To explore the significance of phosphatidylinositol-3-kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild-type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over-expressed wild-type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage-independent growth activity and migration activity of immortalized airway epithelium 16HBE14o-cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild-type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

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Benzo[a]pyrene diol epoxide (B[a]PDE), the ultimate carcinogenic metabolite of benzo[a] pyrene, has been implicated in the mutagenesis of the p53 gene involved in smoking-associated lung cancer. To further understand the role of B[a]PDE in lung tumour progression, we investigated its effect on the numerical integrity of centrosomes and chromosome stability in lung cancer cells lacking p53. Exposure of p53-deficient H1299 lung cancer cells to B[a]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. Analysis of H1299 cells stably expressing fluorescence-tagged centrin (a known centriolar marker) revealed that the centrosome amplification was primarily attributable to excessive centrosome duplication rather than to centriole splitting. Forced expression of POLK DNA polymerase, which has the ability to bypass B[a]PDE-guanine lesions in an error-free manner, suppressed the B[a]PDE-induced centrosome amplification. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2, 3, and 16 revealed that B[a]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. We extended these findings to primary lung carcinomas containing non-functional p53, and found a strong association between centrosome amplification and a high level of B[a]PDE-DNA accumulation. Therefore B[a]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization as well as its mutagenic activity.

BackgroundGenomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.Methods and ResultsA genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.ConclusionThese results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

These results suggested that TNK2 amplification is an independent predictor of a poor prognosis in patients with GC and leads to an increase in the malignant potential of GC cells.