Honami Naora

Ovarian Cancer Metastasis: Integrating insights from disparate model organisms

Despite considerable efforts to improve early detection, and advances in chemotherapy, metastasis remains a major challenge in the clinical management of ovarian cancer. Studies of new murine models are providing novel insights into the pathophysiology of ovarian cancer, but these models are not readily amenable to genetic screens. Genetic analysis of border-cell migration in the Drosophila melanogaster ovary provides clues that will improve our understanding of ovarian cancer metastasis at the molecular level, and also might lead to potential therapeutic targets.

Lineage infidelity of epithelial ovarian cancers is controlled by HOX genes that specify regional identity in the reproductive tract

Although epithelial ovarian cancers (EOCs) have been thought to arise from the simple epithelium lining the ovarian surface or inclusion cysts, the major subtypes of EOCs show morphologic features that resemble those of the müllerian duct–derived epithelia of the reproductive tract. We found that HOX genes, which normally regulate müllerian duct differentiation, are not expressed in normal ovarian surface epithelium (OSE), but are expressed in different EOC subtypes according to the pattern of müllerian-like differentiation of these cancers. Ectopic expression of Hoxa9 in tumorigenic mouse OSE cells gave rise to papillary tumors resembling serous EOCs. In contrast, Hoxa10 and Hoxa11 induced morphogenesis of endometrioid-like and mucinous-like EOCs, respectively. Hoxa7 showed no lineage specificity, but promoted the abilities of Hoxa9, Hoxa10 and Hoxa11 to induce differentiation along their respective pathways. Therefore, inappropriate activation of a molecular program that controls patterning of the reproductive tract could explain the morphologic heterogeneity of EOCs and their assumption of müllerian-like features.

Activated Signal Transducer and Activator of Transcription (STAT) 3 Localization in Focal Adhesions and Function in Ovarian Cancer Cell Motility

Constitutive activation of the Janus-activated kinase/signal transducer and activator of transcription (STAT) pathway promotes the proliferation and survival of cancer cells in culture and is associated with various cancers, including those of the ovary. We found that constitutively activated STAT3 levels correlated with aggressive clinical behavior of ovarian carcinoma specimens. Furthermore, inhibition of STAT3 reduced the motility of ovarian cancer cells in vitro. Surprisingly, we found that activated STAT3 localized not only to nuclei but also to focal adhesions in these cells. Activated STAT3 coimmunoprecipitated with phosphorylated paxillin and focal adhesion kinase and required paxillin and Src for its localization to focal adhesions. These results suggest that Janus-activated kinase/STAT signaling may contribute to ovarian cancer cell invasiveness.

Deregulation of the HOXA10 Homeobox Gene in Endometrial Carcinoma: Role in Epithelial-Mesenchymal Transition

Homeobox genes encode transcription factors that control cell differentiation and play essential roles in developmental patterning. Increasing evidence indicates that many homeobox genes are aberrantly expressed in cancers, and that their deregulation significantly contributes to tumor progression. The homeobox gene HOXA10 controls uterine organogenesis during embryonic development and functional endometrial differentiation in the adult. We investigated whether HOXA10 expression is deregulated in endometrial carcinomas, and how counteracting this aberrant expression modifies tumor behavior. We found that down-regulation of HOXA10 expression in endometrial carcinomas strongly correlates with increased tumor grade and is associated with methylation of the HOXA10 promoter. Enforced expression of HOXA10 in endometrial carcinoma cells inhibited invasive behavior in vitro and tumor dissemination in nude mice. The inhibitory effect of HOXA10 on invasive behavior was attributable at least in part to the ability of HOXA10 to induce expression of the epithelial cell adhesion molecule E-cadherin by down-regulating expression of Snail, a repressor of E-cadherin gene transcription. These findings reveal a novel role for HOXA10 deregulation in the progression of endometrial carcinoma by promoting epithelial-mesenchymal transition.

HOXA9 promotes ovarian cancer growth by stimulating cancer-associated fibroblasts

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-β2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

Inhibition of ovarian cancer growth by a tumor-targeting peptide that binds eukaryotic translation initiation factor 4E.

Purpose: A critical step of protein synthesis involves the liberation of the mRNA cap-binding translation initiation factor eIF4E from 4EBP inhibitory binding proteins, and its engagement to the scaffolding protein eIF4G. eIF4E is a candidate target for cancer therapy because it is overexpressed or activated in many types of tumors and has tumorigenic properties. Our aim was to design and evaluate 4EBP-based peptides for their antitumor activity in ovarian cancer. Experimental Design: The ability of peptides to bind and inhibit eIF4E was determined by immunoprecipitation and by assaying cap-dependent reporter synthesis. To target ovarian tumors, the lead candidate 4EBP peptide was fused to an analog of gonadotropin-releasing hormone (GnRH). Cellular uptake of peptide, and effects on cell viability and cell death were determined. The antitumor activity of fusion peptide was evaluated in female nude mice bearing i.p. ovarian tumor xenografts. Results: 4EBP-based peptides bound eIF4E, prevented eIF4E from binding eIF4G, and inhibited cap-dependent translation. GnRH agonist-4EBP fusion peptide was taken up by, and inhibited the growth of, GnRH receptor-expressing tumor cells, but not receptor-negative cells. Intraperitoneal tumor burden was significantly smaller in mice treated with fusion peptide than in mice treated with saline (P < 0.001). Ascites was also reduced in peptide-treated mice. Significant cytotoxic effects to host tissues were not observed. On the other hand, treatment with GnRH agonist alone did not inhibit tumor growth or ascites. Conclusion: Because ovarian cancer is rarely cured by conventional chemotherapies, GnRH-4EBP fusion peptide may be of therapeutic potential for treatment of this disease.

Homeodomain protein DLX4 counteracts key transcriptional control mechanisms of the TGF-Β cytostatic program and blocks the antiproliferative effect of TGF-Β

The antiproliferative activity of transforming growth factor-β (TGF-β) is essential for maintaining normal tissue homeostasis and is lost in many types of tumors. Gene responses that are central to the TGF-β cytostatic program include activation of the cyclin-dependent kinase inhibitors, p15Ink4B and p21WAF1/Cip1, and repression of c-myc. These gene responses are tightly regulated by a repertoire of transcription factors that include Smad proteins and Sp1. The DLX4 homeobox patterning gene encodes a transcription factor that is absent from most normal adult tissues, but is expressed in a wide variety of malignancies, including lung, breast, prostate and ovarian cancers. In this study, we demonstrate that DLX4 blocks the antiproliferative effect of TGF-β. DLX4 inhibited TGF-β-mediated induction of p15Ink4B and p21WAF1/Cip1 expression. DLX4 bound and prevented Smad4 from forming complexes with Smad2 and Smad3, but not with Sp1. However, DLX4 also bound and inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc independently of TGF-β/Smad signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-β cytostatic program could explain, in part, the resistance of tumors to the antiproliferative effect of TGF-β.

Developmental patterning in the wrong context: The paradox of epithelial ovarian cancers

In contrast to many other types of cancer, epithelial ovarian cancers often exhibit differentiation patterns that are more specialized than those of the tissue of origin. Many epithelial ovarian cancers assume morphologic features reminiscent of the müllerian duct-derived epithelia, but are thought to arise from cells of the simple ovarian surface epithelium that is not of müllerian duct origin. This article discusses recent studies that implicate aberrant activation of embryonic pathways in ovarian cancer histogenesis, and highlights the intimate relationship between developmental plasticity and neoplasia.

Steroid receptor coactivator-3, a homolog of Taiman that controls cell migration in the Drosophila ovary, regulates migration of human ovarian cancer cells.

Border cell migration is a process that occurs during Drosophila ovarian development in which cells derived from a simple epithelium migrate and invade neighboring tissue. This process resembles the behavior of cancerous cells that derive from the simple epithelium of the human ovary. One important regulator of border cell migration is Taiman, a homolog of steroid receptor coactivator-3 (SRC-3). Because increasing evidence indicates that similarities exist between the molecular control of migration of border cells and of cancer cells, we investigated whether SRC-3 controls ovarian cancer cell migration. Little or no SRC-3 expression was detected in normal ovarian surface epithelium, ovarian cysts and borderline ovarian tumors that lack stromal invasion. In contrast, SRC-3 was abundantly expressed in high-grade ovarian carcinomas. Inhibiting SRC-3 expression in ovarian cancer cells markedly reduced cell spreading and migration, and altered intracellular localization of focal adhesion kinase. This inhibitory effect on cell migration was independent of the estrogen receptor (ER) status of the cells. These studies reveal a novel role for SRC-3 in ovarian cancer progression by promoting cell migration, independently of its role in estrogen receptor signaling.

Expression of the Homeobox Gene HOXA9 in Ovarian Cancer Induces Peritoneal Macrophages to Acquire an M2 Tumor-Promoting Phenotype

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8(+) tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages.