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

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.

Mentions: We previously profiled the gene expression alterations in prostate fibroblasts following DNA damage and identified a spectrum of highly induced transcripts of which a subset encoded secreted proteins including GDNF [6]. To confirm and quantify these findings in independent experiments, we exposed PSC27 prostate myofibroblasts [14] to agents known to cause DNA damage: 0.6 mM H202, 10 μg/ml bleomycin, 100 nM mitoxantrone and 10 Gy radiation (IR). We confirmed the induction of DNA damage in the treated cells by assessing the phosphorylation status of serine 139 on H2A histone family, member X (γ-H2AX), indicative of DNA double strand breaks (Fig 1A). We also treated PSC27 fibroblasts with the microtubule poison docetaxel, a chemotherapeutic widely used in the treatment of advanced prostate cancer. Exposure to 50 nM docetaxel also resulted in DNA damage with γ-H2AX phosphorylation detected at levels roughly equivalent to that observed with IR (Fig 1A), a finding concordant with previous findings showing docetaxel induces DNA damage, though indirectly via replication-mediated double strand breaks [15, 16]. Using microarray-based methods to quantitate gene expression alterations we determined that GDNF expression increased substantially (> 6-fold) regardless of the agent used to induce DNA damage (Fig 1B). To determine the temporal pattern of GDNF up-regulation after DNA damage, we measured GDNF transcript levels by qRT-PCR from one to 16 days post treatment and observed a gradual increase in GDNF transcripts over time, from 2.5-fold at 5 days to 5-fold by 16 days after IR, and from 3.5-fold to 11.5-fold after mitoxantrone treatment (p<0.001) (Fig 1C, D). However, when analyzing the amount of intracellular GDNF protein in prostate fibroblasts after these treatments by ELISA, protein induction to above detection limit was found as early as five days post exposure, and the concentration did not further increase over time despite further increases in GDNF transcript levels (Fig 1E), suggesting that GDNF protein may be secreted. We also compared GDNF transcript levels by microarray analysis in micro-dissected cancer-associated stromal tissue before and after exposure to chemotherapy in men with prostate cancer enrolled in a neoadjuvant clinical trial combining mitoxantrone and docetaxel [17, 18]. In the majority of cases analyzed (7/10), GDNF transcripts were elevated after therapy in these paired clinical samples (Fig 1 F) consistent with the findings in cultured prostate fibroblasts.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.
Mentions: We previously profiled the gene expression alterations in prostate fibroblasts following DNA damage and identified a spectrum of highly induced transcripts of which a subset encoded secreted proteins including GDNF [6]. To confirm and quantify these findings in independent experiments, we exposed PSC27 prostate myofibroblasts [14] to agents known to cause DNA damage: 0.6 mM H202, 10 μg/ml bleomycin, 100 nM mitoxantrone and 10 Gy radiation (IR). We confirmed the induction of DNA damage in the treated cells by assessing the phosphorylation status of serine 139 on H2A histone family, member X (γ-H2AX), indicative of DNA double strand breaks (Fig 1A). We also treated PSC27 fibroblasts with the microtubule poison docetaxel, a chemotherapeutic widely used in the treatment of advanced prostate cancer. Exposure to 50 nM docetaxel also resulted in DNA damage with γ-H2AX phosphorylation detected at levels roughly equivalent to that observed with IR (Fig 1A), a finding concordant with previous findings showing docetaxel induces DNA damage, though indirectly via replication-mediated double strand breaks [15, 16]. Using microarray-based methods to quantitate gene expression alterations we determined that GDNF expression increased substantially (> 6-fold) regardless of the agent used to induce DNA damage (Fig 1B). To determine the temporal pattern of GDNF up-regulation after DNA damage, we measured GDNF transcript levels by qRT-PCR from one to 16 days post treatment and observed a gradual increase in GDNF transcripts over time, from 2.5-fold at 5 days to 5-fold by 16 days after IR, and from 3.5-fold to 11.5-fold after mitoxantrone treatment (p<0.001) (Fig 1C, D). However, when analyzing the amount of intracellular GDNF protein in prostate fibroblasts after these treatments by ELISA, protein induction to above detection limit was found as early as five days post exposure, and the concentration did not further increase over time despite further increases in GDNF transcript levels (Fig 1E), suggesting that GDNF protein may be secreted. We also compared GDNF transcript levels by microarray analysis in micro-dissected cancer-associated stromal tissue before and after exposure to chemotherapy in men with prostate cancer enrolled in a neoadjuvant clinical trial combining mitoxantrone and docetaxel [17, 18]. In the majority of cases analyzed (7/10), GDNF transcripts were elevated after therapy in these paired clinical samples (Fig 1 F) consistent with the findings in cultured prostate fibroblasts.

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