Mullerian Inhibiting Substance Type Research Articles

Mullerian inhibiting substance type II receptor as a potential target for antineoplastic therapy

The review considers properties of the type II anti-Mullerian hormone receptor (mullerian inhibiting substance receptor type II, MISRII), a transmembrane sensor with its own serine/threonine protein kinase activity, triggering apoptosis of the Mullerian ducts in mammalian embryogenesis and providing formation of the male type reproductive system. According to recent data, MISRII overexpression in the postnatal period is found in cells of a number of ovarian, mammary gland, and prostate tumors, and anti-Mullerian hormone (AMH) has a pro-apoptotic effect on MISRII-positive tumor cells. This fact makes MISRII a potential target for targeted anti-cancer therapy. Treatment based on targeting MISRII seems to be a much more effective alternative to the traditional one and will significantly reduce the drug dose. However, the mechanism of MISRII-AMH interaction is still poorly understood, so the development of new anticancer drugs is complicated. The review analyzes MISRII molecular structure and expression levels in various tissues and cell lines, as well as current understanding of the AMH binding mechanisms and data on the possibility of using MISRII as a target for the action of AMH-based antineoplastic drugs.

Anti-Proliferative and Apoptotic Activities of Müllerian Inhibiting Substance Combined with Calcitriol in Ovarian Cancer Cell Lines.

PurposeThis study aimed to investigate whether Müllerian inhibiting substance (MIS) in combination with calcitriol modulates proliferation and apoptosis of human ovarian cancer (OCa) cell lines (SKOV3, OVCAR3, and OVCA433) and identify the signaling pathway by which MIS mediates apoptosis.Materials and MethodsOCa cell lines were treated with MIS in the absence or presence of calcitriol. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay and apoptosis was evaluated by DNA fragmentation assay. Western blot and enzyme-linked immunosorbent assay were used to determine the signaling pathway. ResultsThe cells showed specific staining for the MIS type II receptor. Treatment of OCa cells with MIS and calcitriol led to dose- and time-dependent inhibition of cell growth and survival. The combination treatment significantly suppressed cell growth, down-regulated the expression of B-cell lymphoma 2 (Bcl-2), and up-regulated the expressions of Bcl-2 associated X protein, caspase-3, and caspase-9 through the extracellular signal-regulated kinase signaling pathway.ConclusionThese results, coupled with a much-needed decrease in the toxic side effects of currently employed therapeutic agents, provide a strong rationale for testing the therapeutic potential of MIS, alone or in combination with calcitriol, in the treatment of OCa.

Abstract AS25: An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer

Abstract Purpose: Ovarian cancer is the most lethal malignancy of the female reproductive tract; as such, effective treatments are needed to improve patient survival. MIS has previously been shown to inhibit growth of a stem-like subset of the total cell population in ovarian cancer cell lines (Wei et al, 2010; Meirelles et al, 2012). We have recently engineered novel peptide modifications to human MIS (LR-MIS), which increase production and potency in vitro and in vivo (Pepin et al, 2013), and inserted it into an AAV vector. AAV delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression (Zhang et al, 2011). Therefore, we assessed the efficacy of a single intraperitoneal (ip) dose of AAV9-LR-MIS to inhibit tumor growth of an established cell line for subsequent evaluation in patient-derived ovarian cancer xenografts (PDX). Methods: 6 week old female nude mice were treated with a single ip injection of 3e11 or 1e12 virions of AAV9 LR-MIS or GFP. Serum levels of human MIS were monitored weekly by ELISA. After serum steady state was achieved, the established ovarian cancer cell line, OVCAR5 was xenografted subcutaneously at 1e6 cells per animal. Tumors treated with AAV were measured twice weekly. Additionally, Patient ascites were processed to isolate the cancer cell population, which was specifically amplified in vitro to create low passage primary cell lines. Results: AAV9-LR-MIS treatment resulted in elevated and sustained blood concentrations of MIS (Fig1A) in nude mice without any signs of toxicity, and significantly inhibited OVCAR5 tumor growth (p=0.0003) (Fig1B) in two separate experiments. AAV9-GFP revealed that the pancreas, omentum, gut mesentery, and body wall muscles were the primary sites of infection. Treatment with MIS induced downregulation of CCND1 in tumors. A panel of 15 primary ovarian cancer cell lines, all expressing the MIS type II receptor were generated from patient ascites and cells are being tested for their ability to grow as PDX in Nude, NOD/SCID, and NSG mice. Figure 1.AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA).Figure 1. AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA). Conclusions: AAV9 virally delivered human LR-MIS can produce adequate sustained levels of MIS that inhibited the growth of the human ovarian cancer cell line, OVCAR 5. AAV9 LR MIS provides a well-tolerated, low toxicity treatment of ovarian cancer, which can now be evaluated in a phase 0 trial using PDX models being prepared from human ovarian cancer ascites. Citation Format: David Pepin, Amanda Sosulski, Katherine Hendren, Li Hua Zhang, Fotini Nicolaou, Dan Wang, Guangping Gao, Patricia K Donahoe. An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS25.

AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.

Transcriptome analysis reveals that Müllerian inhibiting substance regulates signaling pathways that contribute to endometrial carcinogenesis.

Müllerian inhibiting substance (MIS) has been shown to inhibit growth of a number of tumors in vitro and/or in vivo, but the downstream pathways which it regulates are not fully understood. In the present study we show that MIS type II receptor was highly expressed in AN3CA cells, a cell line derived from human endometrial cancer cell in which MIS-treatment caused a reduction of cell viability, and induced cellular apoptosis and genes involved cell cycle arrest. To understand the genome-wide effects of MIS on gene regulation, we performed serial gene expression analyses from 0 to 96 h at 24 h intervals after treating AN3CA cells with MIS. Transcriptomic analysis of molecular changes induced by MIS identified 2,688 differentially expressed genes that were significantly up- or down-regulated during the 96 h study period. When the 2,688 differentially expressed genes were mapped to known biological processes, Wnt-, cancer-, proteolysis-, cytoskeleton-, cell cycle-, apoptosis-, and MAPK-signaling pathways emerged as the functions most significantly changed by MIS in AN3CA cells. Furthermore, western blot analysis validated that protein expression of cell cycle inhibitory genes, apoptotic protease activating factor-1 (APAF-1), β-catenin-interacting protein (ICAT), Rb related protein 130 (p130), and inhibitor of disheveled Dvl and Axin complex (IDAX), were gradually increased over the time of the study, whereas downstream cell cycle activating genes, cyclin-dependent kinase 2 (CDK2) and phospho-c-Jun were downregulated in MIS-treated AN3CA cells. These transcriptome analyses support previous observations that MIS functions as a tumor suppressor, potentially by regulating signaling pathways that could contribute to endometrial carcinogenesis, and indicating that MIS should be considered as a potential treatment for endometrial cancer.

Müllerian Inhibiting Substance Induces Apoptosis of Human Endometrial Stromal Cells in Endometriosis

Müllerian inhibiting substance (MIS) is produced in Sertoli cells of fetal testis and causes regression of müllerian ducts in male embryos. MIS also can induce the cell cycle arrest and apoptosis in müllerian duct-derived tumors in vivo and in vitro. Our objective was to investigate the expression of MIS type II receptor (MISR II) and whether MIS can inhibit the proliferation and induce apoptosis in primary cultures of endometrial stromal cells (ESC) of endometriosis. In vitro experiments were performed in the university research laboratory. Tissue samples from 12 patients who had undergone evisceration for ovarian endometrial cysts were included in this study. The expression of MISR II in ESC was investigated by immunohistochemistry. The cell viability and apoptosis in ESC treated with MIS was measured by methylthiazoletetrazolium assay and annexin V analysis. The expression of regulatory proteins in ESC treated with MIS was shown by Western blotting. ESC showed specific immunostaining for the MISR II. ESC treated with MIS exhibited 32% growth inhibition (P = 0.0001). The changes in cell cycle distribution after MIS exposure at 72 h demonstrated that S and G(2)M phases were decreased; G(0)G(1) and sub-G(0)G(1) phases were increased. ESC treated with MIS showed 13.72% annexin V-fluorescein isothiocyanate positivity. In the ESCs, which contain defective p16, MIS increased the expression of pocket proteins p107 and p130 and decreased E2F transcription factor 1. The results support a central role for MIS in endometriosis. Although the precise mechanism of MIS-mediated inhibition of ESC growth has not been fully defined, these data suggest that MIS has activity against ESC in vitro and may also be an effective targeted therapy for endometriosis.

Mullerian inhibiting substance (MIS) pathway as a therapeutic target in ovarian cancer: Deciphering the necessary components

Objective: Activation of mullerian inhibiting substance type II receptor (T2R) leads to dimerization with one of three candidate type I receptors (T1R) and growth inhibition of mullerian tissues. While T2R is commonly expressed in epithelial ovarian cancer (EOC), the prevalence of T1R is unknown; this data is critical because our in vitro studies suggest that biologic response is dependent upon which candidate T1R is involved in signaling. The aims of this research were to characterize the frequency of candidate T1R expression in EOC and determine if clinical outcomes are dependent on receptor expression.

Identification of characteristic molecular signature of M�llerian inhibiting substance in human HPV-related cervical cancer cells

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone (AMH), is a member of the transforming growth factor-β (TGF-β) superfamily that plays an important role in the mesenchymal-epithelial interaction, cell growth and proliferation, extracellular matrix production and tissue remodeling. Previously, we demonstrated that MIS suppressed ovarian cancer cell growth and suggested large-scale genetic elements that could be responsible for anti-neoplastic effects of MIS on ovarian cancer cells. In this study, we demonstrated the expression of MIS type II receptor (MISRII) in the human papillomavirus (HPV)-16-related cervical cancer cell lines CaSki and SiHa, and a non-HPV-related cervical cancer cell line, C33A. We also showed that MIS inhibited growth of cervical cancer cells, and induced cellular apoptosis of C33A. In addition, we identified a characteristic molecular signature of MIS in CaSki cells by using whole genome expression analysis. Of the 1,690 genes that showed significant expression changes by MIS, 21 genes were related to cell cycle; 13 genes to apoptosis; and 52 genes to the cancer pathway. On performing a search for cell cycle pathways in the KEGG pathway database, several gene expressions at the G1/S checkpoint were found. In particular, the expression of p16 and p107 increased and that of E2F2 and E2F3 decreased at an early stage, whereas the expression of E2F4 and E2F5 decreased at a later stage after MIS treatment. These data suggest that MIS produces activity against HPV16-related cervical cancers in vitro, and MIS may also be an effective targeted therapy for HPV16-related cervical cancer. Genetic data obtained here could be useful in determining the treatment strategy of MISR-expressing cervical tumors in the future.

Abstract 3262: Role of the mullerian ducts in the development of genito-urinary organs and coelomic epithelial tissues in female mice

Abstract Ovarian carcinoma is the most lethal carcinoma arising in female reproductive organ, but the exact site of origin is unclear. It has long been believed that the mesothelial cell layer covers the ovarian surface is the site of origin of these tumors and its role in ovarian tumorigenesis is still being debated. A central issue in this debate is the potential relationship between the coelomic epithelium, from which the ovarian surface mesothelium is derived, and the mullerian ducts, from which most of the female reproductive organs are derived. We created two transgenes, both driven by the Mullerian inhibiting substance type II receptor (MisIIr) promoter (initially obtained from Dr. Denise Connolly, Fox Chase Cancer Center, Philadelphia, PA), respectively expressing beta-galactosidase (LacZ) and Cre recombinase. A minimal promoter sequence, hsp68, was introduced in the beta-galactosidase construct. Expression of MisIIr is known to be present in the mullerian ducts and some of their adult derivatives. We crossed mice carrying the MisIIr-Cre transgene with ROSA 26 (R26R) reporter mice and compared the results of colorimetric assays for beta-galactosidase activity on histological sections from MisIIr-hsp68-LacZ and MisIIr-Cre;R26R transgenic mice of various ages. Detection of enzyme activity in tissues from MisIIr-hsp68-LacZ mice indicated MisIIr promoter activity at the time of the examination whereas expression in tissues derived from MisIIr-Cre;R26R mice also indicated prior activity in ancestral cells, regardless of whether this promoter is active or not at the time of the examination. The upper portion of the vagina, the uterine horns, and the oviducts, which are known to be derived from the mullerian ducts, stained positive for LacZ in MisIIr-Cre;R26R mice as expected. Also positive were ovarian follicles, suggesting an embryological relationship between these structures and the mullerian ducts. No detectable LacZ positivity was present in either the ovarian surface mesothelium or the coelomic epithelium, suggesting a lack of relationship between these tissues and the mullerian ducts. Unexpectedly, a portion of the renal collecting system stained positively in female, but not in male MisIIr-Cre;R26R transgenic mice, raising the possibility of an embryological relationship between the mullerian ducts and some renal tubular cells found in adult kidneys. LacZ positivity was only found in vagina, uterine horns, and oviducts of mice carrying the MisIIr-hsp68-LacZ transgene, suggesting that although ovarian follicular cells and renal tubular cells may be embryologically derived from the mullerian ducts, the MisIIr promoter is either inactive or minimally active in these tissues in adult mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3262.

Abstract 1586: Expression of MIS type II receptor in ovarian cancer influences survival in vitro and in vivo

Abstract Objective: Mullerian inhibiting substance type II receptor (MISRII) is a tissue-specific target for therapy in gynecologic cancers. MISRII plays a specialized role in regression of the mullerian tracts embryologically. Analogous to other TGF-β family members, ligand (MIS) binding to the type II receptor leads to a heteromeric complex with type I receptors, downstream Smad signaling, cell cycle arrest and regression. We previously published that the majority of epithelial ovarian cancers (EOC) and endometrial cancers (EC) express MISRII in a tissue specific manner. The purpose of these studies was to investigate the biologic significance of MISRII expression in EOC. Methods: For in vitro studies, we developed both chimeric and endogenous cell models to study MISRII. Chimeric MIS type I and II receptors were created by fusing the ligand binding domain of GM-CSF (α or β subunit) to the transmembrane and cytoplasmic domains of the type I/II receptor; these receptors were stably transfected into Ovcar8 cells. The chimera system allows specific signaling of the receptor complex and the ability to separately analyze candidate type I receptors. The endogenous cell model was created in SKOV3 (MISRII- EOC cell line) by stably expressing full-length MISRII. Growth inhibition was assessed by colony formation in soft agar in response to receptor activation and cisplatin. A BRE-luciferase reporter construct was used to measure Smad 1/5 activation after exposure to MIS. FACS analysis was used to assess cell cycle progression. Results: In the chimeric model, receptor activation resulted in up to 60% inhibition of colony formation (dependent on which candidate type I receptor was targeted). Additionally, receptor activation sensitized cells to the effects of cisplatin (1μM) with nearly 4-fold inhibition of colony formation. In the endogenous MISRII expressing EOC cell model, functional signaling was first confirmed using a BRE-luciferase reporter assay to document induction of Smad activation after exposure to rhMIS (3.2 relative light units increase after 20nM MIS). This model was then assessed for cell cycle progression in the presence of MIS. We observed a strong G1 arrest (62% vs. 43% untreated) after 15h exposure to 25nM MIS in cells expressing the receptor with no change in the MIS- parental cell line. These in vitro data are consistent with our survival results in EOC demonstrating a significantly improved overall survival (p=0.04) in MISRII expressing cancers. Conclusions: The majority of gynecologic cancers express MISRII. Our in vitro studies demonstrate that receptor activation results in cell cycle arrest, reduced colony formation, and sensitization to cisplatin. Confirming this in vitro data is the observation that EOC expressing MISRII have improved response to therapy and longer survival. Together, these results support further targeting of the MISRII pathway as an anticancer strategy in gynecologic malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1586.

Development of an efficiently cleaved, bioactive, highly pure FLAG-tagged recombinant human Mullerian Inhibiting Substance

Mullerian Inhibiting Substance (MIS), a member of the TGF-β family, causes regression of the Mullerian duct in male embryos, after binding to Mullerian Inhibiting Substance Receptor II (MISRII). It has also been extensively demonstrated that it can inhibit proliferation of various cancer cell lines such as ovarian, prostate, and breast cancer in vitro and in vivo. Hence, the availability of a recombinant, epitope tagged, bioactive MIS is important for the selection of patients for treatment and for probing novel molecular targets for MIS in various tissues. To this end, we have expressed a recombinant, internally FLAG-tagged form of hMIS with the tag (DYKDDDDK) immediately after the cleavage site (427–428) of MIS at the C-terminus with a modified dibasic cleavage motif sequence. We show that this construct results in a highly pure, endogenously processed (cleaved) FLAG MIS, that causes complete regression of the Mullerian Duct in an organ culture assay. In addition, purified FLAG MIS was able to bind and affinity purify both transfected and endogenous MIS type II receptor. The availability of this fully functional, epitope tagged form of MIS should facilitate scale-up for preclinical and clinical use and should also be used for the study of MIS binding proteins and for tracking in pharmacokinetic studies.

A Single Base Pair Mutation Encoding a Premature Stop Codon in the MIS Type II Receptor Is Responsible for Canine Persistent Müllerian Duct Syndrome

Müllerian inhibiting substance (MIS), a secreted glycoprotein in the transforming growth factor-beta family of growth factors, mediates regression of the Müllerian ducts during embryonic sex differentiation in males. In persistent Müllerian duct syndrome (PMDS), rather than undergoing involution, the Müllerian ducts persist in males, giving rise to the uterus, fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited autosomal recessive trait. Previous studies indicated that a defect in the MIS receptor or its downstream signaling pathway was likely to be causative of the canine syndrome. In this study, the canine PMDS phenotype and clinical sequelae are described in detail. Affected and unaffected members of this pedigree are genotyped, identifying a single base pair substitution in MISRII that introduces a stop codon in exon 3. The homozygous mutation terminates translation at 80 amino acids, eliminating much of the extracellular domain and the entire transmembrane and intracellular signaling domains. Findings in this model could enable insights to be garnered from correlation of detailed clinical descriptions with molecular defects, which are not otherwise possible in the human syndrome.

Evidence for a Müllerian-inhibiting substance autocrine/paracrine system in adult human endometrium

To determine if adult human endometrium possesses an intact müllerian-inhibiting substance (MIS) signal transduction system and, if so, whether MIS can modulate endometrial cell viability. Adult human endometrial tissue was subjected to reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. In addition, cultured human endometrial stromal cells were treated with recombinant MIS or transiently transfected with MIS and/or MIS type II receptor (MISRII) expression plasmids to assess for effects upon endometrial cell viability and apoptosis. The MIS in conditioned media was quantified by specific ELISA. Academically affiliated medical center. Reproductive-age women undergoing routine infertility evaluation. Endometrial sampling. Assessment of MIS gene transcription and protein translation in human endometrial tissue in vivo and in vitro. 1) The RT-PCR revealed that adult human endometrium expresses the genes for MIS, MISRII, ALK3, and Smads 1 and 9. 2) Immunohistochemistry reveals that MIS and MISRII protein are expressed in human endometrium. 3) Immunocytochemistry of cultured human endometrial cells reveals that MIS and MISRII protein are primarily restricted to mitosing cells. 4) ELISA reveals that MIS is secreted by human endometrial cells in vitro and that this process is increased by sex steroids. 5) Increasing local MIS concentration in cultured human endometrial stromal cells either by exogenous administration or transient transfection significantly decreases cell viability and increases apoptosis. Adult human endometrium possesses a functional MIS signal transduction system which negatively regulates cellular viability.

Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-beta superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2'-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

C-Jun N-terminal Kinase Inhibitor II (SP600125) Activates Müllerian Inhibiting Substance Type II Receptor-Mediated Signal Transduction

Müllerian inhibiting substance (MIS), the hormone required for Müllerian duct regression in fetal males, is also expressed in both adult males and females, but its physiological role in these settings is not clear. The expression of the MIS type II receptor (MISRII) in ovarian cancer cells and the ability of MIS to inhibit proliferation of these cells suggest that MIS might be a promising therapeutic for recurrent ovarian cancer. Using an MISRII-dependent activity assay in a small-molecule screen for MIS-mimetic compounds, we have identified the c-Jun N-terminal kinase inhibitor SP600125 as an activator of the MIS signal transduction pathway. SP600125 increased the activity of a bone morphogenetic protein-responsive reporter gene in a dose-dependent manner and exerted a synergistic effect when used in combination with MIS. This effect was specific for the MISRII and was not seen with other receptors of the TGFbeta family. Moreover, treatment of mouse ovarian cancer cells with a combination of SP600125 and paclitaxel, an established chemotherapeutic agent used in the treatment of ovarian cancer, or with MIS enabled inhibition of cell proliferation at a lower dose than with each treatment alone. These results offer a strong rationale for testing the therapeutic potential of SP600125, alone or in combination with already established drugs, in the treatment of recurrent ovarian cancer with a much-needed decrease in the toxic side effects of currently employed therapeutic agents.

Activin receptor-like kinase-2 inhibits activin signaling by blocking the binding of activin to its type II receptor

Activin receptor-like kinase-2 (Alk2) has been shown to be a promiscuous type I receptor for the transforming growth factor beta (TGFbeta) family of growth and differentiation factors, such as activin, bone morphogenetic proteins, and Müllerian inhibiting substance (MIS). We have studied the putative role of Alk2 in activin signaling using MA-10 cells, a mouse transformed Leydig cell line, in which endogenous expression of cytochrome P450 c17 hydroxylase/C17-20 lyase mRNA is inhibited by both MIS and activin A. Overexpression of Alk2 in MA-10 cells inhibited the activation of the activin-responsive CAGA-luciferase reporter and, conversely, transfection of siRNA for Alk2 increased the response. In contrast, overexpression of the MIS type II receptor in MA-10 cells increased the activin-mediated induction of CAGA-luciferase approximately fivefold, which we hypothesized occurs by MIS type II receptor sequestering endogenous Alk2. Binding experiments with (125)I-labeled activin show that the underlying mechanism of Alk2-mediated inhibition of activin signaling involves Alk2 blocking the access of activin to its type II receptor, which we show can bind Alk2 in the absence of ligand. These results show that the complement of other type I receptors in addition to the ligand-specific type I receptor can provide an important mechanism for modulating cell-specific responses to members of the TGFbeta family.

Mullerian Inhibiting Substance enhances subclinical doses of chemotherapeutic agents to inhibit human and mouse ovarian cancer

Mullerian Inhibiting Substance (MIS), a biological modifier that causes regression of Mullerian ducts in male embryos, is effective as a single agent in vitro and in vivo against human and mouse ovarian cancer cell lines expressing MIS type II receptor; however, little is known about how recombinant human MIS (rhMIS), now being scaled for preclinical trials, could be used in combination with cytotoxic or targeted chemotherapeutic agents. Mouse serous and endometrioid ovarian carcinoma cell lines were tested in vitro against rhMIS alone and with doxorubicin, paclitaxel, or cisplatin as agents in clinical use. Because MIS releases FK506 binding protein (FKBP12), which activates the mammalian target of rapamycin (mTOR) downstream of Akt, rhMIS and rapamycin combinations were tested. MIS increases p16 protein levels, and 5'-Aza-2'-deoxycytidine (AzadC) induces p16 mRNA; therefore, they were used in combination in vitro and in vivo with a human ovarian cancer cell line. A paclitaxel-resistant human ovarian cancer cell line and its parental line both respond to rhMIS in vitro. Additivity, synergy, or competition was observed with MIS and rapamycin, AzadC, doxorubicin, cisplatin, and paclitaxel, suggesting that MIS in combination with selective targeted therapies might achieve greater activity against ovarian cancer than the use of each individual agent alone. These assays and statistical analyses could be useful in selecting rhMIS and chemotherapeutic agent combinations that enhance clinical efficacy and reduce toxicity.

Müllerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Müllerian duct regression

Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stage- and tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor (MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-to-mesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by differentially regulating their expression.

Mullerian inhibiting substance suppresses tumor growth in the C3(1)T antigen transgenic mouse mammary carcinoma model

Mullerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro. To extend the use of MIS to treat breast cancer, it is essential to test the responsiveness of mammary tumor growth to MIS in vivo. Mammary tumors arising in the C3(1) T antigen mouse model expressed the MIS type II receptor, and MIS in vitro inhibited the growth of cells derived from tumors. Administration of MIS to mice was associated with a lower number of palpable mammary tumors compared with vehicle-treated mice (P=0.048), and the mean mammary tumor weight in the MIS-treated group was significantly lower compared with the control group (P=0.029). Analysis of proliferating cell nuclear antigen (PCNA) expression and caspase-3 cleavage in tumors revealed that exposure to MIS was associated with decreased proliferation and increased apoptosis, respectively, and was not caused by a decline in T antigen expression. The effect of MIS on tumor growth was also evaluated on xenografted human breast cancer cell line MDA-MB-468, which is estrogen receptor- and retinoblastoma-negative and expresses mutant p53, and thus complements the C3(1)Tag mouse mammary tumors that do not express estrogen receptor and have functional inactivation of retinoblastoma and p53. In agreement with results observed in the transgenic mice, MIS decreased the rate of MDA-MB-468 tumor growth and the gain in mean tumor volume in severe combined immunodeficient mice compared with vehicle-treated controls (P=0.004). These results suggest that MIS can suppress the growth of mammary tumors in vivo.

Endometrial cancer is a receptor-mediated target for Mullerian Inhibiting Substance.

Mullerian Inhibiting Substance (MIS), a 140-kDa homodimer glycoprotein member of the TGF-beta superfamily of biological-response modifiers, causes regression of the Mullerian ducts in developing male embryos. MIS also can induce growth arrest and apoptosis in ovarian and cervical cancer cell lines. The embryonic progenitor of the ovarian and cervical epithelium is the coelomic epithelium, the same tissue that regresses under the direction of MIS in the male. The endometrium and uterus also arise from the coelomic epithelium and the Mullerian ducts. Here, we show that both normal human endometrium and endometrial cancers express the receptor for MIS and that MIS can inhibit the proliferation of a number of human endometrial cancer cell lines that express the MIS type II receptor. In the representative endometrial cancer cell line AN3CA, MIS affects the expression of key cell-cycle regulatory proteins. This work broadens the scope of tumors that MIS can potentially control and, by elucidating the MIS signaling pathway, identifies other potential avenues for intervention.