Endometrial Cancer Stem Cells Research Articles

Characteristics of Cancer Stem Cells and Their Potential Role in Endometrial Cancer.

Endometrial cancer is one of most common types of gynaecological tumours in developing countries. It has been suggested that cancer stem cells play an important role in the development of endometrial cancer. These are a subset of highly tumorigenic cells with similar features to normal stem cells (unlimited proliferation, multi-potential differentiation, self-renewal, aggressiveness, invasion, recurrence, and chemo- and endocrine therapy resistance). Wnt/β-catenin, Hedghog, and Notch1 are the most frequently activated pathways in endometrial cancer stem cells. The presence of cancer stem cells is associated with the resistance to chemotherapy caused by different mechanisms. Various markers, including CD24, CD40, CD44, CD9, CD133, and CD 166, have been identified on the surface of these cells. A higher expression of such markers translates into enhanced tumorigenicity. However, there is no strong evidence showing that any of these identified markers can be used as the universal marker for endometrial cancer stem cells. Growing data from genomic and proteomic profiling shed some light on the understanding of the molecular basis of cancers in humans and the role of cancer stem cells. However, there is much left to discover. Therefore, more studies are needed to fully uncover their functional mechanisms in order to prevent the development and recurrence of cancer, as well as to enhance treatment effectiveness.

ESMO 2023 Abstract Highlights

The following highlights spotlight selected abstracts presented at the European Society for Medical Oncology (ESMO) Annual Congress 2023. They cover key topics, including the importance of nutrients in lung cancer survival rates; a new, minimally invasive therapy which targets endometrial cancer stem cells; and new potential for identifying bladder cancer using genomic sequencing.

Endometrial Cancer Stem Cells Related Signaling Pathways

Abstract: Endometrial cancer is gynecologic cancer that occurs in the uterus. Endometrial cancer stem cells (ECSC) are a small population of cancer cells that represent a crucial role in the metastasis of endometrial cancer cells to other organs in the body. ECSC can proliferate and give rise to mature cancer cells, which are found to participate in the aggressiveness of metastatic lesions. Therefore, targeting ECSC can be a valuable strategy for drug development against the metastasis of endometrial cancer. Previous studies have demonstrated that several signaling pathways, including Wnt, mTOR, EGFR, NOTCH, STAT3, VEGF, and SHH show modest effects and regulate the growth, epithelial-to-mesenchymal transition (EMT), and tumorigenesis of ECSC. Non-coding RNAs (ncRNAs) also play an important role in ECSC self-renewal, progression, and drug resistance. Hence, targeting these pathways might be a novel therapeutic approach for endometrial cancer diagnosis and therapy. This mini-review aims to characterize the main signaling pathways involved in the stimulation of ECSCs proliferation and tumorigenesis.

43P Looking for therapeutic targets on the proteome profile of endometrial cancer stem cells

43P Looking for therapeutic targets on the proteome profile of endometrial cancer stem cells

42P Developing a photodynamic therapy strategy targeted to endometrial cancer stem cells

42P Developing a photodynamic therapy strategy targeted to endometrial cancer stem cells

PVT1/miR-136/Sox2/UPF1 axis regulates the malignant phenotypes of endometrial cancer stem cells

Tumor stem cells (TSCs) are thought to contribute to the progression and maintenance of cancer. Previous studies have suggested that plasmacytoma variant translocation 1 (PVT1) has a tumor-promoting effect on endometrial cancer; however, its mechanism of action in endometrial cancer stem cells (ECSCs) is unknown. Here, we found that PVT1 was highly expressed in endometrial cancers and ECSCs, correlated with poor patient prognosis, promoted the malignant behavior and the stemness of endometrial cancer cells (ECCs) and ECSCs. In contrast, miR-136, which was lowly expressed in endometrial cancer and ECSCs, had the opposite effect, and knockdown miR-136 inhibited the anticancer effects of down-regulated PVT1. PVT1 affected miR-136 specifically binding the 3’ UTR region of Sox2 by competitively “sponging” miR-136, thus positively saving Sox2. Sox2 promoted the malignant behavior and the stemness of ECCs and ECSCs, and overexpression Sox2 inhibited the anticancer effects of up-regulated miR-136. Sox2 can act as a transcription factor to positively regulate Up-frameshift protein 1 (UPF1) expression, thereby exerting a tumor-promoting effect on endometrial cancer. In nude mice, simultaneously downregulating PVT1 and upregulating miR-136 exerted the strongest antitumor effect. We demonstrate that the PVT1/miR-136/Sox2/UPF1 axis plays an important role in the progression and maintenance of endometrial cancer. The results suggest a novel target for endometrial cancer therapies.

Knockdown of the stem cell marker Musashi-1 inhibits endometrial cancer growth and sensitizes cells to radiation

BackgroundEndometrial carcinoma is the most common gynecological cancer in Europe. Musashi-1 is known to be a key regulator of endometrial cancer stem cells and a negative prognostic marker. In the present study, we aimed to understand growth and gene expression patterns in endometrial carcinoma after Musashi-1 knockdown in vitro and in vivo. Changes in therapeutic resistance were also assessed.MethodsFirst, we performed analyses to understand Musashi-1 expression patterns using The Cancer Genome Atlas database. We then proceeded to assess effects of small interfering RNA-based Musashi-1 targeting in two endometrial carcinoma cell lines, Ishikawa and KLE. After quantifying baseline changes in cell metabolism, we used MTT tests to assess chemotherapy effects and colony formation assays to understand changes in radioresistance. For mechanistic study, we used quantitative polymerase chain reaction (qPCR) and western blotting of key Musashi-1 target genes and compared results to primary tissue database studies. Finally, xenograft experiments in a mouse model helped understand in vivo effects of Musashi-1 knockdown.ResultsMusashi-1 is aberrantly expressed in primary tumor tissues. In vitro, silencing of Musashi-1 resulted in a strong decline in cell proliferation and radioresistance, while chemoresistance remained unchanged. Loss of Musashi-1 led to downregulation of telomerase, DNA-dependent protein kinase, the Notch pathway and overexpression of cyclin-dependent kinase inhibitor p21, the latter of which we identified as a key mediator of Msi-1 knockdown-related anti-proliferative signaling. In vivo, the anti-proliferative effect was confirmed, with Msi-1 knockdown tumors being about 40% reduced in size.ConclusionsMusashi-1 knockdown resulted in a strong decrease in endometrial cancer proliferation and a loss of radioresistance, suggesting therapeutic potential.

Endometrial Cancer Stem Cells: Where Do We Stand and Where Should We Go?

Endometrial cancer is one of the most common malignant diseases in women worldwide, with an incidence of 5.9%. Thus, it is the most frequent cancer of the female genital tract, with more than 34,000 women dying, in Europe and North America alone. Endometrial Cancer Stem Cells (CSC) might be drivers of carcinogenesis as well as metastatic and recurrent disease. Therefore, targeting CSCs is of high interest to improve prognosis of patients suffering of advanced or recurrent endometrial cancer. This review describes the current evidence of molecular mechanisms in endometrial CSCs with special emphasis on MYC and NF-κB signaling as well as mitochondrial metabolism. Furthermore, the current status of immunotherapy targeting PD-1 and PD-L1 in endometrial cancer cells and CSCs is elucidated. The outlined findings encourage novel therapies that target signaling pathways in endometrial CSCs as well as immunotherapy as a promising therapeutic approach in the treatment of endometrial cancer to impede cancer progression and prevent recurrence.

UPF1 contributes to the maintenance of endometrial cancer stem cell phenotype by stabilizing LINC00963

Endometrial cancer stem cells (ECSCs) play a vital role in endometrial cancer (EC) metastasis, relapse, and chemoresistance. However, the molecular mechanisms that sustain ECSCs remain elusive. Here, we showed that the expression of UPF1 was upregulated in EC tissues and ECSCs and correlated with poor clinicopathological characteristics. UPF1 silencing suppressed ECSC hallmarks, such as sphere formation ability, carboplatin resistance, migration and invasion, and cell cycle progression. UPF1 regulated the behavior and fate of ECSCs by stabilizing LINC00963. LINC00963 further shares the same miRNA response element with the core transcription factor SOX2 and relieved the suppression of SOX2 by miR-508-5p in self-renewing ECSCs. Notably, inhibition of UPF1 and LINC00963 in combination severely impaired the in vivo tumorigenic potential of ECSCs. We demonstrate that the UPF1/LINC00963/miR-508-5p/SOX2 axis has potential value in modulating ECSC maintenance, chemoresistance, and tumorigenesis in EC, which highlights a novel promising target for EC treatment.

LncRNA MONC suppresses the malignant phenotype of Endometrial Cancer Stem Cells and Endometrial Carcinoma Cells by regulating the MiR-636/GLCE axis

BackgroundEmerging evidence shows that abnormal expression of long non-coding RNA is involved in the occurrence and development of various tumors. LncRNA MONC is abnormally expressed in head and neck squamous cell carcinoma, lung cancer, colorectal cancer, and acute megakaryocytic leukemia, but the biological function and potential regulatory mechanism of MONC in endometrial cancer stem cells (ECSCs) and endometrial cancer cells (ECCs) have not been studied. In this study, we aimed to explore the tumor suppressive effect and mechanism of MONC in regulating ECSCs and ECCs.MethodsWe used qRT-PCR to detect the expression of MONC, miR-636 and GLCE in normal human endometrial tissues and endometrial carcinoma (EC) tissues. Luciferase assay was used to verify the binding sites between MONC and miR-636 and between miR-636 and GLCE. Double fluorescence in situ hybridization was used to locate MONC and miR-636 in cells. ECSCs were obtained by flow cytometry sorting assay. Sphere formation assay, CCK-8 assay, transwell invasion assay, cell cycle analysis and apoptosis assay were used to detect the effects of MONC/miR-636/GLCE axis on the malignant biological behavior of ECSCs and ECCs. The effect of MONC on the epithelial-to-mesenchymal transition (EMT) process was detected using western blot. Finally, we conducted in vivo verification through Tumor xenografts in BALB/C nude mice.ResultsIn this study, we found MONC is low expression in endometrial carcinoma (EC) and patients in the MONC high-expression group had a better prognosis. MONC and miR-636 are relatively co-localized in the cytoplasm. MONC directly inhibits the malignant biological behavior of ECSCs and ECCs by directly inhibiting miR-636. Simultaneously, miR-636 may indirectly reduce the expression of MONC. Down-regulation of miR-636 may promote GLCE expression by targeting the 3′-untranslated region (UTR) of the downstream gene GLCE, thereby inhibiting the progression of ECSCs. MONC combined with miR-636 inhibited tumor epithelial-to-mesenchymal transition (EMT) process. In addition, we verified the tumor suppressive effect of MONC in nude mice, miR-636 can rescue the tumor suppressive effect of overexpressing MONC.ConclusionsIn conclusion, this study showed that MONC inhibits the malignant phenotypes of ECSCs and ECCs by regulating the miR-636/GLCE axis. Thus the MONC/miR-636/GLCE axis may provide novel treatment avenues for human EC.

YBX2 and cancer testis antigen 45 contribute to stemness, chemoresistance and a high degree of malignancy in human endometrial cancer

Y-box binding protein 2 (YBX2) has been associated with the properties of both germ cells and cancer cells. We hypothesized that YBX2 might contribute to the characteristics of cancer stem cells (CSCs). In this study, we clarified the function of YBX2 in endometrial cancer stem cells. We established a human YBX2-expressing Ishikawa (IK) cell line (IK-YBX2 cells). We analyzed gene expression associated with stemness and isolated SP cells from IK-YBX2 cells. The SP population of IK-YBX2 cells, the expression of ALDH1 and serial sphere-forming capacity were associated with levels of YBX2 expression. IK-YBX2 cells were resistant to anti-cancer drugs. In gene expression analysis, a gene for cancer testis antigen, CT45, was generally overexpressed in IK-YBX2 cells. YBX2-mediated CT45 expression was associated with increased levels of self-renewal capacity and paclitaxel resistance. The level of CT45 expression was enhanced in high-grade and/or advanced stages of human endometrial cancer tissues. We conclude that expression of YBX2 is essential for the stem cell-like phenotype. CT45 contributes to stemness associated with YBX2 and might be related to the progression of endometrial cancer.

Hypoxia induces an endometrial cancer stem-like cell phenotype via HIF-dependent demethylation of SOX2 mRNA

Endometrial cancer stem cells (ECSCs) are stem-like cells endowed with self-renewal and differentiation abilities, and these cells are essential for cancer progression in endometrial cancer (EC). As hallmarks of the tumour microenvironment (TME), hypoxia and hypoxia-inducing factors (HIFs) give rise to the dysregulation of tumour stemness genes, such as SOX2. Against this backdrop, we investigated the regulatory mechanisms regulated by HIFs and SOX2 in ECSCs during EC development. Here, ECSCs isolated from EC cell lines and tissues were found to express stemness genes (CD133 and aldehyde dehydrogenase, ALDH1) following the induction of their ECSC expansion. Notably, m6A methylation of RNA and HIF-1α/2α-dependent AlkB homologue 5 (ALKBH5) participate in the regulation of HIFs and SOX2 in EC, as confirmed by the observations that mRNA levels of m6A demethylases and ALKBH5 significantly increase under hypoxic conditions in ECSCs. Moreover, hypoxia and high ALKBH5 levels restore the stem-like state of differentiated ECSCs and increase the ECSC-like phenotype, whereas the knockdown of HIFs or ALKBH5 significantly reduces their tumour initiation capacity. In addition, our findings validate the role of ALKBH5 in promoting SOX2 transcription via mRNA demethylation, thereby maintaining the stem-like state and tumorigenicity potential of ECSCs. In conclusion, these observations demonstrate a critical role for m6A methylation-mediated regulation of the HIF-ALKBH5-SOX2 axis during ECSC expansion in hypoxic TMEs.

Endometrial Stem/Progenitor Cells

To summarize endometrial stem/progenitor cell biology and endometrial cancer stem cells. The subject is relatively new that only about 15 years ago, colony-forming units, as putative endometrial stem/progenitor cells, have been demonstrated in an endometrial sample. Earlier and some recent studies have justifiably focused on endometriosis as the serious gynecologic problem of women. However, on the side of endometrial stem cells research, many current studies are still working on in vitro methods, and the majority of the rest are on animal models. Reported cells as endometrial stem/progenitor cell populations have been heterogeneous such as endometrial mesenchymal stem cells, endometrial epithelial progenitor cells, and side population cells. After the recognition of putative endometrial stem cells, quite a few stem cell markers have been identified. Thereafter, cancer stem cell speculations have raised; in endometrial cancer, CD133+ and CXCR4+ cells, with potential cancer stem cell characteristics, have been shown to have higher antineoplastic resistance than that of other cell populations. Endometrial mesenchymal stem cells have plasticity characteristics and may differentiate into mature cells other than endometrium. On the other hand, bone marrow-derived stem cells can differentiate into endometrial cells. Since the endometrial stem/progenitor cells are easy to access, they can be an attractive source for regenerative medicine studies. Even though many markers have been identified for endometrial stem cells, more specific and sensitive marker combinations are yet to be defined for endometrial cancer stem cell in order to aim for more effective treatment approaches.

Endometrial Cancer Stem Cells: Role, Characterization and Therapeutic Implications.

Endometrial cancer (EC) is the most frequent gynecological cancer. In patients with relapsed and advanced disease, prognosis is still dismal and development of resistance is common. In this context, endometrial Cancer Stem Cells (eCSC), stem-like cells capable to self-renewal and differentiation in mature cancer cells, represent a potential field of expansion for drug development. The aim of this review is to characterize the role of eCSC in EC, their features and how they could be targeted. CSC are involved in progression, invasiveness and metastasis (though epithelial to mesenchimal transition, EMT), as well as chemoresistance in EC. Nevertheless, isolation of eCSC is still controversial. Indeed, CD133, Aldheyde dehydrogenase (ALDH), CD117, CD55 and CD44 are enriched in CSCs but there is no universal marker nowadays. The most frequently activated pathways in eCSC are Wingless-INT (Wnt)/β-catenin, Notch1, and Hedghog, with a high expression of self-renewal transcription factors like Octamer binding transcription factor 4 (OCT), B Lymphoma Mo-MLV Insertion Region 1 Homolog (BMI1), North American Network Operations Group Homebox protein (NANOG), and SRY-Box 2 (SOX2). These pathways have been targeted with selective drugs alone or in combination with chemotherapy and immunotherapy. Unfortunately, although preclinical results are encouraging, few clinical data are available.

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

SummaryUterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

Targeting Endometrial Cancer Stem Cell Activity with Metformin Is Inhibited by Patient-Derived Adipocyte-Secreted Factors.

Advanced endometrial cancer continues to have a poor prognosis, due to limited treatment options, which may be further adversely impacted by obesity. Endometrial cancer stem cells have been reported to drive metastasis, chemotherapy resistance and disease relapse, but have yet to be fully characterised and no specific targeted therapies have been identified. Here, we describe the phenotype and genotype of aldehyde dehydrogenase high (ALDHhigh) and CD133+ve endometrial cancer stem cells and how adipocyte secreted mediators block the inhibitory effect of metformin on endometrial cancer stem cell activity. Ishikawa and Hec-1a cell lines were used to characterise ALDHhigh and CD133+ve endometrial cancer cells using flow cytometry, functional sphere assays and quantitative-Polymerase Chain Reaction. The comparative effect of metformin on endometrial cancer stem cell activity and bulk tumour cell proliferation was determined using an Aldefluor and cytotoxicity assay. The impact of adipocyte secreted mediators on metformin response was established using patient-derived conditioned media. ALDHhigh cells demonstrated greater endometrial cancer stem cell activity than CD133+ve cells and had increased expression of stem cell and epithelial-mesenchymal transition genes. Treatment with 0.5–1 mM metformin reduced the proportion and activity of both endometrial cancer stem cell populations (p ≤ 0.05), without affecting cell viability. This effect was, however, inhibited by exposure to patient-derived adipocyte conditioned media. These results indicate a selective and specific effect of metformin on endometrial cancer stem cell activity, which is blocked by adipocyte secreted mediators. Future studies of metformin as an adjuvant therapy in endometrial cancer should be adequately powered to investigate the influence of body mass on treatment response.

Targeting cancer stem cell signature gene SMOC-2 Overcomes chemoresistance and inhibits cell proliferation of endometrial carcinoma.

BackgroundEndometrial cancer is one of the most common gynecological malignancies and has exhibited an increasing incidence rate in recent years. Cancer stem cells (CSCs), which are responsible for tumor growth and chemoresistance, have been confirmed in endometrial cancer. However, it is still challenging to identify endometrial cancer stem cells to then target for therapy.MethodsFlow cytometry was used to identify the endometrial cancer stem cells. Sphere formation assay, western blotting, qRT-PCR assay, cell viability assay, xenograft assay and immunohistochemistry staining analysis were utilized to evaluate the effect of SPARC-related modular calcium binding 2 (SMOC-2) on the cells proliferation and drug resistance. Cell viability assay, qRT-PCR assay, immunofluorescence staining, Co-IP assay and luciferase reporter gene assay were performed to explore the possible molecular mechanism by which SMOC-2 activates WNT/β-catenin pathway.FindingsWe found the expression of SPARC-related modular calcium binding 2 (SMOC-2), a member of SPARC family, was higher in endometrial CSCs than that in non-CSCs. SMOC-2 was also more highly expressed in spheres than in monolayer cultures. The silencing of SMOC-2 suppressed cell sphere ability; reduced the expression of the stemness-associated genes SOX2, OCT4 and NANOG; and enhanced chemosensitivity in endometrial cancer cells. By co-culture IP assay, we demonstrated that SMOC-2 directly interacted with WNT receptors (Fzd6 and LRP6), enhanced ligand-receptor interaction with canonical WNT ligands (Wnt3a and Wnt10b), and finally, activated the WNT/β-catenin pathway in endometrial cancer. SMOC-2 expression was closely correlated with CSC markers CD133 and CD44 expression in endometrial cancer tissue.InterpretationTaken together, we conclude that SMOC-2 might be a novel endometrial cancer stem cell signature gene and therapeutic target for endometrial cancer.FundNational Natural Science Foundation of China, Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, Scientific and Technological Innovation Act Program of Fengxian Science and Technology Commission, Natural Science Foundation of Shanghai.

PO-270 Signalling pathways WNT, hedgehog and NOTCH in breast cancer with presence and absence of cancer stem cells

IntroductionEndometrial carcinoma is the most common malignancy of the female genital tract in developed countries. These tumours, usually are early diagnosed and associated to a good prognosis, although 20% of the cases, present an aggressive and invasive behaviour. Cancer stem cells (CSC) are defined as an undifferentiated tumour cell subpopulation involved in tumour initiation, resistance to therapy and metastatic phenotype. Several mechanisms are associated to resistance to therapy, such as pathways of aldehyde dehydrogenase (ALDH), an enzyme related with tumour size, lymphatic nodal invasion, recurrent disease, and poor prognosis. In endometrial cancer, the ALDH1 expression was related to tumorigenesis and chemoresistance. In our previous work, ALDH expression was associated with endometrial CSC phenotype.Material and methodsRL95-2, a human endometrial cancer cell line, was submitted to ALDH inhibitors, diethylaminobenzaldehyde (DEAB) 50–100 µM, all-trans retinoic acid (ATRA) 5–10 µM and JQ1 100–250 nM for 48 hours. The cytotoxicity was evaluated through MTT assay. ALDH expression was evaluated through western blot. Sphere formation assay was performed, cells were cultured in coated plates with poly(2-hydroxyethyl-methacrylate), with serum-free DMEM F12 medium with or without 100 µM DEAB, for 5 days. Every two days, spheres were supplemented with EGF and bFGF at a concentration of 10 ng/mL. To evaluate the sphere projection area, spheres obtained were photographed at a magnification of 400x and analysed with Image J software.Results and discussionsThe metabolic activity in of RL95-2 cells was not influenced by the presence of ALDH inhibitors. Cells submitted to 50 and 100 µM DEAB presented a decrease in ALDH relative expression compared with the control cells, of about 36% and 27%, respectively. Regarding the evaluation of sphere projection area, preliminary results showed that spheres submitted to 100 µM DEAB appears to harbour decreased dimensions than control spheres.ConclusionDEAB was the most promising ALDH inhibitor in endometrial cancer cells. This inhibitor also seems to influence the sphere profile considering spheres dimensions. In further studies, we aim to evaluate its influence in the response to cytostatics.FundingFCT (Portugal) PEst-UID/NEU/04539/2013 and FEDER-COMPETE (FCOMP-01–0124-FEDER-028417 and POCI-01–0145-FEDER-007440), Bolsa Liga Portuguesa Contra o Cancro/CIMAGO, CIMAGO n° 02/2017, Sociedade Portuguesa de Ginecologia/Bayer.

PO-269 Targeting ALDH inhibition in endometrial cancer cells

IntroductionEndometrial carcinoma is the most common malignancy of the female genital tract in developed countries. These tumours, usually are early diagnosed and associated to a good prognosis, although 20% of the cases, present an aggressive and invasive behaviour. Cancer stem cells (CSC) are defined as an undifferentiated tumour cell subpopulation involved in tumour initiation, resistance to therapy and metastatic phenotype. Several mechanisms are associated to resistance to therapy, such as pathways of aldehyde dehydrogenase (ALDH), an enzyme related with tumour size, lymphatic nodal invasion, recurrent disease, and poor prognosis. In endometrial cancer, the ALDH1 expression was related to tumorigenesis and chemoresistance. In our previous work, ALDH expression was associated with endometrial CSC phenotype.Material and methodsRL95-2, a human endometrial cancer cell line, was submitted to ALDH inhibitors, diethylaminobenzaldehyde (DEAB) 50–100 µM, all-trans retinoic acid (ATRA) 5–10 µM and JQ1 100–250 nM for 48 hours. The cytotoxicity was evaluated through MTT assay. ALDH expression was evaluated through western blot. Sphere formation assay was performed, cells were cultured in coated plates with poly(2-hydroxyethyl-methacrylate), with serum-free DMEM F12 medium with or without 100 µM DEAB, for 5 days. Every two days, spheres were supplemented with EGF and bFGF at a concentration of 10 ng/mL. To evaluate the sphere projection area, spheres obtained were photographed at a magnification of 400x and analysed with Image J software.Results and discussionsThe metabolic activity in of RL95-2 cells was not influenced by the presence of ALDH inhibitors. Cells submitted to 50 and 100 µM DEAB presented a decrease in ALDH relative expression compared with the control cells, of about 36% and 27%, respectively. Regarding the evaluation of sphere projection area, preliminary results showed that spheres submitted to 100 µM DEAB appears to harbour decreased dimensions than control spheres.ConclusionDEAB was the most promising ALDH inhibitor in endometrial cancer cells. This inhibitor also seems to influence the sphere profile considering spheres dimensions. In further studies, we aim to evaluate its influence in the response to cytostatics.FundingFCT (Portugal) PEst-UID/NEU/04539/2013 and FEDER-COMPETE (FCOMP-01–0124-FEDER-028417 and POCI-01–0145-FEDER-007440), Bolsa Liga Portuguesa Contra o Cancro/CIMAGO, CIMAGO n° 02/2017, Sociedade Portuguesa de Ginecologia/Bayer.

Nuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells

Radiation therapy has become an important tool in the treatment of cancer patients, but most patients relapse within 5 years. Relapse is due to the presence of cancer stem cells (CSCs), but the molecular mechanism of radioresistance in CSCs remains largely elusive. Here, we found that irradiation-resistant (IR) cells exhibited increased stem cell-like properties together with elevated anchorage-independent growth and metastasis ability. EGFR not only leads to increased acquisition of endometrial cancer stem cell markers in radioresistant sublines but is critical for the cancer stem-cell phenotype and tumorigenicity. Moreover, PKM2 functions as an interacting partner of EGFR, which induces the EMT phenotype and stem cell-like properties in IR cells. Finally, we found that the regulatory function of the EGFR-PKM2 axis is dependent on nuclear EGFR. In sum, our study indicated that EGFR and PKM2 directly interact and bind with each other to regulate the transcription of stemness-related genes and promote the stem-like phenotype, thus promoting invasion and metastasis.