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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Drug Resistance
Colorectal Neoplasms
Intercellular Signaling Peptides and Proteins
Cytokines
Interleukin-8
Drug Therapy
Therapeutics
Messenger RNA
Mitogen-Activated Protein Kinase Kinases
Conditioned Culture Medium
Luciferases
Fluorouracil
Caspase 3
Melanoma
Cell Survival
Flow Cytometry
Cell Death
Clone Cells
Western Blotting
Enzyme-Linked Immunosorbent Assay

Cite this

Forsythe, N., Celtikci, B., Johnston, P., & Van Schaeybroeck, S. (2015). The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. In The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer (Vol. A221). National Cancer Research Institute.
Forsythe, Nicholas ; Celtikci, Basak ; Johnston, Patrick ; Van Schaeybroeck, Sandra. / The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. Vol. A221 National Cancer Research Institute, 2015.
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title = "The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer",
abstract = "BackgroundMutations 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.MethodIsogenic 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.ResultsWe 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.ConclusionConditioned 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.",
author = "Nicholas Forsythe and Basak Celtikci and Patrick Johnston and {Van Schaeybroeck}, Sandra",
year = "2015",
month = "11",
language = "English",
volume = "A221",
booktitle = "The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer",
publisher = "National Cancer Research Institute",

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Forsythe, N, Celtikci, B, Johnston, P & Van Schaeybroeck, S 2015, The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. in The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. vol. A221, National Cancer Research Institute.

The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. / Forsythe, Nicholas; Celtikci, Basak; Johnston, Patrick; Van Schaeybroeck, Sandra.

The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. Vol. A221 National Cancer Research Institute, 2015.

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

TY - GEN

T1 - The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer

AU - Forsythe, Nicholas

AU - Celtikci, Basak

AU - Johnston, Patrick

AU - Van Schaeybroeck, Sandra

PY - 2015/11

Y1 - 2015/11

N2 - BackgroundMutations 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.MethodIsogenic 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.ResultsWe 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.ConclusionConditioned 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.

AB - BackgroundMutations 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.MethodIsogenic 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.ResultsWe 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.ConclusionConditioned 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.

M3 - Conference contribution

VL - A221

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

PB - National Cancer Research Institute

ER -

Forsythe N, Celtikci B, Johnston P, Van Schaeybroeck S. The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. In The role of Oncogenic BRAF in regulating growth factor/cytokine shedding and drug resistance in colorectal cancer. Vol. A221. National Cancer Research Institute. 2015