M.I. Lerman

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

Previous reports showed that the loss of DNA sequences on the short arm of chromosome 3 (3p) is consistently found in sporadic renal cell carcinomas. To evaluate the significance of this genetic change, we looked for the loss of 3p alleles in hereditary renal cell carcinomas and other tumors from patients with von Hippel-Lindau disease. Specific loss of alleles from chromosome 3p was detected with polymorphic DNA markers in 11 renal cell carcinomas, one pheochromocytoma, two spinal hemangioblastomas and one cerebellar hemangioblastoma from von Hippel-Lindau patients. Multiple renal cell carcinomas in individuals with von Hippel-Lindau disease showed loss of the same chromosome 3p alleles, which demonstrated that the same chromosome was deleted in each tumor. Analysis of haplotypes indicated that the loss of chromosome 3p alleles was from the chromosome bearing the balancing, wild-type allele of the VHL gene. These results are consistent with the concept that the VHL gene is a recessive oncogene. Renal cell carcinoma, pheochromocytoma, and spinal and cerebellar hemangioblastomas develop in predisposed family members when somatic mutational events lead to loss of chromosome 3p sequences bearing the wild-type allele of the VHL gene.

The deletion of the short arm of chromosome 3 is frequently observed in lung cancer. To determine whether the von Hippel-Lindau (VHL) disease tumor suppressor gene located at 3p25 is responsible for oncogenesis in lung cancer, we searched the known open reading frame using the single-strand conformation polymorphism (SSCP) technique for mutations in the VHL gene in 72 cancer cell lines including small cell (SCLC) and non-small cell (NSCLC) lung cancers, carcinoids, and mesotheliomas. SSCP analysis showed that four cell lines have altered SSCP patterns within the coding region and one in an intron of the VHL gene. SCLC line NCI-H1672 had a somatic mutation, G to A at nucleotide (nt) 530, leading to amino acid substitution (glycine to aspartic acid) compared to normal DNA from the same patient. Mesothelioma line NCI-H28 had T to A mutation at nt 479 leading to leucine to histidine amino acid change. We found one frequent polymorphism A (0.72) or G (0.28) at nt 19 resulting in either serine or glycine at this position, changes also found in normal peripheral blood cell DNA, often in a heterozygous state. In addition, we found single rare polymorphisms which did not alter the coding region including: C to G at nt 396, G to T at nt 843, and C to T change in an intron. These results suggest that the VHL gene is only rarely mutated in thoracic malignancies.