We evaluated protective pathways in pulmonary papillary adenocarcinoma, a histomorphologically distinctive subtype of lung adenocarcinoma. We found that the frequency of high expression levels of P53, ERCC1, and RRM1 are similar in cases of pulmonary papillary to those observed previously in other types of pulmonary adenocarcinoma. However, we found a remarkably high KEAP1
mutation frequency (12/20 cases, 60%) in pulmonary papillary adenocarcinoma, which is significantly greater than our previously reported rates of 18% for pulmonary non-small cell carcinoma in general and 28% for adenocarcinoma [16
], and strikingly higher than the frequency of approximately 3% recently reported for NSCLC [18
]. As expected, KEAP1
mutations were associated with increased nuclear expression of the NRF2 transcription factor in these papillary cancers, but some cases without KEAP1
mutations also displayed increased NRF2 expression and nuclear localization. These high rates of KEAP1
mutations and NRF2 overexpression in true papillary adenocarcinoma of the lung could be related to the reported poor prognosis and chemotherapy resistance of these cancers, and we propose that this distinctively high frequency of KEAP1
mutations supports the recognition of true papillary adenocarcinoma as a diagnostic entity.
The KEAP1 alterations found in pulmonary papillary adenocarcinoma tumors included insertions, deletions, and frameshifts, with the most frequent type of mutation in pulmonary papillary adenocarcinoma (involving 58.3% (7/12) of the mutations) consisting of a single base insertion that would result in a premature stop codon and thus a truncated KEAP1 protein. Remarkably, 25% (3/20) of the cases with mutations were found to have more than one mutation affecting the KEAP1 gene. Taken together with our previous data and others, our present study indicates that KEAP1 gene alterations are frequent in pulmonary adenocarcinomas, particularly in papillary adenocarcinomas.
We also found that cancer cells of pulmonary papillary adenocarcinoma demonstrate an increased accumulation of NRF2 by IHC staining, and in tumors with KEAP1
mutations, both nuclear and cytoplasmic accumulations of NRF2 are markedly increased. These data suggest that frequent mutations of KEAP1
in pulmonary papillary adenocarcinoma could significantly reduce the sensitivity of these cancers to chemotherapy. Increased NRF2 staining was also demonstrated in several cases of pulmonary papillary adenocarcinoma with wild-type KEAP1
, suggesting that there are other mechanisms that could contribute to stabilization and induction of NRF2. Possible mechanisms for this include: 1) increased levels of the nuclear oncoprotein prothymosin, which regulates the intranuclear dissociation of KEAP1-NRF2 complex [19
]; 2) somatic mutations in Nrf2
gene which impair its binding with Keap1 [19
]; 3) alternative splicing of KEAP1
, resulting in a nonfunctional KEAP1 protein in cancer cells [21
]; 4) methylation of CpG-rich regions of KEAP1
promoter, resulting in decreased expression of this protein [22
]; and 5) phosphorylation of NRF2 by protein kinase(s) associated with the MAPK/ERK signaling cascade, leading to stabilization of NRF2 [23
]. Evidently, mechanisms for regulation of KEAP1-NRF2 pathway are complex, and further studies are needed to understand exaggerated NRF2 responses in cancer cells even in the absence of KEAP1
In summary, our finding of frequent KEAP1 mutations in pulmonary papillary adenocarcinoma and the association between loss of functional KEAP1 and increased NRF2 activity suggest important roles for the KEAP1-NRF2 pathway in the regulation of antioxidants, detoxification enzymes, and drug transporters activity in this subset of pulmonary adenocarcinoma. The relatively disproportionate frequency of KEAP1 mutations in pulmonary papillary adenocarcinoma supports the recognition of these cancers as a unique subset of lung adenocarcinoma.