A Novel Integrated VMAT/IMRT Technique for the Treatment of Non-Small Cell Lung Cancer

Abstract

Purpose/Objective(s): Casitas B-lineage lymphoma (CBL) is an E3 ubiquitin ligase and adaptor molecule that is important in cancer. It belongs in the family with CBL-B and CBL-3. Our previous studies detected CBL mutations, loss of heterozygosity, and low protein expression in nonsmall cell lung cancer (NSCLC). We also determined the genetic variations of CBL, their relationship to receptor tyrosine kinases (RTK) such as EGFR and MET, and their functionality in NSCLC. However, ubiquitination of EGFR showed no difference between CBL wild type (WT) and mutants (Mts). Therefore, we undertook to investigate whether MET is a potential target of CBL and better therapeutic strategy. Materials/Methods: CBL alterations were determined in NSCLC patients with next generation sequencing. Immunoprecipitation was performed to detect the ubiquitination of MET by CBL WT and Mts. MET inhibitor SU11274 was utilized to compare cell viability of CBL WT cells with viability of CBL Mt cells. Cell motility after SU11274 treatment was examined by wound healing assay. Soft agar assay was performed to investigate colony formation of shRNA knockdown (sh-CBL) cells and SU11274 treatment. PamGene Chip analysis was used to detect and predict the RTK phosphorylation difference between CBL WT and Mt/sh-CBL cells. In vivo mouse study was performed to examine tumor growth and metastasis of sh-CBL cells. Results: CBL had 6% (8/135) mutation rate in NSCLC patient population. When CBL and MET protein expression levels were analyzed by immunoblot, CBL WT cells had lower MET protein expression than CBL Mt cells. The ubiquitination of MET was decreased in cells that transiently expressed CBL Mt relative to CBL WT cells. In addition, CBL Mt cells were more sensitive toMET inhibitor SU11274 than CBLWT cells. Wound healing assay demonstrated sh-CBL cells had an increase in cell motility and more sensitive with SU11274 migration inhibition. Furthermore, soft agar assay showed an increase in colony number and size of sh-CBL cells compared to control cells. After treatment with SU11274, sh-CBL cells showed inhibition in colony formation. In vivo study showed sh-CBL cells had lower tumor growth but more metastasis than control cells. PamGene results predicted an effective cell signaling pathway involving MET, paxillin, EPHA2, and VEGFR. Conclusions: Ubiquitination assay result suggested MET is one of the direct targets of CBL. The cell viability and motility results demonstrated that CBL mutants and sh-CBL cells are more sensitive than CBLWT cells to MET inhibitor SU11274, suggesting they have higher MET protein expression. Moreover, the PamGene results indicated that MET is involved in CBL cell signaling pathway. Thus, CBL gene status could be a potential indication for lung cancer therapy utilizing MET and other inhibitors. Author Disclosure: Y.C. Tan: None. C. Rolle: None. L. Zhu: None. M.K. Srivastava: None. S. Sharma: None. R. Salgia: None.