Renal cell carcinoma accounted for an estimated 57,760 new cases and estimated 12,980 deaths in the United States in 2009. Current treatment options for systemic renal cell carcinoma yield markedly low response percentages; however, recent cytokine therapy experiments have produced promising results. A novel adenovirus, Ad.5/3-mda-7, has been synthesized to efficiently infect renal cancer cells with the mda-7 gene. This gene encodes for the cytokine MDA-7/IL-24 that has the ability to specifically target transformed cells. Assays performed with this adenovirus resulted in an increased percentage of cell death in renal cancer cells when compared to infection with Ad.5/3-cmv empty vector. Further assays that combined Ad.5/3-mda-7 infection with treatments of small molecule inhibitors increased the percentages of cell death by upregulating JNK and p38 MAPK pathways, downregulating the ERK1/2 MAPK pathway, and downregulating the PI3K pathway. Western blots confirmed upregulation and downregulation of these pathways by probing for key proteins. Renal cancer cells responded best to infection with Ad.5/3-mda-7 and treatment with PD184352, PX866, and Rapamycin. This combinatorial treatment caused a greater percentage of cell death than the sum of the two individual treatments, suggesting a synergistic inhibition of cell growth pathways. These findings suggest that the combination of Ad.5/3-mda-7 and specific small molecule inhibitors has developmental potential as a novel and more efficient treatment option for systemic renal cell carcinoma.

Glioblastoma multiforme (GBM) is an aggressive cancer that affects millions of patients per year. Conventional therapies combining chemotherapeutic agents with radiation can only extend survival by a few months; therefore, there is a dire need for an effective means of treating this deadly disease. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), currently in the early stages of FDA pre-IND drug trials, has proven to be an effective cancer specific cytokine, able to trigger the onset of mitochondrial dysfunction and/or autophagy. GBM’s have mutations that often result in the activation of cytoprotective cell signaling pathways, preventing cancer therapeutics and even MDA-7/IL-24 treatments from being effective. Since the discovery of MDA-7/IL-24 a number of groups have shown toxic effects in a variety of tumor cells. However, the lethality of MDA-7/IL-24 is not enough to eradicate the tumor. We hypothesized two xxiii rationales for this minimalistic effect. First, the MDA-7/IL-24 gene delivery mechanisms are not efficient or second, active pro-survival pathways are playing a role in protection. Here we have shown that the inhibition of cytoprotective cell-signaling pathways using small molecule inhibitors of mitogen-activated extracellular regulated kinase (MEK)1/2 and phosphatidyl inositol 3-kinase (PI3K) or AKT; mammalian target of rapamycin (mTOR) and MEK1/2; HSP90 inhibitor 17AAG; and the autophagy-inducing drug OSU-03012 (AR-12), enhances the toxicity of MDA-7/IL-24. In addition, the use of a modified recombinant adenovirus comprised of the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, proved to be a more effective, CAR-independent means of infecting and killing GBM cells in vitro and in vivo when compared to Ad.5-mda-7. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.

Ovarian cancer is the most lethal gynecological malignancy among women. The current first-line treatments for ovarian cancer are cisplatin, carboplatin and paclitaxel. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Therefore, the resistance to cisplatin therapy has been a critical hurdle in the management of recurrent ovarian cancer. The mechanisms responsible for cisplatin resistance are not completely understood. In the search for new therapies to overcome/bypass cisplatin resistance, melanoma differentiation gene-7 (MDA-7) IL-24, which is a new cytokine, has anti-cancer efficacy by suppressing cell growth and inducing apoptosis in a broad range of tumor cells and does not induce any toxicity in normal cells, thus, making it a potentially effective therapeutic gene for ovarian cancer. The purpose of this study was to evaluate the potential therapeutic efficacy of MDA-7 to treat ovarian carcinoma. Since adenoviral-mediated MDA-7 gene therapy has been shown to be well tolerated and showed biological activity in clinical studies in the context of other carcinomas we assessed the anticancer effects of Ad.mda-7 and in combination with cis-platinum on ovarian cancer cells. Our results show that the purified recombinant MDA-7 protein, GST-MDA-7, and Ad.mda7 virus (5) induced growth arresst and apoptosis in ovarian cancer cells. However, the apoptosis induction was low and directly correlated with infectivity of Ad.mda-7 virus (5). The use of a modified Ad.mda-7 virus type5, Ad.mda-7 virus type(5/3), inhanced infectivity and significantly enhanced ovarian cancer cell killing in human ovarian cancer cell lines in vitro compared to unmodified Ad.mda-7 virus, Ad.mda-7 virus type5. Also Ad-mda7 synergizes with cis-platinum in vitro and enhances ovarian cancer cell death. Taken together, these findings demonstrate that MDA-7 is capable of promoting growth suppression and inducing cell death in ovarian cancer cells, at least OVCAR cells and support the pharmacological interest of the combination of MDA-7 and cis-platinum.

Melanoma differentiation associated gene-7 (mda-7) was discovered in the Fisher laboratory by subtraction hybridization of temporally spaced subtracted cDNA libraries prepared from terminally differentiated human melanoma cells treated with human fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ), an approach called ‘differentiation induction subtraction hybridization’ (DISH). mda-7 is located in human chromosome 1q32–33 and based on sequence homology, chromosomal localization, and its functional properties, the mda-7 gene is now classified as a member of the IL-10 family of cytokines and named IL-24. The mda-7/IL-24 cDNA encodes a protein of 206-amino acids with a predicted size of ~24-kDa, which contains an interleukin (IL)-10 signature motif at amino acids 101–121 (SDAESCYLVHTLLEFYLKTVF) shared by other members of the IL-10 family of cytokines. Sequence analysis revealed the presence of a 49-amino acid signal peptide suggesting that the molecule could be cleaved and secreted. Expression of MDA-7/IL-24 protein was detected in cells of the immune system (mainly by expression in tissues associated with the immune system, such as spleen, thymus and PBMC) and normal human melanocytes. Of interest, a progressive loss of MDA-7/IL-24 expression during melanoma progression suggests an inverse relationship between MDA-7/IL-24 expression and the evolution of melanocytes to various stages of melanoma. mda-7/IL-24 induces growth suppression in human melanoma and other cancer cells, without affecting normal cells. Subsequent studies provided consistent evidence that ectopic expression of mda-7/IL-24 employing a replication incompetent adenovirus (Ad.mda-7) resulted in apoptosis induction and cell death in a wide variety of solid tumors including melanoma, malignant glioma, carcinomas of the breast, kidney, cervix, colorectum , liver, lung, ovary and prostate sparing normal cellular counterparts, i.e., such as normal melanocytes, astrocytes, fibroblasts, and mesothelial and epithelial cells. The in vitro antitumor activity of mda-7/IL-24 readily translated into the in vivo situation in animal models containing human breast, prostate, lung and colorectal carcinomas and in malignant glioma xenografts. Moreover, the ability of mda-7/IL-24 to induce a potent “bystander cancer-specific killing effect” provides an unprecedented opportunity to use this molecule to target for destruction not only primary tumors, but also metastases. Based on its profound cancer-selective tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a Phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant (44%) clinical activity. This review focuses on the recent enhancements in our understanding of the mode of action of mda-7/IL-24 and its potential applications as a unique and promising effective cytokine-based gene therapy for human cancers. The first chapter explored the efficacy of a tropism-modified Ad-based cancer gene therapy approach for eradicating low CAR colorectal cancer cells. We show that in low CAR human colorectal cancer cells (RKO), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficient than Ad.5 delivering mda-7 (Ad.5-mda-7) in expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis and inhibiting in vivo tumor growth in a nude mouse xenograft model. Additionally, our in vitro and in vivo data confirms that BI-97C1 (Sabutoclax) profoundly sensitizes mda-7/IL-24 mediated toxicity in colorectal cancer. Thus, Ad.5/3-mda-7, alone and/or in combination with BI-97C1 (Sabutoclax), might represent an improved and more effective therapeutic approach for colorectal and other cancers. In view of the essential roles of anti-apoptotic Bcl-2 family proteins in tumorigenesis and chemoresistance, efforts are focused on developing small molecule inhibitors of Bcl-2 family proteins as potential therapeutics for cancer. Unfortunately, due to the unique structure of Mcl-1 as compared with Bcl-2 and Bcl-xL, currently employed inhibitors, such as ABT-737 or its clinical counterpart, ABT-263, display limited affinity for Mcl-1. Using nuclear magnetic resonance (NMR) binding assays and computational docking studies, we have recently identified a series of new Apogossypol derivatives, compound 3 (BI-79D10) and compound 11 (BI-97C1), with pan-Bcl-2- inhibitory potency. BI-79D10 binds to Bcl- xL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively. BI-97C1 (Sabutoclax) is an optically pure individual Apogossypol derivative that retains all the properties of BI-79D10 along with superior in vitro and in vivo efficacy. Because Mcl-1 is over-expressed in the majority of PCs, we hypothesized that suppressing Mcl-1 by treating human PC cells with BI-97C1 (Sabutoclax) would sensitize them to mda-7/IL-24-mediated cytotoxicity. The second chapter study highlights the noteworthy potential of a combinatorial approach involving mda-7/IL-24, a broad-acting anticancer gene, and BI-97C1 (Sabutoclax), which targets Mcl-1, to sensitize PC to mda-7/IL-24-mediated cytotoxicity, thereby enhancing therapeutic efficacy. Our data suggests that treatment with the combination regimen of mda-7/IL-24 and BI-97C1 (Sabutoclax) induces autophagy that facilitates apoptosis in association with up regulation of NOXA, accumulation of Bim, and activation of Bax and Bak. Treatment with mda-7/IL-24 and BI-97C1 (Sabutoclax) inhibited the growth of PC xenografts and suppressed PC development in an immunocompetent transgenic mouse model of PC. The third chapter study explored the efficacy of a tropism-modified CRCA cancer gene therapy approach for eradicating low CAR prostate cancer cells. We showed that in low CAR PC3 cells Ad.5/3-CTV is more efficient than Ad.5-CTV in delivering transgene (mda-7/IL-24), infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor ‘bystander’ effect in a nude mouse human prostate cancer xenograft and suppressed PC development in an immunocompetent transgenic mouse model of PC model.

Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.