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Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth - PubMed

  • ️Sun Jan 01 2017

Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth

X Deng et al. Oncogene. 2017.

Abstract

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

PRMT5 regulates prostate cancer cell growth in an AR-dependent manner. (a–d) Induction of PRMT5 knockdown by doxycycline (Dox+) inhibited cell proliferation in AR-expressing LNCaP and C4-2 cells but not in DU145 and RWPE-1 cells that do not express AR. (e) PRMT5 knockdown induced by Dox decreased AR expression in LNCaP and C4-2 stable cell lines. (f) Knockdown of PRMT5 in LNCaP-shPRMT5 cells reduced the mRNA level of the indicated AR target genes measured by qRT-PCR. (g) Restored cell growth by exogenous expression of FLAG-AR in LNCaP cells transiently co-transfected with SC, or pLKO-Tet-On-shPRMT5 (KD) in combination with pFLAG-CMV (Vec) or pFLAG-CMV-AR (AR). (h) Representative Western blots from g to verify the expression of FLAG-AR and the knockdown of PRMT5. *P<0.05; **P<0.01; and ***P<0.001.

Figure 2
Figure 2

Epigenetic activation of AR transcription by PRMT5 in LNCaP cells. (a) Transient knockdown of PRMT5 (KD) reduced AR mRNA level when compared with SC. (b) Transient knockdown of PRMT5 had no effect on the AR-luciferase reporter gene (AR-Luc) activity. (c) Enrichment of H4R3me2s, but not H3R8me2s and H2AR3me2s, on the proximal promoter region of the AR gene in LNCaP cells. (d) Transient knockdown of PRMT5 reduced symmetric dimethylation of H4R3 (H4R3me2s). (e) Knockdown of PRMT5 induced by doxycycline (Dox+) reduced PRMT5 binding to the proximal promoter region of the AR gene when compared with cells without Dox (Dox−). (f) Knockdown of PRMT5 induced by doxycycline (Dox+) reduced the enrichment of H4R3me2s on the proximal promoter region of the AR gene when compared with cells without Dox (Dox−).

Figure 3
Figure 3

PRMT5 interacts with Sp1 and Brg1 on the proximal promoter region of the AR gene in LNCaP cells. (a) Co-immunoprecipitation of Sp1 with PRMT5. (b) Co-immunoprecipitation of Brg1 with PRMT5. (c) Knockdown of Sp1 induced by doxycycline (Dox+) reduced AR expression in Dox-inducible stable cell line LNCaP-shSp1. (d–f) Dox-induced knockdown of Sp1 reduced the binding of Sp1, PRMT5 and Brg1 to the same proximal promoter region of the AR gene. *P<0.05 and ***P<0.001.

Figure 4
Figure 4

PRMT5 expression correlates positively with AR expression in prostate cancer. (a) Shown are representative immunohistochemistry staining images (magnification × 400) of PRMT5 in benign tissue (N5), Gleason 6 prostate cancer tissue (6T1) and Gleason 7 prostate cancer tissue (7T8). The total expression score of PRMT5 is significantly higher in prostate cancer tissues (PCa) when compared with BPH. Scale bar, 30 μm. (b) PRMT5 expression correlates positively with AR expression at the protein level in the same TMA from a. (c) Representative images of PRMT5 and AR expression from serial sections of prostate cancer tissues. The upper panels show higher expression of both PRMT5 and AR in the nucleus and the lower panels show weaker expression of both PRMT5 and AR in the nucleus. Scale bar, 30 μm. (d) PRMT5 expression correlates positively with AR expression at the transcript level. The data were retrieved from Oncomine database.

Figure 5
Figure 5

Knockdown of PRMT5 suppresses the growth of xenograft tumors in mice. (a) LNCaP-shPRMT5 cells were implanted subcutaneously into the right lower flanks of 10 nude mice per group, and the tumor growth was monitored twice weekly in Dox-treated (Dox+) and untreated (Dox−) mice. (b) Similar experiment was performed as described in a for LNCaP-SC cell line. (c and d) Representative images showing inhibition of PRMT5 and AR expression in Dox-treated tumor nodules. No effect on PRMT5 and AR expression in xenograft tumors derived from LNCaP-SC was observed. Scale bar: 10 μm.

Figure 6
Figure 6

Proposed model for epigenetic activation of AR transcription by PRMT5.

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