Brief Overview of Three On-going Cancer Biology Projects in the Laboratory



Introduction: One in nine women will be diagnosed with breast cancer. A significant percentage of women will already have metastatic cancers at the time of diagnosis and even though the 5-year survival rates have improved, a majority of women will still succumb to recurrent disease. To develop metastatic disease, the extracellular matrix surrounding the cancer must be degraded, allowing cancer cells to travel to distant sites. Matrix degradation is mediated by proteases, one of which is urokinase plasminogen activator (uPA). A specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), regulates uPA activity. Surprisingly, an elevated level of PAI-1 is a poor prognostic factor for breast cancer patients. It is unclear why a substance that blocks matrix degradation would be linked to a poor prognosis. Our objective is to understand the "paradox" of why too much PAI-1 is detrimental to women with breast cancer. We believe that expression of PAI-1 confers a survival advantage upon breast cancer cells. Our results will shed valuable information on the role of PAI-1 in one of the most significant problems hindering the treatment of women with breast cancer, invasion and metastasis.

Understanding recurrent disease is one of the most critical areas of ovarian cancer biology and fundamental for the development of effective treatment regimens. The vast majority of women diagnosed with ovarian cancer will experience recurrent disease within 5 years. Treatment options for these patients are often limited. The seeding of metastatic tumor masses throughout the peritoneal cavity marks recurrent ovarian cancer. Metastatic cancers are the result of the migration of cells from the initial tumor mass and occur following both loss of adhesion between cells and the extracellular matrix and significant alterations in the cell architecture. The destruction of adhesive contacts is made in part by members of the plasminogen activator family. Expression of urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) have been shown to be up-regulated in the majority (>50%) of ovarian cancers and are independent poor prognostic factors for patient survival. As with other cancers, uPA expression and activity is believed to be critical for metastasis. We are studying ovarian tumor cells lines and their ability to adhere, migrate and invade in an in vitro environment, and the signaling process that is transmitted by the plasminogen activator system. We are studying by immunohistochemistry, the occurrence of serpins and their proteases in primary and metastatic ovarian cancer. We believe that the results of this work will ultimately benefit women with metastatic, recurrent ovarian cancer.

PPARγ, Adipocytes, Breast Cancer and Serpins-Plasminogen Activator System: We hypothesize that adipocytes as stromal cells exert effects on breast tumor epithelial cells to support invasion.  There is increasing epidemiological evidence between consumption of a high-fat Western diet and the incidence of breast cancer.  Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors expressed by adipocytes.  PPARγ is expressed by adipocytes and is found in several breast tumor cell lines.  Natural ligands of PPARγ (ω-3 and ω-6 fatty acids, 15-deoxyΔ12, 14-prostaglandin J2, and some phytoestrogens) modulate adipocyte function and differentiation.  We also propose that adipocytes have the potential to modulate breast tumor cell invasion through the plasminogen activator system.  PPARγ activation in adipocytes increases the expression of urokinase plasminogen activator (uPA) and the inhibitor plasminogen activator inhibitor-1 (PAI-1).  We want to prove that adipocytes contribute to the invasive process in breast cancer, and that modulation of PPARγ activity in adipocytes, by diet as such, could help prevent or limit invasion.  We hypothesize that PPARγ ligands upregulate PAI-1, uPA, and (urokinase plasminogen activator receptor (uPAR) in adipocytes to promote invasion-linked signaling pathways.  We also hypothesize that increased expression of PAI-1 confers a survival advantage upon breast cancer cells by altering migration and invasive characteristics. Potential Outcome and Benefits of the Research: This research ascribes novel activities to tumor-associated adipocytes, to PAI-1, and to PPARγ ligands that could promote an invasive phenotype in breast cancer.  In addition, our potential results have translational implications in the prevention of breast cancer through diet. 

"PAI-1 Cycle" in Breast Cancer:  We are studying the hypothesis that the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is up-regulated as a consequence of the Metabolic Syndrome, which exerts a detrimental effect on both breast tumor epithelial cells and vascular endothelial cells to support invasion.  We propose the existence of a “PAI-1 cycle”, sustained by the Metabolic Syndrome, whose action is manifest in the tumor microenvironment that promotes numerous pathological consequences.  The incidence of type 2 diabetes and breast cancer is increased in the elderly, and they both share obesity as a common risk factor.  We are studying how adipocytokines produced due to the Metabolic Syndrome alter PAI-1 expression to promote angiogenesis, tumor-cell migration/invasion, procoagulant microparticle formation from breast tumor and endothelial cells that generate thrombin, which then further propagates PAI-1 synthesis, all of which culminates to provide a chemotherapy-resistant breast tumor microenvironment.

Down-regulation of Phosphatidylinositol 3-Kinase/Akt Increases PAI-1 Expression to Inhibit Migration and Invasion of Cancer Cells: Increased levels of urokinase-type plasminogen activator (uPA) are associated with shortened overall survival in ovarian cancer patients.  Additionally, elevated levels of the serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1), an uPA inhibitor, have also been correlated with an unfavorable prognosis in ovarian cancer.  Therefore, it is critical to understand the signaling pathways that regulate PAI-1 and uPA expression in cancer cell migration-invasion.  We are studying the PI3K/Akt, RhoA/Rho-kinase, p38 MAPK and ERK 1/2 MAPK pathways and their modulation of PAI-1 and uPA expression and wound-induced cell migration in SKOV-3 ovarian cancer cells using cell and molecular biology techniques.  The PI3K/Akt pathway is being examined using pharmacological inhibitors (LY294002 and Wortmannin), Akt siRNA, and constitutively active Akt adenovirus in the SKOV-3 cells.  We have found that the PI3K/Akt pathway in SKOV-3 cells negatively regulates PAI-1 expression.  We also confirm that the level of PI3K/Akt activity positively correlates with migratory abilities and uPA expression.  A reduction in active Akt results in an increase in PAI-1 expression coupled with a decrease in uPA expression to ultimately result in reduced cell migration and invasion.  By contrast, an increase in Akt activity reduces PAI-1 expression and results in an increase in SKOV-3 wound-induced cell migration.  These results suggest an overall ovarian tumor-protective role for PAI-1, and these results demonstrate a new relationship with the PI3K/Akt system and PAI-1 expression that further denotes the complex role that PAI-1 has in cancer.