Recessive oncogenes (tumor suppressor genes) play a major role in the pathogenesis of human lung cancer. However, 3p is the most frequently involved chromosomal region in lung cancer suggesting the location of one or more new recessive oncogene(s). Cytogenetic and molecular evidence for allele loss of 3p occurs in >80% of small cell (SCLC) and >50% of non-small cell (NSCLC) lung cancers. In fact the evidence is suggestive of several 3p distinct recessive oncogenes located at 3p25, 3p21.3 (two sites), 3p14.2, and 3p12-13. Studies of preneoplasia indicate the loss of 3p21 alleles is one of the earliest alterations found in preneoplastic lesions (occurring at the stage of hyperplasia) suggesting one or more 3p recessive oncogenes functions as """"""""gatekeepers"""""""" in the molecular pathogenesis of lung cancer.
The specific aims of this project are: (specific aim #1) to isolate by positional cloning a new recessive oncogene residing at 3p21.3. This will be done using the unique reagents including a complete cosmid contig and cDNA clones for >25 different genes, that have been assembled by Dr. Minna and his collaborators covering the shortest region of overlap in a series of nested homozygous deletions found in SCLC genomic DNAs. The gene will be identified by searching for mutations in the open reading frame of these cDNAs using SSCP and DNA sequencing techniques.
Specific aim #2 is to determine the functional characteristics of this locus by introducing, through microcell mediated chromosome transfer, a portion of human chromosome 3p into a human lung cancer line bearing a homozygous deletion for the 3p21.3 region and then testing for suppression of tumorigenicity in nude mice, soft agarose colony formation in cell culture, and induction of apoptosis (programmed cell death) by Dr. Killary.
Specific aim #3 proposed to test for suppression of telomrase activity, development of telomere shortening, and loss of immortal cell growth in lung cancer, following introduction of candidate cosmids and cDNAs from the 3p21.3 homozygous deletion as well as portions of chromosome 3 using unique assays developed by Dr. Shay's lab. The translational goal is to apply this information to develop new methods for identification of genetic changes in preneoplastic lesions for very early lung cancer diagnosis; use as a surrogate molecular marker; search for genetic predisposition via germline mutations in the gene; and potentially to develop tumor specific therapy. The research being translated involves cytogenetic, allele loss, genetic changes in preneoplasia, homozygous deletion discovery, and positional cloning information. The steps required are identification of candidate 3p21.3 recessive oncogenes in the homozygous deletion, determining their open reading frame sequence and expression in lung cancer, screening for mutations that alter the primary sequence, demonstration of mutations in tumor cell lines, primary tumors, and preneoplastic lesions, testing for whether the presence of the mutation identifies very high risk individuals and the reversibility of the lesions with chemoprevention (surrogate markers), tests for germline inheritance of mutant genes and whether these lead to an inherited cancer predisposition syndrome; and, based on studies of the function of the gene, designing other diagnostic tests and potential therapies. Thus, this project interacts with Project #2 (Genetic Susceptibility to Lung Cancer), Project #3 (Molecular early Detection of Lung Cancer), and Project #4 (Chemoprevention of Lung Cancer).
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