Müllerian Inhibiting Substance Inhibits Ovarian Cell Growth through an Rb-independent Mechanism

Abstract

Mü llerian inhibiting substance (MIS), a transforming growth factor-␤ family member, causes regression of the Mü llerian duct in male embryos. MIS overexpression in transgenic mice ablates the ovary, and MIS inhibits the growth of ovarian cancer cell lines in vitro, suggesting a key role for this hormone in postnatal development of the ovary. This report describes a mechanism for MISmediated growth inhibition in both a human epithelial ovarian cancer cell line and a cell line derived from normal ovarian surface epithelium, which is the origin of human epithelial ovarian cancers. MIS-treated cells accumulated in the G 1 phase of the cell cycle and subsequently underwent apoptosis. MIS up-regulated the cyclin-dependent kinase inhibitor p16 through an MIS type II receptor-mediated mechanism and inhibited growth in the absence of detectable or inactive Rb protein. Prolonged treatment with MIS down-regulated the Rb-related protein p130 and increased the Rb familyregulated transcription factor E2F1, overexpression of which inhibited growth. These findings demonstrate that p16 is required for MIS-mediated growth inhibition in ovarian epithelial cells and tumor cells and suggest that up-regulation of E2F1 also plays a role in this process.MIS, 1 a member of the TGF-␤ family of hormones, induces regression of the epithelial-mesenchymal unit of the Mü llerian duct in the embryonic urogenital ridge in males. In the absence of MIS, differentiation of the Mü llerian duct into the uterus, fallopian tubes, and upper vagina in female embryos occurs autonomously (1). The 140-kDa MIS homodimer is enzymatically cleaved into two distinct fragments. The carboxylterminal fragment composed of a dimer with subunits of M r 12,500 retains bioactivity, whereas a noncleavable mutant is devoid of biological function (2). MIS is produced at high levels by Sertoli cells of the testis even after the regression of the Mü llerian duct and decreases at adolescence. In females, it is synthesized by granulosa cells of the ovary. Measurement of circulating serum MIS levels in females indicates that MIS in females is produced postnatally, increases at the onset of puberty, and is undetectable at menopause (3, 4). It is hypothesized that binding of MIS ligand to the MIS type II receptor, a serine threonine kinase (5-7), leads to heterodimerization with a type I receptor, initiating a signaling cascade.The MIS type II receptor gene contains 11 exons and encodes a 63-kDa protein, which is expressed at very high levels in the uterus, testis, and ovary (5-7). Male mice that lack both alleles of the MIS type II receptor have a persistent Mü llerian duct, which differentiates into a uterus and oviducts, a phenotype reminiscent of MIS ligand null mice (8). Imbeaud et al. (9) have identified MIS type II receptor mutations in male patients with Persistent Mü llerian Duct syndrome, which reaffirms the developmental significance of its expression in humans. Transgenic female mice that overexpress MIS ligand, demonstrate complete ablation of the ovary, along wit...

Identification of large-scale characteristic genes of Müllerian inhibiting substance in human ovarian cancer cells

“…Regarding G1/S cell cycle regulation by MIS, we found that MIS did not induce most of the CDK inhibitors, but p19 INK4D and p21 Cip1 were slightly induced at 96 h. It is interesting to know that the transcript level of p16 INK4A was not changed by MIS in a previous study (19) and the same result was obtained from our analysis (Fig. 4B).…”

“…These findings are significant since the MIS type II receptor has been detected in an increasing number of human gynecological tumors (35). In addition, it also suggests that MIS inhibits growth inhibition through the activation of p16, a negative cell cycle regulator (19), and consequently disrupts cell cycle progression and leads to apoptosis. In order to confirm the anti-neoplastic activity of MIS, we assessed the growth inhibition and apoptosis of MIS on OVCAR-8 cells, which was previously demonstrated to express MISRII and to respond to MIS by growth inhibition.…”

“…In previous studies, it has been demonstrated that recombinant human MIS inhibits the growth of ovarian cancer cells and various primary tumors in vitro and in vivo (19,(32)(33)(34). These findings are significant since the MIS type II receptor has been detected in an increasing number of human gynecological tumors (35).…”

“…It was also demonstrated that OVCAR-8, an epithelial ovarian cancer cell line, expresses MISRII and responds to MIS by growth inhibition mediated through a retinoblastoma protein (p-RB)-independent mechanism involving the up-regulation of p16 at the protein level. This suggests that MIS requires p16 for growth inhibition in ovarian cancer cells, which in turn arrests cell growth at the G1/S phase and subsequently augments apoptosis (19). Although several reports have described the anti-neoplastic properties of MIS, little insight into the molecular nature of anti-tumor growth or apoptosis has been gained.…”

“…Since most ovarian tumors originate from the surface epithelium of the ovary, which is of Mullerian duct origin (15,16), the effect of MIS on the growth of ovarian cancer cells has been an area of intense study. Many previous studies have demonstrated that MIS inhibits the growth of Müllerian duct-derived tumors (17)(18)(19) and several cancer cell lines or primary cells in vitro (20)(21)(22)(23). It was also demonstrated that OVCAR-8, an epithelial ovarian cancer cell line, expresses MISRII and responds to MIS by growth inhibition mediated through a retinoblastoma protein (p-RB)-independent mechanism involving the up-regulation of p16 at the protein level.…”