DNA damage induces GDNF secretion in the tumor microenvironment with paracrine effects promoting prostate cancer treatment resistance

Abstract

Though metastatic cancers often initially respond to genotoxic therapeutics, acquired resistance is common. In addition to cytotoxic effects on tumor cells, DNA damaging agents such as ionizing radiation and chemotherapy induce injury in benign cells of the tumor microenvironment resulting in the production of paracrine-acting factors capable of promoting tumor resistance phenotypes. In studies designed to characterize the responses of prostate and bone stromal cells to genotoxic stress, we found that transcripts encoding glial cell line-derived neurotrophic factor (GDNF) increased several fold following exposures to cytotoxic agents including radiation, the topoisomerase inhibitor mitoxantrone and the microtubule poison docetaxel. Fibroblast GDNF exerted paracrine effects toward prostate cancer cells resulting in enhanced tumor cell proliferation and invasion, and these effects were concordant with the expression of known GDNF receptors GFRA1 and RET. Exposure to GDNF also induced tumor cell resistance to mitoxantrone and docetaxel chemotherapy. Together, these findings support an important role for tumor microenvironment damage responses in modulating treatment resistance and identify the GDNF signaling pathway as a potential target for improving responses to conventional genotoxic therapeutics.

Figure 1. DNA damage induces GDNF expression in human prostate fibroblasts

(A) Western blot probing for DNA damage marker y-H2AX post Docetaxel (DOC) (50 nM) and irradiation (IR) (10 Gy) in PSC27 cell lysates. (B) Gene expression microarray data of GDNF in human prostate stromal cells treated with hydrogen peroxide (H₂O₂), Bleomycin (Bleo) and irradiation (IR) on log scale. qPCR data showing up-regulation of GDNF after (C) irradiation between 6 and 16 days post treatment and after (D) mitoxantrone (MIT) treatment between days 7 and 15 post treatment. (E) ELISA assay measuring GDNF protein in cell lysates (Ly) 5d, 10d and 15d after DNA damage induced by irradiation (10 Gy) compared to non-irradiated control (CTRL). (F) GDNF transcript level changes measured by microarrays in micro-dissected CaP stroma after treatment with DOC and MIT in 10 paired patient samples.

Figure 2. DNA damages induces GDNF secretion producing autocrine effects in prostate fibroblasts

GDNF protein levels measured by ELISA in (A) conditioned medium (CM) and (B) cell lysates (Ly) of PSC27 prostate stromal fibroblasts after DNA damage by treatment with Docetaxel (DOC; 50 nM), Mitoxantrone (MIT; 100 nM), or irradiation (IR; 10 Gy) 15d after treatment. (C) GDNF specific ELISA measuring GDNF levels in Ly and CM of virally transduced PSC27 cells and (D) western blot analysis of both CM and Ly of the same cell line. (E) Western blot analysis of signaling pathways in PSC27 cells after stimulation with 100 ng/ml hrGDNF or CM of GDNF over-expressing cells. (F) Parental PSC27 (black, 9 passages) were transduced with TurboGFP (white, #1) or GDNF-V5 (blue, #2) and passaged to replicative exhaustion. Passage numbers are shown as absolute counts. (G) PSC27 cells stimulated with hrGDNF for 5 days, relative cell counts are shown.

Figure 3. Bone fibroblasts induce GDNF following DNA damage but lack autocrine signaling

GDNF protein levels measured by ELISA in cell lysates of (A) HS5 and (B) HS27a human bone stromal cells after DNA damage by treatment with Docetaxel (DOC; 1 nM), Mitoxantrone (MIT; 100 nM), or irradiation (IR; 10 Gy) 15d after treatment. (C) ELISA analysis of GDNF expression in cell lysate and secretion in to CM of bone stromal cells virally transduced to over-express GDNF tagged with a V5-epitope. (D) Western blot analysis of GDNF expression (Ly) and secretion in to the conditioned medium (CM) of HS-GDNF-V5 cells. (E) Bone stromal cells HS5 and HS27a were stimulated with full serum (FBS) or 100 ng/ml hrGDNF and signaling pathways were analyzed by western blot. Cell counts for (F) HS5 and (G) HS27a bone stormal cells stimulated with increasing concentrations of GDNF after 5d of culturing. Significant changes (p≤0.05) are shown as red bars.

Figure 4. Prostate cancer cells respond to GDNF stimulation and activate SRC and ERK pathways

(A) Cell proliferation assay counting viable cells after 5d of stimulation with 5-10 ng/ml hrGDNF in serum free conditions. (B) Transcript level analysis for RET receptor and GFRA-familiy members in CaP cell lines, one array probe per box are shown. (C) Western blot analysis of signaling pathway activation in GDNF responsive cells (M12 and M2205) and GDNF insensitive cells (DU145 and LNCaP). (D) Protein staining for GDNF family receptors and RET from the Protein Atlas data base in CaP patient samples. (E) Cell invasion assay using 100 ng/ml of hrGDNF as chemo-attractant in serum free conditions.

Figure 5. GDNF promotes tumor cell resistance to genotoxic chemotherapy

Epithelial CaP cells were stimulated with 100 ng/ml hrGDNF in serum free conditions and treated for 5d with (A) 50 nM Docetaxel or (B) 100 nM Mitoxantrone. Cell numbers and viability were analyzed and are shown as normalized values compared to Tx w/o GDNF stimulation as baseline.

Figure 6. GDNF induces gene expression changes via the activation of transcription factor networks

Heat map profiles of gene expression changes upon GDNF stimulation in (A) epithelial and stromal prostate cancer cells, (B) in M12 epithelial cells alone, and (C) in PSC27 PPFs alone. (D) Activation scores for transcription factor target gene groups after GDNF stimulation in PSC27 (black) and M12 (blue) cells. (E) Activation scores for RB, E2F1 and AR target gene groups after GDNF stimulation in epithelial CaP cells. (F) Gene expression changes of known E2F1 and AR target genes with enhancer modules regulated by GDNF stimulation.