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. Author manuscript; available in PMC: 2018 Aug 15.
Published in final edited form as: Clin Cancer Res. 2017 May 10;23(16):4885–4896. doi: 10.1158/1078-0432.CCR-17-0528

RET signaling in prostate cancer

Kechen Ban 1, Shu Feng 1, Longjiang Shao 1, Michael Ittmann 1
PMCID: PMC5609835  NIHMSID: NIHMS875723  PMID: 28490466

Abstract

Purpose

Large diameter perineural prostate cancer is associated with poor outcomes. GDNF, with its co-receptor GFRα1, binds RET and activates downstream pro-oncogenic signaling. Since 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.

Keywords: prostate cancer, signal transduction, RET, p70 S6 kinase

INTRODUCTION

Prostate cancer (PCa) is the second-leading cancer cause of cancer in American men, with 27,540 deaths expected to occur due to PCa in 2015 (1). While the prognosis for early stage PCa is generally excellent, few effective therapeutic options exist for advanced PCa. It has been appreciated for many years that the tumor microenvironment plays an important role in the initiation and progression of prostate and other cancers. One important component of this microenvironment is nerves. It is well known that PCa has a propensity to grow in perineural locations, as do a number of other cancers such as pancreatic cancer. Perineural invasion (PNI) is defined as the presence of cancer infiltration in, around and/or through the nerves (2) and is the result of reciprocal interactions between cancer cells and adjacent nerves(3). PNI is an adverse prognostic factor for many cancers, including prostate, pancreatic, head and neck, colon, skin and salivary cancers (4–8). While PNI per se is not predictive of aggressive disease in PCa, large diameter perineural tumor is one of the most significant pathological predictors of poor outcome (9) following radical prostatectomy. Furthermore, PNI is associated with poor outcomes following radiation therapy(10,11), suggesting a pro-survival effect of PCa cell interactions with nerves. These clinical observations show that the interactions between nerves and PCa cells can have a significant impact on treatment outcomes in men with PCa which ultimately must be related to the underlying biology.

Recent functional studies in vitro and correlative studies in vivo have shown significant interactions between nerves and adjacent cancer cells that promote cell survival, proliferation and migration of PCa cells (2,3,12). For example, PCa cells adjacent to nerves display increased proliferation and decreased apoptosis compared to cells away from nerves(12), indicating local microenvironmental influence on the cancer cells in this niche. Similar findings have been reported in other neurotrophic cancer such as pancreatic cancer(13). Studies in rats have shown that denervation of the prostate leads to almost complete loss of epithelium (14), indicating a strong trophic effect of nerves on normal prostate epithelium. Similarly, men with complete spinal cord injury had significantly smaller prostates than controls(15). Studies by Magnon et al(16) have shown that chemical or surgical ablation of nerves inhibits tumorigenesis and metastasis in both xenograft and transgenic mouse models of PCa, unequivocally establishing that nerve-PCa cell interactions play a significant role in PCa initiation and progression but the molecular basis of these interactions is still unclear.

We have carried out expression microarray analysis of laser captured PCa reactive stroma (17) and shown that among the upregulated genes is glial cell line-derived neurotrophic factor (GDNF). Interestingly, GDNF levels are increased during androgen induced regrowth of the prostate after castration(18). GDNF is present in the peripheral nerves of normal prostate and in reactive stroma in PCas where it can be secreted and potentially interact with PCa cells. Of course, GDNF is expressed in nerves in potential metastatic sites as well. Functional studies in pancreatic cancer implicate GDNF as a key factor promoting perineural migration in vitro in this disease (19,20). It has also been shown in breast cancer that inflammatory cytokines can induce expression of GDNF by fibroblastic cells and tumor cells and GDNF increases proliferation and motility(21), indicating that GDNF is also expressed away from nerves in some contexts.

GDNF binds to RET, a transmembrane receptor tyrosine kinase, in conjunction with its co-receptor GFRα1 and activates cellular signaling (20). Robinson et al have shown RET is expressed in the three PCa cell lines tested (PC3, DU145 and LNCaP) as well as the CWR series of xenografts (22). Given that RET is present on at least some PCa cells and GDNF is expressed in nerves and reactive stroma cells we sought to determine the potential role of RET signaling in PCa. We have found that GDNF is not only expressed in the tumor microenvironment but is also expressed as an autocrine factor by PCa cell lines. Our data indicates that the rate limiting factor for RET signaling in most PCa is GFRα1, which is secreted by nerves, creating a niche promoting RET signaling in the perineural space. Furthermore, RET signaling can enhance cellular phenotypes associated with cancer progression in vitro and tumorigenesis in vivo. We have found that PI3K and ERK signaling downstream of RET induce activation of p70 S6 kinase (p70S6K), which is required for RET induced proliferation, invasion and soft agar colony formation. Finally, we have found that RET is expressed in subset of localized prostate adenocarcinomas as well as small cell neuroendocrine cancers. Given that agents targeting RET signaling are already available in the clinic our findings suggest that RET is a potential therapeutic target on both adenocarcinoma and small cell carcinoma of the prostate.

MATERIALS AND METHODS

Tissue Culture

Human PCa cells LNCaP, 22Rv1, DU145 and PC3 cells, and PNT1A, an immortalized normal prostate cell line, were all maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). LAPC4 cells were cultured in RPMI-1640 medium with 10% FBS supplemented with 10nM R1881 (Sigma). VCaP and 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) with 10% FBS. All cell culture medium contained 1x Antibiotic-Antimycotic (Gibco). PNT1A cells were obtained from the European Type Culture Collection. All other cell lines were obtained from the American Type Culture Collection. Cells were obtained between 2001 and 2012, expanded, frozen and stored as stocks in liquid nitrogen. Cell lines were tested monthly for mycoplasma contamination. All cell lines are authenticated by STR analysis at MD Anderson Cancer Center Characterized Cell Line Core Facility at the time of use.

For GDNF and GFRα1 treatments, cells were cultured with FBS-free medium for 24 hours and then treated with recombinant human GDNF at 100 ng/ml (ProSpec) or recombinant human GFRα1 at 100 ng/ml (R&D Systems) for 30 minutes. For inhibitor treatments, cells were treated with the PI3K inhibitor LY294002 at 20 μM (Cell Signaling), the MEK inhibitor U0126 at 10 μM (EMD Millipore) or vehicle for 1 hour and cell pellets collected for Western blot analysis.

Prostate and prostate cancer tissues

Tissue samples were obtained from Baylor College of Medicine Human Tissue Acquisition and Pathology Core under an Institutional Review Board approved protocols.

Western blot

For Western blot analysis, cell pellets were resuspended with T-PER tissues protein extraction reagent (Thermo Scientific) containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (PhosSTOP EASYpack, Roche Diagnostics), sonicated for 10 seconds and clarified by centrifugation. Extracted proteins were quantified by the Bio-Rad Protein Assay. Proteins were subjected to electrophoresis in PAGEr Gold Precast Protein Gels (Tris-Glycine) (Lonza) and transferred to membranes. Membranes were then blocked with 5% nonfat dry milk for 1 hour at room temperature, incubated with primary antibody overnight at 4°C followed by a secondary antibody conjugated horseradish peroxidase for 1 hour at room temperature. Protein-antibody complexes were detected by Pierce ECL Western Blotting Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (both from Thermo Scientific). The following antibodies were used: GFRα1 (H-70; Santa Cruz); RET (Novus); AKT, phospho-AKT Ser 473), p44/42 MAPK (ERK1/2), phospho-p44/42 ERK1/2, p70S6K, phospho-p70 S6K (T421/S424 #9204), β-actin (all from Cell Signaling). Ponceau S staining was carried out on membranes by briefly rinsing in distilled water and then incubating the membrane in Ponceau S Staining Solution (Cell Signaling) for 2 minutes at room temperature, washed in distilled water until distinct reddish-pink protein bands were visible and a photograph of the stained membrane was taken.

Reverse transcriptase polymerase chain reaction (RT-PCR)

To examine the mRNA expression of RET and GFRα1 in human PCa cell lines, we carried out conventional RT-PCR and quantitative real-time RT-PCR (Q-RT-PCR). Total RNA was extracted using the RNeasy kit (Qiagen). cDNA was synthesized using the amfiRivert cDNA synthesis Platinum Master Mix kit (GenDEPOT). Primers used for conventional RT-PCR were: RET: 5′-ACA GGG GAT GCA GTA TCT GG-3′ (RET-2767F) and 5′-CTG GCT CCT CTT CAC GTA GG-3′ (RET-2920R) and GFRα1: 5′-TCC GGG TGG TCC CAT TCA TA-3′ (GFRα1-1039F) and 5′-AGC ATT CCG TAG CTG TGC TT-3′ (GFRα1-1333R). HPRT was used for endogenous control using primers: HPRT-F: 5-GCAGACTTTGCTTTCCTTGG-3 (HPRT-F) and 5-TCAGGGATTTGAATCATGTTTG-3(HPRT-R). Quantitative PCR was performed according to standard TaqMan Fast protocol (Applied Biosystems). TaqMan primer/probe combinations for specific RET gene used was RET Hs01120030 (Life Technologies). β-Actin (ACTB Hs01060665) (Life Technologies) was used for endogenous control.

GDNF Enzyme-linked Immunosorbent Assay (ELISA)

Cells were cultured in FBS free medium for 5 days. The conditioned medium was concentrated by Amicon Ultra-15 (UFC900324). ELISA was performed using GDNF Emax® ImmunoAssay Systems (Promega) according to the manufacturer’s instructions.

Immunohistochemistry

Immunohistochemistry was carried out on sections of formalin fixed paraffin-embedded tissues by standard procedures on a Leica Bond III autostainer. Antigen retrieval was carried out with heat retrieval using Bond Epitope Retrieval Solution 2 (AR9640; Leica). A rabbit monoclonal anti-RET antibody (Cell Signaling, clone E1N8X) was used at 1:100 dilution for 30 minutes at room temperature. Detection was carried out using Leica’s Bond Polymer Refine kit (DS9800) for 16 minutes followed by chromogen for 5 minutes. Counterstain was hematoxylin

Cell proliferation assay

Cells were plated at 4X103 cells/well in 100μl medium in 96-well plates. The next day, cells were cultured with FBS-free medium. Cells were then treated with or without GFRα1 (100 ng/ml), recombinant human GDNF (100 ng/ml), anti-GFRα1 (2 μg/ml) (Santa Cruz), anti-GDNF antibody (Santa Cruz), normal goat IgG at 2 μg/ml (Santa Cruz) or 10 μl of concentrated conditioned media with/without dorsal root ganglia (DRG) for 6 days, with media changed one time on day three. Cell proliferation was then assessed by Cell Counting Kit-8 assay (Dojindo Molecular Technologies).

Cell invasion assay

Cell invasion assay was performed using the BD BioCoat™ Matrigel™ Invasion Chamber (BD Biosciences). Cells were starved for 24 hours in serum-free medium prior to seeding and 1×105 cells were then seeded into each insert in 0.5 ml of serum-free medium. Below the insert, 0.7 ml of serum-free medium with/without GFRα1 (100 ng/ml), GDNF (100 ng/ml), anti-GFRα1 (2 μg/ml) (Santa Cruz), normal goat IgG (2 μg/ml) (Santa Cruz) or 10 μl of concentrated conditioned media with or without DRG was added into each well. After 24 hours, the invasion cells on the bottom surface of the membrane were fixed with 100% methanol and stained with 0.3% crystal violet in 2% ethanol for 20 minutes and cells counted under a microscope.

Colony formation assay

To evaluate anchorage-independent growth, 2.5 X 103 cells/well were seeded in 0.4% agar on top of a base layer containing 0.6% agar in the 6-well plate. Cells were treated 2 times per week with 2 ml of cell culture media with/without GFRα1 (100 ng/ml), GDNF (100 ng/ml), anti-GFRα1 (2 μg/ml) (Santa Cruz), normal goat IgG (2 μg/ml) (Santa Cruz) or 50 μl of concentrated conditioned media with/without DRG. The plate was incubated at 37 °C at 5% CO2 in a humidified incubator for 21 days. At the end of the experiment, colonies were fixed with methanol, stained with 0.5% crystal violet, and counted.

Studies with lenvatinib

The RET inhibitor lenvatinib was obtained from Selleckchem (Cat#. S1164). Cell invasion was assessed as above using 0.7 ml of 10% FBS-medium below the well in the absence or presence of lenvatinib (100 nM or 500 nM). After 24 hours, the invasion cells on the bottom surface of the membrane were counted as described above. To evaluate anchorage-independent growth, 2.5X103 cells/well were seeded in 0.4% agar on top of a base layer containing 0.6% agar in the 6-well plate. Cells were treated 2 times per week with 2 ml of cell culture media in the absence or presence of lenvatinib (100 nM or 500 nM) for 21 days. At the end of the experiment, colonies were fixed with methanol, stained with 0.5% crystal violet, and counted.

Lentivirus infection

LNCaP or 22Rv1 cells were plated in 12-well plates at a density of 1×105 cells/well. The next day, the growth media was removed and 1 ml of fresh medium with 8 μg/ml polybrene (Sigma) and 100 μl scrambled (catalog # LVP015-G), GDNF (catalog # iV008619b) or RET (catalog # iV028816a) siRNA GFP Lentivirus (Applied Biological Materials) was added. The virus was removed after 24 hours of incubation and fresh medium was added to the wells. Stable transfectant populations of cells were selected by treatment with 2 μg/ml puromycin (Sigma) for 15 days.

Transient transfection

Cells were transiently transfected with scrambled siRNA (catalog # sc-37007), GFRα1 siRNA (catalog # sc-35469), RET siRNA (catalog # sc-36404) (all from Santa Cruz) or p70S6K siRNA (catalog # #6572, Cell signaling) using the Lipofectamine 2000 transfection reagent (Invitrogen). Twenty-four or 48 hours after transfection, cells were used for further analyses.

Collection of conditioned media from dorsal root ganglion (DRG) culture

DRG were harvested from C57BL mice and were placed in growth factor-depleted Matrigel matrix (BD Bioscience) in a 100-mm dish with 10% FBS medium at 37°C at 5% CO2 in a humidified incubator for 3 days. DRG were then cultured with 0.1% FBS medium for additional 3 days, and medium were collected and concentrated using centrifugal filter (Merck Millipore) to 200 μl volume. Control conditioned medium was obtained from identical conditions and procedure except there were no DRG in the Matrigel.

In vitro dorsal root ganglion (DRG) co-culture model of perineural invasion

The in vitro model of nerve invasion is originally described by Ayala et al (3) DRG were harvested from C57BL mice and placed in 6-well plates and covered with 20 μl of growth factor reduced Matrigel (BD Biosciences). After growth in RPMI 1640 medium with 10% FBS for 7 days, 2X105 cancer cells were added into the well. For GFRα1 blockage, anti-GFRα1 antibody (2 μg/ml) was added to media. Normal goat IgG (2 μg/ml) was used as a control. Twenty-four hours after the cancer cells were added, images were acquired using an Axiovert 40 CFL inverted microscope (ZEISS) and invasion cells were counted.

Mouse xenograft studies

All experiments were carried out on 8–10 week-old male SCID mice in accordance with the IACUC approved protocol. Tumor xenografts were established by subcutaneous injection over each flank using either LNCaP cells with stable RET knockdown (si-RET) or vector controls LNCaP cells (si-V) (5X106 cells/site) mixed 1:1 with Matrigel (Becton Dickinson). The tumor size was measured twice weekly using calipers and the tumor volume in mm3 calculated as volume= (height × width × length)/2. Tumors were harvested 35 days after the cell inoculation and tumors were excised and weighed.

Statistical analysis

Numerical values were compared using Student’s t-test, two-tailed with p<.05 considered significant when two groups were compared. For multiple groups one-way ANOVA was used and when significant, pairwise comparison carried out with p<.05 considered significant for pairwise comparisons. The chi-squared test was used to compare the RET expression in benign prostate and cancer tissues.

RESULTS

Expression of RET in PCa cell lines

Given that GDNF is secreted in the tumor microenvironment, we initially sought to determine if RET is expressed in a broad array of PCa cell lines. RT-PCR of RNAs from all PCa cell lines and the immortalized prostate epithelial cell line PNT1A shows that all PCa cell lines and prostate epithelial cells express RET mRNA at variable levels (Fig 1A). Western blot analysis confirmed RET expression at the protein level that was concordant with mRNA levels measured by Q-RT-PCR (Fig 1B and 1C). Both GDNF and GFRα1 are released by nerves and have been previously shown to be able to induce cancer cell proliferation and invasion (23). We therefore treated PCa cell lines with GDNF, GFRα1 or both and evaluated proliferation (Fig 1D). Somewhat surprisingly, all cell lines responded to GFRα1 alone but only 22Rv1 cells responded to GDNF alone. Furthermore, combined treatment with GDNF and GFRα1 was no more potent than GFRα1 alone. Similar results were seen when invasion and soft agar colony formation were assessed (Fig 1E). These findings implied that GDNF was present in the PCa cell lines since all cell lines responded to exogenous GFRα1. Furthermore, only 22Rv1 cells can respond to exogenous GDNF, suggesting that they are the only PCa cell line that expresses GFRα1. Indeed, as seen in Fig 2A and 2B, by Western blotting and ELISA for GDNF, all PCa cell lines expressed variable levels of GDNF protein. This indicates that GDNF can act as an autocrine factor in PCa, in addition to its role as a potential paracrine factor from nerves. Furthermore, RT-PCR analysis showed that only 22Rv1 expressed significant amounts of GFRα1 (Fig 2C) and Western blot shows that 22Rv1 are the only PCa cell line to express GFRα1 protein (Fig 2D). It should be noted that even 22Rv1 responded to exogenous GFRα1, indicating that exogenous GFRα1 protein can enhance the response of PCa cells to GDNF even in the presence endogenous GFRα1

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

Figure 1

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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.

Figure 2

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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.

GFRα1 secreted by dorsal root ganglia promotes cancer phenotypes and perineural invasion

It is known that nerves can release soluble GFRα1 (24). We therefore examined the ability of dorsal root ganglia (DRG) excised from mice to enhance cancer phenotypes in PCa cells in vitro. As expected, GFRα1 is present in the conditioned media (CM) of DRG in culture (Fig 3A). CM from DRG was able to significantly enhance proliferation, invasion and soft agar colony formation in both LNCaP and 22Rv1 cells (Fig 3B). This was significantly blocked by neutralizing anti-GFRα1 antibody (Fig 3C).

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

Figure 3

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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)

To evaluate the impact of GFRα1 on perineural invasion, we used the DRG in vitro coculture model of perineural invasion. We found that LNCaP and 22Rv1 cancer cells invaded along the neurites and that perineural invasion was significantly attenuated by anti-GFRα1 antibody (Fig 3D), indicating that GFRα1 also plays an important role in the perineural invasion. To confirm that perineural invasion was increased by RET signaling we carried out similar experiments using LNCaP and 22Rv1 cells with RET knockdown and showed significant decreases in perineural invasion (Fig 3E).

To confirm the role of RET in the biological effects of GFRα1, LNCaP and 22Rv1 cell lines were stably infected with siRNA lentivirus targeting RET or nontargeting control. Q-RT-PCR analysis confirmed that the expression RET mRNA was decreased by 81% in both targeted cell lines compared to scrambled control cells (Supplementary Fig 1A). Knockdown of RET expression reduced the increased cell proliferation (Supplementary Fig 1B), invasion (Supplementary Fig. 1C) and colony formation (Supplementary Fig 1D) by GFRα1 in both LNCaP and 22Rv1 cell lines, indicating that RET mediates the effects of GFRα1 on these cell biological responses. This data was confirmed by transient transfections using a second siRNA (Supplementary Fig 2)

RET signaling promotes tumor growth in vivo

To determine whether RET signaling can promote tumor growth in vivo we inoculated LNCaP with stable RET knockdown or vector controls as subcutaneous xenografts in SCID mice. As can be seen in Figure 4, RET knockdown significantly decreased tumor growth in vivo, confirming the oncogenic impact of RET signaling in vivo. Relevant to this observation, we have found that lenvatinib, which inhibits RET, can significantly inhibit both invasion and soft agar colony formation in both LNCaP and 22RV1 cells (Supplementary Figure 3). Lenvatinib, is a multikinase inhibitor that inhibits several other receptor tyrosine kinases in addition to RET(25). Thus, it is not clear that the effects of lenvatinib are attributable to RET inhibition alone, but these results, along with our in vivo studies, suggest that RET is potentially targetable in in vivo using known agents that are currently in clinical use for targeting RET in other cancers.

Figure 4. RET knockdown decreases tumor growth in vivo.

Figure 4

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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

GFRα1 activates p70S6 kinase

To determine the signaling pathways downstream of RET signaling we treated LNCaP and 22Rv1 cells with GDNF, GFRα1 or both proteins and analyzed activation of key signaling pathways by Western blotting. In LNCaP cells, GFRα1 (and GDNF plus GFRα1) significantly stimulated phosphorylation of ERK1/2, but did not increase AKT phosphorylation, which is already high in these cells due to PTEN inactivation (Fig. 5A). In 22Rv1 cells, GDNF, GFRα1 and GDNF plus GFRα1 stimulated AKT phosphorylation, with GFRα1 and GDNF plus GFRα1 showing stronger effects (Fig. 5A). ERK1/2 phosphorylation was not increased in 22Rv1 cells, which have intrinsically high ERK activation. However, in both cell lines there was a marked increase on phosphorylation of p70S6K. Knockdown of RET confirmed that the effects of GFRα1 are mediated via RET signaling (Fig 5B). This was further confirmed with a second RET siRNA (Supplementary Fig 4A)

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

Figure 5

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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.

To dissect the role of AKT and ERK phosphorylation in the activation of p70S6K we treated each cell lines with either the selective PI3K inhibitor, LY294002 or the selective inhibitor of the MAPK pathway, U0126. Both drugs showed the expected activities in both cell lines (Fig 5C). Interestingly, treatment with either LY294002 or U0126 almost completely abolished activation of p70S6K in both cell lines. These results indicate that activities of both PI3K and MAPK pathways are required for activation of p70S6K, although the dominant pathway activated by GDNF/GFRα1/RET depends on the cellular context.

Transfection of p70S6K targeting siRNA in LNCaP and 22Rv1 cells was used to evaluate the role of p70S6K in the effect of GFRα1 on cell phenotypes. Knockdown of p70S6K almost completely abolished cell proliferation (Fig 5D), invasion (Fig 5E) and colony formation (Fig. 5F) in response to GFRα1 treatment. This was confirmed with a second p70S6k siRNA (Supplementary Fig 4B and 4C). Moreover, inhibition of p70S6K also decreased perineural invasion (Supplementary Fig 5A–D). These findings indicate that p70S6K plays a pivotal role in the effect of GFRα1/RET on these cellular behaviors.

Expression of RET protein in PCa

Our data shows that RET is expressed in all PCa cell lines tested, all of which were established from advanced PCas. To examine the expression of RET in clinically localized prostate adenocarcinomas we examined a tissue microarray containing 325 clinically localized cancers from radical prostectomies that has been described previously(26) using immunohistochemistry. Positive control tissue was adrenal medulla (Supplementary Fig 6A). Intensity (0–3) and extent (0–3) of staining was scored to derive a multiplicative index ranging from 0–9, with 9 representing strong diffuse staining (Fig 6A) as described previously(26). A total of 61 cancers (18.8%) showed staining. Some had weak staining (≤ 3, shown in Fig 6B), but this staining is almost certainly biologically significant since LNCaP xenografts, which are inhibited by RET knockdown, show similar intensity of staining by immunohistochemistry (Fig 6C). Interestingly, staining was highly cancer specific, with only a single case showing staining in benign luminal epithelium (p<.001; chi-squared). Nerves and ganglia in the stroma of occasional tissue microarray cores were stained as expected as an internal positive control (Fig 6D). Supporting these findings, examination of TCGA databases in cBioportal show that PCa has the third highest RET mRNA levels after pheochromocytoma/paragangioma and breast cancer (Supplementary Figure 7). Of course, pheochromocytoma and paraganglioma would be expected to express high levels of RET since their tissues of origin intrinsically express high levels of RET. It has also been shown that more than half of all breast cancers express RET by immunohistochemistry (27).

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

Figure 6

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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).

Recent reports have shown that RET mRNA is expressed in lung small cell carcinoma cell lines (28) and in small intestinal neuroendocrine tumors (29). We therefore examined whether RET protein is expressed in small cell neuroendocrine cancers of the prostate, which are highly aggressive tumors that can be present at initial diagnosis or arise from adenocarcinomas after prolonged treatment of with androgen blockade. We examined a radical prostatectomy which contained extensive areas of small cell carcinoma as well as high grade prostate adenocarcinoma. The majority of the adenocarcinoma component was negative for RET protein while the neuroendocrine component stained strongly (Fig 6E). Some foci were observed with clear cut glandular features and showed weaker RET staining (Fig 6F). We also examined two “channel TURPs” from patients with small cell neuroendocrine carcinoma. Both stained positively for RET in a heterogeneous pattern (Fig 6G and Supplementary Fig 6B). To determine if RET expression was also present in neuroendocrine tumors from other sites we also analyzed a primary small intestinal neuroendocrine tumor (carcinoid), a carcinoid metastatic to liver and a primary lung small cell carcinoma. The metastatic carcinoid stained strongly (Fig 6H) while the primary small intestinal carcinoid and the pulmonary small cell carcinoma stained heterogeneously (Supplementary Fig 6C and 6D).

DISCUSSION

In this study we demonstrate that RET is ubiquitously expressed in PCa cell lines, which are derived from advanced PCas. In addition, it is expressed in cancer cells in 18% of clinically localized adenocarcinomas as assessed by immunohistochemistry of a tissue microarray. It should be noted that PCa is heterogeneous disease and other important molecular alterations, such as SPOP mutation or SPINK1 expression, are present in less than 15% of cases (30,31). A smaller study of 30 whole mount prostatectomy sections by Dawson et al (32) showed RET expression in the majority of cases by immunohistochemistry. It should be noted that examination of whole mount specimens is more likely to detect areas of RET expression if there is significant heterogeneity in its expression, since TMAs present only a fraction of the cancer present in any given prostate. Thus, our TMA study may underestimate the number of cases with at least focal staining in the radical prostatectomy specimen. Of note, we only observed a single case (of 325) which showed staining of benign luminal epithelial cells, so staining was significantly increased in PCa compared to benign epithelium. Dawson et al (32) observed no staining in benign luminal cells, although they did observe weak staining in basal cells, which we did not observe. Thus RET is expressed at increased levels in a significant fraction of prostate adenocarcinomas, although determining the exact percentage in various cancer stages will require further investigation.

In addition, to prostate adenocarcinomas, RET was expressed in three of three small cell neuroendocrine prostate cancer tested, although in a heterogeneous pattern. Drake et al (33) have previously identified a small cell carcinoma of the prostate with activation of RET signaling based on phosphoproteomics, so it is likely that the RET signaling is active in some or all small cell carcinomas of the prostate. In addition, we observed RET protein expression in neuroendocrine cancers of other origins, consistent with mRNA studies by other groups (28,29) so RET expression is almost certainly a more general property of neuroendocrine cancers. Recent studies have shown that the ASCL1 transcription factor plays a key role in pulmonary neuroendocrine cancers and RET is a downstream target of this gene (28).

Our studies show that RET signaling can enhance proliferation, invasion and soft agar colony formation in vitro and tumor growth in vivo in PCa. In GDNF-RET signaling pathway, GDNF first binds to GFRα1 and then binds to and activates RET. GDNF is expressed by nerves and we have previously shown that GDNF is expressed in PCa stroma (17). Normal prostatic stroma is rich in nerve fibers and nerves are increased in the peritumoral stroma in PCa (34). We have now shown that PCa cell lines express and secrete GDNF. Thus GDNF is available within PCa tumors to stimulate RET signaling as an autocrine and/or paracrine growth factor if GFRα1 is present. Of the cell lines tested, only 22Rv1 cells express sufficient GFRα1 to support GDNF/RET signaling. Huber et al (35) have previously noted that only PCa cell lines making GFRα1 can respond to exogenous GDNF. Our data indicates that secreted GFRα1 from nerves can provide this limiting factor in a paracrine manner. The expression of both GDNF and GFRα1 by nerves creates a perineural niche in which RET signaling can occur for PCas expressing RET. Given that 22Rv1 cells express GFRα1, it seems likely that a subset of PCas express GFRα1. The relative contribution of nerves and PCa cells to GFRα1 protein expression is not completely clear and will require further studies. In addition, PCa cell lines express GDNF, again implying that at least a subset of advanced PCas express this growth factor. Thus in addition to RET signaling in the perineural niche there may be autocrine signaling or combined autocrine/paracrine signaling via RET in PCa. Again, further studies are needed to define the extent of expression GFRα1 and GDNF and the cells expressing these factors in various stages of PCa.

RET signaling can activate both the PI3K/AKT and MAPK/ERK signaling pathways through phosphorylation of tyrosine 1062 in RET(36). It has been well documented that PI3K/AKT/mTOR and MAPK/ERK signaling pathways play a fundamental role in the regulation of cell growth, division, survival and migration in cancer. In this study, we found that in LNCaP cells, RET signaling stimulated ERK1/2 phosphorylation while in 22Rv1 cells, RET signaling stimulated AKT phosphorylation. Thus the exact pathways stimulated by RET signaling are dependent on the cellular context. However, in both cell lines p70S6K phosphorylation was markedly increased by RET signaling. Inhibitor studies showed that activation of both ERK and AKT signaling was needed for RET induced p70S6K phosphorylation. p70S6K is well known as a key downstream target of P13K/AKT/mTOR pathway. It is less appreciated the ERK signaling can also enhance p70S6K phosphorylation (37). Mechanistically, this has been shown to be due to phosphorylation of TSC2 by ERK, which abrogates TCS2 inhibition of mTOR (38). In addition, RSK, which is downstream of ERK, can also directly target mTORC1 and increase its kinase activity (37). Of note, our studies demonstrate that knockdown of p70S6K markedly decreased proliferation, invasion and soft agar colony formation induced by RET signaling, indicating that it plays a critical role in promoting these phenotypes. Interestingly, Horii et al (39)have shown a strong correlation of phospho-p70S6K with both phospho-ERK and phospho-4EBP1 (another mTOR target) and with recurrent disease in breast cancer. Whether phospho-p70S6K has prognostic significance in PCa is unknown but will be important to determine.

Our studies suggest that RET may be a therapeutic target in the subset of prostate adenocarcinomas expressing RET. Perineural invasion is associated with poor outcomes following radiation therapy (10,11,40), suggesting a pro-survival effect of prostate cancer cell interactions with nerves during radiation therapy. Of note, decreasing phospho-p70S6K using an mTOR inhibitor has been shown to enhance radiation sensitivity in lung cancer (41). Both ERK and AKT phosphorylation have also been strongly implicated in resistance to radiation therapy (42,43). Treatment with vandetanib, which inhibits RET, results in improved responses to radiation in head and neck cancer (44), which is also neurotropic. We hypothesize that PCas expressing RET may be particularly resistant to radiation therapy due to prosurvival RET signaling in the perineural niche and that targeting RET signaling could potential improve outcomes of radiation therapy. Of note, Huber et al (35) have shown that DNA damage by radiation or chemotherapy enhances GDNF secretion by fibroblasts from the prostate and bone, which could further enhance RET signaling in this context. In addition, men with advanced cancer, including small cell cancers might also benefit from targeting RET signaling. There are a variety of agents in the clinic or under development that can target RET signaling (36) or RET (27), including lenvatinib, which has been approved for treatment of thyroid and renal cancer (25). Further studies are needed to explore this potential therapeutic approach in PCa.

Supplementary Material

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STATEMENT OF TRANSLATIONAL RELEVANCE.

Agents targeting RET signaling are already available in the clinic. Our findings suggest that RET is a potential therapeutic target on both adenocarcinoma and small cell carcinoma of the prostate

Acknowledgments

Grant Support: This work was supported by grants from the Dept of Defense Prostate Cancer Research Program (W81XWH-14-1-0505; MI); the Prostate Cancer Foundation (MI); the Dept of Veterans Affairs Merit Review program (5-IO1 BX002560-02; MI); the National Cancer Institute to the Dan L. Duncan Cancer (P30 CA125123) supporting the Human Tissue Acquisition and Pathology Shared Resource and by the use of the facilities of the Michael E. DeBakey VAMC.

We gratefully acknowledge the skilled assistance of Julie Zhao with RET immunohistochemi

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Supplementary Materials

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