Disrupting Ovarian Cancer Metastatic Colonization: Insights from Metastasis Suppressor Studies

Ovarian cancer affects approximately 25,000 women in the United States each year and remains one of the most lethal female malignancies. A standard approach to therapy is surgical cytoreduction, after which the remaining microscopic residual disease is treated with chemotherapy. The vast majority of patients have disease recurrence, underscoring the crucial need for approaches to control the regrowth, or colonization, of tissues after local treatment. Improved therapies require mechanistic information about the process of metastatic colonization, the final step in metastasis, in which cancer cells undergo progressive growth at secondary sites. Studies of metastasis suppressors are providing insights into events controlling metastatic colonization. This paper reviews our laboratory's approach to the identification, characterization, and functional testing of the JNKK1/MKK4 metastasis suppressor in ovarian cancer metastatic colonization. Specifically, we demonstrate that interaction of ovarian caner cells with the omental microenvironment activates JNKK1/MKK4 resulting in decreased proliferation without affecting apoptosis. The potential role of the omental microenvironment, specifically milky spot structures, is also described. It is our goal to provide this work as a usable paradigm that will enable others to study metastasis suppressors in clinical and experimental ovarian cancer metastases

FYN is overexpressed in human prostate cancer

To test the hypothesis that FYN , a member of the SRC family of kinases (SFKs), is up-regulated in prostate cancer, as FYN is functionally distinct from other SFKs, and interacts with FAK and paxillin (PXN), regulators of cell morphology and motility. MATERIALS AND METHODS Through data-mining in Oncomine ( http://www.oncomine.org ), cell-line profiling with immunoblotting, quantitative reverse transcription and polymerase chain reaction (RT-PCR) and immunohistochemical analysis, we described FYN expression in prostate cancer. The analysis included 32 cases of prostate cancer, nine of prostatic intraepithelial neoplasia (PIN) and 19 normal prostates. Samples were scored for the percentage of stained glands and intensity of staining (from 0 to 3). Each sample was assigned a composite score generated by multiplying percentage and intensity. RESULTS Data-mining showed an eight times greater FYN expression in prostate cancer than in normal tissue; this was specific to FYN and not present for other SFKs. Expression of FYN in prostate cancer cell lines (LNCaP, 22Rv1, PC3, DuPro) was detected using quantitative RT-PCR and immunoblotting. Expression of FYN and its signalling partners FAK and PXN was detected in human tissue. Comparing normal with cancer samples, there was a 2.1-fold increase in median composite score for FYN ( P  < 0.001) 1.7-fold increase in FAK ( P  < 0.001), and a doubling in PXN ( P  < 0.05). There was a 1.7-fold increase in FYN ( P  < 0.05) and a 1.6-fold increase in FAK ( P  < 0.01) in cancer compared with PIN. CONCLUSIONS These studies support the hypothesis that FYN and its related signalling partners are up-regulated in prostate cancer, and support further investigation into the role of the FYN as a therapeutic target.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71987/1/j.1464-410X.2008.08009.x.pd

<em>In Vivo</em> and <em>Ex Vivo</em> Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases

Building on the foundation of daring hypotheses: Using the MKK4 metastasis suppressor to develop models of dormancy and metastatic colonization

AbstractThe identification of a novel metastasis suppressor function for the MAP Kinase Kinase 4 protein established a role for the stress-activated kinases in regulating the growth of disseminated cancer cells. In this review, we describe MKK4’s biological mechanism of action and how this information is being used to guide the development of new models to study cancer cell dormancy and metastatic colonization. Specifically, we describe the novel application of microvolume structures, which can be modified to represent characteristics similar to those that cancer cells experience at metastatic sites. Although MKK4 is currently one of many known metastasis suppressors, this field of research started with a single daring hypothesis, which revolutionized our understanding of metastasis, and opened up new areas of exploration for basic research. The combination of our increasing knowledge of metastasis suppressors and such novel technologies provide hope for possible clinical interventions to prevent suffering from the burden of metastatic disease

Abstract A82: Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization

Abstract Introduction: The omentum is the primary site of metastasis in both ovarian cancer models and clinical disease. It is composed predominantly of adipose studded with lymphoreticular organs (milky spots), distinguishing it from other peritoneal adipose. Milky spots are specialized for immune cell trafficking and peritoneal surveillance. We and others have shown that ovarian cancer cells exploit the physiologic function(s) of these structures for omental metastatic colonization. The purpose of this study was to identify cellular and molecular mechanisms responsible for ovarian cancer homing to and growth within milky spot structures. Experimental Procedures: Quantitative in vivo and ex vivo assays were used to evaluate human (SKOV3ip.1, HEYA8, and CaOV3) and murine (ID8) ovarian cancer cell localization to milky spots on the murine omental fat band. Assays were conducted using C57/Bl6 mice or those lacking B cells (Igh6-/-); T cells (Nude); B and T cells (Rag1-/-); or B, T, and NK cells (BN XID). In vitro assays were used to assess the migration-promoting activity of omentum- and macrophage-conditioned media. Standard approaches were used to assess protein expression, cell growth and viability, etc. Rationale: Milky spots provide resident tissue macrophages and lymphocytes needed for peritoneal homeostasis. Macrophages and stromal cells secrete chemokines promoting peritoneal lymphocyte homing to the omentum. In response to peritoneal irritants, activated CD11b+ milky spot macrophages organize coordinated expansion of vascular and stromal compartments. The increase in both the number and size of milky spots is needed to process particulates, resolve infections, and encapsulate foreign bodies. Hypothesis: CD11b+ cells secrete homeostatic chemokines promoting G-protein-dependent migration of ovarian cancer cells to milky spots. Ovarian cancer cell binding to adhesion molecules on the milky spot surface activates CD11b+ cell-dependent tissue remodeling, creating a microenvironment promoting ovarian cancer growth. New Findings: Consistent with our hypothesis, in vivo assays showed that macrophage depletion prior to injection of ID8 and SKOV3ip.1 cells prevented microscopic metastasis formation. In vitro assays found that macrophages are required for ovarian cancer cell localization to milky spots. These data prompt the hypothesis that CD11b+ cells produce a factor(s) responsible for the migration-promoting ability of omentum-conditioned media. To test this, media was conditioned by omental adipose isolated from mice after macrophage depletion. In support of our hypothesis, the migration-promoting activity of macrophage-depleted omentum-conditioned media was on a par with that of media conditioned by milky spot-deficient adipose. Further, media conditioned by CD11b+ cells recapitulates the migration-promoting activity exhibited by omentum-conditioned media. In vitro and ex vivo assays were used to test whether ovarian cancer cells utilize mechanisms analogous to lymphocyte trafficking for milky spot homing. Specifically, cells were pretreated with pertussis toxin or vehicle alone, and then evaluated for migration in response to omentum-conditioned media and milky spot localization ex vivo. Consistent with data reported for lymphocyte homing, pertussis toxin pre-treatment caused a 40% to 50% reduction in ovarian cancer cell homing. Conclusions and Current Efforts: Our data support a model in which CD11b+ macrophages secrete one or more chemokines promoting G-protein receptor-dependent ovarian cancer cell migration, and potentially integrin activation, which mediate milky spot homing. Current experiments focus on identification of integrin-ligand interactions, CD11b+ cell activation, and defining the link between ovarian cancer cell growth and increase in milky spot size and number. Citation Format: Venkatesh Krishnan, Kelly Mitchell, Jason Miska, Sophia George, Patricia Shaw, Victoria Seewaldt, Maciej Lesniak, Marina Chekmareva, Lev Becker, Vinita Parkash, Cindy Miranti, Carrie Rinker-Schaeffer. Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A82