Inhibition of HER-2/neu Kinase Impairs Androgen Receptor Recruitment to the Androgen Responsive Enhancer

Abstract

Advanced prostate cancer invariably recurs despite androgen deprivation therapy. The androgen receptor (AR) likely plays a key role in this progression and in the continued survival and proliferation of prostate cancer cells in the low androgen environment. Cross-talk with growth factor receptors, such as epidermal growth factor receptor (EGFR) family, has been postulated as a potential mechanism to activate AR in recurrent prostate cancer. We have investigated the role of HER-2/neu (ErbB-2) tyrosine kinase in AR function by characterizing the effect of inhibiting endogenous HER-2 activity in LNCaP cells. We used two independent methods, expression of intracellular single-chain antibody against HER-2 and treatment with a novel dual EGFR/HER-2 kinase inhibitor GW572016 (lapatinib). Expression of intracellular HER-2 antibody scFv-5R and treatment with GW572016 inhibited HER-2 signaling. This HER-2 inhibition led to impairment of AR-mediated functions, such as androgen-stimulated growth and the induction of endogenous prostate-specific antigen (PSA) mRNA and protein. Androgen-stimulated recruitment of AR and histone acetylation at the androgen responsive enhancer of the PSA gene, detected by chromatin immunoprecipitation analysis, were impaired by HER-2 inhibition. GW572016 was more potent in its ability to inhibit PSA expression and AR recruitment and histone acetylation than the EGFR-selective kinase inhibitor ZD1839 (gefitinib), consistent with the HER-2 kinase playing the major role in AR regulation. These results show that HER-2 signaling is required for optimal transcriptional activity of AR in prostate cancer cells and suggest that HER-2 inhibition may provide a novel strategy to disrupt AR function in prostate cancer. (Cancer Res 2005; 65(8): 3404-9)

Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation

“…These results suggest that Ack1 is a critical intermediate of HER2 signaling in prostate cancer cells and its activation of ARregulated genes (13). The effect of Ack1 knockdown on androgen target gene expression such as PSA and hK2 and recruitment and DNA binding of AR is similar to the reported effect of HER2 inhibition or knockdown (14,15). Cells expressing activated Ack1 exhibit enhanced recruitment and DNA binding of AR and increased androgen target gene expression.…”

“…We and others have demonstrated that heregulin-dependent HER2/HER3 receptor tyrosine kinase signaling enhances AR activity (12)(13)(14)(15); the effect of activated Ack1 on AR-dependent gene expression was similar to that which we observed by heregulin-dependent activation of the HER2/HER3 heterodimer (13). Therefore, we tested whether Ack1 may act as a downstream mediator of HER2/ HER3 on AR.…”

“…Receptor tyrosine kinase HER2 has been previously shown to modulate AR signaling, in part, through its effect on the recruitment and binding of AR to the androgen-responsive enhancer elements (14,15). Ligand-activated HER2 led to AR tyrosine phosphorylation, which was abrogated by Ack1 knockdown.…”

“…Pooled LNCaP cells stably expressing myc-tagged caAck, kdAck, and wild-type wAck were derived by retrovirus transduction and selection in puromycin and were implanted in nude mice (5) (24) were transfected by using Effectine (QIAGEN, Valencia, CA) or Fugene (Roche Diagnostics, Indianapolis, IN) as per the manufacturer's protocol, and immunoprecipitation and immunoblotting were performed as described earlier (5). ChIP analysis was performed by using an antibody against AR (Santa Cruz Technology, Santa Cruz, CA) or Ack1 (5) as described earlier (14). The GST-fusion protein purification and in vitro kinase assay were performed as described earlier (25).…”

“…3A). To address the role of HER2 in heregulin-dependent Ack1 activation, we used LNCaP-scFv-5R cells, in which HER2 signaling was abolished by expression of a single-chain intracellular antibody against HER2 that sequesters nascent HER2 in the endoplasmic reticulum (14). Heregulin-induced activation of Ack1 was abolished in LNCaP-scFv-5R cells (Fig.…”