PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knock-down (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 anti-angiogenic isoform VEGF165b in PC3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of prostate malignancy. 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 into the use of SRPK1 inhibition as a potential anti-angiogenic therapy in prostate malignancy. C duplicate examples of extracts; C quantification from three replicates with normalization on tubulin transmission for equal loading. C. RT-PCR analysis shows presence of VEGF165b splicing isoforms in PC3 cells with SRPK1-KD (1, 2, 3 C plasmid controls; 4,5 C RT-PCRs). D. Effect of SRPK1-KD on VEGF165b protein expression in PC3 cells To establish whether the SRPK1-VEGF splicing regulation was present in PC-3 cells we generated a stable knock-down of SRPK1. PC-3 cells were transduced with lentivirus made up of shRNAi to SRPK1 or scrambled shRNAi, selected in puromycin for 3 weeks and mRNA and protein extracted. The extent of knock-down was assessed both by qRT-PCR and Western blotting (Supplementary Physique 2). RT-PCR analysis, Western blot and ELISA exhibited that there was a switch towards VEGF165b isoform in cells with stable SRPK1-KD (Physique 2 C,D and Supplementary Physique 3). Interestingly, the fold-increase in VEGF165b at protein level (2D) seems to be higher than at the RNA level (2C) suggesting a possible additional post-transcriptional layer of regulation PP1 Analog II, 1NM-PP1 (see conversation). To determine whether SRPK1-KD in PC3 cells influenced SR protein expression and/or phosphorylation, western blot analysis was performed. Supplementary Physique 4 shows that expression of different SR proteins was not affected but there was a pronounced decrease in phosphorylation in SRSF 1, 2 and 5 in KD cells compared to controls. SRPK1 PP1 Analog II, 1NM-PP1 knock-down does not impact cell growth, proliferation, invasion and migration of PC-3 cells – examples of microscopic fields of PC-3 cells double-stained with Hoechst and Ki-67; – quantification of Ki-67 fluorescence in control and SRPK1-KD cells at 24, 48 and 72 hours after plating equivalent figures; C. Matrigel migration-invasion assay. Quantification of cells migrated on the bottom a 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 scrape wound, 24 and 48 hours after the initial scrape. These data taken together suggest that SRPK1-KD does not result in an effect on PC-3 cells, by regulating VEGF or other genes splicing, that would influence their rate of growth, proliferation, migration or invasion in vitro. SRPK1 knock-down reduces subcutaneous PC-3 tumour growth through inhibition of angiogenesis in a manner dependent on VEGF splicing PP1 Analog II, 1NM-PP1 Since SRPK1-KD induced a splicing switch towards VEGF anti-angiogenic isoforms we investigated whether this would impact the rate of tumour growth in which we asked whether VEGF165 cDNA overexpression driven by a VEGF-promoter (which would mimic endogenous VEGF but HBGF-4 be insensitive to alternate splicing) could rescue the tumour growth in SRPK1-KD cells. SRPK1-KD or control cells were transfected with a plasmid made up of the VEGF165 cDNA under the control of the VEGF promoter. SRPK1-KD did not impact VEGF promoter activity in PC3 cells, as assessed in vitro using a luciferase reporter plasmid driven by the endogenous VEGF promoter sequence (Supplementary Physique 7). One million PC-3 SRPK1-KD/VEGF165 and CTRL KD/VEGF165 cells were injected subcutaneously in the flank of male nude mice and tumour volume was monitored. As a control, 1106 PC-3 SRPK1-KD/pCDNA3 and CTRL/pCDNA3 cells (transfected with vacant plasmid) were injected in parallel. The ability of the.PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. that this selective upregulation of pro-angiogenic VEGF in prostate malignancy may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knock-down (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 anti-angiogenic isoform VEGF165b in PC3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of prostate malignancy. 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 into the use of SRPK1 inhibition as a potential anti-angiogenic therapy in prostate malignancy. C duplicate examples of extracts; C quantification from three replicates with normalization on tubulin transmission for equal loading. C. RT-PCR analysis shows presence of VEGF165b splicing isoforms in PC3 cells with SRPK1-KD (1, 2, 3 C plasmid controls; 4,5 C RT-PCRs). D. Effect of SRPK1-KD on VEGF165b protein expression in PC3 cells To establish whether the SRPK1-VEGF splicing regulation was present in PC-3 cells we generated a stable knock-down of SRPK1. PC-3 cells were transduced with lentivirus made up of shRNAi to SRPK1 or scrambled shRNAi, selected in puromycin for 3 weeks and mRNA and protein extracted. The extent of knock-down was assessed both by qRT-PCR and Western blotting (Supplementary Physique 2). RT-PCR analysis, Western blot and ELISA exhibited that there was a switch towards VEGF165b isoform in cells with stable SRPK1-KD (Physique 2 C,D and Supplementary Physique 3). Interestingly, the fold-increase in VEGF165b at protein level (2D) seems to be higher than at the RNA level (2C) suggesting a possible additional post-transcriptional layer of regulation (see conversation). To determine whether SRPK1-KD in PC3 cells influenced SR protein expression and/or phosphorylation, western blot analysis was performed. Supplementary Physique 4 shows that expression of different SR proteins was not affected but there was a pronounced decrease in phosphorylation in SRSF 1, 2 and 5 in KD cells compared to controls. SRPK1 knock-down does not impact cell growth, proliferation, invasion and migration of PC-3 cells – examples of microscopic fields of PC-3 cells double-stained with Hoechst and Ki-67; – quantification of Ki-67 fluorescence in control and SRPK1-KD cells at 24, 48 and 72 hours after plating equivalent figures; 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. These data taken together suggest that SRPK1-KD does not result in an effect on PC-3 PP1 Analog II, 1NM-PP1 cells, by regulating VEGF or other genes splicing, that would influence their rate of growth, proliferation, migration or invasion in vitro. SRPK1 knock-down reduces subcutaneous PC-3 tumour growth through inhibition of angiogenesis in a manner dependent on VEGF splicing Since SRPK1-KD induced a splicing switch towards VEGF anti-angiogenic isoforms we investigated whether this would affect the rate of tumour growth in which we asked whether VEGF165 cDNA overexpression driven by a VEGF-promoter (which would mimic endogenous VEGF but be insensitive to alternative splicing) could rescue the tumour growth in SRPK1-KD cells. SRPK1-KD or control cells were transfected with a plasmid containing the VEGF165 cDNA under the control of the VEGF promoter. SRPK1-KD did not affect VEGF promoter activity in PC3 cells, as assessed in vitro using a luciferase reporter plasmid driven by the endogenous VEGF promoter sequence (Supplementary Figure 7). One million PC-3 SRPK1-KD/VEGF165.
