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Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Advani SJ, Camargo MF, Seguin L, Mielgo A, Anand S, Hicks AM, Aguilera J, Franovic A, Weis SM, Cheresh DA - Nat Commun (2015)

Bottom Line: Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance.CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response.Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Medicine and Applied Sciences at the UC San Diego Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

No MeSH data available.


Related in: MedlinePlus

CRAF pS338 is induced by genotoxic stress and protects tumour cells from DNA damage.(a) Embryonic fibroblasts isolated from wild-type (WT), BRAF−/− or CRAF−/− mice were irradiated (6 Gy), and DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci/cell±s.e.m. for n=6 fields analysed per group. ‘*' indicates P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data shown are representative of two independent experiments. (b) HCT-116 cells were transfected with siRNA to BRAF and CRAF. Cells were irradiated (2 Gy) and cell survival was measured using clonogenic survival assay. Graph shows mean surviving fraction±s.e.m. ‘*' indicates P<0.05 from two-sided t-test comparing si-CRAF to si-BRAF, si-CTRL or non-transfected with n=3 wells per group. Data shown are representative of three independent experiments. (c) HCT-116 cells treated with KG5 (1μm) overnight, irradiated (6 Gy) and whole cell lysates collected. Immunoblotting to indicate phospho CRAF and BRAF sites. Data shown are representative of three independent experiments. (d) Immunoblotting for pS338 CRAF following 6 Gy or 0.5 μM etoposide. Immunostaining for CRAF pS338 (green) with dose range of IR in HCT-116 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of five fields per group for two independent experiments. (e) HCT-116 xenograft tumours were irradiated (6 Gy) and then immunostained for CRAF pS338 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (f) Immunostaining for CRAF pS338 (green) in HCT-116 cells treated with KG5 (1 μM) overnight then irradiated (6 Gy). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of n=5 fields per group for two independent experiments. (g) HCT-116 cells were treated with KG5 and then irradiated. DNA double strand breaks were measured by neutral comet tail assay (n=100+ cells per group). Cell survival was measured using a clonogenic assay (n=3 wells per group). Bars represent mean±s.e.m. *P<0.05 from two-sided t-test comparing vehicle control and KG5. Data are representative of two independent experiments.
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f1: CRAF pS338 is induced by genotoxic stress and protects tumour cells from DNA damage.(a) Embryonic fibroblasts isolated from wild-type (WT), BRAF−/− or CRAF−/− mice were irradiated (6 Gy), and DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci/cell±s.e.m. for n=6 fields analysed per group. ‘*' indicates P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data shown are representative of two independent experiments. (b) HCT-116 cells were transfected with siRNA to BRAF and CRAF. Cells were irradiated (2 Gy) and cell survival was measured using clonogenic survival assay. Graph shows mean surviving fraction±s.e.m. ‘*' indicates P<0.05 from two-sided t-test comparing si-CRAF to si-BRAF, si-CTRL or non-transfected with n=3 wells per group. Data shown are representative of three independent experiments. (c) HCT-116 cells treated with KG5 (1μm) overnight, irradiated (6 Gy) and whole cell lysates collected. Immunoblotting to indicate phospho CRAF and BRAF sites. Data shown are representative of three independent experiments. (d) Immunoblotting for pS338 CRAF following 6 Gy or 0.5 μM etoposide. Immunostaining for CRAF pS338 (green) with dose range of IR in HCT-116 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of five fields per group for two independent experiments. (e) HCT-116 xenograft tumours were irradiated (6 Gy) and then immunostained for CRAF pS338 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (f) Immunostaining for CRAF pS338 (green) in HCT-116 cells treated with KG5 (1 μM) overnight then irradiated (6 Gy). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of n=5 fields per group for two independent experiments. (g) HCT-116 cells were treated with KG5 and then irradiated. DNA double strand breaks were measured by neutral comet tail assay (n=100+ cells per group). Cell survival was measured using a clonogenic assay (n=3 wells per group). Bars represent mean±s.e.m. *P<0.05 from two-sided t-test comparing vehicle control and KG5. Data are representative of two independent experiments.

Mentions: Given the well-established role for RAS activity in tumour cell resistance to therapy, we examined the relative contribution of the RAF family kinases BRAF and CRAF to radioresistance. Mouse embryonic fibroblasts isolated from BRAF−/− or CRAF−/− mice were exposed to radiation and monitored for DNA damage by examining the level of nuclear γH2AX foci. Only those cells deficient in CRAF showed radiosensitivity (Fig. 1a, Supplementary Fig. 1a), suggesting that CRAF but not BRAF contributed to radioresistance. We extended these studies by knocking down either CRAF or BRAF in HCT-116 human colorectal adenocarcinoma and PANC-1 human pancreatic adenocarcinoma cells and measured cell survival and DNA damage following irradiation (Fig. 1b, Supplementary Fig. 1b,c). In irradiated HCT-116 cells, knockdown of CRAF decreased cell survival. In addition, knockdown of CRAF in PANC-1 cells increased DNA damage as measured by neutral comet assay. In contrast, knockdown of BRAF in HCT-116 and PANC-1 cells had no such effect. While CRAF was required for radioresistance, MEK inhibition did not result in radiosensitization consistently (Supplementary Fig. 2). These findings suggest that, while CRAF protects cells from DNA damage, this function may not require CRAF kinase activity or MEK activation.


Kinase-independent role for CRAF-driving tumour radioresistance via CHK2.

Advani SJ, Camargo MF, Seguin L, Mielgo A, Anand S, Hicks AM, Aguilera J, Franovic A, Weis SM, Cheresh DA - Nat Commun (2015)

CRAF pS338 is induced by genotoxic stress and protects tumour cells from DNA damage.(a) Embryonic fibroblasts isolated from wild-type (WT), BRAF−/− or CRAF−/− mice were irradiated (6 Gy), and DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci/cell±s.e.m. for n=6 fields analysed per group. ‘*' indicates P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data shown are representative of two independent experiments. (b) HCT-116 cells were transfected with siRNA to BRAF and CRAF. Cells were irradiated (2 Gy) and cell survival was measured using clonogenic survival assay. Graph shows mean surviving fraction±s.e.m. ‘*' indicates P<0.05 from two-sided t-test comparing si-CRAF to si-BRAF, si-CTRL or non-transfected with n=3 wells per group. Data shown are representative of three independent experiments. (c) HCT-116 cells treated with KG5 (1μm) overnight, irradiated (6 Gy) and whole cell lysates collected. Immunoblotting to indicate phospho CRAF and BRAF sites. Data shown are representative of three independent experiments. (d) Immunoblotting for pS338 CRAF following 6 Gy or 0.5 μM etoposide. Immunostaining for CRAF pS338 (green) with dose range of IR in HCT-116 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of five fields per group for two independent experiments. (e) HCT-116 xenograft tumours were irradiated (6 Gy) and then immunostained for CRAF pS338 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (f) Immunostaining for CRAF pS338 (green) in HCT-116 cells treated with KG5 (1 μM) overnight then irradiated (6 Gy). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of n=5 fields per group for two independent experiments. (g) HCT-116 cells were treated with KG5 and then irradiated. DNA double strand breaks were measured by neutral comet tail assay (n=100+ cells per group). Cell survival was measured using a clonogenic assay (n=3 wells per group). Bars represent mean±s.e.m. *P<0.05 from two-sided t-test comparing vehicle control and KG5. Data are representative of two independent experiments.
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f1: CRAF pS338 is induced by genotoxic stress and protects tumour cells from DNA damage.(a) Embryonic fibroblasts isolated from wild-type (WT), BRAF−/− or CRAF−/− mice were irradiated (6 Gy), and DNA damage was assessed using γH2AX staining. Graph shows mean γH2AX foci/cell±s.e.m. for n=6 fields analysed per group. ‘*' indicates P<0.05 from two-sided t-test comparing WT and CRAF−/−. Data shown are representative of two independent experiments. (b) HCT-116 cells were transfected with siRNA to BRAF and CRAF. Cells were irradiated (2 Gy) and cell survival was measured using clonogenic survival assay. Graph shows mean surviving fraction±s.e.m. ‘*' indicates P<0.05 from two-sided t-test comparing si-CRAF to si-BRAF, si-CTRL or non-transfected with n=3 wells per group. Data shown are representative of three independent experiments. (c) HCT-116 cells treated with KG5 (1μm) overnight, irradiated (6 Gy) and whole cell lysates collected. Immunoblotting to indicate phospho CRAF and BRAF sites. Data shown are representative of three independent experiments. (d) Immunoblotting for pS338 CRAF following 6 Gy or 0.5 μM etoposide. Immunostaining for CRAF pS338 (green) with dose range of IR in HCT-116 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of five fields per group for two independent experiments. (e) HCT-116 xenograft tumours were irradiated (6 Gy) and then immunostained for CRAF pS338 (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (f) Immunostaining for CRAF pS338 (green) in HCT-116 cells treated with KG5 (1 μM) overnight then irradiated (6 Gy). Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. Data shown are representative of n=5 fields per group for two independent experiments. (g) HCT-116 cells were treated with KG5 and then irradiated. DNA double strand breaks were measured by neutral comet tail assay (n=100+ cells per group). Cell survival was measured using a clonogenic assay (n=3 wells per group). Bars represent mean±s.e.m. *P<0.05 from two-sided t-test comparing vehicle control and KG5. Data are representative of two independent experiments.
Mentions: Given the well-established role for RAS activity in tumour cell resistance to therapy, we examined the relative contribution of the RAF family kinases BRAF and CRAF to radioresistance. Mouse embryonic fibroblasts isolated from BRAF−/− or CRAF−/− mice were exposed to radiation and monitored for DNA damage by examining the level of nuclear γH2AX foci. Only those cells deficient in CRAF showed radiosensitivity (Fig. 1a, Supplementary Fig. 1a), suggesting that CRAF but not BRAF contributed to radioresistance. We extended these studies by knocking down either CRAF or BRAF in HCT-116 human colorectal adenocarcinoma and PANC-1 human pancreatic adenocarcinoma cells and measured cell survival and DNA damage following irradiation (Fig. 1b, Supplementary Fig. 1b,c). In irradiated HCT-116 cells, knockdown of CRAF decreased cell survival. In addition, knockdown of CRAF in PANC-1 cells increased DNA damage as measured by neutral comet assay. In contrast, knockdown of BRAF in HCT-116 and PANC-1 cells had no such effect. While CRAF was required for radioresistance, MEK inhibition did not result in radiosensitization consistently (Supplementary Fig. 2). These findings suggest that, while CRAF protects cells from DNA damage, this function may not require CRAF kinase activity or MEK activation.

Bottom Line: Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance.CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response.Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Medicine and Applied Sciences at the UC San Diego Moores Cancer Center, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

No MeSH data available.


Related in: MedlinePlus