Date of Award
Master of Science
Jennifer A. Doll
Dean Nardelli, Carol Mitchell
Background: Prostate cancer (PCa) is the most common cancer that occurs in men in the United States. To grow, tumors need to induce new blood vessels growth, a process called angiogenesis. Thus, tumors down regulate molecules that inhibit this process. Two such molecules that are down-regulated in PCa are thrombospondin-1 (TSP-1) and pigment epithelium-derived factor (PEDF). Interestingly, both of these proteins also function in regulating lipid metabolism. Our lab has data to suggest that TSP-1 induces PEDF expression and lipid catabolism (lipolysis), the hydrolysis of triglycerides into fatty acids and a glycerol, in PCa cells; however, the molecular pathway of this regulation has not been elucidated. Previous studies in PCa cells have also shown that TSP-1 treatment increases the levels of activated signaling mediators, JNK and Src kinase. However, it is unclear if induction of JNK or Src is necessary for TSP-1- induced PEDF expression and/or lipolytic activity. In this study, I tested the hypothesis that Src and/or JNK signaling is required for TSP-1- induced PEDF expression and lipolytic activity in PCa cells.
Methods: PCa cell lines, PC-3 (androgen insensitive) and LNCaP (androgen sensitive), and the normal prostate epithelial cell line, RWPE-1, were used in these studies. Cells were treated for 48 h with TSP-1 (5 or 20 nM) with or without chemical inhibitor treatment, either a JNK [SP600125] or Src family kinase [PP2] inhibitor (20 μM). After 48 h, conditioned media (CM) and cell lysate (CL) samples were collected. CM was used to evaluate lipolysis activity via the free glycerol assay. PEDF levels were quanitified in both CM and CL by enzyme linked immunosorbent assay (ELISA) and Western blot analysis.
Results: In RWPE-1, LNCaP, and PC-3 cells, no significant difference was observed in cell proliferation in response to TSP-1 or to either the JNK or Src inhibitor. Viability was decreased in response to TSP-1 in the presence of JNK inhibitor or Src inhibitor in LNCaP cells; whereas, in PC-3 cells, there was a slight increase in viability with TSP-1 in the presence of the JNK inhibitor treatment (P-value <0.010). In RWPE-1 cells, both JNK inhibition and Src inhibition decreased lipolytic activity. Likewise, in the PCa cell lines, lipolytic activity was significantly decreased with either JNK or Src inhibitor treatment as compared to untreated cells (P-value <0.001). In LNCaP cells, PEDF expression levels were significantly increased with TSP-1 at 5 nM with either JNK or Src inhibitor treatment. However in RWPE-1 CL, PEDF expression was decreased with TSP-1 at 5 nM in the presence of JNK inhibitor treatment.
Conclusion: My data support that TSP-1- induced lipolytic activity is based on the activity of JNK and/or Src family kinase in RWPE-1 cells. The activation of JNK and/or Src family kinase could regulate PEDF expression in RWEP-1 and LNCaP cells. This study suggests that the JNK and/or Src kinase pathways could be a clinically relevant therapeutic target in PCa.
Khamjan, Nizar, "Thrombospondin-1 Signaling Mechanisms Regulating Pigment Epithelium-Derived Factor Expression and Lipolytic Activity in Prostate Cancer" (2014). Theses and Dissertations. 702.