{"paper_id":"25491e10-cee7-497d-9f06-c5b19ec250ce","body_text":"Abstract\nPluripotent, very small embryonic-like stem cells (VSELs) and the ‘progenitors’ endometrial stem cells (EnSCs) along with associated molecular changes in endometrial cancer, that developed seven months after neonatal exposure to estradiol in one of the sixty mice, were studied in the present study. Endocrine disruption affected both endometrium and myometrium, there was accumulation of endometrial fluid and significant hyperplasia. Disrupted endometrial-myometrial junction resulted in mobilization of myometrial cells into endometrium and epithelial and stromal cells into myometrium suggestive of adenomyosis. Markers specific for VSELs/ EnSCs (OCT-4, NANOG, SSEA-1, SCA-1, c-KIT) showed increased expression in uterine sections and marked upregulation of corresponding transcripts (Oct-4A, Oct-4, Sox-2, Nanog, Sca-1, c-Kit) was noted in RNA extracted from both uterine tissue and stem cells enriched from endometrial fluid. Hormonal receptors (ER-α, ER-β, PR, FSHR) were upregulated in both tumor sections and in endometrial fluid. ER-β and FSHR (Fshr3) expression was prominent suggesting a major role in endometrial cancer. Cancer cells showed global hypomethylation (reduced expression of 5-methyl cytosine), reduced expression of tumor suppressor gene (PTEN) and increased expression of cancer stem cells marker (CD166) which suggested dysregulation and aberrant oncogenic events. Increased expression of PCNA, Ki67, SOX-9 suggested excessive proliferation and hyperplasia which are predominant signs of endometrial cancer. Results suggest that VSELs increase in numbers and possibly transform into cancer stem cells (co-express CD166 and OCT-4) in endometrial cancer. Expression of OCT-4, CD133, ALDHA1 and CD166 in side-population cells from human endometrial cancer samples suggests a possible role of VSELs in human endometrial cancer as well.\nGraphical abstract\nSimilar content being viewed by others\nData availability\nAdditional data is provided as supplement.\nCode availability\nNA.\nAbbreviations\n- VSELs:\n-\nVery small embryonic like stem cells\n- EnSCs:\n-\nEndometrial stem progenitor cells\n- OCT-4:\n-\nOctamer-binding transcription factor 4\n- SOX-2:\n-\nSex determining region Y-box 2\n- SSEA-1:\n-\nStage specific embryonic antigen-1\n- SCA-1:\n-\nStem cells antigen-1\n- cKIT:\n-\nReceptor tyrosine kinase\n- CD166 /ALCAM:\n-\nCluster of differentiation166 / Activated Leukocyte Cell Adhesion Molecule\n- ALDHA1:\n-\nAldehyde dehydrogenases\n- PTEN:\n-\nPhosphatase and Tensin Homolog deleted on Chromosome 10\n- 5mC:\n-\n5-Methylcytosine\n- ER:\n-\nEstrogen receptors\n- FSHR:\n-\nFollicle stimulating hormone receptors\n- PCNA:\n-\nProliferating cell nuclear antigen\nReferences\nLu, K. H., & Broaddus, R. R. (2020). Endometrial cancer. New England Journal of Medicine, 383(21), 2053–2064. https://doi.org/10.1056/nejmra1514010\nDoherty, M. T., Sanni, O. B., Coleman, H. G., Cardwell, C. R., Mccluggage, W. G., Quinn, D., Mcmenamin, Ú. C. (2020). Concurrent and future risk of endometrial cancer in women with endometrial hyperplasia: A systematic review and meta-analysis. Plos One, 15(4). https://doi.org/10.1371/journal.pone.0232231\nGhazarian, A. A., & Mcglynn, K. A. (2020). Increasing incidence of testicular germ cell tumors among racial/ethnic minorities in the United States. Cancer Epidemiology Biomarkers & Prevention, 29(6), 1237–1245. https://doi.org/10.1158/1055-9965.epi-20-0107\nKaushik, A., Anand, S., & Bhartiya, D. (2020). Altered biology of testicular VSELs and SSCs by neonatal endocrine disruption results in defective spermatogenesis, reduced fertility and tumor initiation in adult mice. Stem Cell Reviews and Reports, 16(5), 893–908. https://doi.org/10.1007/s12015-020-09996-3\nNewbold, R. R., Jefferson, W. N., & Padilla-Banks, E. (2007). Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract. Reproductive Toxicology, 24(2), 253–258. https://doi.org/10.1016/j.reprotox.2007.07.006\nRatajczak, M. Z., Ratajczak, J., & Kucia, M. (2019). Very small embryonic-like stem cells (VSELs): An update and future directions. Circulation Research, 124, 208–210.\nBhartiya, D., Shaikh, A., Anand, S., Patel, H., Kapoor, S., et al. (2016). Endogenous, very small embryonic-like stem cells: Critical review, therapeutic potential and a look ahead. Human Reproduction Update, 23, 41–76.\nSingh, P., Metkari, S. M., & Bhartiya, D. (2021). Additional evidence to support OCT-4 positive VSELs and EnSCs as the elusive tissue-resident stem/progenitor cells in adult mice uterus. Stem Cell Research & Therapy. https://doi.org/10.1186/s13287-022-02703-8\nSingh, P., & Bhartiya, D. (2020). Pluripotent stem (VSELs) and progenitor (EnSCs) cells exist in adult mouse uterus and show cyclic changes across estrus cycle. Reproductive Sciences, 28(1), 278–290. https://doi.org/10.1007/s43032-020-00250-2\nJames, K., Bhartiya, D., Ganguly, R., Kaushik, A., Gala, K., Singh, P., & Metkari, S. M. (2018). Gonadotropin and steroid hormones regulate pluripotent very small embryonic-like stem cells in adult mouse uterine endometrium. Journal of Ovarian Research, 11(1). https://doi.org/10.1186/s13048-018-0454-4\nBhartiya, D., & James, K. (2017). Very small embryonic-like stem cells (VSELs) in adult mouse uterine perimetrium and myometrium. Journal of Ovarian Research, 10(1). https://doi.org/10.1186/s13048-017-0324-5\nGunjal, P., Bhartiya, D., Metkari, S., Manjramkar, D., & Patel, H. (2015). Very small embryonic-like stem cells are the elusive mouse endometrial stem cells- a pilot study. Journal of Ovarian Research, 8(1). https://doi.org/10.1186/s13048-015-0138-2\nDing, D.-C., Liu, H.-W., Chang, Y.-H., & Chu, T.-Y. (2017). Expression of CD133 in endometrial cancer cells and its implications. Journal of Cancer, 8(11), 2142–2153. https://doi.org/10.7150/jca.18869\nDarvishi, B., Boroumandieh, S., Majidzadeh-A, K., Salehi, M., Jafari, F., & Farahmand, L. (2020). The role of activated leukocyte cell adhesion molecule (ALCAM) in cancer progression, invasion, metastasis and recurrence: A novel cancer stem cell marker and tumor-specific prognostic marker. Experimental and Molecular Pathology, 115, 104443. https://doi.org/10.1016/j.yexmp.2020.104443\nPaczkowska, E., Kawa, M., Klos, P., Staniszewska, M., Sienko, J., & Dabkowska, E. (2011). Aldehyde dehydrogenase (ALDH) – A promising new candidate for use in preclinical and clinical selection of pluripotent very small embryonic-like stem cells (VSEL SCs) of high long-term repopulating hematopoietic potential. Annals of Transplantation, 16(3), 59–71. https://doi.org/10.12659/aot.881996\nSingh, P., Metkari, S. M., & Bhartiya, D. (2021). Mice uterine stem cells are affected by neonatal endocrine disruption & initiate uteropathies in adult life independent of circulatory ovarian hormones. Stem Cell Reviews and Reports. https://doi.org/10.1007/s12015-021-10279-8\nCarpinello, O. J., DeCherney, A. H., & Hill, M. J. (2018). Developmental Origins of health and disease: The history of the barker hypothesis and assisted reproductive technology. Seminars in Reproductive Medicine, 36(3–04), 177–182. https://doi.org/10.1055/s-0038-1675779\nRyznar, R. J., Phibbs, L., & Van Winkle, L. J. (2021). Epigenetic modifications at the center of the Barker Hypothesis and their transgenerational implications. International Journal of Environmental Research and Public Health, 18(23), 12728. https://doi.org/10.3390/ijerph182312728\nRatajczak, M. Z., Bujko, K., Mack, A., Kucia, M., & Ratajczak, J. (2018). Cancer from the perspective of stem cells and misappropriated tissue regeneration mechanisms. Leukemia, 32(12), 2519–2526. https://doi.org/10.1038/s41375-018-0294-7\nRatajczak, MZ., Tarnowski, M., Borkowska, S., & Serwin, K. (2013). The Embryonic Rest Hypothesis of cancer development: 150 years later. Trends in Stem Cell Proliferation and Cancer Research, 51–63. https://doi.org/10.1007/978-94-007-6211-4_3\nJohnson, D. N., Barroeta, J. E., Antic, T., & Lastra, R. R. (2017). Cytomorphologic features of metastatic endometrioid carcinoma by fine needle aspiration. Diagnostic Cytopathology, 46(2), 105–110. https://doi.org/10.1002/dc.23855\nToledo, G., & Oliva, E. (2008). Smooth muscle tumors of the uterus: A practical approach. Archives of Pathology & Laboratory Medicine, 132(4), 595–605. https://doi.org/10.5858/2008-132-595-smtotu\nGonzalez, G., Mehra, S., Wang, Y., Akiyama, H., & Behringer, R. R. (2016). Sox9 overexpression in uterine epithelia induces endometrial gland hyperplasia. Differentiation, 92(4), 204–215. https://doi.org/10.1016/j.diff.2016.05.006\nGuo, F., Levine, L., & Berenson, A. (2021). Trends in the incidence of endometrial cancer among young women in the United States, 2001 to 2017. Journal of Clinical Oncology, 39(15_suppl):5578–5578. https://doi.org/10.1200/jco.2021.39.15_suppl\nBirnbaum, L. S., & Fenton, S. E. (2003). Cancer and developmental exposure to endocrine disruptors. Environmental Health Perspectives, 111(4), 389–394. https://doi.org/10.1289/ehp.5686\nMallozzi, M., Leone, C., Manurita, F., Bellati, F., & Caserta, D. (2017). Endocrine disrupting chemicals and endometrial cancer: An overview of recent laboratory evidence and epidemiological studies. International Journal of Environmental Research and Public Health, 14(3), 334. https://doi.org/10.3390/ijerph14030334\nNewbold, R. R., Bullock, B. C., & Mclachlan, J. A. (1992). Hormone-dependent uterine adenocarcinoma following developmental treatment with diethylstilbestrol: A murine model for hormonal carcinogenesis. Hormonal Carcinogenesis, 309–312,. https://doi.org/10.1007/978-1-4613-9208-8_47\nLi, D., Li, H., Wang, Y., Eldomany, A., Wu, J., Yuan, C., Liu, D. (2018). Development and characterization of a polarized human endometrial cell epithelia in an air–liquid interface state. Stem Cell Research & Therapy, 9(1). https://doi.org/10.1186/s13287-018-0962-6\nLlarena, N. C., Richards, E. G., Priyadarshini, A., Fletcher, D., Bonfield, T., & Flyckt, R. L. (2020). Characterizing the endometrial fluid cytokine profile in women with endometriosis. Journal of Assisted Reproduction and Genetics, 37(12), 2999–3006. https://doi.org/10.1007/s10815-020-01989-y\nRaffone, A., Seracchioli, R., Raimondo, D., Maletta, M., Travaglino, A., Raimondo, I., & Zullo, F. (2020). Prevalence of adenomyosis in endometrial cancer patients: a systematic review and meta-analysis. Archives of Gynecology and Obstetrics, 303(1), 47–53. https://doi.org/10.1007/s00404-020-05840-8\nTerzic, M., Aimagambetova, G., Kunz, J., Bapayeva, G., Aitbayeva, B., Terzic, S., & Laganà, A. S. (2021). Molecular basis of endometriosis and endometrial cancer: Current knowledge and future perspectives. International Journal of Molecular Sciences, 22(17), 9274. https://doi.org/10.3390/ijms22179274\nHenderson, B. E., & Feigelson, H. S. (2000). Hormonal carcinogenesis. Carcinogenesis, 21(3), 427–433. https://doi.org/10.1093/carcin/21.3.427\nSarno, L. (2019). Endometrial carcinoma and Bisphenol A: A pilot case-control study. Biomedical Journal of Scientific & Technical Research, 21(4). https://doi.org/10.26717/bjstr.2019.21.003641\nCarvalho, M. J., Laranjo, M., Abrantes, A. M., Casalta-Lopes, J., Sarmento-Santos, D., Costa, T., & Oliveira, C. (2018). Endometrial cancer spheres show cancer stem cells phenotype and preference for oxidative metabolism. Pathology & Oncology Research, 25(3), 1163–1174. https://doi.org/10.1007/s12253-018-0535-0\nNakamura, M., Kyo, S., Zhang, B., Zhang, X., Mizumoto, Y., Takakura, M., & Inoue, M. (2010). Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer. Human Pathology, 41(11), 1516–1529. https://doi.org/10.1016/j.humpath.2010.05.006\nKato, K. (2012). Endometrial cancer stem cells: a new target for cancer therapy. Anticancer Res, 32(6), 2283–93\nRutella, S., Bonanno, G., Procoli, A., Mariotti, A., Corallo, M., Prisco, M. G., … Ferrandina, G. (2009). Cells with characteristics of cancer stem/progenitor cells express the cd133 antigen in human endometrial tumors. Clinical Cancer Research, 15(13), 4299–4311. https://doi.org/10.1158/1078-0432.ccr-08-1883\nFriel, A. M., Zhang, L., Curley, M. D., Therrien, V. A., Sergent, P. A., Belden, S. E., … Rueda, B. R. (2010). Epigenetic regulation of CD133 and tumorigenicity of CD133 positive and negative endometrial cancer cells. Reproductive Biology and Endocrinology, 8(1). https://doi.org/10.1186/1477-7827-8-147\nRahadiani, N., Ikeda, J.-I., Mamat, S., Matsuzaki, S., Ueda, Y., Umehara, R., & Morii, E. (2011). Expression of aldehyde dehydrogenase 1 (ALDH1) in endometrioid adenocarcinoma and its clinical implications. Cancer Science, 102(4), 903–908. https://doi.org/10.1111/j.1349-7006.2011.01864.x\nCousins, F. L., Pandoy, R., Jin, S., & Gargett, C. E. (2021). The elusive endometrial epithelial stem/progenitor cells. Frontiers in Cell and Developmental Biology, 9. https://doi.org/10.3389/fcell.2021.640319\nSenbanjo, L. T., & Chellaiah, M. A. (2017). CD44: A multifunctional cell surface adhesion receptor is a regulator of progression and metastasis of cancer cells. Frontiers in Cell and Developmental Biology, 5. https://doi.org/10.3389/fcell.2017.00018\nGonzalez G. Role of SOX9 in uterine gland development and disease initiation. The University of Texas MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences Dissertations & Theses. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/254\nRatajczak, M. Z., Zuba-Surma, E. K., Shin, D.-M., Ratajczak, J., & Kucia, M. (2008). Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity. Experimental Gerontology, 43(11), 1009–1017. https://doi.org/10.1016/j.exger.2008.06.002\nZuba-Surma, E. K., Klich, I., Greco, N., Laughlin, M. J., Ratajczak, J., & Ratajczak, M. Z. (2010). Optimization of isolation and further characterization of umbilical cord blood-derived very small embryonic/ epiblast-like stem cells (VSELs). European Journal of Haematology, 84(1), 34–46. https://doi.org/10.1111/j.1600-0609.2009.01352.x\nSzukiewicz, D., Stangret, A., Ruiz-Ruiz, C., Olivares, E. G., Soriţău, O., Suşman, S., & Szewczyk, G. (2021). Estrogen- and progesterone (P4)-mediated epigenetic modifications of endometrial stromal cells (EnSCs) and/or mesenchymal stem/stromal cells (MSCs) in the etiopathogenesis of endometriosis. Stem Cell Reviews and Reports, 17(4), 1174–1193. https://doi.org/10.1007/s12015-020-10115-5\nSyed, S. M., Kumar, M., Ghosh, A., Tomasetig, F., Ali, A., Whan, R. M., Tanwar, P. S. (2020). Endometrial Axin2 cells drive epithelial homeostasis, regeneration, and cancer following oncogenic transformation. Cell Stem Cell, 26(1). https://doi.org/10.1016/j.stem.2019.11.012\nSyed, S. M., & Tanwar, P. S. (2020). Axin2 endometrial stem cells: the source of endometrial regeneration and cancer. Molecular & Cellular Oncology, 7(3), 1729681. https://doi.org/10.1080/23723556.2020.1729681\nHapangama, D., Kamal, A., & Bulmer, J. (2014). Estrogen receptor β: the guardian of the endometrium. Human Reproduction Update, 21(2), 174–193. https://doi.org/10.1093/humupd/dmu053\nSong, K., Dai, L., Long, X., Wang, W., & Di, W. (2020). Follicle-stimulating hormone promotes the proliferation of epithelial ovarian cancer cells by activating sphingosine kinase. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-70896-0\nOlejar, T., Vetvicka, D., Boucek, J., Zabrodsky, M., Benes, J., Kabesova, M., & Pouckova, P. (2019). The FSHR expression in head and neck squamous cell cancer. a pilot immunohistochemical study. Anticancer Research, 40(1), 349–356.\nRadu, A., Pichon, C., Camparo, P., Antoine, M., Allory, Y., Couvelard, A., & Ghinea, N. (2011). Expression of follicle-stimulating hormone receptor in tumor blood vessels. Obstetrical & Gynecological Survey, 66(2), 99–101. https://doi.org/10.1097/ogx.0b013e3182168\nChoi, J.-H., Choi, K.-C., Auersperg, N., & Leung, P. C. K. (2004). Overexpression of follicle-stimulating hormone receptor activates oncogenic pathways in preneoplastic ovarian surface epithelial cells. The Journal of Clinical Endocrinology & Metabolism, 89(11), 5508–5516. https://doi.org/10.1210/jc.2004-0044\nStilley, J. A., Christensen, D. E., Dahlem, K. B., Guan, R., Santillan, D. A., England, S. K., Segaloff, D. L. (2014). FSH Receptor (FSHR) Expression in human extragonadal reproductive tissues and the developing placenta, and the impact of its deletion on pregnancy in mice. Biology of Reproduction, 91(3). https://doi.org/10.1095/biolreprod.114.118562\nMarca, A. L., Artenisio, A. C., Stabile, G., Rivasi, F., & Volpe, A. (2005). Evidence for cycle-dependent expression of follicle-stimulating hormone receptor in human endometrium. Gynecological Endocrinology, 21(6), 303–306. https://doi.org/10.1080/09513590500402756\nPonikwicka-Tyszko, D., Chrusciel, M., Stelmaszewska, J., Bernaczyk, P., Sztachelska, M., Sidorkiewicz, I., & Rahman, N. A. (2016). Functional expression of FSH receptor in endometriotic lesions. The Journal of Clinical Endocrinology & Metabolism, 101(7), 2905–2914. https://doi.org/10.1210/jc.2016-1014\nRobin, B., Planeix, F., Sastre-Garau, X., Pichon, C., Olesen, T. K., Gogusev, J., & Ghinea, N. (2015). Follicle-stimulating hormone receptor expression in endometriotic lesions and the associated vasculature. Reproductive Sciences, 23(7), 885–891. https://doi.org/10.1177/1933719115623647\nWang, L., Felix, J. C., Lee, J. L., Tan, P. Y., Tourgeman, D. E., O’Meara, A. T., & Amezcua, C. A. (2003). The proto-oncogene c-kit is expressed in leiomyosarcomas of the uterus. Gynecologic Oncology, 90(2), 402–406. https://doi.org/10.1016/s0090-8258(03)00274-9\nFouquet, B., Santulli, P., Noel, J.-C., & Misrahi, M. (2016). Ovarian-like differentiation in eutopic and ectopic endometrioses with aberrant FSH receptor, INSL3 and GATA4/6 expression. BBA Clinical, 6, 143–152. https://doi.org/10.1016/j.bbacli.2016.11.002\nPacchiarotti, A., Caserta, D., Sbracia, M., & Moscarini, M. (2011). Expression of oct-4 and c-kit antigens in endometriosis. Fertility and Sterility, 95(3), 1171–1173. https://doi.org/10.1016/j.fertnstert.2010.10.029\nBhartiya, D., Patel, H., Kaushik, A., Singh, P., & Sharma, D. (2021). Endogenous, tissue-resident stem/progenitor cells in gonads and bone marrow express FSHR and respond to FSH via FSHR-3. Journal of Ovarian Research, 14(1). https://doi.org/10.1186/s13048-021-00883-0\nBhartiya, D., & Patel, H. (2021). An overview of FSH-FSHR biology and explaining the existing conundrums. Journal of Ovarian Research, 14(1). https://doi.org/10.1186/s13048-021-00880-3\nWang, K.-Y., Chen, C.-C., Tsai, S.-F., & Shen, C.-K. J. (2016). Epigenetic enhancement of the post-replicative DNA mismatch repair of mammalian genomes by a hemi-mCpG-Np95-Dnmt1 Axis. Scientific Reports, 6(1). https://doi.org/10.1038/srep37490\nVirant-Klun, I., Kenda-Suster, N., & Smrkolj, S. (2016). Small putative NANOG, SOX2, and SSEA-4-positive stem cells resembling very small embryonic-like stem cells in sections of ovarian tissue in patients with ovarian cancer. Journal of Ovarian Research, 9(1). https://doi.org/10.1186/s13048-016-0221-3\nAcknowledgements\nHelp from Praveen, Swati and Shobha from NIRRH central facilities is acknowledged. The study was supported by Indian Council of Medical Research, Government of India, New Delhi. PS acknowledges DST-INSPIRE fellowship (IF170144).\nFunding\nThe study was funded by core support provided to the corresponding author by Indian Council of Medical Research, Government of India, New Delhi.\nAuthor information\nAuthors and Affiliations\nContributions\nPS performed all experiments, data interpretation and manuscript preparation. DB designed the study, financial support, results interpretation, manuscript preparation. Both authors agree and approve final version of the article.\nCorresponding author\nEthics declarations\nEthics approval\nThe study was approved by Institute Animal Ethics Committee.\nConsent to participate\nNot applicable.\nConsent for publication\nNIRRH manuscript number is RA/1116/09–2021.\nConflicts of interest/Competing interests\nAuthors declare no conflict of interest whatsoever that could be perceived as prejudicing the impartiality of the research reported.\nAdditional information\nPublisher's Note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nThis article belongs to the Topical Collection: Special Issue on Tissue-resident Stem/Progenitor Cells Endowed with Broader Germ Layer Specification Potential in Normal and Cancerous Tissues\nSupplementary Information\nESM 1 (download PDF )\n(PDF 879 kb)\nRights and permissions\nAbout this article\nCite this article\nSingh, P., Bhartiya, D. Molecular Insights into Endometrial Cancer in Mice. Stem Cell Rev and Rep 18, 1702–1717 (2022). https://doi.org/10.1007/s12015-022-10367-3\nAccepted:\nPublished:\nVersion of record:\nIssue date:\nDOI: https://doi.org/10.1007/s12015-022-10367-3","source_license":"public-domain-us","license_restricted":false}