Booth Laurence, Roberts Jane L, Poklepovic Andrew, Dent Paul
Departments of Biochemistry and Molecular Biology.
Departments of, Medicine, Virginia Commonwealth University, Richmond, VA 23298-0035.
Oncol Signal. 2018 Jun;1(1):19-30. doi: 10.1016/j.onsig.2017.07.001. Epub 2017 Aug 16.
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.
我们专注于确定E3泛素连接酶NEDD4在非小细胞肺癌（NSCLC）细胞对阿法替尼耐药中的作用。与对照克隆相比，阿法替尼耐药的H1975克隆中NEDD4和c-MET过表达，而ERBB1、ERBB3、ERBB4和PTEN的表达低于对照克隆。敲低NEDD4可增强PTEN、ERBB1/3/4和c-MET的表达。这还与mTOR表达和mTOR磷酸化增强约3倍以及磷酸化ULK-1 S757水平升高约4倍有关。在缺乏NEDD4或自噬调节蛋白Beclin1的情况下，[培美曲塞+西地那非]的药物组合或组蛋白去乙酰化酶（HDAC）抑制剂丙戊酸钠均无法：降低ERBB1/3/4的表达；降低ULK-1 S757的磷酸化；或增强ULK-1 S317和ATG13 S318的磷酸化。[培美曲塞+西地那非]通过自噬降解降低了多种HDAC的表达。I类HDAC表达的降低降低了ERBB1/3/4和PTEN的表达。用[培美曲塞+西地那非]或丙戊酸钠处理缺乏NEDD4的阿法替尼耐药克隆，导致自噬体和自溶酶体形成的诱导作用较弱，细胞杀伤作用降低。敲低NEDD4降低了[培美曲塞+西地那非]的致死率；敲低PTEN增强了药物诱导的杀伤作用。联合敲低NEDD4和PTEN将单独敲低PTEN引起的杀伤增加量降低至基础水平。总体而言，我们的数据表明NEDD4在维持我们的耐药H1975克隆中的阿法替尼耐药表型方面起着至关重要的作用。
