Tyrosine Phosphorylation Of Androgen Receptor Research Articles

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression

Androgen receptor (AR) signaling plays important roles in breast cancer progression. We show here that Kindlin-2, a focal adhesion protein, is critically involved in the promotion of AR signaling and breast cancer progression. Kindlin-2 physically associates with AR and Src through its two neighboring domains, namely F1 and F0 domains, resulting in formation of a Kindlin-2-AR-Src supramolecular complex and consequently facilitating Src-mediated AR Tyr-534 phosphorylation and signaling. Depletion of Kindlin-2 was sufficient to suppress Src-mediated AR Tyr-534 phosphorylation and signaling, resulting in diminished breast cancer cell proliferation and migration. Re-expression of wild-type Kindlin-2, but not AR-binding-defective or Src-binding-defective mutant forms of Kindlin-2, in Kindlin-2-deficient cells restored AR Tyr-534 phosphorylation, signaling, breast cancer cell proliferation and migration. Furthermore, re-introduction of phosphor-mimic mutant AR-Y534D, but not wild-type AR reversed Kindlin-2 deficiency-induced inhibition of AR signaling and breast cancer progression. Finally, using a genetic knockout strategy, we show that ablation of Kindlin-2 from mammary tumors in mouse significantly reduced AR Tyr-534 phosphorylation, breast tumor progression and metastasis in vivo. Our results suggest a critical role of Kindlin-2 in promoting breast cancer progression and shed light on the molecular mechanism through which it functions in this process.

MP84-15 PREVENT METASTASIS BY DOUBLE CHIMERIC ANTIGEN RECEPTOR T CELLS TARGETING CIRCULATING CANCER CELLS FOR PROSTATE CANCER TREATMENT

INTRODUCTION AND OBJECTIVES: We and others have demonstrated that the androgen receptor (AR) is essential for the development and progression of prostate cancer. Although androgendeprivation therapy (ADT) is effective initially, unfortunately, metastatic prostate cancer often recurs as so-called castration resistant prostate cancer (CRPC). Overexpression of AR, AR hypersensitivity, c-terminal truncated AR variants, as well as altered steroid metabolism are considered the main mechanisms for CRPC. Current anti-AR treatments (Abiraterone and Enzalutamide) for CRPC only extend patient survival for approximately 5 months, indicating novel therapeutic agents are urgently needed. It has been shown that AR variants possess an intact and functional N-terminal domain that contains several key motifs and tyrosine residues involved in AR transactivation. METHODS: We recently designed a 14-amino acid peptide spanning the 355-368 residues (ARi14 peptide) on AR N-terminal, consisting of the partial transactivation activation unit-1 and three key tyrosine residues involved in CRPC progression. Multiple prostate cancer cell lines were used in the experiments. Trypan blue and Alamar Blue assays were used to assess cell proliferation. Western blot and anti-AR immunoprecipitation were conducted to assess AR protein levels and tyrosine phosphorylation. Quantitative RT-PCR was performed to assess PSA mRNA expression. RESULTS: Treatment of multiple AR-positive prostate cancer cells with this short peptide induced a significant reduction of AR protein, as well as AR transactivation in a luciferase reporter assay in a time-dependent manner. However, AR gene expression at the mRNA level was not altered. Further analysis revealed that ARi14 peptide abolished androgen-stimulated AR tyrosine phosphorylation in LNCaP cells, indicating a disruption of AR with its tyrosine kinase. In 22RV1 cells that harbor AR variants the ARi14 peptide dramatically reduced the protein levels of full length AR and AR variants, abolished AR-target PSA gene expression and also suppressed 22RV1 cell proliferation that Enzalutamide failed to do so. Sequence mutation analysis demonstrated that the C-terminal 8 residue portion (361-368aa) of the ARi14 is the key element for its anti-AR activities. CONCLUSIONS: Our data demonstrated that the novel short peptide ARi14 exerts a potent anti-AR activity by inducing AR protein degradation possibly through reduced tyrosine phosphorylation in Enzalutamide-resistant prostate cancer cells.

MP84-14 OVERCOMING ENZALUTAMIDE-RESISTANT WITH A NOVEL ANTI-AR PEPTIDE IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-14 OVERCOMING ENZALUTAMIDE-RESISTANT WITH A NOVEL ANTI-AR PEPTIDE IN PROSTATE CANCER CELLS Daniel Zainfeld, Ruibao Chen, J Brantley Thrasher, and Benyi Li Daniel ZainfeldDaniel Zainfeld More articles by this author , Ruibao ChenRuibao Chen More articles by this author , J Brantley ThrasherJ Brantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2238AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We and others have demonstrated that the androgen receptor (AR) is essential for the development and progression of prostate cancer. Although androgen-deprivation therapy (ADT) is effective initially, unfortunately, metastatic prostate cancer often recurs as so-called castration resistant prostate cancer (CRPC). Overexpression of AR, AR hypersensitivity, c-terminal truncated AR variants, as well as altered steroid metabolism are considered the main mechanisms for CRPC. Current anti-AR treatments (Abiraterone and Enzalutamide) for CRPC only extend patient survival for approximately 5 months, indicating novel therapeutic agents are urgently needed. It has been shown that AR variants possess an intact and functional N-terminal domain that contains several key motifs and tyrosine residues involved in AR transactivation. METHODS We recently designed a 14-amino acid peptide spanning the 355-368 residues (ARi14 peptide) on AR N-terminal, consisting of the partial transactivation activation unit-1 and three key tyrosine residues involved in CRPC progression. Multiple prostate cancer cell lines were used in the experiments. Trypan blue and Alamar Blue assays were used to assess cell proliferation. Western blot and anti-AR immunoprecipitation were conducted to assess AR protein levels and tyrosine phosphorylation. Quantitative RT-PCR was performed to assess PSA mRNA expression. RESULTS Treatment of multiple AR-positive prostate cancer cells with this short peptide induced a significant reduction of AR protein, as well as AR transactivation in a luciferase reporter assay in a time-dependent manner. However, AR gene expression at the mRNA level was not altered. Further analysis revealed that ARi14 peptide abolished androgen-stimulated AR tyrosine phosphorylation in LNCaP cells, indicating a disruption of AR with its tyrosine kinase. In 22RV1 cells that harbor AR variants the ARi14 peptide dramatically reduced the protein levels of full length AR and AR variants, abolished AR-target PSA gene expression and also suppressed 22RV1 cell proliferation that Enzalutamide failed to do so. Sequence mutation analysis demonstrated that the C-terminal 8 residue portion (361-368aa) of the ARi14 is the key element for its anti-AR activities. CONCLUSIONS Our data demonstrated that the novel short peptide ARi14 exerts a potent anti-AR activity by inducing AR protein degradation possibly through reduced tyrosine phosphorylation in Enzalutamide-resistant prostate cancer cells. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1095 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Daniel Zainfeld More articles by this author Ruibao Chen More articles by this author J Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Novel Membrane-associated Androgen Receptor Splice Variant Potentiates Proliferative and Survival Responses in Prostate Cancer Cells

Progression from the androgen-sensitive to androgen-insensitive (or castration-resistant) stage is the major obstacle for sustained effectiveness of hormonal therapy for prostate cancer. The androgen receptor (AR) and its splice variants play important roles in regulating the transcription program essential for castration resistance. Here, we report the identification of a novel AR splice variant, designated as AR8, which is up-regulated in castration-resistant prostate cancer cells. AR8 is structurally different from other known AR splice variants because it lacks a DNA binding domain and therefore, unlikely functions as a transcription factor on its own. Immunofluorescence staining revealed that AR8 was primarily localized on the plasma membrane, possibly through palmitoylation of two cysteine residues within its unique C-terminal sequence. Mutation of these putative palmitoylation sites in AR8 led to loss of its plasma membrane localization. In addition, we demonstrated that overexpression of AR8 in prostate cancer cells promoted association of Src and AR with the EGF receptor in response to EGF treatment and enhanced tyrosine phosphorylation of AR. Conversely, specific knockdown of AR8 expression in prostate cancer cells compromised EGF-induced Src activation and AR phosphorylation. This effect was accompanied with attenuation of proliferation and increased apoptosis in prostate cancer cells cultured in androgen-depleted medium. We also showed that AR8 was required for optimal transcriptional activity of AR in response to treatment of both androgen and EGF. Taken together, our results demonstrate that the membrane-associated AR8 isoform may contribute to castration resistance by potentiating AR-mediated proliferative and survival responses to hormones and growth factors.

Abstract 4533: Interaction between androgen receptor and its corepressor SLIRP is regulated by Ack1 tyrosine kinase

Abstract Reactivation of androgen receptor (AR) is critical to castration resistant progression of prostate cancer. We previously demonstrated that Ack1 (activated cdc42-associated kinase) promotes growth of prostate xenograft tumors in castrated animals by activation of AR through phosphorylation of the Tyr-267 site. However, the molecular mechanisms underlying activation of AR by Ack1 need to be fully elucidated. Using the proteomics-based approach of Differential In-Gel Electrophoresis, we searched for proteins whose interaction with AR was regulated by Ack1 by characterizing proteins coimmunoprecipitating with AR in transfected 293T cells in the absence or in the presence of activated Ack1 kinase. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. We confirmed that the interaction between AR and SLIRP is abrogated by Ack1 expression and dihydrotestosterone (DHT) in 293T cells and in LNCaP prostate cancer cells. Additionally, treatment with heregulin, a ligand activating HER-2/HER-3 and Ack1 downstream, also led to dissociation of AR and SLIRP in LNCaP cells. Tyrosine kinase activity of Ack1 was shown to be required for its ability to dissociate AR and SLIRP through the use of a kinase-dead mutant in 293T cells and the use of an Ack1 inhibitor dasatinib in LNCaP cells treated with heregulin. However, disruption of interaction between AR and SLIRP by Ack1 cannot be attributed to any known AR tyrosine phosphorylation sites since Ack1 expression led to disruption of binding between SLIRP and AR Y267F, Y363F, and Y534F mutants. Since SLIRP does not contain tyrosine residues, the substrate of Ack1 involved in AR-SLIRP dissociation is not known yet. We showed that SLIRP functions as a physiologically relevant corepressor of AR in prostate cancer cells. Chromatin immunoprecipitation analysis demonstrated that SLIRP was bound to the androgen regulated enhancers of AR target genes in the absence of androgen and that androgen or heregulin treatment led to loss of SLIRP binding at the androgen regulated enhancers. AR-dependent reporter activity was inhibited by overexpression of SLIRP and was increased by SLIRP knockdown. SLIRP knockdown also increased the expression of endogenous AR target mRNA levels of PSA and hK2. In summary, our data suggest that Ack1 kinase inhibits the interaction between AR and the SLIRP corepressor and causes dissociation of SLIRP from the AR target gene enhancers and thereby enhance AR transcriptional activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4533. doi:10.1158/1538-7445.AM2011-4533

Compensatory Upregulation of Tyrosine Kinase Etk/BMX in Response to Androgen Deprivation Promotes Castration-Resistant Growth of Prostate Cancer Cells

We previously showed that targeted expression of non-receptor tyrosine kinase Etk/BMX in mouse prostate induces prostate intraepithelial neoplasia, implying a possible causal role of Etk in prostate cancer development and progression. Here, we report that Etk is upregulated in both human and mouse prostates in response to androgen ablation. Etk expression seems to be differentially regulated by androgen and interleukin 6 (IL-6), which is possibly mediated by the androgen receptor (AR) in prostate cancer cells. Our immunohistochemical analysis of tissue microarrays containing 112 human prostate tumor samples revealed that Etk expression is elevated in hormone-resistant prostate cancer and positively correlated with tyrosine phosphorylation of AR (Pearson correlation coefficient rho = 0.71, P < 0.0001). AR tyrosine phosphorylation is increased in Etk-overexpressing cells, suggesting that Etk may be another tyrosine kinase, in addition to Src and Ack-1, which can phosphorylate AR. We also showed that Etk can directly interact with AR through its Src homology 2 domain, and such interaction may prevent the association of AR with Mdm2, leading to stabilization of AR under androgen-depleted conditions. Overexpression of Etk in androgen-sensitive LNCaP cells promotes tumor growth while knocking down Etk expression in hormone-insensitive prostate cancer cells by a specific shRNA that inhibits tumor growth under androgen-depleted conditions. Taken together, our data suggest that Etk may be a component of the adaptive compensatory mechanism activated by androgen ablation in prostate and may play a role in hormone resistance, at least in part, through direct modulation of the AR signaling pathway.

Shepherding AKT and androgen receptor by Ack1 tyrosine kinase

Ack1 (also known as ACK, TNK2, or activated Cdc42 kinase) is a structurally unique non-receptor tyrosine kinase that is expressed in diverse cell types. It integrates signals from plethora of ligand-activated receptor tyrosine kinases (RTKs), for example, MERTK, EGFR, HER2, PDGFR and insulin receptor to initiate intracellular signaling cascades. Ack1 transduces extracellular signals to cytosolic and nuclear effectors such as the protein kinase AKT/PKB and androgen receptor (AR), to promote cell survival and growth. While tyrosine phosphorylation of AR at Tyr267 regulates androgen-independent recruitment of AR to the androgen-responsive enhancers and transcription of AR target genes to drive prostate cancer progression, phosphorylation of an evolutionarily conserved Tyrosine 176 in the kinase domain of AKT is essential for mitotic progression and positively correlates with breast cancer progression. In contrast to AR and AKT, Ack1-mediated phosphorylation of the tumor suppressor Wwox at Tyr287 lead to rapid Wwox polyubiquitination followed by degradation. Thus, by its ability to promote tumor growth by negatively regulating tumor suppressor such as Wwox and positively regulating pro-survival factors such as AKT and AR, Ack1 is emerging as a critical player in cancer biology. In this review, we discuss recent advances in understanding the physiological functions of Ack1 signaling in normal cells and the consequences of its hyperactivation in various cancers.

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

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.

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

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

The neuroendocrine-derived peptide parathyroid hormone-related protein promotes prostate cancer cell growth by stabilizing the androgen receptor.

During progression to an androgen-independent state following androgen ablation therapy, prostate cancer cells continue to express the androgen receptor (AR) and androgen-regulated genes, indicating that AR is critical for the proliferation of hormone-refractory prostate cancer cells. Multiple mechanisms have been proposed for the development of AR-dependent hormone-refractory disease, including changes in expression of AR coregulatory proteins, AR mutation, growth factor-mediated activation of AR, and AR protein up-regulation. The most prominent of these progressive changes is the up-regulation of AR that occurs in >90% of prostate cancers. A common feature of the most aggressive hormone-refractory prostate cancers is the accumulation of cells with neuroendocrine characteristics that produce paracrine factors and may provide a novel mechanism for the regulation of AR during advanced stages of the disease. In this study, we show that neuroendocrine-derived parathyroid hormone-related protein (PTHrP)-mediated signaling through the epidermal growth factor receptor (EGFR) and Src pathways contributes to the phenotype of advanced prostate cancer by reducing AR protein turnover. PTHrP-induced accumulation of AR depended on the activity of Src and EGFR and consequent phosphorylation of the AR on Tyr(534). PTHrP-induced tyrosine phosphorylation of AR resulted in reduced AR ubiquitination and interaction with the ubiquitin ligase COOH terminus of Hsp70-interacting protein. These events result in increased accumulation of AR and thus enhanced growth of prostate cancer cells at low levels of androgen.

Activated Cdc42-associated kinase Ack1 promotes prostate cancer progression via androgen receptor tyrosine phosphorylation: Mahajan NP, Liu Y, Majumder S, Warren MR, Parker CE, Mohler JL, Earp HS, Whang YE, Lineberger Comprehensive Cancer Center, Departments of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC

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.

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

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.

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

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

Regulation of androgen receptor activity by tyrosine phosphorylation

The androgen receptor (AR) is essential for the growth of prostate cancer cells. Here, we report that tyrosine phosphorylation of AR is induced by growth factors and elevated in hormone-refractory prostate tumors. Mutation of the major tyrosine phosphorylation site in AR significantly inhibits the growth of prostate cancer cells under androgen-depleted conditions. The Src tyrosine kinase appears to be responsible for phosphorylating AR, and there is a positive correlation of AR tyrosine phosphorylation with Src tyrosine kinase activity in human prostate tumors. Our data collectively suggest that growth factors and their downstream tyrosine kinases, which are elevated during hormone-ablation therapy, can induce tyrosine phosphorylation of AR and such modification may be important for prostate tumor growth under androgen-depleted conditions.