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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

Stress-induced PAK1 triggers CRAF pS338 to prevent DNA damage.(a) HCT-116 cells treated with 6 Gy or 0.5 μM etoposide were lysed and analysed for total and phosphorylated PAK1/PAK2. Data are representative of three independent experiments. (b) HCT-116 xenograft tumours from mice exposed to Etoposide (5 mg kg−1) were harvested, immunostained and imaged by confocal microscopy to assess levels of PAK pS141 (green). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (c) Expression of PAK1, 2 and 4 were silenced using siRNA in HCT-116. PAK and CRAF pS338 levels were assessed by immunoblotting. Data shown are representative of two different siRNAs per target, for two independent experiments. (d) Expression of PAK1 was silenced using siRNA in HCT-116 cells and then cells were treated with 6 Gy. DNA damage was assessed using neutral comet assay. Mean comet tail length±s.e.m., *P<0.0001 from two-sided t-test comparing irradiated si-CTRL versus si-PAK1 with n=100+ cells per group. Data shown are representative of two different siRNAs per target, for two independent experiments. (e) HCT-116 cells were transfected with WT or active PAK1 (L107F) for 72 h and then irradiated. Cells were immunoblotted for pS338 CRAF and survival was measured by clonogenic assay. Mean surviving fraction±s.e.m., *P<0.05 from two-sided t-test comparing WT versus active PAK1 with n=6 wells per group.
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f3: Stress-induced PAK1 triggers CRAF pS338 to prevent DNA damage.(a) HCT-116 cells treated with 6 Gy or 0.5 μM etoposide were lysed and analysed for total and phosphorylated PAK1/PAK2. Data are representative of three independent experiments. (b) HCT-116 xenograft tumours from mice exposed to Etoposide (5 mg kg−1) were harvested, immunostained and imaged by confocal microscopy to assess levels of PAK pS141 (green). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (c) Expression of PAK1, 2 and 4 were silenced using siRNA in HCT-116. PAK and CRAF pS338 levels were assessed by immunoblotting. Data shown are representative of two different siRNAs per target, for two independent experiments. (d) Expression of PAK1 was silenced using siRNA in HCT-116 cells and then cells were treated with 6 Gy. DNA damage was assessed using neutral comet assay. Mean comet tail length±s.e.m., *P<0.0001 from two-sided t-test comparing irradiated si-CTRL versus si-PAK1 with n=100+ cells per group. Data shown are representative of two different siRNAs per target, for two independent experiments. (e) HCT-116 cells were transfected with WT or active PAK1 (L107F) for 72 h and then irradiated. Cells were immunoblotted for pS338 CRAF and survival was measured by clonogenic assay. Mean surviving fraction±s.e.m., *P<0.05 from two-sided t-test comparing WT versus active PAK1 with n=6 wells per group.

Mentions: Previous studies have shown that CRAF S338 phosphorylation depends on one or more members of the PAK family22232425. Therefore, we considered whether radiation or etoposide treatment of tumour cells would stimulate PAK activation that, in turn, would lead to CRAF pS338 and its capacity to trigger the DNA damage response. Accordingly, we found that treatment of HCT-116 cells either in vivo or in vitro with IR or etoposide resulted in enhanced activation of PAK1 and PAK2 as measured by pS141 immunoreactivity (Fig. 3a,b). Interestingly, knockdown of PAK1 (but not PAK2 or PAK4) completely abolished CRAF pS338 (Fig. 3c), and this was accompanied by a dramatic increase in IR-mediated DNA damage (Fig. 3d). Moreover, expression of constitutively active PAK1 (L017F), which increased pS338 CRAF, enhanced cell survival following radiation (Fig. 3e). Together, these findings indicate that DNA damage leads to PAK1 activation, resulting in CRAF pS338 and DNA repair.


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)

Stress-induced PAK1 triggers CRAF pS338 to prevent DNA damage.(a) HCT-116 cells treated with 6 Gy or 0.5 μM etoposide were lysed and analysed for total and phosphorylated PAK1/PAK2. Data are representative of three independent experiments. (b) HCT-116 xenograft tumours from mice exposed to Etoposide (5 mg kg−1) were harvested, immunostained and imaged by confocal microscopy to assess levels of PAK pS141 (green). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (c) Expression of PAK1, 2 and 4 were silenced using siRNA in HCT-116. PAK and CRAF pS338 levels were assessed by immunoblotting. Data shown are representative of two different siRNAs per target, for two independent experiments. (d) Expression of PAK1 was silenced using siRNA in HCT-116 cells and then cells were treated with 6 Gy. DNA damage was assessed using neutral comet assay. Mean comet tail length±s.e.m., *P<0.0001 from two-sided t-test comparing irradiated si-CTRL versus si-PAK1 with n=100+ cells per group. Data shown are representative of two different siRNAs per target, for two independent experiments. (e) HCT-116 cells were transfected with WT or active PAK1 (L107F) for 72 h and then irradiated. Cells were immunoblotted for pS338 CRAF and survival was measured by clonogenic assay. Mean surviving fraction±s.e.m., *P<0.05 from two-sided t-test comparing WT versus active PAK1 with n=6 wells per group.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4559870&req=5

f3: Stress-induced PAK1 triggers CRAF pS338 to prevent DNA damage.(a) HCT-116 cells treated with 6 Gy or 0.5 μM etoposide were lysed and analysed for total and phosphorylated PAK1/PAK2. Data are representative of three independent experiments. (b) HCT-116 xenograft tumours from mice exposed to Etoposide (5 mg kg−1) were harvested, immunostained and imaged by confocal microscopy to assess levels of PAK pS141 (green). Scale bar, 100 μm. Data shown are representative of n=4 mice per group, four fields per mouse, for two independent experiments. (c) Expression of PAK1, 2 and 4 were silenced using siRNA in HCT-116. PAK and CRAF pS338 levels were assessed by immunoblotting. Data shown are representative of two different siRNAs per target, for two independent experiments. (d) Expression of PAK1 was silenced using siRNA in HCT-116 cells and then cells were treated with 6 Gy. DNA damage was assessed using neutral comet assay. Mean comet tail length±s.e.m., *P<0.0001 from two-sided t-test comparing irradiated si-CTRL versus si-PAK1 with n=100+ cells per group. Data shown are representative of two different siRNAs per target, for two independent experiments. (e) HCT-116 cells were transfected with WT or active PAK1 (L107F) for 72 h and then irradiated. Cells were immunoblotted for pS338 CRAF and survival was measured by clonogenic assay. Mean surviving fraction±s.e.m., *P<0.05 from two-sided t-test comparing WT versus active PAK1 with n=6 wells per group.
Mentions: Previous studies have shown that CRAF S338 phosphorylation depends on one or more members of the PAK family22232425. Therefore, we considered whether radiation or etoposide treatment of tumour cells would stimulate PAK activation that, in turn, would lead to CRAF pS338 and its capacity to trigger the DNA damage response. Accordingly, we found that treatment of HCT-116 cells either in vivo or in vitro with IR or etoposide resulted in enhanced activation of PAK1 and PAK2 as measured by pS141 immunoreactivity (Fig. 3a,b). Interestingly, knockdown of PAK1 (but not PAK2 or PAK4) completely abolished CRAF pS338 (Fig. 3c), and this was accompanied by a dramatic increase in IR-mediated DNA damage (Fig. 3d). Moreover, expression of constitutively active PAK1 (L017F), which increased pS338 CRAF, enhanced cell survival following radiation (Fig. 3e). Together, these findings indicate that DNA damage leads to PAK1 activation, resulting in CRAF pS338 and DNA repair.

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