Issue No. 7, July 2017
VCU Institute of Molecular Medicine (VIMM) NEWS & VIEWS
The VIMM, established in 2008 by Paul B. Fisher, M.Ph., Ph.D., the Founding Director, is comprised of outstanding scientists/clinicians from VCU School of Medicine focusing on important medical-related research in cancer, neurodegeneration and infectious diseases. The purpose of this NEWS & VIEWS is to highlight the exciting research being performed by the VIMM members.          

Determining cell signaling pathways mediating the cytotoxicity of Melanoma Differentiation Associated gene-7/Interleukin-24 (MDA-7/IL-24) in lung cancer.

  • Lung cancer is the leading cause of cancer death in the US, and non-small cell lung cancer (NSCLC) represents the majority of these cancers.

  • NSCLC carries a poor prognosis/median survival of <12 months, has a 5-year survival rate of ~15%, and current treatment options are palliative.

  • MDA-7/IL-24 is a cytokine that potently inhibits cell growth and induces apoptosis in lung cancers with minimal lethality toward normal cells.

  • The Chalfant, Park, and Fisher Laboratories found that MDA-7/IL-24 downregulates Bcl-x(L) via the production of Bcl-x(s) mRNA through alternative mRNA processing in lung cancer cells of various oncogenotypes.

  • MDA-7/IL-24 exerts cytotoxic effects on lung cancer cells by activating the Bcl-x(s) 5’ splice site via the SRC/PKCd signaling axis.

  • The concept of Bcl-x(s) mRNA downregulating Bcl-x(L) may prove an effective therapeutic avenue to enhance cancer-specific killing.

Novel cell signaling pathways targeted by MDA-7/IL-24 in lung cancer

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*.
MDA-7/IL-24 induces the activation of the Bcl-x(s)/proximal 5’ splice site of Bcl-x pre-mRNA via the SRC/PKCd signaling axis. Schematic of how MDA-7 suppresses cell survival by alteration of Bcl-x splicing via a SRC/PKCd signaling pathway. Specifically, intracellular MDA-7 expression promotes the activation of the Bcl-x(s) 5’ splice site via either an intracellular receptor event or ER stress to induce SRC and PKCd activation, which may involve a direct or indirect effect of PKCd on SAP155 (downregulation) or other RNA trans-factors to up-regulate Bcl-x(s) level and down-regulate Bcl-x(L) level. As SAP155 is downregulated by MDA-7 and cannot conclusively be determined as the regulatory RNA trans-factor, PKCd is likely affecting Bcl-x 5’ splice site selection in an indirect fashion. The overall main theme of the study is that intracellular MDA-7 reduces cell viability through directly manipulating the level of anti-apoptotic Bcl-x(L) via affecting Bcl-x 5’ splice site selection, which requires the SRC/PKCd signaling pathway.

*Shapiro BA, Vu NT, Shultz MD, Shultz JC, Mietla JA, Gouda MM, Yacoub A, Dent P, Fisher PB, Park MA, Chalfant CE. Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis. J Biol Chem. 2016 Oct 7;291(41):21669-21681. PMID: 27519412 PMCID: PMC5076836
About the Investigators
Charles E. Chalfant, PhD. is a Research Career Scientist in the Veterans Administration, Vice Chair and Professor of Biochemistry and Molecular Biology at VCU-SOM, and a member of VIMM. He holds the Paul M. Corman, M.D. Chair in Cancer Research from the VCU Massey Cancer Center (MCC), and is the Deputy Director of the VCU Johnson Center for Critical Care and Pulmonary Research and Co-leader of the Cancer Cell Signaling Program for the MCC. Paul B. Fisher, MPh, PhD, is Professor and Chair of Human and Molecular Genetics (HMG), Director of the VCU Institute of Molecular Medicine (VIMM) and Thelma Newmeyer Corman Chair in Cancer Research in the MCC. Margaret Park, PhD is an assistant professor of Biochemistry and Molecular Biology at VCU-SOM and a member of the MCC. Paul Dent, PhD, is a Professor of Biochemistry and Molecular Biology at VCU-SOM. Dr. Adly Yacoub, PhD, associated with the Dent Laboratory. Drs. Brian A. Shapiro, Jacqueline Shultz, and Jennifer Mietla are former postdoctoral trainees in Dr. Chalfant’s laboratory at VCU-SOM. Ngoc Vu, PhD, is a former PhD student in the Chalfant laboratory, and is currently an assistant professor in Vietnam. Michael D. Shultz, PhD. was a CDA-2 fellow in the Veterans Administration, and is currently a staff member in the Safety Division at the McGuire VAMC. Mr. Mazen Gouda was an undergraduate research assistant in the Chalfant laboratory.