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. 2015 Aug;14(8):1884-95.
doi: 10.1158/1535-7163.MCT-14-1057. Epub 2015 Jun 8.

NF-κB2/p52:c-Myc:hnRNPA1 Pathway Regulates Expression of Androgen Receptor Splice Variants and Enzalutamide Sensitivity in Prostate Cancer

Nagalakshmi Nadiminty  1 , Ramakumar Tummala  2 , Chengfei Liu  2 , Wei Lou  2 , Christopher P Evans  3 , Allen C Gao  4
Affiliations

NF-κB2/p52:c-Myc:hnRNPA1 Pathway Regulates Expression of Androgen Receptor Splice Variants and Enzalutamide Sensitivity in Prostate Cancer

Nagalakshmi Nadiminty et al. Mol Cancer Ther. 2015 Aug.

Abstract

Castration-resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signaling. Alternative splicing of the AR to generate constitutively active, ligand-independent variants is one of the principal mechanisms that promote the development of resistance to next-generation antiandrogens such as enzalutamide. Here, we demonstrate that the splicing factor heterogeneous nuclear RNA-binding protein A1 (hnRNPA1) plays a pivotal role in the generation of AR splice variants such as AR-V7. hnRNPA1 is overexpressed in prostate tumors compared with benign prostates, and its expression is regulated by NF-κB2/p52 and c-Myc. CRPC cells resistant to enzalutamide exhibit higher levels of NF-κB2/p52, c-Myc, hnRNPA1, and AR-V7. Levels of hnRNPA1 and AR-V7 are positively correlated with each other in prostate cancer. The regulatory circuit involving NF-κB2/p52, c-Myc, and hnRNPA1 plays a central role in the generation of AR splice variants. Downregulation of hnRNPA1 and consequently of AR-V7 resensitizes enzalutamide-resistant cells to enzalutamide, indicating that enhanced expression of hnRNPA1 may confer resistance to AR-targeted therapies by promoting the generation of splice variants. These findings may provide a rationale for cotargeting these pathways to achieve better efficacy through AR blockade.

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Conflict of interest statement

Conflicts of interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
HnRNPA1 promotes generation of AR splice variants. qPCR to determine the expression levels of AR-V7 and FL AR in A) 22Rv1 and B) VCaP cells transfected with hnRNPA1 or hnRNPA2 siRNAs. C) Western analysis in 22Rv1 and VCaP cells transfected with hnRNPA1 or hnRNPA2 siRNAs. D) Western blotting for AR-V7 in LNCaP cells transfected with hnRNPA1 cDNA. E) qPCR analysis in LNCaP cells transfected with hnRNPA1 cDNA. Results are presented as means ± SD of 3 experiments performed in triplicate. * denotes p≤0.05.
Figure 2
Figure 2
Recruitment of hnRNPA1 and hnRNPA2 to splice sites in AR pre-mRNA is enhanced in 22Rv1-Enza-R and C4-2B-Enza-R enzalutamide-resistant PCa cells compared to 22Rv1 and C4-2B parental cells respectively. RIP assays for splice sites of A) AR-V7, B) AR in 22Rv1-Enza-R compared to 22Rv1 and C) AR-V7, D) AR in C4-2B-Enza-R compared to C4-2B. Results are presented as means ± SD of 2 experiments performed in duplicate. * denotes p≤0.05.
Figure 3
Figure 3
Expression levels of hnRNPA1 and AR-V7 are positively correlated with each other. A) Representative immunoblot for hnRNPA1, hnRNPA2 and AR-V7 in lysates from 27 paired benign and tumor patient samples. qRT-PCR of mRNA levels of (B) hnRNPA1 and (C) AR-V7 in 10 paired normal and tumor clinical prostate samples. Results are presented as means ± SD of 2 experiments performed in triplicate. * denotes p≤0.05. Relative expression levels of hnRNPA1 (D) and hnRNPA2 (E) in the representative Singh_prostate (n=102) dataset from Oncomine. Relative expression levels of hnRNPA1 in GDS1439 dataset (F) and in GDS2545 dataset (G) from GEO.
Figure 4
Figure 4
Reciprocal regulation between c-Myc and hnRNPA1 is responsible for the generation of AR splice variants. A) Immunoblotting for hnRNPA1 and AR-V7 in 22Rv1 and VCaP cells transfected with c-Myc shRNA. B) Immunoblotting for c-Myc in LNCaP, 22Rv1 and VCaP cells transfected with hnRNPA1 or hnRNPA2 siRNAs. qRT-PCR for mRNA levels of full length AR and AR-V7 in 22Rv1 (C) and VCaP (D) cells transfected with c-Myc shRNA. Insets show the expression of c-Myc mRNA in cells transfected with c-Myc shRNA. (E) Protein and (F) mRNA levels of AR-V7, FL AR, c-Myc and hnRNPA1 in 22Rv1 cells transfected with c-Myc shRNA with or without overexpression of hnRNPA1. Results are presented as means ± SD of 2 experiments performed in triplicate. * denotes p≤0.05.
Figure 5
Figure 5
NF-kappaB2/p52 regulates expression of c-Myc and hnRNPA1. A) Left panel, Immunoblotting for hnRNPA1, c-Myc and AR-V7 in LN-p52 cells. Right panel, Immunoblotting for hnRNPA1, c-Myc and AR-V7 in LN/TR/p52 cells. B) Left panel, Western blotting for AR-V7 in LN-p52 cells transfected with hnRNPA1 siRNA. Middle panel, Immunoblotting for AR-V7 and hnRNPA1 in LN-p52 cells transfected with c-Myc shRNA. Right panel, Immunoblotting for AR-V7, hnRNPA1 and c-Myc in 22Rv1 cells transfected with p52 shRNA. C) Left panel, 22Rv1 cells resistant to enzalutamide (22Rv1-Enza-R) express higher levels of AR-V7, hnRNPA1, c-Myc and NF-kappaB2/p52. Middle panel, C4-2B cells resistant to enzalutamide (C4-2B-Enza-R) express higher levels of hnRNPA1, AR-V7, c-Myc and NF-kappaB2/p52. Right panel, Xenografts from C4-2B-Enza-R cells exhibit higher levels of AR-V7, hnRNPA1 and c-Myc. D) Left panel, Western analysis of AR-V7 in 22Rv1-Enza-R cells transfected with hnRNPA1 siRNA. Right panel, Expression of AR-V7 and hnRNPA1 in 22Rv1-Enza-R cells transfected with c-Myc shRNA. All results are shown as representative images from 2 experiments performed in duplicate. E) Left panel, Chart depicting the positive correlation between mRNA levels of NF-kappaB2/p52, c-Myc, hnRNPA1 and AR-V7 in 10 paired benign and tumor prostate clinical samples. Right panel, Chart depicting the correlation between relative protein levels of NF-kappaB2/p52, c-Myc, hnRNPA1 and AR-V7 in 27 paired benign and tumor prostate clinical samples. Band intensities in immunoblots were quantified using ImageJ software and plotted as arbitrary units. The horizontal lines represent the median of each series.
Figure 6
Figure 6
Suppression of hnRNPA1 restores enzalutamide sensitivity of enzalutamide-resistant PCa cells. A) Left panel, Cell survival in 22Rv1-Enza-R cells transfected with hnRNPA1 or hnRNPA2 siRNAs and treated with vehicle or 20 μM enzalutamide. Cell numbers were counted after 48 h. Right panel, immunoblots confirm the downregulation of hnRNPA1 or hnRNPA2 and of AR-V7. B) Left panel, Cell survival in LN-p52 cells transfected with hnRNPA1 or hnRNPA2 siRNAs and treated with vehicle or 20 μM enzalutamide. Cell numbers were counted after 48 h. Right panel, immunoblots confirm the downregulation of hnRNPA1 or hnRNPA2 and AR-V7. C) Left panel, Cell survival in VCaP cells transfected with hnRNPA1 or hnRNPA2 siRNAs and treated with vehicle or 20 μM enzalutamide. Cell numbers were counted after 48 h. Right panel, immunoblots confirm the downregulation of hnRNPA1 or hnRNPA2 and of AR-V7. Results are presented as means ± SD of 3 experiments performed in triplicate. * denotes p≤0.05. D) Schematic representation of the alternative splicing of AR mRNA regulated by the NF-kappaB2:c-Myc:hnRNPA1 axis.
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