Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression
Owing to the marked sexual dimorphism of hepatocellular carcinoma (HCC), sex hormone receptor signaling has been implicated in numerous aspects of liver cancer pathogenesis. We sought to reconcile the clear contribution of androgen receptor (AR) activity that has been established in preclinical models of HCC with the clinical failure of AR antagonists in patients with advanced HCC by evaluating potential resistance mechanisms to AR-targeted therapy. The AR locus was interrogated for resistance-causing genomic modifications using publicly available primary HCC datasets (1,019 samples). Analysis of HCC tumor and cell line RNA-seq data revealed enriched expression of constitutively active, treatment-refractory AR splice variants (AR-SV). HCC cell lines expressed C-terminal-truncated AR-SV; 28 primary HCC samples abundantly expressed AR-SV. Low molecular weight AR species were nuclear localized and constitutively active. Furthermore, AR/AR-SV signaling promoted AR-mediated HCC cell progression and conferred resistance to AR antagonists. Ligand-dependent and -independent AR signaling mediated HCC epithelial-to-mesenchymal transition by regulating the transcription factor SLUG. These data suggest that AR-SV expression in HCC drives HCC progression and resistance to traditional AR antagonists. Novel therapeutic approaches that successfully target AR-SVs may be therapeutically beneficial for HCC. SIGNIFICANCE: Treatment-refractory, constitutively active androgen receptor splice variants promote hepatocellular carcinoma progression by regulating the epithelial-to-mesenchymal transition pathway.
Figure: AR-FL and AR-SVs expression in HCC primary samples and cell lines. (a, left) Top 100 (of 372) combined per-patient (x-axis) AR-FL (blue) and ligand-independent AR-SVs (red, as described in Supplementary Table 2). Numbers of patients with abundant AR-SV expression noted (inset) (a, right) RNA-Seq data from TCGA LIHC cohort were interrogated for AR-SVs transcript expression in female (n=121) and male (n=251). Statistical significance for AR-Svs expression in males vs females were evaluated using Mann-Whitney test **** p<0.0001 versus female. (b) Analyses of tumor RNA from 12 HCC majority cirrhotic and chronic hepatitis infected patients who underwent liver resection (male=10, female=2). Levels are compared to negative control THLE-2, normal liver cells, and positive control VCaP, PCa cells, to show abundant patient AR-FL and AR-v7 expression. Bars represent average technical duplicates and are matched for each patient. (c) Transcript abundance in transcript per million (TPM) of protein coding androgen receptor transcripts in 2 prostate cancer and 18 HCC cell lines from Cancer Cell Line Encyclopedia (CCLE) database. AR-FL (blue) and AR-SVs (red), as in Figure 1A, are presented. HCCLM3 cell data are not present in the CCLE. HCC cell AR transcript and protein expression were further validated by RT-PCR (d, h) and Western Blot (f, g), respectively. (d). RT-PCR analyses of AR-FL and AR-SVs transcripts in AR-positive prostate cancer (VCaP), AR-negative prostate cancer (DU145), AR-positive HCC (HCCLM3, SNU-423), AR-negative HCC (HepG2, PLC/PRF/5) and immortalized normal liver (THLE2) cell lines. (n=3, geometric mean ± SD). ARv4 and ARv12 were undetectable (supplementary Figure 5A). (e) Comparison of mean AR-FL and AR-v7 mRNA in primary samples as compared to the most abundant AR-SV expressing AR-positive HCC cells, HCCLM3, demonstrating robust AR isoform expression in primary HCC. (f) Western blot analysis with an N-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3 and SNU-423 cells and low molecular weight (MW) AR species in HCCLM3 cells migrating similarly to known AR-SVs in VCaP PCa cells. No AR-FL or lower MW species of AR were detected in HepG2, PLC/PRF/5, DU145, or THLE-2 cells. AR-negative HCC cell line, PLC/PRF/5, was transfected with either AR-FL expressing plasmid (PLC5_pAR-FL) or AR-v7 expressing plasmid (PLC5_pAR-v7) as positive controls for AR-FL and AR-v7, respectively. (g) Western blot analysis with a C-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3, SNU-423 and PLC5_pAR-FL cells. However, N-terminal directed monoclonal AR antibody detectable AR-SVs in VCaP and HCCLM3 cells are not detectable with c-terminal directed monoclonal AR antibody. WB performed with 35μg total protein lysate for all liver cell lines and 10μg for VCaP and DU145 and with primary N-terminal (CS#5153, Cell Signaling) or C-terminal AR mAb (ab52615, Abcam). (h). RT-PCR analyses of AR-FL and AR-SVs transcripts namely AR-v1, v3, and v7 in AR-positive HCC (HCCLM3, SNU-423, SNU475), AR-negative HCC (PLC/PRF/5) and immortalized normal liver (THLE2) cell lines (performed on low passage cells from ATCC Liver Cancer Panel TCP-1011, n=3, geometric mean ± SD). (i) Western blot analysis with an AR-v7 specific monoclonal AR antibody shows AR-v7 protein in 22Rv1, PLC5_pAR-v7, VCaP and SNU-475 cells. No AR-v7 reactive lower MW species of AR were detected in HCCLM3 cells. No AR-FL protein was detected in any of these cells. (j) To further confirm that the low molecular weight species that were detected by an AR-v7 specific AR mAb are C-terminal truncated splice variants, the blot presented in Figure 1I performed with a C-terminal targeting AR mAb was stripped, blocked and incubated with an N-terminal targeting AR mAb revealing abundant AR-FL in 22Rv1, VCaP and HCCLM3. The GAPDH blot from (i) is presented again here for convenience. No AR-FL isoform was detected in SNU-475 or PLC5_pAR-v7. However, low molecular weight AR species were detected in HCCLM3. (k) WGS of SNU-475 cells revealed a large ~48-kb hemizygous deletion in the AR-locus which included exons 4–8 of the AR-FL gene. This deletion is consistent with AR-v7 but not AR-FL expression and is strongly supported by sequencing data which included 56 read pairs with split reads and/or discordant pair alignments.
