TRANSFORMING FUNCTIONS OF MUTANT COLON CANCER ONCOGENES

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The goal of this proposal is to define in human colon epithelium the transforming activity of five genes which in human colorectal cancers are common sites of somatic mutation. These genes are p53, K-ras, MCC, APC, and DCC. Specifically, we will test altered forms of these putative colon cancer genes for their ability to cause malignant progression of a unique nontransformed colon adenoma cell line, VACO- 330. VACO-330, established from a benign human colon adenoma, retains many features of a benign colon epithelium. VACO-330 is nontumorigenic in the nude mouse, incapable of anchorage independent growth in soft agar, and has well differentiated epithelial morphology. It is growth factor dependent, requiring for growth stimulation by TGF-alpha. We have shown that VACO-330 bears one mutant and one wild type p53 allele, bears only germ line ras alleles, and expresses at very low levels MCC, APC, and DCC transcripts. Common genetic alterations present in colon cancers include mutations of the K-ras-p53, MCC and APC genes, and deletions of p53, DCC, APC and MCC genes. Major questions now remaining are: 1) what functional features of the malignant phenotype are imparted by these altered genes; 2) are these altered genes dominant or recessive, encoding active oncogenes or inactive suppressor genes? We will answer these questions by study of progression from adenoma to carcinoma in VACO-330 transfected with additional altered forms of these five genes. The general approach will be to determine by sequence analysis which alleles are in VACO-330 already mutant and which remain wild type. We will then: 1) turn off in VACO-330 expression of the wild type alleles (assaying suppressor activities), 2) add exogenous mutant alleles to the VACO-330 wild type alleles (assaying dominant oncogenic activity), and 3) add exogenous wild type alleles to the VACO-330 mutant alleles (assaying suppressor activities). Progression of VACO-330 induced by transfected genes will be determined by assay of transfectants for: acquisition of tumorigenicity and anchorage independent growth, for changes in morphology and growth rate, and for acquisition of independence from the requirement for stimulation by TGF- alpha. Specific studies proposed are: 1) To determine by transfection the activity of coexpressed mutant K-ras and mutant p53 alleles in promoting VACO-330 progression; 2) To determine the genotype of the VACO-330 APC, DCC and MCC alleles; 3) To determine suppressor gene activity of DCC, APC, and MCC by assay of progression induced by antisense constructs which will turn off expression of these transcripts in VACO-330; 4) To determine the suppressor activity of transfected wild type MCC, APC and DCC by assay of their ability to revert colon neoplasia; 5) To determine the dominant transforming activity of specific mutant MCC and APC alleles which have been cloned from colon carcinomas by transfecting these mutant MCC and APC cDNAs into VACO-330.