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

Huber RM, Lucas JM, Gomez-Sarosi LA, Coleman I, Zhao S, Coleman R, Nelson PS - Oncotarget (2015)

Bottom Line: Though metastatic cancers often initially respond to genotoxic therapeutics, acquired resistance is common.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

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.

Show MeSH

Related in: MedlinePlus

Bone fibroblasts induce GDNF following DNA damage but lack autocrine signalingGDNF 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385841&req=5

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

Mentions: DNA damaging regimens are used focally in the case of radiation to the primary site or discrete metastatic foci, regionally in the case of bone-directed radioisotopes such as RAD-223, and systemically when using genotoxic chemotherapeutics for the treatment of CRPC which often metastasizes to bone [19]. To determine if DNA damaging regimens also induce GDNF in benign cells residing in the bone marrow niche, we measured GDNF expression after treatment with IR (10 Gy), docetaxel (1 nM) and mitoxantrone (100 nM) in two bone stromal cell lines (HS5 and HS27a, [20]). In both cell lines, GDNF was below detection limit in control settings. However, exposure to IR, docetaxel, or mitoxantrone strongly induced GDNF protein production as measured by a GDNF specific ELISA (Fig 3A, B). To isolate GDNF effects from the spectrum of other damage-induced factors, we generated GDNF over-expressing cell lines for HS5 and HS27a cells (Fig 3C, D). The proliferation rates of the GDNF over-expressing bone fibroblast lines did not differ from the control cells (data not shown). Subsequent analysis of the signaling pathways which are known to be down-stream of GDNF and which were activated in prostate fibroblasts, such as SRC and ERK phosphorylation, did not show any activation in HS5 or HS27a cells (Fig 3E). There were also no changes in AKT phosphorylation status or activation of down-stream S6 kinase, indicating that the bone stromal cells, unlike prostate fibroblasts, are not sensitive to autocrine/paracrine stimulation with GDNF, despite the fact that they do substantially increase GDNF expression after genotoxic damage. While exposure to GDNF did not produce changes in SRC, ERK and S6K activation patterns, increasing GDNF concentrations (25-200 ng/ml) did have an anti-proliferative effect on HS5 fibroblasts when compared to control conditions or to cells stimulated with low concentrations of GDNF (5-10 ng/ml; p<0.05) (Fig 3F). HS27a fibroblasts did not show significant changes in cell growth or proliferation in these assays (Fig 3G).


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

Huber RM, Lucas JM, Gomez-Sarosi LA, Coleman I, Zhao S, Coleman R, Nelson PS - Oncotarget (2015)

Bone fibroblasts induce GDNF following DNA damage but lack autocrine signalingGDNF 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385841&req=5

Figure 3: Bone fibroblasts induce GDNF following DNA damage but lack autocrine signalingGDNF 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.
Mentions: DNA damaging regimens are used focally in the case of radiation to the primary site or discrete metastatic foci, regionally in the case of bone-directed radioisotopes such as RAD-223, and systemically when using genotoxic chemotherapeutics for the treatment of CRPC which often metastasizes to bone [19]. To determine if DNA damaging regimens also induce GDNF in benign cells residing in the bone marrow niche, we measured GDNF expression after treatment with IR (10 Gy), docetaxel (1 nM) and mitoxantrone (100 nM) in two bone stromal cell lines (HS5 and HS27a, [20]). In both cell lines, GDNF was below detection limit in control settings. However, exposure to IR, docetaxel, or mitoxantrone strongly induced GDNF protein production as measured by a GDNF specific ELISA (Fig 3A, B). To isolate GDNF effects from the spectrum of other damage-induced factors, we generated GDNF over-expressing cell lines for HS5 and HS27a cells (Fig 3C, D). The proliferation rates of the GDNF over-expressing bone fibroblast lines did not differ from the control cells (data not shown). Subsequent analysis of the signaling pathways which are known to be down-stream of GDNF and which were activated in prostate fibroblasts, such as SRC and ERK phosphorylation, did not show any activation in HS5 or HS27a cells (Fig 3E). There were also no changes in AKT phosphorylation status or activation of down-stream S6 kinase, indicating that the bone stromal cells, unlike prostate fibroblasts, are not sensitive to autocrine/paracrine stimulation with GDNF, despite the fact that they do substantially increase GDNF expression after genotoxic damage. While exposure to GDNF did not produce changes in SRC, ERK and S6K activation patterns, increasing GDNF concentrations (25-200 ng/ml) did have an anti-proliferative effect on HS5 fibroblasts when compared to control conditions or to cells stimulated with low concentrations of GDNF (5-10 ng/ml; p<0.05) (Fig 3F). HS27a fibroblasts did not show significant changes in cell growth or proliferation in these assays (Fig 3G).

Bottom Line: Though metastatic cancers often initially respond to genotoxic therapeutics, acquired resistance is common.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

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.

Show MeSH
Related in: MedlinePlus