTCTGCTCCTTAGC TTTTTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-14</td><td>GAGACTGAGTAGTCAACTTCCATGATTTT CCCTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-15</td><td>CGGATGGCCAGTTCTGGTCGGGGTATTAC ACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-16</td><td>CGAAGAGCTGCCGGAGGTCGTCGGCGTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-17</td><td>ACGGCGGGGCTCAGGTTCCCAATGTATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-18</td><td>CCTCATTCTCGTCTGGCGTTTGGTCACTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-19</td><td>GTTCACGGTTTTCCCGCCCTTGCCAATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-20</td><td>GAAGTTTTCTTCCTTCAGTTTCCCAAAGAT CCTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-lgf2bp2-21</td><td>CCCTGAGCCTTAAACTGGGCTTCAGGTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Mm-Igf2bp2-22</td><td>GACCAGTGATGATGACCATCCTCTCACTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Igf2bp2-23</td><td>TGATGTGGGAATGGGCCGAAATGGTGAT GAGTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Igf2bp2-24</td><td>TAGCCCTGGGATCAGATTGGCTTGTTGG TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-01</td><td>CTTCGTGGTACTTGGCTGACAGCCAGGTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-02</td><td>ACCCGGATGCTGATCTTATCTTTCTTTAGA TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-03</td><td>TTCTCTGGGCGGCTGGTCCGGATGAATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-04</td><td>AAATGCCCTTTGTGTTGGCTGCTAACTCTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-05</td><td>GATGGCGGCCATGCGAATCTGGTCGATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-06</td><td>CAGAAGGGCACTGTCCGGTCACGTGTG TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-07</td><td>GGAATTCTTGGTGTCTCCATCCAGGGC TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-08</td><td>GCAACGAGGTTGTCCAGCTTGTAAATTCC AGTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-09</td><td>CAGGGAGCCAGTGGCCACATCGGTGATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-10</td><td>GCTTGTTTTCGGGACAGGATGCCCCGTCTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-11</td><td>AGTCAGAGCTGATCTTCCTCAGGTTCA GTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-12</td><td>GGCCTCGGGTAGGAAGCCAGCTTCAGTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-13</td><td>CAGGCGATTGGCTGTGTCCTTCAGGGTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-14</td><td>CTAGCCCTTGGTGACAAGGCCTTTCAC TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-15</td><td>CTTGCACAATGGCGTCCTTGTCATAACC TTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-16</td><td>AGTGACCGTCACTCCAGGTTCACCCATT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-17</td><td>GCTCGGGCAGAGTCTAGGATGAGTTT CCTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-18</td><td>TCCAGGGGCTTGATAGTGAAGGGATCC ATTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-19</td><td>CAGCAGCCAAGGCCTCATGCAGAGTTTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-20</td><td>CTTGCTCTTCAGGACCACCTTGGCTTTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-21</td><td>CCGACCTGGAAATCCTCATTGTTGCTA TAATTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-22</td><td>CTGCCTTCTCTGTTGCAACTCCATCGTT ACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-23</td><td>GGAGCCACAGAGGTTGATGTTGCTCT CATTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr><tr><td>Mm-Tkt-24</td><td>GGGTGCTCCTTTTTGAAGAGCTCCGAG AAGTTACACTCGGACCTCGTCGACATGCATT</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>FLAP-Y</td><td>AATGCATGTCGACGAGGTCCGAGTGTAA</td><td>N/A</td></tr><tr><td>elf3d human</td><td>Ambion</td><td>s16499</td></tr><tr><td>elf3d human</td><td>Ambion</td><td>s16500</td></tr><tr><td>elf3d mouse</td><td>Ambion</td><td>S79900</td></tr><tr><td>elf3d mouse</td><td>Ambion</td><td>s79901</td></tr><tr><td>elf3e human</td><td>Ambion</td><td>s7485</td></tr><tr><td>elf3e human</td><td>Ambion</td><td>s7483</td></tr><tr><td>elf3e mouse</td><td>Ambion</td><td>s68369</td></tr><tr><td>elf3e mouse</td><td>Ambion</td><td>s68370</td></tr><tr><td>elf3h human</td><td>Ambion</td><td>s16509</td></tr><tr><td>elf3h human</td><td>Ambion</td><td>s16507</td></tr><tr><td>elf3h mouse</td><td>Ambion</td><td>s86276</td></tr><tr><td>elf3h mouse</td><td>Ambion</td><td>s86274</td></tr><tr><td>elf3k mouse</td><td>Ambion</td><td>s92041</td></tr><tr><td>elf3k mouse</td><td>Ambion</td><td>S92039</td></tr><tr><td>IGF2BP2 mouse</td><td>Ambion</td><td>s115360</td></tr><tr><td>IGF2BP2 mouse</td><td>Ambion</td><td>s115361</td></tr><tr><td>TKT mouse</td><td>Sigma</td><td>241972</td></tr><tr><td>TKT mouse</td><td>Sigma</td><td>241973</td></tr><tr><td>v-Src human</td><td>Ambion</td><td>s13413</td></tr><tr><td>v-Src human</td><td>Ambion</td><td>s13414</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>elf3h-GFP</td><td>Vector Builder</td><td>230803</td></tr><tr><td>elf3e-GFP</td><td>Vector Builder</td><td>221031</td></tr><tr><td>TKT-DDK</td><td>Origen</td><td>RC203497</td></tr><tr><td>stdMCP-stdGFP</td><td>Add Gene</td><td>98916</td></tr><tr><td>HaloTag-β-actin</td><td>Add Gene</td><td>102718</td></tr><tr><td>Src-GFP</td><td>Add Gene</td><td>110496</td></tr><tr><td>SrcY527F</td><td>Add Gene</td><td>17675</td></tr><tr><td>β-CateninS33Y</td><td>Add Gene</td><td>13371</td></tr><tr><td>cMycT58A-His</td><td>Add Gene</td><td>45598</td></tr></table>

Software and algorithms

<table><tr><td>FIJI</td><td>ImageJ</td><td>https://imagej.net/software/fiji/downloads</td></tr><tr><td>GraphPad Prism v9</td><td>GraphPad</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>Image Lab 6.1</td><td>Bio-Rad</td><td>https://www.bio-rad.com/fr-fr/product/image-lab-software?ID=KRE6P5E8Z</td></tr><tr><td>IPA</td><td>Qiagen</td><td>https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/</td></tr></table>

Other

<table><tr><td>ChemiDoc MP Imaging System</td><td>Biorad</td><td>N/A</td></tr><tr><td>Laser microdissector Palm type 4</td><td>Zeiss</td><td>N/A</td></tr><tr><td>Nanodrop 2000</td><td>ThermoFischer</td><td>N/A</td></tr><tr><td>Lab Chip GX touch HT</td><td>Perkin Elmer</td><td>N/A</td></tr><tr><td>RNA Chip DNA 5K/RNA CZE</td><td>Perkin Elmer</td><td>N/A</td></tr><tr><td>Illumina Stranded mRNA Prep Ligation</td><td>Illumina</td><td>N/A</td></tr><tr><td>NEBNext library quant kit</td><td>New England BioLab</td><td>N/A</td></tr><tr><td>Transwell chambers 8.0µm pore size</td><td>Falcon</td><td>353097</td></tr></table>
# EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

## Cell culture

NIH-3T3 WT, NIH-3T3-Src cells were a generous gift from Sara A. Courtneidge (Burnham Institute for Medical Research, LaJolla, CA). NIH-3T3 WT, NIH-3T3-Src and Huh7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose Glutamax-I (Invitrogen) supplemented with 10% fetal calf serum (SigmaAldrich). All the cell lines were confirmed for the absence of mycoplasma by PCR.

## Patient samples

All patients gave informed consent, and this study was approved by our local committee "Direction de la Recherche Clinique et de l'Innovation" of Bordeaux University Hospital, Bordeaux Liver Biobank BB-0033-00036. The processing and use of data collected as part of this study were performed following the General Data Protection Regulation (GDPR-EU 2016/679) and complied with the Reference Methodology" (MR-004) of French Data Protection laws in force. Liver FFPE samples were clinically and histologically reviewed by Prof. Bioulac-Sage. Liver FFPE slices were performed using microtome instrument then immunochemistry was performed with antibodies listed in STAR Methods.

# METHOD DETAILS

## Transfection

The plasmids eIF3h-GFP (Vector Builder #230803) and eIF3e-GFP (Vector Builder #221031) were transduced in NIH-3T3-Src and NIH-3T3-Src Lifeact-mRuby cells by lentiviral infection. Transduced cells were sorted (BD FACSARIA III cell sorter) and re-plated in culture to be used. TKT-DDK plasmid was purchased from Origen and transfected (1µg) using JetPrime (PolyPlus transfection) following the provider's instructions. siRNA oligonucleotides (50nM) were transfected by Lipofectomine RNAiMax (Invitrogen). siRNAs were listed in STAR Methods.

## Western Blot

Cells were washed twice in ice-cold PBS and scraped into ice-cold RIPA lysis buffer (Sigma) completed with Complete and PhoStop inhibitor (Roche Applied Science) and lysed for 30 min at 4°C. Lysates were then clarified by centrifugation at 13,000 g for 10 min at 4°C. Protein concentration was measured using Pierce BCA protein assay (ThermoFisher). Proteins (20µg) were boiled and separated using TGX Stain-Free FastCast gels Acrylamide 10% (Biorad) at 120V for 90min. Proteins were transferred into nitrocellulose membrane (Biorad) using Trans-blot Turbo System (Biorad). Membranes were blocked in 5% BSA TBS-T (50 mM Tris, 150 mM NaCl and 0.1% Tween 20 supplemented with 5% BSA) during 1h at room temperature before to be incubated with primary antibodies (1/2000 in 5% BSA TBS-T) during overnight at 4°C. After 3 washes in TBS-T, membranes were incubated with fluorochrome-conjugated secondary antibodies (1/5000 in 5% BSA TBS-T) during 1h at room temperature. After 3 washes in TBS-T, membranes were exposed to right excitation sources using ChemiDoc MP Imaging System (Biorad). Uncropped membranes are available in Data S1.

## eIF4E immunoprecipitation and eIF4G blot analysis

1mg of cell lysate were incubated with 4 µg of eIF4E antibody during overnight at 4°C with constant agitation. The same amount of mouse IgG has been used as control. Protein G Dynabeads (Life Technologies) were washed twice with PBS and once with RIPA lysis buffer before incubation for 3h at 4°C with samples containing antibodies or IgG. After 5 time washing with RIPA buffer, precipitated proteins were eluted in 2x LDS sample buffer containing 50 mM dithiothreitol (DTT) and boiled at 95°C for 10 min before analysis via SDS-PAGE electrophoresis and western blotting.

## Sunset assay

Cells were treated with puromycin (10mg/ml) during 10min at 37°C then washed twice in ice-cold PBS for protein extraction as described above in Western Blot section. For negative control we pre-treated cells with the translation inhibitor cycloheximide (35µM) during 10min at 37°C.

## Degradation assay

Sterile coverslips were coated with Oregon Green 488-conjugated Gelatin (Invitrogen) for 20 min, then fixed with 0.5% glutaraldehyde (Sigma) for 40 min. After twice washing with PBS, coated-coverslip were placed into 24 wells/plate and cells were seeded (20K/well). Degradation area was measured 24h after seeding and a total of 20 images were acquired for each condition and normalized by number of nuclei in each image. Quantification has been done using ImageJ software. For HUH7 degradation assays, cells were pre-treated or not with Transforming Growth Factor β 1 (TGF-β1, 3ng/ml) during 48h as previously described.³⁰ The pre-coated gelatin coverslips were incubated with type 1 collagen (0.5mg/ml) diluted in DPBS during 4 h at 37°C. Excess of collagen was removed then cells were seeded (40K/well) treated or not with TGF-β1 (3ng/ml) during 24h.
## Invasion assay

Boyden chambers (8.0µm pore size, Falcon) were coated with 150µL of Matrigel (80µg/ml) in a 24-well plate. For HUH7 cells, cells were treated and Boyden chambers were also coated with type 1 collagen as described before in degradation assay section. Cells were pre-treated with mitomycin (10µg/mL) during 2h at 37°C before to be seeded in fresh serum-free medium (50K/insert). As a chemotactant, 20% FBS complete medium was used in the lower part of the transwell in the 24-well plate. After 24 h, the media was carefully removed, cells were fixed with 4% PFA (Electron Microscopy Sciences) for 10 min and stained with crystal violet (Sigma-Aldrich) for 10 min at room temperature. After twice washing with distilled water, cells in the upper part of the transwell membrane were removed by wiping with a cotton swab. The crystal violet has been eluted by adding 400µL of 33% acetic acid into each insert and shaking for 10min. The absorbance of eluted crystal violet has been measured at 590nm and the number of invaded cells has been quantified following established standard curve.

## Polysome profiling and bioinformatical analyses

NIH-3T3-Src cells were cultivated in 15 mm culture dishes and treated with PP2 at sub-confluence as described above. Cells were treated with 100 µM cycloheximide (CHX) for 5 min before being harvested on ice. Cells were washed twice with cold PBS with 100 µM cycloheximide and lysed in 350 µL hypotonic lysis buffer (5 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 1.5 mM KCl, 100 µg/mL cycloheximide, 2 mM DTT, 0.5% Triton X-100, 0.5% sodium deoxycholate and 1mM Ribonucleoside vanadyl complexes). After normalizing the RNA concentration, lysates were loaded onto a 5 mL linear 10%-45% gradient, and centrifuged at 45,000 rpm for 45 min at 4°C. The absorbance at 254 nm was continuously recorded using an ISCO fractionator. For mRNA translation analysis,⁵⁵ 250 µL of lysates were loaded onto a 5 mL discontinuous 5%-34%-55% sucrose gradient (20 mM HEPES-KOH pH 7.6, 100 mM KCl, 5 mM MgCl2) and centrifuged at 45,000 rpm (SW 55 Ti rotor, Beckman Coulter, Inc.). Fractions containing mRNAs associated to more than 3 ribosomes, were pooled and purified using TRIzol-LS (Thermo Fisher Scientific) in parallel to 50 µL of the total lysate. RNAs were treated with DNAse-I using RNA Clean & Concentrator kit (Zymo Research). The quality (DV200 > 60%) and the quantity (concentration) of all mRNA samples were evaluated using a Fragment Analyser (Agilent) prior sequencing. Changes in translational efficiency were quantified using analysis of partial variance as implemented in the anota2seq R/Bioconductor package version 1.24.0.⁵⁶ Both preprocessing and differential expressions of cytosolic and polysome-associated RNA was assessed using the anota2seq package after a correlation and PCA initial exploration. Sequencing batch was considered for correction while TMM-log2 normalization was utilized in the built-in parameters. For such analyses, Benjamini-Hochberg correction was used to account for multiple testing with a 95% FDR threshold. A random variance model was used to increase statistical power.⁵⁷,⁵⁸ All regulatory modes were considered for the analysis.

## Immunofluorescence and imaging

Cells were fixed using 4% PFA for 10min at room temperature then rinsed 3 times with PBS. Cells were permeabilized with 0.1% Triton X-100 (Sigma) for 10 min at room temperature before being rinsed twice with PBS. Cells were then incubated with primary antibodies (1:100 diluted in 4% BSA-PBS) for 40 min at room temperature, rinsed 3 times with PBS and incubated with secondary antibodies (1:100 diluted in 4% BSA-PBS) for 30 min at room temperature. Nuclei were stained with DAPI (1:200 diluted) and actin was stained with phalloidin (1:200 diluted). Coverslips were mounted on microscope slides using Fluoromount-G mounting media (SouthernBiotech) and were imaged using SP5 confocal microscope (Leica). Images were analyzed using ImageJ software. Invadosomes counting was performed on F-actin-stained cells 24h after seeding and a total of 20 images were acquired for each condition and normalized by number of nuclei in each image. Quantification has been done using ImageJ software.

## Co-staining β-actin mRNA and coding protein

Coding sequences for tandem-dimer stdMCP-stdGFP (Add Gene #98916) and HaloTag-β-actin (Add Gene #102718) were cloned into the lentivirus expression vector then used to transfect NIH-3T3 Src cells as previously described.⁵⁹ To visualize β-actin protein, we incubated cells with Halo-ligand JF549 (Promega) at final concentration of 1µM during 30 min following provider's instructions before cell fixation and imaging as described see above.

## Single Molecule Fluorescence In Situ Hybridization

Single Molecule Fluorescence In Situ Hybridization (smFISH) was performed as described.⁶⁰ Briefly, NIH-3T3-Src cells expressing LifeAct-GFP were fixed in 4% paraformaldehyde and permeabilized in 70% ethanol. Cells were then hybridized using two types of probes: (i) 24 unlabeled primary probes containing both an Igf2bp2 or a Tkt mRNA targeting sequence and a shared sequence (FLAP); (ii) a secondary probe conjugated to two Cy3 moieties, pre-hybridized in vitro to the primary probes via the FLAP sequence. Probes for Igf2bp2 and Tkt mRNAs were designed using Oligostan R-Scrip (https://bitbucket.org/muellerflorian/fishader/src/master/Oligostan/). All probes were purchased from Integrated DNA Technologies (Belgium); the sequences are listed in STAR Methods. Nuclei were stained with Hoechst and cells were mounted in CitiFluor (Electron Microscopy Sciences, USA). Image acquisition was performed on a Zeiss Axio Imager Z2 equipped with an Xcite 120LED Boost excitation source and a Orca Flash 4.0 V2+ Hama-matsu camera, using a 63X Plan Apochromat 1.4 NA oil objective controlled by Zen Software. Images were acquired as 0.24 µm z-stacks and processed with ImageJ.
## Laser microdissection

Invadosomes were microdissected from PFA fixed NIH-3T3 Src Lifeact-mRuby cells with a PALM type 4 (Zeiss) automated laser mi-
cro dissector.

## Label-free quantitative proteomics

Three independent biological replicates on total protein extracts were compared by label-free protein quantification. Proteins were loaded on a 10% acrylamide SDS-PAGE gel and proteins were visualized by Colloidal Blue staining. Migration was stopped when samples had just entered the resolving gel and the unresolved region of the gel was cut into only one segment. Each SDS-PAGE band was cut into 1 mm × 1 mm gel pieces. Gel pieces were destained in 25 mM ammonium bicarbonate (NH₄HCO₃), 50% Aceto-nitrile (ACN) and shrunk in ACN for 10 min. After ACN removal, gel pieces were dried at room temperature. Proteins were first reduced in 10 mM dithiothreitol, 100 mM NH₄HCO₃ for 60 min at 56°C then alkylated in 100 mM iodoacetamide, 100 mM NH₄HCO₃ for 60 min at room temperature and shrunk in ACN for 10 min. After ACN removal, gel pieces were rehydrated with 50 mM NH₄HCO₃ for 10 min at room temperature. Before protein digestion, gel pieces were shrunk in ACN for 10 min and dried at room temperature. Proteins were digested by incubating each gel slice with 10 ng/µL of trypsin (V5111, Promega) in 40 mM NH₄HCO₃, rehydrated at 4°C for 10 min, and finally incubated overnight at 37°C. The resulting peptides were extracted from the gel by three steps: a first incubation in 40 mM NH₄HCO₃ for 15 min at room temperature and two incubations in 47.5% ACN, 5% formic acid for 15 min at room temperature. The three collected extractions were pooled with the initial digestion super-natant, dried in a SpeedVac, and resuspended with 0.1% formic acid for a final concentration of 0.05 µg/µL. NanoLC-MS/MS analysis were performed using an Ultimate 3000 RSLC Nano-UPHLC system (Thermo Scientific, USA) coupled to a nanospray Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific, California, USA). Each peptide extracts were loaded on a 300 µm ID x 5 mm PepMap C₁₈ precolumn (Thermo Scientific, USA) at a flow rate of 10 µL/min. After a 3 min desalting step, pep-tides were separated on a 50 cm EasySpray column (75 µm ID, 2 µm C₁₈ beads, 100 Å pore size, ES903, Thermo Fisher Scientific) with a 4–40% linear gradient of solvent B (0.1% formic acid in 80% ACN) in 57 min. The separation flow rate was set at 300 nL/min. The mass spectrometer operated in positive ion mode at a 2.0 kV needle voltage. Data was acquired using Xcalibur 4.4 software in a data-dependent mode. MS scans (m/z 375–1500) were recorded at a resolution of R = 120000 (@ m/z 200), a standard AGC target and an injection time in automatic mode, followed by a top speed duty cycle of up to 3 s for MS/MS acquisition. Precursor ions (2–7 charge states) were isolated in the quadrupole with a mass window of 1.6 Th and fragmented with HCD@28% normalized collision energy. MS/MS data was acquired in the ion trap with rapid scan mode, a 20% normalized AGC target and a maximum injection time in dynamic mode. Selected precursors were excluded for 60 s. Protein identification was done in Proteome Discoverer 2.5. Mascot 2.5 algorithm was used for protein identification in batch mode by searching against an UniProt *Mus musculus* protein database (55,334 entries, release December 7, 2022; https://www.uniprot.org/website). Two missed enzyme cleavages were allowed for the trypsin. Mass tolerances in MS and MS/MS were set to 10 ppm and 0.6 Da. Oxidation (M) and acetylation (K) were searched as dynamic modifications and carbamidomethylation (C) as static modification. Raw LC-MS/MS data were imported in Proline Studio for feature detection, alignment, and quantification.⁶¹ Proteins identification was accepted only with at least 2 specific peptides with a pretty rank = 1 and with a protein FDR value less than 1.0% calculated using the “decoy” option in Mascot. Label-free quantification of MS1 level by extracted ion chromatograms (XIC) was carried out with parameters indicated previously.⁶² The normalization was carried out on median of ratios. The inference of missing values was applied with 5% of the background snoise. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁶³ partner repository with the dataset identifier PXD071367.

## RNA extraction

Isolation of the total RNA from each sample was performed with Direct-zol RNA miniprep (Zymo) following the supplier procedures.
The concentration of RNA was measured by spectrophotometry (NanoDrop 2000, ThermoFisher) for each sample. RNA quality was
checked using Lab Chip GX touch HT (PerkinElmer) and RNA chip DNA 5K/RNA CZE (PerkinElmer).

## Libraries synthesized

cDNA libraries were synthesized using 500 ng of RNA from each sample with the Illumina Stranded mRNA Prep Ligation (Illumina) kit following the supplier procedures. Briefly, the first step consists on capturing mRNAs with magnetic beads targeting their poly-A tail. Then, mRNAs were fragmented, reverse transcribed into double strand cDNA and 3' adenylated adapters and anchors were ligated to both ends. Finally, the cDNA library was amplified by PCR with a number of PCR cycles adapted to the start material quantity. Libraries quality was checked with Xmark chip on the Lab Chip GX touch HT (PerkinElmer). After quantification of individual libraries by q-PCR (Roche LC480) using the NEBNext library quant kit (New England BioLabs), all the samples were pooled in an equimolar manner. The quality and quantity of the pool were checked by qPCR (Roche LC480) with the same kit and Lab Chip GX touch HT (PerkinElmer) before sequencing. mRNA sequencing was performed using Nextseq 2000 Illumina (paired-end 2x100 bp) with a minimum of 30 million reads per sample in the PGTB facility (INRAE - Pierroton). The transcriptomic data have been deposited to the Science DataBank partner repository with the dataset identifier CSTR:31253.11.sciencedb.33451.
**Human database**

The Human database related to patient survival and expression level of targeted genes were extracted from TCGA Pan-Cancer Atlas 2018.

**Bioinformatics analysis**

Proteins and mRNAs lists provided in **STAR Methods** were used for Ingenuity Pathways Analysis (Qiagen), partner interaction network (STRING)⁶⁴ and Gene Ontology classification from the PANTHER database (https://pantherdb.org/).

**QUANTIFICATION AND STATISTICAL ANALYSIS**

Statistical significance was determined using Student's t test and calculated using GraphPad Prism software. Graph bars represent the mean ± SEM. Significant differences (*p* < 0.05) are indicated in the figures.
Supplemental information

Src promotes tumor cell invasion

by hijacking the translation machineries

Benjamin Bonnard, Anouk Chatefau, Cyril Dourthe, Sylvaine Di Tommaso, Jean-William Dupuy, Isabelle Mahouche, Jacobo Solorzano, Morgane Le Bras, Anne-Aurélie Raymond, Violaine Moreau, Arnaud Jabouille, Anne Blangy, Yvan Martineau, and Frédéric Saltel
<figure><img src="image_28.png" /><figcaption>**Figure S1:** Src kinase activity regulates protein synthesis and specific eIF3 subunit expression, related to Figure 1. (A) Confocal microscopy images of NIH-3T3 Src cells stained for F-actin (red) and puromycin-labeled nascent proteins (green). Scale bar: 40µm. (B) NIH-3T3 Src cells were treated with cycloheximide (CHX) during 1h. The bar graph shows the number of invadosomes per nuclei. (C-E) NIH-3T3 Src cells were treated with the Src inhibitor SU6656 during 1h (SU 1H) then cells were replaced into fresh medium during recovery period (SU 1H + R6H). DMSO treatment has been used as control group (Ctrl). (C) The bar graph shows the quantification of Src phosphorylation. (D) The left panel shows representative images of invadosomes stained with phalloidin (red); the bar graph shows the number of invadosomes per nuclei. Scale bar: 20µm. Error bars (mean ± SEM, n=20 fields, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis). (E) The SUnSET assay was performed, and CHX treatment was used as a negative control. Bar graph shows the quantification of puromycin incorporation. (F) NIH-3T3 Src cells were transfected with siRNA control (si Ctrl) or with two independent siRNAs targeting Src. SUnSET assay performed on transfected NIH-3T3 Src cells. Bar graph shows the quantification of puromycin incorporation. (G) Polysome profiles from NIH-3T3 Src cells treated or not with PP2 during 1h. Absorbance at 254nm is shown as a function of sedimentation. Free mRNAs, the large eukaryotic ribosome subunit (60S), monosome (80S) and heavy polysomes fractions are indicated. (H) The volcano plot displays downregulated (blue) and upregulated (red) proteins in NIH-3T3 Src cells treated or not with Src inhibitor (PP2) during 24 hours. Protein expression of eIF3c/e/h/i/l subunits is indicated in purple. (I-L) Protein expression of eIF3 subunits in WT and NIH-3T3 Src cells treated or untreated with Src inhibitor (PP2 or SU) for 24 hours. Protein expression was normalized with total protein content using Stain Free technology (SF). Bar graphs for western blot quantification were presented with error bars (mean ± SEM, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis).</figcaption></figure>
A

<figure><img src="image_29.png" /></figure>

B

<figure><img src="image_30.png" /><figcaption>**Figure S2:** Only the activated form of Src regulates the formation of mature invadosomes in NIH 3T3 WT, related to Figure 1. (A) Representative western blot performed with cell lysates from NIH 3T3 WT cells transfected with control-plasmid expressing GFP; Src Y527F; β catenin S33Y; cMyc T58A. (B) Fluorescence microscopy images of NIH 3T3 WT cells transfected with control-plasmid expressing GFP; Src Y527F; β catenin S33Y; cMyc T58A. F-actin (white), oncogenes and GFP (red) were stained. The right panel displays fluorescent gelatin matrix (ECM, green) with the degradation area depicted in black. (Scale bar: 20µm).</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Figure S3:** Only the activated form of *Src* demonstrated a subcellular enrichment of *eIF3* subunit in NIH 3T3 WT, related to Figure 1. (A-C) Fluorescence microscopy images of sublocalization of *eIF3e* (A), *eIF3h* (B) and *eIF3d* (C) in NIH 3T3 WT cells transfected with control-plasmid expressing GFP; *Src* Y527F; *b* catenin S33Y; *cMyc* T58A; *F-actin* (white), oncogenes and *GFP* (green) and *eIF3* subunits (red) were stained. (Scale bar: 20µm).</figcaption></figure>
<figure><img src="image_32.png" /><figcaption>**Figure S4:** Key role of Src-regulated eIF3 subunits in invadosome formation and ECM degradation, related to Figure 2. (A-H) NIH-3T3 Src cells were transfected with siRNA control (si Ctrl) or with two independent siRNAs targeting eIF3h or eIF3e or eIF3d or eIF3k. (A-B) Bar graphs show the relative number of invaded NIH-3T3 Src cells treated with siRNAs targeting eIF3h or eIF3e into Boyden chambers. Scale bar: 400mm. Error bars (mean ± SEM, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis). (C-D) Protein expression of eIF3 a/b/c/d/e/h/k subunits was evaluated by western blot. Error bars (mean ± SEM, three independent experiments, \*P<0.05 compare to si Ctrl, paired t-test was used for statistical analysis). (E) Bar graph shows the relative number of invaded NIH-3T3 Src cells treated with siRNAs targeting eIF3d into Boyden chambers. Scale bar: 400mm. Error bars (mean ± SEM, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis). (F) siIF3k-transfected NIH-3T3 Src cells were seeded on a fluorescent gelatin matrix. The upper panel shows representative images of invadosomes stained with phalloidin; the bar graph shows the number of invadosomes per nuclei. The lower panel shows representative images of degradation area (black); the bar graph shows the quantification of matrix degradation area per nuclei after 24 hours. Scale bar: 20mm. Error bars (mean ± SEM, *n*=20 fields, three independent experiments, \*P<0.05, paired t-test was used for statistical analysis). (G-H) Protein expression of eIF3 a/b/c/d/e/h/k subunits was evaluated by western blot in NIH-3T3 Src cells treated with siRNAs targeting eIF3d (G) or eIF3k (H). Protein expression was normalized with total protein content using Stain Free technology (SF). Error bars (mean ± SEM, three independent experiments, \*P<0.05 compare to si Ctrl, paired t-test was used for statistical analysis).</figcaption></figure>
<figure><img src="image_33.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_38.png" /></figure>

**Figure S5: Specific mRNAs and product proteins eIF3-dependent colocalize in invadosomes and control ECM degradation and invasion, related to Figure 3.** (A) The bar graph shows the protein classes of the most mRNAs enriched (top 30) into invadosomes compare to whole cells. (B) Confocal microscopy images of F-actin (red) and exogen TKT (green) in NIH-3T3 Src cells. Scale bar: 20mm. (C-D) NIH-3T3 Src cells were transfected with siRNA control (si Ctrl) or with two independent siRNAs targeting IGF2BP2 or TKT. Protein expression of IGFBP2 and TKT was assessed by western blot. Protein expression was normalized with total protein content using Stain Free technology (SF). Error bars (mean ± SEM, three independent experiments, \*P<0.05 compare to si Ctrl, paired t-test was used for statistical analysis). (D) TKT-depleted NIH-3T3 Src cells were seeded on a fluorescent gelatin matrix. The upper panel presents representative images of invadosomes stained with phalloidin, with the bar graph indicating the number of invadosomes per nuclei. The lower panel displays representative images of the degradation area (depicted in black), with the bar graph quantifying the matrix degradation area per nuclei after 24 hours. Scale bar: 40µm. Error bars (mean ± SEM, n=20 fields, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis). (E) Single molecule Fluorescence In Situ Hybridization was used to localize TKT mRNAs in NIH-3T3 Src cells. Z-projections of 3 out of 12 slices of the z-stacks encompassing the whole cell height, scale bar: 10µm. Right panel showed pixel quantification of TKT mRNAs in invadosomes and whole cells (n=16 cells with associated invadosomes were considered, \*P<0.05, paired t-test was used for statistical analysis). (F) Bar graphs show the relative number of invaded NIH-3T3 Src cells transfected with siRNA control or with two independent siRNAs targeting IGF2BP2 or TKT into Boyden chambers. Scale bar: 400mm. Error bars (mean ± SEM, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis).
<figure><img src="image_39.png" /><figcaption>**Figure S6:** Src kinase activity enhances canonical and non-canonical eukaryotic translation initiation leading to invadosome formation, related to Figure 4. (A) Representation of String network based on kinases regulated by Src activity. (B) NIH-3T3 WT (WT) and NIH-3T3 Src (Src) cells were treated or not with Src inhibitors (PP2 or SU). The phosphorylation status of protein kinase B (AKT), ribosomal protein S6 kinase beta-1 (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were assessed by western blot and normalized to WT cells (dashed red line). Error bars (mean ± SEM, four independent experiments, \*P<0.05 compare to WT cells and \#P<0.05 compare to Src cells, paired t-test was used for statistical analysis). (C) Cap-column experiment was performed using protein lysate from NIH-3T3 Src cells treated with Src inhibitor (PP2), PI3K inhibitor (LY294002), mTOR inhibitor (Rapamycin, PP242 or Torin1) and inhibitor of eIF4e/eIF4g interaction (4EGi-1). DMSO treatment has been used as control group (Ctrl). (D) Protein expression of eIF3d in WT cells and Src cells treated with Src inhibitors (PP2 or SU). Protein expression was normalized with total protein content using Stain Free technology (SF). Error bars (mean ± SEM, four independent experiments, \*P<0.05, paired t-test was used for statistical analysis).</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>**Figure S7:** The Src - eIF3h/3e/3d axis is enriched in HCC patients and enhanced the invasiveness of HCC cell line, related to Figure 5. (A) Heat map shows the correlation between survival prognosis status with high expression level of targeted mRNAs in selected types of cancers (B) Heat map shows the mRNA expression correlation between Src and eIF3 subunits in three selected types of cancers (endometrial, prostate and testis). (C-E) Huh7 cells were transfected with siRNA control (si Ctrl) or with two independent siRNAs targeting eIF3h or eIF3e or eIF3d treated with TGF-b1 then seeded on Boyden chambers coated with collagen type I. Bar graph shows the relative number of invaded cells. Scale bar: 400mm. Error bars (mean ± SEM, four independent experiments, \*P<0.05 compare to si Ctrl, paired t-test was used for statistical analysis).</figcaption></figure>
<table><thead><tr><th colspan="3">Translational proteins</th></tr><tr><th>Src-<br>dependent</th><th colspan="2">Enriched in invadosomes</th></tr></thead><tbody><tr><td>AARS1</td><td>AIMP1</td><td>RPL12</td></tr><tr><td>CARS1</td><td>CARS1</td><td>RPL22</td></tr><tr><td>EEF1A1</td><td>DARS</td><td>RPL23</td></tr><tr><td>EEF2</td><td>EEF1A1</td><td>RPL26</td></tr><tr><td>EIF2B3</td><td>EEF1A2</td><td>RPL28</td></tr><tr><td>EIF3A</td><td>EEF1D</td><td>RPL30</td></tr><tr><td>EIF3B</td><td>EEF2</td><td>RPL31</td></tr><tr><td>EIF3C</td><td>EFTUD2</td><td>RPL34</td></tr><tr><td>EIF3E</td><td>EIF2A</td><td>RPL35A</td></tr><tr><td>EIF3F</td><td>EIF2S1</td><td>RPL36A</td></tr><tr><td>EIF3G</td><td>EIF3C</td><td>RPS11</td></tr><tr><td>EIF3H</td><td>EIF3E</td><td>RPS12</td></tr><tr><td>EIF3L</td><td>EIF3F</td><td>RPS13</td></tr><tr><td>EIF4B</td><td>EIF3G</td><td>RPS14</td></tr><tr><td>EIF4G1</td><td>EIF3H</td><td>RPS15A</td></tr><tr><td>EIF5B</td><td>EIF3I</td><td>RPS16</td></tr><tr><td>GARS1</td><td>EIF3K</td><td>RPS19</td></tr><tr><td>GSPT1</td><td>EIF3L</td><td>RPS20</td></tr><tr><td>IARS1</td><td>EIF4B</td><td>RPS26</td></tr><tr><td>KARS1</td><td>EIF5A</td><td>RPS27</td></tr><tr><td>LARS1</td><td>ELAVL1</td><td>RPS3</td></tr><tr><td>PSMD7</td><td>EPRS</td><td>RPS3A</td></tr><tr><td>RPL19</td><td>FARSA</td><td>RPS4X</td></tr><tr><td>SARS1</td><td>FXR1</td><td>RPSA</td></tr><tr><td>TARS1</td><td>RARS</td><td>VARS</td></tr><tr><td>VARS1</td><td>RPL10</td><td>YARS</td></tr><tr><td></td><td>RPL10A</td><td></td></tr></tbody></table>

**Table S1:** Translational proteins classification by Gene Ontology analysis of proteins regulated by Src and enriched in invadosomes.

**Bold** = commonly regulation of translational factors
<table><caption>Table S2: Most significant enriched mRNAs in invadosomes
**Bold**=mRNAs coding for cytoskeletal proteins</caption><thead><tr><th>ENSEMBL ID</th><th>Gene name</th><th>Protein name</th><th>Protein class</th></tr></thead><tbody><tr><td>ENSMUSG00000031972</td><td>Acta1</td><td>Actin, alpha skeletal muscle</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000035783</td><td>Acta2</td><td>Actin, aortic smooth muscle</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000059430</td><td>Actg2</td><td>Actin, gamma-enteric smooth muscle</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000003033</td><td>Ap1m1</td><td>AP-1 complex subunit mu-1</td><td>membrane traffic protein (PC00150)</td></tr><tr><td>ENSMUSG00000026280</td><td>Atg4b</td><td>Autophagy Related 4B Cysteine Peptidase</td><td>protein modifying enzyme (PC00260)</td></tr><tr><td>ENSMUSG00000040298</td><td>Btbd16</td><td>BTB/POZ domain-containing protein 16</td><td>unclassified</td></tr><tr><td>ENSMUSG000000073077</td><td>Cfap47</td><td>Cilia and flagella-associated protein 47</td><td>structural protein (PC00211)</td></tr><tr><td>ENSMUSG00000033658</td><td>Ddx19b</td><td>RNA helicase</td><td>RNA metabolism protein (PC00031)</td></tr><tr><td>ENSMUSG00000058454</td><td>Dhcr7</td><td>7-dehydrocholesterol reductase</td><td>metabolite interconversion enzyme (PC00262)</td></tr><tr><td>ENSMUSG00000016349</td><td>Eef1a2</td><td>Elongation factor 1-alpha 2</td><td>translational protein (PC00263)</td></tr><tr><td>ENSMUSG00000069495</td><td>Epc2</td><td>Enhancer of polycomb homolog 2</td><td>chromatin/chromatin-binding, or -regulatory protein (PC00077)</td></tr><tr><td>ENSMUSG00000022040</td><td>Ephx2</td><td>Epoxide Hydrolase 2</td><td>metabolite interconversion enzyme (PC00262)</td></tr><tr><td>ENSMUSG00000061099</td><td>Gapdhs</td><td>Glyceraldehyde-3-Phosphate Dehydrogenase</td><td>metabolite interconversion enzyme (PC00262)</td></tr><tr><td>ENSMUSG00000049649</td><td>Gpr3</td><td>G-protein coupled receptor 3</td><td>transmembrane signal receptor (PC00197)</td></tr><tr><td>ENSMUSG00000046675</td><td>Lyset</td><td>Lysosomal Enzyme Trafficking Factor</td><td>unclassified</td></tr><tr><td>ENSMUSG00000007480</td><td>Mc5r</td><td>Melanocortin receptor 5</td><td>transmembrane signal receptor (PC00197)</td></tr><tr><td>ENSMUSG00000025271</td><td>Pfkfb1</td><td>6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 1</td><td>metabolite interconversion enzyme (PC00262)</td></tr><tr><td>ENSMUSG00000000632</td><td>Sez6</td><td>Seizure protein 6</td><td>unclassified</td></tr><tr><td>ENSMUSG00000021892</td><td>Sh3bp5</td><td>SH3 domain-binding protein 5</td><td>scaffold/adaptor protein (PC00226)</td></tr><tr><td>ENSMUSG00000014606</td><td>Slc25a11</td><td>Mitochondrial 2-oxoglutarate/malate carrier protein</td><td>transporter (PC00227)</td></tr><tr><td>ENSMUSG000000067377</td><td>Tspan6</td><td>Tetraspanin 6</td><td>scaffold/adaptor protein (PC00226)</td></tr><tr><td>ENSMUSG00000067702</td><td>Tuba3a</td><td>Tubulin alpha-3A chain</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000067338</td><td>Tuba3b</td><td>Tubulin alpha-3B chain</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000058672</td><td>Tubb2a</td><td>Tubulin beta-2A chain</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG000000045136</td><td>Tubb2b</td><td>Tubulin beta-2B chain</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000062380</td><td>Tubb3</td><td>Tubulin beta-3 chain</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000001525</td><td>Tubb5</td><td>Tubulin beta-5 chain</td><td>cytoskeletal protein (PC00085)</td></tr><tr><td>ENSMUSG00000019505</td><td>Ubb</td><td>Polyubiquitin-B</td><td>unclassified</td></tr><tr><td>ENSMUSG00000008348</td><td>Ubc</td><td>Polyubiquitin-C</td><td>unclassified</td></tr><tr><td>ENSMUSG00000030403</td><td>Vasp</td><td>Vasodilator-stimulated phosphoprotein</td><td>scaffold/adaptor protein (PC00226)</td></tr></tbody></table>
<table><caption>**Table S3:** Common enriched mRNAs and proteins in invadosomes

**Bold**= downregulated proteins by sieIF3E and found to be enriched in invadosomes (mRNA and protein level)</caption><thead><tr><th>Ensembl ID</th><th>Gene name</th><th>Protein name</th><th>Protein class</th></tr></thead><tbody><tr><td>ENSMUSG00000029580</td><td>Ahcy</td><td>Adenosylhomocysteinase</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000026341</td><td>Chmp4b</td><td>Charged multivesicular body protein 4b</td><td>membrane traffic protein</td></tr><tr><td>ENSMUSG00000027597</td><td>Cnn3</td><td>Calponin-3</td><td>unclassified</td></tr><tr><td>ENSMUSG00000025428</td><td>Dars</td><td>Aspartate-tRNA ligase</td><td>translational protein</td></tr><tr><td>ENSMUSG00000006273</td><td>Dhx9</td><td>ATP-dependent RNA helicase A</td><td>RNA metabolism protein</td></tr><tr><td>ENSMUSG000000114133</td><td>Dlst</td><td>Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000015733</td><td>Eef1a1</td><td>Elongation factor 1-alpha 1</td><td>translational protein</td></tr><tr><td>ENSMUSG00000006699</td><td>Eef1a2</td><td>Elongation factor 1-alpha 2</td><td>translational protein</td></tr><tr><td>ENSMUSG00000038467</td><td>Eif3e</td><td>Eukaryotic translation initiation factor 3 subunit E</td><td>translational protein</td></tr><tr><td>ENSMUSG00000004665</td><td>Fxr1</td><td>Fragile X mental retardation syndrome-related protein 1</td><td>translational protein</td></tr><tr><td>ENSMUSG000000053931</td><td>Hadhb</td><td>Trifunctional enzyme subunit beta</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000074643</td><td>Hnrpc</td><td>Heterogeneous nuclear ribonucleoproteins C1/C2</td><td>RNA metabolism protein</td></tr><tr><td>ENSMUSG00000026356</td><td>Hspa5</td><td>Endoplasmic reticulum chaperone BiP</td><td>chaperone</td></tr><tr><td>ENSMUSG00000042699</td><td>Igf2bp2</td><td>Insulin-like growth factor 2 mRNA-binding protein 2</td><td>RNA metabolism protein</td></tr><tr><td>ENSMUSG00000004789</td><td>Matr3</td><td>Matrin-3</td><td>RNA metabolism protein</td></tr><tr><td>ENSMUSG00000037742</td><td>Mcm2</td><td>Serine protease inhibitor A3K</td><td>protein-binding activity modulator</td></tr><tr><td>ENSMUSG00000016349</td><td>Mvp</td><td>V-type proton ATPase 16 kDa proteolipid subunit</td><td>scaffold/adaptator protein</td></tr><tr><td>ENSMUSG000000020929</td><td>Myof</td><td>Myoferlin</td><td>membrane traffic protein</td></tr><tr><td>ENSMUSG00000030738</td><td>Ndufs1</td><td>NADH-ubiquinone oxidoreductase 75 kDa subunit</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000022336</td><td>Nedd4l</td><td>E3 ubiquitin-protein ligase NEDD4-like</td><td></td></tr><tr><td>ENSMUSG00000022337</td><td>Phb2</td><td>Prohibitin-2</td><td>unclassified</td></tr><tr><td>ENSMUSG00000027680</td><td>Ppp3ca</td><td>Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform</td><td>protein modifying enzyme</td></tr><tr><td>ENSMUSG00000026103</td><td>Prdx2</td><td>Peroxiredoxin-2</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000026879</td><td>Psmd13</td><td>26S proteasome non-ATPase regulatory subunit 13</td><td>protein modifying enzyme</td></tr><tr><td>ENSMUSG000000059447</td><td>Rab11a</td><td>Ras-related protein Rab-11A</td><td>protein-binding activity modulator</td></tr><tr><td>ENSMUSG000000060373</td><td>Rack1</td><td>Receptor of activated protein C kinase 1</td><td>unclassified</td></tr><tr><td>ENSMUSG00000015165</td><td>Rars</td><td>Arginine-tRNA ligase</td><td>translational protein</td></tr><tr><td>ENSMUSG00000026864</td><td>Rbmxl1</td><td>RNA binding motif protein</td><td></td></tr><tr><td>ENSMUSG000000033581</td><td>Rnh1</td><td>Ribonuclease H1</td><td>RNA metabolism protein</td></tr><tr><td>ENSMUSG000000037236</td><td>Rpl28</td><td>60S ribosomal protein L28</td><td>translational protein</td></tr><tr><td>ENSMUSG00000002870</td><td>Snx3</td><td>Sorting nexin-3</td><td>unclassified</td></tr><tr><td>ENSMUSG000000052105</td><td>Spata5</td><td>ATPase family protein 2 homolog</td><td>transporter</td></tr><tr><td>ENSMUSG00000030681</td><td>Syncr1p</td><td>Heterogeneous nuclear ribonucleoprotein Q</td><td>RNA metabolism protein</td></tr><tr><td>ENSMUSG00000048612</td><td>Tkt</td><td>Transketolase</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000026234</td><td>Tpm1</td><td>Tropomyosin alpha-1 chain</td><td>cytoskeleton protein</td></tr><tr><td>ENSMUSG00000025968</td><td>Ugdh</td><td>UDP-glucose 6-dehydrogenase</td><td>metabolite interconversion enzyme</td></tr><tr><td>ENSMUSG00000024589</td><td>Vdac2</td><td>Voltage-dependent anion-selective channel protein 2</td><td>transporter</td></tr></tbody></table>
<table><thead><tr><td>Protein ID</td><td>Protein name</td><td>Protein class</td></tr></thead><tbody><tr><td>Acat2</td><td>ACETYL-COA ACETYLTRANSFERASE, CYTOSOLIC</td><td>acyltransferase</td></tr><tr><td>Actn1</td><td>ALPHA-ACTININ-1</td><td>actin or actin-binding cytoskeletal protein</td></tr><tr><td>Actr2</td><td>ACTIN-RELATED PROTEIN 2</td><td>actin and actin related protein</td></tr><tr><td>Ap2b1</td><td>AP-2 COMPLEX SUBUNIT BETA</td><td>membrane traffic protein</td></tr><tr><td>Ap2m1</td><td>AP-2 COMPLEX SUBUNIT MU</td><td>membrane traffic protein</td></tr><tr><td>Arhgap1</td><td>RHO GTPASE-ACTIVATING PROTEIN 1</td><td>GTPase-activating protein</td></tr><tr><td>Atp6v1a</td><td>V-TYPE PROTON ATPASE CATALYTIC SUBUNIT A</td><td>ATP synthase</td></tr><tr><td>Btf3</td><td>TRANSCRIPTION FACTOR BTF3</td><td>DNA-binding transcription factor</td></tr><tr><td>Casp3</td><td>CASPASE-3</td><td>protease</td></tr><tr><td>Cat</td><td>CATALASE</td><td>peroxidase</td></tr><tr><td>Cbr3</td><td>CARBONYL REDUCTASE [NADPH] 3</td><td>reductase</td></tr><tr><td>Cdk4</td><td>CYCLIN-DEPENDENT KINASE 4</td><td>non-receptor serine/threonine protein kinase</td></tr><tr><td>Cmpk1</td><td>UMP-CMP KINASE</td><td>nucleotide kinase</td></tr><tr><td>Crot</td><td>PEROXISOMAL CARNITINE O-OCTANOYLTRANSFERASE</td><td>acyltransferase</td></tr><tr><td>Csrp1</td><td>CYSTEINE AND GLYCINE-RICH PROTEIN 1</td><td>actin or actin-binding cytoskeletal protein</td></tr><tr><td>Ctcf1</td><td>TRANSCRIPTIONAL REPRESSOR CTCFL</td><td>C2H2 zinc finger transcription factor</td></tr><tr><td>Ctsl</td><td>PROCATHEPSIN L</td><td>cysteine protease</td></tr><tr><td>Dctn2</td><td>DYNACTIN SUBUNIT 2</td><td>microtubule binding motor protein</td></tr><tr><td>Ddx17</td><td>ATP-DEPENDENT RNA HELICASE DDX17-RELATED</td><td>RNA helicase</td></tr><tr><td>Ddx6</td><td>ATP-DEPENDENT RNA HELICASE DDX6-RELATED</td><td>RNA helicase</td></tr><tr><td>Dpp3</td><td>DIPEPTIDYL PEPTIDASE 3</td><td>metalloprotease</td></tr><tr><td>Eif3d</td><td>EUKARYOTIC TRANSLATION INITIATION FACTOR 3 SUBUNIT D (PTHR12399:SF0)</td><td>translation initiation factor</td></tr><tr><td>Eif3e</td><td>EUKARYOTIC TRANSLATION INITIATION FACTOR 3 SUBUNIT E</td><td>translation initiation factor</td></tr><tr><td>Esyt2</td><td>EXTENDED SYNAPTOTAGMIN-2</td><td>membrane trafficking regulatory protein</td></tr><tr><td>Fdps</td><td>FARNESYL PYROPHOSPHATE SYNTHASE</td><td>acyltransferase</td></tr><tr><td>G3bp1</td><td>RAS GTPASE-ACTIVATING PROTEIN-BINDING PROTEIN 1</td><td>RNA metabolism protein</td></tr><tr><td>G6pdx</td><td>GLUCOSE-6-PHOSPHATE 1-DEHYDROGENASE</td><td>dehydrogenase</td></tr><tr><td>Gfm1</td><td>ELONGATION FACTOR G, MITOCHONDRIAL</td><td></td></tr><tr><td>Gnai2</td><td>GUANINE NUCLEOTIDE-BINDING PROTEIN G(I) SUBUNIT ALPHA-2</td><td>heterotrimeric G-protein</td></tr><tr><td>Gsta4</td><td>GLUTATHIONE S-TRANSFERASE A4</td><td>transferase</td></tr><tr><td>Hmgb1</td><td>HIGH MOBILITY GROUP PROTEIN B1</td><td>HMG box transcription factor</td></tr><tr><td>Hmgcs1</td><td>HYDROXYMETHYLGLUTARYL-COA SYNTHASE, CYTOPLASMIC</td><td></td></tr><tr><td>Idi1</td><td>ISOPENTENYL-DIPHOSPHATE DELTA-ISOMERASE 1</td><td>isomerase</td></tr><tr><td>Igf2bp2</td><td>INSULIN-LIKE GROWTH FACTOR 2 MRNA-BINDING PROTEIN 2</td><td>RNA metabolism protein</td></tr><tr><td>Igf2bp3</td><td>INSULIN-LIKE GROWTH FACTOR 2 MRNA-BINDING PROTEIN 3</td><td>RNA metabolism protein</td></tr><tr><td>Itgb1</td><td>INTEGRIN BETA-1</td><td>integrin</td></tr><tr><td>Kpna2</td><td>IMPORTIN SUBUNIT ALPHA-1</td><td>transporter</td></tr><tr><td>Lxn</td><td>LATEXIN</td><td>protease inhibitor</td></tr><tr><td>Mat2a</td><td>S-ADENOSYLMETHIONINE SYNTHASE ISOFORM TYPE-2</td><td>nucleotidyltransferase</td></tr><tr><td>Mdh1</td><td>MALATE DEHYDROGENASE, CYTOPLASMIC</td><td>dehydrogenase</td></tr><tr><td>Myo1e</td><td>UNCONVENTIONAL MYOSIN-IE</td><td>actin binding motor protein</td></tr></tbody></table>
<table><caption>**Table S4:** Downregulated proteins by sieIF3E in NIH-3T3 Src cells</caption><tr><td>Nln</td><td>NEUROLYSIN, MITOCHONDRIAL</td><td>metalloprotease(PC00153)</td></tr><tr><td>Npps</td><td>PUROMYCIN-SENSITIVE AMINOPEPTIDASE-RELATED</td><td>metalloprotease(PC00153)</td></tr><tr><td>Nqo1</td><td>NAD(P)H DEHYDROGENASE [QUINONE] 1</td><td>oxidoreductase(PC00176)</td></tr><tr><td>Ogt</td><td>UDP-N-ACETYLGLUCOSAMINE-PEPTIDE N-ACETYLGLUCOSAMINYLTRANSFERASE 110 KDA SUBUNIT</td><td>protein modifying enzyme(PC00260)</td></tr><tr><td>P4hb</td><td>PROTEIN DISULFIDE-ISOMERASE</td><td>chaperone(PC00072)</td></tr><tr><td>Pa2g4</td><td>PROLIFERATION-ASSOCIATED PROTEIN 2G4</td><td>protease(PC00190)</td></tr><tr><td>Pafah1b3</td><td>PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE IB SUBUNIT ALPHA1</td><td>protein modifying enzyme(PC00260)</td></tr><tr><td>Pgm1</td><td>PHOSPHOGLUCOMUTASE-1</td><td>mutase(PC00160)</td></tr><tr><td>Ppib</td><td>PEPTIDYL-PROLYL CIS-TRANS ISOMERASE B</td><td>chaperone(PC00072)</td></tr><tr><td>Prkar2b</td><td>CAMP-DEPENDENT PROTEIN KINASE TYPE II-BETA REGULATORY SUBUNIT</td><td>kinase modulator(PC00140)</td></tr><tr><td>Psmb1</td><td>PROTEASOME SUBUNIT BETA TYPE-1</td><td>metalloprotease(PC00153)</td></tr><tr><td>Psmb5</td><td>PROTEASOME SUBUNIT BETA TYPE-5</td><td>protease(PC00190)</td></tr><tr><td>Psmd12</td><td>26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 12</td><td>protease(PC00190)</td></tr><tr><td>Psme3</td><td>PROTEASOME ACTIVATOR COMPLEX SUBUNIT 3</td><td></td></tr><tr><td>Psph</td><td>PHOSPHOSERINE PHOSPHATASE</td><td>phosphatase(PC00181)</td></tr><tr><td>Slc2a1</td><td>SOLUTE CARRIER FAMILY 2, FACILITATED GLUCOSE TRANSPORTER MEMBER 1</td><td>secondary carrier transporter(PC00258)</td></tr><tr><td>Slc5a3</td><td>SODIUM_MYO-INOSITOL COTRANSPORTER</td><td>secondary carrier transporter(PC00258)</td></tr><tr><td>Sptbn1</td><td>SPECTRIN BETA CHAIN, NON-ERYTHROCYTIC 1</td><td>actin or actin-binding cytoskeletal protein(PC00041)</td></tr><tr><td>Srpra</td><td>SIGNAL RECOGNITION PARTICLE RECEPTOR SUBUNIT ALPHA</td><td>G-protein(PC00020)</td></tr><tr><td>Tfg</td><td>PROTEIN TFG</td><td>membrane traffic protein(PC00150)</td></tr><tr><td>Tkt</td><td>TRANSKETOLASE</td><td>transketolase(PC00221)</td></tr><tr><td>Tpp2</td><td>TRIPEPTIDYL-PEPTIDASE 2</td><td>serine protease(PC00203)</td></tr><tr><td>Vat1</td><td>SYNAPTIC VESICLE MEMBRANE PROTEIN VAT-1 HOMOLOG</td><td></td></tr><tr><td>Vcp</td><td>TRANSITIONAL ENDOPLASMIC RETICULUM ATPASE</td><td>primary active transporter(PC00068)</td></tr></table>
<table><caption>PTK ASSAY</caption><thead><tr><th colspan="4">SRC VS WT</th><th colspan="4">SRC + PP2 VS SRC</th></tr><tr><th>Kinase Name</th><th>Kinase Family</th><th>Mean Specificity Score</th><th>Median Kinase Statistic</th><th>Kinase Name</th><th>Kinase Family</th><th>Mean Specificity Score</th><th>Median Kinase Statistic</th></tr></thead><tbody><tr><td>LTK</td><td>Alk</td><td>0.6</td><td>18.7</td><td>CTK</td><td>Csk</td><td>0.82</td><td>-1.95</td></tr><tr><td>Tyro3/Sky</td><td>Axl</td><td>1.58</td><td>20.26</td><td>EphB1</td><td>Eph</td><td>1.19</td><td>-1.7</td></tr><tr><td>EphA1</td><td>Eph</td><td>0.58</td><td>20.03</td><td>EphA5</td><td>Eph</td><td>1.05</td><td>-1.65</td></tr><tr><td>FAK2</td><td>Fak</td><td>0.54</td><td>17.97</td><td>EphA10</td><td>Eph</td><td>0.85</td><td>-1.59</td></tr><tr><td>IGF1R</td><td>InsR</td><td>0.65</td><td>19.2</td><td>EphB2</td><td>Eph</td><td>0.82</td><td>-1.52</td></tr><tr><td>JAK1~b</td><td>JakA</td><td>0.93</td><td>24.86</td><td>FAK2</td><td>Fak</td><td>1.48</td><td>-1.92</td></tr><tr><td>JAK2</td><td>JakA</td><td>0.57</td><td>18.12</td><td>FAK1</td><td>Fak</td><td>1.1</td><td>-1.84</td></tr><tr><td>Met</td><td>Met</td><td>1.02</td><td>20.22</td><td>FGFR1</td><td>FGFR</td><td>1.43</td><td>-2.89</td></tr><tr><td>Ron</td><td>Met</td><td>0.76</td><td>21.31</td><td>FGFR4</td><td>FGFR</td><td>0.57</td><td>-2.13</td></tr><tr><td>ROR1</td><td>Ror</td><td>1.35</td><td>33.37</td><td>JAK1~b</td><td>JakA</td><td>1.06</td><td>-1.64</td></tr><tr><td>RYK</td><td>Ryk</td><td>1.01</td><td>25.08</td><td>Met</td><td>Met</td><td>1.22</td><td>-1.97</td></tr><tr><td>MEKK6/MAP3K6</td><td>STE</td><td>1.18</td><td>29.83</td><td>PDGFR[alpha]</td><td>PDGFR</td><td>0.94</td><td>-1.54</td></tr><tr><td>ASK/MAP3K5</td><td>STE</td><td>1.18</td><td>29.83</td><td>Brk</td><td>Src</td><td>1.06</td><td>-1.57</td></tr><tr><td>MAP2K7</td><td>STE</td><td>0.76</td><td>21.89</td><td>MEKK6/MAP3K6</td><td>STE</td><td>0.98</td><td>-1.61</td></tr><tr><td>MKK3/MAP2K3</td><td>STE</td><td>0.59</td><td>19.86</td><td>ASK/MAP3K5</td><td>STE</td><td>0.98</td><td>-1.61</td></tr><tr><td>MKK6/MAP2K6</td><td>STE</td><td>0.59</td><td>19.86</td><td>SEK1/MAP2K4</td><td>STE</td><td>1.25</td><td>-1.69</td></tr><tr><td>MEK1/MAP2K1</td><td>STE</td><td>0.57</td><td>19.49</td><td>MAP2K7</td><td>STE</td><td>1.1</td><td>-1.6</td></tr><tr><td>SEK1/MAP2K4</td><td>STE</td><td>0.56</td><td>19.32</td><td>MKK3/MAP2K3</td><td>STE</td><td>1.05</td><td>-1.61</td></tr><tr><td>Syk</td><td>Syk</td><td>1.21</td><td>18.57</td><td>MKK6/MAP2K6</td><td>STE</td><td>1.05</td><td>-1.61</td></tr><tr><td>ZAP70</td><td>Syk</td><td>0.96</td><td>18.47</td><td>FLT4</td><td>VEGFR</td><td>0.81</td><td>-1.46</td></tr></tbody></table>

Table S5: Pamgene analysis PTK assay

**Bold** = common deregulated kinases
<table><caption>STK ASSAY</caption><thead><tr><th colspan="4">SRC VS WT</th><th colspan="4">SRC + PP2 VS SRC</th></tr><tr><th>Kinase Name</th><th>Kinase Family</th><th>Mean Specificity Score</th><th>Median Kinase Statistic</th><th>Kinase Name</th><th>Kinase Family</th><th>Mean Specificity Score</th><th>Median Kinase Statistic</th></tr></thead><tbody><tr><td>ADCK3</td><td>ABC1</td><td>1.67</td><td>2.37</td><td>ADCK3</td><td>ABC1</td><td>1.38</td><td>-1.08</td></tr><tr><td>CDK2</td><td>CDK</td><td>1.36</td><td>1.94</td><td>AlphaK1</td><td>Alpha</td><td>0.96</td><td>-1.11</td></tr><tr><td>CDC2/CDK1</td><td>CDK</td><td>1.32</td><td>1.83</td><td>CDK10</td><td>CDK</td><td>0.73</td><td>-0.99</td></tr><tr><td>CDK7</td><td>CDK</td><td>1.2</td><td>1.86</td><td>ROCK1</td><td>DMPK</td><td>1.65</td><td>-1.26</td></tr><tr><td>CDK4</td><td>CDK</td><td>0.78</td><td>1.8</td><td>ROCK2</td><td>DMPK</td><td>1.17</td><td>-1.1</td></tr><tr><td>CDK11</td><td>CDK</td><td>0.76</td><td>1.9</td><td>HIPK1</td><td>DYRK</td><td>1.82</td><td>-1.44</td></tr><tr><td>CDKL2</td><td>CDKL</td><td>0.81</td><td>2.24</td><td>IKK[beta]</td><td>IKK</td><td>1.68</td><td>-1.43</td></tr><tr><td>ROCK1</td><td>DMPK</td><td>2.06</td><td>3.02</td><td>PKC[zeta]</td><td>PKC</td><td>1.91</td><td>-1.15</td></tr><tr><td>ROCK2</td><td>DMPK</td><td>1.15</td><td>2.15</td><td>PKC[beta]</td><td>PKC</td><td>1.53</td><td>-1.07</td></tr><tr><td>HIPK1</td><td>DYRK</td><td>0.93</td><td>2.44</td><td>PKC[iota]</td><td>PKC</td><td>1.5</td><td>-1.01</td></tr><tr><td>p38[delta]</td><td>MAPK</td><td>1.81</td><td>2.06</td><td>PKC[epsilon]</td><td>PKC</td><td>1.43</td><td>-0.94</td></tr><tr><td>JNK1</td><td>MAPK</td><td>1.78</td><td>1.91</td><td>PKC[eta]</td><td>PKC</td><td>1.28</td><td>-0.99</td></tr><tr><td>JNK3</td><td>MAPK</td><td>1.76</td><td>1.91</td><td>PKC[theta]</td><td>PKC</td><td>1.2</td><td>-0.91</td></tr><tr><td>p38[beta]</td><td>MAPK</td><td>1.22</td><td>2.14</td><td>PKC[delta]</td><td>PKC</td><td>1.07</td><td>-0.92</td></tr><tr><td>p38[gamma]</td><td>MAPK</td><td>1.12</td><td>1.94</td><td>RAF1</td><td>RAF</td><td>1.87</td><td>-1.17</td></tr><tr><td>JNK2</td><td>MAPK</td><td>1.06</td><td>1.79</td><td>MSK1</td><td>RSK</td><td>1.65</td><td>-1.17</td></tr><tr><td>MAPK14</td><td>MAPK</td><td>0.99</td><td>1.75</td><td>p70S6K</td><td>RSK</td><td>1.21</td><td>-0.93</td></tr><tr><td>PKC[iota]</td><td>PKC</td><td>1.53</td><td>2.1</td><td>RSK1/p90R SK</td><td>RSK</td><td>1.11</td><td>-1.07</td></tr><tr><td>MSK1</td><td>RSK</td><td>0.85</td><td>1.97</td><td>RSKL1</td><td>RSKL</td><td>1.53</td><td>-1.37</td></tr><tr><td>p70S6K</td><td>RSK</td><td>0.79</td><td>1.72</td><td>HGK/ZC1</td><td>STE20</td><td>0.97</td><td>-1.08</td></tr></tbody></table>

Table S6: Pamgene analysis STK assay

**Bold** = common deregulated kinases