Src signaling plays an important role in prostate cancer (PrCa) progression. It has previously been shown that Src interacts with androgen receptor (AR) and enhances AR transactivation. Although it has been shown that Src promotes AR activity, the underlying pathway has not been defined. To help characterize the Src-AR pathway, the cellular localizations of Src, p-Src, AR, pAR, and Prostate Specific Antigen (PSA, an AR target gene) were analyzed in androgen-dependent (AD) LNCaP cells and in androgen-independent (AI) castration-resistant C4-2B cells. Using sub-cellular fractionation, the data showed that treatment of AD cells with synthetic androgen R1881 increased p-Src, AR, pAR, and PSA in the nucleus, while the levels of c-Src remained unchanged. Treatment of AI cells with R1881 increased pSrc and AR in the nucleus, while the levels of c-Src and PSA remained unchanged. When using immunofluorescence microscopy, R1881 did not appear to increase the nuclear levels of p-Src or c-Src, so perhaps this technique is not as sensitive or quantitative as subcellular fractionation immunoblots. The presence of PSA in the nucleus was unexpected given its well proven role as a secreted protein. Nuclear PSA was observed upon androgen stimulation in AD and AI cells, and in the nucleus of AI cells upon androgen deprivation. Given PSA's ability to induce cell division and decrease apoptosis when transfected into cells, its presence in the nucleus may imply that PSA acts there to help induce tumorigenesis. The effect of Src on AR activity was further studied by transfection of a dominant negative src (SrcK298M) in AD and AI cells. Transfection with SrcK298M did not affect PSA expression in LNCaP cells, but strongly inhibited PSA levels in AI cells. Integrin signaling through Src was investigated in PrCa by ligand binding assay in AD and AI cells. The data showed that alpha v beta 3 integrin (but not alpha v beta 6) upon attachment to fibronectin or TGF-beta-latency associated peptide (TGF- beta-LAP) increases p-Src levels in AD and AI cells, while the levels of c-Src, PSA, and AKT remain unchanged. Thus, alpha v beta 3 integrin facilitates Src signaling, but the activation does not appear to affect AR transactivation. In conclusion, these data show that Src is required for AR activity and, consequently, PSA expression in AI prostate cancer cells, but not in AD cells. These data also suggest that the nuclear co-localization of p-Src, AR and PSA might allow macromolecular interactions, which can further enhance AR transactivation and promote disease progression. With respect to the switch in tumor progression from an AD to AI state, the data indicate that the integrin-Src pathway does not include AKT or PSA (and not AR by deduction), so perhaps other non-AR pathways help facilitate tumor growth at the AI state.
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-theses-1752 |
| Date | 05 May 2010 |
| Creators | Saxena, Parmita |
| Contributors | Samuel M. Politz, Advisor, David S. Adams, Committee Member, Lucia R. Languino, Advisor |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses (All Theses, All Years) |
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