PHLPP1 Expression Through AKT and ERK Dual Signaling Pathways May Slow Down the Resistance to TKI in EGFR-Mutated Lung Adenocarcinoma

Abstract

Background: The epidermal growth factor receptor (EGFR) kinase inhibitors are effective treatments for lung cancers with EGFR activating mutations, but the magnitude of tumor regression varies and drug resistance is unavoidable. Multiple mechanisms of resistance to EGFR-TKIs have been identified, including the occurrence of secondary mutations in the EGFR gene, MET amplification, acquired BRAF rearrangements and activation of bypass pathways. Central to these mechanisms of resistance is the re-activation of AKT and ERK signaling, which enables escape of tumor cells from EGFR inhibitor treatment. However, the mechanisms of reactivation of PI3K-AKT and ERK/MAPK pathway are unclear. Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP) acts as tumor suppressors in various types of human cancer by suppressing cell survival pathways and promoting apoptosis through inhibiting AKT and ERK pathway activation. Here, we hypothesize that PHLPP is a key regulator of EGFR-TKI resistance in lung cancer and a potential treatment target for overcoming resistance to EGFR-TKI treatment. Method: A transcriptomic of PHLPP1 in non-small lung cancer cell according to gefitinib sensitivity obtained from Gene Expression Omnibus (GEO) database under accession number GSE4342 were analysis. The lentivirus-mediated delivery of shRNA was used to generated stable knockdown of PHLPP1 expression lung cancer cells, and retrovirus-mediated delivery was used to generated stable overexpression of PHLPP1 lung cancer cells. Western blotting, real-time PCR (RT-PCR) and immunofluorescence were used to determined PHLPP expression in vitro. PHLPP1 expression in clinical sample was determined by Immunohistochemical (IHC) staining. MTT assay was conducted to determine the cell proliferation. Xenografts bearing PHLPP overexpression and control were evaluated EGFR-TKI induced tumor regression. Result: PHLPP1 gene expression was higher in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines from a GEO public database. In vitro, EGFR mutated NSCLC cell line HCC827 continuously exposing to gefitinib exhibited dramatically reduced expression of PHLPP1 and increased phosphorylation AKT and ERK. Knockdown of PHLPP1 decreased cell death induced by the EGFR-TKI in EGFR-mutant lung cancer cells, overexpression of PHLPP1 enhanced gefitinib-induced apoptosis in gefitinib-resistance EGFR-mutant lung cancer cells. In xenograft model, overexpression of PHLPP showed significantly more tumor regression after gefitinib treatment at 1-week time point compared to control group. In patients, PHLPP1 were highly expressed in tumors with EGFR common mutations pre- and post-development of resistance to EGFR TKIs. PHLPP1 expression were down regulated in the post-relapse tumor samples compared to that of pre-treatment, and patients with higher PHLPP1 expression in pre-treatment had significantly longer progression-free survival (PFS). Conclusion: PHLPP loss may be a key molecule contributing to the resistance of EGFR-TKI by activating PI3K-AKT and ERK/MAPK signaling pathway. PHLPP may serve as a potential predictor of EGFR-TKI treatment response in these patients. Up-regulating PHLPP expression may prevent or /and delay the emergence of EGFR-TKI resistance. Further study is warranted to prove PHLPP as an effective strategy.