Lung cancer is the leading cause of cancer death in industrialized countries (~28% of all cancer deaths in the US). More deaths will occur this year from lung cancer than breast, prostate, and colorectal cancers combined. Non-small cell lung cancers (NSCLC) represent the majority of lung cancers, carry a poor prognosis with a median survival of less than 12 months, and have a cumulative five-year survival rate of approximately 15%. Most patients present with unresectable disease (75% of patients), and current treatment options are palliative at best. Although spiral CT scans can provide an overall survival benefit to “at risk” patients (i.e., 30-pack per year smokers-current or former), improved success and impact on decreasing death from NSCLC will rely on the identification of cancer-specific molecular targets for the development of new and effective therapeutics. Identification of novel and cancer-specific molecular mechanisms controlling the tumorigenicity of NSCLC cells will aid in development of these future targeted therapies, which is the focus of this study.

Through a successful collaboration between the Chalfant, Park, Fisher, and Dent laboratories, we now report that Melanoma Differentiation Associated gene-7/Interleukin-24 (MDA-7/IL-24), a cytokine that exhibits cytotoxic effects on tumor cells while sparing untransformed cells, induces the killing of lung cancer cells of various oncogenotypes via a novel mechanism involving alternative RNA splicing. Specifically, MDA-7/IL-24 reduced the ratio of Bcl-x(L)/Bcl-x(s) mRNA via activation of the Bcl-x(s)-specific 5’ splice site, which reduced Bcl-x(L) protein expression. As the loss of Bcl-x(L) expression is necessary for MDA-7/IL-24 to induce apoptosis, this finding detailed a novel regulatory mechanism to remove this anti-apoptotic factor. Surprisingly, the Bcl-x(L) protein expression was also “rescued” in MDA-7/IL-24-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells exposed to Bcl-x(s) adenovirus exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA. These findings show, for the first time, a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKC completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKC signaling axis in NSCLC cells.

This investigation was a fruitful collaboration between the laboratories and colleagues of Paul B. Fisher, MPh, PhD, Charles E. Chalfant, PhD, Dr. Paul Dent, and Dr. Margaret A. Park, PhD. Dr. Brian A. Shapiro, a former postdoctoral trainee in Dr. Chalfant’s laboratory at VCU, and Dr. Ngoc Vu, a PhD student in the Chalfant laboratory, are co-first authors of this paper, and performed much of the biological work. This work demonstrates the vital type of collaboration between different VCU and VIMM laboratories that can lead to important advances in cancer cell signaling pathways. This work was supported by research grants from the Veteran’s Administration (VA Merit Review, I BX001792 (CEC) and a Research Career Scientist Award, 13F-RCS-002 (CEC)); from the Vietnam Education Foundation (fellowship to NTV); from the National Institutes of Health via HL125353 (CEC), HD087198 (CEC), CA117950 (CEC), CA154314 (CEC), RR031535 (CEC), CA192613 (PD), CA097318 (PBF), CA127641 (PBF), P01 CA104177 (PBF), NH1C06-RR17393 (to Virginia Commonwealth University for renovation), and a T32 Post-Doctoral Fellowship (Postdoctoral Training Program in Cancer Biology, CA085159) (BAS); from the US-Israel Binational Science Foundation via BSF#2011360 (CEC); and from the National Foundation for Cancer Research (PBF). Services and products in support of the research project were generated by the VCU Massey Cancer Center Shared supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059. The study was published in JBC on October 7th, 2016*.