Purpose: The androgen receptor (AR) is a liganddependent transcription factor that mediates gene expression and growth of normal and malignant prostate cells. In prostate tumors that recur after androgen withdrawal, the AR is highly expressed and transcriptionally active in the absence of testicular androgens. In these ''androgenindependent'' tumors, alternative means of AR activation have been invoked, including regulation by growth factors and their receptors in prostate cancer recurrence.Experimental Design and Results: In this report, we show that HER receptor tyrosine kinases 1 through 4 are expressed in the CWR-R1 recurrent prostate cancer cell line; their stimulation by epidermal growth factor (EGF) and heregulin activates downstream signaling, including mitogen-activated protein kinase and phosphatidylinositol-3 kinase and Akt pathways. We show that heregulin activates HER2 and HER3 and increases androgen-dependent AR transactivation of reporter genes in CWR-R1 cells. Tyrosine phosphorylation of HER2 and HER3, AR transactivation, and cell proliferation induced by heregulin were more potently inhibited by the EGFR/HER2 dual tyrosine kinase inhibitor GW572016 (lapatinib) than the EGFR-specific inhibitor ZD1839 (gefitinib). Basal proliferation in the absence of growth factors was also inhibited by GW572016 to a greater extent than ZD1839, suggesting that low level HER2/HER3 activation, perhaps by an autocrine pathway contributes to the proliferation signal.Conclusions: These data indicate that heregulin signaling through HER2 and HER3 increases AR transactivation and alters growth in a recurrent prostate cancer cell line. Therefore, inhibition of low-level HER2 signaling may be a potential novel therapeutic strategy in prostate cancer.

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)

Cell proliferation, differentiation, and survival are highly coordinated processes during the development and maturation of the mammary gland, and control of these mechanisms is critical for the prevention of breast cancers (reviewed in reference 39). Aberrant regulation of the HER/ErbB family of receptor tyrosine kinases (RTKs) and their ligands is a common occurrence in many human cancers, including breast cancer (14,15,45). This family consists of four related members, HER1/ErbB1/EGFR (epidermal growth factor receptor), HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4. Each protein is comprised of a large amino-terminal extracellular domain, a transmembrane domain, and a large intracellular domain with a tyrosine-rich carboxy-terminal region and a tyrosine kinase-like sequence (27,33,56). The tyrosine kinase activity of the ErbBs is induced upon ligand interaction, leading to receptor dimerization (homo-and heterodimerization) and subsequent receptor transphosphorylation. Although HER2 is the preferential heterodimeric partner, HER2 does not bind any conventional ligand within the two major families of ErbB ligands (EGF-like ligands and heregulin [HRG]/neuregulin-like ligands) and therefore relies on HER1, HER3, or HER4 for activation of its tyrosine kinase activity.The well-documented growth stimulatory effects of HER1 and HER2 have driven the investigation of ErbB signaling in breast cancer. HER1 is expressed in nearly all human carcinomas, including breast cancers, while nearly 20 to 25% of breast cancers overexpress HER2 and/or exhibit gene amplification at the her2 locus (26,37,40,48). Expression of either HER1 or HER2 in tumor specimens correlates with a shorter survival period, a higher grade of malignancy, and an overall poor prognosis (10, 12-14, 19, 24). Genetically engineered animal models of breast cancer confirm the role of HER1 and HER2 in driving proliferation of the mammary epithelium (reviewed in reference 39). Recent evidence suggests that increased expression of HER3 in breast cancers also correlates with a poor prognosis. HER3 is overexpressed in about 20% of all breast cancers and is frequently coexpressed with HER2 (2,5,10,23,31,52,53). This has generated the hypothesis that HER2/ HER3 heterodimers may function to simultaneously drive cellular proliferation and survival in breast cancer cells.In contrast, there is evidence that HER4 expression correlates with a more differentiated tumor grade, longer survival, and positive prognostic indicators, such as estrogen receptor expression (1,17,29,38,41,44). Women whose breast tumors express HER4 exhibit the lowest risk of death due to cancer compared to women whose tumors express HER1, HER2, or HER3. During breast development, HER4 expression and activity (measured by tyrosine phosphorylation) are lowest during phases of epithelial cell proliferation and highest during phases of differentiation (35). Mammary glands from mice that lack HER4 activity, either by Cre-Lox technology, cardiac-specific transgene rescue of a HER4 knockout (with the mammary ...