The SMARCD Family of SWI/SNF Accessory Proteins Is Involved in the Transcriptional Regulation of Androgen Receptor-Driven Genes and Plays a Role in Various Essential Processes of Prostate Cancer

Abstract

Previous studies have demonstrated an involvement of chromatin-remodelling SWI/SNF complexes in the development of prostate cancer, suggesting both tumor suppressor and oncogenic activities. SMARCD1/BAF60A, SMARCD2/BAF60B, and SMARCD3/BAF60C are mutually exclusive accessory subunits that confer functional specificity and are components of all known SWI/SNF subtypes. To assess the role of SWI/SNF in prostate tumorigenesis, we studied the functions and functional relations of the SMARCD family members. Performing RNA-seq in LnCAP cells grown in the presence or absence of dihydrotestosterone, we found that the SMARCD proteins are involved in the regulation of numerous hormone-dependent AR-driven genes. Moreover, we demonstrated that all SMARCD proteins can regulate AR-downstream targets in androgen-depleted cells, suggesting an involvement in the progression to castration-resistance. However, our approach also revealed a regulatory role for SMARCD proteins through antagonization of AR-signalling. We further demonstrated that the SMARCD proteins are involved in several important cellular processes such as the maintenance of cellular morphology and cytokinesis. Taken together, our findings suggest that the SMARCD proteins play an important, yet paradoxical, role in prostate carcinogenesis. Our approach also unmasked the complex interplay of paralogue SWI/SNF proteins that must be considered for the development of safe and efficient therapies targeting SWI/SNF.

Keywords: SMARCD1/BAF60A; SMARCD2/BAF60B; SMARCD3/BAF60C; SWI/SNF complex; chromatin-remodeling; prostate cancer.

Figure 1

Alterations of SMARCD1, SMARCD2 and SMARCD3 in two PCa patient cohorts. (A) SMARCD1, SMARCD2 and SMARCD3 were each found altered in 6% of patients (n = 488) suffering from non-metastatic prostate adenocarcinoma of various stages. While mutations were infrequent, the majority of alterations accounted for mRNA up-regulation. (B) In a cohort of patients with metastatic disease (n = 429), alterations of SMARCD1, SMARCD2 and SMARCD3 were detected in 5%, 11% and 9% of cases, respectively. The predominant types of alterations were mRNA up-regulation and gene amplifications.

Figure 2

mRNA and protein levels of SMARCD1, SMARCD2 and SMARCD3 in benign and malignant prostate cell lines. (A) Expression levels of SMARCD1, SMARCD2 and SMARCD3 in various prostate cell lines were measured by qPCR. RNA was isolated from several biological replicates (n = 3). Asterisks indicate the statistical significance of differences in gene expression between the respective cell line and non-malignant RWPE-1 cells. Error bars indicate the standard error of the mean (SEM). Standard deviations were calculated using the relative-fold expression determined in various technical replicates (n = 6). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (B) SMARCD1, SMARCD2 and SMARCD3 protein levels in various prostate cell lines were determined by Western blotting.

Figure 3

SMARCD3 is an androgen-regulated gene. LnCAP cells were induced with various concentrations of DHT for (A) 8 h, (B) 24 h, (C) 48 h or (D) 72 h and SMARCD1, SMARCD2 and SMARCD3 expression levels were measured by qPCR. RNA was isolated from several biological replicates (n = 3). Asterisks indicate the statistical significance of differences in gene expression compared to the androgen-deprived control. Error bars indicate the standard error of the mean (SEM). Standard deviations were calculated using the relative-fold expression determined in various technical replicates (n = 6). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 4

Effects of siRNA-mediated silencing of SMARCD1, SMARCD2 and SMARCD3 in various combinations on cell viability. siRNA-mediated knockdown of SMARCD1, SMARCD2 and SMARCD3 alone or in various combinations was performed in the cell lines (A) LnCAP or (B) C4-2 and cell viability was determined after five days. If not indicated otherwise by brackets, asterisks indicate the statistical significance of differences in cell viability between the respective sample and the negative control. Error bars indicate the standard error of the mean (SEM). Standard deviations were calculated using the relative cell viability determined in various biological replicates (n = 7). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: not significant.

Figure 5

Morphologic effects of siRNA-mediated knockdown of SMARCD1, SMARCD2 and SMARCD3 in LnCAP cells. Immunostaining of β–tubulin and nuclear pore complex (NPC) proteins was performed in LnCAP cells treated with (A) a scrambled control or siRNAs targeting (B) SMARCD1, (C) SMARCD2, (D) SMARCD3, (E) SMARCD1 and SMARCD2, (F) SMARCD1 and SMARCD3 and (G) SMARCD2 and SMARCD3. Arrows indicate binucleated cells.

Figure 6

Morphologic effects of siRNA-mediated knockdown of SMARCD1, SMARCD2 and SMARCD3 in RWPE-1 cells. Immunostaining of β–tubulin and nuclear pore complex (NPC) proteins was performed in RWPE-1 cells treated with (A) a scrambled control or siRNAs targeting (B) SMARCD1, (C) SMARCD2, (D) SMARCD3, (E) SMARCD1 and SMARCD2, (F) SMARCD1 and SMARCD3 and (G) SMARCD2 and SMARCD3. Arrows indicate binucleated cells.

Figure 7

Differentially regulated genes in response to siRNA-mediated silencing of the SMARCD genes and AR performed in the presence or absence of DHT. siRNA-mediated knockdown of (A) SMARCD1, (B) SMARCD2, (C) SMARCD3 and (D) AR was performed in LnCAP cells grown in the presence (left) or absence (center) of DHT. RNA-seq was performed using RNA isolated from several biological replicates (n = 3). Differential gene expression was considered statistically significant for genes with log2 fold changes > ± 1 and adjusted p-values < 0.05. Genes exhibiting differential regulation exclusively in the presence or absence of DHT or under both experimental conditions were assessed (right).