Oncogene-Induced Senescence Is a Crucial Antitumor Defense Mechanism of Human Endometrial Stromal Cells

Being the major cellular component of highly dynamic tissue, endometrial stromal cells (EnSCs) are exposed to cycles of proliferation upon hormonal stimulation, which might pose risks for the accumulation of mutations and malignization. However, endometrial stromal tumors are rare and uncommon. The present study uncovered defense mechanisms that might underlie the resistance of EnSCs against oncogenic transformation. All experiments were performed in vitro using the following methods: FACS, WB, RT-PCR, IF, molecular cloning, lentiviral transduction, and CRISPR/Cas9 genome editing. We revealed that the expression of the mutant HRASG12V leads to EnSC senescence. We experimentally confirmed the inability of HRASG12V-expressing EnSCs to bypass senescence and resume proliferation, even upon estrogen stimulation. At the molecular level, the induction of oncogene-induced senescence (OIS) was accompanied by activation of the MEK/ERK, PI3K/AKT, p53/p21WAF/CIP/Rb, and p38/p16INK4a/Rb pathways; however, inhibiting either pathway did not prevent cell cycle arrest. PTEN loss was established as an additional feature of HRASG12V-induced senescence in EnSCs. Using CRISPR-Cas9-mediated PTEN knockout, we identified PTEN loss-induced senescence as a reserve molecular mechanism to prevent the transformation of HRASG12V-expressing EnSCs. The present study highlights oncogene-induced senescence as an antitumor defense mechanism of EnSCs controlled by multiple backup molecular pathways.


Introduction
The endometrium is the inner lining of the uterus, which is essential for human reproduction.During a women's life, this unique tissue adapts to multiple physiological states, including premenarche, menstrual cycling, pregnancy, and postmenopause [1,2].Among these states, menstrual cycling is almost continuous during the fertile period, with the exception of pregnancy and lactation.Menstrual cycles are regulated by oscillating levels of estrogen and progesterone, which lead to cyclical growth, differentiation/decidualization, shedding, and subsequent regeneration of two-thirds of the endometrium [1].At the histological level, the endometrium is composed of a layer of stromal cells invaginated by epithelial glands and covered by the luminal epithelium [2].The highly dynamic nature of this tissue contributes to the accumulation of somatic mutations in cancer-associated genes, which in turn poses risks for developing cancer in adult women [3,4].Endometrial cancer is one of the most common gynecologic malignancies worldwide [3].Interestingly, despite the fact that both epithelial and stromal cells are affected by cyclic hormonal alterations, endometrial cancer is commonly referred to as endometrial carcinoma, while endometrial stromal tumors seem to be rare and unusual types of tumors [5,6].The latter raises the question regarding the defense mechanisms that might underlie the resistance of endometrial stromal cells (EnSCs) against oncogenic transformation.
Senescence is a well-established tumor-suppressive mechanism [7].At the cellular level, senescence is considered an important intrinsic stress reaction that prevents the propagation of damaged cells via irreversible cell cycle block [7].Different types of senescence are commonly distinguished, including replicative and various stress-induced forms [8].More than 20 years ago, the expression of oncogenes was also shown to trigger senescence [9].Currently, numerous oncogenes such as HRAS G12V , NRAS Q61R , BRAFV 600E are proven to trigger oncogene-induced senescence (OIS) in various types of cells reviewed in [10].OIS shares the same basic features as replicative and stress-induced forms of senescence.These features include irreversible proliferation block, enhanced expression of the inhibitors of cyclin-dependent kinases p21 WAF/CIP and p16 INK4a , DNA damage, increased cell size, elevated levels of intracellular reactive oxygen species, and senescence-associated β-galactosidase (SA-β-gal) activity [10].Plenty of evidence provides strong arguments that OIS serves as the first line of defense against cancer development [11][12][13][14].Indeed, cells exhibiting features of OIS have been identified in early neoplastic and premalignant lesions in various genetically engineered mouse models, as well as in humans [11,12].Interestingly, the further progression of a subset of these lesions to more advanced cancer stages is associated with the loss of senescence features [14].
Previously, we have shown that EnSCs are prone to senescence triggered by various stresses, including oxidative stress, heat shock, radiation, and treatment with genotoxic agents [15][16][17][18].However, the response of EnSCs towards oncogene expression remained uncovered.In the present study, we tested the suggestion that OIS might serve as a defense mechanism for EnSCs against the neoplastic transformation.

Expression of HRAS G12V Results in a Senescence-like Phenotype in EnSCs
Mutations in the RAS gene occur frequently in various human cancers and have been experimentally validated as the drivers of tumor initiation and maintenance.The missense mutation G12V contributes to HRAS oncogenicity by stabilizing this protein in a constitutively active GTP-bound state [3,4].To explore the outcomes of HRAS G12V expression in EnSCs, we utilized the tetracycline-controlled system (Tet-On), which allows the inducible expression of the mutant oncogene HRAS G12V .The system included two lentivectors.
(1) The first one contained the sequence of the mutant oncogene HRAS G12V , preceded by the regulatory Tet-operator (TetO) sequence; (2) the second lentivector contained the coding sequences of the repressor protein TetR and of the fluorescent reporter GFP (Figure 1A).In the absence of tetracycline, TetR binds to the TetO sequence and prevents the expression of HRAS G12V .Upon addition, tetracycline interacts with TetR, which allows the unhindered expression of the oncogene.Using this system, we initially evaluated the proliferation kinetics in HRAS G12V -expressing EnSCs.As shown in Figure 1B, upon tetracycline treatment, EnSCs transduced with the lentivectors for HRAS G12V expression demonstrated a gradual decrease in proliferation rate until its complete loss on day 4 (Figure 1B).Control EnSCs, which carried the same Tet-On lentiviral system but were not treated with tetracycline, preserved normal proliferation rates during the whole observation period (Figure 1B).To validate the acquisition of the senescent phenotype by EnSCs expressing HRAS G12V , we further investigated other hallmarks of senescence.As shown in microphotographs, EnSCs expressing HRAS G12V became flattened and vacuolated (Figure 1C).Moreover, upon the expression of the oncogene, EnSCs gradually increased in size (Figure 1D).Next, we revealed SA-β-gal activity in HRAS G12V -expressing EnSCs (Figure 1E,F).Finally, we observed enhanced expression of the crucial components of the senescence-associated secretory phenotype (SASP), including IL6, MMP2, and STC1, in EnSCs upon HRAS G12V expression (Figure 1G).Together, the set of identified parameters provides clear evidence in favor of senescence induction in EnSCs expressing HRAS G12V .

EnSCs Expressing HRAS G12V Are Unable to Bypass Senescence
The above results indicate that EnSCs enter senescence upon activation of the oncogene HRAS G12V .Although senescence is believed to be an irreversible cell state, there is also evidence indicating that some cells may overcome OIS [14,19].It has been shown that cells that remained in the senescent state for prolonged periods may resume proliferation and develop features of cancer cells [14,19].In accordance with this notion, we observed the emergence of small clones of proliferating cells among the senescent ones on day 13 of tetracycline treatment (Figure 2A).Moreover, around day 40 of tetracycline treatment, small proliferating cells almost completely replaced senescent ones.To verify if these cells indeed escaped from HRAS G12V -induced senescence, we modified our lentivectors and performed additional experiments.The original lentivector containing the HRAS G12V coding sequence also included the puromycin resistance gene sequence.The latter allowed the selection of HRAS G12V -carrying cells on puromycin-containing media; however, this selection may be imperfect.To further control the selection procedure, we cloned the mCherry coding sequence in the same reading frame just before the HRAS G12V sequence.This ensured that both genes were under the same promoter (Figure 2B).Using our modified HRAS G12V -mCherry Tet-On system, we visualized HRAS G12V -expressing EnSCs by mCherry fluorescence.As expected, at day 37 of tetracycline treatment, we observed large and flattened senescent EnSCs, which were mCherry-positive, and colonies of small cells, which did not express mCherry and thus did not carry HRAS G12V (Figure 2B).This result favors the notion that mCherry-negative small cells might remain after puromycin selection.Indeed, an additional round of puromycin treatment led to the significant death of these small cells, which was comparable to that of the control (non-transduced) EnSCs (Figure 2C).Together, these data provide evidence that EnSCs cannot overcome HRAS G12V -induced senescence and suggest a tight molecular regulation of OIS stability in EnSCs.
small proliferating cells almost completely replaced senescent ones.To verify if these cells indeed escaped from HRAS G12V -induced senescence, we modified our lentivectors and performed additional experiments.The original lentivector containing the HRAS G12V coding sequence also included the puromycin resistance gene sequence.The latter allowed the selection of HRAS G12V -carrying cells on puromycin-containing media; however, this selection may be imperfect.To further control the selection procedure, we cloned the mCherry coding sequence in the same reading frame just before the HRAS G12V sequence.This ensured that both genes were under the same promoter (Figure 2B).Using our modified HRAS G12V -mCherry Tet-On system, we visualized HRAS G12V -expressing EnSCs by mCherry fluorescence.As expected, at day 37 of tetracycline treatment, we observed large and flattened senescent EnSCs, which were mCherry-positive, and colonies of small cells, which did not express mCherry and thus did not carry HRAS G12V (Figure 2B).This result favors the notion that mCherry-negative small cells might remain after puromycin selection.Indeed, an additional round of puromycin treatment led to the significant death of these small cells, which was comparable to that of the control (non-transduced) EnSCs (Figure 2C).Together, these data provide evidence that EnSCs cannot overcome HRAS G12V -induced senescence and suggest a tight molecular regulation of OIS stability in EnSCs.

Estrogen Supplementation Does Not Prevent Proliferation Loss in HRAS G12V -Expressing EnSCs
Due to their functional role, EnSCs are exposed to the cyclic influence of estrogen, which stimulates proliferation of these cells crucial for endometrial regrowth during each menstrual cycle [2].At the same time, there is literary evidence indicating that estrogen might reduce the senescence of various cell types, including endothelial progenitor cells, chondrocytes, and human mesenchymal stem cells [20][21][22].Therefore, we assessed the effects of estrogen on the main characteristics of HRAS G12V -expressing EnSCs.To this end, we supplemented tetracycline-containing growth media with β-Estradiol.Importantly, β-Estradiol had no effect on either proliferation or the size and SA-β-gal activity of the HRAS G12V -expressing EnSCs (Figure 3).These results provide additional confirmation of the stability of the senescence reaction of EnSCs upon the oncogene expression.
chondrocytes, and human mesenchymal stem cells [20][21][22].Therefore, we assessed the effects of estrogen on the main characteristics of HRAS G12V -expressing EnSCs.To this end, we supplemented tetracycline-containing growth media with β-Estradiol.Importantly, β-Estradiol had no effect on either proliferation or the size and SA-β-gal activity of the HRAS G12V -expressing EnSCs (Figure 3).These results provide additional confirmation of the stability of the senescence reaction of EnSCs upon the oncogene expression.

Molecular Mechanisms Regulating HRASG12V-Induced Senescence in EnSCs
The main cellular outcomes of RAS activation are proliferation and survival, mediated via the RAS/RAF/MEK/ERK and RAS/PI3K/AKT pathways [23].In line with that, we observed the phosphorylation of the main downstream targets of RAS-ERK1/2 and AKT-shortly after tetracycline addition (Figure 4A).The latter resulted in a brief period of hyperproliferation of oncogene-expressing EnSCs, as indicated in Figure 4B.
Hyperproliferation caused by oncogenic HRAS G12V drives DNA replication stress and constitutive activation of the DNA damage response (DDR) [24].Indeed, we detected the accumulation of γH2AX foci, which mark DNA damage, in HRAS G12V -expressing EnSCs (Figure 4C).Activation of DDR in EnSCs triggered by the expression of the oncogene led to the phosphorylation of Chk2 and the tumor suppressor protein p53 (Figure 4D).The latter was followed by the enhanced expression of the inhibitor of cyclin-dependent kinases p21 WAF/CIP and the establishment of cell cycle arrest (Figure 4E).Another classical molecular pathway responsible for maintaining cell cycle block during senescence is p38/p16 INK4a /Rb [17].As depicted in Figure 4E, phosphorylation of the stress kinase p38 increased after 8 days of tetracycline treatment, which further led to the elevated expression of p16 INK4a .Thus, we detected activation of two major signaling pathways, p53/p21 WAF/CIP /Rb and p38/p16 INK4a /Rb, that mediate cell cycle block in HRAS G12Vexpressing EnSCs.Activation of both pathways was strictly coordinated; expression of p21 WAF/CIP started just after tetracycline addition and remained elevated for 8 days, while expression of p16 INK4a increased only on day 11 (Figure 4E).The activation dynamics revealed suggest that the p53/p21 WAF/CIP /Rb pathway is responsible for initiating cell cycle block, whereas the p38/p16 INK4a /Rb pathway mediates its stabilization during OIS in EnSCs.

Molecular Mechanisms Regulating HRASG12V-Induced Senescence in EnSCs
The main cellular outcomes of RAS activation are proliferation and survival, mediated via the RAS/RAF/MEK/ERK and RAS/PI3K/AKT pathways [23].In line with that, we observed the phosphorylation of the main downstream targets of RAS-ERK1/2 and AKT-shortly after tetracycline addition (Figure 4A).The latter resulted in a brief period of hyperproliferation of oncogene-expressing EnSCs, as indicated in Figure 4B.Having established the crucial molecular pathways that mediate the initiation and progression of HRAS G12V -induced senescence in EnSCs, we further tested the possibility of reversing or alleviating this reaction.To this end, we utilized specific inhibitors for each pathway, which included (1) U0126, an inhibitor of the kinase activity of MEK1/2, which prevents the activation of ERK; (2) LY294002, a blocker of PI3K-dependent AKT phosphorylation and kinase activity; (3) SB203580, an inhibitor of the catalytic activity of p38 that binds to its ATP-binding pocket; (4) pifithrin-α, an inhibitor of p53 activity (Figure 5A).The compounds were used at concentrations that were found to be efficient and nontoxic in our previous studies [15,[25][26][27].As shown in Figure 5A,B, U0126 and LY294002 prevented cell size increase and SA-β-Gal staining of HRAS G12V -expressing EnSCs, which demonstrates the involvement of both pathways in the acquisition of the senescent phenotype during OIS.At the same time, none of the applied compounds could prevent the loss of proliferation induced by the oncogene expression (Figure 5C).Of note, we previously revealed that cell cycle arrest in stress-induced senescent EnSCs could be overcome by p38 inhibition [15].These results suggest that cell cycle arrest in HRAS G12Vexpressing EnSCs is probably controlled by several backup pathways that prevent cell cycle reentry and transformation.

PTEN Loss-Induced Senescence Is a Backup Molecular Mechanism to Prevent the Transformation of HRAS G12V -Expressing EnSCs
Previously, it was shown that the oncogenic HRAS downregulates the expression of the tumor suppressor PTEN, which negatively regulates cell survival by opposing the activation of the PI3K/AKT/mTOR signaling network [23].The expression of the mutant HRAS G12V in EnSCs resulted in decreased mRNA and protein expression of the crucial tumor suppressor PTEN (Figure 6A,B).As shown in Figure 6A,B, both PTEN mRNA and protein levels gradually declined from day 2 to a minimal level on day 6 of tetracycline treatment.To determine the functional role of PTEN downregulation during the development of HRAS G12V -induced senescence of EnSCs, we conducted PTEN knockout experiment.As shown in Figure 6C,D, application of the CRISPR/Cas9 genome editing system resulted in a significant reduction in PTEN gene expression and almost completely abolished its expression at the protein level.We next assessed the main characteristics of En-SCs with PTEN loss.Notably, EnSCs with PTEN knockout demonstrated increased cell size and autofluorescence levels, indicating hypertrophy and lipofucine accumulation, along with complete proliferation loss (Figure 6E-G).Moreover, we detected enhanced SA-β-gal staining of EnSCs with PTEN loss (Figure 6H,I).Together, these data indicate that the loss of PTEN is sufficient to trigger senescence in EnSCs.Thus, the loss PTEN

PTEN Loss-Induced Senescence Is a Backup Molecular Mechanism to Prevent the Transformation of HRAS G12V -Expressing EnSCs
Previously, it was shown that the oncogenic HRAS downregulates the expression of the tumor suppressor PTEN, which negatively regulates cell survival by opposing the activation of the PI3K/AKT/mTOR signaling network [23].The expression of the mutant HRAS G12V in EnSCs resulted in decreased mRNA and protein expression of the crucial tumor suppressor PTEN (Figure 6A,B).As shown in Figure 6A,B, both PTEN mRNA and protein levels gradually declined from day 2 to a minimal level on day 6 of tetracycline treatment.To determine the functional role of PTEN downregulation during the development of HRAS G12V -induced senescence of EnSCs, we conducted PTEN knockout experiment.As shown in Figure 6C,D, application of the CRISPR/Cas9 genome editing system resulted in a significant reduction in PTEN gene expression and almost completely abolished its expression at the protein level.We next assessed the main characteristics of EnSCs with PTEN loss.Notably, EnSCs with PTEN knockout demonstrated increased cell size and autofluorescence levels, indicating hypertrophy and lipofucine accumulation, along with complete proliferation loss (Figure 6E-G).Moreover, we detected enhanced SA-β-gal staining of EnSCs with PTEN loss (Figure 6H,I).Together, these data indicate that the loss of PTEN is sufficient to trigger senescence in EnSCs.Thus, the loss PTEN observed in HRAS G12V -expressing EnSCs might serve as an additional backup mechanism to induce senescence and thus avoid cell transformation.

Discussion
The present study uncovered the defense mechanisms of EnSCs against transformation.We revealed that EnSCs are prone to OIS in response to HRAS G12V oncogene expression.The first evidence of HRAS-induced senescence dates back to 1997, when the authors observed stalled mitotic activity and an enlarged and flattened morphology of human diploid fibroblasts expressing mutant HRAS G12V [9].Later on, this observation was significantly extended, and today, OIS resulting from the expression of different oncogenes is considered an intrinsic antitumor mechanism common to various types of cells [10].Similar to other cell types, upon HRAS G12V expression, EnSCs acquire all features typical for senescent cells, including proliferation block, altered morphology, and SA-β-Gal activity.Commonly, the loss of proliferation and the development of the senescent phenotype are considered as consequences of DDR resulting from hyper-replication of genomic DNA induced by the oncogene expression [24].In line with this notion, we observed a brief period of hyperproliferation of HRAS G12V -expressing EnSCs followed by proliferation arrest and the emergence of DNA damage.
Despite the concrete mechanism of DNA damage caused by oncogene expression, DDR further leads to cell cycle arrest through the activation of the p53/p21 WAF/CIP /Rb and/or p38/p16 INK4a /Rb pathways [17].Although senescence as the outcome of oncogene expression may appear to be independent of cell type, the molecular mechanisms that establish cell cycle arrest during OIS largely depend on the specific cellular context [28].For example, the expression of p53 is crucial for the development of HRAS G12V -induced senescence in normal human fibroblasts because its depletion prevents proliferation arrest in cells expressing the oncogene [9,24,29].In contrast, normal human mammary epithelial cells and esophageal keratinocytes undergo HRAS G12V -induced senescence in a p53-independent manner [30,31].The same controversy holds true for the involvement of p16 INK4a in HRAS G12V -induced senescence.While the progression of OIS in fibroblasts relies on the expression of p16 INK4a , the depletion of p16 INK4a in HRAS G12V -expressing normal human melanocytes had no effect on senescence progression [32,33].Notably, a recent study revealed that the expression of oncogenic HRAS G12V , along with p16 INK4a knockdown, in EnSCs induced high-grade endometrial stromal sarcoma in mice [34].Here, we detected activation of both signaling pathways, p53/p21 WAF/CIP /Rb and p38/p16 INK4a /Rb, in HRAS G12V -expressing EnSCs.However, neither the inhibition of p53 nor the downregulation of p38/p16 INK4a affected OIS progression in EnSCs.Moreover, inhibition of ERK and AKT, which are downstream targets of the major signaling pathways directly activated by RAS, reduced the senescent phenotype but also did not affect the cell cycle block in oncogene-expressing EnSCs.These data suggest the existence of compensatory pathways that regulate cell cycle arrest during OIS in EnSCs.
Further analysis of the molecular consequences of HRAS G12V expression in EnSCs revealed the gradual loss of PTEN expression.Earlier, it was shown that oncogenic RAS downregulates the expression of the proapoptotic tumor suppressor PTEN in fibroblasts and epithelial cells through a p53-independent pathway [23].The authors of the above study experimentally verified that oncogenic RAS may suppress PTEN expression via the RAF/MEK/ERK/c-Jun pathway, leading to cellular transformation.However, in the case of HRAS G12V -expressing EnSCs, decreased expression of PTEN did not lead to cellular transformation.By performing an additional set of experiments, we uncovered that the loss of PTEN alone is sufficient to trigger senescence in EnSCs.This result is consistent with the existing literature, which reveals the tumor suppressor loss-induced form of senescence induced by the reduced expression of the PTEN gene [35,36].Furthermore, a recent study uncovered the crucial role of the interplay between p53 and PTEN in cell fate decision, including cell cycle arrest, senescence, autophagy, and apoptosis [37].It should be highlighted that previous studies considered both HRAS G12V -and PTEN-loss-induced forms of senescence separately.However, the present study provides the first evidence of the possible intersection between these forms of senescence.Together, the obtained results allow for the speculation that PTEN loss might serve as a backup mechanism that additionally controls proliferation arrest during HRAS G12V -induced senescence of EnSCs.
Despite the tight molecular control of proliferation arrest during OIS, a growing body of evidence demonstrates that cells might escape from OIS through cell-autonomous and cell-non-autonomous mechanisms, including derepression of the hTERT locus, reorganization of topologically associated domains, stemness-associated reprogramming, and downregulation of histone demethylases [14,19].For example, a previous study revealed that the population of human diploid fibroblasts could spontaneously escape from OIS induced by HRAS G12V [19].The authors demonstrated preserved proliferation and the decreased expression of p16 INK4a in OIS-escaped fibroblasts.Similar to these findings, we detected the emergence of small proliferating EnSCs, which eventually replaced senescent EnSCs from the population.However, we experimentally verified that these small cells originated from imperfect lentiviral transduction and subsequent antibiotic selection.Probably, the results of the above study might also be influenced by the technical batches of the transduction procedure, as OIS-escaped fibroblasts were susceptible to stress-induced senescence and unable to grow in soft agar [19].According to our data, EnSCs that expressed HRAS G12V remained senescent for prolonged periods and could not overcome OIS and resume proliferation.
Another possibility to escape from OIS that should be taken into account might result from the physiological estrogen-mediated regulation of EnSC proliferation within the endometrium.This possibility is reinforced by the fact that estrogen controls telomerase activity and hTERT expression in various estrogen-targeted tissues, including the endometrium [38].Indeed, estrogen deficiency leads to telomere shortening, whereas estrogen hyperstimulation increases the telomerase activity and maintains telomere length [39].Moreover, estrogen supplementation has been found to be effective in reducing the senescence of various types of cells [20][21][22].Notably, HRAS G12V -expressing EnSCs remained stably arrested and preserved all the features of senescent cells despite the addition of estrogen.

EnSC Culture Conditions
The EnSC line used in this study was obtained from the shared research facility "Vertebrate cell culture collection" at the Institute of Cytology of the Russian Academy of Sciences (supported by the Ministry of Science and Higher Education of the Russian Federation, Agreement №075-15-2021-683).Cells were characterized in our previous study [40].Cells were cultured in DMEM/F12 (Gibco BRL, Grand Island, NY, USA) at 37 • C in a humidified incubator containing 5% CO 2 .The cultural medium was supplemented with 10% FBS (HyClone, Logan, UT, USA), 1% penicillin-streptomycin (Gibco BRL, NY, USA), and 1% glutamax (Gibco BRL, NY, USA).Serial passaging was performed when the cells reached 80-90% confluence.Cells at early passages (5-9) were used in all experiments.

Molecular Cloning
Four-steps molecular cloning was performed to insert the sequence encoding the mCherry fluorescent protein into pLenti CMV/TO RasV12 Puro (w119-1) (#22262, Addgene, Watertown, NY, USA).Firstly, the mCherry sequence, along with the P2A sequence, were amplified from the pUltra-hot lentivector (#24130, Addgene, Watertown, NY, USA) using the specific primers listed in Table 1.Secondly, the HRAS G12V sequence was amplified from the pLenti CMV/TO RasV12 Puro (w119-1) using the specific primers listed in Table 1.Of note, the mCherry reverse primer and the HRAS G12V forward primer contained complementary sites for further overlap.The obtained sequences were then combined by amplification using two primers-mCherry forward and HRAS G12V reverse (annealing temperature 67 • C, 2 min elongation).The final product was inserted into pLenti CMV/TO RasV12 Puro (w119-1) through restriction using XbaI (New England Biolabs, Ipswich, MA, USA) and BamHI (New England Biolabs, Ipswich, MA, USA), followed by ligation using Quick Ligation™ Kit (New England Biolabs, Ipswich, MA, USA).The obtained lentivector was named HRAS-mCherry pLenti CMV/TO RasV12 Puro.

Flow Cytometry
Measurements of proliferation, cell size, and autofluorescence (lipofuscin accumulation) were carried out by flow cytometry.Flow cytometry was performed using the CytoFLEX (Beckman Coulter, Brea, CA, USA) and the obtained data were analyzed using CytExpert software version 2.0 (Beckman Coulter, Brea, CA, USA).Adherent cells were rinsed twice with PBS and harvested by trypsinization.Detached cells were pooled, resuspended in fresh medium, and stained with 0.1 µg/mL 4 ,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA).DAPI-negative (living) cells were then counted and analyzed for autofluorescence and forward light scattering (reflecting cell size).

SA-β-Gal Staining
SA-β-gal staining was performed using the senescence β-galactosidase staining kit (Cell Signaling, Danvers, MA, USA) according to the manufacturer's instructions.Quantitative analysis of images was produced with the application of MatLab package.For each experimental point, no less than 50 randomly selected cells were analyzed.

Immunofluorescence
Cells grown on coverslips were fixed with 4% formaldehyde (15 min), permeabilized with 0.1% Triton X-100 (10 min) and blocked with 1% bovine serum albumin (1 h).Cells were incubated with primary anti-γH2AX antibodies (Abcam, Cambridge, USA) overnight at 4 • C, followed by incubation with secondary antibodies-Alexa Fluor 488 goat antimouse (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature.The slides were counterstained with 1 µg/mL DAPI, mounted using 2% propyl gallate, and analyzed using Zeiss LSM Pascal 5 laser scanning microscope (Carl Zeiss, Oberkochen, Germany).ZOE Fluorescent Cell Imager (Bio-Rad Laboratories, Hercules, CA, USA) was used to acquire images of live cells expressing fluorescent reporter proteins.4.9.RNA Extraction, Reverse Transcription, and Real-Time PCR RNA extraction, reverse transcription, and real-time PCR were performed as described in our previous study [17].Reagents for RNA extraction (ExtractRNA reagent), for reverse transcription (MMLV RT kit) and for real time PCR (HS SYBR kit) were obtained from Evrogen, Moscow, Russia.Gene expression levels were assessed using the Realtime PCR BioRad CFX-96 amplifier (Bio-Rad Laboratories, Hercules, CA, USA).The following analysis of the obtained data was performed using the Bio-Rad CFX Manager software (Bio-Rad Laboratories, Hercules, CA, USA).Primer sequences and the corresponding annealing temperatures are listed in Table 2.

Statistical Analysis
To obtain significance in the difference between two groups, Welch's t-test or Wilcoxon test were applied.For comparisons among multiple groups, ANOVA or Kruskel-Wallis tests were used.Statistical analysis was performed using GraphPad Prism version 8.0.5.

Conclusions
To sum up, in the present study, we discovered that senescence is the primary and consistent response of EnSCs to the oncogene expression.The activation of MEK/ERK, PI3K/AKT, p53/p21 WAF/CIP /Rb, and p38/p16 INK4a /Rb signaling cascades governed the initiation and development of OIS in EnSCs.The inhibition of either pathway, as well as estrogen supplementation, did not prevent cell cycle arrest in HRAS G12V -expressing cells.Finally, we revealed that PTEN loss-induced senescence may serve as a possible reserve molecular mechanism to prevent transformation of HRAS G12V -expressing EnSCs.The results obtained suggest that OIS is the defense mechanism underlying the resistance of EnSCs against oncogenic transformation.The limitations of this study include the use of a single EnSCs line and a single model of OIS.Further investigations of the reactions of EnSCs to oncogenic signals should involve replicating the experiments using additional EnSCs lines and different models of OIS.Nevertheless, our findings, at least in part, may explain the rare incidence of endometrial stromal tumors.
Importantly, further precise investigation is required to understand EnSCs senescence in relation to the initiation and progression of endometrial epithelial cancer.A growing body of evidence highlights that senescent stromal cells within the tumor microenvironment are capable of interacting with tumor cells.Though paracrine action, senescent cells might be involved in tumor initiation and progression reviewed in [41].In line with this speculation, a recent study revealed a significant correlation between stromal p16 INK4a expression and endometrial carcinomas, rather than with benign and preneoplastic lesions [42].The authors concluded that stromal expression of p16 INK4a is involved in the development and progression of endometrial carcinoma.Bearing in mind that increased expression of p16 INK4a is the most common marker of senescent cells in vivo, it can be logically assumed that senescent EnSCs might have a protumorigenic influence on the epithelial component of the endometrium.However, this assumption remains to be elucidated in future studies.

Figure 2 .
Figure 2. Identification of colonies of small proliferating cells that appear during long-term culturing of HRAS G12V -expressing EnSCs as an artifact of imperfect puromycin selection.(A,B) Schemes of the original and modified two-component Tet-On systems for inducible expression of HRAS G12V under tetracycline (TET) stimulation.Modification resulted in the expression of the fluorescent protein mCherry under the same promoter as HRAS G12V .Microphotographs show the absence of mCherry fluorescence in colonies of small cells (indicated with yellow arrows) that appear during long-term culturing of HRAS G12V -expressing EnSCs (indicated with white arrows).Scale bars on the microphotographs are 100 µm.(C) Relative viability of long-term clonal culture of HRAS G12V -ex-

Figure 2 .
Figure 2. Identification of colonies of small proliferating cells that appear during long-term culturing of HRAS G12V -expressing EnSCs as an artifact of imperfect puromycin selection.(A,B) Schemes of the original and modified two-component Tet-On systems for inducible expression of HRAS G12V under tetracycline (TET) stimulation.Modification resulted in the expression of the fluorescent protein mCherry under the same promoter as HRAS G12V .Microphotographs show the absence of mCherry fluorescence in colonies of small cells (indicated with yellow arrows) that appear during long-term culturing of HRAS G12V -expressing EnSCs (indicated with white arrows).Scale bars on the microphotographs are 100 µm.(C) Relative viability of long-term clonal culture of HRAS G12Vexpressing (TET+) EnSCs and control non-transduced EnSC under puromycin selection.Data are presented as mean ± SD, n = 3, ns p > 0.05 by two-way ANOVA (p-value corresponds to the interaction "Day":"Type of cells").N = 3 means triplicate culture in parallel.

Figure 3 .
Figure 3. Estrogen supplementation had no effect on the characteristics of HRAS G12V -expressing En-SCs.Similar to HRAS G12V -expressing cells (TET+), HRAS G12V -expressing EnSCs treated with estrogen (TET + E2) were characterized by increased size (A), enhanced SA-β-Gal activity (B), and proliferation loss (C).Untreated control cells are marked as TET−.(A) Data are presented as mean ± SD, n = 3 (means triplicate culture in parallel), ns p ≥ 0.05 by one-way ANOVA with Tukey's HSD.(B) Data are presented as median ± 1.5IQR, n = 50; ns p ≥ 0.05 by Kruskal-Wallis test with pairwise comparisons by Wilcoxon test and corrections for multiple testing by FDR.(C) Data are presented as mean ± SD, n = 3 (means triplicate culture in parallel), ns p ≥ 0.05 by two-way ANOVA with Tukey's HSD.

Figure 3 .
Figure 3. Estrogen supplementation had no effect on the characteristics of HRAS G12V -expressing EnSCs.Similar to HRAS G12V -expressing cells (TET+), HRAS G12V -expressing EnSCs treated with estrogen (TET + E2) were characterized by increased size (A), enhanced SA-β-Gal activity (B), and proliferation loss (C).Untreated control cells are marked as TET−.(A) Data are presented as mean ± SD, n = 3 (means triplicate culture in parallel), ns p ≥ 0.05 by one-way ANOVA with Tukey's HSD.(B) Data are presented as median ± 1.5IQR, n = 50; ns p ≥ 0.05 by Kruskal-Wallis test with pairwise comparisons by Wilcoxon test and corrections for multiple testing by FDR.(C) Data are presented as mean ± SD, n = 3 (means triplicate culture in parallel), ns p ≥ 0.05 by two-way ANOVA with Tukey's HSD.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 9 of 17 observed in HRAS G12V -expressing EnSCs might serve as an additional backup mechanism to induce senescence and thus avoid cell transformation.