The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer

Basak Celtikci, Robbie Carson, Patrick Johnston, Sandra Van Schaeybroeck

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Background

Oncogenic BRAF mutations occur in 8% of advanced colorectal cancer (CRC) patients. Several phase IIII studies like COIN and CRYSTAL showed the worst prognosis in metastatic CRC patients with BRAF mutation. There are also a number of retrospective studies suggesting that activating BRAF mutations predict for non-response to EGFR inhibition. Clear understanding of the vulnerabilities of BRAF mutant (MT) CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC.

Method

BRAFMT/WT CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Cell viability was measured using the MTT assay. Apoptosis was determined by using Western Blotting for PARP and cleaved caspase 3 and Flow Cytometry.

Results

Treatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in increases in STAT3 and JAK activity in BRAFMT CRC cells, but not in BRAFWT cells. c-MET was identified as the receptor tyrosine kinase regulating JAK/STAT3 activity following MEKi in BRAFMT CRC cells. Inhibition of c-MET or JAK/STAT3 activation using siRNA or small molecular inhibitors resulted in synergy and significant increases in apoptosis when combined with AZD6244 in BRAFMT CRC cells. In addition, MEK1/2 inhibition resulted in increased FLIPL expression levels in BRAFMT CRC cells, which was abrogated following treatment with METi or JAK/STAT3 inhibition. By binding to FADD, FLIP inhibits apoptosis by blocking pro-caspase 8 activation. Silencing of c-FLIP and the small molecule HDAC inhibitor vorinostat or entinostat decrease basal and MEK1/2-induced c-FLIP expression and result in potent increases in apoptosis when combined with AZD6244, especially in BRAFMT CRC cells.

Conclusion

We have identified STAT3 and c-FLIP as important escape mechanisms following MEK1/2i in BRAFMT CRC cells. Combinations of MET/MEKi or HDAC/MEKi can be potential novel treatment strategies for poor prognostic BRAFMT advanced CRC patients.
Original languageEnglish
Title of host publicationThe role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer
PublisherNational Cancer Research Institute
Publication statusPublished - Nov 2014

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Mitogen-Activated Protein Kinase Kinases
Colorectal Neoplasms
Apoptosis
Mutation
Western Blotting
Histone Deacetylase Inhibitors
Caspase 8
Receptor Protein-Tyrosine Kinases
Caspase 3
Small Interfering RNA
Cell Survival
Flow Cytometry
Therapeutics

Cite this

Celtikci, B., Carson, R., Johnston, P., & Van Schaeybroeck, S. (2014). The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. In The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer National Cancer Research Institute.
Celtikci, Basak ; Carson, Robbie ; Johnston, Patrick ; Van Schaeybroeck, Sandra. / The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. National Cancer Research Institute, 2014.
@inproceedings{c162c06bfbd4467b9be47c2c681d2462,
title = "The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer",
abstract = "BackgroundOncogenic BRAF mutations occur in 8{\%} of advanced colorectal cancer (CRC) patients. Several phase IIII studies like COIN and CRYSTAL showed the worst prognosis in metastatic CRC patients with BRAF mutation. There are also a number of retrospective studies suggesting that activating BRAF mutations predict for non-response to EGFR inhibition. Clear understanding of the vulnerabilities of BRAF mutant (MT) CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC.MethodBRAFMT/WT CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Cell viability was measured using the MTT assay. Apoptosis was determined by using Western Blotting for PARP and cleaved caspase 3 and Flow Cytometry.ResultsTreatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in increases in STAT3 and JAK activity in BRAFMT CRC cells, but not in BRAFWT cells. c-MET was identified as the receptor tyrosine kinase regulating JAK/STAT3 activity following MEKi in BRAFMT CRC cells. Inhibition of c-MET or JAK/STAT3 activation using siRNA or small molecular inhibitors resulted in synergy and significant increases in apoptosis when combined with AZD6244 in BRAFMT CRC cells. In addition, MEK1/2 inhibition resulted in increased FLIPL expression levels in BRAFMT CRC cells, which was abrogated following treatment with METi or JAK/STAT3 inhibition. By binding to FADD, FLIP inhibits apoptosis by blocking pro-caspase 8 activation. Silencing of c-FLIP and the small molecule HDAC inhibitor vorinostat or entinostat decrease basal and MEK1/2-induced c-FLIP expression and result in potent increases in apoptosis when combined with AZD6244, especially in BRAFMT CRC cells.ConclusionWe have identified STAT3 and c-FLIP as important escape mechanisms following MEK1/2i in BRAFMT CRC cells. Combinations of MET/MEKi or HDAC/MEKi can be potential novel treatment strategies for poor prognostic BRAFMT advanced CRC patients.",
author = "Basak Celtikci and Robbie Carson and Patrick Johnston and {Van Schaeybroeck}, Sandra",
year = "2014",
month = "11",
language = "English",
booktitle = "The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer",
publisher = "National Cancer Research Institute",

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Celtikci, B, Carson, R, Johnston, P & Van Schaeybroeck, S 2014, The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. in The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. National Cancer Research Institute.

The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. / Celtikci, Basak; Carson, Robbie; Johnston, Patrick; Van Schaeybroeck, Sandra.

The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. National Cancer Research Institute, 2014.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

TY - GEN

T1 - The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer

AU - Celtikci, Basak

AU - Carson, Robbie

AU - Johnston, Patrick

AU - Van Schaeybroeck, Sandra

PY - 2014/11

Y1 - 2014/11

N2 - BackgroundOncogenic BRAF mutations occur in 8% of advanced colorectal cancer (CRC) patients. Several phase IIII studies like COIN and CRYSTAL showed the worst prognosis in metastatic CRC patients with BRAF mutation. There are also a number of retrospective studies suggesting that activating BRAF mutations predict for non-response to EGFR inhibition. Clear understanding of the vulnerabilities of BRAF mutant (MT) CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC.MethodBRAFMT/WT CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Cell viability was measured using the MTT assay. Apoptosis was determined by using Western Blotting for PARP and cleaved caspase 3 and Flow Cytometry.ResultsTreatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in increases in STAT3 and JAK activity in BRAFMT CRC cells, but not in BRAFWT cells. c-MET was identified as the receptor tyrosine kinase regulating JAK/STAT3 activity following MEKi in BRAFMT CRC cells. Inhibition of c-MET or JAK/STAT3 activation using siRNA or small molecular inhibitors resulted in synergy and significant increases in apoptosis when combined with AZD6244 in BRAFMT CRC cells. In addition, MEK1/2 inhibition resulted in increased FLIPL expression levels in BRAFMT CRC cells, which was abrogated following treatment with METi or JAK/STAT3 inhibition. By binding to FADD, FLIP inhibits apoptosis by blocking pro-caspase 8 activation. Silencing of c-FLIP and the small molecule HDAC inhibitor vorinostat or entinostat decrease basal and MEK1/2-induced c-FLIP expression and result in potent increases in apoptosis when combined with AZD6244, especially in BRAFMT CRC cells.ConclusionWe have identified STAT3 and c-FLIP as important escape mechanisms following MEK1/2i in BRAFMT CRC cells. Combinations of MET/MEKi or HDAC/MEKi can be potential novel treatment strategies for poor prognostic BRAFMT advanced CRC patients.

AB - BackgroundOncogenic BRAF mutations occur in 8% of advanced colorectal cancer (CRC) patients. Several phase IIII studies like COIN and CRYSTAL showed the worst prognosis in metastatic CRC patients with BRAF mutation. There are also a number of retrospective studies suggesting that activating BRAF mutations predict for non-response to EGFR inhibition. Clear understanding of the vulnerabilities of BRAF mutant (MT) CRC is important, and identification of druggable targets uniquely required by BRAFMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC.MethodBRAFMT/WT CRC cells were used. Changes in protein expression/activity were assessed by Western Blotting. Cell viability was measured using the MTT assay. Apoptosis was determined by using Western Blotting for PARP and cleaved caspase 3 and Flow Cytometry.ResultsTreatment with MEK1/2 inhibitors AZD6244, trametinib, UO126 and PD98059 resulted in increases in STAT3 and JAK activity in BRAFMT CRC cells, but not in BRAFWT cells. c-MET was identified as the receptor tyrosine kinase regulating JAK/STAT3 activity following MEKi in BRAFMT CRC cells. Inhibition of c-MET or JAK/STAT3 activation using siRNA or small molecular inhibitors resulted in synergy and significant increases in apoptosis when combined with AZD6244 in BRAFMT CRC cells. In addition, MEK1/2 inhibition resulted in increased FLIPL expression levels in BRAFMT CRC cells, which was abrogated following treatment with METi or JAK/STAT3 inhibition. By binding to FADD, FLIP inhibits apoptosis by blocking pro-caspase 8 activation. Silencing of c-FLIP and the small molecule HDAC inhibitor vorinostat or entinostat decrease basal and MEK1/2-induced c-FLIP expression and result in potent increases in apoptosis when combined with AZD6244, especially in BRAFMT CRC cells.ConclusionWe have identified STAT3 and c-FLIP as important escape mechanisms following MEK1/2i in BRAFMT CRC cells. Combinations of MET/MEKi or HDAC/MEKi can be potential novel treatment strategies for poor prognostic BRAFMT advanced CRC patients.

M3 - Conference contribution

BT - The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer

PB - National Cancer Research Institute

ER -

Celtikci B, Carson R, Johnston P, Van Schaeybroeck S. The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. In The role of STAT3 and c-FLIP as acute resistance mechanisms to MEK inhibition in BRAF mutant colorectal cancer. National Cancer Research Institute. 2014