Oncogenic mutations of Ras, present in about 50% of colon cancers, regulate cell proliferation and cell survival and alter the sensitivity of cells to chemopreventive agents through modulation of downstream target genes. Using isogenic cell lines that differ only in the presence of mutant Ras, we demonstrated that activating mutations of k-ras inhibit the expression of STAT1 and STAT2, transcription factors required for signaling by IFNs (Klampfer et al, Journal of Biological Chemistry 2003, in appendix). Accordingly, a panel of colon cancer cell lines with k-ras mutation displayed reduced expression of IFN target genes, and inactivation of the mutant k-ras allele in the HCT116 colon cancer cell line was sufficient to restore the expression of STAT1 and STAT2, as well as their downstream target genes. These data led us to the testable hypothesis that activating Ras mutations perturb the balance between STAT1 dependent and STAT1 independent signaling by IFN( and thereby alter its biological activity. Since STAT1 is an important regulator of apoptosis, this raised the possibility that Ras modulates apoptosis in response to chemopreventive agents in a STAT1-dependent manner. We used isogenic cell lines with genetic inactivation of mutant Ras to demonstrate that activated Ras promotes apoptosis in response to the short chain fatty acid butyrate (Klampfer et al, Journal of Biological Chemistry 2004, in appendix). Here we present evidence that, like Ras mutations, STAT1 deficiency promotes butyrate-induced apoptosis and that STAT1 is required for butyrate-induced p21 expression. Finally, we show that loss of p21 favors apoptosis over cell cycle arrest upon butyrate treatment, suggesting that enhanced apoptosis in STAT1 deficient cells may be due to impaired p21 induction.
The aims of this proposal are: 1) to determine the molecular mechanism whereby Ras mutations interfere with IFN/STAT1 signaling, 2) to examine whether targeted expression of mutant Ras interferes with IFN/STAT signaling also in vivo, 3) to define the effect of Ras mutations, and STAT1 deficiency, on the responsiveness of colonic epithelial cells to interferons, and to the prototypic chemopreventive agents for patients with colon cancer predisposition, sulindac and aspirin, and 4) to test the hypothesis that STAT1 is required for transcriptional activation of p21 in response to butyrate and sulindac.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Tumor Cell Biology Study Section (TCB)
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Perloff, Marjorie
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Montefiore Medical Center (Bronx, NY)
New York
United States
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Kaler, Pawan; Augenlicht, Leonard; Klampfer, Lidija (2012) Activating mutations in ýý-catenin in colon cancer cells alter their interaction with macrophages; the role of snail. PLoS One 7:e45462
Klampfer, Lidija (2011) Cytokines, inflammation and colon cancer. Curr Cancer Drug Targets 11:451-64
Kaler, Pawan; Galea, Vincent; Augenlicht, Leonard et al. (2010) Tumor associated macrophages protect colon cancer cells from TRAIL-induced apoptosis through IL-1beta-dependent stabilization of Snail in tumor cells. PLoS One 5:e11700
Kaler, P; Augenlicht, L; Klampfer, L (2009) Macrophage-derived IL-1beta stimulates Wnt signaling and growth of colon cancer cells: a crosstalk interrupted by vitamin D3. Oncogene 28:3892-902
Escandell, Jose M; Kaler, Pawan; Recio, M Carmen et al. (2008) Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21. Biochem Pharmacol 76:198-207
Klampfer, Lidija; Huang, Jie; Shirasawa, Senji et al. (2007) Histone deacetylase inhibitors induce cell death selectively in cells that harbor activated kRasV12: The role of signal transducers and activators of transcription 1 and p21. Cancer Res 67:8477-85
Klampfer, L; Huang, J; Kaler, P et al. (2007) STAT1-independent inhibition of cyclooxygenase-2 expression by IFNgamma;a common pathway of IFNgamma-mediated gene repression but not gene activation. Oncogene 26:2071-81
Stempelj, Mateja; Kedinger, Michele; Augenlicht, Leonard et al. (2007) Essential role of the JAK/STAT1 signaling pathway in the expression of inducible nitric-oxide synthase in intestinal epithelial cells and its regulation by butyrate. J Biol Chem 282:9797-804