Advanced

Discovery of a novel B-Raf fusion protein related to c-Met drug resistance

Dillon, Roslyn; Nilsson, Carol L LU ; Shi, Stone D-H; Lee, Nathan V; Krastins, Bryan and Greig, Michael J (2011) In Journal of Proteome Research 10(11). p.94-5084
Abstract

In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was... (More)

In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was observed in GTL-16, a gastric carcinoma cell line with a constitutively activated c-Met receptor. In this report, mass spectrometry (MS) based quantitative phosphoproteomic analysis was used to determine changes in signaling pathways in the parental cells in response to c-Met inhibition and to investigate the changes in protein levels and related canonical pathways in both parental and PF-04217903 resistant (R3) clones in response to c-Met inhibition. The quantitative MS workflow included phosphoprotein enrichment of cell lysates from six treatment conditions: in-solution digestion, chemical labeling of peptides with a set of 6-plex isobaric tandem mass tags (TMT), HILIC fractionation, phosphopeptide enrichment, and nano LC-MS/MS on a LTQ-Orbitrap mass spectrometer. An investigation of these quantitative datasets using Ingenuity Pathways Analysis (IPA) revealed pathway changes in the various treatments that were consistent with previously observed transcriptomic and phenotypic changes. Proteomic analysis also revealed an increase in B-Raf expression in R3 clones. Expression profiling confirmed that B-Raf gene copy number was up-regulated and also indicated the presence of a mutated form of B-Raf. Using a bottom-up MS approach, SND-1 was identified as the B-Raf fusion partner. The discovery of this novel B-Raf fusion protein presents a novel target with potential clinical implications in the treatment of patients resistant to c-Met inhibitors.

(Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
keywords
Amino Acid Sequence, Antineoplastic Agents, Cell Line, Tumor, Drug Resistance, Neoplasm, Gene Expression, Humans, MAP Kinase Signaling System, Molecular Sequence Data, Nuclear Proteins, Oncogene Proteins, Fusion, Protein Interaction Maps, Proteome, Proto-Oncogene Proteins B-raf, Proto-Oncogene Proteins c-met, Journal Article
in
Journal of Proteome Research
volume
10
issue
11
pages
11 pages
publisher
The American Chemical Society
external identifiers
  • scopus:80655131098
ISSN
1535-3893
DOI
10.1021/pr200498v
language
English
LU publication?
no
id
1e9149e2-1f63-4acb-b374-257ea6598964
date added to LUP
2017-05-16 10:29:22
date last changed
2017-05-21 05:01:35
@article{1e9149e2-1f63-4acb-b374-257ea6598964,
  abstract     = {<p>In recent years, there have been notable advances with the development of anticancer drugs including those targeting protein tyrosine kinases such as the c-Met receptor, which has been implicated in the development and progression of several cancers. However, despite such progress, drug resistance continues to be the single most important cause of cancer treatment failure, and understanding the mechanisms of drug resistance remains a major hurdle in treating patients with recurrent disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that potently inhibits c-Met-driven processes such as cell growth (proliferation and survival), motility, invasion, and morphology of a variety of tumor cells. Resistance to PF-04217903 was observed in GTL-16, a gastric carcinoma cell line with a constitutively activated c-Met receptor. In this report, mass spectrometry (MS) based quantitative phosphoproteomic analysis was used to determine changes in signaling pathways in the parental cells in response to c-Met inhibition and to investigate the changes in protein levels and related canonical pathways in both parental and PF-04217903 resistant (R3) clones in response to c-Met inhibition. The quantitative MS workflow included phosphoprotein enrichment of cell lysates from six treatment conditions: in-solution digestion, chemical labeling of peptides with a set of 6-plex isobaric tandem mass tags (TMT), HILIC fractionation, phosphopeptide enrichment, and nano LC-MS/MS on a LTQ-Orbitrap mass spectrometer. An investigation of these quantitative datasets using Ingenuity Pathways Analysis (IPA) revealed pathway changes in the various treatments that were consistent with previously observed transcriptomic and phenotypic changes. Proteomic analysis also revealed an increase in B-Raf expression in R3 clones. Expression profiling confirmed that B-Raf gene copy number was up-regulated and also indicated the presence of a mutated form of B-Raf. Using a bottom-up MS approach, SND-1 was identified as the B-Raf fusion partner. The discovery of this novel B-Raf fusion protein presents a novel target with potential clinical implications in the treatment of patients resistant to c-Met inhibitors.</p>},
  author       = {Dillon, Roslyn and Nilsson, Carol L and Shi, Stone D-H and Lee, Nathan V and Krastins, Bryan and Greig, Michael J},
  issn         = {1535-3893},
  keyword      = {Amino Acid Sequence,Antineoplastic Agents,Cell Line, Tumor,Drug Resistance, Neoplasm,Gene Expression,Humans,MAP Kinase Signaling System,Molecular Sequence Data,Nuclear Proteins,Oncogene Proteins, Fusion,Protein Interaction Maps,Proteome,Proto-Oncogene Proteins B-raf,Proto-Oncogene Proteins c-met,Journal Article},
  language     = {eng},
  month        = {11},
  number       = {11},
  pages        = {94--5084},
  publisher    = {The American Chemical Society},
  series       = {Journal of Proteome Research},
  title        = {Discovery of a novel B-Raf fusion protein related to c-Met drug resistance},
  url          = {http://dx.doi.org/10.1021/pr200498v},
  volume       = {10},
  year         = {2011},
}