Development and characterization of human fetal female reproductive tract organoids to understand Müllerian duct anomalies.

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.

Bovine and human endometrium-derived hydrogels support organoid culture from healthy and cancerous tissues.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth-Holm-Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting high-throughput, cell culture-based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Establishment and characterization of oviductal organoids from farm and companion animals†.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.

Mass Spectrometry Proteomic Analysis of Four p53 Patterns in Differentiated Vulvar Intraepithelial Neoplasia.

OBJECTIVE: The histopathologic diagnostic criteria of differentiated vulvar intraepithelial neoplasia (dVIN), the precursor of human papillomavirus-independent squamous cell carcinoma, are basal atypia, a negative or non-block-positive p16, and a supportive p53 immunohistochemistry (IHC). Several different patterns of supportive p53 IHC have been described. This study aims to determine the relationship between p53 IHC patterns and mass spectrometry analysis of cellular proteins in dVIN. METHODS: Four patterns of p53 IHC were studied: overexpression, cytoplasmic, wild type, and intermediate expression between wild type and overexpression. For each pattern, tissue samples of 4 examples were subjected to mass spectrometry. RESULTS: The protein profile within each p53 IHC pattern shared common features. Each of the 4 p53 patterns had a distinguishable protein profile when compared with the other 3 patterns. CONCLUSIONS: The distinguishable protein profiles in different p53 IHC patterns suggest diverse mechanisms of TP53 dysfunction. Subtyping dVIN by p53 IHC is worthy of further study because varied protein expression profiles may translate into different clinical behavior.

In vivo genetic cell lineage tracing reveals that oviductal secretory cells self-renew and give rise to ciliated cells.

The epithelial lining of the fallopian tube is vital for fertility, providing nutrition to gametes and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions primarily consist of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing fallopian tube epithelial homoeostasis are unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and various Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple-transgenic mouse models, we showed that Wnt/ß-catenin signaling is essential for self-renewal as well as the differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

Specific deletion of LKB1/Stk11 in the Müllerian duct mesenchyme drives hyperplasia of the periurethral stroma and tumorigenesis in male mice.

Nearly all older men will experience lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH), the etiology of which is not well understood. We have generated Stk11CKO mice by conditional deletion of the liver kinase B1 (LKB1) tumor suppressor gene, Stk11 (serine threonine kinase 11), in the fetal Müllerian duct mesenchyme (MDM), the caudal remnant of which is thought to be assimilated by the urogenital sinus primordial mesenchyme in males during fetal development. We show that MDM cells contribute to the postnatal stromal cells at the dorsal aspect of the prostatic urethra by lineage tracing. The Stk11CKO mice develop prostatic hyperplasia with bladder outlet obstruction, most likely because of stromal expansion. The stromal areas from prostates of Stk11CKO mice, with or without significant expansion, were estrogen receptor positive, which is consistent with both MD mesenchyme-derived cells and the purported importance of estrogen receptors in BPH development and/or progression. In some cases, stromal hyperplasia was admixed with epithelial metaplasia, sometimes with keratin pearls, consistent with squamous cell carcinomas. Mice with conditional deletion of both Stk11 and Pten developed similar features as the Stk11CKO mice, but at a highly accelerated rate, often within the first few months after birth. Western blot analyses showed that the loss of LKB1 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) induces activation of the phospho-5' adenosine monophosphate-activated protein kinase and phospho-AKT serine/threonine kinase 1 signaling pathways, as well as increased total and active ß-catenin. These results suggest that activation of these signaling pathways can induce hyperplasia of the MD stroma, which could play a significant role in the etiology of human BPH.

Oestrogen fuels the growth of endometrial hyperplastic lesions initiated by overactive Wnt/ß-catenin signalling.

Unopposed oestrogen is responsible for approximately 80% of all the endometrial cancers. The relationship between unopposed oestrogen and endometrial cancer was indicated by the increase in the number of endometrial cancer cases due to the widespread use of oestrogen replacement therapy. Approximately 30% of the endometrial cancer patients have mutations in the Wnt signalling pathway. How the unbalanced ratios of ovarian hormones and the mutations in Wnt signalling pathway interact to cause endometrial cancer is currently unclear. To study this, we have developed a uterine epithelial cell-specific inducible cre mouse model and used 3D in vitro culture of human endometrial cancer cell lines. We showed that activating mutations in the Wnt signalling pathway for a prolonged period leads to endometrial hyperplasia but not endometrial cancer. Interestingly, unopposed oestrogen and activating mutations in Wnt signalling together drive the progression of endometrial hyperplasia to endometrial cancer. We have provided evidence that progesterone can be used as a targeted therapy against endometrial cancer cases presented with the activating mutations in Wnt signalling pathway.

Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus.

In mice, implantation always occurs towards the antimesometrial side of the uterus, while the placenta develops at the mesometrial side. What determines this particular orientation of the implanting blastocyst remains unclear. Uterine glands are critical for implantation and pregnancy. In this study, we showed that uterine gland development and active Wnt signaling activity is limited to the antimesometrial side of the uterus. Dkk2, a known antagonist of Wnt signaling, is only present at the mesometrial side of the uterus. Imaging of whole uterus, thick uterine sections (100-1000µm), and individual glands revealed that uterine glands are simple tubes with branches that are directly connected to the luminal epithelium and are only present towards the antimesometrial side of the uterus. By developing a unique mouse model targeting the uterine epithelium, we demonstrated that Wnt/ß-catenin signaling is essential for prepubertal gland formation and normal implantation, but dispensable for postpartum gland development and regeneration. Our results for the first time have provided a probable explanation for the antimesometrial bias for implantation.

Extracellular matrix (ECM) activates ß-catenin signaling in uterine fibroids.

Recent studies showed that genetic aberrations in the MED12 gene, probably through the canonical WNT/ß-catenin pathway, lead to the pathogenesis of uterine fibroids. However, a comprehensive analysis of the WNT pathway in MED12-mutated and MED12-wild-type fibroids has not been performed. The objective of this study was to determine the status of the WNT pathway in human fibroids. We performed Sanger sequencing to define the MED12 mutational status of fibroids and normal myometrium samples. qPCR arrays were carried out to determine the status of the WNT signaling pathway in MED12-mutated and MED12-wild-type fibroids. Liquid chromatography-mass spectrometry (LC-MS), Western blotting and immunohistochemistry were used to monitor the expression of ß-catenin. We showed that ß-catenin expression was increased in fibroids compared to the adjacent myometrium samples. However, ß-catenin expression showed no correlation with MED12 mutation status. Of all the WNT signaling components, WNT inhibitors showed the greatest differences in expression between fibroids and controls. WIF1, a WNT inhibitor, was identified as the most significantly upregulated gene in fibroids. We cultured primary fibroid cells on hydrogels of known stiffness to decipher the influence of biomechanical cues on ß-catenin expression and revealed increased levels of ß-catenin when cells were cultured on a stiffer surface. In conclusion, our data showed that ß-catenin expression in fibroids occurs independently of MED12 mutations. Biomechanical changes upregulate ß-catenin expression in fibroids, providing an attractive avenue for developing new treatments for this disease.

Epithelial Wnt/ßcatenin signalling is essential for epididymal coiling.

Organ shape and size are important determinants of their physiological functions. Epithelial tubes are anlagen of many complex organs. How these tubes acquire their complex shape and size is a fundamental question in biology. In male mice, the Wolffian duct (WD; postnatally known as epididymis) undergoes an astonishing transformation, where a straight tube only a few millimetres long elongates to over 1000 times its original length and fits into a very small space, due to extensive coiling of epithelium, to perform the highly specialized function of sperm maturation. Defective coiling disrupts sperm maturation and leads to male infertility. Recent work has shown that epithelial cell proliferation is a major driver of WD coiling. Still, very little is known about the molecular signals involved in this process. Testicular androgens are known regulators of WD development. However, epithelial androgen receptor signalling is dispensable for WD coiling. In this study, we have shown that Wnt signalling is highly active in the entire WD epithelium during its coiling, and is limited to only a few segments of the epididymis in later life. Pharmacological and genetic suppression of Wnt signalling inhibited WD coiling by decreasing cell proliferation and promoting apoptosis. Comparative gene expression analysis identified Fibroblast growth factor 7 (Fgf7) as a prime Wnt target gene involved in WD coiling and in vitro treatment with Fgf7 protein increased coiling of WDs. In summary, our work has established that epithelial canonical Wnt signalling is a critical regulator of WD coiling and its precise regulation is essential for WD/epididymal differentiation.

Stromal liver kinase B1 [STK11] signaling loss induces oviductal adenomas and endometrial cancer by activating mammalian Target of Rapamycin Complex 1.

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.

Loss of LKB1 and PTEN tumor suppressor genes in the ovarian surface epithelium induces papillary serous ovarian cancer.

Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.

Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis.

Male patients with Peutz-Jeghers syndrome (PJS) have defective spermatogenesis and are at increased risk of developing Sertoli cell tumors. Mutations in the Liver Kinase B1 (LKB1/STK11) gene are associated with the pathogenesis of PJS and have been identified in non-PJS patients with sporadic testicular cancers. The mechanisms controlled by LKB1 signaling in Sertoli cell functions and testicular biology have not been described. We have conditionally deleted the Lkb1 gene (Lkb1(cko)) in somatic testicular cells to define the molecular mechanisms involved in the development of the testicular phenotype observed in PJS patients. Focal vacuolization in some of the seminiferous tubules was observed in 4-week-old mutant testes but germ cell development appeared to be normal. However, similar to PJS patients, we observed progressive germ cell loss and Sertoli cell only tubules in Lkb1(cko) testes from mice older than 10 weeks, accompanied by defects in Sertoli cell polarity and testicular junctional complexes and decreased activation of the MAP/microtubule affinity regulating and focal adhesion kinases. Suppression of AMP kinase and activation of mammalian target of rapamycin (mTOR) signaling were also observed in Lkb1(cko) testes. Loss of Tsc1 or Tsc2 copies the progressive Lkb1(cko) phenotype, suggesting that dysregulated activation of mTOR contributes to the pathogenesis of the Lkb1(cko) testicular phenotype. Pten(cko) mice had a normal testicular phenotype, which could be explained by the comparative lack of mTOR activation detected. These studies describe the importance of LKB1 signaling in testicular biology and the possible molecular mechanisms driving the pathogenesis of the testicular defects observed in PJS patients.

Interferon-epsilon is a novel regulator of NK cell responses in the uterus.

The uterus is a unique mucosal site where immune responses are balanced to be permissive of a fetus, yet protective against infections. Regulation of natural killer (NK) cell responses in the uterus during infection is critical, yet no studies have identified uterine-specific factors that control NK cell responses in this immune-privileged site. We show that the constitutive expression of IFNε in the uterus plays a crucial role in promoting the accumulation, activation, and IFNγ production of NK cells in uterine tissue during Chlamydia infection. Uterine epithelial IFNε primes NK cell responses indirectly by increasing IL-15 production by local immune cells and directly by promoting the accumulation of a pre-pro-like NK cell progenitor population and activation of NK cells in the uterus. These findings demonstrate the unique features of this uterine-specific type I IFN and the mechanisms that underpin its major role in orchestrating innate immune cell protection against uterine infection.

PTEN loss and HOXA10 expression are associated with ovarian endometrioid adenocarcinoma differentiation and progression.

Epithelial ovarian cancer is a heterogeneous disease that is subdivided into five major histotypes but the mechanisms driving their differentiation are not clear. Mutations in adenomatous polyposis coli (APC) and ß-catenin are commonly observed in the human ovarian endometrioid adenocarcinoma (OEA) patients. However, the mechanisms subsequent to APC deletion in ovarian tumorigenesis have not been well characterized. We have conditionally deleted APC in the murine ovarian surface epithelium (OSE) and showed that its loss leads to development of epithelial inclusion cysts. High-grade OEAs with tightly packed villoglandular histology were observed in older APC-deleted mice. Phosphatase and tensin homolog (PTEN) expression was elevated in the early lesions but lost after progression to the more advanced tumors. Knockdown of APC or expression of a gain-of-function ß-catenin similarly induced human OSE cells to develop tumors with endometrioid histology in xenografts. Expression of HOXA10 was induced in both the advanced APC-deleted murine tumors and in the tumor xenografts of human OSE cells with knocked-down APC. These results show that reduced APC activity is sufficient to induce formation of epithelial inclusion cysts and support OEA development and suggest that induced HOXA10 expression and loss of PTEN are key mechanisms driving endometrioid histotype differentiation and progression.

Focal Mullerian duct retention in male mice with constitutively activated beta-catenin expression in the Mullerian duct mesenchyme.

Müllerian-inhibiting substance (MIS), which is produced by fetal Sertoli cells shortly after commitment of the bipotential gonads to testicular differentiation, causes Müllerian duct (MD) regression. In the fetal female gonads, MIS is not expressed and the MDs will differentiate into the internal female reproductive tract. We have investigated whether dysregulated ß-catenin activity affects MD regression by expressing a constitutively activated nuclear form of ß-catenin in the MD mesenchyme. We show that constitutively activated (CA) ß-catenin causes focal retention of MD tissue in the epididymides and vasa deferentia. In adult mutant mice, the retained MD tissues express α-smooth muscle actin and desmin, which are markers for uterine differentiation. MD retention inhibited the folding complexity of the developing epididymides and usually led to obstructive azoospermia by spermatoceles. The MDs of urogenital ridges from mutant female embryos showed less regression with added MIS in organ culture compared with control MDs when analyzed by whole mount in situ hybridization for Wnt7a as a marker for the MD epithelium. CA ß-catenin did not appear to affect expression of either MIS in the embryonic testes or its type II receptor (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nuclear accumulation, suggesting that dysregulated ß-catenin must inhibit MD regression independently of MIS signaling. These studies suggest that dysregulated Wnt/ß-catenin signaling in the MD mesenchyme might also be a contributing factor in persistent Müllerian duct syndrome, a form of male pseudohermaphroditism, and development of spermatoceles.

Extracellular matrix in high-grade serous ovarian cancer: Advances in understanding of carcinogenesis and cancer biology.

High-grade serous ovarian cancer (HGSOC) is notoriously known as the "silent killer" of post-menopausal women as it has an insidious progression and is the deadliest gynaecological cancer. Although a dual origin of HGSOC is now widely accepted, there is growing evidence that most cases of HGSOC originate from the fallopian tube epithelium. In this review, we will address the fallopian tube origin and involvement of the extracellular matrix (ECM) in HGSOC development. There is limited research on the role of ECM at the earliest stages of HGSOC carcinogenesis. Here we aim to synthesise current understanding of the contribution of ECM to each stage of HGSOC development and progression, beginning at serous tubal intraepithelial carcinoma (STIC) precursor lesions and proceeding across key events including dissemination of tumourigenic fallopian tube epithelial cells to the ovary, survival of these cells in peritoneal fluid as multicellular aggregates, and colonisation of the ovary. Likewise, as part of the metastatic series of events, serous ovarian cancer cells survive travel in peritoneal fluid, attach to, migrate across the mesothelium and invade into the sub-mesothelial matrix of secondary sites in the peritoneal cavity. Halting cancer at the pre-metastatic stage and finding ways to stop the dissemination of ovarian cancer cells from the primary site is critical for improving patient survival. The development of drug resistance also contributes to poor survival statistics in HGSOC. In this review, we provide an update on the involvement of the ECM in metastasis and drug resistance in HGSOC. Interplay between different cell-types, growth factor gradients as well as evolving ECM composition and organisation, creates microenvironment conditions that promote metastatic progression and drug resistance of ovarian cancer cells. By understanding ECM involvement in the carcinogenesis and chemoresistance of HGSOC, this may prompt ideas for further research for developing new early diagnostic tests and therapeutic strategies for HGSOC with the end goal of improving patient health outcomes.

Loss of liver kinase B1 in human seminoma.

Testicular cancer is a common malignancy of young males and is believed to be originated from defective embryonic or adult germ cells. Liver kinase B1 (LKB1) is a serine/threonine kinase and a tumor suppressor gene. LKB1 is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, often inactivated in many human cancer types. In this study, we investigated the involvement of LKB1 in the pathogenesis of testicular germ cell cancer. We performed immunodetection of LKB1 protein in human seminoma samples. A 3D culture model of human seminoma was developed from TCam-2 cells, and two mTOR inhibitors were tested for their efficacy against these cancer cells. Western blot and mTOR protein arrays were used to show that these inhibitors specifically target the mTOR pathway. Examination of LKB1 showed reduced expression in germ cell neoplasia in situ lesions and seminoma compared to adjacent normal-appearing seminiferous tubules where the expression of this protein was present in the majority of germ cell types. We developed a 3D culture model of seminoma using TCam-2 cells, which also showed reduced levels of LKB1 protein. Treatment of TCam-2 cells in 3D with two well-known mTOR inhibitors resulted in reduced proliferation and survival of TCam-2 cells. Overall, our results support that downregulation or loss of LKB1 marks the early stages of the pathogenesis of seminoma, and the suppression of downstream signaling to LKB1 might be an effective therapeutic strategy against this cancer type.

Prevalence and clinical significance of co-existing mutations in MED12 and FH in uterine fibroids of Australian women.

Uterine fibroids are exceedingly common benign tumours of the female reproductive system and cause severe symptoms, including acute pain, bleeding, and infertility. Fibroids are frequently associated with genetic alterations affecting mediator complex subunit 12 (MED12), fumarate hydratase (FH), high mobility group AT-hook 2 (HMGA2) and collagen, type IV alpha 5 and alpha 6 (COL4A5-COL4A6). Recently, we reported MED12 exon 2 mutations in 39 out of 65 uterine fibroids (60%) from 14 Australian patients. The aim of this study was to evaluate the status of FH mutations in MED12 mutation-positive and mutation-negative uterine fibroids. FH mutation screening of altogether 65 uterine fibroids and corresponding adjacent normal myometrium (n = 14) was carried out by Sanger sequencing. Three out of 14 patients displayed somatic mutations in FH exon 1 in addition to harbouring MED12 mutation in uterine fibroids. This study is the first to report that the mutations in MED12 and FH co-exist in uterine fibroids of Australian women.

The Müllerian inhibiting substance type 2 receptor suppresses tumorigenesis in testes with sustained ß-catenin signaling.

Dysregulated WNT/ß-catenin signaling in murine testes results in a phenotype with complete germ cell loss that resembles human Sertoli cell-only syndrome. In other systems, including the ovary, dysregulated WNT/ß-catenin induces tumorigenesis but no tumors are observed in the mutant testes without deletion of a tumor suppressor, such as phosphatase and tensin homolog (PTEN). Müllerian inhibiting substance (MIS, also known as AMH), a member of the transforming growth factor-ß family of growth factors responsible for Müllerian duct regression in fetal males, has been shown to inhibit tumor growth in vitro and in vivo but its role as an endogenous tumor suppressor has never been reported. We have deleted the MIS type 2 receptor (MISR2), and thus MIS signaling, in mice with dysregulated WNT/ß-catenin and show that these mice develop testicular stromal tumors with 100% penetrance within a few months postnatal. The tumors are highly proliferative and have characteristics of either Sertoli cell tumors or progenitor Leydig cell tumors based on their marker profiles and histology. Phosphorylated Sma and mothers against decapentaplegic-related homolog 1/5/8 is absent in the tumors and ß-catenin target genes are induced. The tumor suppressor TP53 is also highly expressed in the tumors, as is phosphorylated γH2AX, which is indicative of DNA damage. The phenotype of these tumors closely resembles those observed when PTEN is also deleted in mice with dysregulated WNT/ß-catenin. Tumorigenesis in these mice provides conclusive evidence that physiological MIS signaling is a tumor suppressor mechanism and suggests that targeted treatment of MISR2-expressing cancers with therapeutic MIS should have a beneficial effect on tumor progression.