RET Signaling in Prostate Cancer

Abstract

Purpose: Large diameter perineural prostate cancer is associated with poor outcomes. GDNF, with its coreceptor GFRα1, binds RET and activates downstream pro-oncogenic signaling. Because both GDNF and GFRα1 are secreted by nerves, we examined the role of RET signaling in prostate cancer.Experimental Design: Expression of RET, GDNF, and/or GFRα1 was assessed. The impact of RET signaling on proliferation, invasion and soft agar colony formation, perineural invasion, and growth in vivo was determined. Cellular signaling downstream of RET was examined by Western blotting.Results: RET is expressed in all prostate cancer cell lines. GFRα1 is only expressed in 22Rv1 cells, which is the only line that responds to exogenous GDNF. In contrast, all cell lines respond to GDNF plus GFRα1. Conditioned medium from dorsal root ganglia contains secreted GFRα1 and promotes transformation-related phenotypes, which can be blocked by anti-GFRα1 antibody. Perineural invasion in the dorsal root ganglion assay is inhibited by anti-GFRα antibody and RET knockdown. In vivo, knockdown of RET inhibits tumor growth. RET signaling activates ERK or AKT signaling depending on context, but phosphorylation of p70S6 kinase is markedly increased in all cases. Knockdown of p70S6 kinase markedly decreases RET induced transformed phenotypes. Finally, RET is expressed in 18% of adenocarcinomas and all three small-cell carcinomas examined.Conclusions: RET promotes transformation associated phenotypes, including perineural invasion in prostate cancer via activation of p70S6 kinase. GFRα1, which is secreted by nerves, is a limiting factor for RET signaling, creating a perineural niche where RET signaling can occur. Clin Cancer Res; 23(16); 4885-96. ©2017 AACR.

Figure 1. RET expression and response to GDNF and/or GFRα1 in prostate cancer

A. RET mRNA expression by RT-PCR using RNAs from PCa cell lines as indicated. PNT1A is an immortalized normal epithelial cell line. H₂0 indicates water control (no RNA). HPRT was used as an input control. B. RET mRNA expression by Q-RT-PCR in PCa cell lines. C. RET expression by Western blot analysis in PCa cell lines. Actin is a loading control. D. LNCaP, 22Rv1 or DU145 cells were treated with GDNF and/or GFRα1 and cell proliferation analyzed. The p-values for the differences in cell proliferation between GDNF and control or GDNF and GFRα1 by t-test are shown. All controls versus GFRα1 and GFRα1plus GDNF are also significantly different but values are not shown. E. Analysis of invasion and soft agar colony formation in LNCaP and 22Rv1 treated with GDNF and/or GFRα1. The p-values for the differences in invasion between GDNF and control or GDNF and GFRα1 by t-test are shown. All controls versus GFRα1 and GFRα1 plus GDNF are also significantly different but values are not shown. For soft agar colony formation p-values for significant differences between controls and GDNF or GFRα1 are shown; GFRα1 plus GDNF was also significantly different than controls but values are not shown.

Figure 2. Expression of GDNF and GFRα1 by prostate cancer cell lines

A. GDNF expression as assessed by Western blot of lysates of PCa cell lines. Positive control is recombinant human GDNF (500 ng). Actin is a loading control for cell lysates. B. Analysis of GDNF in conditioned medium from PCa cell lines as determined by GDNF ELISA. C. GFRα1 mRNA expression by RT-PCR in PCa cell lines as indicated. PNT1A is an immortalized normal epithelial cell line. H₂0 indicates water control (no RNA). HPRT was used as an input control. D. Expression of GFRα1 as assessed by Western blot of cell lysates of LNCaP and 22Rv1 cells. Actin is a loading control.

Figure 3. Dorsal root ganglia (DRG) promote transformed phonotypes via GFRα1

A. Western blot of conditioned medium without (CM) or with dorsal root ganglia (DCM). Ponceau S staining indicates equivalence of input protein. B. Effect of CM or DCM on cell proliferation, invasion and soft agar colony formation by LNCaP or 22Rv1 cells. The p values of t-tests comparing CM and DCM are shown. C. The effect of DCM on proliferation, invasion and soft agar colony formation invasion is inhibited by anti-GFRα1 neutralizing antibody. Cells were incubated with DCM and the indicated amount of neutralizing antibody. The p values for pairwise analysis versus control after ANOVA is shown by asterisks * p<.05. ** p<.01; ***p<.001 D. Perineural invasion along DRG by LNCaP cells (left) and 22Rv1 cells (right) is inhibited by anti-GFRα1 antibody. E. Perineural invasion is inhibited by RET knockdown LNCaP cells (left) and 22Rv1 cells (right)

Figure 4. RET knockdown decreases tumor growth in vivo

LNCaP cells with knockdown of RET (si-RET) or vector control cells (si-V) were inoculated subcutaneously in SCID mice and tumor growth monitored for 5 weeks. A. Calculated tumor volume over time. B. Final tumor weights. *** p<.001, t-test versus control

Figure 5. Phosphorylation of p70 S6 kinase is a key downstream target of RET signaling

A. Phosphorylation of AKT, ERK and p70 S6 kinase (p70S6K) in response to treatment with GDNF, GFRα1 or both in LNCaP and 22Rv1 cells as determined by Western blotting. B. Knockdown of RET blocks phosphorylation of AKT, ERK and p70S6K in response to GFRα1. C. Treatment with the PI3K inhibitor blocks AKT activation and U0126 blocks ERK activation. Treatment with either inhibitor blocks phosphorylation of p70S6K, indicating that both ERK and PI3K activation are needed for p70S6K activation. D–F. Knockdown of p70S6K completely blocks increased proliferation (D), invasion (E) and soft agar colony formation (F). Note that control baseline values were also significantly decreased. The p values for t-tests are as indicated.

Figure 6. RET expression in prostate adenocarcinoma and small cell carcinoma

A. Prostate adenocarcinoma with strong expression of RET (100X). B. Prostate adenocarcinoma with weak RET expression. Normal epithelium with no staining is to the left (arrowhead) (200X) C. RET expression in LNCaP xenograft (400X). D. Ganglion cells (large cells) and nerve (arrowhead) present in the tissue microarray serve as an internal positive control (400X). E. Prostate adenocarcinoma (left) and small cell carcinoma (right) from a radical prostatectomy (100X). F. Adenocarcinoma from same specimen as in E with focal RET staining and cribiform glandular architecture (200X). G. Small cell neuroendocrine carcinoma from transurethral resection (400X). H. Small intestinal neuroendocrine carcinoma (carcinoid) metastatic to liver. Liver is to the upper right (100X).