Lung cancer is the leading cause of cancer death worldwide with 2.2 million new cases diagnosed and 1.8 million deaths per year. Lung squamous cell carcinoma (LSCC) is an aggressive histological subtype of non-small cell lung cancers (NSCLC), which is strongly associated with cigarette smoking and exposure to environmental pollutants. In collaboration with the Computational Biomedicine group at Boston University, we identified several putative cancer driver mutations in benign premalignant lung tumors, extracted from upper bronchial airway epithelium. The gene mutations from premalignant tumors are thought to initiate neoplasia but cannot promote malignancy independently. It is hypothesized that additional cooperating mutations will have a compounding effect on tumorigenesis if co-expressed in the same tumor cell. We used cancer genomics data from LSCC primary tumors in the Cancer Genome Atlas (TCGA) database to identify lung pre-malignancy associated genes that are significantly co-mutated. Two of the identified mutant genes, PIK3CA and NFE2L2, were shown to co-occur at a statistically significant rate in LSCC primary tumors.
The PIK3CA gene encodes the PI3K lipid kinase, which regulates the AKT and mTOR kinase signaling pathways, thus promoting cell proliferation and survival. NRF2, the product of NFE2L2 gene, is a transcription factor that regulates the antioxidant response, playing a protective role against oxidizing cellular damage. NRF2 promotes the transcription of key proteins in the antioxidant response such as glutathione S transferase and NADPH oxidase. NRF2 is normally subject to ubiquitin-mediated degradation, which is regulated by the KEAP1 protein. Loss of function KEAP1 gene mutations are common in lung cancer. When cells are exposed to oxidizing agents, KEAP1 is modified by these agents, resulting in release and stabilization of NRF2, and the subsequent transcription of antioxidant response genes.
Studies of PI3K and NRF2, and their downstream effectors have shown that both the PI3K/AKT/mTOR and NRF2/KEAP1 signaling pathways control autophagy, which is a catabolic process that regulates the recycling of macromolecules under conditions of nutrient deprivation. PI3K and NRF2 both control the activity of the SQSTM1/p62 protein, which plays a major role in autophagic degradation of cargo proteins. Autophagy has been implicated as a tumor suppressive mechanism. Both PI3K and NRF2 are known to inhibit autophagy in lung cancer cells. Based on the significant frequency of co-occurrence of PIK3CA and NFE2L2 gene mutations in pre-malignant LSCC lesions, we hypothesize that PI3K and NRF2 cooperate to inhibit autophagy to promote LSCC progression.
To test our hypothesis, we co-expressed mutant forms of PIK3CA (E545K) and NFE2L2 (T80K) into a non-transformed Human Bronchial Epithelial Cell line (HBEC-3KT). We performed a series of Western Blots to verify PI3K and NRF2 protein expression as well as downstream AKT activation and markers of autophagy pathway activation. mTORC1 is an effector of PI3K and plays a central role in the inhibition of autophagy through the PI3K/AKT/mTOR signaling network. Therefore, we performed Western Blot analysis of samples treated with the mTORC1 inhibitor Everolimus to compare the effects of mTORC1 inhibition on autophagy activation in control, single PIK3CA, NFE2L2 and double mutant HBEC3-KT cells. We observed significant suppression of autophagy in the PI3K/NRF2 double mutant cells. Moreover, the studies also showed that the double mutant cells are more sensitive to anti-proliferative effects of Everolimus compared to control and single mutant cells.
Taken together, our studies show that PIK3CA and NFE2L2 mutations cooperate to hyperactivate the AKT kinase and to suppress autophagy pathway activation. This represents a key mechanism of the malignant transformation of benign premalignant LSCC lesions. This warrants further research into the cooperation between PI3K and NRF2 in lung cancer pathogenesis. Our results have important implications both for diagnosis and treatment of LSCC. Though many important advances in the treatment of lung cancer have been made over the past few decades including the use of tyrosine kinase inhibitors (TKIs) such as Erlotinib, there is still much to understand about the biology and mechanisms of the disease.1 Blockers of the T-cell checkpoint, such as anti-PD-1 drugs are currently FDA-approved first lines of therapy for NSCLC. In addition, immunotherapy has shown some efficacy in lung cancer patients.2 Our studies provide rationale for the development of therapeutics that suppress NRF2 and PI3K activity in the treatment of LSCC.3 Since mTORC1 inhibitors cause robust inhibition of PIK3CA/NFE2L2 double mutant cell proliferation, future studies will be aimed at testing combinations of mTORC1, PI3K and NRF2 pathway inhibitors to treat LSCC.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/43377 |
| Date | 17 November 2021 |
| Creators | Guthlein, Caitlin Margaret |
| Contributors | Singh, Anurag |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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