Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor supp ressor genes (TSGs), the expression of which is frequently lost in lung can cers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them i nto lung cancer NCl-H1299 cells, which do not express either gene. Colony f ormation of H1299 cells was reduced 90% after transfection with wild-type S EMA3B compared with the control vector. By contrast, only 30-40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B var iants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apo ptosis. We found that lung cancers (n = 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested condit ioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of sever al lung cancer lines 30-90%, whereas SEMA3B mutants or SEMA3F had little ef fect in the same assay. Sequencing of sodium bisulfite-treated DNA showed d ense methylation of CpG sites in the SEMA3B 5' region of lung cancers not e xpressing SEMA3B but no methylation in SEMA3B-expressing tumors. These resu lts are consistent with SEMA3B functioning as a TSG, the expression of whic h is inactivated frequently in lung cancers by allele loss and promoter reg ion methylation.