Title

The Rho GTPase guanine nucleotide exchange factor Vav3 enhances androgen receptor transcriptional activity through ligand-dependent and independent mechanisms

Date of Award

2005

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmacology

First Committee Member

Keith Webster, Committee Chair

Abstract

The progression of prostate cancer from androgen dependence to androgen independence marks the aggressive form of this disease. To gain an understanding of the mechanisms involved in this progression, we investigated gene-expression differences between the androgen-dependent human prostate cancer cell line, LNCaP, and its androgen-independent (AI) derivative, LNCaP-R1. A striking difference was an 8.5-fold up-regulation of Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), in the AI cells. The Rho family of small GTPases regulates a variety of cellular processes including actin cytoskeleton reorganization and cell proliferation. GEFs promote the exchange of GDP for GTP thereby favoring the active state of Rho proteins. Inhibition of Vav3 reduced Rac1 activity in AI prostate cancer cells. Conversely, expression of a constitutively active Vav3 mutant increased Rac1 activity in LNCaP cells. These findings suggest that Vav3 can function as a GEF in prostate cancer cells. We investigated mechanisms by which Vav3 may facilitate progression to AI. A large body of research supports a continued role for the androgen receptor (AR) in AI disease. Since LNCaP-R1 cells exhibit enhanced AR transcriptional activity, we investigated the effects of Vav3 on AR-regulated gene expression. Reporter gene assays showed that Vav3 enhanced AR transactivation of a variety of androgen-regulated gene promoters and knock down of Vav3 resulted in decreased AR transcriptional activity. Vav3 also increased androgen-induced levels of PSA mRNA. Further, Vav3 increased AR activity at low doses (0.1 nM) of androgen. This finding is particularly relevant, as androgen-ablated patients retain AR signaling and low androgen levels in prostate tissue. Vav3 did not affect AR levels or interact with AR. We mutated several functional domains of Vav3 to define potential mechanisms of Vav3 effects on AR. Mutational analysis revealed that the GEF function is dispensable for Vav3-mediated enhancement of AR activity; however, the pleckstrin homology (PH) domain is required. These findings suggest that Vav3 mediates its effects on AR through protein/protein interactions. Additionally introduction of a constitutively active Vav3 mutant (C.A. Vav3) resulted in ligand-independent activation of AR. In contrast to Vav effects on ligand occupied AR, the effects of C.A. Vav3 on AR were GEF-dependent. These findings suggest that Vav3 may allow continued AR signaling under conditions of low androgen through both ligand dependent and independent mechanisms.

Keywords

Health Sciences, Oncology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3198714