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

Abstract

Activation of the androgen receptor (AR) may play a role in androgen-independent progression of prostate cancer. Multiple mechanisms of AR activation, including stimulation by tyrosine kinases, have been postulated. We and others have recently shown involvement of activated Cdc42-associated tyrosine kinase Ack1 in advanced human prostate cancer. Here we provide the molecular basis for interplay between Ack1 and AR in prostate cancer cells. Activated Ack1 promoted androgen-independent growth of LNCaP and LAPC-4 prostate xenograft tumors, AR recruitment to the androgen-responsive enhancer, and androgen-inducible gene expression in the absence of androgen. Heregulin-stimulated HER2 activation induced Ack1 activation and AR tyrosine phosphorylation. Ack1 knockdown inhibited heregulin-dependent AR tyrosine phosphorylation, AR reporter activity, androgen-stimulated gene expression, and AR recruitment. Ack1 was recruited to the androgen-responsive enhancers after androgen and heregulin stimulation. In 8 of 18 primary androgen-independent prostate tumor samples, tyrosine-phosphorylated AR protein was detected and correlated with the detection of tyrosine-phosphorylated Ack1. Neither was elevated in androgen-dependent tumors or benign prostate samples. Activated Ack1 phosphorylated AR protein at Tyr-267 and Tyr-363, both located within the transactivation domain. Mutation of Tyr-267 completely abrogated and mutation of Tyr-363 reduced Ack1-induced AR reporter activation and recruitment of AR to the androgen-responsive enhancer. Expression of AR point mutants inhibited Ack1-driven xenograft tumor growth. Thus, Ack1 activated by surface signals or oncogenic mechanisms may directly enhance AR transcriptional function and promote androgen-independent progression of prostate cancer. Targeting the Ack1 kinase may be a potential therapeutic strategy in prostate cancer.

Fig. 1.

Activated Ack1 promotes androgen-independent growth of prostate xenograft tumors, androgen-regulated gene expression, and AR recruitment. (A) LNCaP cells (2 × 10⁶ cells per injection) stably expressing caAck or vector control were injected s.c. into the flanks of castrated nude mice. (B) LAPC-4 cells (2 × 10⁶ cells per injection) stably expressing caAck or kinase dead kdAck were injected s.c. into the flanks of castrated nude mice. (C and D) LNCaP cells stably expressing caAck, kdAck, or vector were treated with dihydrotestosterone (DHT) (10 nM) for 12 h. Quantitative RT-PCR for PSA (C) and hK2 (D) mRNA was performed. Data shown are representative of three similar independent experiments. (E) LNCaP cells stably expressing caAck or vector were treated with DHT for 2 h, and ChIP analysis for AR binding to the androgen response element (ARE) III enhancer of the PSA gene was performed by using quantitative PCR. Data shown are representative of three similar independent experiments.

Fig. 2.

Activated Ack1 binds and tyrosine phosphorylates AR in vivo and in vitro; primary human androgen-independent prostate cancer specimens express tyrosine-phosphorylated AR and Ack1. (A and B) LNCaP or LAPC-4 cells expressing myc-tagged Ack1 constructs were immunoprecipitated by using antibodies described, followed by immunoblotting analysis. (C) Schematic of GST-AR and GST-cAR constructs. TAD, transactivation domain; DBD, DNA-binding domain; LBD, ligand-binding domain. (D) Purified GST-AR and GST-cAR proteins were incubated with caAck or kdAck and ATP. Ack and GST-AR proteins were separated, electrophoresed, and subjected to immunoblotting analysis. (E) Equal amounts of protein from lysates of AICaP, ADCaP, and BPH samples were subjected to immunoprecipitation, followed by immunoblotting as indicated. (F) The levels of tyrosine-phosphorylated AR and Ack1 from immunoblots of AICaP samples were quantified by using SCION Image (Frederick, MD) software and plotted in arbitrary units. Pearson's product-moment correlation coefficient (r) and Spearman's rank correlation coefficient (r_s) were calculated by using SAS statistical software, version 9.1 (SAS Institute, Cary, NC).

Fig. 3.

Heregulin-mediated HER2 activation leads to Ack1 activation and AR tyrosine phosphorylation; Ack1 is required for AR target gene expression and recruitment. (A) LNCaP and LAPC-4 cells were treated with heregulin (10 ng/ml) for indicated times. Equal amounts of protein lysates were subjected to immunoprecipitation, followed by immunoblotting as indicated. (B) LNCaP-scFv-5R cells expressing the intracellular antibody against HER2 (14) were treated with EGF (10 ng/ml) or heregulin (10 ng/ml) for indicated time intervals, and protein lysates were subjected to immunoprecipitation followed by immunoblotting as indicated. (C) LNCaP and LAPC-4 cells were treated with heregulin (10 ng/ml) for indicated times. Equal amounts of protein lysates were subjected to immunoprecipitation, followed by immunoblotting as indicated. (D) LNCaP cells were transfected with control or Ack1-specific siRNA (50 nM), and 48 h after transfection cells were treated with heregulin (10 ng/ml) for 90 min. Equal amounts of protein lysates were subjected to immunoprecipitation, followed by immunoblotting as indicated. (E) LAPC-4 cells were transfected with the ARR2PB-luciferase reporter (500 ng) and the AR vector (50 ng) and control or Ack1-specific siRNA (100 nM). Twenty-four hours after transfection, cells were treated with DHT (10 nM) or heregulin (10 ng/ml) for 16 h, and luciferase activity was determined. (F) LNCaP cells were transfected with control or Ack1-specific siRNA sequences (50 nM). (G and H) LAPC-4 cells were transfected with control or Ack1-specific siRNA (50 nM), and 24 h after transfection cells were treated with DHT (10 nM) for 16 h or untreated. Quantitative RT-PCR for PSA (G) and hK2 (H) mRNA was performed. Data are representative of three similar independent experiments. (I and J) LAPC-4 cells transfected with siRNA as above were treated with DHT for 2 h, and ChIP analysis for binding of Ack1 (I) or AR (J) to the ARE III enhancer of the PSA gene was performed by using quantitative PCR.

Fig. 4.

Ack1 phosphorylates AR at Tyr-267 and Tyr-363. (A) FLAG-tagged full-length AR and the gAR-deletion construct (amino acids 142–540) are shown. (B) 293T cells were transfected with the AR or gAR expression vector (2 μg), along with the caAck or kdAck expression vector (2 μg). Twenty-four hours after transfection, protein lysates were subjected to immunoprecipitation, followed by immunoblotting as indicated. (C) Schematic of AR domains and point mutants. (D) 293T cells were transfected with the AR or AR point mutant expression vector (1 or 2 μg for Y267F to keep AR protein expression levels similar) and the caAck or kdAck vector (2 μg). Twenty-four hours after transfection, protein lysates were subjected to immunoprecipitation, followed by immunoblotting as indicated. Relative expression of phosphorylated AR is shown below the top panel.

Fig. 5.

Mutation of AR phosphorylation sites leads to inhibition of Ack1-induced AR transactivation, DNA binding, and androgen-independent growth of xenograft tumors. (A) 293T cells were transfected with the AR or AR point mutant vector (500 ng or 1 μg for Y267F), caAck (or kdAck) (500 ng), and the ARR2PB-luciferase reporter (500 ng). Twenty-four hours after transfection, cells were treated with DHT (10 nM) for 16 h, and luciferase activity was determined. Data shown represent mean + SE of four independent experiments. (B) LNCaP cells were transfected with ARR2PB-luciferase (500 ng) and the AR or AR point mutant vector (200 or 400 ng for Y267F) without Ack1 vector. Twenty-four hours after transfection, cells were treated with DHT (10 nM) for 16 h, and luciferase activity was determined. (C) LNCaP-caAck cells stably expressing FLAG-tagged AR or point mutants were treated with DHT for 2 h, and ChIP analysis was performed by using FLAG antibody. Precipitated DNA was subjected to quantitative PCR analysis targeting the ARE III enhancer of the PSA gene. (D) LNCaP-caAck cells stably expressing FLAG-tagged wild-type AR or Y267F or Y363F mutants of AR were injected s.c. (2 × 10⁶ cells per injection) into the flanks of castrated nude mice.

Fig. 6.

Model of Ack1 and AR activation. Cell surface receptors activated by autocrine, paracrine, or mutational events may activate the Ack1 kinase, as indicated by Ack1 autophosphorylation. Subsequently, Ack1 binds and phosphorylates AR protein. The AR–Ack1 complex translocates to the nucleus and binds to the AREs on DNA, where it activates AR-dependent gene expression in the absence of or at suboptimal levels of androgen. Alternative mechanisms, including Ack1 gene amplification or mutation, may activate intracellular Ack1.