The TAS1R2 sweet taste receptor regulates skeletal muscle mass and fitness.

Muscle fitness and mass deteriorate under the conditions of obesity and aging for reasons yet to be fully elucidated. Herein, we describe a novel pathway linking peripheral nutrient sensing and skeletal muscle function through the sweet taste receptor TAS1R2 and the involvement of ERK2-PARP1-NAD signaling axis. Muscle-specific deletion of TAS1R2 (mKO) in mice produced elevated NAD levels due to suppressed PARP1 activity, improved mitochondrial function, increased muscle mass and strength, and prolonged running endurance. Deletion of TAS1R2 in obese or aged mice also ameliorated the decline in muscle mass and fitness arising from these conditions. Remarkably, partial loss-of-function of TAS1R2 (rs35874116) in older, obese humans recapitulated the healthier muscle phenotype displayed by mKO mice in response to exercise training. Our findings show that inhibition of the TAS1R2 signaling in skeletal muscle is a promising therapeutic approach to preserve muscle mass and function.

The Ile191Val is a partial loss-of-function variant of the TAS1R2 sweet-taste receptor and is associated with reduced glucose excursions in humans.

OBJECTIVE: Sweet taste receptors (STR) are expressed in the gut and other extra-oral tissues, suggesting that STR-mediated nutrient sensing may contribute to human physiology beyond taste. A common variant (Ile191Val) in the TAS1R2 gene of STR is associated with nutritional and metabolic outcomes independent of changes in taste perception. It is unclear whether this polymorphism directly alters STR function and how it may contribute to metabolic regulation. METHODS: We implemented a combination of in vitro biochemical approaches to decipher the effects of TAS1R2 polymorphism on STR function. Then, as proof-of-concept, we assessed its effects on glucose homeostasis in apparently healthy lean participants. RESULTS: The Ile191Val variant causes a partial loss of function of TAS1R2 through reduced receptor availability in the plasma membrane. Val minor allele carriers have reduced glucose excursions during an OGTT, mirroring effects previously seen in mice with genetic loss of function of TAS1R2. These effects were not due to differences in beta-cell function or insulin sensitivity. CONCLUSIONS: Our pilot studies on a common TAS1R2 polymorphism suggest that STR sensory function in peripheral tissues, such as the intestine, may contribute to the regulation of metabolic control in humans.

High-dose saccharin supplementation does not induce gut microbiota changes or glucose intolerance in healthy humans and mice.

BACKGROUND: Non-caloric artificial sweeteners (NCAS) are widely used as a substitute for dietary sugars to control body weight or glycemia. Paradoxically, some interventional studies in humans and rodents have shown unfavorable changes in glucose homeostasis in response to NCAS consumption. The causative mechanisms are largely unknown, but adverse changes in gut microbiota have been proposed to mediate these effects. These findings have raised concerns about NCAS safety and called into question their broad use, but further physiological and dietary considerations must be first addressed before these results are generalized. We also reasoned that, since NCAS are bona fide ligands for sweet taste receptors (STRs) expressed in the intestine, some metabolic effects associated with NCAS use could be attributed to a common mechanism involving the host. RESULTS: We conducted a double-blind, placebo-controlled, parallel arm study exploring the effects of pure saccharin compound on gut microbiota and glucose tolerance in healthy men and women. Participants were randomized to placebo, saccharin, lactisole (STR inhibitor), or saccharin with lactisole administered in capsules twice daily to achieve the maximum acceptable daily intake for 2 weeks. In parallel, we performed a 10-week study administering pure saccharin at a high dose in the drinking water of chow-fed mice with genetic ablation of STRs (T1R2-KO) and wild-type (WT) littermate controls. In humans and mice, none of the interventions affected glucose or hormonal responses to an oral glucose tolerance test (OGTT) or glucose absorption in mice. Similarly, pure saccharin supplementation did not alter microbial diversity or composition at any taxonomic level in humans and mice alike. No treatment effects were also noted in readouts of microbial activity such as fecal metabolites or short-chain fatty acids (SCFA). However, compared to WT, T1R2-KO mice were protected from age-dependent increases in fecal SCFA and the development of glucose intolerance. CONCLUSIONS: Short-term saccharin consumption at maximum acceptable levels is not sufficient to alter gut microbiota or induce glucose intolerance in apparently healthy humans and mice. TRIAL REGISTRATION: Trial registration number NCT03032640 , registered on January 26, 2017. Video abstract.

SIX1 cooperates with RUNX1 and SMAD4 in cell fate commitment of Müllerian duct epithelium.

During female mammal reproductive tract development, epithelial cells of the lower Müllerian duct are committed to become stratified squamous epithelium of the vagina and ectocervix, when the expression of ΔNp63 transcription factor is induced by mesenchymal cells. The absence of ΔNp63 expression leads to adenosis, the putative precursor of vaginal adenocarcinoma. Our previous studies with genetically engineered mouse models have established that fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), bone morphogenetic protein (BMP)/SMAD, and activin A/runt-related transcription factor 1 (RUNX1) signaling pathways are independently required for ΔNp63 expression in Müllerian duct epithelium (MDE). Here, we report that sine oculis homeobox homolog 1 (SIX1) plays a critical role in the activation of ΔNp63 locus in MDE as a downstream transcription factor of mesenchymal signals. In the developing mouse reproductive tract, SIX1 expression was restricted to MDE within the future cervix and vagina. SIX1 expression was totally absent in SMAD4 null MDE and was reduced in RUNX1 null and FGFR2 null MDE, indicating that SIX1 is under the control of vaginal mesenchymal factors: BMP4, activin A and FGF7/10. Furthermore, Six1, Runx1, and Smad4 gene-dose-dependently activated ΔNp63 expression in MDE within the vaginal fornix. Using a mouse model of diethylstilbestrol (DES)-associated vaginal adenosis, we found DES action through epithelial estrogen receptor α (ESR1) inhibits activation of ΔNp63 locus in MDE by transcriptionally repressing SIX1 and RUNX1 in the vaginal fornix.

Ovarian insufficiency and CTNNB1 mutations drive malignant transformation of endometrial hyperplasia with altered PTEN/PI3K activities.

Endometrioid endometrial carcinomas (EECs) carry multiple driver mutations even when they are low grade. However, the biological significance of these concurrent mutations is unknown. We explored the interactions among three signature EEC mutations: loss-of-function (LOF) mutations in PTEN, gain-of-function (GOF) mutations of phosphoinositide 3-kinase (PI3K), and CTNNB1 exon 3 mutations, utilizing in vivo mutagenesis of the mouse uterine epithelium. While epithelial cells with a monoallelic mutation in any one of three genes failed to propagate in the endometrium, any combination of two or more mutant alleles promoted the growth of epithelium, causing simple hyperplasia, in a dose-dependent manner. Notably, Ctnnb1 exon 3 deletion significantly increased the size of hyperplastic lesions by promoting the growth of PTEN LOF and/or PI3K GOF mutant cells through the activation of neoadenogenesis pathways. Although these three mutations were insufficient to cause EEC in intact female mice, castration triggered malignant transformation, leading to myometrial invasion and serosal metastasis. Treatment of castrated mice with progesterone or estradiol attenuated the neoplastic transformation. This study demonstrates that multiple driver mutations are required for premalignant cells to break the growth-repressing field effect of normal endometrium maintained by ovarian steroids and that CTNNB1 exon 3 mutations play critical roles in the growth of preneoplastic cells within the endometrium of premenopausal women and in the myometrial invasion of EECs in menopausal women.

Transient inhibition of p53 homologs protects ovarian function from two distinct apoptotic pathways triggered by anticancer therapies.

Platinum-based chemotherapies can result in ovarian insufficiency by reducing the ovarian reserve, a reduction believed to result from apoptosis of immature oocytes via activation/phosphorylation of TAp63α by multiple kinases including CHEK2, CK1, and ABL1. Here we demonstrate that cisplatin (CDDP) induces oocyte apoptosis through a novel pathway and that temporary repression of this pathway fully preserves ovarian function in vivo. Although ABL kinase inhibitors effectively block CDDP-induced apoptosis of oocytes, oocytic ABL1, and ABL2 are dispensable for damage-induced apoptosis. Instead, CDDP activates TAp63α through the ATR > CHEK1 pathway independent of TAp63α hyper-phosphorylation, whereas X-irradiation activates the ATM > CHEK2 > TAp63α-hyper-phosphorylation pathway. Furthermore, oocyte-specific deletion of Trp73 partially protects oocytes from CDDP but not from X-ray, highlighting the fundamental differences of two pathways. Nevertheless, temporary repression of DNA damage response by a kinase inhibitor that attenuates phosphorylation of ATM, ATR, CHEK1, and CHEK2 fully preserves fertility in female mice against CDDP as well as X-ray. Our current study establishes the molecular basis and feasibility of adjuvant therapies to protect ovarian function against two distinctive gonadotoxic therapeutics, CDDP, and ionizing radiation.

Patient-derived xenograft model for uterine leiomyoma by sub-renal capsule grafting.

Uterine leiomyoma (UL) or fibroid is a benign smooth muscle tumor of the myometrium with a lifetime incidence of approximately 70%. ULs often require medical intervention due to severe symptoms such as heavy menstrual bleeding and abdominal pain. Although the most common and effective management of ULs is surgical removal, the invasive surgical procedure imposes physical and psychological burdens on the patients. Moreover, the economic burden of UL on health care system is enormous due to the high cost of surgeries. Thus, therapeutic options with long-term efficacy to replace surgical management are urgently needed. For the development of such medical options, reliable preclinical research models are imperative. Ex vivo culture of UL cells has been the primary research model for decades. However, recent studies demonstrated that primary cell culture is not a suitable model for UL research, as primary cultures of ULs mostly consist of non-tumor fibroblasts. Here we describe the protocol for patient-derived xenograft of UL, which faithfully replicates the phenotypes of human UL in situ.

Cellular kinetics of MED12-mutant uterine leiomyoma growth and regression in vivo.

Cellular mechanisms of uterine leiomyoma (LM) formation have been studied primarily utilizing in vitro models. However, recent studies established that the cells growing in the primary cultures of MED12-mutant LM (MED12-LM) do not carry causal mutations. To improve the accuracy of LM research, we addressed the cellular mechanisms of LM growth and regression utilizing a patient-derived xenograft (PDX) model, which faithfully replicates the patient tumors in situ The growth and maintenance of MED12-LMs depend on 17ß-estradiol (E2) and progesterone (P4). We determined E2 and P4-activated MAPK and PI3K pathways in PDXs with upregulation of IGF1 and IGF2, suggesting that the hormone actions on MED12-LM are mediated by the IGF pathway. When hormones were removed, MED12-LM PDXs lost approximately 60% of volume within 3 days through reduction in cell size. However, in contrast to general belief, the survival of LM cells was independent of E2 and/or P4, and apoptosis was not involved in the tumor regression. Furthermore, it was postulated that abnormal collagen fibers promote the growth of LMs. However, collagen fibers of actively growing PDXs were well aligned. The disruption of collagen fibers, as found in human LM specimens, occurred only when the volume of PDXs had grown to over 20 times the volume of unstimulated PDXs, indicating disruption is the result of growth not the cause. Hence, this study revises generally accepted theories on the growth and regression of LMs.

Novel SOX17 frameshift mutations in endometrial cancer are functionally distinct from recurrent missense mutations.

Extensive genomic profiling for endometrioid endometrial carcinoma (EEC) has pointed to genes and pathways important in uterine development as critical mediators of endometrial tumorigenesis. SOX17 is a developmental transcription factor necessary for proper endoderm formation that has been implicated as a tumor suppressor and shown to modulate WNT signaling. SOX17 mutation analysis in 539 primary EECs revealed frequent missense and frameshift mutations with an overall 11.5% mutation rate. More than half the mutations identified were frameshifts (32 of 62), and the hotspot missense changes, p.Ala96Gly and p.Ser403Ile, were seen in 14 tumors. None of the cases with a mutation had a second SOX17 mutation or evidence of allelic loss. Immunofluorescence microscopy performed on primary samples showed that there were no changes in SOX17 protein expression associated with mutation. Low/absent SOX17 staining was significantly associated with advanced stage, high tumor grade and reduced recurrence-free survival. Functional assessment of the two hotspot missense mutations and three representative frameshift mutations showed that SOX17-A96G and SOX17-S403I have transcriptional activities similar to SOX17 wild-type (WT), whereas none of the frameshift mutant proteins showed transcriptional activity. Forced expression of SOX17-WT, -A96G or -S403I in EC cell lines moderately increased ß-catenin mediated transcription, which contrasts with previous data showing SOX17 is an inhibitor of TCF/ß-catenin signaling. The proliferation of EC cell lines was expectedly reduced by transfection with SOX17-WT, and further reduced by SOX17-A96G and SOX17-S403I. These data implicate SOX17 mutation as a selected event in EEC, with clear differences between the missense and frameshift mutations.

Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma.

Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here, we report the elucidation of the biological characteristics of the two most prevalent uterine leiomyoma subtypes, MED12-mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) uterine leiomyomas. Because each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-uterine leiomyoma were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of extracellular matrix in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both uterine leiomyoma subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these uterine leiomyoma subtypes emphasize the importance of subtypes and genotypes in designing nonsurgical therapeutic strategies for uterine leiomyoma. Cancer Res; 77(24); 6891-901. ©2017 AACR.

FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct.

Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate.

Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model.

STUDY QUESTION: Does halofuginone (HF) inhibit the growth of human uterine leiomyoma cells in a mouse xenograft model? SUMMARY ANSWER: HF suppresses the growth of human uterine leiomyoma cells in a mouse xenograft model through inhibiting cell proliferation and inducing apoptosis. WHAT IS KNOWN ALREADY: Uterine leiomyomas are the most common benign tumors of the female reproductive tract. HF can suppress the growth of human uterine leiomyoma cells in vitro. The mouse xenograft model reflects the characteristics of human leiomyomas. STUDY DESIGN, SIZE, DURATION: Primary leiomyoma smooth muscle cells from eight patients were xenografted under the renal capsule of adult, ovariectomized NOD-scid IL2Rγ(null) mice (NSG). Mice were treated with two different doses of HF or vehicle for 4 weeks with six to eight mice per group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse body weight measurements and immunohistochemical analysis of body organs were carried out to assess the safety of HF treatment. Xenografted tumors were measured and analyzed for cellular and molecular changes induced by HF. Ovarian steroid hormone receptors were evaluated for possible modulation by HF. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of mice carrying human UL xenografts with HF at 0.25 or 0.50 mg/kg body weight for 4 weeks resulted in a 35-40% (P < 0.05) reduction in tumor volume. The HF-induced volume reduction was accompanied by increased apoptosis and decreased cell proliferation. In contrast, there was no significant change in the collagen content either at the transcript or protein level between UL xenografts in control and HF groups. HF treatment did not change the expression level of ovarian steroid hormone receptors. No adverse pathological effects were observed in other tissues from mice undergoing treatment at these doses. LIMITATIONS, REASONS FOR CAUTION: While this study did test the effects of HF on human leiomyoma cells in an in vivo model, HF was administered to mice whose tolerance and metabolism of the drug may differ from that in humans. Also, the longer term effects of HF treatment are yet unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study showing the effectiveness of HF in reducing UL tumor growth by interfering with the main cellular processes regulating cell proliferation and apoptosis are in agreement with previous studies on the effects of HF on other fibrotic diseases. HF can be considered as a candidate for reducing the size of leiomyomas, particularly prior to surgery. STUDY FUNDING/COMPETING INTERESTS: This project was funded by NIH PO1HD057877 and R01 HD064402. Authors report no competing interests.

Human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b initiate tumors in vivo.

CONTEXT: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Using a dye-exclusion technique, we previously identified a side population of leiomyoma cells exhibiting stem cell characteristics. However, unless mixed with mature myometrial cells, these leiomyoma side population cells did not survive or grow well in vitro or in vivo. OBJECTIVE: The objective of this study was to identify cell surface markers to isolate leiomyoma stem/progenitor cells. DESIGN: Real-time PCR screening was used to identify cell surface markers preferentially expressed in leiomyoma side population cells. In vitro colony-formation assay and in vivo tumor-regeneration assay were used to demonstrate functions of leiomyoma stem/progenitor cells. RESULTS: We found significantly elevated CD49b and CD34 gene expression in side population cells compared with main population cells. Leiomyoma cells were sorted into three populations based on the expression of CD34 and CD49b: CD34(+)/CD49b(+), CD34(+)/CD49b(-), and CD34(-)/CD49b(-) cells, with the majority of the side population cells residing in the CD34(+)/CD49b(+) fraction. Of these populations, CD34(+)/CD49b(+) cells expressed the lowest levels of estrogen receptor-α, progesterone receptor, and α-smooth muscle actin, but the highest levels of KLF4, NANOG, SOX2, and OCT4, confirming their more undifferentiated status. The stemness of CD34(+)/CD49b(+) cells was also demonstrated by their strongest in vitro colony-formation capacity and in vivo tumor-regeneration ability. CONCLUSIONS: CD34 and CD49b are cell surface markers that can be used to enrich a subpopulation of leiomyoma cells possessing stem/progenitor cell properties; this technique will accelerate efforts to develop new therapies for uterine leiomyoma.

Cell autonomous phosphoinositide 3-kinase activation in oocytes disrupts normal ovarian function through promoting survival and overgrowth of ovarian follicles.

In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation.

Anti-miR182 reduces ovarian cancer burden, invasion, and metastasis: an in vivo study in orthotopic xenografts of nude mice.

High-grade serous ovarian carcinoma (HGSOC) is a fatal disease, and its grave outcome is largely because of widespread metastasis at the time of diagnosis. Current chemotherapies reduce tumor burden, but they do not provide long-term benefits for patients with cancer. The aggressive tumor growth and metastatic behavior characteristic of these tumors demand novel treatment options such as anti-microRNA treatment, which is emerging as a potential modality for cancer therapy. MicroRNA-182 (miR182) overexpression contributes to aggressive ovarian cancer, largely by its negative regulation of multiple tumor suppressor genes involved in tumor growth, invasion, metastasis, and DNA instability. In this study, we examined the therapeutic potential of anti-miR182 utilizing the animal orthotopic model to mimic human ovarian cancer using ovarian cancer cells SKOV3 (intrabursal xenografts) and OVCAR3 (intraperitoneal injection). These models provide a valuable model system for the investigation of ovarian cancer therapy in vivo. Through a combination of imaging, histological, and molecular analyses, we found that anti-miR182 treatment can significantly reduce tumor burden (size), local invasion, and distant metastasis compared with its control in both models. The bases of anti-miR182 treatment are mainly through the restoration of miR182 target expression, including but not limited to BRCA1, FOXO3a, HMGA2, and MTSS1. Overall, our results strongly suggest that anti-miR182 can potentially be used as a therapeutic modality in treating HGSOC.

Down-regulation of miR-29b is essential for pathogenesis of uterine leiomyoma.

Uterine leiomyomata (LMs) are the most common tumor affecting the female reproductive organs. The most notable pathophysiologic feature of this tumor is the excessive accumulation of rigid extracellular matrix (ECM) composed mainly of collagen types I and III. It is believed that the rigidity of the collagen-rich ECM causes symptoms such as abnormal bleeding and pelvic pain/pressure. However, the molecular pathogenesis for this ECM-rich tumor has yet to be elucidated. We have established that miR-29b was consistently down-regulated in LM compared with myometrium (MM). Hence, the function of miR-29b in LM was examined in vivo using adult female ovariectomized NOD-scid IL2Rγ(null) mice for subrenal xenograft models. In LM xenografts, restoring miR-29b inhibited the accumulation of ECM and the development of solid tumors. Although the miR-29b knockdown in MM cells increased the expression of collagens, it did not transform MM cells into tumorigenic, indicating that the down-regulation of miR-29b is essential but not sufficient for LM tumorigenesis. In addition, 17ß-estradiol and progesterone down-regulated miR-29b and up-regulated mRNAs for multiple collagens in LM xenografts. Thus, we conclude that ECM production in LMs is regulated by steroid hormones via down-regulation of miR-29b, which is one of the mechanisms underlying the excessive accumulation of ECM.

Paracrine activation of WNT/ß-catenin pathway in uterine leiomyoma stem cells promotes tumor growth.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/ß-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of ß-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of ß-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/ß-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

MK-2206, an AKT inhibitor, promotes caspase-independent cell death and inhibits leiomyoma growth.

Uterine leiomyomas (ULs), benign tumors of the myometrium, are the number one indication for hysterectomies in the United States due to a lack of an effective alternative therapy. ULs show activation of the pro-survival AKT pathway compared with normal myometrium; however, substantial data directly linking AKT to UL cell survival are lacking. We hypothesized that AKT promotes UL cell survival and that it is a viable target for inhibiting UL growth. We used the investigational AKT inhibitor MK-2206, currently in phase II trials, on cultured primary human UL and myometrial cells, immortalized leiomyoma cells, and in leiomyoma grafts grown under the kidney capsule in mice. MK-2206 inhibited AKT and PRAS40 phosphorylation but did not regulate serum- and glucocorticoid-induced kinase and ERK1/2, demonstrating its specificity for AKT. MK-2206 reduced UL cell viability and decreased UL tumor volumes. UL cells exhibited disruption of mitochondrial structures and underwent cell death that was independent of caspases. Additionally, mammalian target of rapamycin and p70S6K phosphorylation were reduced, indicating that mammalian target of rapamycin complex 1 signaling was compromised by AKT inhibition in UL cells. MK-2206 also induced autophagy in UL cells. Pretreatment of primary UL cells with 3-methyladenine enhanced MK-2206-mediated UL cell death, whereas knockdown of ATG5 and/or ATG7 did not significantly influence UL cell viability in the presence of MK-2206. Our data provide molecular evidence for the involvement of AKT in UL cell survival and suggest that AKT inhibition by MK-2206 may be a viable option to consider for the treatment of ULs.

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium.

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5' sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate.

Role of stem cells in human uterine leiomyoma growth.

BACKGROUND: Uterine leiomyoma is the most common benign tumor in reproductive-age women. Each leiomyoma is thought to be a benign monoclonal tumor arising from a single transformed myometrial smooth muscle cell; however, it is not known what leiomyoma cell type is responsible for tumor growth. Thus, we tested the hypothesis that a distinct stem/reservoir cell-enriched population, designated as the leiomyoma-derived side population (LMSP), is responsible for cell proliferation and tumor growth. PRINCIPAL FINDINGS: LMSP comprised approximately 1% of all leiomyoma and 2% of all myometrium-derived cells. All LMSP and leiomyoma-derived main population (LMMP) but none of the side or main population cells isolated from adjacent myometrium carried a mediator complex subunit 12 mutation, a genetic marker of neoplastic transformation. Messenger RNA levels for estrogen receptor-α, progesterone receptor and smooth muscle cell markers were barely detectable and significantly lower in the LMSP compared with the LMMP. LMSP alone did not attach or survive in monolayer culture in the presence or absence of estradiol and progestin, whereas LMMP readily grew under these conditions. LMSP did attach and survive when directly mixed with unsorted myometrial cells in monolayer culture. After resorting and reculturing, LMSP gained full potential of proliferation. Intriguingly, xenografts comprised of LMSP and unsorted myometrial smooth muscle cells grew into relatively large tumors (3.67 ± 1.07 mm(3)), whereas xenografts comprised of LMMP and unsorted myometrial smooth muscle cells produced smaller tumors (0.54 ± 0.20 mm(3), p<0.05, n = 10 paired patient samples). LMSP xenografts displayed significantly higher proliferative activity compared with LMMP xenografts (p<0.05). CONCLUSIONS: Our data suggest that LMSP, which have stem/reservoir cell characteristics, are necessary for in vivo growth of leiomyoma xenograft tumors. Lower estrogen and progesterone receptor levels in LMSP suggests an indirect paracrine effect of steroid hormones on stem cells via the mature neighboring cells.