# Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients

Charly Jehanno, Yann Le Page, Gilles Flouriot, Pascale Le Goff, Denis Michel

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Charly Jehanno, Yann Le Page, Gilles Flouriot, Pascale Le Goff, Denis Michel. Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients. International Journal of Experimental Pathology, 2023, 104 (2), pp.64 - 75. 10.1111/iep.12464. http://hal-04255920

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients

Charly Jehanno<fnref n="1" />,<fnref n="2" /> | Yann Le Page<fnref n="1" /> | Gilles Flouriot<fnref n="1" /> | Pascale Le Goff<fnref n="1" /> |
Denis Michel<fnref n="1" /> ID

**Correspondence**

Denis Michel, University of Rennes, Inserm, EHESP, Irset UMR 1085, Rennes, France.

Email: denis.michel@live.fr

**Funding information**

Ligue inter-regionale de lutte contre le cancer Grand-Ouest

**Abstract**

By depriving cancer cells of blood supplies of oxygen and nutrients, anti-angiogenic therapy is aimed at simultaneously asphyxiating and starving the cells. But in spite of its apparent logic, this strategy is generally counterproductive over the long term as the treatment seems to elicit malignancy. Since a defect of blood supply is expected to deprive tumours simultaneously of oxygen and nutrients naturally, we examine here these two deprivations, alone or in combination, on the phenotype and signalling pathways of moderately aggressive MCF7 cancer cells. Each deprivation induces some aspects of the aggressive and migratory phenotypes through activating several pathways, including HIF1-alpha as expected, but also SRF/MRTFA and TCF4/beta-catenin. Strikingly, the dual deprivation has strong cooperative effects on the upregulation of genes increasing the metastatic potential, such as four and a half LIM domains 2 (FHL2) and HIF1A-AS2 lncRNA, which have response elements for both pathways. Using anti-angiogenic agents as monotherapy is therefore questionable as it may give falsely promising short-term tumour regression, but could ultimately exacerbate aggressive phenotypes.

**KEYWORDS**

breast cancer, epithelial–mesenchymal transition (EMT), gene expression, hypoxia, metastasis

## 1 | INTRODUCTION

When deprived of nutrient and oxygen resources, beyond a critical size, tumours stimulate their own vascularization.<fnref n="1" /> Therefore, naturally anti-angiogenic treatments are expected to forbid the growth of tumours, by preventing neovascularization and the supply of oxygen and nutrients. Despite this rationale, the approach often gives disappointing results because of an increase in

recurrences.<fnref n="2" />⁻⁵ To understand this drawback, we tested the effects of deprivations in (1) oxygen, (2) nutrients and (3) both. Indeed, to be relevant, experiments mimicking anti-angiogenic conditions must carry out this dual deprivation. We found that the withdrawal of each ingredient favours dedifferentiation and acquisition of a migratory phenotype by activating interfering mechanisms involving HIF1-alpha (HIF1A), the central actor of the response to hypoxia, SRF/MRTFA, known to reprogramme cells and

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2023 The Authors. *International Journal of Experimental Pathology* published by John Wiley & Sons Ltd on behalf of Company of the International Journal of Experimental Pathology (CIJEP).

<footnote n="1">¹University of Rennes, Inserm, EHESP, Irset UMR 1085, Rennes, France</footnote>

<footnote n="2">²Department of Biomedicine, University of Basel, Basel, Switzerland</footnote>
to mediate mechanical genetic transduction leading to contractile activities and TCF4/beta-catenin (CTNNB1), also clearly involved in epidermal–mesenchymal transition (EMT). Strikingly, when applied simultaneously, the lack of oxygen and nutrients induces-in a strongly cooperative manner-the expression of genes promoting invasiveness and increasing the metastatic potential, such as the migration gene FHL2 and the mesenchymal marker HIF1A-AS2. Finally, these pathways influence the activity of each other and potentiate the activity of HIF1A. Together, these results clearly suggest that the antiangiogenic strategy could dramatically favour cancer cell dissemination.

2 | MATERIALS AND METHODS

**2.1 | Cell culture and transfection**

The human breast cancer MCF7 cells or MCF7 cells stably expressing MKL1-CA⁶ were grown in Dulbecco's modified Eagle medium (DMEM-GIBCO) containing 10% foetal bovine serum (FCS-Biowest) and antibiotics (GIBCO) at 37°C under 5% CO₂. Before the experiments, the cells were maintained for 24 h in DMEM with 2% dextran/charcoal-stripped FCS (dcFCS-Biowest) and then further incubated for 24 h in the specified medium. The poor medium (PM) is DMEM without pyruvate, glutamine and glucose but with 2% dcFCS. Transfection experiments of plasmid DNA or si-RNA were performed as previously described.⁶ The wound healing assay was performed using an Ibidi® chamber according to the manufacturer's instructions. After verifying that identical results on cell dissociation and induction of EMT characteristics were obtained with gaseous hypoxia and by the addition of 200 µM CoCl₂, we used this latter chemical treatment in the following experiments presented herein to facilitate cotreatments and long-term treatments. Immunofluorescence experiments were performed as previously described.⁶

**2.2 | Plasmid, siRNA and antibodies**

The p3Xflag-MKL1-N200 expression vector was kindly provided by Professor R. Prywes (Colombia University, New York, NY, USA). It encodes a constitutively nuclear and active form of MRTFA (MRTFA-CA). pcDNA3-S33Y CTNNB1 (#19286), M50 Super 8x TOPFlash (TCF4-Luc #12456) and HRE-luciferase (#26731) were purchased from Addgene. Human MRTFA (ON-TARGETplus® SMARTpool®) and esiRNA human HIF1A were purchased from Thermo Scientific and Sigma respectively. Antibodies against the following proteins were used:

CTNNB1 (ab6302 Abcam), CDH1 (ab15148 Abcam), ERK (extracellular signal-regulated kinase 1); K-23 ESR1 (HC-20, SC-543, Santa Cruz Biotechnology), FHL2 (ab12327; Abcam), HIF1A (610958 BD Biosciences), MRTFA (ab113264 Abcam) and phalloidin-iFluor 594 (lab176757; Abcam).

**2.3 | Western blotting**

Cells were lysed by sonication in a denaturing buffer (4% SDS, 200 mM dithiothreitol, 20% glycerol, 0.001% bromophenol blue and 100 mM Tris–HCl pH 6.8). Protein extracts were separated by SDS–PAGE, transferred to nitrocellulose membrane and probed with specific primary antibodies. After incubation with horseradish peroxidase-conjugated secondary antibody, complexes were detected with chemiluminescent HRP substrate (Immobilon Western, Millipore).

**2.4 | RNA extraction and RT-qPCR**

Total RNA was extracted from cells using the RNeasy kit (Qiagen) according to the manufacturer protocol. Five hundred nanogram of RNA were reverse transcribed using random primers and the iScript Reverse Transcription Supermix was purchased from Biorad. Quantitative real-time PCRs were performed with CFX384 touchTM real-time PCR detection system using the iQTM SYBR Green supermix from Biorad. The following primer sequences were used: ACTG2: forward GCGGAAGTACTCAG TCTGG, reverse CACTTCCTGTGGACAATGGA; FHL2: forward GGCAAGAAGTACATCCTGCG reverse CCACC AGTGAGTTTCTGCAC, HIF1A-AS2: forward AAAGC TTGGGCAAATTATTCA, reverse TGAATGGGATGAGT GAAGCA; SRF: forward CATCCCTTGGGCCATCTGT, reverse GCAGTTCAGCTCCACCAGAT; SNAI2: forward CAAGGACACATYAGAACTCACAC, reverse AGGTT TTGGAGCAGTTTTTGCAC; and VEGFA: forward AGGAGGAGGGCAGAATCATCA, reverse CTCGATTG GATGGCAGTAGCT.

**2.5 | ChIP-seq data**

The genomic binding sites for SRF in MCF7 cells were from ENCODE DCC (Stanford, GEO access: GSM1010839). The identification of MRTFA-binding sites was performed using ChIP-seq experiments described in.⁷ The identification of TCF4-binding regions in the human genome was performed using MCF7 cells (Peggy Farnham lab, USC. GEO:GSM945859). The HIF1A-binding sites were
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determined by ChIP-seq in MCF7 cells (David R. Mole lab. University of Oxford GEO access: GSM700944). ChIP-Seq data on the presence of CTNNB1 are available in SW480 cells GSE5392735. For functional annotation, we collected bed files provided in the publication corresponding to genomic regions specifically bound by the corresponding transcription factors or cofactors. Using the online GREAT platform (http://great.stanford.edu/) and using the following settings (5000 bp upstream and 1000 bp downstream, with up to 30 kb extension), the list of genes bound by selected transcription factors and cofactors was generated.

## 2.6 Quantification

Immunostaining signals were quantified from fluorescence microscopy images by averaging the intensity of more than a thousand cells using an automatic Fiji plugin. After nucleus identification using DAPI labelling and background subtraction, total fluorescence was extracted from the picture obtained with the fluorescent antibody. Superscripts on histograms correspond to Mann–Whitney *U* tests. RT-PCR values are expressed as the mean fold induction compared to controls of four independent experiments; *p* < .05 with the Mann–Whitney *U* test.

# 3 RESULTS AND DISCUSSION

## 3.1 Effects of CoCl₂ and poor medium on HIF1A and cellular phenotypes

The molecular pathways involved in the response to hypoxia have long been identified as regular components of EMT, regardless of the primary mode of induction of this multifaceted cellular activity, which explains why hypoxia can itself enhance EMT. Hypoxia is one of the expected consequences of a lack of blood supply, which is itself logically concomitant with nutrient deprivation. These two deprivations are therefore associated in the case of anti-angiogenic treatment, so it is crucial to test their joint action. To examine the impact on EMT of nutrient deficiency, alone or in conjunction with hypoxia, we selected the MCF7 mammary human cell line because it is not aggressive and retains marked characteristics of epithelial differentiation, whose loss can be easily detected. Especially when grown to confluency, these cells form pseudo-epithelial layers with strong intercellular contacts. We tested the possible interferences of poor medium (written in abbreviated form PM and concretely defined herein as a low-serum, glucose-free, glutamine-free and pyruvate-free medium) with the major regulator of hypoxia, namely HIF1A. As chemical treatments with CoCl₂,

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Effects of CoCl₂ and nutrient-poor medium (PM) alone or in combination, on the subcellular location, the cellular content and the transcriptional activity of HIF1A. (A) si-RNA experiment aimed at verifying that CoCl₂ induces HIF1A-mediated expression of the angiogenetic factor VEGFA mRNA measured by RT-PCR. (B) Effects of CoCl₂ and PM on the subcellular distribution of HIF1A. (C) Total cellular content of HIF1A following CoCl₂ and/or PM treatments. (D) HIF1A-responsive element (HRE)-driven transcription in response to CoCl₂ and/or PM treatments. Columns with different superscripts differ significantly (*p* < .05).</figcaption></figure>

known to inhibit prolyl hydroxylases and stabilize HIF-1,⁸ would be technically easier than gaseous hypoxia in our model, we first verified that they mimic hypoxia in these cells through the upregulation of its most representative marker, VEGFA, and that HIF1A is involved in this regulation, as controlled by siRNA. In the RT experiment of Figure 1A quantifying mRNA contents, in the presence of normal HIF1A (control siRNA unrelated to HIF1A), CoCl₂
leads to a 20-fold increase in VEGFA, whereas the anti-HIF1A si-RNA reduces this induction to 5-fold, showing that HIF1A mediates, at least in part, the effect of CoCl₂ on the most emblematic marker for hypoxia VEGFA. Figure 1 shows the different functional facets of HIF1A activation influenced by PM, including its nuclear enrichment, an increase in its cellular content (Figure 1B) and its transcriptional activity (Figure 1C). The increase in its cellular content may result from a stabilization according to its classical mode of activation. The nuclear translocation of HIF1A is clearly associated with its transcriptional activity, as determined by transient expression of an HRE (HIF-responsive element)-Luc reporter plasmid. PM stimulates the effects of CoCl₂ on the nuclear translocation and transcriptional activity of HIF1A but has little impact on its own in the absence of CoCl₂ (Figure 1D).

## 3.2 | Candidate molecular actors mediating the effects of hypoxia and PM

A previous study⁶ and preliminary experiments led us to select the SRF/MRTFA and TCF4/CTNNB1 signalling pathways as possible relays of the effects described above. In the literature, certain phenotypic changes induced by hypoxia are mediated by the SRF/MRTFA-dependent contractile programme, clearly associated with EMT,⁹ either directly through RhoA-ROCK1 signalling already known to be activated by hypoxia,¹⁰ or following actin polymerization.¹¹ In addition, MRTFA has recently been shown to erase the initial cellular differentiation status¹² and to favour the stemness of breast cancer cells.¹³ Another central actor of EMT¹⁴,¹⁵ and invasion¹⁶ is CTNNB1. It has been shown to enhance HIF1A-mediated transcription,¹⁷ but has also been proposed to mediate the effect of 2-deoxyglucose,¹⁸ a competitive inhibitor of glucose uptake by cells, which is expected to mimic to some extent the PM. Moreover, a well-established consequence of tissue disruption during EMT is the release of CTNNB1.¹⁹ Finally, the nuclear location of CTNNB1 has previously been shown to be associated with the acquisition of invasiveness in the MCF7 cells studied here.²⁰ The experimental tests and ChIP-seq data described below strongly support the validity of these candidates. A simple study of the cellular distribution of MRTFA and CTNNB1 was first conducted by immunocytochemistry, which shows interesting behaviours and different effects of CoCl₂ and PM. The nuclear translocation of MRTFA is stimulated by CoCl₂ while that of CTNNB1 is triggered by PM. As shown in Figure 2A, CTNNB1 staining, which is mainly located at the intercellular junctions in control conditions, becomes cytoplasmic in cells treated with CoCl₂ and mainly nuclear in cells cultured in PM.

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** MRTFA and CTNNB1, candidate molecular actors for mediating the effects of CoCl₂ and/or PM. (A) Differential effects of CoCl₂ and/or PM and acidic medium on the nuclear translocation of MRTFA and CTNNB1. Acidosis (pH:6.5) is tested because it is an expected consequence of microenvironmental modifications by mesenchymal cells. (B) Immunoblotting detection of MRTFA and CTNNB1 in cells submitted to CoCl₂ and PM. Note that the long (*) and short (**) MRTFA isoforms are differentially regulated. The three tracks shown on each panel are contiguous in the original transfer membranes. (C) HRE-mediated transcription in cells cotransfected with either MRTFA-CA or CTNNB1. Columns with different superscripts differ significantly (*p* < .05).</figcaption></figure>

These experiments, however, do not fully recapitulate the expected effects of a defect in blood supply and irrigation of tumours because they were carried out in a buffered culture medium. To be complete, it would be necessary to examine the effects of acidosis. Indeed, one
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of the stereotyped facets of EMT is the metabolic reprogramming called the Warburg effect, from respiratory (oxidative phosphorylation) to fermentative (glycolysis) whose main by-product, lactate, is exported in the extracellular space, leading to the acidification of the extracellular medium in the vicinity. In the literature, low pH has been shown to favour the migratory potential of cancer cells in two ways: in the cell, low pH stimulates the action of key enzymes,²¹,²² while after proton export in the medium, low pH contributes to the dismantlement of the extracellular matrix,²³ which can explain how acidosis can promote tumour invasion.²⁴ We have tested the effect of acidosis specifically in the absence of hypoxia by replacing the standard buffered medium with an acidic medium (pH: 6.5). Although CoCl₂ in buffered medium induces the nuclear translocation of MRTFA, no such translocation is obtained with low pH alone. By contrast, CTNNB1 previously located at the cellular contours becomes strongly enriched in the nucleus (Figure 2A). This observation corroborates the model described by Chen et al.²⁵ that the disruption of cell-cell contacts could lead to the intracellular release of CTNNB1 previously trapped in adherens junctions. In addition to their nuclear translocation, we then measured the abundance of MRTFA and CTNNB1 by immunoblotting (Figure 2B). The intracellular content of CTNNB1 strongly increases in cells cultured in PM but less than with CoCl₂. According to the literature, an increase in CTNNB1 concentration may result from a decreased degradation by the ubiquitin proteasome after the inactivation of certain of its ubiquitin ligase(s). Contrary to CTNNB1, the long form of MRTFA is induced more by CoCl₂ than by the PM (Figure 2B). The effects of the treatments on the nuclear translocation and the abundance of CTNNB1 and MRTFA are parallel (compare Figure 2A and Figure 2B). A subtlety, however, appears in our immunoblotting experiment since the anti-MRTFA antibody reveals several forms of this protein. Shorter ones, possibly derived from an internal transcription initiation,²⁶ are increased in PM, in a manner similar to that of CTNNB1. This result will prove remarkably consistent with the genomic recruitment of MRTFA and CTNNB1 (see below). Since the loss of epithelial phenotype of MCF7 cells is also characterized by the disappearance of the oestrogen receptor alpha (ESR1), we verified that this phenomenon is also obtained with CoCl₂ and PM (Figure 2A). Finally, in the absence of treatment, simple cotransfections with MRTFA-CA or CTNNB1 induce HIF1A-mediated transcription such as CoCl₂ or PM respectively (compare Figure 1D and Figure 2C). In these experiments aimed at distinguishing their different signalling pathways, the treatments with CoCl₂ and PM were applied separately, but to test a possible synergistic effect between these pathways, combined treatments were then

tested. The phenotypic modifications observed by microscopy (Figure 3), including intercellular dysjunction and acquisition of a more stellate and less epithelial morphology, evoke epithelial–mesenchymal transition (EMT).

Both CoCl₂ and PM treatments trigger the well-established primary mark of EMT: dismantling of the intercellular contact marker CDH1 (E-cadherin). CDH1 is present in epithelial cell-to-cell junctions close to actin filaments, and its disruption can be responsible for the release of CTNNB1 from cellular junctions observed in Figure 3A. Actin is redistributed accordingly, switching from cell borders to intracellular, sometimes in the form of stress fibres in the most transformed cells. We measured the polymerization of actin which is a well-established mode of activation of MRTFA,¹¹ and accordingly, modifications of the subcellular location of MRTFA are observed. The nuclear translocation of MRTFA clearly follows the intensity of phalloidin staining (Figure 3A). MRTFA, which is mainly cytoplasmic in control cells, is strongly relocated in cells treated with CoCl₂, but less markedly enriched in the nuclei of cells cultured in PM, despite their stronger cell-cell dissociation. However, in some cells intensely stained with fluorescent phalloidin, a nuclear translocation of MRTFA is noticeable. With CoCl₂, the nuclear enrichment of MRTFA is more obvious even when the cells begin to dissociate (Figure 2A). Hence, PM stimulates CoCl₂-induced nuclear translocation of both MRTFA (Figure 3A) and HIF1A (Figure 1B). The long-term treatment with CoCl₂ induces a stabler nuclear location of MRTFA while causing a less pronounced nuclear translocation of HIF1A (Figure 3C). The weaker nuclear relocation of MRTFA in cells cultured in PM, which contrasts with the severe phenotypic impact of this treatment, suggests the involvement of other signalling mechanisms, of which one could be CTNNB1 as supported by the results of Figure 2. PM induces cellular dissociation even more rapidly and markedly than CoCl₂ but is not as potent for inducing the nuclear translocation of MRTFA. Although less effective in inducing acute genetic responses, fluorescence microscopy (Figure 3A, bottom panels) suggests that a long-term treatment with CoCl₂ stabilizes the nuclear location of MRTFA. We, therefore, selected this condition for the following experiments. Very interestingly, the functional relationships between the hypoxia pathways and MRTFA seem to be reciprocal. The activation of HIF1A by CoCl₂ (Figure 1A) is accompanied by a nuclear relocation of MRTFA, while conversely, the forced expression of a constitutively active mutant version of MRTFA induces the nuclear localization of HIF1A (Figure 3B). This cross-talk, to which others will be added later, could create a positive feedback stabilizing the cellular response. The interrelation between HIF1A and MRTFA is clearly illustrated by the behaviour of FHL2, a typical target gene
**FIGURE 3** (A) Effects of short (24 h) or long (LT: >1 month) treatments with CoCl₂, alone or in combination with PM, on the polymerization of filamentous Actin (phalloidin staining) and the redistribution of E cadherin (CDH1). The nuclear enrichment of endogenous MRTFA is shown in parallel. Twenty-four-hour CoCl₂ and PM treatments both lead to cell-cell disjunction. A more pronounced nuclear translocation of MRTFA is obtained with CoCl₂ compared to PM. (B) Stable expression of constitutively active MRTFA leads to the permanent nuclear localization of HIF1A. (C) Effect of short- and long-term treatments with CoCl₂ on HIF1A nuclear accumulation.

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of MRTFA. Anti-MRTFA as well as anti-HIF1A siRNAs significantly decrease the induction of FHL2 by CoCl₂ (Figure 4A), showing that MRTFA actually mediates, at least in part, the effects of hypoxia.

Accordingly, the forced expression of the MRTFA-CA mutant clearly upregulates FHL2 and other SRF/MRTFA target genes as ACTG2 and SRF itself (Figure 4B). FHL2 accumulates precisely in the cells enriched in nuclear MRTFA (Figure 4D), consistent with the regulation of this gene by the SRF/MRTFA pathway. The roles of FHL2 in contractile and migratory phenotypes are reflected by its subcellular organization and its accumulation at the peripheral adhesion points. Indeed, this multifunctional adaptor protein has been shown to simultaneously inhibit the cell cycle and stimulate tumoural invasion: it is (1) a

cog in the mechanical phenomena of migration, working in the focal adhesion points with partners such as filamin; and (2) a transcriptional regulator participating to cell cycle arrest, in part by promoting the expression of CDKN1A (P21). In keeping with the previous results, MCF7 cells overexpressing MRTFA-CA upregulate FHL2 (Figure 4C, bottom left panel) and systematically display a strong FHL2 staining similar to that obtained with CoCl₂ (compare the bottom panels of Figure 4C). The link between migration and FHL2 is further illustrated by the selective presence of FHL2 in the front of migration in wound healing assays (Figure 4C, top right panel), in accordance with its roles in cell migration and wound healing.²⁷ In this respect, the involvement of FHL2 in cancer is not a surprise considering that this pathology has been pertinently compared to a
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MRTFA
E cad

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FIGURE 4 Regulation of FHL2. (A) si-RNAs directed against HIF1A and MRTFA inhibit the expression of FHL2 by CoCl₂. The bottom panels are immunoblots showing the capacity of the si-RNAs to prevent the induction by CoCl₂ of HIF1A and MRTFA. (B) Upregulation of FHL2, ACTG2, SRF and SNAI2 mRNAs in MCF7 cells stably expressing MRTFA-CA. Columns with different superscripts differ significantly (*p* < .05). (C) Immunodetection of FHL2 in various conditions showing its accumulation in the leading migratory cells in wound assays (top right panel) and its accumulation in the nuclei and the borders of cells either overexpressing MRTFA-CA or treated with CoCl₂ (bottom panels). (D) Immunodetection of FHL2 (red) showing its overexpression and accumulation into bundles and focal adhesion points, precisely in the cells with a nuclear enrichment of MRTFA (green).

sort of overhealing,²⁸ a notion confirmed by their largely overlapping genetic programmes.²⁹

## 3.3 | Joint transcriptional effects of CoCl₂ and PM

We quantified by RT-PCR the effects of CoCl₂, PM and both, on the cellular accumulation of the mRNAs encoding VEGFA, the traditional HIF1A target gene, FHL2, the established target of MRTFA and the noncoding RNA HIF1A-AS2 involved in the establishment of a malignant phenotype.³⁰⁻³² We tested short-term (24 h) and long-term (1 month) CoCl₂ treatments because of their different effects on HIF1A and MRTFA nuclear enrichments (Figure 3), together with partial or complete nutrient deprivation. The long treatment, associated with depletions in

glucose, pyruvate and glutamine, slightly overadditively induces VEGFA, but strongly overadditively induces FHL2 and HIF1A-AS2 (Figure 5A). This synergistic induction is particularly clear for HIF1A-AS2 and spectacular with a short treatment with CoCl₂ (Figure 5B).

## 3.4 | Comparison with genomic data

To evaluate the panel of genes that can be subjected to cross-regulation by HIF1A, MRTFA and CTNNB1, we launched a meta-analysis aimed at identifying genes with binding sites for these factors. The mere presence of a transcription factor-binding motif, even near-consensual, is insufficient to prove the actual fixation of this factor because it can be prevented by chromatin organization. Hence, we assessed the existence of functional binding sites extracted
<figure><img src="image_18.png" /><figcaption>**FIGURE 5** Effect of short- and long-term treatments with CoCl₂ and PM on VEGFA, FHL2 and HIF1A-AS2 RNA accumulation. (A) Depletions of glucose, pyruvate and glutamine, in conjunction or not with long-term treatment with CoCl₂. (B) Short-term treatment with CoCl₂ with or without PM. Columns with different superscripts differ significantly (*p* < .05).</figcaption></figure>

from ChIP-seq data performed against these three factors. Using the GREAT online platform, we identified from bed files a shortlist of 98 genes whose regulatory elements are bound by HIF1A, MRTFA and CTNNB1, as shown in the Venn diagram of Figure 6A.

Strikingly, the three genes FHL2, MRTFA and HIF1A-AS2 belong to this subset of genes. VEGFA was

only found bound by HIF1A and CTNNB1 but not by MKL1. Functional annotation of genes sharing these corecruitments using the WikiPathway resource reveals that the top five cellular activities with combined scores higher than 15, including focal adhesion, integrin-mediated cell adhesion, TGF-beta signalling pathway and EGR/EGFR signalling pathway, are all associated with aggressiveness. We then focused our attention on the proximal regions of the few genes identified above to clearly visualize the recruitment peaks. In addition, we looked at the fixation relies of the MRTFA and CTNNB1 factors. Indeed, MRTFA and CTNNB1 are coactivators devoid of DNA-binding domains but endowed with strong transactivation domains. CTNNB1 coactivates DNA-bound TCF4 while MRTFA coactivates DNA-bound SRF, whereas TCF4 and SRF are by themselves poorly transcriptionally active. An additional complication is that different coactivators are possible for SRF. It can, for instance, be activated by pro-mitotic ELK and pro-migratory MRTFA factors, the choice between these optional coactivators seems largely dictated by genespecific contexts,³³ and the genes strongly upregulated in the present case have been shown to be preferentially regulated by MRTFA, as verified by the systematic screen of.⁷ The long noncoding HSF1A-AS2 RNA can naturally not be visualized by immunocytochemistry, but the transcription factor recruitments near its transcriptional initiation site (Figure 6B) suggest that it has a central role in the system studied here. The HSF1A-AS2 gene is located in close vicinity of HIF1A, encoding the master transcriptional regulator of hypoxia; but while HIF1A is itself poorly regulated at the transcriptional level, HSF1A-AS2 is a transcriptional target for many signalling pathways including HIF1A itself (Figure 6B). This combination of DNA-bound factors can explain the powerful synergy between these factors and the synergy between CoCl₂ and PM treatments. Recent functional studies have established that HSF1A-AS2 can control hypoxic pathways even in the absence of hypoxia³⁰ and that the expression of this RNA promotes a marked metastatic mesenchymal phenotype.³¹

These properties may mediate the adverse effects of the dual oxygen and nutrient deprivation. In line with previous reports on the regulation of FHL2 by MRTFA,³⁴ a proximal peak of recruitment of SRF is found in front of the second exon of the FHL2 gene (Figure 6F), centred on a consensual SRF-binding site (5'-ccttatatgg-3') and close to the main transcription start site, as verified by the accumulation of RNAPII in this region. When inserted in an enhancer-less reporter plasmid, this element mediates more than 200-fold induction by activated MRTFA (not shown). TCF4-binding sites are also present in the vicinity, but more clearly in front of the first exon. This upstream TCF4-binding site is validated by the superposed recruitment of CTNNB1 (Figure 6F). The coexistence of
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FIGURE 6 ChIP-seqs. (A) Venn diagram showing the recruitment of MRTFA (MKL1), CTNNB1 (beta-catenin) and HIF1A (HIF1alpha). A subset of 98 genes can be bound by these three factors. (B) Coexistence of recruitment sites for TCF4, CTNNB1, SRF and HIF1A in the proximal regulatory region of the HSF1A-AS2 gene. Genomic recruitment sites in the proximal regulatory regions of (C) VEGFA (D) SRF, (E) MRTFA and FHL2 (F) genes. Accumulation of RNAPII reveals the presence of internal promoters (**) in FHL2 and MRTFA genes.

SRF/MRTFA and TCF4/CTNNB1-binding sites could mediate the synergistic coregulation of FHL2 by these factors (Figure 5B). We did not find a clear HIF1A-binding site near the FHL2 gene although it has been described as a HIF1A target gene.³⁵ Instead, the upregulation of FHL2 by hypoxia could be indirect and mediated, for example, by the SRF/MRTFA pathway also involved in hypoxia. Interestingly, the MRTFA gene itself can be bound by SRF, TCF4 and HIF1A. The RNAPII ChIP-seq also suggests the presence of two transcription initiation sites in the

MRTFA gene. The conventional upstream promoter contains recruitment sites for HIF1A and SRF and a minor binding site for TCF4. In addition, an internal putative promoter between the exons 3 and 4, as in the study of²⁶ and coinciding with accumulation of RNAPII, contains a major recruitment site for TCF4 (Figure 6E). These recruitments fairly agree with our measurements of MRTFA expression (Figure 2B), since CoCl₂, which we have shown to induce MRTFA, mainly regulates the long form, in a self-regulatory mechanism, while the PM rather induces
the short isoform of MRTFA (Figure 2B). This short protein version has been otherwise involved in cellular transformation into myofibroblasts,²⁶ a process requiring the massive synthesis of contractile machineries. The SRF/MRTFA transcription system appears remarkably self-regulated since both SRF and MRTFA genes are targets of SRF (Figure 6D,E). The coexistence of TF recruitments on a promoter is not a sufficient condition for cooperativity which obeys complex and hardly predictable rules, but it explains at least in part the striking transcriptional results obtained for FHL2 and HIF1A-AS2. A more modest cooperativity (factor of 1.3) is obtained for MRTFA.

4 | CONCLUSIONS

Oxygen and nutrient deprivations are shown here to co-
operate for promoting epithelial dedifferentiation and ac-
quisition of a migratory phenotype of MCF7 cells, through
activating at least three different pathways: HIF1A, SRF/
MRTFA and TCF4/CTNNB1, as summarized in Figure 7.

These interfering pathways converge to key genes
critical for the establishment of an aggressive phenotype,
including FHL2 and HIF1A-AS2. In addition to govern-
ing FHL2 and HIF1A-AS2 expression, SRF/MRTFA and
HIF1A are functionally connected. The convergence of
pathways towards common target genes can explain (i)
why they induce similar cellular behaviours and, more
importantly, (ii) how they can act synergically in the in-
duction of EMT in case of dual depletion of oxygen and
nutrients, a situation precisely expected when cutting off
blood supply. Moreover, once initiated, these responses
are then modulated by various cross- and reciprocal
regulations in the system, including positive feedbacks
(Figure 7), and possibly negative feedbacks. For instance,
FHL2, which is a target for MRTFA, has been shown up-
regulated by hypoxia/CoCl₂³⁵ but also reported to inter-
act with HIF1A and repress its transcriptional activity.³⁵
Besides, CTNNB1 enhances the transcriptional activity
of HIF1A, but its own gene is repressed by HIF1A.¹⁷ In
addition to regulate common target genes, the pathways
defined here can reinforce each other. For instance, the
CTNNB1 pathway was shown to be induced by acido-
sis, as confirmed here in Figure 2, which itself may be
a consequence of the initiation of the Warburg effect by
hypoxia, thereby self-enforcing EMT in vivo. The pres-
ent results could provide molecular bases underlying
the questioning of the anti-angiogenic therapy in the
literature.²,³ The pro-tumoral effect of chronic hypoxia
has long been reported: for example, in a study showing
that long-term intermittent hypoxia triggers the emer-
gence of malignant cells starting from normal cells.³⁶

<figure><img src="image_27.png" /><figcaption>**FIG URE 7** Schematized molecular consequences of the dual deprivation of oxygen and nutrients as a result of blood supply interruption.</figcaption></figure>

Pathologists have long noticed that the cells embedded
at the centre of solid tumours, which are the less well
irrigated, display the most aggressive phenotype with
stellate contours and are enriched in polymerized actin.
Recent experiments cancelling blood supply strikingly
confirmed the risk of hypoxia and nutrient deficiency
caused by anti-angiogenic approaches.³⁷ The unintended
consequences of starvation and hypoxia shown in the
study of Wang et al.³⁷ on migratory cellular behaviours
appear in fact logical and adaptive and might have been
anticipated. Indeed, faced with a lack of supply of ox-
ygen and nutrients, the possible responses of cancer
cells for their survival can be either (1) autophagy, en-
abling them to resist temporarily by drawing resources
(carbon and energy) from their own constituents,³⁸ or
(2) mobility, leading them to adopt the nomadic and op-
portunistic consumer strategy of invaders. In this con-
text, the anti-angiogenesis strategy would be seriously
counterproductive by inciting the few surviving cells to
acquire a migratory phenotype and spread in the organ-
ism. Anti-angiogenic treatments can first cause encour-
aging tumour regression, but can then increase the risk
of recurrence possibly mediated by a direct influence of
hypoxia on glycolysis³⁹ and metastasis.⁴,⁵,¹⁰ The identi-
fication of candidate mechanisms involved in all these
observations could help develop new strategies to cir-
cumvent these pitfalls.
<figure><img src="image_28.png" /></figure>

## ACKNOWLEDGEMENTS

This work is supported by the Ligue inter-regionale de lutte contre le cancer des côtes d'Armor.

## CONFLICT OF INTEREST

The authors declare no competing interest.

## ORCID

Denis Michel https://orcid.org/0000-0002-0643-8025

## REFERENCES

1. Sitohy B, Nagy JA, Dvorak HF. Anti-VEGF/VEGFR therapy for cancer: reassessing the target. *Cancer Res.* 2012;72:1909-1914.

2. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. *Cancer Cell.* 2009;15:232-239.

3. Pàez-Ribes M, Allen E, Hudock J, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. *Cancer Cell.* 2009;15:220-231.

4. Rankin EB, Giaccia AJ. Hypoxic control of metastasis. *Science.* 2016;352:175-180.

5. Semenza GL. The hypoxic tumor microenvironment: a driving force for breast cancer progression. *Biochim Biophys Acta.* 1863;2016:382-391.

6. Flouriot G, Huet G, Demay F, Pakdel F, Boujrad N, Michel D. The Actin/MKL1 signalling pathway influences cell growth and gene expression through large-scale chromatin reorganization and histone post-translational modifications. *Biochem J.* 2014;461:257-468.

7. Esnault C, Stewart A, Gualdrini F, et al. Rho-Actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts. *Genes Dev.* 2014;28:943-958.

8. Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-α. *J Biol Chem.* 2003;278:15911-15916.

9. Scharenberg MA, Chiquet-Ehrismann R, Asparuhova MB. Megakaryoblastic leukemia protein-1 (MKL1): increasing evidence for an involvement in cancer progression and metastasis. *Int J Biochem Cell Biol.* 2010;42:1911-1914.

10. Gilkes DM, Xiang L, Lee SJ, et al. Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells. *Proc Natl Acad Sci U S A.* 2014;111:E384-E393.

11. Miralles F, Posern G, Zaromytidou AI, Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. *Cell.* 2003;113:329-342.

12. Ikeda T, Hikichi T, Miura H, et al. Srf destabilizes cellular identity by suppressing cell-type-specific gene expression programs. *Nat Commun.* 2018;9:1387.

13. Kim T, Lim DS. The SRF-YAP-IL6 axis promotes breast cancer stemness. *Cell Cycle.* 2016;15:1311-1312.

14. Medici D, Hay ED, Olsen BR. Snail and slug promote epithelial-mesenchymal transition through beta-catenin-T-cCell factor-4-dependent expression of transforming growth factor-beta3. *M Bronner-Fraser Ed.* *Mol Biol Cell.* 2008;19:4875-4887.

15. Fang L, Zhu Q, Neuenschwander M, et al. A small-molecule antagonist of the beta-catenin/TCF4 interaction blocks the self-renewal of cancer stem cells and suppresses tumorigenesis. *Cancer Res.* 2016;76:891-901.

16. Bandapalli OR, Dihlmann S, Helwa R, et al. Transcriptional activation of the beta-catenin gene at the invasion front of colorectal liver metastases. *J Pathol.* 2009;218:370-379.

17. Kaidi A, Williams AC, Paraskeva C. Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia. *Nat Cell Biol.* 2007;9:210-217.

18. Yu SM, Kim HA, Kim SJ. 2-Deoxy-D-glucose regulates differentiation through beta-catenin pathway in rabbit articular chondrocytes. *Exp Mol Med.* 2010;42:503-513.

19. Orsulic S, Huber O, Aberle H, Arnold S, Kemler R. E-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. *J Cell Sci.* 1999;112:1237-1245.

20. Uchino U, Kojima H, Wada K, et al. Nuclear beta-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells. *BMC Cancer.* 2010;10:414.

21. Rozhin J, Sameni M, Ziegler G, Sloane BF. Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. *Cancer Res.* 1994;54:6517-6525.

22. Kato Y, Ozawa S, Miyamoto C, et al. Acidic extracellular microenvironment and cancer. *Cancer Cell Int.* 2013;13:89.

23. Stock C, Gassner B, Hauck CR, et al. Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange. *J Physiol.* 2005;567:225-238.

24. Gatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ. Acid-mediated tumor invasion: a multidisciplinary study. *Cancer Res.* 1999;66:5216-5223.

25. Chen KH, Tung PY, Wu JC, et al. An acidic extracellular pH induces Src kinase-dependent loss of beta-catenin from the adherens junction. *Cancer Lett.* 2008;267:37-48.

26. Scharenberg MA, Pippenger BE, Sack R, et al. TGF-beta-induced differentiation into myofibroblasts involves specific regulation of two MKL1 isoforms. *J Cell Sci.* 2014;127:1079-1091.

27. Wixler V, Hirner S, Müller JM, et al. Deficiency in the LIM-only protein Fhl2 impairs skin wound healing. *J Cell Biol.* 2007;177:163-172.

28. Haddow A. Molecular repair, wound healing, and carcinogenesis: tumor production a possible overhealing? *Adv Cancer Res.* 1972;16:181-234.

29. Riss J, Khanna C, Koo S, et al. Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma. *Cancer Res.* 2006;66:7216-7224.

30. Mineo M, Ricklefs F, Rooj AK, et al. The long non-coding RNA HIF1A-AS2 facilitates the maintenance of mesenchymal glioblastoma stem-like cells in hypoxic niches. *Cell Rep.* 2016;15:2500-2509.

31. Chen M, Zhuang C, Liu Y, et al. Tetracycline-inducible shRNA targeting antisense long non-coding RNA HIF1A-AS2 represses the malignant phenotypes of bladder cancer. *Cancer Lett.* 2016;376:155-164.

32. Li L, Wang M, Mei Z, et al. lncRNAs HIF1A-AS2 facilitates the up-regulation of HIF-1alpha by sponging to miR-153-3p, whereby promoting angiogenesis in HUVECs in hypoxia. *Biomed Pharmacother.* 2017;96:165-172.
33. Gualdrini F, Esnault C, Horswell S, Stewart A, Matthews N, Treisman R. SRF co-factors control the balance between cell proliferation and contractility. *Mol Cell*. 2016;4:1048-1061.

34. Schmidt LJ, Duncan K, Yadav N, et al. RhoA as a mediator of clinically relevant androgen action in prostate cancer cells. *Mol Endocrinol*. 2016;26:716-735.

35. Hubbi ME, Gilkes DM, Baek JH, Semenza GL. Four-and-a-half LIM domain proteins inhibit transactivation by hypoxia-inducible factor 1. *J Biol Chem*. 2012;287:6139-6149.

36. Goldblatt H, Cameron G. Induced malignancy in cells from rat myocardium subjected to intermittent anaerobiosis during long propagation in vitro. *J Exp Med*. 1953;97:525-552.

37. Wang D, Tan C, Xiao F, et al. The "inherent vice" in the anti-angiogenic theory may cause the highly metastatic cancer to spread more aggressively. *Sci Rep*. 2017;7:2365.

38. Hu YL, Jahangiri A, De Lay M, Aghi MK. Hypoxia-induced tumor cell autophagy mediates resistance to anti-angiogenic therapy. *Autophagy*. 2012;8:979-981.

39. Wenger RH, Stiehl DP, Camenisch G. Integration of oxygen signaling at the consensus HRE. *Sci STKE*. 2005;306:1-13.

**How to cite this article:** Jehanno C, Le Page Y, Flouriot G, Le Goff P, Michel D. Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients. *Int J Exp Path*. 2023;104:64-75. doi:10.1111/iep.12464