The PI3K/Akt pathway is one of the important regulators of mammalian cell proliferation and survival, because several components of this pathway are deregulated in many cancers (13
). We performed a large-scale mutational and copy number gain analysis of lung cancer cell lines and primary tumors followed by functional analyses. Consistent with earlier smaller-scale analyses by others (7
), we found a relatively low percentage of PIK3CA
mutations in lung cancers compared with several other epithelial cancers. Whereas we did not find PIK3CA
mutations in SCLC cell lines of pulmonary origin, we found mutations in two ExPuSC cell lines. To date, mutations in the EGFR pathway genes that are well studied (EGFR, BRAF, KRAS, HER2
, and PIK3CA
) have not been reported in SCLC.
Previously, we reported that mutations of EGFR, KRAS
, and HER2
were mutually exclusive, indicating that at least one activating mutation in the EGFR-RAS-RAF signaling pathway is sufficient for the pathogenesis of many lung cancers (15
). In this study, we added BRAF
to the mutual exclusion list. However, the mutational status of PIK3CA
was not mutually exclusive to EGFR
, which is similar to previous reports (6
). Whereas the other genes targeted adenocarcinomas, PIK3CA
mutations targeted different subpopulations of NSCLC.
PI3K may be activated by receptor kinases and Ras, which in turn activates Akt. However, whereas the PI3K and EGFR signaling pathways closely interact, PI3K signaling has additional activators and downstream targets (4
). Our findings of dual mutations in PI3KCA
and genes within the direct EGFR signaling pathways are consistent with these observations.
We found that PIK3CA
copy number gains in cell lines and tumors were more frequent in squamous cell carcinomas than in adenocarcinomas, which is consistent with previous smaller studies (11
). Array CGH analyses of a subset of our cell lines and an independent smaller tumor set confirmed the qPCR data. Adenocarcinomas with PIK3CA
gains tended to have other gene mutations, suggesting that PIK3CA
gain may not be enough for the pathogenesis of adenocarcinoma. However, most of squamous cell carcinomas with PIK3CA
gains had no other alterations in the genes studied, indicating that PIK3CA
may play a pivotal role in pathogenesis of squamous cell cancers. High-level PIK3CA
gains (copy number, >5) were exclusively present in squamous cell carcinomas. PIK3CA
mutations and copy number gains occurred independently of each other, and either molecular event may be sufficient to drive the cell toward tumorigenesis.
We profiled PIK3CA
mRNA expression, PI3K activity and protein expression status in PI3K/Akt pathway to see if PIK3CA
alterations have a biological effect. Cell lines with PIK3CA
gains had increased mRNA expression, as did lines with EGFR
activating mutations, as previously reported (31
). Somewhat surprisingly, PIK3CA
mutant cell lines did not have high mRNA expression.
However, cell lines having either PIK3CA
mutations or gains (or both) were associated with increased PI3K activity and increased expression of pAkt protein (but not with increased total PIK3CA protein) when compared with wild-type cell lines or immortalized bronchial epithelial cells. EGFR
mutant cell lines had the same effects, whereas KRAS
mutant cell lines did not have these effects. According to previous reports, mutant PIK3CA protein causes a gain of enzymatic function and induces oncogenic transformation when expressed in primary chicken embryo fibroblasts and in NIH 3T3 cells (33
). Samuels and colleagues also reported that mutant PIK3CA promotes cell growth and invasion of human cancer cells (35
). Expression of PTEN, a negative regulator of the PI3K/Akt pathway, was present (as total and phosphoprotein) in most of lung cancer cell lines irrespective of their mutational or copy number status.
To determine whether alterations in PIK3CA contribute toward the oncogenic phenotype, we used RNA interference, both transient (siRNA) and stable (shRNA). Results of both knockdown techniques were similar and showed reduction of PIK3CA and pAkt proteins in mutant and high copy number cell lines accompanied by decreased anchorage-dependent and independent growth. However, knockdown of PIK3CA protein in a wild-type cell line lacking detectable pAkt had no effect on cell growth. These results confirm that mutations or copy number gains in squamous lung cancer cells confer a growth advantage and that inhibition of pAkt may be a better indicator of cellular effect (and perhaps of therapeutic efficacy) than inhibition of PIK3CA protein.
The finding that PI3K, a key regulator of cell growth, metabolism, and survival, is frequently activated in cancers has stimulated widespread interest in identifying potent and selective inhibitors of PI3K isoforms (36
). Whereas several potential therapeutic agents have been identified, most do not show isoform selectivity. Recently, the structure of a human p110α/p85α complex was clarified, and the location of the common mutations at specific interfaces identified (37
). These new insights into the structure of PIK3CA
offer insights into the design of specific inhibitors.
In conclusion, PIK3CA
copy number gains occur at much higher frequencies in lung cancers than do activating mutations and that the gains target squamous cell carcinomas. As these alterations confer a growth advantage to the cancer cells, targeting the PI3K/Akt pathway is a potential therapeutic option for squamous cell lung cancers. It is of particular interest to target squamous cell carcinomas, the commonest form of lung cancer in ever smokers (38
), as most recent trials of targeted agents for lung cancer have focused on adenocarcinomas.