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High-grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirm that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through the activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into the examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.

Dissemination of ovarian tumors involves the implantation of cancer spheroids into the mesothelial monolayer on the walls of peritoneal and pleural cavity organs. Biopsies of tumors attached to peritoneal organs show that mesothelial cells are not present under tumor masses. We have developed a live, image-based in vitro model in which interactions between tumor spheroids and mesothelial cells can be monitored in real time to provide spatial and temporal understanding of mesothelial clearance. In this article, we provide evidence that ovarian cancer spheroids use integrin- and talin-dependent activation of myosin and traction force to promote displacement of mesothelial cells from underneath a tumor cell spheroid. These results suggest that ovarian tumor cell clusters gain access to the submesothelial environment by exerting force on the mesothelial cells lining target organs, driving migration and clearance of the mesothelial cells. Significance: This study uses time-lapse video microscopy to decipher cellular events associated with ovarian tumor cell intercalation of mesothelial cell layers. Ovarian cancer clusters were found to use actomyosin-generated force to physically displace mesothelial cells and gain access to the submesothelial environment. Blockade of force-conducting molecules, including α5 integrin, talin I, and nonmuscle myosin II, in cancer cells abrogated mesothelial displacement from underneath attached cancer spheroids. Cancer Discovery; 1(2); 144–57. ©2011 AACR. Read the Commentary on this article by Kenny et al., p. 100 This article is highlighted in the In This Issue feature, p. 91

Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States(1). Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal(2). It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity(3) (Figure 1, Movie 1). All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells(4-6) (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections(3,5-7) (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells(8), and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)(9,10). This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers(8-16), and was first described in a report showing that ovarian tumor cells utilize an integrin -dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid(17). This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin –dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

Insulin/IGF-signaling (IIS) affects longevity, stress resistance and metabolism in worms, flies, and mammals. The Forkhead transcription factor DAF-16/FOXO is the major downstream effector of IIS and is responsible for the activation and repression of genes that mediate the diverse effects of IIS. We surveyed a set of informatically predicted conserved DAF-16/FOXO target genes and identified the novel DAF-16 direct target hlh-13. hlh-13 is the predicted homolog of the mammalian transcription factor Ptf1a, a critical determinant of pancreatic development. We found that an hlh-13 mutant exits L1 arrest and IIS-dependent dauer diapause faster than control worms, but is not involved in lifespan or resistance to a variety of stresses. Our results have identified a novel DAF-16 target gene and linked its function to known outputs of IIS. Considering the high conservation of IIS in diverse species, our results also hint at an intriguing connection of IIS and Ptf1a in mammals.

Metastatic dissemination of ovarian tumor cells to organs in the peritoneal cavity involves the intercalation of dissociated tumor cells into the mesothelial monolayer covering those organs. We established a cell culture model that uses time-lapse video microscopy to investigate the mechanisms involved in the initial interactions between tumor cells and the mesothelium. Tumor cells are cultured in suspension, where they associate into multicellular spheroids. When spheroids are incubated with a mesothelial monolayer, the tumor cells attach to the mesothelial cells and then displace the mesothelial cells and gain access to the underlying substratum. We believe that force generation by the ovarian cancer cells is required for mesothelial clearance, as inhibition of myosin IIA, IIB, or talin I in the ovarian cancer spheroids attenuates mesothelial clearance. To further elucidate the mechanisms by which ovarian cancer cell aggregates clear a mesothelial monolayer, we have taken several different approaches. First, 21 ovarian cancer cell lines were classified according to their ability to clear a mesothelial monolayer. 14 of the cell lines were able to induce clearance in the monolayer, whereas 8 could not. Western blot analysis of the expression levels of several integrins in the 21 cell lines revealed a positive correlation between α5 integrin expression and clearance ability. Furthermore, blocking α5 integrin function, using a function-blocking antibody, significantly inhibited mesothelial clearance in 6 out of 8 cell lines tested. In addition, data from RNA microarrays on the 21 cell lines were analyzed to identify genes that are differentially expressed in intercalation-competent and -incompetent tumor cell lines. The microarray analysis revealed several candidate genes, including: SEPT6, GJA1, VIM, and SLIT2 (up) as well as IL23A, INHIBB and LCN2 (down). The functional importance of these genes, and others, in mesothelial clearance is currently being investigated through loss-of-function and gain-of-function genetic manipulations. Finally, we have initiated a targeted RNAi screen using one of the ovarian cancer cell lines, OVCA433, that aggressively clears the mesothelial monolayer. We plan to include siRNAs that were shown to regulate migration in a previous screen our lab had performed, as well as, other siRNAs directed against genes known to be involved in migration. In a pilot screen using 54 siRNAs, we found several genes that decrease mesothelial clearance, including EPHB2, IGFR1, and MAP3K11. We are expanding the screen to include genes that are differentially expressed in clearance competent and incompetent ovarian tumor cell lines. The hits form these screens will be validated and characterized to identify the cellular pathways that regulate mesothelial clearance. Lastly, we will determine, in vivo, if these genes regulate mesothelial invasion in a mouse model of peritoneal metastasis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2011-4751