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Analysis of Ras G12V/D Mutation on Downstream Pathways Using Multiplex Immunoassays
Author(s) -
Hwang Joseph,
Saporita Anthony,
Xiao Qiang
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.647.13
Subject(s) - anti apoptotic ras signalling cascade , mutation , cancer research , gtpase , neuroblastoma ras viral oncogene homolog , biology , mapk/erk pathway , effector , mutant , cancer , kinase , cell growth , pi3k/akt/mtor pathway , carcinogenesis , signal transduction , genetics , microbiology and biotechnology , gene , kras
Ras proteins are members of small GTPases that function as molecular switches by alternating between inactive GDP‐bound and active GTP‐bound states. Active Ras‐GTP activate downstream pathways, including MAPK pathway by binding to Raf kinase and phosphatidylinositol 3‐kinase (PI3K) to promote cellular proliferation, survival, growth and differentiation. In the 3 decades since the discovery of RAS mutations in cancer, numerous studies have established mutant RAS as a driver of tumor initiation and progression. Oncogenic mutations in Ras are found in approximately 20% of human cancers. Furthermore, 90% of pancreatic cancers harbor K‐Ras mutations with 80% of K‐Ras mutations occurring at codon 12. RAS mutation is associated with poor prognosis due to very limited therapeutic options. To address the need for better treatment options for Ras driven cancers, the RAS Initiative was established by the National Cancer Institution. Renewed efforts in understanding Ras effectors and regulatory partners, as well as advancement in new technologies for Ras assays, are currently underway. To this end, we have developed a novel multiplex immunoassay capable of simultaneously detecting total Ras, Ras G12V, Ras G12D, phospho‐MEK1, phospho‐MEK2, B‐Raf and C‐Raf in a single well. We have tested the feasibility of using this kit on cancer derived cell lines, mouse embryonic fibroblasts, and tumor tissues. This kit was able to confirm K‐Ras G12V mutation found in the COR‐L23 cell line and N‐Ras G12D mutation found in THP‐1 cell line. Furthermore, commercially purchased colon tumor tissues characterized as Ras G12V and Ras G12D positive were confirmed using our kit. Next, we analyzed downstream pathways using commercially available immunoassay kits, such as the Multi‐pathway panel and Apoptosis panel with respect to understanding differential effect of Ras G12D vs G12V mutation. In conclusion, we have developed a novel multiplex immunoassay tool that can further our understanding of Ras mutations and its effect on downstream targets. Support or Funding Information MilliporeSigma Corp. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .