Abstract
We focused on defining the role of the E3 ligase NEDD4 in NSCLC cell afatinib resistance. Afatinib resistant H1975 clones over-expressed NEDD4 and c-MET compared to control clones and expressed less ERBB1, ERBB3, ERBB4 and PTEN than control clones. Knock down of NEDD4 enhanced the expression of PTEN, ERBB1/3/4 and c-MET. This was also associated with a ∼3-fold enhancement in both mTOR expression and mTOR phosphorylation and a ∼4-fold elevation in phospho-ULK-1 S757 levels. In the absence of NEDD4 or the autophagy regulatory protein Beclin1, neither the drug combination of [pemetrexed + sildenafil] nor the HDAC inhibitor sodium valproate was as capable of: reducing the expression of ERBB1/3/4; reducing phosphorylation of ULK-1 S757; or at enhancing the phosphorylation of ULK-1 S317 and ATG13 S318. [Pemetrexed + sildenafil] exposure, via autophagic degradation, reduced the expression of multiple HDACs. Reduced expression of Class I HDACs lowered the expression of ERBB1/3/4 and PTEN. Treatment of afatinib resistant clones lacking NEDD4 with [pemetrexed + sildenafil] or sodium valproate resulted in a weaker induction of autophagosome and autolysosome formation and with reduced cell killing. Knock down of NEDD4 reduced [pemetrexed + sildenafil] lethality; knock down of PTEN enhanced drug-induced killing. Combined knock down of NEDD4 and PTEN reduced the elevated amount of killing caused by PTEN knock down alone back to basal levels. Collectively, our data argue that NEDD4 plays an essential role in maintaining the afatinib-resistant phenotype in our resistant H1975 clones.