# TFEB triggers a matrix degradation and invasion program in triple-negative breast cancer cells upon mTORC1 repression

David Remy, Sandra Antoine-Bally, Sophie de Tocqueville, Célia Jolly, Anne-Sophie Macé, Gabriel Champenois, Fariba Nemati, Isabel Brito, Virginie Raynal, Amulya Priya, et al.

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## To cite this version:

David Remy, Sandra Antoine-Bally, Sophie de Tocqueville, Célia Jolly, Anne-Sophie Macé, et al. TFEB triggers a matrix degradation and invasion program in triple-negative breast cancer cells upon mTORC1 repression. Developmental Cell, 2025, 60 (7), pp.1018-1035.e8. 10.1016/j.devcel.2024.12.005. hal-05351327

HAL Id: hal-05351327

https://hal.science/hal-05351327v1

Submitted on 10 Nov 2025

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# Developmental Cell

## TFEB triggers a matrix degradation and invasion program in triple-negative breast cancer cells upon mTORC1 repression

**Graphical abstract**

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

**Authors**

David Remy, Sandra Antoine-Bally,
Sophie de Tocqueville, ...,
Didier Meseure, Elisabetta Marangoni,
Philippe Chavrier

**Correspondence**

david.remy@curie.fr (D.R.),
philippe.chavrier@curie.fr (P.C.)

**In brief**

Remy et al. demonstrate that mTORC1 repression using clinically relevant mTOR inhibitors boosts extracellular matrix degradation by triple-negative breast cancer cells. TFEB is required downstream of mTORC1 repression, which induces a basal-like phenotype and disrupts DCIS organization in a tumor xenograft model.

**Highlights**

* mTORC1 repression induces ~10-fold stimulation of ECM degradation by TNBC cells

* TFEB required for induction of the ECM degradation response

* mTORC1 repression and TFEB activation induce invadopodia hyperfunctionality

* mTORC1 repression induces a basal-like invasive program in a DCIS-like tumor model

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## Article

# TFEB triggers a matrix degradation and invasion program in triple-negative breast cancer cells upon mTORC1 repression

David Remy,<fnref n="1" />,* Sandra Antoine-Bally,<fnref n="1" /> Sophie de Tocqueville,<fnref n="1" /> Célia Jolly,<fnref n="1" /> Anne-Sophie Macé,<fnref n="2" /> Gabriel Champenois,<fnref n="3" /> Fariba Nemati,<fnref n="4" /> Isabel Brito,<fnref n="5" /> Virginie Raynal,<fnref n="6" /> Amulya Priya,<fnref n="1" /> Adèle Berlioz,<fnref n="1" /> Ahmed Dahmani,<fnref n="4" /> André Nicolas,<fnref n="3" /> Didier Meseure,<fnref n="3" /> Elisabetta Marangoni,<fnref n="4" /> and Philippe Chavrier<fnref n="1" />,<fnref n="7" />,*

## SUMMARY

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently hyperactivated in triple-negative breast cancers (TNBCs) associated with poor prognosis and is a therapeutic target in breast cancer management. Here, we describe the effects of repression of mTOR-containing complex 1 (mTORC1) through knockdown of several key mTORC1 components or with mTOR inhibitors used in cancer therapy. mTORC1 repression results in an ~10-fold increase in extracellular matrix proteolytic degradation. Repression in several TNBC models, including in patient-derived xenografts (PDXs), induces nuclear translocation of transcription factor EB (TFEB), which drives a transcriptional program that controls endolysosome function and exocytosis. This response triggers a surge in endolysosomal recycling and the surface exposure of membrane type 1 matrix metalloproteinase (MT1-MMP) associated with invadopodia hyperfunctionality. Furthermore, repression of mTORC1 results in a basal-like breast cancer cell phenotype and disruption of ductal carcinoma *in situ* (DCIS)-like organization in a tumor xenograft model. Altogether, our data call for revaluation of mTOR inhibitors in breast cancer therapy.

## INTRODUCTION

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is often hyperactivated in breast cancers (BCs), including in triple-negative BCs (TNBCs), providing therapeutic options for this poor prognosis subtype, which is immune to endocrine therapy.<fnref n="1" />,<fnref n="2" /> Under nutrient-replete conditions, mechanistic target of rapamycin complex 1 (mTORC1), which includes mTOR kinase and Raptor subunits, is recruited at the endolysosomal surface by RagA (or RagB) complexed with RagC (or RagD) GTPases, and is activated by Ras homolog enriched in the brain (Rheb) GTPase.<fnref n="3" /> Rag nucleotide-binding state is controlled by amino acid (aa) levels by endolysosomal aa transporter, SLC38A9, vacuolar-type proton adenosine triphosphatase (v-ATPase), and pentameric LAMTOR complex, acting as a guanine exchange factor for RagA and RagB. Cytosolic multiprotein complexes called GATOR1 and folliculin (FLCN)-folliculin-interacting protein (FNIP) complex, function as GTPase-activating protein (GAP)

for RagA (RagB) and RagC (RagD), respectively.<fnref n="4" /> aa sufficiency triggers LAMTOR and FLCN-FNIP complex activity, resulting in accumulation of active RagA<sup>GTP</sup>(or RagB<sup>GTP</sup>)-RagC<sup>GDP</sup>(or RagD<sup>GDP</sup>) complexes on endolysosomes, which tether mTORC1 to these membranes. Raptor interacts with and presents protein substrates to mTOR kinase, including ribosomal kinase S6K, translation regulator 4E-BP1, and autophagy initiator, ULK1.<fnref n="4" />,<fnref n="5" /> In the absence of aas, GATOR1 switches RagA (or B) to a guanine diphosphate (GDP)-bound conformation, promoting recruitment of the tuberous sclerosis complex (TSC) Rheb-GAP complex, Rheb inactivation, and mTORC1 release from endolysosomes and inactivation.<fnref n="4" />,<fnref n="6" />

In addition, active mTORC1 phosphorylates and represses transcription factor EB (TFEB).<fnref n="3" />,<fnref n="4" /> Upon aa starvation and mTORC1 repression, non-phosphorylated TFEB translocates into the nucleus, promoting expression of genes that are related to lysosome function and autophagy (CLEAR gene network).<fnref n="7" />,⁸ FLCN is key to maintaining a GDP-bound state of RagC (and RagD), serving as an unconventional recruitment mechanism

<footnote n="1">¹Institut Curie, CNRS UMR 144, PSL University, 75005 Paris, France</footnote>

<footnote n="2">²CurieCoreTech Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, PSL University, 75005 Paris, France</footnote>

<footnote n="3">³Experimental Pathology Platform, Institut Curie, 75005 Paris, France</footnote>

<footnote n="4">⁴Laboratory of Preclinical Investigation, Institut Curie, PSL University, 26 Rue d'Ulm, 75005 Paris, France</footnote>

<footnote n="5">⁵CurieCoreTech Bioinformatics (CUBIC) Platform, Institut Curie, PSL University, 75005 Paris, France</footnote>

<footnote n="6">⁶CurieCoreTech Next Generation Sequencing (ICGex) Platform, Institut Curie, PSL University, 75005 Paris, France</footnote>

<footnote n="7">⁷Lead contact</footnote>

<footnote>*Correspondence: david.remy@curie.fr (D.R.), philippe.chavrier@curie.fr (P.C.)</footnote>

<footnote>https://doi.org/10.1016/j.devcel.2024.12.005</footnote>
<caption>A MDA-MB-231</caption>

Gelatin<sup>OG488</sup>/Cortactin

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pS6K/GAPDH
(Fold change to siCtrl)

Gelatin degradation
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Area of endosomal mTOR/cell area
(Fold change to siCtrl)

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<figure><img src="image_9.png" /><figcaption>**Figure 1.** mTORC1 repression in BC cells promotes invadopodia-dependent gelatinolysis</figcaption></figure>

(A) MDA-MB-231 cells were silenced for RagC expression and plated on fluorescently labeled gelatin (green) for 6 h. Samples were stained for cortactin (red). Higher magnification of the boxed region is shown in insets. Dotted lines, cell contour.

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that enables phosphorylation by mTORC1 and tethers inactive phosphorylated (p)TFEB to endolysosomes.⁵,⁹,¹⁰ When FLCN is missing, an atypical RagA(or B)GTP-RagC(or D)GTP complex is generated that retains capacity to activate mTORC1 on endolysosomes. Contrarily, this complex is unable to bind and phosphorylate TFEB, which translocates into the nucleus leading to dysregulated transcriptional activity and pathological conditions, including tumor formation.⁵,⁸

Scavenging and lysosomal degradation of extracellular proteins, including components of the extracellular matrix (ECM), can serve as aa source for pancreatic adenocarcinoma and BC cells under nutrient scarce conditions and mTORC1 regulation.¹¹,¹² Invadopodia are cancer-specific ECM-degrading structures forming at plasma membrane-matrix contact sites, which are key to cancer cell invasion.¹³,¹⁴ The adaptor protein, TKS5 (SH3PXD2A), is crucial for invadopodia function, which require surface exposure and accumulation of membrane type 1 matrix metalloproteinase (MT1-MMP) by exocytosis from storage endolysosomal compartments.¹⁵,¹⁶ Local polymerization of an invadopodial F-actin network nucleated by a TKS5/CDC42/ Arp2/3 complex signaling cascade generates pushing forces against constricting ECM fibrils, which, concurrent with MT1-MMP-dependent collagenolytic activity, drives invasive track formation and promotes tumor cell dissemination.¹⁷,¹⁸ Along this line, MT1-MMP plays a key role for basement membrane breaching and local invasion during the transition of ductal carcinoma *in situ* (DCIS) to invasive carcinoma in BC.¹⁹⁻²² In addition, recent work, including ours, highlighted the contribution of MT1-MMP and TKS5 in starvation-induced ECM degradation response of BC cells.²³,²⁴ Whether and how mTORC1 signaling can intersect with and control ECM remodeling by BC cells are overarching, yet unexplored questions. In this article, using question-driven and discovery-based approaches, we present congruent evidence that repression of mTORC1, including with pharmacological mTOR inhibitors used for BC management, promotes a TFEB-dependent transcriptional program that stimulates invadopodia-mediated ECM degradation in TNBC cell lines and patient-derived xenograft (PDX) cells *ex vivo*. At the mechanistic level, we report that mTORC1 repression promotes MT1-MMP exposure at the cell surface by stimulating TFEB-induced exocytic recycling of MT1-MMP from endolysosomes to generate hyperfunctional invadopodia supporting enhanced ECM degradation and invasive track formation. Moreover, this mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> program results in induction of a p63<sup>High</sup>/MT1-MMP<sup>High</sup> phenotype and disease progression characteristic of an invasive basal-like program in human DCIS tumor xenografts. Our work unveils a mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> response relevant for TNBCs leading to increased MT1-MMP/invadopodia-mediated matrix degradation and invasive potential.

## RESULTS

### mTORC1 repression stimulates invadopodia-mediated matrix degradation in BC cells

To dissect molecular mechanisms underlying the control of invadopodia-mediated matrix degradation by mTORC1, key mTORC1 signaling components were silenced in human MDA-MB-231 TNBC cells and gelatin degradation capacity was assessed. Cells treated with control non-targeting small interfering RNA (siRNA) showed cortactin-positive structures associated with dark gelatin degradation spots, typical of invadopodia-mediated matrix degradation (Figure 1A).¹³ Silencing of MT1-MMP or TKS5 abolished gelatin degradation (Figure 1B; Figure S1A).¹⁸,²⁵ These cells showed mTOR association with CD63-positive endolysosomes and high levels of pS6K and p4E-BP1 (Figures 1B and 1C; Figure S1C).²³,²⁶ Knockdown (KD) of mTORC1 subunit, Raptor, led to ~70% reduction in pS6K and p4E-BP1 levels and mTOR dissociation from endolysosomes, and drastic 10-fold increase in gelatin degradation as compared with cells treated with control siRNA (Figures 1B and 1C; Figures S1A and S1C). Conversely, KD of mTORC2 component, Rictor, did not affect pS6K and p4E-BP1 levels, endolysosomal mTOR, or gelatin degradation (Figures 1B and 1C; Figures S1A-S1C). These results indicated that repression of mTORC1 (mTORC1<sup>OFF</sup>), not mTORC2, correlated with robust enhancement of matrix degradation by TNBC cells.

As expected, Rheb KD did not affect mTOR endolysosomal recruitment in contrast to RagA or RagC KD, which caused mTOR dissociation (Figure 1C).²⁷ Silencing of RagA, RagC, or Rheb GTPases significantly impaired mTORC1 activity, and upregulated matrix degradation by ~1-log (Figures 1A-1C; Figures S1A and S1B). In contrast, silencing of RagB or RagD had no significant impact on mTOR association with CD63+ endolysosomes and on gelatin degradation (Figures 1B and 1C; Figures S1A and S1B). In addition, KD of aa-sensing transporter, SLC38A9,²⁸ repressed mTORC1 activity and stimulated a robust matrix degradation response (Figure 1B; Figures S1A-S1C). Collectively, these data indicated that RagA/RagC heterodimer is the major regulator of mTORC1 localization and activity in MDA-MB-231 cells and that similarly to Raptor, its loss robustly increased gelatinolysis. Critically, double KD (DKD) of RagC (or Raptor) and TKS5 abolished stimulation of matrix degradation, demonstrating that mTORC1<sup>OFF</sup> activity status unleashed a strong invadopodia-dependent ECM degradation program (Figure 1D).

Silencing of NRPL2 and DEPDC5, two subunits of the RagA GAP GATOR1 complex, increased mTOR lysosomal recruitment, but did not significantly change mTORC1 activity nor it affected gelatin degradation as compared with control treatment

(B) Green bars represent mean and standard error of the mean (SEM) pS6K levels (Figure S1A), normalized to GAPDH levels in cells treated with non-targeting siRNA (siCtrl) from at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test. Pink bars represent mean gelatin degradation levels and SEM of cells treated with indicated siRNAs normalized to level of cells treated with non-targeting siRNA from at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(C) Distribution of mTOR (cyan), CD63 (magenta), and F-actin (green) in MDA-MB-231 cells treated with indicated siRNAs. Higher magnification of boxed regions is shown in insets. Mean area of mTOR signal in association with CD63-positive vesicles normalized to cell area and to siCtrl-treated cell value ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(D) Mean gelatin degradation ± SEM by MDA-MB-231 cells treated with indicated siRNAs as in (A) from at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test. All numerical data and statistical tests are provided in Table S1; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001; ns, not significant. Scale bars, 10 and 5 µm (insets).
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A

TFEB<sup>GFP</sup> intensity

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B

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C

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D

TFEB<sup>GFP</sup> intensity

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E

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F

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G

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H

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MCF10DCIS

I

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MCF10DCIS

J

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MCF10DCIS

<caption>**Figure 2.** TFEB activation is required for matrix degradation upon mTORC1 repression</caption>

(A) TFEB<sup>GFP</sup> distribution (fire lookup table (LUT)) was assessed in MDA-MB-231 cells treated with the indicated siRNAs. Dotted line, cell and nucleus contour based on DAPI and phalloidin staining (data not shown).

(B) Graph represents mean intensity ratio ± SEM of nuclear versus cytoplasmic TFEB<sup>GFP</sup> in cells treated with indicated siRNAs from three independent experiments. Cells grown in complete (CM) or aa-starved medium (EBSS) for 6 h were used as controls.

(C) Mean gelatin degradation levels ± SEM of MDA-MB-231 cells treated with indicated siRNAs normalized to levels in siCtrl-treated cells from at least three independent experiments.

(D) TFEB<sup>GFP</sup> distribution in MDA-MB-231 cells treated with indicated drugs as in (A).

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(Figures 1B and 1C; Figures S1A–S1C). As expected, DKD of Lamtor1 and Lamtor4 subunits (LAMTOR Rag-GEF complex) decreased endolysosomal recruitment and activity of mTOR (Figures 1B and 1C; Figures S1A–S1C). Puzzlingly, Lamtor1/4 DKD did not significantly affect ECM degradation (Figure 1B), corresponding to the only tested condition, in which mTORC1 repression did not correlate with stimulation of ECM proteolysis.

Finally, KD of FLCN, which controls RagC$^{GDP}$ (and RagD$^{GDP}$) levels and TFEB function, induced a robust ECM degradation response (~15-fold), while it did not affect mTOR recruitment to the lysosomal surface, nor pS6K and p4E-BP1 levels in agreement with published reports<sup>5,9</sup> (Figures 1B and 1C; Figures S1A, and S1C). Collectively, these data revealed that mTORC1<sup>OFF</sup> state promoted a robust proteolytic, invadopodia-mediated ECM degradation program. The stimulation by FLCN KD suggested a potential contribution of TFEB to this program, possibly irrespective of the phosphorylation status of main mTORC1 substrates, that we sought to address in more details.

## TFEB activation accounts for ECM degradation upon mTORC1 repression

TFEB localization and activity are regulated by mTORC1-dependent phosphorylation on Ser142 and Ser211 residues.<sup>29,30</sup> As expected, pTFEB<sup>Ser211</sup> level decreased upon aa and growth factor starvation or RagC silencing (Figure S2A). TFEB<sup>GFP</sup> was mostly cytoplasmic in MDA-MB-231 cells grown in nutrient-replete conditions, while it translocated into the nucleus of cells silenced for Raptor, FLCN, or RagC, or upon starvation (Figures 2A and 2B; Figure S2B). In addition, RagC (or Raptor, or FLCN) silencing increased CD63+ endolysosome burden and the autophagy response highlighted by accumulation of cytoplasmic LC3-II+ vesicular structures (Figures S2C–S2E). Concomitant silencing of ATG5, a central component of autophagy, obliterated LC3-II+ vesicles, while having no effect on gelatin degradation (Figures S2D–S2F). Altogether, these data indicated that treatments that repressed mTORC1 and stimulated gelatinolysis, correlated with characteristic nuclear translocation and activation of TFEB and endolysosome biogenesis but independent of the autophagy response.<sup>31</sup>

TFEB contribution to the regulation of matrix degradation in response to mTORC1 repression was investigated. Strikingly, when Raptor, FLCN, or RagC were silenced simultaneously with TFEB, induction of gelatinolysis was abolished (Figure 2C; Figure S2B). Of note, mere TFEB silencing did not affect basal matrix degradation in MDA-MB-231 cells cultured in nutrient-replete conditions (Figure 2C; Figure S2B). Matrix degradation induced by RagC KD was not affected by depletion of related

transcription factor E3 (TFE3), indicating a specific role for TFEB (Figure S2G).

To further explore the relationship between mTORC1 repression and upregulation of the ECM degradation program, mTOR was pharmacologically inhibited upon treatment with Torin-1, rapamycin (Rapa), or everolimus (Evo), a therapeutic Rapa analog (Figure S2H). Overnight drug treatment induced significant TFEB<sup>GFP</sup> redistribution into the nucleus (Figure 2D; Figure S2I). Nuclear TFEB binds CLEAR motifs in the promoter region of its target genes.<sup>7</sup> Activity of a 4XCLEAR-LUC reporter promoter increased upon Rapa treatment, asserting transcriptional activity of nuclear TFEB upon mTORC1 inhibition (Figure S2J). Strikingly, all three inhibitors unleashed a strong gelatin degradation response, which was blunted upon TFEB KD (Figure 2E).

We confirmed that 4XCLEAR-LUC reporter activity increased in cells transiently overexpressing TFEB<sup>GFP</sup>,<sup>32</sup> concomitant of increased collagenolysis (Figures S2K and S2L). Additionally, TFEB was pre-activated using apilimod, an inhibitor of phosphatidylinositol-3-phosphate 5-kinase, PIKfyve, whose activity is required for endolysosomal retention of inactive pTFEB.<sup>33,34</sup> As reported, while it did not affect mTORC1 activity toward S6K and 4EBP1, apilimod induced TFEB nuclear translocation (Figure 2F; Figure S2M), which correlated with a ~1-log increase in gelatin degradation that could be abolished by TFEB KD (Figure 2G).

MCF10DCIS.com cells (MCF10DCIS herein) is a human TNBC cell line with a gain-of-function H1047R mutation within the kinase domain of PI3K catalytic subunit (PIK3CA), corresponding to a hot-spot mutation in the PI3K/AKT/mTOR axis in TNBC.<sup>35</sup> RagC KD in MCF10DCIS cells reduced pS6K and p4E-BP1 levels and induced TFEB nuclear translocation (Figures S3A and S3B). Detection of cleaved type I collagen by Col1-3/4 C neo-epitope immunostaining<sup>36,37</sup> revealed that RagC KD stimulated a robust collagenolytic activity by MCF10DCIS cells, which was abolished upon TFEB silencing (Figure 2H). Additionally, Torin-1, Rapa, or Evo treatment promoted nuclear translocation of TFEB and induced collagenolysis that was abolished upon TFEB KD (Figures S3C–S3F). Contrasting with the situation in MDA-MB-231 cells, collagen degradation and TFEB nuclear translocation were unaffected upon Raptor or FLCN KD in MCF10DCIS cells, although these treatments reduced pS6K and p4E-BP1 levels (Figures S3G–S3I). At this stage, the reason why FLNC or Raptor silencing did not affect TFEB nuclear localization in MCF10DCIS cells remains unclear, possibly reflecting genetic differences in these two cell lines. Yet, our results clearly identified TFEB activation as a conserved, key event in the inducible ECM degradation program of TNBC cells.

(E) Mean gelatin degradation levels ± SEM of siTFEB- or siCtrl-treated MDA-MB-231 cells treated with indicated drugs for 16 h. Levels are normalized to cells treated with corresponding vehicle from at least three independent experiments.

(F) Mean intensity ratio ± SEM of nuclear versus cytoplasmic TFEB<sup>GFP</sup> in MDA-MB-231 cells treated with apilimod or vehicle from three independent experiments.

(G) Mean gelatin degradation levels ± SEM of siTFEB- or siCtrl-treated MDA-MB-231 cells treated with indicated with apilimod or vehicle. Levels are normalized to cells treated with siCtrl and vehicle from at least three independent experiments.

(H) Type I collagen cleavage by indicated MCF10DCIS cell populations. Data are normalized to control siRNA-treated cells (fold change). Mean ± SEM are shown from three biologically independent experiments.

(I) Mean nuclear versus cytoplasmic TFEB intensity ratio ± SEM in MCF10DCIS treated with indicated drugs or vehicle for 16 h.

(J) Mean type I collagen cleavage ± SEM by indicated MCF10DCIS cell populations from three biologically independent experiments. Data are normalized to control vehicle and siNT-treated cells (fold change). All statistical analyses were performed using one-way ANOVA using Kruskal-Wallis comparison test. All numerical data and statistical tests are provided in Table S1. *p* < 0.05; **p* < 0.01; ***p* < 0.001; ****p* < 0.0001; ns, not significant. Scale bars, 10 µm.
A MDA-MB-231
MT1-MMP<sup>pHLuo</sup>/Collagen I

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## TFEB controls an ECM degradation program in response to DNA damage

We recently reported that DNA damage in MCF10DCIS cells stimulated a strong ECM degradation response and correlated with local invasion in an intraductal xenograft model.³⁸ Studies investigated interrelations between the DNA damage response (DDR) pathway and mTORC1 activity and found that DDR led to mTORC1 repression and TFEB activation, while mTORC1 repression interfered with DDR.³⁹,⁴⁰ As expected, MCF10DCIS cell treatment with DNA-damaging drug, doxorubicin (Doxo), resulted in a robust increase in γH2AX-positive DNA damage foci and ~40% decrease in mTORC1 activity, independent on TFEB (Figures S3J and S3K). Similarly, mTORC1 repression upon Torin-1 treatment also increased DNA damage foci, although to a much lower extent (Figures S3J and S3K). Doxo-mediated mTORC1 repression resulted in significant nuclear translocation of TFEB comparable to Torin-1 treatment (Figure 2I). Altogether, these data indicated that genotoxic stress induced mTORC1 inhibition and TFEB activation in MCF10DCIS cells alike observations in other cell types.⁴⁰ Strikingly, Doxo treatment caused a ~4-fold increase in collagen cleavage,³⁸ which was abolished upon TFEB silencing (Figure 2J). Taken together, these experiments revealed that genotoxic stress increased collagenolysis in a relevant TNBC model based on the generic TFEBON program.

As an additional approach, RagC was knocked out in MDA-MB-231 cells using CRISPR-Cas9 (Figure S4A). RRAGC KO prevented mTOR association with endolysosomes and promoted constitutive nuclear localization of TFEBGFP (Figures S4B and S4C). Confirming above-described results, MDA-MB-231 RRAGC KO cells were highly gelatinolytic in a TFEB-dependent manner (Figure S4D). Similarly, MCF10DCIS cells KO for RRAGC displayed prominent TFEB nuclear localization and were highly collagenolytic as compared with parental counterpart (Figures S4E-S4H).

## TFEB stimulates MT1-MMP exocytosis and invadopodia dynamics and function

Immunoblotting analysis revealed that in MDA-MB-231 and MCF10DCIS cells, RagC KD resulted in a ~1.5-fold increase in

total MT1-MMP level, the key invadopodial ECM degradation effector, which was not seen upon RagC + TFEB KD (Figures S5A-S5C). A potential effect of RagC KD and TFEB activation on MT1-MMP exocytosis at invadopodia was assessed by live cell imaging of MDA-MB-231 cells expressing MT1-MMP<sup>pHLuorin</sup> (ectodomain-inserted super-ecliptic GFP becoming fluorescent upon exposure to neutral pH in extracellular space).⁴¹ Vesicular MT1-MMP<sup>pHLuorin</sup> signal was detected in vicinity of contacted collagen fibers and at fiber-plasma membrane contact sites (Video S1; gallery in Figure 3A). We previously established that MT1-MMP<sup>pHLuorin</sup>-containing endolysosomes remained connected with the plasma membrane through membrane tethers, allowing MT1-MMP transfer to the surface.¹⁶,³⁷ RagC KD led to a 3-fold increase in number of fluorescent MT1-MMP<sup>pHLuorin</sup>-positive endolysosomes, and 1.5-fold increase of static (≥ 14 min), likely tethered, MT1-MMP delivery carriers³⁷ (Figures 3A-3C; Video S1). These data are consistent with RagC KD/mTORC1<sup>OFF</sup> signaling inducing a burst of MT1-MMP exocytic insertion events. Cell surface labeling confirmed a 1.5-fold increase of endogenous surface-exposed MT1-MMP upon RagC KD, which was abolished by TFEB KD (Figures 3D and 3E; Figure S5A). Similarly, RagC KD induced a 2-fold increase in surface level of lysosomal CD63 (Figure 3F). Additionally, a 20%-40% increase in surface MT1-MMP was found in MCF10DCIS cells treated with Doxo or Torin-1 (Figure S5D). Collectively, these data indicated that mTORC1 repression triggered TFEB-dependent MT1-MMP surface exposure as consequence of increased endolysosome exocytosis at cell-matrix contact sites.

In agreement with MT1-MMP's role as initiator of invadopodia formation,¹⁷ TKS5-positive invadopodia increased by 70% upon RagC KD, and this increase was TFEB dependent (Figure 3G). In-vadopodia combine actin-based force production and proteolytic fiber degradation for invasive track formation.¹⁷ We used particle image velocimetry (PIV) to assess global movement (velocity) and direction of TKS5<sup>GFP</sup>-positive invadopodia and contacting fibers. We observed that the speed of outward invadopodia expansion significantly increased upon RagC KD as compared with control treatment, while concomitant TFEB KD abolished it (Figures 3H and 3I; Video S2). Additionally,

<caption>**Figure 3.** Active TFEB promotes MT1-MMP exocytosis and invadopodia dynamics</caption>

(A) Still images from representative movies of MDA-MB-231 cells expressing MT1-MMP<sup>pHLuorin</sup> (green) treated with control or RagC siRNA cultured on fluorescently labeled type I collagen (red). Right panels are galleries from video sequences (Video S1) documenting dynamics of exocytosis of MT1-MMP<sup>pHLuorin</sup> vesicles (inverted gray LUT, red arrows) on the collagen fibers (red). Double arrowheads, vesicle-to-plasma membrane connecting tether.

(B and C) Mean number ± SEM of exocytic events per min per cell (B) and percentage of static MT1-MMP<sup>pHLuorin</sup> vesicles for ≥ 14 min ± SEM in cells treated with indicated siRNA from two independent experiments (C). Statistical analysis was performed using Mann-Whitney test.

(D) Endogenous surface-labeled MT1-MMP (LUT fire) in MDA-MB-231 cells treated with indicated siRNAs.

(E) Mean intensity ± SEM of surface MT1-MMP per cell with indicated siRNA treatment. Data from three independent experiments are normalized to siCtrl. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(F) Mean intensity ± SEM of surface CD63 as in (E). Statistical analysis was performed using Mann-Whitney test.

(G) Mean number ± SEM of TKS5<sup>GFP</sup>-positive invadopodia in MDA-MB-231 cells treated with indicated siRNAs and normalized to number in siCtrl-treated cells from three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(H) Galleries of still images from video sequences of MDA-MB-231 expressing TKS5<sup>GFP</sup> treated with control or RagC siRNA on type I collagen (not shown, see Video S2). Green arrows are velocity vectors representing direction and speed of invadopodia motion computed by PIVLab. Orange arrows are interpolated velocity vectors.

(I) Mean velocity (µm/min) ± SEM of TKS5<sup>GFP</sup>-positive invadopodia in cells treated with indicated siRNAs measured from three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(J) Color-coded time projection of 10-min-interval images showing dynamics of TKS5<sup>GFP</sup>-positive invadopodia and fluorescent type I collagen fibers in cells treated with indicated siRNAs. All numerical data and statistical tests are provided in Table S1. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001; ns, not significant. Scale bars, 10 and 5 µm (H).
invadopodia expansion, fiber remodeling and matrix pore
enlargement induced by RagC KD were lost upon silencing of
TFEB or MT1-MMP as revealed by time projection of time-lapse
image series (Figure 3J). Altogether, these data indicated that a
surge in MT1-MMP exocytosis and activity was induced upon
TFEB activation, promoting formation of highly dynamic, hyper-
functional invadopodia.

Identification of mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> transcriptomic
program controlling invadopodia function

Among known TFEB targets,³³ core retromer complex compo-
nents, Vps35 and Vps26, regulate MT1-MMP exocytic recycling
and ECM degradation.⁴²,⁴³ We confirmed that Vps35 KD signif-
icantly diminished basal gelatinolysis activity and found that it
abolished mTORC1<sup>OFF</sup>-induced ECM degradation (Figure S6A).
These data identified retromer complex as a shared component
of the basal and inducible ECM degradation programs. Addition-
ally, TFEB regulates expression of the endolysosomal calcium
channel, mucolipin-1 (MCOLN1, aka TRPML1), which promotes
endolysosomal exocytosis.³³ Activation of MCOLN1 using ML-
SA1 agonist promoted gelatin degradation, while its silencing
prevented induction of ECM degradation upon RagC KD
(Figures S6B and S6C).

Next, we performed RNA sequencing (RNA-seq) analysis of MDA-MB-231 cells treated with two independent RagC siRNAs to identify genes differentially regulated upon mTORC1 repression. Gene set enrichment and gene ontology analysis revealed signatures common to both siRagC#1 and #2 related to lysosomal function and transport, regulation of exocytosis, MMP activity, and TFEB transcriptional activity (Figures 4A and 4B; Figure S6D). Negative enrichment scores were found for cell-cycle and protein translation genes, consistent with mTORC1 repression (Figure 4A; Figure S6D). Differential gene expression (DEG) analysis identified 155 and 105 genes that were differentially regulated upon treatment with siRagC#1 or siRagC#2, respectively (fold change (FC) = 1.4) (Figure 4C; Figure S6E). Among 40 co-regulated genes in siRagC#1 and #2 datasets, known TFEB targets were found such as ACP5, APOE, ATP6V0D2, COL16A1, FABP3, GDF15, SIGLEC15, SPON2, and SULT1C2.44-47 We confirmed transcriptional upregulation of some of these genes in MDA-MB-231 and MCF10DCIS cells knocked down for RagC or treated with mTORC1 inhibitors (Figure 4D; Figures S6F and S6G).

Acidification of the pericellular microenvironment is related to
increased invadopodia activity and tumor invasion.⁴⁸ We as-
sessed the contribution of v-ATPase subunit, ATP6V0D2, to
ECM degradation as it was upregulated, and v-ATPase contrib-
utes to extracellular space acidification (Figures 4C and 4D;
Figures S6F and S6G). Interestingly, gelatin degradation induced
upon RagC KD was abolished upon ATP6V0D2 silencing (Fig-
ure 4E). Similarly, inhibition of v-ATPase using Bafilomycin A1
repressed gelatin degradation induced by Torin-1 treatment
(Figure 4F), identifying ATP6V0D2, and v-ATPase, as an impor-
tant component of the mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> ECM degradation
program.

Recently, in a drug repurposing strategy, eltrombopag (EIO), a
United States Food and Drug Administration (FDA)-approved
compound used for treatment of chronic immune thrombocyto-
penia, was identified as a small molecule able to bind TFEB with

a K<sub>d</sub> of ~350 nM. EIO inhibited TFEB interaction with CLEAR-box
motifs in target genes, repressing its transcriptional activity.⁴⁹
Remarkably, upregulation of Torin-1-induced genes was in-
hibited in a dose-dependent manner by EIO (Figure S6H). Basal
gelatinolysis augmented upon increasing doses of EIO although
differences were not statistically significant (Figure 4G). Most
strikingly, EIO treatment counteracted Torin-1-induced gelati-
nolysis, in a dose-dependent manner (Figure 4G). These data
directly supported a pivotal contribution of TFEB transcriptional
activity to the inducible ECM degradation response of TNBC
cells and suggested a potent countermeasure to control an un-
wanted program in cancer management.

mTORC1 repression induces a collagenolytic pro-
invasive program in TNBC preclinical models ex vivo

Consequences of mTORC1 repression on 3D invasion in a dense
type I collagen matrix were investigated using MDA-MB-231 and
MCF10DCIS multicellular spheroids. Compared with control
spheroids, we observed a 30%–40% increase in invasion capac-
ity of RagC-depleted spheroids with parallel 2.5-fold increased
collagenolysis (Figures 5A–5G). These data indicated that
mTORC1 repression resulted in stronger invasive potential of
TNBC cells in 3D matrix *in vitro*.

For generalization to disease-closed models, we used PDXs
generated from primary TNBC tumors that are relevant regarding
clinicopathological parameters such as molecular subtypes and
drug response.²³,⁵⁰,⁵¹ Tumor cells dissociated from resected
TNBC PDX tumors were plated on a fibrillar type I collagen layer
ex vivo. Cortactin-positive structures closely associated with
cleaved fibers were visible, representing genuine invadopodia
(compare Figure 5H and Figure 3H; Ferrari et al.¹⁷). All together,
these observations indicated that freshly explanted PDX cells
formed bona fide active invadopodia when in contact with
collagen fibrils. Treatment of ex vivo-cultured PDX cells for
16 h with Rapa, Torin-1, or Evo strongly reduced mTORC1 activ-
ity toward S6K and 4E-BP1 (Figures S7A–S7C). In addition,
mTOR inhibition increased (CD63/mTOR-positive) endolyso-
some burden in HBCx-60 and HBX-90 PDX-derived cells (Fig-
ure S7D). Moreover, chemical inhibition of mTORC1 repressed
pTFEB<sup>S211</sup> levels and promoted its nuclear translocation,
consistent with activation of a transcriptomic program leading
to above-mentioned expansion of the endolysosomal network
(Figures S7E and S7F). Furthermore, MT1-MMP expression
increased in two out of three PDX models upon Evo treatment
(Figures S7G and S7H). Strikingly, pharmacological inhibition
of mTORC1 and TFEB activation induced a significant increase
in collagen cleavage in the three PDX models, fully confirmatory
of results in TNBC cell lines (Figures 5I and 5J). Similarly, we
observed that Doxo-induced DNA damage promoted collagen
cleavage by HBCx-60 TNBC PDX cells ex vivo (Figures 5K and
5L). Taken together, these experiments revealed that mTORC1
inhibition or genotoxic stress led to increased collagenolysis by
clinico-pathologically relevant TNBC models that involve the
generic TFEB<sup>ON</sup> program.

To evaluate whether Evo and mTORC1 inhibition impacted in-
vasion pattern of PDX explants, we generated PDX-derived or-
ganoids (PDXOs) that were embedded in fibrillar collagen,
mimicking solid tumors. PDXOs were cultured for 3 days in the
collagen gel (day 3), and drug or vehicle treatments were applied
<figure><img src="image_34.png" /><figcaption>**Figure 4. mTORC1<sup>OFF</sup> transcriptomic program in BC cells**</figcaption></figure>

(A) Selected ontology gene sets from the Molecular Signatures Database (MSigDB) based on normalized enrichment score (NES) in GSEA analysis of RNA-seq datasets form siRagC#1 and #2 versus siCtrl-treated cells.

(B) Enrichment plots from GSEA analysis performed on ranked siRagC #1 list.

(C) Volcano plots of gene-expression fold change from RNA-seq analysis of MDA-MB-231 cells treated with siRagC #1 or siRagC #2 as compared with siCtrl from six independent experiments. Dotted lines, fold change (FC) of 1.6 and *p* value of 0.05. Blue dots, differentially expressed genes (DEGs) specific to a single RagC siRNA; red dots, DEG common to both RagC siRNAs; gray dots, non-DEG.

(D) Mean RNA levels ± SEM determined by quantitative real time PCR analysis of selected genes in MDA-MB-231 cells treated with indicated siRNA or drugs from at least four independent experiments. Data are normalized to siCtrl or vehicle. Statistical analysis was performed using Mann-Whitney test.

(E–G) Graphs represent mean gelatin degradation levels ± SEM of MDA-MB-231 cells treated with indicated siRNAs or drugs normalized to siCtrl or vehicle, respectively, from three independent experiments. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test. All numerical data and statistical tests are provided in Table S1. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001; ns, not significant.
MDA-MB-231

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

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

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

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

HBCx-60 PDX (explant)
Cortactin/Col1-3/4C/Col I

<figure><img src="image_39.png" /><figcaption>I</figcaption></figure>

HBCx-90 PDX (explant)
Phalloidin/Col1-3/4C

<figure><img src="image_40.png" /><figcaption>J</figcaption></figure>

pS6K/GAPDH
(Fold change to vehicle)

<figure><img src="image_41.png" /><figcaption>M</figcaption></figure>

HBCx-8 PDX (Organoids)
Phalloidin/Col1-3/4C

<figure><img src="image_42.png" /></figure>

MCF10DCIS.com

<figure><img src="image_43.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_45.png" /><figcaption>K</figcaption></figure>

HBCx-60 PDX
(explant)

<figure><img src="image_46.png" /><figcaption>L</figcaption></figure>

<figure><img src="image_47.png" /></figure>

(legend on next page)
for 4 days (day 7). After 3 days, PDXOs were relatively small, with low pericellular collagen cleavage activity and minimal invasion (Figure 5M). In control conditions at day 7, PDXO size increased as cells proliferated. Overall, PDXOs remained compact although some cells escaped the organoids and invaded through the collagen gel (Figure 5M). Evo treatment impeded organoid growth⁵² and promoted strong pericellular collagenolysis by disseminated cells in the surrounding matrix (Figure 5M). Invasion and collagenolysis were abolished by treatment with pan-MMP inhibitor, GM6001 (Figure 5M). Altogether, these observations supported the conclusion that mTORC1 repression by therapeutic inhibitors in short term *ex vivo* culture of TNBC PDX cells triggered an invasion program based on MT1-MMP-mediated collagenolysis.

## RAGGC KO and TFEB activation affect basement membrane and DCIS-like tumor progression *in vivo*

The relevance of mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> response on matrix remodeling and local invasion was assessed *in vivo*, first by evaluating the effect of Evo treatment on mice-implanted HBCx-60 PDX. 3 weeks after implantation, mice were treated with Evo for 3 or 12 days. Increased necrosis was already visible after 3 days of treatment, and reduction of tumor size was observed after 12 days (Figures S8A and S8B). mTORC1 proxies, pmTOR<sup>Ser2448</sup> and pS6<sup>Ser240/Ser244</sup>, were strongly repressed already after 3 days of Evo treatment (Figures 6A and 6B; Figures S8C–S8E). Analysis of TFEB localization revealed a significant increase in nuclear translocation both at days 3 and 12 of Evo treatment (Figures 6C and 6D; Figure S8F). Collectively, these data uncovered Evo capacity to repress mTORC1 and induce TFEB activation in TNBC PDX-implanted mice *in vivo*.

Absence of clear locoregional invasion in HBCx-60 PDX precluded analysis of the effect of mTORC1 repression in this model. We turned to a human DCIS xenograft model consisting of MCF10DCIS cells, which generate DCIS-like tumors upon subcutaneous injection in nude mice, and progress with local ECM remodeling and invasion (Figure S9A).⁵³ RAGGC KO did

not affect MCF10DCIS cell proliferation *in vitro* nor tumor growth *in vivo* (Figures S4F and S9B). As previously described, MCF10DCIS tumor xenografts consisted of DCIS-like nodules of pan-cytokeratin (CK)-positive epithelial tumor cells surrounded by an outer layer of basal, (myoepithelial)-like p63-positive cells facing the separating collagen-IV-rich, basement membrane-like stroma (Figures 6E and 6K; Figures S9C–S9E).⁵³ DCIS-like nodule organization with basal p63+ cells at the periphery persisted over time (see semi-quantitative H-score, Figure 6F), although reduction of the separating stroma was visible over the course of the 5-week experiment coinciding with tumor growth and local invasion (Figure 6K; Figures S9C–S9E).⁵³ Cells positive for p63 forming the outer layer of the nodules were strongly positive for MT1-MMP (Figures 6G and 6H), reminiscent of p63<sup>High</sup>/MT1-MMP<sup>High</sup> myoepithelial cell organization in normal breast tissue and characteristic of DCIS tumors in patients.²⁰,⁵³ In addition, TFEB was cytoplasmic in control MCF10DCIS tumor nodules, showing a perinuclear, granular distribution, likely corresponding to inactive TFEB associated with endolysosomes (Figures 6I and 6J). In contrast, in tumor xenografts generated by MCF10DCIS cells KO for RAGGC, tumor cells were massively positive for nuclear p63 and membrane-associated MT1-MMP within the tumor nodules starting from week 1 and on (Figures 6E–6H; Figure S9F–S9H). In addition, TFEB was predominantly nuclear within the entire tumor nodules generated by RAGGC KO cells (Figures 6I and 6J). All together, these findings uncovered that DCIS-like organization was disrupted in RAGGC KO xenografts, while the delimitating ECM-rich stroma was reduced and a collagen-IV-rich matrix progressively accumulated between individual tumor cells inside the tumor masses (Figure 6K, arrowheads; Figures S9F–S9H). Collectively, these data indicated that mTORC1 repression and consequent TFEB activation correlated with appearance of basal-like p63<sup>High</sup>/MT1-MMP<sup>High</sup> tumors concomitant with disrupted DCIS-like organization and induction of ECM-remodeling capacity of tumor cells, suggestive of local invasion and cancer progression.

## Figure 5. mTORC1 inhibition unleashes a 3D invasion program

(A) RagC expression in MDA-MB-231 cells treated with control or RagC siRNA. Vinculin, loading control.

(B) Multicellular spheroids of MDA-MB-231 cells treated with control or RagC siRNA cultured in 3D type I collagen for 48 h and stained for F-actin (inverted gray LUT). Insets, spheroids fixed 45 min after inclusion in collagen as control.

(C and D) Mean invasion ± SEM (C) and mean collagen cleavage ± SEM by multicellular spheroids treated with indicated siRNAs in 3D collagen from three (C) or two (D) independent experiments. Data are normalized to siCtrl. Statistical analysis was performed using Mann-Whitney test.

(E–G) MCF10DCIS cells were treated and analyzed as in (A) and (B), and 3D invasion was quantified as in (C). Means invasion ± SEM are shown. Data are normalized to siCtrl and are from three independent experiments.

(H) TNBC PDX cells (HBCx-90) cultured on fibrillar type I collagen (red) for 6 h in complete medium and stained for cortactin (green) and collagen cleavage (orange). Insets, higher magnification of collagenolytic invadopodia in boxed region.

(I) TNBC PDX cells (HBCx-90) embedded in fibrillar type I collagen gel and cultured with indicated mTORC1 inhibitors or vehicle for 16 h. Samples were stained for F-actin (cyan) and cleaved collagen revealing pericellular collagenolysis (orange).

(J) Green bars represent mean ± SEM pS6K levels (see Figures S7A–S7C), normalized to GAPDH levels (fold change) in three distinct TNBC PDXs (HBCx-60, -90, and -8) treated with indicated drugs or vehicles. Statistical analysis was performed using one-sample t test. Pink bars represent mean collagen cleavage ± SEM in the three PDXs from three independent experiments. Data are normalized to corresponding vehicle for each drug. Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(K) Mean γH2AX foci number per nucleus ± SEM in HBCx-60 PDX cells treated with Torin-1 or with DNA-damaging agent, doxorubicin (Doxo). Data from two independent experiments are normalized to DMSO (vehicle). Statistical analysis was performed using one-way ANOVA with Kruskal-Wallis test.

(L) Mean collagen cleavage ± SEM of HBCx-60 cells treated with indicated drugs. Data from three independent experiments are normalized to DMSO (vehicle).

(M) Organoids of cells collected from HBCx-8 embedded in 3D type I fibrillar collagen for 3 days in complete medium (day 3, insets). Indicated inhibitors were added to cultures for 4 more days (day 7). Samples were stained for F-actin (inverted gray LUT) and cleaved collagen (red). Collagenolysis increased in Evo-treated PDXOs as compared with control or PDXOs treated with combination of Evo and GM6001. All numerical data and statistical tests are provided in Table S1.

*p* < 0.05; **p* < 0.01; ***p* < 0.001; ****p* < 0.0001; ns, not significant. Scale bars, 200 and 50 µm (M).
<figure><img src="image_48.png" /><figcaption>Figure 6. mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> promotes DCIS-like tumor progression *in vivo*</figcaption></figure>

(A) HBCx-60 PDX tumors were implanted and after 3 weeks, mice were treated for 3 or 12 days with Evo or vehicle (Ctrl). Tumors analyzed by immunohistochemistry for pS6<sup>Ser240/244</sup> expression. Corresponding low magnification sections are shown in Figure S8E.

(B) Mean H-score of pS6 ± SEM in indicated conditions. Statistical analysis was performed using Holm-Sídák's test.

(C) TFEB staining after 12 days of treatment. Low magnification sections are shown in Figure S8E. Right row shows AI-assisted nucleus segmentation and nuclear (n)TFEB intensity scoring.
DISCUSSION

We demonstrate that various situations converging in nuclear
translocation and activation of TFEB, boosted by ~1-log an in-
vadopodia-, MT1-MMP-dependent ECM proteolytic degrada-
tion program and enhanced BC cells' invasive potential. These
conditions included (1) mTORC1 repression in MDA-MB-231
cells through genetic silencing of key mTORC1 components
that are key for controlling TFEB nuclear localization and activ-
ity⁵ (Figures 7A-7C), (2) mTORC1 inhibitors, including Evo,
currently evaluated as potential targeted therapy in TNBCs
with alterations in the PI3K/AKT/mTOR axis (Figure 7B), and
(3) genotoxic stress (Figure 7D).¹,⁵,²⁴,⁵⁵ Moreover, our findings
indicate that direct TFEB activation (overexpression, apilimod,
FLCN KD) triggered the matrix degradation program (Figures
1B, 2F, and 2G; Figure S2L).⁹ On the contrary, TFEB KD or phar-
macological inhibition (EIO) abolished ECM degradation in
response to mTORC1 repression (Figures 2Eand 4G). Collec-
tively these data demonstrate that invadopodia control,
downstream of mTORC1 repression, solely depends on
TFEB, irrespective of other mTORC1 substrates. Potential clin-
icopathologic relevance is grounded on converging observa-
tions in well-established clinically relevant TNBC models,
namely several TNBC cell lines and explanted PDX cells
(Figures 5I-5M), Evo treatment of a TNBC PDX in vivo, as well
as a TNBC DCIS-like xenograft model based on MCF10DCIS
cells KO for RRAGC (Figure 6).

Transcriptomic profiling revealed a gene signature consistent
with hallmarks of endolysosome biogenesis and function, endo-
lysosome exocytosis, MMP activity, and TFEB transcriptional
activity (Figure 4). Interestingly, some of the identified genes
were not required for basal ECM degradation in BC cells, such
as v-ATPase subunit ATP6V0D2 (Figure 4E). In contrast, the
core retromer component, VPS35, and known TFEB target,
was required both for basal and mTORC1<sup>OFF</sup>-induced matrix
degradation responses (Figure S6A).<sup>42,43</sup> In addition, very
consistently, TFEB silencing did not impact basal ECM degrada-
tion nor did it affect basal surface MT1-MMP and invadopodia
levels (Figures 2C, 2E, 2G, 2H, 3E, and 3G). Collectively, these
data suggest that the mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> program is not the
mere amplification of the basal matrix degradation program
but represents an emerging mechanism promoting invadopodia
hyperfunctionality and invasive track formation.<sup>13,16,17</sup> In a
broader perspective, besides implications as a pro-invasive
and metastasis mechanism, the mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> response
may contribute to metabolic reprogramming and capacity of
cancer cells to benefit from alternative ECM-derived aa sources
in nutrient-poor environment.<sup>11,12,23,56</sup>

Relevance and contribution of the mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> ECM
degradation response could be established in relation with local
invasion using a human TNBC DCIS-like xenograft model
(Figure 6; Figure S9). In this model, due to their bipotential pro-
genitor property *in vivo*, MCF10DCIS cells form tumor nodules
surrounded by a rim of basal (myoepithelial)-like p63<sup>High</sup>/
MT1-MMP<sup>High</sup> cells separated by a basement membrane-like
stroma.<sup>53,57</sup> Strikingly, TFEB activation upon mTORC1 repres-
sion in RRAGC KO MCF10DCIS tumor xenograft correlated
with conversion of the entire tumor cell population to a basal-
like phenotype, disruption of the DCIS organization, collagen-
IV-rich basement membrane remodeling and tumor progression
(Figure 6; Figure S9). Myoepithelial cells of the breast (and
MCF10DCIS cells<sup>20</sup>) express the ΔNp63α isoform required for
mammary gland development and function.<sup>58,59</sup> Myoepithelial
cells control basement membrane deposition and remodeling.<sup>60</sup>
In addition, we reported that ΔNp63α contributes to local inva-
sion in basal-like TNBC through MT1-MMP transcriptional upre-
gulation, facilitating basement membrane breaching during the
DCIS-to-invasive transition.<sup>20</sup> ΔNp63α is also shown to control
collective invasion in BC organoids by promoting a basal epithe-
lial program.<sup>61,62</sup> Based on these and additional findings, it is
likely that in MCF10DCIS tumors, direct contact with the
collagen-IV-rich stroma instructs cells at the nodule rim to adopt
a basal-like p63<sup>High</sup>/MT1-MMP<sup>High</sup> phenotype reminiscent of
DCIS organization. Upon RRAGC KO and TFEB activation, over-
all enhancement of matrix remodeling possibly drives a positive
feedback loop mechanism leading to (1) disruption of DCIS-like
organization and (2) increased exposure of tumor cells to
collagen cleavage fragments, promoting global acquisition of a
basal-like phenotype, which further stimulates ECM deposition
and turnover inside the tumor mass. This phenotype is reminiscent
of the aggressive p63<sup>High</sup>/MT1-MMP<sup>High</sup> basal-like (BL2)
subtype reported in human TNBC patients.<sup>20,63</sup> Therefore, our
data based on TNBC models including PDXs support the possi-
bility that mTORC1 repression using mTOR inhibitors, a potential
therapeutic option for TNBC management, may induce a pro-
invasive basal-like program.

Our data show that treatment with DNA double-strand break
inducer, Doxo, triggers the generic mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> ECM
degradation response in MCF10DCIS and TNBC PDX cells
(Figures 2I, 2J, 5K, and 5L). It is well established that genome
stability control is linked to cell growth signaling. mTORC1 activ-
ity is repressed upon genotoxic stress through p53 activation,
which upregulates several mTORC1 negative regulators such
as PTEN, TSC2, sestrins, and AMPK.<sup>39,64</sup> In addition, DNA dam-
age can activate TFEB/TFE3 in a mTORC1 and p53-dependent
manner.<sup>40</sup> Along this line, we found that Doxo treatment resulted

(D) Mean percentage of TFEB-positive nuclei ± SEM in indicated conditions. Statistical analysis was performed using Holm-Sídák's test.
(E, G, I, and K) MCF10DCIS control (Ctrl) or RRAGC KO #C3 cells were injected subcutaneously in nude mice. At weeks 1, 3, or 5 post-inoculation, tumors were dissected, and sections stained with indicated markers. Representative images of week 5 tissue sections of different tumor xenografts labeled for basal marker, p63 (E), MT1-MMP (G), TFEB (I), and basement membrane collagen IV (K). Bottom row in (K) show week 3 sections labeled for collagen IV. Arrowheads point to p63-positive (E) and high-level MT1-MMP-expressing cells (G) at the tumor nodule rim. Arrowheads point to collagen IV deposition between RRAGC KO cells (K). N, nucleus; n, necrosis; s, stroma.

(F, H, and J) Al-based semi-quantitative intensity histoscore (H-score) of nuclear p63 (F), membrane-associated (mb)MT1-MMP (H), and nuclear TFEB (J). Distribution of H-score values corresponding to each time point is represented using Whisker plots. Median, upper and lower quartiles and extremes are shown. However, due to limited sample size for individual time points, statistical analysis comparing control and RRAGC KO tumor xenografts was performed after pooling H-scores from the three time points. Statistical analysis was performed using Mann-Whitney tests. All numerical data and statistical tests are provided in Table S1. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001. Scale bars, 100 and 50 µm (K, week 3).
<figure><img src="image_49.png" /><figcaption>**Figure 7.** Summary scheme of matrix degradation control by the mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> program</figcaption></figure>

(A) In basal, nutrient-replete conditions, mTORC1 is active (mTORC1<sup>ON</sup>), and phosphorylated, inactive TFEB is retained in the cytoplasm (TFEB<sup>OFF</sup>).<sup>6</sup> A basal invadopodia response is activated in the presence of ECM fibers, enabling matrix remodeling and invasive track formation based on MT1-MMP trafficking from endolysosomes to forming invadopodia.<sup>13</sup>

(B and C) Repression of mTORC1 activity (mTORC1<sup>OFF</sup>) upon gene silencing or pharmacological inhibition of mTORC1 (B), or loss of function of the RagC/D GAP, FLCN (C), leads to activation of a TFEB transcriptional program (TFEB<sup>ON</sup>). This program stimulates the exocytic surface exposure of MT1-MMP at invadopodia, enhancing ECM proteolytic cleavage and facilitating tumor cell invasion. Eltrombopag (EIO) blocks TFEB interaction with the promoter of its target genes and abolishes the pro-invasive matrix degradation program (B, inset).

(D) DNA damage leads to TFEB activation triggering a burst of invadopodia activity. Figure created using BioRender.com.

both in reduction of mTORC1 activity and TFEB activation in MCF10DCIS cells (Figure 2I; Figures S3G and S3H). In a recent study, we reported that DNA damage induced upon mechanical compression on the nucleus (dependent on nuclear envelope breakdown and nucleus accessibility of DNA exonuclease TREX1), stimulated ECM degradation in MCF10DCIS cells, while TREX1 KO impaired local invasion in the intraductal injection DCIS xenograft model.<sup>38</sup> The mechanism underlying the matrix degradation burst in response to DNA damage remained unidentified.<sup>38</sup> This study linking DNA damage to mTORC1 repression and TFEB activation provides a mechanistic framework to these earlier observations. In a broader context, it is plausible that genome stress (proliferation-induced compressive stress, genotoxic drugs...) may increase aggressivity and capacity of tumor cells to breach ECM barriers based on the mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup> axis. Our findings may also be critically relevant in the context of BC treatment using genotoxic agents and chemoresistance.

TFEB upregulation is a poor prognosis factor for overall survival in BCs.<sup>65,66</sup> Similarly, low expression of FLCN, Raptor, or mTOR is associated with a lower probability of relapse-free survival.<sup>67,68</sup> Additionally, increased MT1-MMP expression is corre-

lated with lower probability of metastasis-free survival.<sup>19,20</sup> Overall, poor prognosis associated with these conditions is fully consistent with our observations wherein repression of mTORC1 activity and TFEB nuclear activation in TNBC cells promote an invadopodia-mediated invasion program. Despite undoubtful benefit on overall tumor burden reduction, we surmise that treatments that target the mTOR axis, including in combo with genotoxic agents, raise concerns, and our data call for cautious examination of currently evaluated therapies in BC and TNBC management. The here-identified counteractive action of FDA-approved TFEB inhibitor, EIO, on mTORC1<sup>OFF</sup>/TFEB<sup>ON</sup>-induced ECM degradation response raises the possibility of therapeutic strategies to efficiently manage this adversary program.

## Limitations of the study

Our data reveal a robust induction of matrix degradation by TNBC cells in response to mTORC1 repression and consequent activation of TFEB. Why does LAMTOR KD that reduces mTOR lysosomal localization and pS6K and p4E-BP1 levels under basal culture conditions fail to induce the ECM degradation response? What is the contribution of P27Kip1/CDKN1B to this
response? Why is Raptor or FLCN KD unable to stimulate ECM degradation in MCF10DCIS cells? We have yet to address these issues.

Furthermore, albeit our study uncovers the capacity of mTORC1 inhibition and TFEB activation in disrupting MCF10DCIS tumor nodule organization and promoting a basal-like program in a DCIS xenograft system, it is limited to a single model, and currently, it does not address whether mTORC1 inhibition can promote BC invasiveness and metastasis in vivo. In addition, while mTOR inhibitor Evo has been approved for the treatment of advanced hormone receptor-positive BC and drugs targeting the PI3K/AKT/mTOR pathway are under evaluation in TNBCs, it is not a current line of treatment in this aggressive subtype.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by lead contact Philippe Chavrier, (philippe.chavrier@curie.fr).

### Materials availability

Any requests for resources and reagents should be directed to lead contact.

### Data and code availability

RNA-seq source data are available with the GEO accession number: GEO: GSE242138 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242138). Home-made codes for image analysis used for this study are available online: https://github.com/David-Remy. The pipe-line analysis for RNA-seq data is available online: https://github.com/bioinfo-pf-curie/RNA-seq/tree/v4.0.0. PIVLab, the open-source MATLAB code for PIV analysis is available online: (https://www.mathworks.com/matlabcentral/fileexchange/27659-pivlab-particle-image-velocimetry-piv-tool-with-gui). Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

Authors greatly acknowledge the CurieCore Tech facilities "Cell and Tissue Imaging" facility and "Bioinformatics (CUBIO)" of Institut Curie for technical help, Dr. J. d'Alessandro for help with PIV analysis, Drs. C. Demetriades, H. Stenmark, and C. Raiborg for helpful comments during the preparation of this manuscript. This study was supported by grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from Agence Nationale de la Recherche ("Investissements d'Avenir" program), ITMO-Cancer Aviesan (Plan Cancer III), and SiRIC-Curie program (INCa-DGOS-465 and INCa-DGOS-Inserm_12554) to ICGex NGS platform of Institut Curie, and generous donations from Association Ruban Rose (Grand Prix 2022) and Mr. T. Paulsen (InvaCell Project), funding by Fondation ARC (ARCPGA12019120000973_1574) and INCa PLBIO 2019-1-PL BIO-07-ICR-1 to P.C. Funders had no role in study design, data collection and analysis, and decision to publish or preparation of the manuscript.

## AUTHOR CONTRIBUTIONS

Conceptualization, D.R. and P.C.; experimentation, D.R., S.A.-B., S.d.T., C.J., G.C., F.N., V.R., A.P., and A.D.; software, D.R. and A.-S.M.; data analysis, D.R., S.A.-B., S.d.T., C.J., G.C., A.P., and A.B.; formal analysis, I.B. and D.M.; drafting manuscript, D.R. and P.C.; manuscript approval, all authors; resources and supervision, D.R., A.N., E.M., and P.C.; project administration, D.R. and P.C.; funding acquisition, P.C.

## 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

* Patient-derived xenografts

* Ex vivo culture of patient-derived xenograft cells

* Subcutaneous xenografts

* Human breast cancer cell lines

* METHOD DETAILS

* Antibodies

* Drugs

* Transfection and siRNA treatment

* Generation of KO cell lines and proliferation assay

* Fluorescent gelatin degradation assay

* Polymerization of type I collagen gel and quantification of pericellular collagenolysis

* Multicellular tumor spheroid invasion assays

* Western blot analysis

* Surface MT1-MMP and CD63 staining

* DNA damage detection

* MT1-MMP<sup>pHLuorin</sup> exocytosis

* Particle Image Velocimetry analysis of TK5<sup>GFP</sup>-positive invasion

* Nuclear translocation of TFEB

* Luciferase Assay

* Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

* RNA Sequencing (RNA-Seq) and expression analysis

* Immunohistochemistry analysis

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2024.12.005.

Received: November 13, 2023

Revised: August 14, 2024

Accepted: December 2, 2024

Published: December 26, 2024

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59. Forster, N., Saladi, S.V., van Bragt, M., Sfondouris, M.E., Jones, F.E., Li, Z., and Ellisen, L.W. (2014). Basal cell signaling by p63 controls luminal progenitor function and lactation via NRG1. Dev. Cell 28, 147-160. https://doi.org/10.1016/j.devcel.2013.11.019.

60. Gudjonsson, T., Adriance, M.C., Sternlicht, M.D., Petersen, O.W., and Bissell, M.J. (2005). Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia. J. Mammary Gland Biol. Neoplasia 10, 261-272. https://doi.org/10.1007/s10911-005-9586-4.

61. Fisher, M.L., Balinth, S., and Mills, A.A. (2023). ΔNp63alpha in cancer: importance and therapeutic opportunities. Trends Cell Biol. 33, 280-292. https://doi.org/10.1016/j.tcb.2022.08.003.

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66. Giatromanolaki, A., Sivridis, E., Kalamida, D., and Koukourakis, M.I. (2017). Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis. Clin. Breast Cancer 17, e119-e125. https://doi.org/10.1016/j.clbc.2016.11.006.
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74. Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. https://doi.org/10.1038/nmeth.2089.

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Antibodies</td><td></td><td></td></tr><tr><td>4E-BP1</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#9452; RRID: AB_331692</td></tr><tr><td>Actin</td><td>Sigma</td><td>Cat#A1978; RRID: AB_476692</td></tr><tr><td>ATG5</td><td>Novus Biologicals</td><td>Cat#NB110-53818; RRID: AB_828587</td></tr><tr><td>CD63 (LAMP3)</td><td>BD Biosciences</td><td>Cat#556019; RRID: AB_396297</td></tr><tr><td>Collagen IV</td><td>Abcam</td><td>Cat#ab6586; RRID: AB_305584</td></tr><tr><td>Collagen cleavage site (Col1-3/4C)</td><td>Immunoglobe GmbH</td><td>Cat#0217-050; RRID: AB_2893279</td></tr><tr><td>Cortactin (p80 / p85)</td><td>Millipore</td><td>Cat#05-180-1; RRID: AB_309647</td></tr><tr><td>Cadherin-1 (clone 4A2C7)</td><td>Thermo Fisher Scientific</td><td>Cat#33-4000; RRID: AB_2533118</td></tr><tr><td>FLCN</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#3697; RRID: AB_2231646</td></tr><tr><td>phospho-Histone H2A.X (Ser139)</td><td>Millipore</td><td>Cat#05-636; RRID: AB_309864</td></tr><tr><td>GAPDH</td><td>Santa Cruz Biotechnology</td><td>Cat#sc-47724; RRID: AB_627678</td></tr><tr><td>GFP</td><td>Abcam</td><td>Cat#ab13970; RRID: AB_300798</td></tr><tr><td>GFP</td><td>Abcam</td><td>Cat#ab6556; RRID: AB_305564</td></tr><tr><td>LAMTOR 1</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#8975; RRID: AB_10860252</td></tr><tr><td>LAMTOR 4</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#13140; RRID: AB_2798129</td></tr><tr><td>LC3</td><td>MBL International</td><td>Cat#M152-3; RRID: AB_1279144</td></tr><tr><td>MT1-MMP</td><td>Millipore</td><td>Cat#MAB3328; RRID: AB_570599</td></tr><tr><td>MT1-MMP (clone 2D7)</td><td>Dr C.L. Tomasetto, IGBMC, Illkirch, France</td><td>N/A</td></tr><tr><td>mTOR</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#2983; RRID: AB_2105622</td></tr><tr><td>NPRL2</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#37344; RRID: AB_2799113</td></tr><tr><td>p27 Kip1/CDKN1B</td><td>Santa Cruz Biotechnology</td><td>Cat#sc-1641; RRID: AB_628074</td></tr><tr><td>p63 (clone 4A4)</td><td>Biocare Medical</td><td>Cat#VP 163 G5; RRID: AB_10582729</td></tr><tr><td>p70 S6 kinase</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#2708; RRID: AB_390722</td></tr><tr><td>Cytokeratin</td><td>Agilent</td><td>Cat#M3515; RRID: AB_2132885</td></tr><tr><td>phospho-4E-BP1</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#13443; RRID: AB_2728761</td></tr><tr><td>phospho-mTOR (Ser2448) (clone 49F9)</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#2976; RRID: AB_490932</td></tr><tr><td>phospho-p70 S6 kinase</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#9205; RRID: AB_330944</td></tr><tr><td>phospho-S6 (Ser240/244) (clone D68F8)</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#5364; RRID: AB_10694233</td></tr><tr><td>phospho-TFEB (Ser211)</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#37681; RRID: AB_2799117</td></tr><tr><td>RagA</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#4357; RRID: AB_10545136</td></tr><tr><td>RagB</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#8150; RRID: AB_11178806</td></tr><tr><td>RagC</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#9480; RRID: AB_10614716</td></tr><tr><td>Raptor</td><td>Santa Cruz Biotechnology</td><td>Cat#sc-81537; RRID: AB_2130791</td></tr><tr><td>TFE3</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#14779; RRID: AB_2687582</td></tr><tr><td>TFEB</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#4240; RRID: AB_11220225</td></tr><tr><td>TFEB</td><td>Thermo Fisher Scientific</td><td>Cat#A303-673A; RRID: AB_11204751</td></tr><tr><td>TKS5</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#16619; RRID: AB_2798767</td></tr><tr><td>Vinculin, clone VIIF9</td><td>Millipore</td><td>Cat#MAB3574; RRID: AB_2304338</td></tr><tr><td>Vps35 (clone B5)</td><td>Santa Cruz Biotechnology</td><td>Cat#sc-374372; RRID: AB_10988942</td></tr><tr><td>HRP</td><td>Sigma-Aldrich</td><td>Cat#A0545; RRID: AB_257896</td></tr><tr><td>HRP</td><td>Jackson ImmunoResearch Labs</td><td>Cat#115-035-062; RRID: AB_2338504</td></tr><tr><td>Goat Anti-Chicken IgG Antibody, AF488</td><td>Molecular Probes</td><td>Cat#A11039; RRID: AB_142924</td></tr></tbody></table>

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**Continued**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Donkey Anti-Mouse IgG Antibody, AF488</td><td>Molecular Probes</td><td>Cat#A21202; RRID: AB_141607</td></tr><tr><td>Cy3-AffiniPure Donkey Anti-Mouse IgG</td><td>Jackson ImmunoResearch Labs</td><td>Cat#715-165-151; RRID: AB_2315777</td></tr><tr><td>Donkey Anti-Mouse IgG Polyclonal Antibody</td><td>Molecular Probes</td><td>Cat#A31571; RRID: AB_162542</td></tr><tr><td>Donkey Anti-Rabbit IgG Antibody, AF488</td><td>Molecular Probes</td><td>Cat#A-21206; RRID: AB_2535792</td></tr><tr><td>Cy3-AffiniPure Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch Labs</td><td>Cat#711-165-152; RRID: AB_2307443</td></tr></tbody></table>

**Bacterial and virus strains**

<table><tr><td>E. coli One Shot™ TOP10<br/>[F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74<br/>recA1 araD139 Δ(araLEU) 7697 galU<br/>galK rpsL (StrR) endA1 nupG]</td><td>Invitrogen</td><td>Cat#C404010</td></tr></table>

**Biological samples**

<table><tr><td>Patients Derived Xenografts (PDXs)</td><td>Preclinical Investigation Laboratory,<br/>Translational Research Department,<br/>Institut Curie (Paris, France)</td><td>https://institut-curie.org/plateforme/<br/>curiecoretech-vivo-preclinical-experiments</td></tr></table>

**Chemicals, peptides, and recombinant proteins**

<table><tr><td>Apilimod</td><td>Euromedex</td><td>Cat#S6414</td></tr><tr><td>Bafilomycin A1</td><td>Sigma</td><td>Cat#196000</td></tr><tr><td>Doxorubicin (Doxo)</td><td>Cell Signaling TECHNOLOGY</td><td>Cat#5927</td></tr><tr><td>Eltrombopag (EIO)</td><td>Sigma</td><td>Cat#SML3182</td></tr><tr><td>Everolimus (Evo)</td><td>Novartis Pharma SAS</td><td>Cat#AFINITOR 10 mg Cpr Plq/30</td></tr><tr><td>GM6001</td><td>Merck Millipore</td><td>Cat#CC1100</td></tr><tr><td>Rapamycin (Rapa)</td><td>Tocris Biotechnne</td><td>Cat#1292</td></tr><tr><td>Torin-1</td><td>Tocris Biotechnne</td><td>Cat#4247</td></tr><tr><td>Phalloidin AF488</td><td>Molecular Probes</td><td>Cat#A12379</td></tr><tr><td>Phalloidin AF546</td><td>Molecular Probes</td><td>Cat#A22283</td></tr><tr><td>Phalloidin AF633</td><td>Molecular Probes</td><td>Cat#A22284</td></tr></table>

**Deposited data**

<table><tr><td>Raw and analyzed RNA-Seq data</td><td>This paper</td><td>GEO: GSE242138</td></tr></table>

**Experimental models: Cell lines**

<table><tr><td>MDA-MB-231</td><td>ATCC</td><td>HTB-26</td></tr><tr><td>MCF10DCIS.com</td><td>Asterand</td><td>Miller et al.<sup>69</sup></td></tr><tr><td>MCF10DCIS.com RRAGC KO</td><td>This paper</td><td>N/A</td></tr></table>

**Experimental models: Organisms/strains**

<table><tr><td>Mouse: Crl:NU(Ico)-Foxn1<sup>nu</sup></td><td>Charles River Laboratories</td><td>Strain Code: 620</td></tr></table>

**Oligonucleotides**

<table><tr><td>SMARTpool siRNA Non-targeting (siCtrl)</td><td>horizon</td><td>D-001810-01</td></tr><tr><td>SMARTpool siRNA: siACP5 (Gene id: 54)</td><td>horizon</td><td>L-009615-00-0005</td></tr><tr><td>SMARTpool siRNA: siATG5 (Gene id: 9474)</td><td>horizon</td><td>L-004374-00-0005</td></tr><tr><td>SMARTpool siRNA: siATP6V0D2 (Gene id: 245972)</td><td>horizon</td><td>L-005105-00-0005</td></tr><tr><td>SMARTpool siRNA: siCDKN1B (Gene id: 1027)</td><td>horizon</td><td>L-003472-00-0005</td></tr><tr><td>SMARTpool siRNA: siDEPDC5 (Gene id: 9681)</td><td>horizon</td><td>L-020708-00-0005</td></tr><tr><td>SMARTpool siRNA: siFLCN (Gene id: 201163)</td><td>horizon</td><td>L-009998-02-0005</td></tr><tr><td>SMARTpool siRNA: siGDF15 (Gene id: 9518)</td><td>horizon</td><td>L-019875-00-0005</td></tr><tr><td>SMARTpool siRNA: siLAMTOR1 (Gene id: 55004)</td><td>horizon</td><td>L-020916-02-0005</td></tr><tr><td>SMARTpool siRNA: siLAMTOR4 (Gene id: 389541)</td><td>horizon</td><td>L-028918-02-0005</td></tr><tr><td>SMARTpool siRNA: siMCOLN1 (Gene id: 57192)</td><td>horizon</td><td>L-006281-00-0005</td></tr></table>

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<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>SMARTpool siRNA: siMT1-MMP (Gene id: 4323)</td><td>horizon</td><td>L-004145-00-0005</td></tr><tr><td>SMARTpool siRNA: siNPRL2 (Gene id: 10641)</td><td>horizon</td><td>L-015645-00-0005</td></tr><tr><td>SMARTpool siRNA: siRICTOR (Gene id: 253260)</td><td>horizon</td><td>L-016984-00-0005</td></tr><tr><td>SMARTpool siRNA: siRHEB (Gene id: 6009)</td><td>horizon</td><td>L-009692-00-0005</td></tr><tr><td>SMARTpool siRNA: siRAPTOR (Gene id: 74370)</td><td>horizon</td><td>L-004107-00-0005</td></tr><tr><td>SMARTpool siRNA: siRRAGA (Gene id: 10670)</td><td>horizon</td><td>L-016070-00-0005</td></tr><tr><td>SMARTpool siRNA: siRRAGB (Gene id: 10325)</td><td>horizon</td><td>L-012189-01-0005</td></tr><tr><td>SMARTpool siRNA: siRRAGC (Gene id: 64121)</td><td>horizon</td><td>L-017822-01-0005</td></tr><tr><td>siRNA: siRRAGC #1 (Gene id: 64121)</td><td>horizon</td><td>J-017822-11-0002</td></tr><tr><td>siRNA: siRRAGC #2 (Gene id: 64121)</td><td>horizon</td><td>J-017822-10-0002</td></tr><tr><td>SMARTpool siRNA: siRRAGD (Gene id: 58528)</td><td>horizon</td><td>L-016120-00-0005</td></tr><tr><td>SMARTpool siRNA: siSIGLEC15 (Gene id: 284266)</td><td>horizon</td><td>L-032127-01-0005</td></tr><tr><td>SMARTpool siRNA: siSLC38A9 (Gene id: 153129)</td><td>horizon</td><td>L-007337-02-0005</td></tr><tr><td>SMARTpool siRNA: siTFE3 (Gene id: 7030)</td><td>horizon</td><td>L-009363-00-0005</td></tr><tr><td>SMARTpool siRNA: siTFEB (Gene id: 7942)</td><td>horizon</td><td>L-009798-00-0005</td></tr><tr><td>SMARTpool siRNA: siTKS5 (Gene id: 9644)</td><td>horizon</td><td>L-006657-00-0005</td></tr><tr><td>SMARTpool siRNA: siVPS35 (Gene id: 55737)</td><td>horizon</td><td>L-010894-00-0005</td></tr><tr><td>Oligonucleotide: sgRRAGC_8_For [5' caccgATCGGCCGCGCCGTAACTGC]</td><td>Sigma</td><td>N/A</td></tr><tr><td>Oligonucleotide: sgRRAGC_8_Rev [5' aaacGCAGTTACGGCGCGGCCGATc]</td><td>Sigma</td><td>N/A</td></tr><tr><td>Oligonucleotide: sgRRAGC_14_For [5' caccgGATATACGTCATTGACGCAC]</td><td>Sigma</td><td>N/A</td></tr><tr><td>Oligonucleotide: sgRRAGC_14_Rev [5' aaacGTGCGTCAATGACGTATATCc]</td><td>Sigma</td><td>N/A</td></tr><tr><td>TaqMan Gene Expression Assay: GAPDH</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs02758991_g1</td></tr><tr><td>TaqMan Gene Expression Assay: ACP5</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00356261_m1</td></tr><tr><td>TaqMan Gene Expression Assay: ATP6V0D2</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs01084784_m1</td></tr><tr><td>TaqMan Gene Expression Assay: GDF15</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00171132_m1</td></tr><tr><td>TaqMan Gene Expression Assay: SIGLEC15</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs01386395_m1</td></tr><tr><td>TaqMan Gene Expression Assay: SLC38A9</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00940336</td></tr><tr><td>TaqMan Gene Expression Assay: RRAGC</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00900846_m1</td></tr><tr><td>TaqMan Gene Expression Assay: MCOLN1/TRPML1</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs01100653_m1</td></tr><tr><td>TaqMan Gene Expression Assay: RICTOR</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00380903_m1</td></tr><tr><td>TaqMan Gene Expression Assay: DEPDC5</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00823262_m1</td></tr><tr><td>TaqMan Gene Expression Assay: RHEB</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs04187711_m1</td></tr><tr><td>TaqMan Gene Expression Assay: RRAGD</td><td>ThermoFisher SCIENTIFIC</td><td>Assay ID: Hs00222001_m1</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>pEGFP-N1-TFEB</td><td>Roczniak-Ferguson et al.<sup>70</sup></td><td>Addgene #38119</td></tr><tr><td>4XCLEAR-luciferase reporter</td><td>Cortes et al.<sup>71</sup></td><td>Addgene #66800</td></tr><tr><td>pGL3 BRE Luciferase</td><td>Korchynskyi and ten Dijke<sup>72</sup></td><td>Addgene #45126</td></tr><tr><td>pGL4.73[hRluc SV40]</td><td>Gift from F. Toledo (Institut Curie, Paris, France)</td><td>N/A</td></tr><tr><td>pSpCas9(BB)-2A-Puro (PX459) V2.0</td><td>Ran et al.<sup>73</sup></td><td>Addgene #62988</td></tr></table>

Software and algorithms

<table><tr><td>ImageJ</td><td>Schneider et al.<sup>74</sup></td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Prism</td><td>GraphPad Software</td><td>https://www.graphpad.com</td></tr></table>

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**Continued**

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>PIVLab</td><td>Thielicke and Stamhuis<sup>75</sup></td><td>https://www.mathworks.com/matlabcentral/fileexchange/27659-pivlab-particle-image-velocimetry-piv-tool-with-gui</td></tr><tr><td>HALO-AI software v3.5</td><td>Indica Labs</td><td>https://learn.indicalab.com/tag/halo-3-5/</td></tr><tr><td>NIS Elements Software</td><td>Nikon</td><td>https://www.microscope.healthcare.nikon.com/fr_AMS/products/software/nis-elements</td></tr><tr><td>MetaMorph</td><td>Molecular Devices</td><td>https://fr.moleculardevices.com/products/cellular-imaging-systems/high-content-analysis/metamorph-microscopy</td></tr><tr><td>CollagenDegradationIndex</td><td>https://github.com/David-Remy</td><td>https://doi.org/10.5281/zenodo.14045677</td></tr><tr><td>Spheroid_3DInvasion</td><td>https://github.com/David-Remy</td><td>https://doi.org/10.5281/zenodo.14045684</td></tr><tr><td>Intensity_perCell</td><td>https://github.com/David-Remy</td><td>https://doi.org/10.5281/zenodo.14045666</td></tr><tr><td>NuclearTranslocation</td><td>https://github.com/David-Remy</td><td>https://doi.org/10.5281/zenodo.14045690</td></tr><tr><td>GelatinDegradationIndex</td><td>https://github.com/David-Remy</td><td>https://doi.org/10.5281/zenodo.14045688</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Patient-derived xenografts

Patients' breast tumor samples were engrafted with informed consent from the patient into the interscapular fat pad of 8- to 12-week-old female Swiss nude mice obtained from Charles River Laboratories, which were maintained under specific pathogen-free conditions. The housing facility was kept at 22 °C (±2 °C) with a relative humidity of 30-70%. The light/dark cycle was 12 hrs light/12 hrs dark. Mice were maintained on a standard diet (4RF25, Mucedola SRL, Italy) and were given free access to food and water. For Evo treatment of PDX in the mice, three weeks after implantation, Evo (or water as control, Ctrl) was administered orally at a dose of 2.5 mg/kg every two days for three or twelve days. Mice were sacrificed 4 hrs after the last treatment and xenografted tumors were harvested, fixed in 10% neutral buffered formalin and embedded in paraffin. All animals used for this study were maintained under pathogen-free conditions, in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved by the ethics committee of the Curie Institute CEEA-IC #118 (Authorization APAFIS#26879-2020081315161665-v1 given by the National Authority) in compliance with the international guidelines. The protocol also complied with the internationally established 3R principles, in accordance with the UKCCCR guidelines.

### Ex vivo culture of patient-derived xenograft cells

The procedure for collecting tumor cells from PDX has been previously described.<sup>23</sup> For the pericellular collagenolysis assay, non-attached PDX tumor cells in the culture supernatant were resuspended in a 2.2 mg/ml collagen I solution as described above and incubated for 16 hrs in DMEM-F12 medium (Gibco) supplemented with 10% FCS (complete medium) and with indicated drugs. For PDX-derived organoids (PDXOs), the same protocol was used except that samples were treated with collagenase/hyaluronidase for 30 min and disperse treatment was omitted to keep cell junctions intact. At the filtration step on Cell Strainer, organoids were recovered in DMEM-F12 complete medium. Finally, samples were centrifuged briefly (1500 rpm, 2-5 s, 3 times) and supernatants containing single cells were discarded. Organoids were embedded in 40 µl drops of type I collagen drops as described above in DMEM-F12 complete medium.

### Subcutaneous xenografts

5x10⁵ Ctrl or RRAGC KO #C3 MCF10DCIS cells were resuspended in 100 µl of a mixture of 50% medium and 50% Growth Factor-Reduced Matrigel (Corning) and injected subcutaneously in the flank of 6-8-week-old virgin female Swiss Nude mice (Charles River Laboratories). Tumor size was measured with a caliper 1-2 times per week. Mice were sacrificed after 1-, 3- or 5 weeks after inoculation, and tumors were fixed and processed for immunohistochemistry staining. In the first experiment, three mice were injected per timepoint and for each cell population. In the second experiment, two mice were injected per timepoint and for each cell population. Animal care and housing and the experimental protocol were specifically approved by the ethics committee of the Curie Institute CEEA-IC #118 (Authorization APAFIS#25909-2020060410487032).
## Human breast cancer cell lines

Human MDA-MB-231 breast adenocarcinoma cells obtained from ATCC (ATCC HTB-26) were grown in L-15 medium (Sigma-Aldrich) supplemented with 15% fetal calf serum (FCS) and 2 mM glutamine (ThermoFisher) at 37°C in 1% CO₂. The MCF10DCIS.com (MCF10DCIS) cell line⁶⁹ was purchased from Asterand and grown in DMEM-F12 supplemented with 10% horse serum (ThermoFisher) and 2 mM glutamine (ThermoFisher) at 37°C in 5% CO₂. MDA-MB-231 cells stably expressing TKS5<sup>GFP</sup> or MT1-MMP<sup>pHLuorin</sup> were generated by lentiviral transduction.¹⁷,³⁷ All cell lines were routinely tested for mycoplasma contamination.

## METHOD DETAILS

### Antibodies

The following antibodies were used in this study. Cell Signaling TECHNOLOGY: 4E-BP1 (9452, 1:1000 for WB), FLCN (3697, 1:1000 for WB), LAMTOR1 (8975, 1:100 for IF, 1:1000 for WB), LAMTOR4 (13140, 1:250 for IF, 1:10000 for WB), mTOR (2983, 1:400 for IF), NPRL2 (37344, 1:1000 for WB), p70 S6 kinase (2708, 1:1000 for WB), p4E-BP1 (13443, 1:1000 for WB), pmTOR Ser2448 (2976, 1:1000 for IHC), pp70 S6 kinase (9205, 1:1000 for WB), pS6 Ser240/244 (5364, 1:1000 for IHC), pTFEB Ser211 (37681, 1:100 for IF, 1:1000 for WB), RagA (4357, 1:100 for IF, 1:1000 for WB), RagB (8150, 1:1000 for WB), RagA (9480, 1:100 for IF, 1:1000 for WB), TFE3 (14779, 1:1000 for WB), TFEB (4240, 1:100 for IF, 1:1000 for WB), TKS5 (16619, 1:100 for IF, 1:1000 for WB); Sigma: Actin (A1978, 1:10000 for WB); Novus Biologicals: ATG5 (NB110-53818, 1:500 for WB); BD Pharmingen: CD63 (556019, 1:200 for IF); Abcam: Collagen IV (ab6586, 1:400 for IHC), GFP (ab13970, 1:500 for IF), GFP (ab6556, 1:500 for WB); Santa Cruz: GAPDH (25788, 1:10000 for WB), p27Kip1/CDKN1B (1641, 1:1000 for WB), Raptor (81537, 1:1000 for WB), Vps35 (sc-374372, 1:500 for WB); Immunoglobe: Collagen cleavage site/Col1⁻³/⁴C (0217-050, 1:100 for IF); Merck: Cortactin (05-180-l, 1:200 for IF), gH2AX (05-636, 1:100 for IF), MT1-MMP (MAB3328, 1:500 for IF, 1:1000 for WB), Vinculin (MAB374-C, 1:1000 for WB); Invitrogen: Cadherin-1 (33-400, 1:100 for IHC); MBL: LC3 (M152-3, 1:1000 for IF); BiocareMedical: p63 (CM 163 A, B, C); Dako: pan-cytokeratins (M3515, undiluted); Bethyl Transduction: TFEB (A303-673A-M, 1:200 for IHC).

### Drugs

The following drugs were used in this study: Apilimod (Euromedex, S6414, 1 µM), Bafilomycin A1 (Sigma, 196000, 100 nM), Doxorubicin (Cell Signaling Technology, 5927S, 80 nM, Eltrombopag (Sigma, SML3182, 5-20 µM), Everolimus (Novartis Pharma SAS, AFINITOR 10 mg Cpr Plq/30, 100 nM), GM6001 (Merck Millipore, CC1100, 40 µM), Rapamycin (Tocris Biotechnne, 1292, 20 nM), Torin-1 (Tocris Biotechnne, 4247, 350 nM). Drugs were added for 16 hrs.

### Transfection and siRNA treatment

For transient expression, MDA-MB-231 cells were transfected with plasmid constructs using Lipofectamine 3000 (ThermoFisher) for 24 hrs according to the manufacturer's instructions. Expression plasmids pEGFP-N1-TFEB (Addgene #38119),⁷⁰ 4XCLEAR-luciferase reporter (Addgene #66800)⁷¹ and pGL3 BRE Luciferase (Addgene #45126)⁷² were purchased from Addgene and kindly provided by Drs S. Ferguson, A. La Spada, and M. Roussel and P. ten Dijke, respectively. Plasmid pGL4.73[hRluc SV40] was a kind gift of Franck Toledo (Institut Curie, Paris, France). For RNA interference, cells were treated with indicated ON-TARGETplus SMART-pool siRNA reagent (50 nM, horizon) using Lullaby reagent (OZ Biosciences) according to manufacturer's instructions. Cells were analyzed 72 hrs later or when required, collected 48 hrs later for transfection.

### Generation of KO cell lines and proliferation assay

RRAGC knock-out (KO) in MDA-MB-231 and MCF10DCIS cells were generated using the CRISPR/Cas9 system developed by the Zhang's lab⁷³ and single guide RNAs (sgRNAs) designed by the Demetriades lab.⁷⁶ Briefly, guides were inserted into PX459 vector digested with BbsI. Cells were seeded in 6-well plates and transfected the following day using Lipofectamin 3000 according to the manufacturer's instructions. After puromycin selection, independent clones were picked by single-cell dilution (MDA-MB-231) or by colony picking (MCF10DCIS). In MDA-MB-231 cells, one sgRNA was used (sgRRAGC_8), while in MCF10DCIS, two sgRNAs were combined (sgRRAGC_8 and sgRRAGC_14). For MCF10DCIS RRAGC KO, clone #C3 was used for all experiments.

For MTT proliferation assay, 5x10³ cells were plated in flat bottom 96-well plates (6 technical replicates), MMT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, 500 µg/ml, Fischer Scientific) was added for 3 hrs. Insoluble formazan converted from MTT by mitochondria of metabolically active cells was solubilized in DMSO and measured by optical density at 570 nm with a plate reader (Clariostar, BMG Labtech). Data were averaged over the six replicates and normalized to the time '0' timepoint of each cell population (Ctrl or RRAGC KO #C3).

### Fluorescent gelatin degradation assay

MDA-MB-231 cells treated with siRNA or drugs were plated on Oregon Green 488 (OG⁴⁸⁸)-conjugated cross-linked gelatin (ThermoFisher) as previously described.³⁶ For contactin staining, cells were pre-extracted after 6 hrs on gelatin with 0.1% Triton X-100 in 4% PFA in PBS for 90 s at 37°C and fixed in 4% PFA in PBS for 30 min at 37°C. For mTOR and CD63 staining, cells were fixed after 3 hrs on gelatin in 4% PFA in PBS for 30 min and permeabilized with 0.1% Triton X-100 in PBS or 15 min. After extensive washes and 1 hrs blocking in 10% FCS in PBS, samples were stained with appropriate antibodies or with fluorescently-labeled phalloidin to stain F-actin. Images were acquired with a wide-field microscope (Eclipse 90i Upright; Nikon) using a 100x Plan Apo VC
1.4 oil objective and a cooled interlined charge-coupled device (CCD) camera (CoolSnap HQ²; Roper Scientific). A z-dimension series of images was taken every 0.2 µm by means of a piezoelectric motor (Physik Instrumente). The system was steered by Metamorph software. Al-assisted quantification of the degradation index (DI) is explained elsewhere.³⁶ mTOR association with endolysosomes was quantified using a homemade Fiji macro as previously described.²⁶

**Polymerization of type I collagen gel and quantification of pericellular collagenolysis**

Coverslips were covered with a thin layer of fluorescently-labeled type I collagen gel as described elsewhere,³⁶ and cells were plated for 60 min (MDA-MB-231) or 4 hrs (MCF10DCIS). Samples were stained for cortactin and cleaved collagen, and images were acquired with a wide-field microscope as described above. Quantification of Col1-³/₄C signal (cleaved collagen) was performed with a homemade ImageJ macro.³⁶,⁷⁴ For visualization purposes, images were deconvoluted using the Nikon NIS-Elements software (3D-deconvolution module; Lucy-Richardson algorithm).

**Multicellular tumor spheroid invasion assays**

MDA-MB-231 and MCF10DCIS cells were transfected with 50 nM of siRNA by nucleofection (Kit V, Lonza) and plated in 6-well plates. The day after, cells were collected and transfected again with 50 nM of siRNA by Lullaby reagent as described above. Multicellular spheroids were prepared immediately after with the hanging droplet method.⁷⁷ After 3 days, spheroids were embedded in a 40 µl drop of 2.2 mg/ml fluorescently-labeled type I collagen and left to polymerize for 90 min at 37°C. Spheroids were fixed immediately in 4% PFA in PBS (T0) or left to grow in a complete medium for two days before fixation (T2). Samples were either stained with DAPI and Phalloidin-Alexa Fluor488 or permeabilized and stained with Col1-³/₄C antibody as described above, and then counterstained with Phalloidin-Alexa Fluor488. Image acquisition was performed with a spinning-disk microscope (Inverted Eclipse Ti-2, Nikon) equipped with a W1 Yokogawa Spinning head using a 4x objective and an ORCA-Flash4.0 V2 sCMOS camera (Hamamatsu) for a wide field of view and a large dynamical range. A z-dimension series of images were taken every 4 µm by means of a Nano z500-N piezoelectric motor (Mad City Labs). The system was steered by NIS Elements software.

For quantification of invasion, stacks of images were projected along the z-axis and background intensity of Phalloidin was measured as a reference value. The mean spheroid diameter was measured using a homemade ImageJ macro.⁷⁴,⁷⁸ To record the Col1-³/₄C signal on the entire spheroid, an Apo LWD 20X water objective was used to acquire a 3x3 mosaic with a 10% overlap stitch between images. For each frame of the mosaic, a z-dimension series of images were taken every 2 µm and the collagen cleavage index was quantified for each frame.

**Western blot analysis**

Treated cells were washed twice with PBS and then collected in 4x Laemmli Sample Reducing buffer and heated for 10 min at 95°C or collected in a homemade RIPA lysis buffer (50 mM Tris HCl pH 8.0, 137 mM NaCl, 1% Triton X-100, 10 mM MgCl₂, 10% glycerol, 1 tablet of cOmplete Protease Inhibitor cocktail (Roche) and 10% phosphatase inhibitor cocking 2 (Sigma). Samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 4-12% Tris-glycine gels (ThermoFisher Scientific). Proteins were transferred on a nitrocellulose membrane using the iBlot2 Dry Blotting System (Invitrogen). After incubating the membranes in 5% BSA or 5% skimmed milk in TBS-Tween 1%, proteins were detected by immunoblotting analysis with the indicated antibodies. Antibodies were detected using the Enhanced Chemiluminescence reagent (Amersham) on the ChemiDoc MP Imaging System (Bio-Rad).

**Surface MT1-MMP and CD63 staining**

Cells were surface labeled with mouse monoclonal anti-MT1-MMP (clone 2D7) or anti-CD63 primary antibodies diluted in complete medium for 30 min at 4°C. After extensive washes with complete medium and once with PBS, cells were fixed in 4% PFA in PBS. To ensure surface labeling, all solutions were ice-cold, and the cells were kept on ice during all incubation steps. Samples were counterstained with Alexa Fluor 488-conjugated anti-mouse IgG secondary antibodies and Cy5-conjugated Phalloidin to visualize cell shape. Image acquisition was performed with a confocal spinning-disk microscope (Inverted Eclipse Ti-2, Nikon) as described above using a 60x water objective. A z-dimension series of images were taken every 0.2 µm by means of a Nano z500-N piezoelectric motor (Mad City Labs). The system was steered by NIS Elements software. Cells were delimited using the Phalloidin signal and the mean intensity of MT1-MMP or CD63 at the plasma membrane was measured and normalized to the mean intensity of MT1-MMP or CD63 in control cells.

**DNA damage detection**

MCF10DCIS cells or HBCx-60 PDX cells treated with Torin-1 or Doxorubicin for 16 hrs (on collagen I for PDX cells) were fixed in 4% PFA in PBS, permeabilized with 0.1% Triton X-100 in PBS. After saturation the coverslips in 10% FBS in PBS, samples were stained with anti- in 10% 4% orin-1 or Doxorubicin for 16 sAlexa Fluor488-conjugated anti-mouse IgG antibodies. Image acquisition was performed with a confocal spinning-disk microscope (Inverted Eclipse Ti-2, Nikon) as described above using a 60x water objective. A z-dimension series of images were taken every 0.5 µm by means of a Nano z500-N piezoelectric motor (Mad City Labs). The system was steered by NIS Elements software. The number of ernjugated anti-mouse IgG antibodies. Image acquisition was performed witthe BioVoxxel 3D toolbox on ImageJ (https://github.com/biovoxxel/bv3dbox).⁷⁴
## MT1-MMP<sup>PHLuorin</sup> exocytosis

MDA-MB-231 MT1-MMP<sup>PHLuorin</sup> cells treated with siRNA were plated on glass bottom dishes (MatTek Corporation) layered with a thin layer of fibrilar fluorescently-labeled type I collagen.<sup>36</sup> Cells were imaged by confocal spinning disk microscopy (one images every 20 s). The number and lifetime of MT1-MMP<sup>PHLuorin</sup> exocytic events (i.e., GFP flashes) were measured per min and per cell.

## Particle Image Velocimetry analysis of TKS5<sup>GFP</sup>-positive invadopodia

MDA-MB-231 TKS5<sup>GFP</sup> cells treated with siRNA were plated on glass bottom dishes (MatTek Corporation) layered with a thin layer of fluorescently-labeled polymerized type I collagen and imaged by confocal spinning disk microscopy (one image/min). PIV analysis was performed on one fixed-size region per cell using PIVLab,<sup>75</sup> an open-source MATLAB tool (https://www.mathworks.com/matlabcentral/fileexchange/27659-pivlab-particle-image-velocimetry-piv-tool-with-gui). Quantification of TKS5<sup>GFP</sup>-positive invadopodia and color-coded time projections have been previously described.<sup>17</sup>

## Nuclear translocation of TFEB

MDA-MB-231 cells were transfected with pEGFP-N1-TFEB (TFEB<sup>GFP</sup>) for 24 hrs either 48h after siRNA transfection or 8h prior to drug supplementation. MCF10DCIS cells were treated with siRNA for 72 hrs of with mTOR drugs for 16 hrs as described above. PDX cells were treated with mTOR drugs for 16 h. Cells were then fixed with 4% PFA in PBS for 20 min. After permeabilization with 0.1% Triton X-100 in PBS for 10 min and blocking with 10% FCS in PBS for 1 h, MDA-MB-231 samples were stained with anti-mTOR, anti-CD63, and anti-GFP primary antibodies and counterstained with AF488-conjugated anti-chicken, Cy3-conjugated anti-rabbit, and AF647-conjugated anti-mouse secondary antibodies. MCF10DCIS samples were stained with anti-TFEB and anti-CD63 primary antibodies and counterstained with Cy3-conjugated anti-rabbit, and AF647-conjugated anti-mouse secondary antibodies as well as Phalloidin-AF488. Samples were mounted in Prolong DAPI for nucleus visualization. A z-dimension series of images were taken on confocal spinning-disk microscopy as described above. Quantification of the nuclear translocation of TFEB (MCF10DCIS) or TFEB<sup>GFP</sup> (MDA-MB-231) was done using a homemade ImageJ macro.<sup>74</sup> For PDX cells, number of nuclei positive for TFEB was counted and normalized to the number of nuclei in the field of acquisition. CD63-positive vesicles were detected using the AutoThreshold and Analyze Particles commands of Fiji excluding regions <50 px to avoid background signals and the total area was divided by the cell area. Values were normalized to the area ratio of control cells.

## Luciferase Assay

MDA-MB-231 were transiently co-transfected with a plasmid containing the pGL3-Luciferase or the pGL3-4X-CLEAR Luciferase-reporter constructs, and SV40 pRL Renilla Luc Control Reporter Vector as an internal control (Promega) using JetPrime transfection reagent (Polyplus-transfection). For TFEB<sup>GFP</sup> overexpression assays, cells were additionally transfected with pEGFP empty vector or pEGFP-N1-TFEB vector. Transfected cells were seeded on 12-well plates for 24 hrs at 37°C. After 4 h, medium was refreshed, with or without rapamycin. Cells were lysed 24 hrs later and assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions and normalized to constitutive Renilla luciferase activity. Normalized data were reported to pGL3 empty vector and plotted as such.

## Quantitative reverse transcription polymerase chain reaction (RT-qPCR)

MDA-MB-231 cells were treated with indicated siRNA for 72 hrs or with indicated drugs for 16 hrs and RNA isolation was carried out with the RNeasy kit according to the manufacturer's instructions (Qiagen). Reverse transcription was performed on 1 µg of RNA with the High-Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions (Applied Biosystems). To assess mRNA levels by real-time RT-qPCR, we used the LightCycler® 480 Probes Master (Roche) and the pre-designed probes and primers from TaqMan Gene Expression assays (Applied Biosystems) on a LightCycler® 480 Instrument II (Roche). All samples were run in duplicate and the amounts of mRNAs of the genes of interest were normalized to that of the reference gene GAPDH according to the 2DCt method.<sup>79</sup>

## RNA Sequencing (RNA-Seq) and expression analysis

MDA-MB-231 cells were treated with two independent siRNAs against RRAGC or control siRNA (6.25 nM, horizon) using Lullaby reagent (OZ Biosciences) and total RNA isolation was performed 72 hrs later as above. Six independent experiments were performed. RNA quality was evaluated with Agilent 2100 BioAnalyzer (Agilent Technologies). RNA sequencing libraries were prepared from 500ng of total RNA using the Illumina® Stranded mRNA Prep Ligation library preparation kit, which allows to perform a strand specific sequencing. This protocol includes a first step of polyA selection using magnetic beads to focus sequencing on polyadenylated transcripts. After RNA fragmentation, cDNA synthesis was then performed and resulting fragments were used for dA-tailing followed by ligation of RNA Index Anchors. PCR amplification with indexed primers (IDT for Illumina RNA UD Indexes) was finally achieved, with 13 cycles, to generate the final cDNA libraries. Individual library quantification and quality assessment was performed using Qubit fluorometric assay (Invitrogen) with dsDNA HS (High Sensitivity) Assay Kit and LabChip GX Touch using a High Sensitivity DNA chip (Perkin Elmer). Libraries were then equimolarly pooled and quantified by qPCR using the KAPA library quantification kit (Roche). Sequencing was carried out on the NovaSeq 6000 instrument (Illumina) using paired-end 2 x 100 bp, to obtain around 30 million clusters (60 million raw paired-end reads) per sample. Pre-analysis and quality control of the data was carried out with the Curie
BioInformatics pipeline (https://github.com/bioinfo-pf-curie/RNA-seq/tree/v4.0.0). For analysis, low-expressed genes (<1 transcript per million (TPM) in all samples) were first filtered out and DEG were obtained by applying the limma methodology with Trimmed Mean of M-values (TMM) normalization factors and voom estimation of the mean-variance relationship of the log-counts.⁸⁰ siCtrl vs. siRagC#1 or siRagC#2 comparisons were carried out and differential expressed genes were obtained with following cut-offs: FDR DRRagC#1 or siRagC#2 comparisons were carried out and differential expressed genes were obtained with following cut-offs: FDR DRRagC#1 or siRagC#2 comparisons were performed on the list of filtered genes. Genes were ranked according to the absolute value of Moderated Welch Test metric using NES (Normalized Enrichment Score) which accounts for differences in gene set size.⁸¹

## Immunohistochemistry analysis

Routinely fixed, paraffin-embedded, 3 µm-thick tissue sections were de-paraffinized, rehydrated and pretreated by heat induced epitope retrieval (HIER pH6 or pH9, 20 min). Epitope detection by horseradish peroxidase technique and DAB chromogenic substrate revelation was performed on the Leica BOND RX Autostainer using the Bond Polymer Refine Detection Kit (Leica) according to manufacturer's instructions. Briefly, after blocking endogenous peroxidase activity (10 min) and inhibiting non-specific staining (20 min), slides were incubated with diluted antibody for 60 min at room temperature. After washing with PBS, sections were incubated with post primary IgG linker reagent to localize mouse antibodies and Poly-HRP IgG reagent localizes rabbit antibodies. Immunoreactive signals were detected using diaminobenzidine (DAB) chromogen. Finally, the sections were lightly counterstained with Mayer's Hematoxylin. Slides were scanned at 40x magnification with Ultra-Fast IntelliSite slide scanner (Philips Healthcare). Virtual slide creation was performed using the Image Management System (IMS) (Philips Healthcare) at 400X mode. Digital image analysis was performed with HALO-Al software v3.5 (Indica Labs) and Deep Learning (DL) models at resolution of 0.25 µm/px. Viable, necrotic, or irrelevant areas of tissue were manually annotated under supervision of an experienced pathologist. DL training model was performed on 3 slides with different IHC intensities (strong, moderate and weak) for the marker. HALO-Al operator stopped the algorithm once an error rate/cross-entropy rate of less than 0.01 had been reached. Then HALO-Al analyzed all the cases blindly and randomly. For each tumor section, a histoscore (H-score) was determined using the formula: $H-score=3 \times %[strong intensity stained cells] + 2 \times %[moderate intensity stained cells] + 1 \times %[weak intensity stained cells]$ giving a 0-300 semi-quantitative value.⁸² Results were compared to pathologist's semi-quantitative assessment. Individual H-scores are provided in Table S1. Number of tumors analyzed for each cell population and timepoint from two independent experiments was limited (up to five), thus samples from three different timepoints were pooled for statistical analysis.

## QUANTIFICATION AND STATISTICAL ANALYSIS

All data are presented as mean ± S.E.M. from at least three independent experiments. GraphPad Prism software was used for statistical analysis. Data were tested for normal distribution using the D'Agostino-Pearson normality test and nonparametric tests were applied otherwise. All numerical data and statistical tests are provided in Table S1. One-way ANOVA, Kruskal-Wallis or Mann-Whitney tests were applied as indicated in Table S1. Statistical significance was defined as *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant.
Supplemental information

TFEB triggers a matrix degradation
and invasion program in triple-negative
breast cancer cells upon mTORC1 repression

David Remy, Sandra Antoine-Bally, Sophie de Tocqueville, Célia Jolly, Anne-Sophie Macé, Gabriel Champenois, Fariba Nemati, Isabel Brito, Virginie Raynal, Amulya Priya, Adèle Berlioz, Ahmed Dahmani, André Nicolas, Didier Meseure, Elisabetta Marangoni, and Philippe Chavrier
A

<figure><img src="image_50.png" /></figure>

B

<figure><img src="image_51.png" /></figure>

C

<figure><img src="image_52.png" /></figure>

**Supplemental Figure 1 related to Figure 1. Quantification of mTORC1 activity.** (A, B) MDA-MB-231 cells treated with the indicated siRNA were analyzed by immunoblotting with the indicated antibodies including total and phosphorylated S6K
(see quantification in Figure 1C) and p4E-BP1 levels. Silencing of targeted mTORC1 components was assessed by immunoblotting analysis with indicated antibodies (A) or by RTqPCR (B). For silencing LAMTOR or GATOR1 complexes, LAMTOR1 and LAMTOR4 or DEPDC5 and NRPL2 subunits were simultaneously silenced, respectively. Vinculin was used as loading control. (C) Quantification of p4E-BP1 levels in cells treated with indicated siRNAs. Data are normalized to control non-targeting siRNA (siCtrl). Numerical data and statistical tests are detailed in Supplemental Table 5.
<figure><img src="image_53.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>E</figcaption></figure>

LC3-II

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_59.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_62.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_63.png" /></figure>

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_66.png" /><figcaption>K</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>L</figcaption></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /><figcaption>M</figcaption></figure>

<figure><img src="image_70.png" /></figure>
**Supplemental Figure 2 related to Figure 2. mTORC1 repression promotes TFEB activity.**

(A) Immunoblots showing levels of total and phosphorylated (p)TFEB (Ser211) in lysates of MDA-MB-231 cells treated with indicated siRNA. Cells treated with control non-targeting siRNA (siCtrl) or cells cultured in EBSS starvation medium were used as control. Vinculin was used as a loading control. (B) Immunoblots showing the depletion of siRNA targets. Vinculin was used as a loading control. (C) Quantification of normalized area of CD63-positive vesicles in MDA-MB-231 cells treated with indicated siRNAs. (D) Immunoblots showing the depletion of RagC and ATG5 in MDA-MB-231 cells treated with indicated siRNA. (E) Indirect immunofluorescence analysis showing the accumulation of LC3-II-positive vesicles (orange) in cells treated with control (siCtrl) or RagC siRNA. The graph shows the area of LC3-II-positive vesicles normalized to the cell area in cells treated with indicated siRNA. (F) Quantification of gelatin degradation by MDA-MB-231 cells treated with indicated siRNAs. (G) Immunoblots showing the depletion of RagC and TFE3 in MDA-MB-231 cells treated with indicated siRNA. The graph shows the quantification of gelatin degradation by MDA-MB-231 cells treated with indicated siRNAs. (H) Immunoblotting analysis and quantification of pS6K and p4E-BP1 levels in MDA-MB-231 cells treated with indicated siRNA and drugs or corresponding vehicle. (I) The graph represents the mean intensity ratio and S.E.M. of nuclear versus cytoplasmic TFEB in cells treated with indicated drugs as in panel H. (J,K) Luciferase activity in MDA-MB-231 cells transfected with reporter 4X-CLEAR LUC construct upon overnight treatment with Rapa or vehicle (J), or TFEBGFP overexpression (K). Promoter-less Luciferase construct (LUC) was used as a negative control. (L) MDA-MB-231 cells transiently expressing GFP or TFEBGFP, cultured on a fibrillar type I collagen network (blue) for 4 hrs. Cleaved collagen I is stained with anti-Col1-¾C1 antibodies (orange).
Inserts show higher magnification of Col1-¾C1 signal in boxed regions (Gray inverted LUT). Graph shows collagen cleavage. (M) Immunoblots and quantification of pS6K and p4E-BP1 and total TFEB levels in MDA-MB-231 cells treated with indicated siRNAs and TFEB activatory drug, Apilimod or vehicle. All numerical data and statistical tests are detailed in Supplemental Table 5. Scale bars, 10 µm, 2 µm (insets).
A MCF10DCIS

<figure><img src="image_71.png" /></figure>

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B TFEB intensity

<figure><img src="image_74.png" /></figure>

<figure><img src="image_75.png" /></figure>

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C

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D TFEB intensity

<figure><img src="image_78.png" /></figure>

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<figure><img src="image_81.png" /></figure>

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E

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F

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G

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H

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I MCF10DCIS

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J gH2AX/Phalloidin

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K

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<figure><img src="image_96.png" /></figure>

<figure><img src="image_97.png" /></figure>
**Supplemental Figure 3 related to Figure 2.** mTORC1 repression and TFEB activation promote ECM degradation in MCF10DCIS cells. (A) Immunoblotting analysis showing RagC and TFEB depletion and levels of pS6K and p4E-BP1 in MCF10DCIS treated with the indicated siRNAs. Graphs show pS6K and p4E-BP1 levels normalized to GAPDH. Data are normalized to siCtrl-treated cells. (B) TFEB immunostaining (Fire LUT) in MCF10DCIS cells treated with control or RagC siRNA. Dotted lines, cell and nucleus contour. The graph represents the mean intensity ratio and S.E.M. of nuclear versus cytoplasmic TFEB in MCF10DCIS cells treated with control or RagC siRNA. (C) Immunoblotting analysis showing levels of pS6K and p4E-BP1 in MCF10DCIS cells treated with indicated siRNAs and drugs. pS6K and p4E-BP1 levels have been quantified and normalized to vinculin levels used as a loading control and mean values from three independent experiments are shown below the blots. (D) TFEB immunostaining (Fire LUT) in MCF10DCIS cells treated with indicated drugs or vehicle. Dotted lines, cell and nucleus contour. (E) Mean intensity ratio and S.E.M. of nuclear versus cytoplasmic TFEB in MCF10DCIS cells treated as indicated. Starvation in EBSS medium was used as a positive control for TFEB translocation as opposed to cells grown in nutrient-replete medium (CM). (F) Quantification of collagen I degradation by MCF10DCIS cells treated with control or TFEB siRNA and cultured with indicated drugs or vehicles. (G) Immunoblotting analysis of levels of pS6K and p4E-BP1 in MCF10DCIS cells treated with indicated siRNAs. GAPDH was used as a loading control. Graphs show quantification of pS6K and p4E-BP1 levels in indicated conditions. Data are normalized to levels in cells treated with siCtrl. (H) Mean collagen I degradation and S.E.M. by MCF10DCIS cells treated with indicated siRNAs. (I) Mean intensity ratio and S.E.M. of nuclear versus cytoplasmic TFEB in MCF10DCIS cells treated as indicated. (J) Confocal images of MCF10DCIS cells treated with control
siRNA and DNA-damaging agent Doxorubicin (Doxo) or DMSO (vehicle) and stained for γH2AX (orange) and F-actin (Phalloidin, cyan). Graph shows the number of γH2AX foci per nucleus in indicated siRNA and drug conditions normalized to siCtrl or DMSO treatment. (K) Immunoblotting analysis of levels of TFEB and pS6K and p4EBP1 in MCF10DCIS cells treated with indicated siRNA and drugs. Vinculin was used as a loading control. Graphs show quantification of mean p-S6K and p4EBP1 levels and S.E.M. in indicated conditions. Data are normalized to levels in cells treated with siCtrl and DMSO. All numerical data and statistical tests are detailed in Supplemental Table 5. Scale bars, 10 µm.
<figure><img src="image_98.png" /></figure>

Supplemental Figure 4 related to Figure 2. Characterization of RRAGC KO in MDA-MB-231 and MCF10DCIS TNBC cells. (A) Immunoblot analysis showing the loss of RagC expression in several independent clones of MDA-MB-231 cells treated with the sgRRAGC_8 guid with CRISPR/Cas9 strategy. Vinculin was used as a loading control. (B) Quantification of the area of mTOR associated with CD63-positive vesicles normalized to cell area in indicated MDA-MB-231 clones KO for RRAGC. Data are normalized to parental WT cells. (C) TFEBGFP immunostaining (Fire LUT), and quantification of nuclear translocation of TFEBGFP in WT or indicated MDA-MB-231 clones KO for RRAGC. Dotted lines, cell and nucleus contour. (D) Gelatin degradation
by WT or indicated MDA-MB-231 clones KO for RRAGC treated with control or TFEB
siRNA. Data are normalized to WT MDA-MB-231 cells treated with siCtrl. (E)
Immunoblot analysis showing the loss of RagC expression in several independent
clones of MCF10DCIS cells treated with the sgRRAGC_8 and _14 guids with
CRISPR/Cas9 strategy. Vinculin was used as a loading control. (F) Mean growth curve
of MCF10DCIS WT or RRAGC KO (clone 3) cells. (G) TFEB immunostaining (Fire
LUT), and quantification of nuclear translocation of TFEB in MCF10DCIS WT or
RRAGC KO(clone 3). Dotted lines, cell and nucleus contour. Starvation (EBSS
treatment) in WT cells was used as control. (G) Collagen cleavage in WT or clone 3 of
RRAGC KO MCF10DCIS cells. Data are normalized to WT cells. Collagen cleavage
by WT or indicated MCF10DCIS clones KO for RRAGC. Data are normalized to WT
MCF10DCIS cells. All numerical data and statistical tests are detailed in Supplemental
Table 5. Scale bars, 10 µm.
A

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B

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C

MCF10DCIS.com

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D

MCF10DCIS.com

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**Supplemental Figure 5 related to Figure 3.** TFEB activation promotes invadopodia dynamics. (A) Representative immunoblots of pS6K and p4E-BP1 levels (quantified in the graphs) and RagC, TFEB and MT1-MMP levels in MDA-MB-231 cells treated with indicated siRNAs. GAPDH and Vinculin were used as loading control. (B, C) Quantification of MT1-MMP levels based on immunoblotting analysis of MDA-MB-231 (B) or MCF10DCIS (C) cells treated with indicated siRNA. Data are normalized to siCtrl. (D) Quantification of mean intensity of surface MT1-MMP per cell in MCF10DCIS cells treated with indicated drugs. Data are normalized to DMSO. All numerical data and statistical tests are detailed in Supplemental Table 5.
<figure><img src="image_104.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_105.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_106.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_107.png" /></figure>

<figure><img src="image_108.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_109.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_110.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_111.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_112.png" /><figcaption>H</figcaption></figure>

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**Supplemental Figure 6 related to Figure 4. TFEBON transcriptional program controlling ECM degradation.**

**(A)** Immunoblot analysis showing the depletion of RagC and Vps35 as well as the levels of pS6K and p4E-BP1 in MDA-MB-231 cells treated with indicated siRNAs. GAPDH and Vinculin were used as loading controls.

The graph shows the quantification of gelatin degradation in cells treated with indicated siRNAs. Data are normalized to siCtrl. **(B)** Gelatin degradation in cells treated with indicated drugs. Data are normalized to DMSO. **(C)** MCOLN1 mRNA levels assessed by RTqPCR in MDA-MB-231 cells treated with indicated siRNAs (left). Gelatin degradation in cells treated with indicated siRNAs (right). Data are normalized to siCtrl.

**(D)** GSEA analysis was performed on the ontology gene set collection from the Molecular Signatures Database (MSigDB) and selected sets are represented as boxplots for siRagC #1 and siRagC #2 versus siCtrl. Red and blue colors are respectively attributed to gene sets with a positive or negative normalized enrichment score (NES). **(E)** Boxplots showing forty differentially expressed genes (DEG) with log₂(FC) ≥ 1.6 (dotted lines) common to siRagC #1 and siRagC #2 versus siCtrl. **(F)** Depletion of the mRNAtargets of the indicated siRNAs was assessed by RTqPCR in MDA-MB-231 cells. Data are normalized to siCtrl. **(G)** mRNA expression levels assessed by RTqPCR for the indicated genes in MCF10DCIS cells treated with indicated siRNA or drugs. Data are normalized to siCtrl or to vehicle. **(H)** mRNA levels of selected genes in MDA-MB-231 cells treated with Torin or DMSO (vehicle) and indicated concentrations of Eltrompobag (EIO) measured by RTqPCR. Data are normalized to DMSO without EIO. All numerical data and statistical tests are detailed in Supplemental Table 5.
<figure><img src="image_114.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_115.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_116.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_117.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_118.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_119.png" /><figcaption>G</figcaption></figure>

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<figure><img src="image_122.png" /><figcaption>H</figcaption></figure>

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Supplemental Figure 7 related to Figure 5. mTORC1<sup>OFF</sup> promotes TNBC cell invasion in vivo. (A, B, C) Representative immunoblots showing the levels of pS6K and p4E-BP1 (quantified in graphs) in tumor cells harvested from three independent TNBC PDXs treated overnight with the indicated drugs or corresponding vehicle. GAPDH was used as loading control. Data are normalized to corresponding vehicle. (D) Quantification of the area of mTOR associated with CD63-positive structures, normalized to cell area in cells obtained from HBCx-60 and HBCx-90 PDXs treated with indicated drugs or vehicles. Data are normalized to vehicle. (E) Quantification of the mean intensity of pTFEB (Ser211) per cell in HBCx-60 and HBCx-90 PDX treated
with indicated drugs or vehicles. Data are normalized to vehicle. (F) Quantification of the percentage of nuclei positive for TFEB in HBCx-60 PDX treated with indicated drugs or vehicles. (G) Representative immunoblots and quantification of MT1-MMP levels in TNBC PDXs in comparison to MDA-MB-231 and MCF10DCIS cell lysates. Vinculin was used a loading control. (H) Representative immunoblots and quantification of MT1-MMP in Evo-treated PDXs (or vehicle), with GAPDH as a loading control. All numerical data and statistical tests are detailed in Supplemental Table 5.
<figure><img src="image_124.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_125.png" /><figcaption>C</figcaption></figure>

HBCx-60 (in vivo)/pmTOR<sup>Ser2448</sup>

<figure><img src="image_126.png" /><figcaption>D</figcaption></figure>

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HBCx-60 (in vivo)
pS6<sup>Ser240/244</sup>

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HBCx-60 (in vivo)
TFEB

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**Supplemental Figure 8 related to Figure 6.** Evo treatment of HBCx-60 TNBC PDX tumors. (A) HBCx-60 TNBC PDX tumors were implanted in nude mice, and after three weeks mice were treated with Evo or vehicle (Ctrl) for three (d3) or twelve (d12) days. The graph shows the mean percentage ± S.E.M. of necrotic areas in the PDX tumors treated as indicated. (B) Mean size (mm³) ± S.E.M. of PDX tumors treated as indicated. (C, D) Immunohistochemistry staining (C) and mean H-Score (D) of phospho-(p)mTOR<sup>Ser2448</sup> signal in sections of HBCx-60 PDX in mice treated for three (d3) or twelve (d12) days with Evo or vehicle. (E) Representative immunohistochemistry
staining of pS6Ser240/244. Boxed regions correspond to higher magnification images shown in Figure 6A. (F) Representative immunohistochemistry staining of TFEB. Boxed regions correspond to higher magnification images shown in Figure 6C. The right row shows Al-assisted nucleus segmentation and intensity scoring of nuclear (n)TFEB. All numerical data and statistical tests are detailed in Supplemental Table 5. Scale bars, 1 mm, 100 µm (panel C, insets).
<figure><img src="image_130.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_131.png" /><figcaption>C</figcaption></figure>

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<figure><img src="image_133.png" /><figcaption>F</figcaption></figure>

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<figure><img src="image_135.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_136.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_137.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_138.png" /><figcaption>H</figcaption></figure>

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Supplemental Figure 9 related to Figure 6. Immunohistological analysis of
subcutaneous tumor xenograft of MCF10DCIS cells KO for RRAGC. (A)

Experimental scheme of DCIS-like tumor xenograft analysis after subcutaneous
injection of MCF10DCIS cells. Ctrl and RRAGC KO #C3 cells were injected
subcutaneously in nude mice. At Week 1-, -3 and -5 post-inoculation, tumors were
dissected, and tissue sections were labeled. This panel was created with help of
BioRender.com. (B) Xenograft tumor growth (mm³) over time after inoculation (Day 0).

(C-H) Representative images of Week 1 (C, F), Week 3 (D, G) and Week 5 (E, H) consecutive sections labeled for pan-cytokeratin (CK), p53-family basal-like marker, p63, MT1-MMP and basement membrane component, collagen IV (Coll IV). Higher magnification images corresponding to boxed regions are shown in the lower rows. n, necrosis. Scale bars, 100 µm, 50 µm (insets).