Role and expression of FRS2 and FRS3 in prostate cancer

Abstract

Background: FGF receptor substrates (FRS2 and FRS3) are key adaptor proteins that mediate FGF-FGFR signalling in benign as well as malignant tissue. Here we investigated FRS2 and FRS3 as a means of disrupting global FGF signalling in prostate cancer.

Methods: FRS2 and FRS3 manipulation was investigated in vitro using over-expression, knockdown and functional assays. FRS2 and FRS3 expression was profiled in cell lines and clinical tumors of different grades.

Results: In a panel of cell lines we observed ubiquitous FRS2 and FRS3 transcript and protein expression in both benign and malignant cells. We next tested functional redundancy of FRS2 and FRS3 in prostate cancer cells. In DU145 cells, specific FRS2 suppression inhibited FGF induced signalling. This effect was not apparent in cells stably over-expressing FRS3. Indeed FRS3 over-expression resulted in enhanced proliferation (p = 0.005) compared to control cells. Given this functional redundancy, we tested the therapeutic principle of dual targeting of FRS2 and FRS3 in prostate cancer. Co-suppression of FRS2 and FRS3 significantly inhibited ERK activation with a concomitant reduction in cell proliferation (p < 0.05), migration and invasion (p < 0.05). Synchronous knockdown of FRS2 and FRS3 with exposure to cytotoxic irradiation resulted in a significant reduction in prostate cancer cell survival compared to irradiation alone (p < 0.05). Importantly, this synergistic effect was not observed in benign cells. Finally, we investigated expression of FRS2 and FRS3 transcript in a cohort of micro-dissected tumors of different grades as well as by immunohistochemistry in clinical biopsies. Here, we did not observe any difference in expression between benign and malignant biopsies.

Conclusions: These results suggest functional overlap of FRS2 and FRS3 in mediating mitogenic FGF signalling in the prostate. FRS2 and FRS3 are not over-expressed in tumours but targeted dual inhibition may selectively adversely affect malignant but not benign prostate cells.

Figure 1

FRS2 and FRS3 mRNA expression in prostate cell lines. A: FRS2 and FRS3 mRNA expression in benign and malignant cell lines as well as protein expression by western blot using FRS2 and FRS3 specific antibodies. B: FRS2 and FRS3 and PSA (positive control) expression in LNCaP cells treated with androgens and assayed at different time points.

Figure 2

Mitogenic effect of FRS3 over-expression and functional redundancy. A: Immunoblot analysis showing FRS3-Myc protein over-expression in DU145 cell line compared to empty vector control cell line following stable transfection with FRS3. B: FRS3 over-expression enhances cell proliferation in DU145 in response to full media and different FGFs. C: FRS2 knock down is compensated for by FRS3 over-expression in proliferation assay in response to different FGF stimulation. Inset image shows immunoblot representative of FRS2 down-regulation following siRNA transfection in DU145 cells. D: FRS3 over-expression reverses the inhibitory effect of FRS2 suppression by siRNA on pERK activation. *p < 0.005.

Figure 3

Effect of dual targeting of FRS2 and FRS3 in cancer and benign cells. A: Immunoblot analysis showing representative dual down-regulation of FRS2 and FRS3 protein in PC3 cell line and corresponding proliferation assay stimulated with full media. B: Down-regulation of FRS2 and FRS3 in DU145 cell line and corresponding proliferation assay stimulated with full media. C: Down-regulation of FRS2 and FRS3 in benign PNT2 cell line and corresponding proliferation assay stimulated with full media. *p < 0.005. siFRS2/3- siRNA against FRS2 and FRS3

Figure 4

Dual targeting of FRS2 and FRS3 is a global inhibitor of FGF induction. A and B: Migration experiments in PC3 and DU145 stimulated with different FGFs. C and D: Invasion assays for PC3 and DU145 cancer prostate cell lines in the presence of different FGFS and EGF. E: Silencing FRS2 and FRS3 inhibits FGF-simulated ERK activation of PC3 cells by FGF1 and FGF2 (*p < 0.005). siFRS2/3- siRNA against FRS2 and FRS3

Figure 5

Dual targeting of FRS2/FRS3 as an adjunct to radiotherapy. A and B: Percentage change in the number of colony forming units in PC3 and DU145 prostate cancer cell lines. C: Percentage change in the number of colony forming units in PNT2 prostate cell lines. (* <0.005; **p <0.05). D: Effect of altering FRS2 and FRS3 levels on PARP cleavage in PC3 cells. siFRS2/3- siRNA against FRS2 and FRS3

Figure 6

Expression of FRS2 and FRS3 mRNA in clinical prostate cancer. A: Relative FRS2 and FRS3 mRNA expression in micro-dissected benign and tumours of different Gleason grades. Each profile represents a tumour derived from an individual patient. B. Relative alterations in FRS2 and FRS3 expression between benign and malignant prostate samples profiled in the MSKCC Prostate Oncogenome dataset (n = 131 tumours) (26). Here only a small minority of tumours had altered FRS2 and FRS3 mRNA with similar numbers exhibiting up-regulation or down-regulation of each.

Figure 7

FRS2 and FRS3 protein expression in clinical prostate tissue. A and B: FRS2 and 3 expression respectively in benign prostate glands C and D: FRS2 expression in malignant prostate biopsies. E and F: FRS3 expression in malignant prostate biopsies. G: Data table on FRS2 protein expression in human prostate tissue stratified by benign or cancer biopsies. H: Data table on FRS3 protein expression in human prostate tissue stratified by benign or cancer biopsies.