The CXCL12/CXCR4 axis promotes ligand-independent activation of the androgen receptor

https://doi.org/10.1016/j.mce.2011.12.015Get rights and content

Abstract

The molecular mechanisms responsible for the transition of some prostate cancers from androgen ligand-dependent to androgen ligand-independent are incompletely established. Molecules that are ligands for G protein coupled receptors (GPCRs) have been implicated in ligand-independent androgen receptor (AR) activation. The purpose of this study was to examine whether CXCL12, the ligand for the GPCR, CXCR4, might mediate prostate cancer cell proliferation through AR-dependent mechanisms involving functional transactivation of the AR in the absence of androgen. The results of these studies showed that activation of the CXCL12/CXCR4 axis promoted: The nuclear accumulation of both wild-type and mutant AR in several prostate epithelial cell lines; AR-dependent proliferative responses; nuclear accumulation of the AR co-regulator SRC-1 protein; SRC-1:AR protein:protein association; co-localization of AR and SRC-1 on the promoters of AR-regulated genes; AR- and SRC-1 dependent transcription of AR-regulated genes; AR-dependent secretion of the AR-regulated PSA protein; P13K-dependent phosphorylation of AR; MAPK-dependent phosphorylation of SRC-1, and both MAPK- and P13K-dependent secretion of the PSA protein, in the absence of androgen. Taken together, these studies identify CXCL12 as a novel, non-steroidal growth factor that promotes the growth of prostate epithelial cells through AR-dependent mechanisms in the absence of steroid hormones. These findings support the development of novel therapeutics targeting the CXCL12/CXCR4 axis as an ancillary to those targeting the androgen/AR axis to effectively treat castration resistant/recurrent prostate tumors.

Highlights

► The CXCL12/CXCR4 axis functionally activates the AR in the absence of androgen. ► CXCL12/CXCR4 promotes nuclear accumulation of the AR and SRC-1 co-regulator. ► CXCL12/CXCR4 stimulates AR-regulated gene transcription and cellular proliferation. ► CXCL12/CXCR4 activates AR and SRC-1 viaPI3K- and MAPK-mediated phosphorylation. ► CXCL12/CXCR4-mediated AR activation may provide a new therapeutic target.

Introduction

Prostate cancer is the leading cause of newly diagnosed cancers, and the second leading cause of cancer-related deaths, in American men (Jemal et al., 2010). Prostate tumors are initially dependent on androgen signaling and can be successfully controlled by a series of strategies that deplete endogenous androgen expression or interfere with androgen receptor (AR)-mediated signaling. These include androgen ablation and anti-androgen therapeutics, such as finasteride, which inhibits the conversion of testosterone to the active form, dihydrotestosterone, or bicalutimde, which directly antagonizes androgen signaling at the level of the receptor (Debes and Tindall, 2002, Pienta and Bradley, 2006). Therapies that directly target the AR are most commonly used either as single therapy or in combination with pharmaceutical or surgical castration or ionizing radiation. However, all of these therapeutic approaches are only effective for early stage androgen-dependent prostate cancer, as progressive prostate tumors develop alternative strategies to survive and grow despite anti-androgen therapy. Eventually, such tumors develop into lethal, metastatic androgen-refractory prostate cancers (Pienta and Bradley, 2006).

The molecular mechanisms responsible for the transition from androgen-dependent to androgen-independent prostate cancer are incompletely established. However, it is known that, despite their insensitivity to anti-androgen and androgen-deprivation therapies, the survival and growth of castration resistant/recurrent prostate cancers (hereafter referred to as CRPCs) are likely dependent upon AR signaling. Evidence that AR activity may be required for the growth and progression of castration resistant prostate tumors is elicited from studies that describe AR gene transcript and protein over-expression; AR gene mutations conferring receptor hyperactivity; AR co-regulator protein dysregulation producing increased AR activity, and chromatin remodeling conducive to more efficient transcription of AR-regulated genes, in such tumors (Chen et al., 2002, Cronauer et al., 2003, Culig et al., 1998, Debes and Tindall, 2002, Jia et al., 2006, Pienta and Bradley, 2006, Scher et al., 2004, Taplin and Balk, 2004). Taken together, these studies suggest that AR signaling plays an important role in progressive castration resistant disease, and that these activities can, and do, occur in the presence of very low levels, or absence of, circulating androgen.

Several studies have reported hormone-independent AR signaling in prostate cancer cells. Diverse types of molecules, including peptide growth factors (EGF, KGF, and IGF1) (Craft et al., 1999, Culig et al., 1994, Shi et al., 2001, Tørring et al., 2000, Wen et al., 2000, Yeh et al., 1999), neuropeptides, including neurotensin and bombesin (Dai et al., 2002, Debes and Tindall, 2002, Lee et al., 2001), and inflammatory mediators such as the interleukins IL-4 and IL-6 (Culig et al., 2005, Hobisch et al., 1998, Lee et al., 2003, Lee et al., 2005, Lee et al., 2008, Lee et al., 2009, Malinowska et al., 2009, Ueda et al., 2002), and the chemokine CXCL8 (IL-8) (Araki et al., 2007, Lee et al., 2004, MacManus et al., 2007, Seaton et al., 2008), have been implicated in hormone-independent AR activation. A common thread among some of these molecules is that they are ligands for G protein coupled receptors (GPCRs). Neuropeptides as well as interleukins and chemokines are ligands for GPCRs, and activation of GPCRs has been linked to hormone-independent AR activation and prostate tumor growth (Begley et al., 2005, Daaka, 2004). Of particular interest to this study is evidence that interactions between the CXC-type chemokine, CXCL8, and the GPCRs that recognize it, CXCR1 and CXCR2, promote the hormone-independent proliferation of prostate cancer LNCaP cells. Moreover, CXCL8-mediated hormone-independent prostate cancer cell proliferation can be inhibited by pretreatment with bicalutamide, implying that AR activation is involved in this response (Seaton et al., 2008). Previous work accomplished in our laboratory has shown that several CXC-type chemokines, including CXCL1, CXCL5, CXCL6, and CXCL12, promote the proliferation of both non-transformed and transformed prostate epithelial cells (Begley et al., 2005, Begley et al., 2007). Like CXCL8, CXCL1, CXCL5 and CXCL6 recognize and effect proliferation through binding the GPCRs CXCR1 and/or CXCR2 (Rot and von Andrian, 2004). However, CXCL12 is the ligand for a different CXCR-type GPCR, CXCR4 (and, to a lesser extent, CXCR7) (Bleul et al., 1996, Burns et al., 2006). This raises the possibility that CXCL12/CXCR4 interactions might also mediate prostate cancer cell proliferation through AR-dependent mechanisms. To test this hypothesis, we have examined whether CXCL12/CXCR4 interactions functionally transactivate the AR to mediate cellular proliferation in the absence of androgen, and determined which intracellular signaling events mediate this process. The results of these studies provide new directions to develop therapeutics to effectively halt the progression, and consequent mortality, of castration resistant prostate tumors.

Section snippets

Cell culture

The androgen-sensitive LNCaP and 22Rv1 cell lines were maintained in 10% RPMI media or serum free, phenol-red free RPMI supplemented with antibiotics as described (Horoszewicz et al., 1980, Sramkoski et al., 1999), VCaP cells were maintained in 10% DMEM or 5% DMEM (without phenol red, and with charcoal stripped serum) supplemented with antibiotics as described (Korenchuk et al., 2001), and LAPC4 cells (kindly provided by Dr. Charles L. Sawyers, Memorial Sloan-Kettering Cancer Center) were

CXCL12/CXCR4 axis activation mediates AR accumulation in the nucleus

We have previously shown that activation of the CXCL12/CXCR4 axis stimulates the proliferation of both androgen-sensitive and androgen-insensitive cells (Begley et al., 2005, Begley et al., 2008). However, we had not previously investigated a potential mechanistic role for AR in CXCL12/CXCR4-mediated proliferation in androgen-sensitive cells in the presence or absence of hormone. Therefore, an initial study was pursued to test whether activation of the CXCL12/CXCR4 axis may promote AR

Discussion

This study was designed to determine whether CXCL12/CXCR4 interactions functionally transactivate the AR to mediate cellular proliferation in the absence of androgen, and to begin to elucidate which intracellular signaling events mediate this process. The results of these studies showed that activation of the CXCL12/CXCR4 axis promoted: The nuclear accumulation of both wild-type and mutant AR in several prostate epithelial cell lines; AR-dependent proliferative responses; nuclear accumulation

Conclusions

In summary, the data reported here identify the activated CXCL12/CXCR4 axis as a novel AR-dependent mechanism that promotes the growth of androgen-dependent prostate epithelial cells by functionally transactivating the AR in the absence of androgen. The implication of these findings is that therapeutically targeting the CXCL12/CXCR4 axis may be warranted to develop therapeutics to effectively halt the progression, and consequent mortality, of castration resistant prostate tumors.

Acknowledgements

We thank Dr. Diane Robins for her helpful discussions during the course of these studies. This work was supported by National Institutes of Health awards NIH/NIDDK 1 R01 DK081841 (J.A.M.), and fellowship awards from The American Urological Association Foundation (S.K.) and the American Foundation for Aging Research (S.K.).

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