The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer

Nicholas Forsythe, Basak Celtikci, Patrick Johnston, Sandra Van Schaeybroeck

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

Abstract

Background
Mutations in BRAF occur in around 8% of colorectal cancer(CRC) patients, are associated with poor prognosis irrespective of treatment and have been associated with resistance to EGFR-targeted therapies. In contrast to BRAF mutant(MT) melanoma, BRAF and MEK inhibition has shown no efficacy in BRAFMT CRC.
This study aimed to investigate the impact of mutant BRAF on the secretome of cancer cells and assessed the effect of these growth factors/cytokines on survival signalling and sensitivity to chemotherapy and MAPKi in BRAFMT CRC.

Method
Isogenic BRAFMT(V600E/WT) and WT(-/WT) CRC cells were used. Cytokine secretion was measured using a cytokine array and validated by ELISA and q-rtPCR. Cell survival was analysed by Flow Cytometry and Western blotting for PARP/caspase 3 cleavage. NF?B activity was measured using a luciferase assay.

Results
We found that conditioned medium derived from BRAFMT CRC cells resulted in increases in EGFR and Akt signalling and protected BRAFWT cells from cell death following 5-FU treatment. We further found higher expression of 12 cytokines in BRAFMT VACO432 cells compared its WT clone, including IL-8, CXCL-1 and TFF3. Further studies showed that treatment of BRAFMT cells with the MEKi AZD6244 or the BRAFi Vemurafenib resulted in early decreases in IL-8 mRNA expression, indicating that IL-8 is transcriptionally regulated by the MAPK pathway in BRAFMT cells. Interestingly, significant increases in IL-8 mRNA and protein levels were observed in the BRAFMT cells, but not in BRAFWT cells at later timepoints. We are currently further investigating the role of the NF?B pathway in regulating this resistance mechanism.

Conclusion
Conditioned medium from BRAFMT CRC cells results in increased EGFR/Akt signalling and chemotherapy-resistance. BRAFMT cells respond to chemotherapy treatment with significant increases in IL-8 levels. This data may indicate a role for IL-8 in resistance to chemotherapy in BRAFMT CRC cells.
Original languageEnglish
Title of host publicationThe role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer
PublisherNational Cancer Research Institute
VolumeA221
Publication statusPublished - Nov 2015

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