Dasatinib inhibits site-specific tyrosine phosphorylation of androgen receptor by Ack1 and Src kinases

Abstract

Activation of androgen receptor (AR) may play a role in the development of castration resistant prostate cancer. Two intracellular tyrosine kinases, Ack1 (activated cdc42-associated kinase) and Src, phosphorylate and enhance AR activity and promote prostate xenograft tumor growth in castrated animals. However, the upstream signals that activate these kinases and lead to AR activation are incompletely characterized. In this study, we investigated AR phosphorylation in response to non-androgen ligand stimulation using phospho-specific antibodies. Treatment of LNCaP and LAPC-4 cells with epidermal growth factor (EGF), heregulin, Gas6 (ligand binding to Mer receptor tyrosine kinase and activating Ack1 downstream), interleukin (IL)-6 or bombesin stimulated cell proliferation in the absence of androgen. Treatment of LNCaP and LAPC-4 cells with EGF, heregulin, or Gas6 induced AR phosphorylation at Tyr-267; IL-6 or bombesin treatment did not. AR phosphorylation at Tyr-534 was induced by treatment with EGF, IL-6 or bombesin, but not by heregulin or Gas6. siRNA-mediated knockdown of Ack1 or Src showed that Ack1 mediates heregulin- and Gas6-induced AR Tyr-267 phosphorylation whereas Src mediates Tyr-534 phosphorylation induced by EGF, IL-6, and bombesin. Dasatinib, a Src inhibitor, blocked EGF-induced Tyr-534 phosphorylation. In addition, we show dasatinib also inhibited Ack1 kinase. Dasatinib inhibited heregulin-induced Ack1 kinase activity and AR Tyr-267 phosphorylation. Dasatinib inhibited heregulin-induced AR-dependent reporter activity. Dasatinib also inhibited heregulin-induced expression of endogenous AR target genes. Dasatinib inhibited Ack1-dependent colony formation and prostate xenograft tumor growth in castrated mice. Interestingly, Ack1 or Src knockdown or dasatinib did not inhibit EGF-induced AR Tyr-267 phosphorylation or EGF-stimulated AR activity, suggesting the existence of an additional tyrosine kinase that phosphorylates AR at Tyr-267. These data suggest that specific tyrosine kinases phosphorylate AR at distinct sites and that dasatinib may exert anti-tumor activity in prostate cancer through inhibition of Ack1.

Loss of Androgen-Regulated MicroRNA 1 Activates SRC and Promotes Prostate Cancer Bone Metastasis

“…We show that SRC is a direct miR-1 target and that SRC RNA and protein levels are modulated by miR-1 in various prostate cancer model systems. SRC expression responsiveness to the SRC inhibitor dasatanib (5,16). We show here that in experimental models, AR directly and positively contributes to the regulation of miR-1 transcription.…”

“…Experimental models have shown that SRC interacts with and phosphorylates AR, resulting in enhanced AR activity (11,16). In addition, SRC is downstream of receptors, such as epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), and upstream of signaling molecules, such as AKT and extracellular signal-regulated kinase (ERK), that have been implicated in PC survival in the absence of sufficient AR signaling (13,(17)(18)(19).…”

Regulation of the androgen receptor by post-translational modifications

“…Tyr267 appears to be the dominant functional site as mutation studies demonstrated that, whereas mutation of Tyr363 can inhibit AR activity, mutation of Tyr267 completely abolished HRG-stimulated AR activation (Mahajan et al 2007). EGF and Gas6 can also result in Tyr267 phosphorylation (Liu et al 2010b). Anti-androgens have no effect on tyrosine phosphorylation at 267 (Mahajan et al 2010); however, inhibition can be achieved using Dasatinib, a Src and Ack1 inhibitor (Liu et al 2010b).…”

“…EGF and Gas6 can also result in Tyr267 phosphorylation (Liu et al 2010b). Anti-androgens have no effect on tyrosine phosphorylation at 267 (Mahajan et al 2010); however, inhibition can be achieved using Dasatinib, a Src and Ack1 inhibitor (Liu et al 2010b). The AR isoform, AR8, has recently been identified.…”

Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression

“…In androgen-depleted conditions, tyrosine kinase, non-receptor, 2 (TNK2 or ACK1), SRC, and erythroblastic leukemia viral oncogene homolog 2 [ERBB2 (HER-2/neu)] tyrosine kinase activity can restore AR function in prostate cancer cells (14)(15)(16)(17). Increased expression of the tyrosine kinase SRC and AR can synergistically drive frank carcinoma of the mouse prostate (18).…”