MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways.
OBJECTIVE
Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines.


METHODS
Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting.


RESULTS
GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1.


CONCLUSIONS
GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
Adriana
A
Estrada-Bernal
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
Moumita
M
Chatterjee
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
S Jaharul
SJ
Haque
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
Linlin
L
Yang
b University of Michigan Comprehensive Cancer Center ; Ann Arbor , MI , USA.
Meredith A
MA
Morgan
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
Shweta
S
Kotian
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
David
D
Morrell
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
Arnab
A
Chakravarti
a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA.
Terence M
TM
Williams
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics* / Q000532:radiotherapy; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000532:radiotherapy; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D011728:Pyridones / Q000627:therapeutic use*; D011744:Pyrimidinones / Q000627:therapeutic use*; D011836:Radiation Tolerance / Q000235:genetics
D000970:Antineoplastic Agents; C117307:KRAS protein, human; D047428:Protein Kinase Inhibitors; D011728:Pyridones; D011744:Pyrimidinones; C560077:trametinib; D016283:Proto-Oncogene Proteins p21(ras)
DNA repair; GSK1120212; KRAS; MEK inhibitor; Mekinist; pancreatic cancer; radiation; trametinib