Serine-arginine protein kinase 1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer

Abstract

Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor A (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa.

Figure 1. SRPK1 and SRSF1 expression are altered in PIN and malignant prostatectomy samples

A. Immunohistochemical staining for SRPK1 in benign, PIN and cancerous prostate tissue sections. B. Quantification of the staining intensity from 17 patients (*=p<0.05, ***=p<0.001, Kruskal–Wallis and Dunn’s post–test). C. Examples of SRSF1 staining for benign, PIN and malignant prostate samples. D. Quantification of the staining intensity from 17 patients.

Figure 2. SRPK1 is increased in prostate cancer cell lines and SRPK1 knockdown switches expression of VEGF₁₆₅ into VEGF₁₆₅b in PC-3 cells

A. SRPK1 expression quantified by qRT-PCR in several PCa cell lines compared to primary prostate epithelial cells (*=p<0.05 PreC vs PC-3, One-way ANOVA). B. Western blot analysis of SRPK1 in PC3 (P), DU145 (D) and LNCaP (L); upper panel – duplicate examples of extracts; lower panel – quantification from three replicates with normalization on tubulin signal for equal loading. C. RT-PCR analysis shows presence of VEGF₁₆₅b splicing isoforms in PC3 cells with SRPK1-KD (lanes 1, 2, 3 – plasmid controls; lanes 4,5 – RT-PCRs). D. Effect of SRPK1-KD on VEGF₁₆₅b protein expression in PC3 cells

Figure 3. SRPK1-KD has no effect on proliferation, migration and invasion of PC-3 cells in vitro

A. Growth of PC-3 cells stably transfected with control and SRPK1 shRNA at 24, 48 and 72 hours after plating equal numbers; B. Ki-67 staining; left panels - examples of microscopic fields of PC-3 cells double-stained with Hoechst and Ki-67; right panels - quantification of Ki-67 fluorescence in control and SRPK1-KD cells at 24, 48 and 72 hours after plating equal numbers; C. Matrigel migration-invasion assay. Quantification of cells migrated on the bottom part of membranes after 24h. D. Scratch-wound assay. Migration potential of cells was calculated as a measure of the distance (mm) covered by the cells to the middle of the scratch wound, 24 and 48 hours after the initial scratch.

Figure 4. SRPK1-KD reduces tumour growth in PC3 xenografts in nude mice

A. Quantitations of the tumour volumes in control and SRPK1-KD mice. B. Examples of tumour growth in both mice groups (tumours outlined in black). C. Examples of microscopic fields of control and SRPK1-KD tumours stained for CD31. Arrows indicate blood vessels. D. Quantitation of microvessel density in control and SRPK1-KD tumours. E. Western blot analysis of SRPK1 and VEGF in duplicate tumours from control and SRPK1-KD groups

Figure 5. Exogenous expression of VEGF cDNA from a VEGF promoter rescues the effect of SRPK1-KD on tumour growth in vivo

A. Tumour growth curves for four groups of mice injected s.c with the following stably-transfected cells: open circles - control shRNA and VEGF₁₆₅ plasmid; filled circles - SRPK1-KD and VEGF₁₆₅ plasmid; open squares - control shRNA and empty vector; filled circles - SRPK1-KD and empty vector; B. Examples of tumour growth in all mice groups (tumours outlined in black).

Figure 6

Expression analysis by Western blot of SRPK1 (A.) and VEGF (B.) levels in the four sets of tumours from the rescue experiment

Figure 7. Small molecule inhibitors of SRPK1

A. SPHINX and SRPIN340 are able to block phosphorylation of SR proteins induced by EGF in PC3 cells and (B.) to switch VEGF terminal exon splicing. C. Intravital imaging of RFP-PC3 tumours in mice treated with saline or SPHINX (i.p. 50μg, triweekly) by fluorescence imaging. D. Intraperitoneal administration of SPHINX significantly decreases tumour growth in orthotopic xenografts of PC3 cells (p<0.05, two-way Anova).