Lack of extracellular matrix switches TGF-β-induced apoptosis of endometrial cells to Epithelial-to-Mesenchymal transition through AKT, ERK and SMAD2/3-depdendent mechanisms

Lack of extracellular matrix switches TGF-β-induced apoptosis of endometrial cells to Epithelial-to-Mesenchymal transition through AKT, ERK and SMAD2/3-depdendent mechanisms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lack of extracellular matrix switches TGF-β-induced apoptosis of endometrial cells to Epithelial-to-Mesenchymal transition through AKT, ERK and SMAD2/3-depdendent mechanisms Anna Ruiz-Mitjana, Raúl Navaridas, Maria Vidal-Sabanés, Aida Perramon-Güell, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1465554/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF- β. It is well known that TGF-β is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-β remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-β-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-β-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-β-induced apoptosis. On the other hand, we demonstrate that TGF-β-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-β to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF- β in normal versus tumoral cells. Apoptosis EMT endometrial cancer TGF-β extracellular matrix cell polarity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Cell-to-cell, cell-to extracellular matrix (ECM) interactions and establishment of a correct cell polarity play a pivotal role in epithelial cell homeostasis. Mounting evidences support that the three dimensional (3D) structure of epithelial tissue plays an important role in deciding cell fate and is involved in the regulation of proliferation, survival and apoptosis of individual epithelial cells 1 – 3 . Cell polarity acts as a tumor suppressor mechanism and its loss is a hallmark of cancer 2 Over the past decades, 3D organoid cultures derived from epithelial tissues have been developed to recreate structures that reassemble their in vivo architecture 4 . Such culture models have emerged as powerful cell-based scenarios to investigate many aspects of cancer pathobiology, such as the role of cell polarity and ECM, the function of tumor suppressors or oncogenes, and the importance of pathways in cancer 5 . To investigate the pathobiology of cancer it is biologically relevant to maintain the features of the 3D architecture of the epithelial tissue in culture. It is known that significant differences are observed between experiments assessed with cells cultured in a 3D polarity structures and experiments using classical two-dimensional (2D) cultures. Consequently, it has been proposed 3D polarity as a non-canonical mechanism of tumor suppression since it may function as an inhibitor of primary tumor development 6 . In normal epithelial tissues, Transforming Growth Factor-β (TGF-β) regulates a plethora of cell functions and processes depending on cell type, cellular context and microenvironment 7 – 9 . TGF-β is secreted as an inactive latent disulfide-linked homodimer (LTGF-β) polypeptide. Mature bioactive TGF-β ligands are produced upon proteolytic cleavage of the latent complex and directly bind to TGF-β type II receptor (TGF-βRII). TGF-βRII phosphorylates and activates the TGF-β receptor type I which, in turn, phosphorylates members of the SMAD family 10 . The SMAD family consists of nine members, which form 3 subfamilies: receptor-activated (R-)SMADs (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8 and SMAD9), a single common-mediator (Co-)SMAD (SMAD4) and two inhibitory (I-)SMADs (SMAD6 and SMAD7). TGF-β also induces non-SMAD pathways, including mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K) and Rho GTPases 11 . Dysregulation of TGF-β signaling has been implicated in the progression of diseases such as fibrosis or cancer. In most normal epithelial tissues, TGF-β acts as tumor suppressor by inhibiting cell cycle progression or activating apoptosis but, during carcinogenesis, TGF-β switches its tumor suppressive functions to promoter ones 12 – 15 . However, the mechanisms of these functional changes of TGF-β are not fully understood and may vary among cell types and contexts. As a tumor promoter, TGF-β stimulation is associated with epithelial-to-mesenchymal transition (EMT) and acquisition of cell migration, invasion and metastatic potential of malignant epithelial cells 14 , 15 . EMT is a well-coordinated process during embryonic development and a pathological feature in neoplasia and fibrosis. Cells undergoing EMT lose the expression of epithelial cell markers such as cytokeratins, E-cadherin or β-catenin and gain the expression of mesenchymal markers such as vimentin 16 . However, over the past few years, numerous studies demonstrate that EMT is a dynamic process in which cells undergoing partial or hybrid EMT display intermediate cell states retaining expression of both epithelial and mesenchymal markers 17 – 19 . Although EMT has traditionally been associated with increased metastatic properties, recent studies have activated the debate on the requirement of EMT and its different EMT states for cancer metastasis 17 – 19 . Here, using a 3D organoid culture system of mouse endometrial epithelial cells, we have investigated the effect of extracellular matrix-induced 3D cell organization on biological responses to TGF-β. We have demonstrated that the lack of ECM and subsequent loss of cell polarity switches TGF-β-induced apoptosis to an EMT-compatible phenotype. We have also inferred the molecular mechanism involved in this swapped function. On the one hand, we demonstrated that the absence of ECM controls sensitivity to TGF-β-induced apoptosis through regulation of AKT signaling. On the other hand, both ERK and SMAD2/3 signaling regulate TGF-β-triggered EMT in absence of ECM. In summary, our results support that ECM-induced cell polarity acts as a non-canonical tumor suppressor for endometrial epithelial cells. Material And Methods Reagents and antibodies The recombinant basement membrane Matrigel was purchased from BD Biosciences (San Jose, CA). Epidermal Growth Factor, hydrocortisone, dexamethasone, RU-486, ICI 182170 and LY 294002 were obtained from Sigma (St Louis, MO), Insulin-Transferrin-Sodium Selenite supplement was obtained from Invitrogen (Invitrogen, Inc., Carlsbad, CA, USA), PD0325901 and U0126 were purchased from Calbiochem (Calbiochem-Novabiochem, UK, Ltd). Antibodies to E-Cadherin, β-Catenin and vimentin were from BDbiosciences; bisBenzimide H 33342 trihydrochloride (Hoechst), and tubulin were obtained from Sigma and anti-cytokeratin was from Abcam (Cambridge UK). Anti-phospho Akt, anti-phospho-p70SK6, cleaved caspase-3 and PTEN antibodies were from Cell Signalling Technology (Beverly, MA). Anti-phospho FAK was from Thermo fisher. Anti-phospo ERK was from Biolegend. Alexa-Flour anti-Rabbit and anti-mouse antibodies were from Invitrogen Peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Jackson ImmunoResearch Europe Ltd (Suffolk, UK). All other regents were obtained from Sigma unless specified. Animals and Isolation of endometrial epithelial cells Mice were housed in a barrier facility and pathogen-free procedures were used in all mouse rooms. Animals were kept in a 12-hour light-dark cycle with ad libitum access to a standard 2014 Teklad Global 14% Protein Rodent Maintenance Diet ( www.harlan.com ) and water. The animal rooms were environmentally controlled (20 ± 2°C, relative humidity 50 ± 5%). The study complied with Law 5/1995 and Act 214/1997 of the Autonomous Community (Generalitat of Catalonia) and EU Directive EEC 63/2010, and was approved by the Ethics Committee on Animal Experiments of the University of Lleida and the Ethics Commission in Animal Experimentation of the Generalitat de Catalunya. CRE:ER (B6.Cg-Tg(CAG-CRE/Esr1* 5Amc/J) mice was obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Double Smad2 fl/fl Smad3 fl/fl mice was provided by Dr.Martin M Matzuk (Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA). CRE:ER mice genotyping PCR was carried out with the following primer: forward primer 5’-CAC TCC CAG AGA CAT ATA CAC-3’ and reverse primer 5’-ACG AAC CTG GTC GAA ATC GT GCG-3’ . Smad3 fl/fl mice genotyping PCR was carried out with the following primer: forward primer 5’-CTC CAG ATC GTG GGC ATA CAG C-3’; smad3 fl/fl reverse primer 5’-GGT CAC AGG GTC CTC TGT GCC-3’. Smad2 fl/fl mice genotyping PCR was carried out with the following primer: forward primer 5’-CAT CAG ATT CCA TTA GAG ATG G-3’; smad2 fl/fl reverse primer 5’-TGA GAC TTC TCT GTA CCC GAT − 3’. CRE:ER +/− Smad2 fl/fl Smad3 fl/fl mice were bred in a mixed background (C57BL6; 129S4) by crossing Smad2 fl/fl Smad3 fl/fl mice with CRE:ER +/− mice. To obtain mice carrying both Smad2 and Smad3 floxed allelles and a single CRE:ER (CRE:ER +/− ), CRE:ER +/− were backcrossed with Smad2 fl/fl ; Smad2 fl/fl mice. The isolation of endometrial epithelial cells was processed as described previously 20 . Briefly, uterine horns were dissected from 3–4 weeks old C57BL6 mice. Uterus were washed with HBSS and digested with Trypsin (Invitrogen). After trypsin digestion epithelial sheets were squeezed-out of the uterine pieces. Epithelial sheets were washed twice with PBS and resuspended in 1 ml of DMEM/F12 (Invitrogen) supplemented with 1% of Sodium Pyruvate (Sigma), 1% of penicillin/streptomycin (Sigma) and 0,1% of fungizone (Invitrogen) (basal medium). Epithelial sheets were mechanically disrupted in basal medium until small clumps of cells were observed. 2D endometrial monolayers and 3D Endometrial organoids cultures Growth of endometrial epithelial cells in 2D cultures was performed by platting cells in basal medium with 2% of dextran-coated charcoal-stripped serum (Invitrogen) and leaved twenty-four hours. To induce deletion of floxed alleles, Tamoxifen (Sigma-Aldrich T5648, St. Louis, MO, USA) was dissolved in 100% EtOH at 1mM and used at 0,5 µl/ml of medium for 24 hours. For immunofluorescence, cells were seeded in a volume of 40 µl /well in 96 well plates black with micro-clear bottom (Greiner Bio-one). For western blotting, cells were placed in a volume of 200 µl in 24-well plates (BDBiosciences). Growth of endometrial epithelial cells in 3D cultures was performed as described previously 20 . Twenty-four hours after platting in plastic, cells were washed with HBSS and incubated with trypsin/EDTA solution (Sigma) for 5 min at 37ºC. Trypsin was stopped by adding DMEM 10% FBS and clumps of 2–8 cells were obtained. Cells were centrifuged at 1000 rpm for 3 minutes and diluted in basal medium containing 3% of matrigel to obtain 4 x 10 4 cell clumps/ml. For immunofluorescence, cells were seeded in a volume of 40 µl /well in 96 well plates (black with micro-clear bottom) (Greiner Bio-one). For western blotting, cells were placed in a volume of 200 µl in 24-well plates (BDBiosciences). In all cases, 24 hours after plating medium was replaced by Basal medium supplemented with 5 ng/ml EGF and 1/100 dilution of Insulin-Transferrin-Sodium Selenite (ITS) Supplement (Invitrogen) and 3% of fresh Matrigel (referred as BIE). Medium was replaced every 2–3 days. Immunofluorescence Immunofluorescence was performed as previously described 20 , 21 .Cultures were fixed with formalin for 5 minutes at room temperature (RT) and washed twice with PBS. Depending on primary antibody, cells were permeabilized with 0,2% triton X-100 in PBS for 10 minutes (indicated as T) or permeabilized with 100% methanol for 2 minutes (indicated as M). Next, cultures were incubated overnight at 4ºC with the indicated dilutions of antibodies: anti-Cytokeratin 1/500(M), β-Catenin 1/100(M), E-Cadherina 1/100(M), Rhodamine conjugated-Phalloidin 1/500(T), anti-Cleaved Caspase-3 1/100(T), anti-Vimentin 1/100 (T) and anti-phospho FAK 1/100 (T). After one day, cells were washed twice with PBS and incubated with PBS containing a 5 µg/ml of Hoechst dye and 1/500 dilution of Alexa Fluor secondary anti-mouse or anti-rabbit antibodies for 2 hours at RT in the case of 2D cultures or overnight at 4ºC for 3D cultures. For double immunofluorescence staining, cells were incubated with the second round of primary and secondary antibodies. We would like to point out that in all double immunofluorescence stains, first and second primary antibodies were from different isotope. Immunofluorescence staining was visualized and analyzed using a confocal microscopy (Olympus). Edition of confocal images was performed using Fluoview software (Olympus). For evaluation of apoptosis and caspase-3 processing, endometrial epithelial glands were analyzed on a confocal microscope Fluoview FV1000. The presence of apoptotic or positive caspase-3 processing (more than 5 cells per gland) was revealed by Hoescht/Cleaved Caspase-3 immunotaining. For each experiment we quantified at least 100 glands. Cell polarity of epithelial cells forming glandular structures was evidenced by double immunostainings as indicated in each figure. Western Blot analysis 2D endometrial monolayers or endometrial organoid 3D cultures stimulated for the indicated periods of time, were washed with HBSS and incubated with trypsin/EDTA solution for 5 min at 37ºC. Incubation with trypsin was done to allow us to separate the glandular structures from Matrigel. Trypsin was stopped by adding DMEM10% FBS and the cells were lysed with lysis buffer (2% SDS, 125 mM Tris-HCL pH6.8). Relative protein concentrations were determined loading an 8% acrylamide gel, transferred to PVDF membranes and blotted with anti-tubulin antibody. Equal amounts of proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA). Non-specific binding was blocked by incubation with TBST (20 mM Tris-Hcl pH7.4, 150 mM NaCl, 0.1% Tween-20) plus 5% of non-fat milk. Membranes were incubated overnight at 4ºC with the indicated dilution of antibodies: PTEN 1/1000, pERK 1/1000, pAKT 1/1000, p-p70S6K 1/1000, E-cadherin 1/1000, β-catenin 1/1000, vimentin 1/1000 and tubulin 1/10000. The procedure was followed by 1-hour incubation with secondary antibody 1/10000 in TBST at RT. Signal was detected with Immobilon Forte Western HRP Substrate (EMD Millipore Corporation, Burlington). Real-time PCR RNA extraction and Real time PCR was performed as previously described 21 .Total RNA was prepared using NZYol from Nzytech according to the manufacturer’s protocol. Reverse transcription reaction was performed using a total 1µg total RNA with TaqMan® Reverse Transcription Kit from Applied Biosystems. Quantitative real-time PCR detection of gene expression was performed with the ABI Prism 7000 Sequence Detection System using the TaqMan® Universal PCR Master Mix (Applied Biosystems). The sequences of primers used for PCR were obtained commercially from Applied biosystems Assay-on-demand Gene Vimentin (Mm01333430_m1) and Gadph (Mm99999915_g1). Relative expression was determinated from cycle threshold (Ct) values, which were normalized to Gadph as the endogenous control. Experiments were performed at least three times and statistical significance was determined by student’s test with p.value ≤ 0.05 Statistical analysis Experiments were performed at least three times and statistical significance was determined by student’s test with p-value ≤ 0.01. Unless otherwise asterisks indicate different p-value. Statistical analysis was performed with GraphPad Prism 8.0. Differences between two groups were assessed by Student’s t test (unpaired or paired as needed depending on the study design). Differences between more than two groups were assessed by one-way ANOVA, followed by the Tukey’s multiple comparison test or two-way ANOVA, followed by the Bonferroni post hoc comparison test. A p-value ≤ 0.05 was considered statistically significant. All data examined are expressed as mean ± SEM. Results Loss of cell polarity caused by absence of ECM impairs TGF-β-induced apoptosis. We have previously demonstrated that TGF-β triggers apoptosis of polarized endometrial epithelial cells grown as organoids in 3D conditions 21 . Disruption of cell polarity is a hallmark of cancer and is intimately involved in cancer progression 6 . Therefore, we decided to analyze the effects of cellular polarization on pro-apoptotic response induced by TGF-β on endometrial epithelial cells. For this purpose, isolated endometrial cells were plated in presence or absence of Matrigel. As we have previously reported, Matrigel elicits a three-dimensional organization of endometrial cells into 3D organoids in which endometrial epithelial cells display a high grade of polarization 20 . In contrast, lack of Matrigel resulted in a conventional 2D monolayer endometrial cells culture. Although endometrial epithelial cells in 2D retained the expression of epithelial markers such as E-cadherin, cytokeratin or β-catenin and were negative for the mesenchymal marker vimentin, they did not show polarized morphology (Fig. 1 A). To address the effects of Matrigel-induced cell polarization on TGF-β responses, both endometrial organoids and endometrial cell monolayers were treated with TGF-β. As we previously demonstrated, addition of TGF-β on endometrial cells cultured as 3D organoids caused a massive increase of cells displaying activation of caspase-3 21 .Surprisingly, treatment of 2D monolayers of epithelial endometrial cells lacking Matrigel with TGF-β to did not lead to caspase-3 activation (Fig. 1 B), indicating that lack of ECM and subsequent loss of cell polarization impairs TGF-β-induced apoptosis. Lack of ECM (Matrigel) increases PI3K/AKT signaling which causes resistance to TGF-β-induced apoptosis. The PI3K/AKT signaling pathway plays a pivotal role in the regulation of cell survival and apoptosis of endometrial epithelial cells. Our previous studies demonstrate that PTEN deficiency leads to increased PI3K/Akt signaling which hampers TGF-β-induced apoptosis of endometrial organoids 21 , 22 .These results enabled us to investigate whether ECM may regulate PI3K/AKT signaling pathway activation and thus sensibility to TGF-β-induced apoptosis. To address this hypothesis, Akt phosphorylation was analyzed by western blotting on 2D and 3D organoid lysates. As shown in Fig. 2 A, 3 D organoids displayed a markedly reduced level of Akt phosphorylation compared to 2D monolayers with no difference in PTEN expression. Next, we wondered whether such a difference in AKT phosphorylation could be the result of Matrigel stimulation itself or, otherwise, be the result of the acquisition of polarized glandular structure. To address this question, 2D monolayers of non-polarized endometrial epithelial cells were stimulated with increasing doses of Matrigel for 48 hours and cell lysates were subjected to western blot analysis. Increasing of Matrigel concentration caused a marked reduction of AKT phosphorylation and its downstream target p70S6K with no changes in PTEN expression (Fig. 2 B). Having demonstrated that the absence of Matrigel results in increased Akt phosphorylation, we investigated whether such an increase was the cause of resistance to TGF-β induced apoptosis in 2D monolayers of endometrial cells. For this purpose, 2D monolayers were pre-treated with the PI3K inhibitor LY294002 and then stimulated with TGF-β. Quantification of cleaved caspase-3 immunofluorescence revealed that inhibition of PI3K/AKT by LY294002 restored TGF-β-induced apoptosis in 2D monolayers of endometrial epithelial cells (Fig. 2 C). TGF-β induces EMT-like changes in non-polarized 2D monolayers of epithelial cells. Phase-contrast observation of non-polarized cells treated TGF-β for 72 hours evidenced a significant change in cell morphology, with transition from typical cobblestones morphology to mesenchymal spindle-shaped and fusiform feature (Fig. 3 A). Since TGF-β is also a potent EMT inducer 15 , 23 , we intended to investigate whether such TGF-β morphological changes were the result of an EMT process. To address this point, we performed an immunofluorescence analysis for the epithelial markers cytokeratin, E-cadherin and β-catenin and for the mesenchymal marker vimentin. Treatment of non-polarized 2D endometrial cells with TGF-β resulted in loss of expression of the epithelial markers E-cadherin, β-Catenin (Fig. 3 B) and cytokeratin (Fig. 3 C) and the acquisition of the mesenchymal marker expression vimentin (Fig. 3 C), suggesting that endometrial cells were undergoing EMT. The results obtained by immunofluorescence analysis were further confirmed by western blot (Fig. 3 D). Inhibition of ERK signaling blocks TGF-β-induced EMT in non-polarized 2D monolayers of endometrial epithelial cells. Having demonstrated that treatment with TGF-β of non-polarized cells induced EMT, we sought to investigate the intracellular signaling pathways involved in EMT signal transduction following TGF-β treatment in non-polarized cells. We have previously demonstrated that ERK/MAPK is required for EMT of endometrial cancer cell lines transduced with a mutant BRAF-V600E mutant 24 . Such evidence enabled us to investigate the role of ERK in TGF-β-induced EMT. For this purpose, non-polarized 2D monolayers of endometrial cells were treated with TGF-β alone or with TGF-β plus the MEK inhibitor U0126 and the expression of EMT markers was analyzed by immunofluorescence. U0126 inhibited the loss of cytokeratin, E-cadherin or β-catenin expression and the increase of the mesenchymal marker vimentin (Fig. 4 A). Loss of E-cadherin and increased vimentin expression were further confirmed by western blot (Fig. 4 B). These results indicate that inhibition ERK signaling by U0126 prevented TGF-β-induced EMT. Given that the presence of Matrigel as an ECM prevents TGF-β-induced EMT, we hypothesized that Matrigel may be regulating ERK activation. To address this point, 2D monolayers of non-polarized endometrial epithelial cells were stimulated with increasing doses of Matrigel for 48 hours and cell lysates were subjected to western blot analysis.. Increasing of Matrigel concentration caused a marked reduction of ERK phosphorylation, suggesting that the presence of ECM downregulates ERK signaling and TGF-β-induced EMT (Fig. 4 C). SMAD2/3 deficiency leads to partial EMT independently of TGF-β stimulation but inhibits TGF-β-induced EMT. SMADs play an important role as transducers of cellular responses after TGF-β receptor engagement 11 . To address the role of these transcription factors inTGF-β-triggered EMT, we isolated endometrial epithelial cells from tamoxifen-inducible double SMAD2/3 knock-out mice (Cre:ER +/− ;SMAD2 f/f ;SMAD3 f/f ). Isolated endometrial epithelial cells were plated in presence of tamoxifen to induce SMAD2/3 ablation and 24 hours later cells were treated with TGF-β. Unexpectedly, SMAD2/3 deficient 2D endometrial cultures displayed a dramatic increase of vimentin expression in absence of TGF-β, while retaining the expression of epithelial markers such as cytokeratin (Fig. 5 A), β-catenin (Fig. 5 B) and E-cadherin (Fig. 5 C). These results suggest that in the absence of ECM SMAD2/3 expression is required to maintain a full epithelial phenotype, and its loss causes partial EMT interpedently of TGF-β stimuli. Next, we analyzed the effect of SMAD2/3 deficiency on TGF-β-induced EMT. Proficient or SMAD2/3-deficient endometrial epithelial cells were treated with TGF-β 20 ng/ml for 72 hours and the expression of vimentin and the epithelial markers β-catenin, cytokeratin and E-cadherin was assessed by immunofluorescence. In opposition to SMAD2/3 expressing cells, cells lacking SMAD2/3 did not show a significant reduction of epithelial markers cytokeratin (Fig. 6 A), β-catenin (Fig. 6 B) and E-cadherin (Fig. 6 C). This result suggests that, in absence of ECM, SMAD2/3 deficiency blocks downregulation of epithelial cell markers related to TGF-β-triggered EMT changes. SMAD2/3 deficiency blocks the acquisition of migratory phenotype induced by TGF-β. Once we analyzed the effects of SMAD2/3 deficiency on TGF-β-induced EMT markers expression, we investigated whether it may correlate with migratory capability of SMAD2/3-deficient cells. To address this issue, endometrial epithelial cells from Cre:ER +/− ;SMAD2 f/f ;SMAD3 f/f were plated in presence of tamoxifen to induce SMAD2/3 ablation and 24 hours later were treated with TGF-β. After TGF-β treatment, the morphology of actin cytoskeleton was evidenced by phalloidin staining and the phosphorylation of FAK by immunofluorescence. Phalloidin staining revealed that TGF-β induced stress fibers formation, indicative of migratory phenotype. TGF-β switched the scattered diffuse phospho-FAK staining to an increased punctuate staining pattern that co-localized with the spreading edge of the stress fibers. These staining patterns suggest that TGF-β triggers migration and formation of new focal adhesions of endometrial epithelial cells (Fig. 7 ). In contrast, TGF-β treatment of endometrial cells lacking SMAD2/3 failed to change the pattern of phalloidin expression or phospho-FAK staining, indicating that deletion of SMAD2/3 impairs TGF-β-induced migration (Fig. 7 ). ECM blocks increased vimentin expression caused by SMAD2/3 deficiency. Next, we investigated whether the partial EMT observed in 2D endometrial monolayers lacking SMAD2/3 was caused by the lack of ECM signals. For this purpose, we performed immunofluorescence analysis of vimentin and epithelial markers in SMAD2/3 deficient 3D organoid cultures. Three-dimensional organoids displayed positive staining for epithelial markers cytokeratin, β-catenin and absence of expression of vimentin, suggesting that ECM signals provided by Matrigel inhibit expression of vimentin (Fig. 8 A). To further demonstrate that ECM is required to preserve the full epithelial phenotype in absence of SMAD2/3, 2D monolayers of SMAD2/3-deficient endometrial cells were acutely stimulated with 5% Matrigel diluted in culture medium and expression of vimentin was assessed by immunofluorescence and RT-qPCR. Indeed, Matrigel stimulation caused a marked reduction of vimentin immunostaining (Fig. 8 B) and expression (Fig. 8 C). ERK activation is required for SMAD2/3-induced vimentin expression. The above presented results demonstrated that TGF-β-induced EMT-like changes require ERK activation and, on the other hand, loss of SMAD2/3 resulted in acquisition of vimentin expression in unstimulated cells, but inhibited TGF-β-induced downregulation of epithelial markers. The next question we addressed was whether expression of vimentin in SMAD2/3-deficient endometrial cells was also reliant on ERK activation. For this purpose, endometrial epithelial cells isolated from tamoxifen-inducible double SMAD2/3 knock-out mice (Cre:ER +/− ;SMAD2 f/f ;SMAD3 f/f ) were plated in presence of tamoxifen to induce SMAD2/3 ablation and 24 hours later were treated with MEK inhibitor U0126 for further 72 hours and the expression of EMT markers was analyzed by immunofluorescence. Treatment with U0126 resulted in completely loss of vimentin expression without affecting cytokeratin expression of endometrial cells lacking SMAD2/3 (Fig. 9 ). This result indicates that ERK activation is also required for increased vimentin downstream of SMAD2/3 loss. Discussion Knowledge of biological determinants deciding cellular responses after TGF-β stimulation are a complex and not completely resolved issue. In uterus, TGF-β plays an important role in its development, structure and function 25 , 26 . Genetically modified mouse models harboring alterations in TGF-β signaling elements have supported role of TGF-β in uterine physiology and pathology. Conditional deletion of TGF-βRI in the uterus leads to structural and functional defects in this organ 27 . Besides its role in uterine development, disruption of TGF-β signalling plays an important role in endometrial carcinogenesis 28 , 29 . As in other malignancies, TGF-β can play a dual function in endometrial cancer progression, acting as tumor suppressor in early stages and as a tumor promoter in later stages. Endometrial cancer display disabled TGF-β signalling 30 leading to loss of growth inhibition 31 acquisition of an invasive phenotype 32 and correlates with poor prognosis 33 . Moreover, TGF-β has been shown to increase aggressiveness of endometrial cancer cell lines 32 , 34 . The role of TGF- β/SMAD pathway in endometrial carcinogenesis has been also revealed by conditional abrogation of TGF-β signaling elements through conditional deletion of TβRI 35 , conditional double deletion of SMAD2 and SMAD3 36 or conditional deletion of TβRI in combination with PTEN-inactivated endometrium 37 .All of them result in metastatic endometrial carcinoma mice. In agreement with these results, we have previously demonstrated that TGF-β/SMAD signaling is an important tumor suppressive mechanism in mouse endometrial cells 21 , 22 . In the past, our laboratory established a 3D culture method to grow endometrial epithelial cells as organoids in presence of Matrigel as artificial ECM 20 . Using this approach, we demonstrated that TGF-β induces apoptosis of mouse 3D endometrial organoids, which is completely impaired by the genetic deletion of SMAD2 and SMAD3 21,22 . In the present work, we show that culture of endometrial epithelial cells without Matrigel as artificial ECM leads to growth of 2D traditional monolayers that retain epithelial phenotype, as indicated by immunofluorescence of epithelial and mesenchymal markers. However, treatment of these 2D cultures with TGF-β induced EMT-like changes instead of the apoptotic cell death observed in 3D organoids. These results support the hypothesis that polarized 3D epithelial organization could act as non-canonical tumor suppressor that prevents the manifestation of neoplasic features. Disruption of glandular structure, including loss of apicobasal polarity, is a hallmark of epithelial cancers. Several crucial cell-polarity proteins are recognized proto-oncogenes or tumor suppressors, and basic mechanisms of cell polarity are often objectives of oncogenic signaling pathways 6 . Among all the factors involved in correct establishment of cell polarity and glandular organization, ECM plays a pivotal role. It has been shown that it plays a critical role in carcinogenesis 38 and in the regulation of TGF-β functions 39 . Our results suggest that the availability of ECM may be one of the determinants of cell context-dependent cellular outcome after exposure to TGF-β in normal endometrial epithelial cells. To this regard, ECM and cell polarity have also been shown to regulate cellular responses to TGF-β of endometrial cancer cell lines 40 . Our results support the hypothesis that polarized 3D epithelial organization acts as a non-canonical tumor suppressor that prevents the manifestation of neoplastic features such as EMT. More importantly, the ECM can be considered as one of the determinants that turns tumor suppressor functions of TGF-β to tumor promoter ones. We have further investigated the mechanisms involved in ECM-mediated cell outcome change after TGF-β treatment. We have found that the absence of ECM enhances AKT and ERK phosphorylation with different functional consequences: increased AKT phosphorylation leads to inhibition of TGF-β induced apoptosis and increased ERK phosphorylation results in EMT. The PI3K/AKT plays a crucial role in the regulation of endometrial cell survival and apoptosis and, therefore, in endometrial carcinogenesis 41 . In fact, our previous results demonstrate that enhanced PI3K/AKT signaling caused by the loss of PTEN is sufficient to confer resistance of endometrial epithelial cells to TGF-β-induced apoptosis 21 . ERK signaling plays a central role in regulation of EMT in many tumoral types, including endometrial epithelial cells 42 , 43 . In endometrial cancers, we have previously demonstrated that enhanced ERK signaling is triggered by constitutively active BRAF mutations 24 . Although it is widely accepted that ECM impacts on intracellular signaling 44 , its functional consequences are still controversial. On the one hand, ECM-activated intracellular pathways such as PI3K/AKT and RAS/ERK have been associated with increased cell survival, proliferation, and anoikis suppression. On the other hand, ECM-induced acquisition of cell polarity acts as non-canonical tumor suppressor mechanism 6 . Our findings demonstrate that the presence of ECM and acquisition of cell polarity determines TGF-β responses, but it can also be important for cancer cell drug sensitivity. For instance, the development of 3D cultures of colorectal cancer cell lines reduced both ERK and AKT phosphorylation, and enhanced sensibility to anti-cancer drugs 45 . Our results support a tumor suppressor function for ECM, as its absence results in apoptosis resistance and EMT, both hallmarks of tumoral phenotype. Another issue that deserves discussion is the role of SMAD signaling on ECM-regulated TGF-β responses. Our previous results demonstrated that loss of SMAD2/3 completely impairs TGF-β-induced apoptosis of endometrial 3D organoids 21 , 22 . Here, we have demonstrated that deletion of SMAD2/3 also influences TGF-β-induced EMT on 2D monolayers. SMAD2/3 can play multiple and opposing TGF-β signaling functions depending of cellular context 8 , 9 .SMADs interact with other transcriptional regulators that influence on TGF-β-induced transcriptional program. These SMAD-interacting transcription factors are tissue-specific and can integrate inputs from other signaling pathways, thereby generating TGF-β versatility in the transcriptional response in context-dependent manner. Interestingly, our results demonstrate that loss of SMAD2/3 causes expression of vimentin even in absence of TGF-β and completely blocks downregulation of epithelial markers, resulting in cells expressing both vimentin and epithelial markers. This phenomenon known as partial or hybrid EMT and has been received special attention because of its involvement in cancer metastasis and therapy resistance 19 , 46 , 47 . Such hybrid phenotype caused by SMAD2/3 deficiency was suppressed by the inhibition of ERK signalling, indicating that ERK is required for acquisition of a full EMT phenotype. In summary, our results demonstrate that ECM is one of the context factors that participates in the cellular responses to TGF-β in endometrial cells. Appropriate ECM-cell contacts can act as non-canonical tumor suppressor mechanism, which can collaborate with other traditional tumor suppressor mechanisms to maintain endometrial homeostasis. Declarations AUTHOR CONTRIBUTION Conceptualization, JE, ME, XMG and X.D.; methodology, NE, RN, ARM, IF, APG and X.D.; investigation, NE, RN, ARM, IF, MVS; validation, all authors; formal analysis, JE, ME, XMG, XD; resources, NE, XMG, XD.; writing, XD.; supervision, XD.; main project administration, XD.; funding acquisition, XD. FUNDING Supported by grants and PID2019-104734RB-I00 from Spanish Ministerio de Ciencia, Innovación y Universidades, Grupos estables de la Asociación Española Contra el Cancer, AECC. INSTITUTIONAL REVIEW BOARD STATEMENT For animal studies, we complied with Law 5/1995 and Act 214/1997 of the Autonomous Community (Generalitat of Catalonia) and EU Directive EEC 63/2010, and was approved by the Ethics Committee on Animal Experiments of the University of Lleida and the Ethics Commission in Animal Experimentation of the Generalitat de Catalunya AKNOWELDGEMENTS We also want to thank Dr. Akihiko Yoshimura (Keio University School of Medicine) for providing us Smad2 fl/fl and Dr Martin M Matzuk (Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA) for providing us SMAD3 fl/fl mice. DATA AVAILABILITY STATEMENT Data Availability Statement: The datasets analyzed during the current study are available from the corresponding author upon reasonable request. ARRIVE STATEMENT. All animal procedures were performed in accordance with ARRIVE guidelines. CONFLICTS OF INTEREST The authors declare no conflict of interest. References Bissell, M. 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Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses. J Cell Sci 130 , 203–218 (2017). Nieto, M. A., Huang, R. Y.-J., Jackson, R. A. & Thiery, J. P. EMT: 2016. Cell 166, 21–45 (2016). Yang, J. et al. Guidelines and definitions for research on epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 21 , 341–352 (2020). Additional Declarations No competing interests reported. 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12:14:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1465554/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1465554/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19634829,"identity":"6ae5e6e6-8f49-44f5-809d-77d92210e58b","added_by":"auto","created_at":"2022-03-25 22:20:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2715246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Cytokeratin, E-cadherin, β-catenin and vimentin immunofluorescence on epithelial endometrial cells grown as 2D monolayers or 3D organoids show no differences in epithelial markers expression. Cells were counterstained with Hoechst to visualize nuclei. \u003cstrong\u003eB. \u003c/strong\u003eConfocal images and quantification of cleaved caspase-3 immunofluorescence on endometrial cells grown as 2D monolayers or 3D organoids untreated (UN) or treated with 10 or 20 ng/ml TGF-β. Cells were counterstained with phalloidin to visualize actin cytoskeleton and morphology and Hoechst to visualize nuclei. Values are mean and error bars represent mean ± S.E.M. **P\u0026lt;0.01, ***P\u0026lt;0.001 by one-way ANOVA, followed by the Tukey’s multiple comparison test.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/e6a62e35b4c3b8454e532d18.png"},{"id":19633876,"identity":"ddc6d7d6-f3b1-4fe0-92c4-08b76a197594","added_by":"auto","created_at":"2022-03-25 22:10:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1386390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Western blot analysis of phosphorylated AKT (p-AKT) and PTEN on lysates obtained from 2D monolayer or 3D organoid cultures. Membrane was re-blotted with tubulin to check for equal protein loading. \u003cstrong\u003eB.\u003c/strong\u003e Western blot analysis of phosphorylated AKT (p-AKT) and PTEN, and phosphorylated p70S6K on lysates obtained from 2D monolayer stimulated for 48 hours with the indicated percentage of Matrigel diluted in basal culture medium. Membrane was re-blotted with tubulin to check for equal protein loading. \u003cstrong\u003eC.\u003c/strong\u003e Representative confocal images and quantification of cleaved-caspase-3 immunofluorescence corresponding to 2D monolayer cultures untreated (UN), treated with 20 ng/ml of TGF-β, 10 μM of LY294002 (LY) or combination (TGF-β+LY) for 48 hours. Cells were counterstained with phalloidin to visualize actin cytoskeleton and morphology, and Hoechst to visualize nuclei. To check the inhibition of AKT phosphorylation by LY294002, matched 2D monolayer cultures were lysed and p-AKT phosphorylation was analyzed by western blot. Membrane was re-blotted with tubulin to check for equal protein loading. Values are mean and error bars represent mean ± S.E.M. ***P\u0026lt;0.001 by one-way ANOVA, followed by the Tukey’s multiple comparison test.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/164bcab187d463f04a7e661a.png"},{"id":19634478,"identity":"c7466244-dd49-48e5-bc37-3638014e45db","added_by":"auto","created_at":"2022-03-25 22:15:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3361727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Phase contrast images of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 hours with TGF-β 20ng/ml. \u003cstrong\u003eB.\u003c/strong\u003e Representative confocal images of β-catenin and E-cadherin immunofluorescences corresponding to 2D endometrial epithelial monolayers untreated (UN) or treated for 72 hours with TGF-β 20 ng/ml. Cells were counterstained with Hoechst to visualize nuclei. \u003cstrong\u003eC.\u003c/strong\u003e Representative confocal images of double cytokeratin and vimentin immunofluorescence of 2D endometrial epithelial monolayers untreated (UN) or treated for 72 hours with TGF-β 20 ng/ml. Cells were counterstained with Hoechst to visualize nuclei. \u003cstrong\u003eD.\u003c/strong\u003e Western blot analysis of E-cadherin, β-catenin and vimentin expression on lysates obtained from 2D monolayer stimulated for 72 hours with TGF-β 20 ng/ml. Membrane was re-blotted with tubulin to check for equal protein loading.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/8cf8f4cea7cd83ffea14dcd6.png"},{"id":19634481,"identity":"8e388fb9-bdbe-4ccd-bad3-53635bdcea87","added_by":"auto","created_at":"2022-03-25 22:15:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5028094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative confocal images of β-catenin, E-cadherin, cytokeratin and vimentin immunofluorescences corresponding to 2D endometrial epithelial monolayers untreated (UN) or treated for 72 hours with TGF-β 20 ng/ml, 10µM of U0126 (U0) or the combination (TGF-β+UO). Cells were counterstained with Hoechst to visualize nuclei. \u003cstrong\u003eB. \u003c/strong\u003eWestern blot analysis of E-cadherin and vimentin expression on lysates obtained from 2D monolayer stimulated for 72 hours with TGF-β 20 ng/ml, 10 µM of U0126 (U0) or the combination (TGF-β+UO). Membrane was re-blotted with tubulin to check for equal protein loading. \u003cstrong\u003eC. \u003c/strong\u003eWestern blot analysis of phosphorylated ERK (pERK) on lysates obtained from 2D monolayer stimulated for 48 hours with the indicated percentage of Matrigel diluted in basal culture medium. Membrane was re-blotted with tubulin to check for equal protein loading.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/935927008109e241433a3b9b.png"},{"id":19633877,"identity":"94ef734f-ae50-4340-aad6-e2ced1f1fbd2","added_by":"auto","created_at":"2022-03-25 22:10:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4256803,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal images of double cytokeratin and vimentin \u003cstrong\u003e(A)\u003c/strong\u003e, β-catenin \u003cstrong\u003e(B)\u003c/strong\u003e or E-cadherin \u003cstrong\u003e(C)\u003c/strong\u003e immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003ef/f \u003c/sup\u003e;SMAD3\u003csup\u003ef/f\u003c/sup\u003e, plated and treated with tamoxifen (TAM)\u0026nbsp;to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles or left untreated (NO TAM).\u0026nbsp;Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/cf225af2b3a88653c801b85a.png"},{"id":19634479,"identity":"f7f86906-3079-4c88-87b1-21334b22d53d","added_by":"auto","created_at":"2022-03-25 22:15:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3602903,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative confocal images of double cytokeratin and vimentin \u003cstrong\u003e(A)\u003c/strong\u003e, β-catenin \u003cstrong\u003e(B)\u003c/strong\u003e or E-cadherin \u003cstrong\u003e(C)\u003c/strong\u003e immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003ef/f \u003c/sup\u003e;SMAD3\u003csup\u003ef/f\u003c/sup\u003e. Cells were plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles or left untreated (NO TAM) and then stimulated for 72 hours with 20 ng/ml of TGF-β.\u0026nbsp;Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/fd280586e64972041cfecc86.png"},{"id":19633882,"identity":"cb3c7722-caca-4c85-ae1f-badf9ecbc905","added_by":"auto","created_at":"2022-03-25 22:10:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3425979,"visible":true,"origin":"","legend":"\u003cp\u003ePhalloidin and phosphorylated FAK (p-FAK) immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003ef/f \u003c/sup\u003e;SMAD3\u003csup\u003ef/f\u003c/sup\u003e , plated and treated tamoxifen (TAM) to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles or left untreated (NO TAM) and then stimulated for 72 hours with 20 ng/ml of TGF-β.\u0026nbsp;Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/ac23c1373f4f08ff79a987f5.png"},{"id":19633885,"identity":"c725d4ac-5150-4c21-bc52-f96f4d0a51d9","added_by":"auto","created_at":"2022-03-25 22:10:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1443293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative confocal images cytokeratin, vimentin, β-catenin andE-cadherin on 3D organoid cultures from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003eF/F \u003c/sup\u003e;SMAD3\u003csup\u003eF/F\u003c/sup\u003e endometrial epithelial cells treated with tamoxifen to induce SMAD2/3 deletion. \u003cstrong\u003eB.\u003c/strong\u003e Immunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003ef/f \u003c/sup\u003e;SMAD3\u003csup\u003ef/f\u003c/sup\u003e, plated and treated with tamoxifen (TAM)\u0026nbsp;to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles then treated\u0026nbsp;for 72 hours with 5% Matrigel diluted in basal culture medium or left untreated (NO ECM). Images were captured after 3 days of tamoxifen-induced deletion. Cells were counterstained with Hoechst to visualize nuclei. \u003cstrong\u003eC.\u003c/strong\u003e RT-qPCR relative quantification of vimentin mRNA expression of 2D endometrial epithelial monolayers isolated from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003ef/f \u003c/sup\u003e;SMAD3\u003csup\u003ef/f\u003c/sup\u003e. Cells were plated and treated (TAM) or not (NO TAM) with\u0026nbsp;tamoxifen to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles, then treated\u0026nbsp;for 72 hours with 5% Matrigel diluted in basal culture medium or left untreated (NO ECM). Results are expressed as mRNA expression relative to untreated (NO ECM). Values are mean ± S.E.M. *P\u0026lt;0.01, by t-test analysis.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/ae9db9a9d9a64a7a2209a098.png"},{"id":19633879,"identity":"041183e4-509a-4242-87e3-2b220fb9d32d","added_by":"auto","created_at":"2022-03-25 22:10:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1965757,"visible":true,"origin":"","legend":"\u003cp\u003eVimentin and cytokeratinimmunofluorescence corresponding to 2D endometrial epithelial monolayers isolated from Cre:ER\u003csup\u003e+/- \u003c/sup\u003e;SMAD2\u003csup\u003ef/f \u003c/sup\u003e;SMAD3\u003csup\u003ef/f\u003c/sup\u003e , plated and treated with tamoxifen (TAM) to induce Cre:ER-mediated deletion of SMAD2 and SMAD3 alleles. Cells were counterstained with Hoechst to visualize nuclei.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/e31339d4e02863e7296e9e1b.png"},{"id":19634831,"identity":"15389d5c-9f1f-4099-84fd-1eb9ff3cc4ce","added_by":"auto","created_at":"2022-03-25 22:20:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":523441,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/739e3f8b-9577-4176-ac72-d04606b38319.pdf"},{"id":19633884,"identity":"a886a133-1bff-4b71-ae71-22f1305aa427","added_by":"auto","created_at":"2022-03-25 22:10:20","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1440482,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedWesternblots.pptx","url":"https://assets-eu.researchsquare.com/files/rs-1465554/v1/d034afcc40d5c22da5c25653.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lack of extracellular matrix switches TGF-β-induced apoptosis of endometrial cells to Epithelial-to-Mesenchymal transition through AKT, ERK and SMAD2/3-depdendent mechanisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCell-to-cell, cell-to extracellular matrix (ECM) interactions and establishment of a correct cell polarity play a pivotal role in epithelial cell homeostasis. Mounting evidences support that the three dimensional (3D) structure of epithelial tissue plays an important role in deciding cell fate and is involved in the regulation of proliferation, survival and apoptosis of individual epithelial cells \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Cell polarity acts as a tumor suppressor mechanism and its loss is a hallmark of cancer \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOver the past decades, 3D organoid cultures derived from epithelial tissues have been developed to recreate structures that reassemble their \u003cem\u003ein vivo\u003c/em\u003e architecture \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Such culture models have emerged as powerful cell-based scenarios to investigate many aspects of cancer pathobiology, such as the role of cell polarity and ECM, the function of tumor suppressors or oncogenes, and the importance of pathways in cancer \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To investigate the pathobiology of cancer it is biologically relevant to maintain the features of the 3D architecture of the epithelial tissue in culture. It is known that significant differences are observed between experiments assessed with cells cultured in a 3D polarity structures and experiments using classical two-dimensional (2D) cultures. Consequently, it has been proposed 3D polarity as a non-canonical mechanism of tumor suppression since it may function as an inhibitor of primary tumor development \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn normal epithelial tissues, Transforming Growth Factor-β (TGF-β) regulates a plethora of cell functions and processes depending on cell type, cellular context and microenvironment \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. TGF-β is secreted as an inactive latent disulfide-linked homodimer (LTGF-β) polypeptide. Mature bioactive TGF-β ligands are produced upon proteolytic cleavage of the latent complex and directly bind to TGF-β type II receptor (TGF-βRII). TGF-βRII phosphorylates and activates the TGF-β receptor type I which, in turn, phosphorylates members of the SMAD family \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The SMAD family consists of nine members, which form 3 subfamilies: receptor-activated (R-)SMADs (SMAD1, SMAD2, SMAD3, SMAD5, SMAD8 and SMAD9), a single common-mediator (Co-)SMAD (SMAD4) and two inhibitory (I-)SMADs (SMAD6 and SMAD7). TGF-β also induces non-SMAD pathways, including mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K) and Rho GTPases \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDysregulation of TGF-β signaling has been implicated in the progression of diseases such as fibrosis or cancer. In most normal epithelial tissues, TGF-β acts as tumor suppressor by inhibiting cell cycle progression or activating apoptosis but, during carcinogenesis, TGF-β switches its tumor suppressive functions to promoter ones \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms of these functional changes of TGF-β are not fully understood and may vary among cell types and contexts. As a tumor promoter, TGF-β stimulation is associated with epithelial-to-mesenchymal transition (EMT) and acquisition of cell migration, invasion and metastatic potential of malignant epithelial cells \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEMT is a well-coordinated process during embryonic development and a pathological feature in neoplasia and fibrosis. Cells undergoing EMT lose the expression of epithelial cell markers such as cytokeratins, E-cadherin or β-catenin and gain the expression of mesenchymal markers such as vimentin \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, over the past few years, numerous studies demonstrate that EMT is a dynamic process in which cells undergoing partial or hybrid EMT display intermediate cell states retaining expression of both epithelial and mesenchymal markers \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although EMT has traditionally been associated with increased metastatic properties, recent studies have activated the debate on the requirement of EMT and its different EMT states for cancer metastasis \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, using a 3D organoid culture system of mouse endometrial epithelial cells, we have investigated the effect of extracellular matrix-induced 3D cell organization on biological responses to TGF-β. We have demonstrated that the lack of ECM and subsequent loss of cell polarity switches TGF-β-induced apoptosis to an EMT-compatible phenotype. We have also inferred the molecular mechanism involved in this swapped function. On the one hand, we demonstrated that the absence of ECM controls sensitivity to TGF-β-induced apoptosis through regulation of AKT signaling. On the other hand, both ERK and SMAD2/3 signaling regulate TGF-β-triggered EMT in absence of ECM. In summary, our results support that ECM-induced cell polarity acts as a non-canonical tumor suppressor for endometrial epithelial cells.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eThe recombinant basement membrane Matrigel was purchased from BD Biosciences (San Jose, CA). Epidermal Growth Factor, hydrocortisone, dexamethasone, RU-486, ICI 182170 and LY 294002 were obtained from Sigma (St Louis, MO), Insulin-Transferrin-Sodium Selenite supplement was obtained from Invitrogen (Invitrogen, Inc., Carlsbad, CA, USA), PD0325901 and U0126 were purchased from Calbiochem (Calbiochem-Novabiochem, UK, Ltd). Antibodies to E-Cadherin, β-Catenin and vimentin were from BDbiosciences; bisBenzimide H 33342 trihydrochloride (Hoechst), and tubulin were obtained from Sigma and anti-cytokeratin was from Abcam (Cambridge UK). Anti-phospho Akt, anti-phospho-p70SK6, cleaved caspase-3 and PTEN antibodies were from Cell Signalling Technology (Beverly, MA). Anti-phospho FAK was from Thermo fisher. Anti-phospo ERK was from Biolegend. Alexa-Flour anti-Rabbit and anti-mouse antibodies were from Invitrogen Peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Jackson ImmunoResearch Europe Ltd (Suffolk, UK). All other regents were obtained from Sigma unless specified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Isolation of endometrial epithelial cells\u003c/h2\u003e \u003cp\u003eMice were housed in a barrier facility and pathogen-free procedures were used in all mouse rooms. Animals were kept in a 12-hour light-dark cycle with \u003cem\u003ead libitum\u003c/em\u003e access to a standard 2014 Teklad Global 14% Protein Rodent Maintenance Diet (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.harlan.com\" target=\"_blank\"\u003ewww.harlan.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.harlan.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and water. The animal rooms were environmentally controlled (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). The study complied with Law 5/1995 and Act 214/1997 of the Autonomous Community (Generalitat of Catalonia) and EU Directive EEC 63/2010, and was approved by the Ethics Committee on Animal Experiments of the University of Lleida and the Ethics Commission in Animal Experimentation of the Generalitat de Catalunya.\u003c/p\u003e \u003cp\u003eCRE:ER (B6.Cg-Tg(CAG-CRE/Esr1* 5Amc/J) mice was obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Double Smad2\u003csup\u003efl/fl\u003c/sup\u003e Smad3\u003csup\u003efl/fl\u003c/sup\u003e mice was provided by Dr.Martin M Matzuk (Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA).\u003c/p\u003e \u003cp\u003eCRE:ER mice genotyping PCR was carried out with the following primer: forward primer 5\u0026rsquo;-CAC TCC CAG AGA CAT ATA CAC-3\u0026rsquo; and reverse primer 5\u0026rsquo;-ACG AAC CTG GTC GAA ATC GT GCG-3\u0026rsquo; .\u003c/p\u003e \u003cp\u003eSmad3\u003csup\u003efl/fl\u003c/sup\u003e mice genotyping PCR was carried out with the following primer: forward primer 5\u0026rsquo;-CTC CAG ATC GTG GGC ATA CAG C-3\u0026rsquo;; \u003cem\u003esmad3\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e reverse primer 5\u0026rsquo;-GGT CAC AGG GTC CTC TGT GCC-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003eSmad2\u003csup\u003efl/fl\u003c/sup\u003e mice genotyping PCR was carried out with the following primer: forward primer 5\u0026rsquo;-CAT CAG ATT CCA TTA GAG ATG G-3\u0026rsquo;; \u003cem\u003esmad2\u003c/em\u003e\u003csup\u003efl/fl\u003c/sup\u003e reverse primer 5\u0026rsquo;-TGA GAC TTC TCT GTA CCC GAT \u0026minus;\u0026thinsp;3\u0026rsquo;.\u003c/p\u003e \u003cp\u003eCRE:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e Smad2\u003csup\u003efl/fl\u003c/sup\u003e Smad3\u003csup\u003efl/fl\u003c/sup\u003e mice were bred in a mixed background (C57BL6; 129S4) by crossing Smad2\u003csup\u003efl/fl\u003c/sup\u003e Smad3\u003csup\u003efl/fl\u003c/sup\u003e mice with CRE:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mice. To obtain mice carrying both Smad2 and Smad3 floxed allelles and a single CRE:ER (CRE:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e), CRE:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e were backcrossed with Smad2\u003csup\u003efl/fl\u003c/sup\u003e; Smad2\u003csup\u003efl/fl\u003c/sup\u003e mice. The isolation of endometrial epithelial cells was processed as described previously \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, uterine horns were dissected from 3\u0026ndash;4 weeks old C57BL6 mice. Uterus were washed with HBSS and digested with Trypsin (Invitrogen). After trypsin digestion epithelial sheets were squeezed-out of the uterine pieces. Epithelial sheets were washed twice with PBS and resuspended in 1 ml of DMEM/F12 (Invitrogen) supplemented with 1% of Sodium Pyruvate (Sigma), 1% of penicillin/streptomycin (Sigma) and 0,1% of fungizone (Invitrogen) (basal medium). Epithelial sheets were mechanically disrupted in basal medium until small clumps of cells were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2D endometrial monolayers and 3D Endometrial organoids cultures\u003c/h2\u003e \u003cp\u003eGrowth of endometrial epithelial cells in 2D cultures was performed by platting cells in basal medium with 2% of dextran-coated charcoal-stripped serum (Invitrogen) and leaved twenty-four hours. To induce deletion of floxed alleles, Tamoxifen (Sigma-Aldrich T5648, St. Louis, MO, USA) was dissolved in 100% EtOH at 1mM and used at 0,5 \u0026micro;l/ml of medium for 24 hours. For immunofluorescence, cells were seeded in a volume of 40 \u0026micro;l /well in 96 well plates black with micro-clear bottom (Greiner Bio-one). For western blotting, cells were placed in a volume of 200 \u0026micro;l in 24-well plates (BDBiosciences).\u003c/p\u003e \u003cp\u003eGrowth of endometrial epithelial cells in 3D cultures was performed as described previously \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Twenty-four hours after platting in plastic, cells were washed with HBSS and incubated with trypsin/EDTA solution (Sigma) for 5 min at 37\u0026ordm;C. Trypsin was stopped by adding DMEM 10% FBS and clumps of 2\u0026ndash;8 cells were obtained. Cells were centrifuged at 1000 rpm for 3 minutes and diluted in basal medium containing 3% of matrigel to obtain 4 x 10\u003csup\u003e4\u003c/sup\u003e cell clumps/ml. For immunofluorescence, cells were seeded in a volume of 40 \u0026micro;l /well in 96 well plates (black with micro-clear bottom) (Greiner Bio-one). For western blotting, cells were placed in a volume of 200 \u0026micro;l in 24-well plates (BDBiosciences). In all cases, 24 hours after plating medium was replaced by Basal medium supplemented with 5 ng/ml EGF and 1/100 dilution of Insulin-Transferrin-Sodium Selenite (ITS) Supplement (Invitrogen) and 3% of fresh Matrigel (referred as BIE). Medium was replaced every 2\u0026ndash;3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.Cultures were fixed with formalin for 5 minutes at room temperature (RT) and washed twice with PBS. Depending on primary antibody, cells were permeabilized with 0,2% triton X-100 in PBS for 10 minutes (indicated as T) or permeabilized with 100% methanol for 2 minutes (indicated as M). Next, cultures were incubated overnight at 4\u0026ordm;C with the indicated dilutions of antibodies: anti-Cytokeratin 1/500(M), β-Catenin 1/100(M), E-Cadherina 1/100(M), Rhodamine conjugated-Phalloidin 1/500(T), anti-Cleaved Caspase-3 1/100(T), anti-Vimentin 1/100 (T) and anti-phospho FAK 1/100 (T). After one day, cells were washed twice with PBS and incubated with PBS containing a 5 \u0026micro;g/ml of Hoechst dye and 1/500 dilution of Alexa Fluor secondary anti-mouse or anti-rabbit antibodies for 2 hours at RT in the case of 2D cultures or overnight at 4\u0026ordm;C for 3D cultures. For double immunofluorescence staining, cells were incubated with the second round of primary and secondary antibodies. We would like to point out that in all double immunofluorescence stains, first and second primary antibodies were from different isotope. Immunofluorescence staining was visualized and analyzed using a confocal microscopy (Olympus). Edition of confocal images was performed using Fluoview software (Olympus).\u003c/p\u003e \u003cp\u003eFor evaluation of apoptosis and caspase-3 processing, endometrial epithelial glands were analyzed on a confocal microscope Fluoview FV1000. The presence of apoptotic or positive caspase-3 processing (more than 5 cells per gland) was revealed by Hoescht/Cleaved Caspase-3 immunotaining. For each experiment we quantified at least 100 glands. Cell polarity of epithelial cells forming glandular structures was evidenced by double immunostainings as indicated in each figure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot analysis\u003c/h2\u003e \u003cp\u003e2D endometrial monolayers or endometrial organoid 3D cultures stimulated for the indicated periods of time, were washed with HBSS and incubated with trypsin/EDTA solution for 5 min at 37\u0026ordm;C. Incubation with trypsin was done to allow us to separate the glandular structures from Matrigel. Trypsin was stopped by adding DMEM10% FBS and the cells were lysed with lysis buffer (2% SDS, 125 mM Tris-HCL pH6.8). Relative protein concentrations were determined loading an 8% acrylamide gel, transferred to PVDF membranes and blotted with anti-tubulin antibody. Equal amounts of proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA). Non-specific binding was blocked by incubation with TBST (20 mM Tris-Hcl pH7.4, 150 mM NaCl, 0.1% Tween-20) plus 5% of non-fat milk. Membranes were incubated overnight at 4\u0026ordm;C with the indicated dilution of antibodies: PTEN 1/1000, pERK 1/1000, pAKT 1/1000, p-p70S6K 1/1000, E-cadherin 1/1000, β-catenin 1/1000, vimentin 1/1000 and tubulin 1/10000. The procedure was followed by 1-hour incubation with secondary antibody 1/10000 in TBST at RT. Signal was detected with Immobilon Forte Western HRP Substrate (EMD Millipore Corporation, Burlington).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR\u003c/h2\u003e \u003cp\u003eRNA extraction and Real time PCR was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.Total RNA was prepared using NZYol from Nzytech according to the manufacturer\u0026rsquo;s protocol. Reverse transcription reaction was performed using a total 1\u0026micro;g total RNA with TaqMan\u0026reg; Reverse Transcription Kit from Applied Biosystems. Quantitative real-time PCR detection of gene expression was performed with the ABI Prism 7000 Sequence Detection System using the TaqMan\u0026reg; Universal PCR Master Mix (Applied Biosystems). The sequences of primers used for PCR were obtained commercially from Applied biosystems Assay-on-demand Gene Vimentin (Mm01333430_m1) and Gadph (Mm99999915_g1). Relative expression was determinated from cycle threshold (Ct) values, which were normalized to Gadph as the endogenous control. Experiments were performed at least three times and statistical significance was determined by student\u0026rsquo;s test with p.value\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperiments were performed at least three times and statistical significance was determined by student\u0026rsquo;s test with p-value\u0026thinsp;\u0026le;\u0026thinsp;0.01. Unless otherwise asterisks indicate different p-value. Statistical analysis was performed with GraphPad Prism 8.0. Differences between two groups were assessed by Student\u0026rsquo;s t test (unpaired or paired as needed depending on the study design). Differences between more than two groups were assessed by one-way ANOVA, followed by the Tukey\u0026rsquo;s multiple comparison test or two-way ANOVA, followed by the Bonferroni post hoc comparison test. A p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant. All data examined are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLoss of cell polarity caused by absence of ECM impairs TGF-β-induced apoptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe have previously demonstrated that TGF-β triggers apoptosis of polarized endometrial epithelial cells grown as organoids in 3D conditions \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Disruption of cell polarity is a hallmark of cancer and is intimately involved in cancer progression \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, we decided to analyze the effects of cellular polarization on pro-apoptotic response induced by TGF-β on endometrial epithelial cells. For this purpose, isolated endometrial cells were plated in presence or absence of Matrigel. As we have previously reported, Matrigel elicits a three-dimensional organization of endometrial cells into 3D organoids in which endometrial epithelial cells display a high grade of polarization \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In contrast, lack of Matrigel resulted in a conventional 2D monolayer endometrial cells culture. Although endometrial epithelial cells in 2D retained the expression of epithelial markers such as E-cadherin, cytokeratin or β-catenin and were negative for the mesenchymal marker vimentin, they did not show polarized morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To address the effects of Matrigel-induced cell polarization on TGF-β responses, both endometrial organoids and endometrial cell monolayers were treated with TGF-β. As we previously demonstrated, addition of TGF-β on endometrial cells cultured as 3D organoids caused a massive increase of cells displaying activation of caspase-3 \u003csup\u003e21\u003c/sup\u003e.Surprisingly, treatment of 2D monolayers of epithelial endometrial cells lacking Matrigel with TGF-β to did not lead to caspase-3 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating that lack of ECM and subsequent loss of cell polarization impairs TGF-β-induced apoptosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLack of ECM (Matrigel) increases PI3K/AKT signaling which causes resistance to TGF-β-induced apoptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe PI3K/AKT signaling pathway plays a pivotal role in the regulation of cell survival and apoptosis of endometrial epithelial cells. Our previous studies demonstrate that PTEN deficiency leads to increased PI3K/Akt signaling which hampers TGF-β-induced apoptosis of endometrial organoids \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.These results enabled us to investigate whether ECM may regulate PI3K/AKT signaling pathway activation and thus sensibility to TGF-β-induced apoptosis. To address this hypothesis, Akt phosphorylation was analyzed by western blotting on 2D and 3D organoid lysates. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD organoids displayed a markedly reduced level of Akt phosphorylation compared to 2D monolayers with no difference in PTEN expression. Next, we wondered whether such a difference in AKT phosphorylation could be the result of Matrigel stimulation itself or, otherwise, be the result of the acquisition of polarized glandular structure. To address this question, 2D monolayers of non-polarized endometrial epithelial cells were stimulated with increasing doses of Matrigel for 48 hours and cell lysates were subjected to western blot analysis. Increasing of Matrigel concentration caused a marked reduction of AKT phosphorylation and its downstream target p70S6K with no changes in PTEN expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eHaving demonstrated that the absence of Matrigel results in increased Akt phosphorylation, we investigated whether such an increase was the cause of resistance to TGF-β induced apoptosis in 2D monolayers of endometrial cells. For this purpose, 2D monolayers were pre-treated with the PI3K inhibitor LY294002 and then stimulated with TGF-β. Quantification of cleaved caspase-3 immunofluorescence revealed that inhibition of PI3K/AKT by LY294002 restored TGF-β-induced apoptosis in 2D monolayers of endometrial epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTGF-β induces EMT-like changes in non-polarized 2D monolayers of epithelial cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePhase-contrast observation of non-polarized cells treated TGF-β for 72 hours evidenced a significant change in cell morphology, with transition from typical cobblestones morphology to mesenchymal spindle-shaped and fusiform feature (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Since TGF-β is also a potent EMT inducer \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we intended to investigate whether such TGF-β morphological changes were the result of an EMT process. To address this point, we performed an immunofluorescence analysis for the epithelial markers cytokeratin, E-cadherin and β-catenin and for the mesenchymal marker vimentin. Treatment of non-polarized 2D endometrial cells with TGF-β resulted in loss of expression of the epithelial markers E-cadherin, β-Catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and cytokeratin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and the acquisition of the mesenchymal marker expression vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), suggesting that endometrial cells were undergoing EMT. The results obtained by immunofluorescence analysis were further confirmed by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition of ERK signaling blocks TGF-β-induced EMT in non-polarized 2D monolayers of endometrial epithelial cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHaving demonstrated that treatment with TGF-β of non-polarized cells induced EMT, we sought to investigate the intracellular signaling pathways involved in EMT signal transduction following TGF-β treatment in non-polarized cells. We have previously demonstrated that ERK/MAPK is required for EMT of endometrial cancer cell lines transduced with a mutant BRAF-V600E mutant \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Such evidence enabled us to investigate the role of ERK in TGF-β-induced EMT. For this purpose, non-polarized 2D monolayers of endometrial cells were treated with TGF-β alone or with TGF-β plus the MEK inhibitor U0126 and the expression of EMT markers was analyzed by immunofluorescence. U0126 inhibited the loss of cytokeratin, E-cadherin or β-catenin expression and the increase of the mesenchymal marker vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Loss of E-cadherin and increased vimentin expression were further confirmed by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results indicate that inhibition ERK signaling by U0126 prevented TGF-β-induced EMT. Given that the presence of Matrigel as an ECM prevents TGF-β-induced EMT, we hypothesized that Matrigel may be regulating ERK activation. To address this point, 2D monolayers of non-polarized endometrial epithelial cells were stimulated with increasing doses of Matrigel for 48 hours and cell lysates were subjected to western blot analysis.. Increasing of Matrigel concentration caused a marked reduction of ERK phosphorylation, suggesting that the presence of ECM downregulates ERK signaling and TGF-β-induced EMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSMAD2/3 deficiency leads to partial EMT independently of TGF-β stimulation but inhibits TGF-β-induced EMT.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSMADs play an important role as transducers of cellular responses after TGF-β receptor engagement \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To address the role of these transcription factors inTGF-β-triggered EMT, we isolated endometrial epithelial cells from tamoxifen-inducible double SMAD2/3 knock-out mice (Cre:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e;SMAD2\u003csup\u003ef/f\u003c/sup\u003e ;SMAD3\u003csup\u003ef/f\u003c/sup\u003e). Isolated endometrial epithelial cells were plated in presence of tamoxifen to induce SMAD2/3 ablation and 24 hours later cells were treated with TGF-β. Unexpectedly, SMAD2/3 deficient 2D endometrial cultures displayed a dramatic increase of vimentin expression in absence of TGF-β, while retaining the expression of epithelial markers such as cytokeratin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results suggest that in the absence of ECM SMAD2/3 expression is required to maintain a full epithelial phenotype, and its loss causes partial EMT interpedently of TGF-β stimuli.\u003c/p\u003e \u003cp\u003eNext, we analyzed the effect of SMAD2/3 deficiency on TGF-β-induced EMT. Proficient or SMAD2/3-deficient endometrial epithelial cells were treated with TGF-β 20 ng/ml for 72 hours and the expression of vimentin and the epithelial markers β-catenin, cytokeratin and E-cadherin was assessed by immunofluorescence. In opposition to SMAD2/3 expressing cells, cells lacking SMAD2/3 did not show a significant reduction of epithelial markers cytokeratin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This result suggests that, in absence of ECM, SMAD2/3 deficiency blocks downregulation of epithelial cell markers related to TGF-β-triggered EMT changes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSMAD2/3 deficiency blocks the acquisition of migratory phenotype induced by TGF-β.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOnce we analyzed the effects of SMAD2/3 deficiency on TGF-β-induced EMT markers expression, we investigated whether it may correlate with migratory capability of SMAD2/3-deficient cells. To address this issue, endometrial epithelial cells from Cre:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e ;SMAD2\u003csup\u003ef/f\u003c/sup\u003e ;SMAD3\u003csup\u003ef/f\u003c/sup\u003e were plated in presence of tamoxifen to induce SMAD2/3 ablation and 24 hours later were treated with TGF-β. After TGF-β treatment, the morphology of actin cytoskeleton was evidenced by phalloidin staining and the phosphorylation of FAK by immunofluorescence. Phalloidin staining revealed that TGF-β induced stress fibers formation, indicative of migratory phenotype. TGF-β switched the scattered diffuse phospho-FAK staining to an increased punctuate staining pattern that co-localized with the spreading edge of the stress fibers. These staining patterns suggest that TGF-β triggers migration and formation of new focal adhesions of endometrial epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In contrast, TGF-β treatment of endometrial cells lacking SMAD2/3 failed to change the pattern of phalloidin expression or phospho-FAK staining, indicating that deletion of SMAD2/3 impairs TGF-β-induced migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eECM blocks increased vimentin expression caused by SMAD2/3 deficiency.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we investigated whether the partial EMT observed in 2D endometrial monolayers lacking SMAD2/3 was caused by the lack of ECM signals. For this purpose, we performed immunofluorescence analysis of vimentin and epithelial markers in SMAD2/3 deficient 3D organoid cultures. Three-dimensional organoids displayed positive staining for epithelial markers cytokeratin, β-catenin and absence of expression of vimentin, suggesting that ECM signals provided by Matrigel inhibit expression of vimentin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). To further demonstrate that ECM is required to preserve the full epithelial phenotype in absence of SMAD2/3, 2D monolayers of SMAD2/3-deficient endometrial cells were acutely stimulated with 5% Matrigel diluted in culture medium and expression of vimentin was assessed by immunofluorescence and RT-qPCR. Indeed, Matrigel stimulation caused a marked reduction of vimentin immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) and expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eERK activation is required for SMAD2/3-induced vimentin expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe above presented results demonstrated that TGF-β-induced EMT-like changes require ERK activation and, on the other hand, loss of SMAD2/3 resulted in acquisition of vimentin expression in unstimulated cells, but inhibited TGF-β-induced downregulation of epithelial markers. The next question we addressed was whether expression of vimentin in SMAD2/3-deficient endometrial cells was also reliant on ERK activation. For this purpose, endometrial epithelial cells isolated from tamoxifen-inducible double SMAD2/3 knock-out mice (Cre:ER\u003csup\u003e+/\u0026minus;\u003c/sup\u003e ;SMAD2\u003csup\u003ef/f\u003c/sup\u003e ;SMAD3\u003csup\u003ef/f\u003c/sup\u003e) were plated in presence of tamoxifen to induce SMAD2/3 ablation and 24 hours later were treated with MEK inhibitor U0126 for further 72 hours and the expression of EMT markers was analyzed by immunofluorescence. Treatment with U0126 resulted in completely loss of vimentin expression without affecting cytokeratin expression of endometrial cells lacking SMAD2/3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This result indicates that ERK activation is also required for increased vimentin downstream of SMAD2/3 loss.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eKnowledge of biological determinants deciding cellular responses after TGF-β stimulation are a complex and not completely resolved issue. In uterus, TGF-β plays an important role in its development, structure and function \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Genetically modified mouse models harboring alterations in TGF-β signaling elements have supported role of TGF-β in uterine physiology and pathology. Conditional deletion of TGF-βRI in the uterus leads to structural and functional defects in this organ \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Besides its role in uterine development, disruption of TGF-β signalling plays an important role in endometrial carcinogenesis \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. As in other malignancies, TGF-β can play a dual function in endometrial cancer progression, acting as tumor suppressor in early stages and as a tumor promoter in later stages. Endometrial cancer display disabled TGF-β signalling \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e leading to loss of growth inhibition \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e acquisition of an invasive phenotype \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and correlates with poor prognosis \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, TGF-β has been shown to increase aggressiveness of endometrial cancer cell lines \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The role of TGF- β/SMAD pathway in endometrial carcinogenesis has been also revealed by conditional abrogation of TGF-β signaling elements through conditional deletion of TβRI \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, conditional double deletion of SMAD2 and SMAD3 \u003csup\u003e36\u003c/sup\u003e or conditional deletion of TβRI in combination with PTEN-inactivated endometrium \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.All of them result in metastatic endometrial carcinoma mice. In agreement with these results, we have previously demonstrated that TGF-β/SMAD signaling is an important tumor suppressive mechanism in mouse endometrial cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the past, our laboratory established a 3D culture method to grow endometrial epithelial cells as organoids in presence of Matrigel as artificial ECM \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Using this approach, we demonstrated that TGF-β induces apoptosis of mouse 3D endometrial organoids, which is completely impaired by the genetic deletion of SMAD2 and SMAD3 \u003csup\u003e21,22\u003c/sup\u003e. In the present work, we show that culture of endometrial epithelial cells without Matrigel as artificial ECM leads to growth of 2D traditional monolayers that retain epithelial phenotype, as indicated by immunofluorescence of epithelial and mesenchymal markers. However, treatment of these 2D cultures with TGF-β induced EMT-like changes instead of the apoptotic cell death observed in 3D organoids. These results support the hypothesis that polarized 3D epithelial organization could act as non-canonical tumor suppressor that prevents the manifestation of neoplasic features. Disruption of glandular structure, including loss of apicobasal polarity, is a hallmark of epithelial cancers. Several crucial cell-polarity proteins are recognized proto-oncogenes or tumor suppressors, and basic mechanisms of cell polarity are often objectives of oncogenic signaling pathways \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Among all the factors involved in correct establishment of cell polarity and glandular organization, ECM plays a pivotal role. It has been shown that it plays a critical role in carcinogenesis \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and in the regulation of TGF-β functions \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Our results suggest that the availability of ECM may be one of the determinants of cell context-dependent cellular outcome after exposure to TGF-β in normal endometrial epithelial cells. To this regard, ECM and cell polarity have also been shown to regulate cellular responses to TGF-β of endometrial cancer cell lines \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Our results support the hypothesis that polarized 3D epithelial organization acts as a non-canonical tumor suppressor that prevents the manifestation of neoplastic features such as EMT. More importantly, the ECM can be considered as one of the determinants that turns tumor suppressor functions of TGF-β to tumor promoter ones.\u003c/p\u003e \u003cp\u003eWe have further investigated the mechanisms involved in ECM-mediated cell outcome change after TGF-β treatment. We have found that the absence of ECM enhances AKT and ERK phosphorylation with different functional consequences: increased AKT phosphorylation leads to inhibition of TGF-β induced apoptosis and increased ERK phosphorylation results in EMT. The PI3K/AKT plays a crucial role in the regulation of endometrial cell survival and apoptosis and, therefore, in endometrial carcinogenesis \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In fact, our previous results demonstrate that enhanced PI3K/AKT signaling caused by the loss of PTEN is sufficient to confer resistance of endometrial epithelial cells to TGF-β-induced apoptosis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. ERK signaling plays a central role in regulation of EMT in many tumoral types, including endometrial epithelial cells \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In endometrial cancers, we have previously demonstrated that enhanced ERK signaling is triggered by constitutively active BRAF mutations \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Although it is widely accepted that ECM impacts on intracellular signaling \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, its functional consequences are still controversial. On the one hand, ECM-activated intracellular pathways such as PI3K/AKT and RAS/ERK have been associated with increased cell survival, proliferation, and anoikis suppression. On the other hand, ECM-induced acquisition of cell polarity acts as non-canonical tumor suppressor mechanism \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrate that the presence of ECM and acquisition of cell polarity determines TGF-β responses, but it can also be important for cancer cell drug sensitivity. For instance, the development of 3D cultures of colorectal cancer cell lines reduced both ERK and AKT phosphorylation, and enhanced sensibility to anti-cancer drugs \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Our results support a tumor suppressor function for ECM, as its absence results in apoptosis resistance and EMT, both hallmarks of tumoral phenotype.\u003c/p\u003e \u003cp\u003eAnother issue that deserves discussion is the role of SMAD signaling on ECM-regulated TGF-β responses. Our previous results demonstrated that loss of SMAD2/3 completely impairs TGF-β-induced apoptosis of endometrial 3D organoids \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Here, we have demonstrated that deletion of SMAD2/3 also influences TGF-β-induced EMT on 2D monolayers. SMAD2/3 can play multiple and opposing TGF-β signaling functions depending of cellular context \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.SMADs interact with other transcriptional regulators that influence on TGF-β-induced transcriptional program. These SMAD-interacting transcription factors are tissue-specific and can integrate inputs from other signaling pathways, thereby generating TGF-β versatility in the transcriptional response in context-dependent manner. Interestingly, our results demonstrate that loss of SMAD2/3 causes expression of vimentin even in absence of TGF-β and completely blocks downregulation of epithelial markers, resulting in cells expressing both vimentin and epithelial markers. This phenomenon known as partial or hybrid EMT and has been received special attention because of its involvement in cancer metastasis and therapy resistance\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Such hybrid phenotype caused by SMAD2/3 deficiency was suppressed by the inhibition of ERK signalling, indicating that ERK is required for acquisition of a full EMT phenotype.\u003c/p\u003e \u003cp\u003eIn summary, our results demonstrate that ECM is one of the context factors that participates in the cellular responses to TGF-β in endometrial cells. Appropriate ECM-cell contacts can act as non-canonical tumor suppressor mechanism, which can collaborate with other traditional tumor suppressor mechanisms to maintain endometrial homeostasis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, JE, ME, XMG and X.D.; methodology, NE, RN, ARM, IF, APG and X.D.; investigation, NE, RN, ARM, IF, MVS; validation, all authors; formal analysis, JE, ME, XMG, XD; resources, NE, XMG, XD.; writing, XD.; supervision, XD.; main project administration, XD.; funding acquisition, XD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSupported by grants and PID2019-104734RB-I00 from Spanish Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades, Grupos estables de la Asociaci\u0026oacute;n Espa\u0026ntilde;ola Contra el Cancer, AECC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINSTITUTIONAL REVIEW BOARD STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor animal studies, we complied with Law 5/1995 and Act 214/1997 of the Autonomous Community (Generalitat of Catalonia) and EU Directive EEC 63/2010, and was approved by the Ethics Committee on Animal Experiments of the University of Lleida and the Ethics Commission in Animal Experimentation of the Generalitat de Catalunya\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAKNOWELDGEMENTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also want to thank Dr. Akihiko Yoshimura (Keio University School of Medicine) for providing us Smad2\u003csup\u003efl/fl\u003c/sup\u003e and Dr Martin M Matzuk (Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, USA) for providing us SMAD3\u003csup\u003efl/fl\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData Availability Statement: The datasets analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eARRIVE STATEMENT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were performed in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBissell, M. 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EMT: 2016. \u003cem\u003eCell\u003c/em\u003e \u0026lt;bvertical-align:super;\u0026gt;166\u0026lt;/bvertical-align:super;\u0026gt;, 21\u0026ndash;45 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, J. \u003cem\u003eet al.\u003c/em\u003e Guidelines and definitions for research on epithelial-mesenchymal transition. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 341\u0026ndash;352 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Apoptosis, EMT, endometrial cancer, TGF-β, extracellular matrix, cell polarity","lastPublishedDoi":"10.21203/rs.3.rs-1465554/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1465554/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF- β. It is well known that TGF-β is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-β remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-β-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-β-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-β-induced apoptosis. On the other hand, we demonstrate that TGF-β-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-β to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF- β in normal versus tumoral cells.\u003c/p\u003e","manuscriptTitle":"Lack of extracellular matrix switches TGF-β-induced apoptosis of endometrial cells to Epithelial-to-Mesenchymal transition through AKT, ERK and SMAD2/3-depdendent mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-25 22:10:17","doi":"10.21203/rs.3.rs-1465554/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-04-18T17:14:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-04-11T05:03:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74e6b6d4-bd87-4840-98af-ed56969d85b6","date":"2022-04-07T23:18:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-04-07T22:49:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-07T10:11:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-03-23T12:25:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-03-23T12:19:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-03-18T12:04:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f43e195d-6156-4470-bee0-a1d2a61da592","owner":[],"postedDate":"March 25th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-08-23T05:14:19+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-25 22:10:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1465554","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1465554","identity":"rs-1465554","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}