(97a) Experimental And Computational Analysis Of Ras Mutation Effects In Apoptosis Signaling

Authors: 
Kreeger, P. K., Massachusetts Institute of Technology
Haigis, K., Massachusetts General Hospital
Jacks, T., Massachusetts Institute of Technology


Mutations in the RAS family of GTPases are frequently identified in colon cancers, with 40% of tumors having a K-RAS mutation. To examine the consequences of K-RAS mutation, we are developing a partial least squares regression model of apoptosis in an isogenic panel of colon carcinoma cells lines with varying RAS genotypes. Our hypothesis is that cell fate decisions are regulated through a quantitative interplay between the intracellular signaling pathways downstream of apoptotic stimuli and RAS, and that elucidation of this function will enable us to discern the role of mutant K-RAS in apoptosis. To develop this model, we are constructing an extensive dataset of apoptotic responses to a combination of extracellular cytokine stimuli and RAS genotype variations, along with the corresponding intracellular signal measurements downstream of RAS and TNFRI. At various times after stimulation, cells were analyzed for levels of phosphorylated proteins (ERK1, ERK2, Akt, IΚBα, JNK, HSP27) using the Luminex multiplex suspension array system, cleaved caspase 8 by quantitative western blot and apoptotic markers by multi-color flow cytometry. These diverse measures provide readouts of signaling downstream of the TNF receptor and RAS. Our results indicate that mutant K-RAS sensitizes cells to TNFα stimulated apoptosis, enhancing apoptosis nearly 33% by 48 hours. Analysis of the accompanying signaling datasets indicates that despite similar basal levels, cells with the K-RAS mutation had depressed pERK2, pJNK, and pHSP27 signaling dynamics (i.e. reduced extent of activation or the absence of initial signaling peaks) following TNFα treatment. Future work will extend this systems biology approach to N-RAS mutations, found in 5% of colon cancers, and animal models that conditionally express RAS mutations in the intestinal epithelium. These studies will help to increase our understanding of the role oncogenic RAS exerts on the apoptotic decision, which could provide novel targets for the treatment of colon cancer.