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Metastasis is the primary determinant of death in patients with diverse solid tumors and MDA-9/Syntenin (SDCBP), a pro-metastatic and pro-angiogenic gene, contributes to this process. Recently, we documented that by physically interacting with IGF-1R, MDA-9/Syntenin activates STAT3 and regulates prostate cancer pathogenesis. These observations firmly established MDA-9/Syntenin as a potential molecular target in prostate cancer. MDA-9/Syntenin contains two highly homologous PDZ domains predicted to interact with a plethora of proteins, many of which are central to the cancerous process. An MDA-9/Syntenin PDZ1 domain-targeted small molecule (PDZ1i) was previously developed using fragment-based drug discovery (FBDD) guided by NMR spectroscopy and was found to be well-tolerated in vivo, had significant half-life (t1/2 = 9 hours) and displayed substantial anti-prostate cancer preclinical in vivo activity. PDZ1i blocked tumor cell invasion and migration in vitro, and metastasis in vivo. Hence, we demonstrate that PDZ1i an MDA-9/Syntenin PDZ1 target-specific small-molecule inhibitor displays therapeutic potential for prostate and potentially other cancers expressing elevated levels of MDA-9/Syntenin.

Prostate cancer is a principal cause of cancer-associated morbidity in men. Although 5-year survival of patients with localized prostate cancer approaches 100%, survival decreases precipitously after metastasis. Bone is the preferred site for disseminated prostate cancer cell colonization, altering the equilibrium of bone homeostasis resulting in weak and fragile bones. Currently, no curative options are available for prostate cancer bone metastasis. Melanoma differentiation associated gene-7 (MDA-7)/IL24 is a well-studied cytokine established as a therapeutic in a wide array of cancers upon delivery as a gene therapy. In this study, we explored the potential anticancer properties of MDA-7/IL24 delivered as a recombinant protein. Using bone metastasis experimental models, animals treated with recombinant MDA-7/IL24 had significantly less metastatic lesions in their femurs as compared with controls. The inhibitory effects of MDA-7/IL24 on bone metastasis resulted from prostate cancer–selective killing and inhibition of osteoclast differentiation, which is necessary for bone resorption. Gain- and loss-of-function genetic approaches document that prosurvival Akt and Mcl-1 pathways are critically important in the antibone metastatic activity of MDA-7/IL24. Our previous findings showed that MDA-7/IL24 gene therapy plus Mcl-1 inhibitors cooperate synergistically. Similarly, an Mcl-1 small-molecule inhibitor synergized with MDA-7/IL24 and induced robust antibone metastatic activity. These results expand the potential applications of MDA-7/IL24 as an anticancer molecule and demonstrate that purified recombinant protein is nontoxic in preclinical animal models and has profound inhibitory effects on bone metastasis, which can be enhanced further when combined with an Mcl-1 inhibitory small molecule. Mol Cancer Ther; 17(9); 1951–60. ©2018 AACR.

PDZ1i does not affect the IL-6 mediated IGF-1R/STAT3 signaling axis.

Supplementary Information and figure legends.

Supplementary Figure 1: Preliminary study defined optimum dose of MDA-7/IL-24 and the number of injections to achieve a maximum effect with or without BI-97D6 (1.5 mg/kg). Supplementary Figure 2: Effect of MDA-7/IL-24 on normal primary mouse bone marrow cells. Supplementary Figure 3: Effect of His-MDA-7 on osteoclast differentiation. Supplementary Figure 4: Effect of MDA-7/IL-24 on the osteoclastic gene regulation pattern and growth of RAW 264.7 and DU-145 cells. Supplementary Figure 5: Quantification of the Western Blots shown in figure 3 using image J software (n=3). Supplementary Figure 6: Effect of Mcl-1 inhibitors on osteoclast differentiation and effect of MDA-7/IL-24 on the osteoclast differentiation process. Supplementary Figure 7: Effect of Akt and Mcl-1 expression on osteoclast differentiation. Supplementary Figure 8: Western blots showing the downstream signaling cascades in Akt overexpressing stable PC3-ML cells. Clone 1 showed significant up regulation in p-Akt, p-GSK3β, and cyclin-D1 expression. Supplementary Figure 9: Mice received intra-cardiac injection of PC3-ML luc cells (1 X105) and were treated with recombinant MDA-7 protein (5mg/kg) for 6 doses as described in materials and methods. Mice were imaged by IVIS imaging system.

Antigen-specific immunotherapy can be limited by induced tumor immunoediting (e.g., antigen loss) or through failure to recognize antigen-negative tumor clones. Melanoma differentiation–associated gene-7/IL24 (MDA-7/IL24) has profound tumor-specific cytotoxic effects in a broad spectrum of cancers. Here we report the enhanced therapeutic impact of genetically engineering mouse tumor-reactive or antigen-specific T cells to produce human MDA-7/IL24. While mock-transduced T cells only killed antigen-expressing tumor cells, MDA-7/IL24-producing T cells destroyed both antigen-positive and negative cancer targets. MDA-7/IL24-expressing T cells were superior to their mock-engineered counterparts in suppressing mouse prostate cancer and melanoma growth as well as metastasis. This enhanced antitumor potency correlated with increased tumor infiltration and expansion of antigen-specific T cells as well as induction of a Th1-skewed immunostimulatory tumor environment. MDA-7/IL24-potentiated T-cell expansion was dependent on T-cell–intrinsic STAT3 signaling. Finally, MDA-7/IL24-modified T-cell therapy significantly inhibited progression of spontaneous prostate cancers in Hi-Myc transgenic mice. Taken together, arming T cells with tumoricidal and immune-potentiating MDA-7/IL24 confers new capabilities of eradicating antigen-negative cancer cell clones and improving T-cell expansion within tumors. This promising approach may be used to optimize cellular immunotherapy for treating heterogeneous solid cancers and provides a mechanism for inhibiting tumor escape.Significance:This research describes a novel strategy to overcome the antigenic heterogeneity of solid cancers and prevent tumor escape by engineering T lymphocytes to produce a broad-spectrum tumoricidal agent.

Although prostate cancer is clinically manageable during several stages of progression, survival is severely compromised once cells invade and metastasize to distant organs. Comprehending the pathobiology of invasion is required for developing efficacious targeted therapies against metastasis. Based on bioinformatics data, we predicted an association of melanoma differentiation-associated gene-9 [syntenin, or syndecan binding protein (SDCBP)] in prostate cancer progression. Using tissue samples from various Gleason stage prostate cancer patients with adjacent normal tissue, a series of normal prostate and prostate cancer cell lines (with differing tumorigenic/metastatic properties), mda-9/syntenin-manipulated variants (including loss-of-function and gain-of-function cell lines), and CRISPR/Cas9 stable MDA-9/Syntenin knockout cells, we now confirm the relevance of and dependence on MDA-9/syntenin in prostate cancer invasion. MDA-9/Syntenin physically interacted with insulin-like growth factor-1 receptor following treatment with insulin-like growth factor binding protein-2 (IGFBP2), regulating downstream signaling processes that enabled STAT3 phosphorylation. This activation enhanced expression of MMP2 and MMP9, two established enzymes that positively regulate invasion. In addition, MDA-9/syntenin-mediated upregulation of proangiogenic factors including IGFBP2, IL6, IL8, and VEGFA also facilitated migration of prostate cancer cells. Collectively, our results draw attention to MDA-9/Syntenin as a positive regulator of prostate cancer metastasis, and the potential application of targeting this molecule to inhibit invasion and metastasis in prostate cancer and potentially other cancers.Significance: This study provides new mechanistic insight into the proinvasive role of MDA-9/Syntenin in prostate cancer and has potential for therapeutic application to prevent prostate cancer metastasis. Cancer Res; 78(11); 2852–63. ©2018 AACR.

Supplementary Figure 1: miRNA expression in MDA-MB-231 cells infected with either Ad.null or Ad.mda-7. Supplementary Figure 2: miR 221 expression in normal cell lines Supplementary Figure 3: miR 221 expression and ROS production following treatment with ROS inducers. Supplementary Figure 4: Beclin-1 expression in miR-221 or anti-miR-221 treated cells. Supplementary Figure 5: Beclin-1, p27, and PUMA expression in anti-miR-221 treated cells. Supplementary Figure 6: Beclin-1 expression in Rapamycin and /or miR-221 treated cells. Supplementary Figure 7: Quantification of MDA-7/IL-24 and Beclin-1 in immunohistochemistry images. Supplementary Figure 8: Immunohistochemical analysis of p27 and PUMA in tumor sections.

MDA-9/Syntenin activates IGF-1R

Supplementary Figure S8. Upregulation of endogenous MDA-7 in mouse cancer cells is not caused by human MDA-7 produced by engineered T cells.

Melanoma differentiation-associated gene-7/IL-24 (mda-7/IL-24) displays broad-spectrum anticancer activity in vitro, in vivo in preclinical animal models, and in a phase I/II clinical trial in patients with advanced cancers without harming normal cells or tissues. Here we demonstrate that mda-7/IL-24 regulates a specific subset of miRNAs, including cancer-associated miR-221. Either ectopic expression of mda-7/IL-24 or treatment with recombinant His-MDA-7 protein resulted in downregulation of miR-221 and upregulation of p27 and PUMA in a panel of cancer cells, culminating in cell death. Mda-7/IL-24–induced cancer cell death was dependent on reactive oxygen species induction and was rescued by overexpression of miR-221. Beclin-1 was identified as a new transcriptional target of miR-221, and mda-7/IL-24 regulated autophagy through a miR-221/beclin-1 feedback loop. In a human breast cancer xenograft model, miR-221–overexpressing MDA-MB-231 clones were more aggressive and resistant to mda-7/IL-24–mediated cell death than parental clones. This is the first demonstration that mda-7/IL-24 directly regulates miRNA expression in cancer cells and highlights the novelty of the mda-7/IL-24–miR-221–beclin-1 loop in mediating cancer cell–specific death. Cancer Res; 77(4); 949–59. ©2016 AACR.

Figure legends for supplementary figures 1 to 8

MDA-9/Syntenin mediated effects are rescued by STAT3.

Supplemental figure legend for figure S1 through S8

Supplementary Figure 1. Expression of adenoviral receptors in neuroblastoma cells. Supplementary Figure 2. Ad.5/3-CTV induces pro-apoptotic molecules and downregulates anti-apoptotic molecules. Supplementary Figure 3. Ad.5/3-CTV induces caspase-independent cell death in neuroblastoma cells. Supplementary Figure 4. Ad.5/3-CTV enhances p53 levels in neuroblastoma cells. Supplementary Figure 5. Nuclear translocation of γâ^'H2AX in Ad.5/3-CTV-treated neuroblastoma cells. Supplementary Figure 6. Ad.5/3-CTV infection in neuroblastoma cells promotes nuclear translocation of pATM. Supplementary Figure 7. ATM inhibitor rescues neuroblastoma cells from Ad.5/3-CTV-induced cell death. Supplementary Figure 8. Effect of Ad.5/3-CTV on MDA-7/IL-24 and AIF expression in breast cancer and melanoma cells.

Metastasis poses significant obstacles to effective therapy and drug development. Melanoma differentiation-associated gene-9/Syntenin-1/Syndecan Binding Protein (MDA-9) is a pro-metastatic gene robustly and differentially expressed in cancer cells in comparison with corresponding adjacent normal/primary cells and represents an attractive therapeutic target to inhibit cancer cell dissemination and tumor growth. MDA-9 contains two tandem PDZ domains that provide a nexus for interactions with multiple proteins that can initiate the transcription of metastasis-associated genes. The goal of this project was to evaluate the efficiency of a unique first-in-class, second-generation MDA-9 dual-PDZ targeted pharmacological inhibitor generated using a combination of a PDZ1 small molecule and a PDZ2 selective peptide. Fragment-based drug discovery and NMR identified PDZ1i, an inhibitor of the PDZ1 domain, that effectively blocks cancer invasion in vitro and in vivo in multiple experimental animal models. To maximize the disruption of MDA-9 signaling an inhibitor has now been developed that simultaneously binds and blocks the activity of both PDZ domains. PDZ1i was joined to the second PDZ binding peptide (TNYYFV) with a PEG linker, resulting in IVMT-Rx-3 that simultaneously engages both PDZ domains of MDA-9. Mechanistic studies involved a combination of molecular and cell biology approaches and included both established and early passaged patient-derived melanoma cell lines. To test the efficacy of potential combinatorial effects in vivo we used a murine syngeneic model and B16F10, an immunotherapy-resistant murine cell line. After confirming synthesis using multiple analytical methods, e.g., HPLC, LC/MS, and NMR, the dissociation constant (determined by microscale thermophoresis (MST) analysis, of the IVMT-Rx-3 recombinant MDA-9 complex was 63 +/- 11 μM. This compound is not toxic to normal melanocytes or melanoma cells, but significantly inhibits invasion in vitro in a wide range of melanomas. IVMT-Rx-3 interrupts c-Src binding to MDA-9 and downregulates NF-kB activation. Inactivation of this transcription factor causes suppression of MMP2/MMP9 and inhibits the secretion of cytokines/growth factors. These molecular changes block cellular invasiveness and facilitate immune cytotoxicity by converting an unfavorable into a favorable tumor microenvironment culminating in the repression of melanoma metastasis. In addition, the in vivo anti-metastatic properties of IVMT-Rx-3 are enhanced when combined with an immune-checkpoint inhibitor. IVMT-Rx-3 is a unique MDA-9 antagonist in terms of composition that effectively inhibits melanoma metastasis as does our small molecule PDZ1i and holds potential for developing novel therapeutic strategies effectively targeting melanoma and in principle, a broad spectrum of human cancers that also overexpress MDA-9. Citation Format: Anjan K. Pradhan, Jinkal Modi, Santanu Maji, Amit Kumar, Praveen Bhoopathi, Padmanabhan Mannangatti, Chunqing Guo, Daniel K. Afosah, Mark Mochel, Nitai D. Mukhopadhyay, John M. Kirkwood, Xiang-Yang Wang, Umesh R. Desai, Devanand Sarkar, Luni Emdad, Swadesh K. Das, Paul B. Fisher. Simultaneous targeting of the PDZ1 and PDZ2 domains of MDA-9 inhibits melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3394.

Apoptosis and autophagy play seminal roles in maintaining organ homeostasis. Apoptosis represents canonical type I programmed cell death. Autophagy is viewed as pro-survival, however, excessive autophagy can promote type II cell death. Defective regulation of these two obligatory cellular pathways is linked to various diseases, including cancer. Biologic or chemotherapeutic agents, which can reprogram cancer cells to undergo apoptosis- or toxic autophagy-mediated cell death, are considered effective tools for treating cancer. Melanoma differentiation associated gene-7 (mda-7) selectively promotes these effects in cancer cells. mda-7 was identified more than two decades ago by subtraction hybridization showing elevated expression during induction of terminal differentiation of metastatic melanoma cells following treatment with recombinant fibroblast interferon and mezerein (a PKC activating agent). MDA-7 was classified as a member of the IL-10 gene family based on its chromosomal location, and the presence of an IL-10 signature motif and a secretory sequence, and re-named interleukin-24 (MDA-7/IL-24). Multiple studies have established MDA-7/IL-24 as a potent anti-cancer agent, which when administered at supra-physiological levels induces growth arrest and cell death through apoptosis and toxic autophagy in a wide variety of tumor cell types, but not in corresponding normal/non-transformed cells. Furthermore, in a phase I/II clinical trial, MDA-7/IL-24 administered by means of a non-replicating adenovirus was well tolerated and displayed significant clinical activity in patients with multiple advanced cancers. This review examines our current comprehension of the role of MDA-7/IL-24 in mediating cancer-specific cell death via apoptosis and toxic autophagy.

Metastasis is the primary determinant of death in patients with diverse solid tumors and MDA-9/Syntenin (SDCBP), a pro-metastatic and pro-angiogenic gene, contributes to this process. Recently, we documented that by physically interacting with IGF-1R, MDA-9/Syntenin activates STAT3 and regulates prostate cancer pathogenesis. These observations firmly established MDA-9/Syntenin as a potential molecular target in prostate cancer. MDA-9/Syntenin contains two highly homologous PDZ domains predicted to interact with a plethora of proteins, many of which are central to the cancerous process. An MDA-9/Syntenin PDZ1 domain-targeted small molecule (PDZ1i) was previously developed using fragment-based drug discovery (FBDD) guided by NMR spectroscopy and was found to be well-tolerated in vivo, had significant half-life (t1/2 = 9 hours) and displayed substantial anti-prostate cancer preclinical in vivo activity. PDZ1i blocked tumor cell invasion and migration in vitro, and metastasis in vivo. Hence, we demonstrate that PDZ1i an MDA-9/Syntenin PDZ1 target-specific small-molecule inhibitor displays therapeutic potential for prostate and potentially other cancers expressing elevated levels of MDA-9/Syntenin.

melanoma differentiation associated gene-7 or Interleukin-24 (mda-7, IL-24) displays expansive anti-tumor activity without harming corresponding normal cells/tissues. This anticancer activity has been documented in vitro and in vivo in multiple preclinical animal models, as well as in patients with advanced cancers in a phase I clinical trial. To enhance the therapeutic efficacy of MDA-7 (IL-24), we engineered a designer cytokine (a “Superkine”; IL-24S; referred to as M7S) with enhanced secretion and increased stability to engender improved “bystander” antitumor effects. M7S was engineered in a two-step process by first replacing the endogenous secretory motif with an alternate secretory motif to boost secretion. Among four different signaling peptides, the insulin secretory motif significantly enhanced the secretion of MDA-7 (IL-24) protein and was chosen for M7S. The second modification engineered in M7S was designed to enhance the stability of MDA-7 (IL-24), which was accomplished by replacing lysine at position K122 with arginine. This engineered “M7S Superkine” with increased secretion and stability retained cancer specificity. Compared to parental MDA-7 (IL-24), M7S (IL-24S) was superior in promoting anti-tumor and bystander effects leading to improved outcomes in multiple cancer xenograft models. Additionally, combinatorial therapy using MDA-7 (IL-24) or M7S (IL-24S) with an immune checkpoint inhibitor, anti-PD-L1, dramatically reduced tumor progression in murine B16 melanoma cells. These results portend that M7S (IL-24S) promotes the re-emergence of an immunosuppressive tumor microenvironment, providing a solid rationale for prospective translational applications of this therapeutic designer cytokine.

Background Aloe vera extract and its bioactive compounds possess anti-proliferative properties against cancer cells. However, no detailed molecular mechanism of action studies has been reported. We have now employed a computational approach to scrutinize the molecular mechanism of lead bioactive compounds from Aloe vera that potentially inhibit DNA synthesis. Methods Initially, the anti-proliferative activity of Aloe vera extract was examined in human breast cancer cells (in vitro/in vivo). Later on, computational screening of bioactive compounds from Aloe vera targeting DNA was performed by molecular docking and molecular dynamics simulation. Results In-vitro and in-vivo studies confirm that Aloe vera extract effectively suppresses the growth of breast cancer cells without significant cytotoxicity towards non-cancerous normal immortal cells. Computational screening predicts that growth suppression may be due to the presence of DNA intercalating bioactive compounds (riboflavin, daidzin, aloin, etc.) contained in Aloe vera. MM/PBSA calculation showed that riboflavin has a higher binding affinity at the DNA binding sites compared to standard drug daunorubicin. Conclusions These observations support the hypothesis that riboflavin may be exploited as an anti-proliferative DNA intercalating agent to prevent cancer and is worthy of testing for the management of cancer by performing more extensive pre-clinical and if validated clinical trials.

Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1β pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.

Simple Summary An optimum cancer therapeutic virus should embody unique properties, including an ability to: Selectively procreate and kill tumor but not normal cells; produce a secreted therapeutic molecule (with broad-acting anti-cancer effects on primary and distant metastatic cells because of potent “bystander” activity); and monitor therapy non-invasively by imaging primary and distant metastatic cancers. We previously created a broad-spectrum, cancer-selective and replication competent therapeutic adenovirus that embodies two of these properties, i.e., specifically reproduces in cancer cells and produces a therapeutic cytokine, MDA-7/IL-24, a “cancer terminator virus” (CTV). We now expand on this concept and demonstrate the feasibility of producing a tripartite CTV (TCTV) selectively expressing three genes from three distinct promoters that replicate in the cancer cells while producing MDA-7/IL-24 and an imaging gene (i.e., luciferase). This novel first-in-class tripartite “theranostic” TCTV expands the utility of therapeutic viruses to non-invasively image and selectively destroy primary tumors and metastases. Abstract Combining cancer-selective viral replication and simultaneous production of a therapeutic cytokine, with potent “bystander” anti-tumor activity, are hallmarks of the cancer terminator virus (CTV). To expand on these attributes, we designed a next generation CTV that additionally enables simultaneous non-invasive imaging of tumors targeted for eradication. A unique tripartite CTV “theranostic” adenovirus (TCTV) has now been created that employs three distinct promoters to target virus replication, cytokine production and imaging capabilities uniquely in cancer cells. Conditional replication of the TCTV is regulated by a cancer-selective (truncated PEG-3) promoter, the therapeutic component, MDA-7/IL-24, is under a ubiquitous (CMV) promoter, and finally the imaging capabilities are synchronized through another cancer selective (truncated tCCN1) promoter. Using in vitro studies and clinically relevant in vivo models of breast and prostate cancer, we demonstrate that incorporating a reporter gene for imaging does not compromise the exceptional therapeutic efficacy of our previously reported bipartite CTV. This TCTV permits targeted treatment of tumors while monitoring tumor regression, with potential to simultaneously detect metastasis due to the cancer-selective activity of reporter gene expression. This “theranostic” virus provides a new genetic tool for distinguishing and treating localized and metastatic cancers.

Progression-elevated gene-3 (PEG-3) and rat growth arrest and DNA damage-inducible gene-34 (GADD34) display significant sequence homology with regulation predominantly transcriptional. The rat full-length (FL) and minimal (min) PEG-3 promoter display cancer-selective expression in rodent and human tumors, allowing for cancer-directed regulation of transgenes, viral replication and in vivo imaging of tumors and metastases in animals, whereas the FL- and min-GADD34-Prom lack cancer specificity. Min-PEG-Prom and min-GADD34-Prom have identical sequences except for two single-point mutation differences (at −260 bp and +159 bp). Engineering double mutations in the min-GADD34-Prom produce the GAPE-Prom. Changing one base pair (+159) or both point mutations in the min-GADD34-Prom, but not the FL-GADD34-Prom, results in cancer-selective transgene expression in diverse cancer cells (including prostate, breast, pancreatic and neuroblastoma) vs. normal counterparts. Additionally, we identified a GATA2 transcription factor binding site, promoting cancer specificity when both min-PEG-Prom mutations are present in the GAPE-Prom. Taken together, introducing specific point mutations in a rat min-GADD34-Prom converts this non-cancer-specific promoter into a cancer-selective promoter, and the addition of GATA2 with existing AP1 and PEA3 transcription factors enhances further cancer-selective activity of the GAPE-Prom. The GAPE-Prom provides a genetic tool to specifically regulate transgene expression in cancer cells.

Antigen-specific immunotherapy can be limited by induced tumor immunoediting (e.g., antigen loss) or through failure to recognize antigen-negative tumor clones. Melanoma differentiation–associated gene-7/IL24 (MDA-7/IL24) has profound tumor-specific cytotoxic effects in a broad spectrum of cancers. Here we report the enhanced therapeutic impact of genetically engineering mouse tumor-reactive or antigen-specific T cells to produce human MDA-7/IL24. While mock-transduced T cells only killed antigen-expressing tumor cells, MDA-7/IL24-producing T cells destroyed both antigen-positive and negative cancer targets. MDA-7/IL24-expressing T cells were superior to their mock-engineered counterparts in suppressing mouse prostate cancer and melanoma growth as well as metastasis. This enhanced antitumor potency correlated with increased tumor infiltration and expansion of antigen-specific T cells as well as induction of a Th1-skewed immunostimulatory tumor environment. MDA-7/IL24-potentiated T-cell expansion was dependent on T-cell–intrinsic STAT3 signaling. Finally, MDA-7/IL24-modified T-cell therapy significantly inhibited progression of spontaneous prostate cancers in Hi-Myc transgenic mice. Taken together, arming T cells with tumoricidal and immune-potentiating MDA-7/IL24 confers new capabilities of eradicating antigen-negative cancer cell clones and improving T-cell expansion within tumors. This promising approach may be used to optimize cellular immunotherapy for treating heterogeneous solid cancers and provides a mechanism for inhibiting tumor escape. Significance: This research describes a novel strategy to overcome the antigenic heterogeneity of solid cancers and prevent tumor escape by engineering T lymphocytes to produce a broad-spectrum tumoricidal agent.

Tumor metastasis comprises a series of coordinated events that culminate in dissemination of cancer cells to distant sites within the body representing the greatest challenge impeding effective therapy of cancer and the leading cause of cancer-associated morbidity. Cancer cells exploit multiple genes and pathways to colonize to distant organs. These pathways are integrated and regulated at different levels by cellular- and extracellular-associated factors. Defining the genes and pathways that govern metastasis can provide new targets for therapeutic intervention. Melanoma differentiation associated gene-9 (mda-9) (also known as Syntenin-1 and SDCBP (Syndecan binding protein)) was identified by subtraction hybridization as a novel gene displaying differential temporal expression during differentiation of melanoma. MDA-9/Syntenin is an established Syndecan binding protein that functions as an adaptor protein. Expression of MDA-9/Syntenin is elevated at an RNA and protein level in a wide-range of cancers including melanoma, glioblastoma, neuroblastoma, and prostate, breast and liver cancer. Expression is increased significantly in metastatic cancer cells as compared with non-metastatic cancer cells or normal cells, which make it an attractive target in treating cancer metastasis. In this review, we focus on the role and regulation of mda-9 in cancer progression and metastasis.

The primary cause of cancer-related death from solid tumors is metastasis. While unraveling the mechanisms of this complicated process continues, our ability to effectively target and treat it to decrease patient morbidity and mortality remains disappointing. Early detection of metastatic lesions and approaches to treat metastases (both pharmacological and genetic) are of prime importance to obstruct this process clinically. Metastasis is complex involving both genetic and epigenetic changes in the constantly evolving tumor cell. Moreover, many discrete steps have been identified in metastatic spread, including invasion, intravasation, angiogenesis, attachment at a distant site (secondary seeding), extravasation and micrometastasis and tumor dormancy development. Here, we provide an overview of the metastatic process and highlight a unique pro-metastatic gene, melanoma differentiation associated gene-9/Syntenin (MDA-9/Syntenin) also called syndecan binding protein (SDCBP), which is a major contributor to the majority of independent metastatic events. MDA-9 expression is elevated in a wide range of carcinomas and other cancers, including melanoma, glioblastoma multiforme and neuroblastoma, suggesting that it may provide an appropriate target to intervene in metastasis. Pre-clinical studies confirm that inhibiting MDA-9 either genetically or pharmacologically profoundly suppresses metastasis. An additional benefit to blocking MDA-9 in metastatic cells is sensitization of these cells to a second therapeutic agent, which converts anti-invasion effects to tumor cytocidal effects. Continued mechanistic and therapeutic insights hold promise to advance development of truly effective therapies for metastasis in the future.

Despite some progress, treating advanced prostate cancer remains a major clinical challenge. Recent studies have shown that prostate cancer can originate from undifferentiated, rare, stem cell-like populations within the heterogeneous tumor mass, which play seminal roles in tumor formation, maintenance of tumor homeostasis and initiation of metastases. These cells possess enhanced propensity toward chemoresistance and may serve as a prognostic factor for prostate cancer recurrence. Despite extensive studies, selective targeted therapies against these stem cell-like populations are limited and more detailed experiments are required to develop novel targeted therapeutics. We now show that MDA-9/Syntenin/SDCBP (MDA-9) is a critical regulator of survival, stemness and chemoresistance in prostate cancer stem cells (PCSCs). MDA-9 regulates the expression of multiple stem-regulatory genes and loss of MDA-9 causes a complete collapse of the stem-regulatory network in PCSCs. Loss of MDA-9 also sensitizes PCSCs to multiple chemotherapeutics with different modes of action, such as docetaxel and trichostatin-A, suggesting that MDA-9 may regulate multiple drug resistance. Mechanistically, MDA-9-mediated multiple drug resistance, stemness and survival are regulated in PCSCs through activation of STAT3. Activated STAT3 regulates chemoresistance in PCSCs through protective autophagy as well as regulation of MDR1 on the surface of the PCSCs. We now demonstrate that MDA-9 is a critical regulator of PCSC survival and stemness via exploiting the inter-connected STAT3 and c-myc pathways.

Although prostate cancer is clinically manageable during several stages of progression, survival is severely compromised once cells invade and metastasize to distant organs. Comprehending the pathobiology of invasion is required for developing efficacious targeted therapies against metastasis. Based on bioinformatics data, we predicted an association of melanoma differentiation-associated gene-9 [syntenin, or syndecan binding protein (SDCBP)] in prostate cancer progression. Using tissue samples from various Gleason stage prostate cancer patients with adjacent normal tissue, a series of normal prostate and prostate cancer cell lines (with differing tumorigenic/metastatic properties), mda-9/syntenin-manipulated variants (including loss-of-function and gain-of-function cell lines), and CRISPR/Cas9 stable MDA-9/Syntenin knockout cells, we now confirm the relevance of and dependence on MDA-9/syntenin in prostate cancer invasion. MDA-9/Syntenin physically interacted with insulin-like growth factor-1 receptor following treatment with insulin-like growth factor binding protein-2 (IGFBP2), regulating downstream signaling processes that enabled STAT3 phosphorylation. This activation enhanced expression of MMP2 and MMP9, two established enzymes that positively regulate invasion. In addition, MDA-9/syntenin-mediated upregulation of proangiogenic factors including IGFBP2, IL6, IL8, and VEGFA also facilitated migration of prostate cancer cells. Collectively, our results draw attention to MDA-9/Syntenin as a positive regulator of prostate cancer metastasis, and the potential application of targeting this molecule to inhibit invasion and metastasis in prostate cancer and potentially other cancers. Significance: This study provides new mechanistic insight into the proinvasive role of MDA-9/Syntenin in prostate cancer and has potential for therapeutic application to prevent prostate cancer metastasis. Cancer Res; 78(11); 2852–63. ©2018 AACR.

Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active β-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation–associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical β-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/β-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/β-catenin signaling, which may offer prognostic and therapeutic opportunities.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a multifunctional cytokine displaying broad-spectrum anticancer activity in vitro or in vivo in preclinical animal cancer models and in a phase 1/2 clinical trial in patients with advanced cancers. mda-7/IL-24 targets specific miRNAs, including miR-221 and miR-320, for down-regulation in a cancer-selective manner. We demonstrate that mda-7/IL-24, administered through a replication incompetent type 5 adenovirus (Ad.mda-7) or with His-MDA-7/IL-24 protein, down-regulates DICER, a critical regulator in miRNA processing. This effect is specific for mature miR-221, as it does not affect Pri-miR-221 expression, and the DICER protein, as no changes occur in other miRNA processing cofactors, including DROSHA, PASHA, or Argonaute. DICER is unchanged by Ad.mda-7/IL-24 in normal immortal prostate cells, whereas Ad.mda-7 down-regulates DICER in multiple cancer cells including glioblastoma multiforme and prostate, breast, lung, and liver carcinoma cells. MDA-7/IL-24 protein down-regulates DICER expression through canonical IL-20/IL-22 receptors. Gain- and loss-of-function studies confirm that overexpression of DICER rescues deregulation of miRNAs by mda-7/IL-24, partially rescuing cancer cells from mda-7/IL-24-mediated cell death. Stable overexpression of DICER in cancer cells impedes Ad.mda-7 or His-MDA-7/IL-24 inhibition of cell growth, colony formation, PARP cleavage, and apoptosis. In addition, stable overexpression of DICER renders cancer cells more resistant to Ad.mda-7 inhibition of primary and secondary tumor growth. MDA-7/IL-24-mediated regulation of DICER is reactive oxygen species-dependent and mediated by melanogenesis-associated transcription factor. Our research uncovers a distinct role of mda-7/IL-24 in the regulation of miRNA biogenesis through alteration of the MITF-DICER pathway.

// Sarmistha Talukdar 1,2 , Swadesh K. Das 1,2,3 , Anjan K. Pradhan 1,2 , Luni Emdad 1,2,3 , Xue-Ning Shen 1,2 , Jolene J. Windle 1,2,3 , Devanand Sarkar 1,2,3 and Paul B. Fisher 1,2,3 1 Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA 2 VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, VA, USA 3 VCU Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA Correspondence to: Paul B. Fisher, email: // Keywords : MDA-9/Syntenin (SDCBP); stemness; glioma stem cells; survival; apoptosis Received : May 31, 2016 Accepted : July 07, 2016 Published : July 26, 2016 Abstract Glioblastoma multiforme (GBM) is an aggressive cancer with current therapies only marginally impacting on patient survival. Glioma stem cells (GSCs), a subpopulation of highly tumorigenic cells, are considered major contributors to glioma progression and play seminal roles in therapy resistance, immune evasion and increased invasion. Despite clinical relevance, effective/selective therapeutic targeting strategies for GSCs do not exist, potentially due to the lack of a definitive understanding of key regulators of GSCs. Consequently, there is a pressing need to identify therapeutic targets and novel options to effectively target this therapy-resistant cell population. The precise roles of GSCs in governing GBM development, progression and prognosis are under intense scrutiny, but key upstream regulatory genes remain speculative. MDA-9/Syntenin (SDCBP), a scaffold protein, regulates tumor pathogenesis in multiple cancers. Highly aggressive cancers like GBM express elevated levels of MDA-9 and contain increased populations of GSCs. We now uncover a unique function of MDA-9 as a facilitator and determinant of glioma stemness and survival. Mechanistically, MDA-9 regulates multiple stemness genes ( Nanog, Oct4 and Sox2 ) through activation of STAT3. MDA-9 controls survival of GSCs by activating the NOTCH1 pathway through phospho-Src and DLL1. Once activated, cleaved NOTCH1 regulates C-Myc expression through RBPJK, thereby facilitating GSC growth and proliferation. Knockdown of MDA-9 affects the NOTCH1/C-Myc and p-STAT3/Nanog pathways causing a loss of stemness and initiation of apoptosis in GSCs. Our data uncover a previously unidentified relationship between MDA-9 and GSCs, reinforcing relevance of this gene as a potential therapeutic target in GBM.

Cancer development and progression to metastasis is a complex process, which largely depends on bidirectional communication between tumor cells and their microenvironment. Melanoma differentiation associated gene-9 (mda-9, also known as Syntenin-1, SDCBP), a gene first cloned by our group, is robustly expressed in multiple cancers including melanoma and contributes to invasion and metastasis in a tumor cell-intrinsic manner. However, the role of MDA-9/Syntenin in the tumor cell-extrinsic microenvironment remains unclear even though MDA-9/Syntenin is ubiquitously expressed in most organs that are active metastatic sites for melanoma, e.g., lung, lymph node, brain, and liver. In this study, we explored the effect of environmental mda-9/syntenin expression on melanoma growth and metastasis using multiple immunocompetent animal models, syngeneic B16 xenograft and intravenous B16 mouse model and a genetically engineered mouse (GEM) model of melanoma. Host-deficient expression of mda-9/syntenin in mice negatively impacted on subcutaneously implanted B16 tumor growth and lung metastasis. Absence of MDA-9/Syntenin in the lung microenvironment suppressed tumor growth by modulating in situ Interleukin 17A (IL17A) expression and impaired the recruitment of myeloid derived suppressor cells (MDSCs) and Th17 cells as compared to genetically wild type animals. Additionally, loss of mda-9/syntenin expression in a spontaneous melanoma model (melanocyte-specific pten loss and BrafV600E mutation) significantly delayed tumor initiation and suppressed metastasis to the lymph nodes and lungs. The present study highlights a novel role of mda-9/syntenin in tumor-promoting inflammation and immune suppression. These observations along with other documented roles of MDA-9/Syntenin in cancer and metastasis support the potential relevance of MDA-9/Syntenin in the carcinogenic process and as a target for developing improved therapies by using either genetic or pharmacologic approaches to treat and prevent melanoma and other cancers.

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in preclinical animal models, and in a phase I clinical trial in patients with advanced cancers without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting endoplasmic reticulum stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival, and angiogenesis. To enhance cancer-selective expression and targeted anticancer activity of mda-7/IL-24, we created a tropism-modified cancer terminator virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24. We now show that Ad.5/3-CTV induces profound neuroblastoma antiproliferative activity and apoptosis in a caspase-3/9–independent manner, both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis-inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV–induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ-H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small-molecule inhibitors that attenuated PARP cleavage by inhibiting γ-H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV–infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24–induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM, and γ-H2AX. Cancer Res; 76(12); 3572–82. ©2016 AACR.

Glioma stem cells (GSCs) comprise a small subpopulation of glioblastoma multiforme cells that contribute to therapy resistance, poor prognosis, and tumor recurrence. Protective autophagy promotes resistance of GSCs to anoikis, a form of programmed cell death occurring when anchorage-dependent cells detach from the extracellular matrix. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of melanoma differentiation associated gene-9/Syntenin (MDA-9) (syndecan binding protein; SDCBP). When MDA-9 is suppressed, GSCs undergo autophagic death supporting the hypothesis that MDA-9 regulates protective autophagy in GSCs under anoikis conditions. MDA-9 maintains protective autophagy through phosphorylation of BCL2 and by suppressing high levels of autophagy through EGFR signaling. MDA-9 promotes these changes by modifying FAK and PKC signaling. Gain-of-function and loss-of-function genetic approaches demonstrate that MDA-9 regulates pEGFR and pBCL2 expression through FAK and pPKC. EGFR signaling inhibits autophagy markers (ATG5, Lamp1, LC3B), helping to maintain protective autophagy, and along with pBCL2 maintain survival of GSCs. In the absence of MDA-9, this protective mechanism is deregulated; EGFR no longer maintains protective autophagy, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. In addition, pBCL2 is down-regulated in the absence of MDA-9, leading to cell death in GSCs under conditions of anoikis. Our studies confirm a functional link between MDA-9 expression and protective autophagy in GSCs and show that inhibition of MDA-9 reverses protective autophagy and induces anoikis and cell death in GSCs.

Subtraction hybridization identified genes displaying differential expression as metastatic human melanoma cells terminally differentiated and lost tumorigenic properties by treatment with recombinant fibroblast interferon and mezerein. This approach permitted cloning of multiple genes displaying enhanced expression when melanoma cells terminally differentiated, called melanoma differentiation associated (mda) genes. One mda gene, mda-7, has risen to the top of the list based on its relevance to cancer and now inflammation and other pathological states, which based on presence of a secretory sequence, chromosomal location, and an IL-10 signature motif has been named interleukin-24 (MDA-7/IL-24). Discovered in the early 1990s, MDA-7/IL-24 has proven to be a potent, near ubiquitous cancer suppressor gene capable of inducing cancer cell death through apoptosis and toxic autophagy in cancer cells in vitro and in preclinical animal models in vivo. In addition, MDA-7/IL-24 embodied profound anticancer activity in a Phase I/II clinical trial following direct injection with an adenovirus (Ad.mda-7; INGN-241) in tumors in patients with advanced cancers. In multiple independent studies, MDA-7/IL-24 has been implicated in many pathological states involving inflammation and may play a role in inflammatory bowel disease, psoriasis, cardiovascular disease, rheumatoid arthritis, tuberculosis, and viral infection. This review provides an up-to-date review on the multifunctional gene mda-7/IL-24, which may hold potential for the therapy of not only cancer, but also other pathological states.

Autophagy is a functionally conserved self-degradation process that facilitates the survival of eukaryotic life via the management of cellular bioenergetics and maintenance of the fidelity of genomic DNA. The first known autophagy inducer was Beclin-1. Beclin-1 is expressed in multicellular eukaryotes ranging throughout plants to animals, comprising a nonmonophyllic group, as shown in this report via aggressive BLAST searches. In humans, Beclin-1 is a haploinsuffient tumor suppressor as biallelic deletions have not been observed in patient tumors clinically. Therefore, Beclin-1 fails the Knudson hypothesis, implicating expression of at least one Beclin-1 allele is essential for cancer cell survival. However, Beclin-1 is frequently monoallelically deleted in advanced human cancers and the expression of two Beclin-1 allelles is associated with greater anticancer effects. Overall, experimental evidence suggests that Beclin-1 inhibits tumor formation, angiogenesis, and metastasis alone and in cooperation with the tumor suppressive molecules UVRAG, Bif-1, Ambra1, and MDA-7/IL-24 via diverse mechanisms of action. Conversely, Beclin-1 is upregulated in cancer stem cells (CSCs), portending a role in cancer recurrence, and highlighting this molecule as an intriguing molecular target for the treatment of CSCs. Many aspects of Beclin-1's biological effects remain to be studied. The consequences of these BLAST searches on the molecular evolution of Beclin-1, and the eukaryotic branches of the tree of life, are discussed here in greater detail with future inquiry focused upon protist taxa. Also in this review, the effects of Beclin-1 on tumor suppression and cancer malignancy are discussed. Beclin-1 holds significant promise for the development of novel targeted cancer therapeutics and is anticipated to lead to a many advances in our understanding of eukaryotic evolution, multicellularity, and even the treatment of CSCs in the coming decades.

Although there is a strong correlation between multinucleated cells (MNCs) and cancer chemo-resistance in variety of cancers, our understanding of how multinucleated cells modulate the tumor micro-environment is limited. We captured multinucleated cells from triple-negative chemo-resistant breast cancers cells in a time frame, where they do not proliferate but rather significantly regulate their micro-environment. We show that oxidatively stressed MNCs induce chemo-resistance in vitro and in vivo by secreting VEGF and MIF. These factors act through the RAS/MAPK pathway to induce chemo-resistance by upregulating anti-apoptotic proteins. In MNCs, elevated reactive oxygen species (ROS) stabilizes HIF-1α contributing to increase production of VEGF and MIF. Together the data indicate, that the ROS-HIF-1α signaling axis is very crucial in regulation of chemo-resistance by MNCs. Targeting ROS-HIF-1α in future may help to abrogate drug resistance in breast cancer.

Prostate cancer is a principal cause of cancer-associated morbidity in men. Although 5-year survival of patients with localized prostate cancer approaches 100%, survival decreases precipitously after metastasis. Bone is the preferred site for disseminated prostate cancer cell colonization, altering the equilibrium of bone homeostasis resulting in weak and fragile bones. Currently, no curative options are available for prostate cancer bone metastasis. Melanoma differentiation associated gene-7 (MDA-7)/IL24 is a well-studied cytokine established as a therapeutic in a wide array of cancers upon delivery as a gene therapy. In this study, we explored the potential anticancer properties of MDA-7/IL24 delivered as a recombinant protein. Using bone metastasis experimental models, animals treated with recombinant MDA-7/IL24 had significantly less metastatic lesions in their femurs as compared with controls. The inhibitory effects of MDA-7/IL24 on bone metastasis resulted from prostate cancer–selective killing and inhibition of osteoclast differentiation, which is necessary for bone resorption. Gain- and loss-of-function genetic approaches document that prosurvival Akt and Mcl-1 pathways are critically important in the antibone metastatic activity of MDA-7/IL24. Our previous findings showed that MDA-7/IL24 gene therapy plus Mcl-1 inhibitors cooperate synergistically. Similarly, an Mcl-1 small-molecule inhibitor synergized with MDA-7/IL24 and induced robust antibone metastatic activity. These results expand the potential applications of MDA-7/IL24 as an anticancer molecule and demonstrate that purified recombinant protein is nontoxic in preclinical animal models and has profound inhibitory effects on bone metastasis, which can be enhanced further when combined with an Mcl-1 inhibitory small molecule. Mol Cancer Ther; 17(9); 1951–60. ©2018 AACR.

MicroRNAs (miRNAs or miRs) are small 19-22 nucleotide long, noncoding, single-stranded, and multifunctional RNAs that regulate a diverse assortment of gene and protein functions that impact on a vast network of pathways. Lin-4, a noncoding transcript discovered in 1993 and named miRNA, initiated the exploration of research into these intriguing molecules identified in almost all organisms. miRNAs interfere with translation or posttranscriptional regulation of their target gene and regulate multiple biological actions exerted by these target genes. In cancer, they function as both oncogenes and tumor suppressor genes displaying differential activity in various cellular contexts. Although the role of miRNAs on target gene functions has been extensively investigated, less is currently known about the upstream regulatory molecules that regulate miRNAs. This chapter focuses on the factors and processes involved in miRNA regulation.

MicroRNAs (miRNAs or miRs) are small 19-22 nucleotide long, noncoding, single-stranded, and multifunctional RNAs that regulate a diverse assortment of gene and protein functions that impact on a vast network of pathways. Lin-4, a noncoding transcript discovered in 1993 and named miRNA, initiated the exploration of research into these intriguing molecules identified in almost all organisms. miRNAs interfere with translation or posttranscriptional regulation of their target gene and regulate multiple biological actions exerted by these target genes. In cancer, they function as both oncogenes and tumor suppressor genes displaying differential activity in various cellular contexts. Although the role of miRNAs on target gene functions has been extensively investigated, less is currently known about the upstream regulatory molecules that regulate miRNAs. This chapter focuses on the factors and processes involved in miRNA regulation.

Background Apoptosis of several host cells induced by parasites/parasite products has been investigated in human filariasis to understand immune hyporesponsiveness. However, apoptosis of monocytes-one of the major antigen presenting cells in peripheral circulation, which are chronically exposed to filarial antigens in infected subjects-is yet to be understood. Methods Apoptosis of human monocytes with Brugia pahangi antigen (BpA) was demonstrated by scoring several apoptotic markers using flow cytometry. Ability of BpA and plasma of infected subjects to suppress lymphocyte proliferation was demonstrated by (3)H thymidine incorporation assay and carboxyfluorescein succinimidyl ester dilution assay. Results BpA induced significant apoptosis of normal human monocytes, primarily through Toll-like receptor 4 (TLR4), and suppressed phytohemagglutinin (PHA)-mediated proliferation of normal human T lymphocytes. However, monocytes of Wuchereria bancrofti-infected subjects were resistant to BpA-induced apoptosis. Plasma of infected subjects also mediated apoptosis of normal monocytes, presumably due to circulating filarial antigens, and resulted in inhibition of PHA-induced proliferation. Conclusion Normal human monocytes were found to be qualitatively different from those of filariasis-infected subjects; whereas filarial antigens mediate apoptosis of normal human monocytes through TLR4, those of infected subjects were found to be resistant.

Melanoma differentiation-associated gene-7/IL-24 (mda-7/IL-24) displays broad-spectrum anticancer activity in vitro, in vivo in preclinical animal models, and in a phase I/II clinical trial in patients with advanced cancers without harming normal cells or tissues. Here we demonstrate that mda-7/IL-24 regulates a specific subset of miRNAs, including cancer-associated miR-221. Either ectopic expression of mda-7/IL-24 or treatment with recombinant His-MDA-7 protein resulted in downregulation of miR-221 and upregulation of p27 and PUMA in a panel of cancer cells, culminating in cell death. Mda-7/IL-24–induced cancer cell death was dependent on reactive oxygen species induction and was rescued by overexpression of miR-221. Beclin-1 was identified as a new transcriptional target of miR-221, and mda-7/IL-24 regulated autophagy through a miR-221/beclin-1 feedback loop. In a human breast cancer xenograft model, miR-221–overexpressing MDA-MB-231 clones were more aggressive and resistant to mda-7/IL-24–mediated cell death than parental clones. This is the first demonstration that mda-7/IL-24 directly regulates miRNA expression in cancer cells and highlights the novelty of the mda-7/IL-24–miR-221–beclin-1 loop in mediating cancer cell–specific death. Cancer Res; 77(4); 949–59. ©2016 AACR.

Glioblastoma multiforme (GBM) is a frequent and aggressive glial tumor, containing a small population of therapy-resistant cells, glioma stem cells (GSCs). Current dogma suggests that tumors regrow from GSCs, and these cells contribute to therapy resistance, poor prognosis, and recurrence; highlighting the importance of GSCs in glioma pathophysiology and therapeutic targeting. Macroautophagy/autophagy-based cellular homeostasis can be changed from pro-survival to pro-cell death by modulating SDCBP/MDA-9/Syntenin (syndecan binding protein)-mediated signaling. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of SDCBP/MDA-9/Syntenin. Conversely, SDCBP/MDA-9/Syntenin silencing induces autophagic death in GSCs, indicating that SDCBP/MDA-9/Syntenin regulates protective autophagy in GSCs under anoikis conditions. This process is mediated through phosphorylation of the anti-apoptotic protein BCL2 accompanied with suppression of high levels of autophagic proteins (ATG5, LAMP1, LC3B) through EGFR signaling. SDCBP/MDA-9/Syntenin-mediated regulation of BCL2 and EGFR phosphorylation is achieved through PTK2/FAK and PRKC/PKC signaling. When SDCBP/MDA-9/Syntenin is absent, this protective mechanism is deregulated, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. Our recent paper reveals a novel functional link between SDCBP/MDA-9/Syntenin expression and protective autophagy in GSCs. These new insights into SDCBP/MDA-9/Syntenin-mediated regulation and maintenance of GSCs present leads for developing innovative combinatorial cancer therapies.

Introduction: The high death rate due to breast cancer is often attributed to aggressive triple negative breast cancer (TNBC). We used the combination of metronomic pegylated liposomal doxorubicin (PLD), and metformin {selectively inhibits cancer stem cells (CSCs)} for effective management of TNBC xenografts generated in mice. Down-regulation of β-catenin is responsible for the efficacy of the combination of metronomic PLD and metformin in treating aggressive TNBC. Methodology: PLD was prepared using the ammonium sulfate gradient method. Morphology, particle size, zeta potential measurement, drug entrapment efficiency, and drug release studies were performed to characterize the prepared liposomal formulation. Cell culture studies were performed on MDA-MB-231 and MDA-MB-468 cell lines (i.e., TNBC cells). MTT assays, mammosphere assays, apoptosis assays, quantitative real-time polymerase chain reactions (qPCR), western blotting experiments were performed. In vivo anticancer efficacy of the combination therapy {antidiabetic dosing of metformin: 150 mg/kg body weight every day, and metronomic dosing of PLD: 1 mg/kg body weight with respect to doxorubicin (Dox) every other day for 3 weeks} was verified in an MDA-MB-231 xenograft model. Results: Monodisperse PLD having dimensions less than 100 nm was successfully prepared (confirmed by AFM, TEM, and DLS). Zeta potential measurement showed that PLD was negatively charged (- 12.1 ± 2.3 mV, n = 3). Encapsulation efficiency and drug release were found to be satisfactory. IC50 (i.e., 50 % inhibitory concentration) of PLD was found to be in nanomolar range with respect to Dox in TNBC cell lines. However, a combination of metformin (500 µM) and PLD resulted in a leftward shift of the concentration-response curve such that the IC50 value of PLD was further reduced in the nanomolar range with respect to Dox. A similar trend was seen in the apoptosis assay. Metformin works together with PLD to reduce non-stem cancer cells, CSCs, and stem cell markers as indicated by the reduction in the number of mammospheres, qPCR results, and western blotting experiments. We also found attenuation of β-catenin in TNBC cells by the combination treatment regimen, as suggested by qPCR and western blotting. Some of the in vitro observations were also confirmed in the MDA-MB-231 xenograft model. Conclusion: Our experiments demonstrate that down-regulation of β-catenin is responsible for the strong anticancer effects of the combination of metformin and PLD in vitro and in vivo. Further studies are warranted with this combination regimen in different cancer types as metformin is an approved anti-diabetic drug with an acceptable safety profile, and metronomic chemotherapy is gaining popularity in treating different cancer types. Citation Format: Indranil Banerjee, Subhayan Das, Chandan Kanta Das, Bikash Ch. Jena, Deblina Bharadwaj, Anjan K. Pradhan, Swadesh K. Das, Paul B. Fisher, Mahitosh Mandal. Combination of metformin and metronomic liposomal doxorubicin exerts a robust anticancer effect in triple negative breast cancer by inhibiting breast cancer stem cells & the Wnt/beta-catenin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3638.

Aberrant expression of the proto-oncogene EVI1 (ecotropic virus integration site1) has been implicated not only in myeloid or lymphoid malignancies but also in colon, ovarian and breast cancers. Despite its importance in oncogenesis, the regulatory factors and mechanisms that potentiate the function of EVI1 and its consequences are partially known. Here we demonstrated that EVI1 is post-translationally modified by SUMO1 at lysine residues 533, 698 and 874. Although both EVI1 and SUMO1 were found to co-localize in nuclear speckles, the sumoylation mutant of EVI1 failed to co-localize with SUMO1. Sumoylation abrogated the DNA binding efficiency of EVI1 and also affected EVI1 mediated transactivation. The SUMO ligase PIASy was found to play a bi-directional role on EVI1, PIASy enhanced EVI1 sumoylation and augmented sumoylated EVI1 mediated repression. PIASy was also found to interact with EVI1 and impaired EVI1 transcriptional activity independent of its ligase activity. Arsenic trioxide (ATO) known to act as an antileukemic agent for acute promyelocytic leukemia (APL) not only enhanced EVI1 sumoylation but also enhanced the co-localization of EVI1 and SUMO1 in nuclear bodies distinct from PML nuclear bodies. ATO treatment also affected the Bcl-xL protein expression in EVI1 positive cell line. Thus, the results showed that arsenic treatment enhanced EVI1 sumoylation, deregulated Bcl-xL, which eventually may induce apoptosis in EVI1 positive cancer cells. The study for the first time explores and reports sumoylation of EVI1, which plays an essential role in regulating its function.

Selective targeting to the tumor niche remains a major challenge in successful cancer therapy. Somatostatin receptor 2 (SSTR2) is overexpressed in breast cancer cells thus making this receptor an attractive target for selective guidance of ligand-conjugated drug liposomes to the tumor site. In this study, a synthetic somatostatin analogue (SST) was used as SSTR2 targeting agent and Diacerein was employed as therapeutic molecule. Diacerein loaded liposomes (DNL) were prepared and they were further decorated with the synthetic and stable analogue of somatostatin (SST-DNL). Fabricated liposomes were nano-size in range and biocompatible. SST-DNL displayed significantly better anti-tumor efficacy as compared to free Diacerein (DN) and DNL in breast cancer models. Enhanced apoptosis in breast cancer cells was detected in SST-DNL treated groups as monitored by cell cycle analysis and changes in expression level of apoptotic/anti-apoptotic proteins Bcl-2, Bax, cleaved Caspase 3 and PARP. SST-DNL more effectively inhibited the oncogenic IL-6/IL-6R/STAT3/MAPK/Akt signalling pathways as compared to DN or DNL in cancer cells. In addition, SST-DNL effectively suppressed angiogenesis and cancer cell invasion. In vivo tumor growth in a MDA-MB-231 mouse xenograft model was significantly suppressed following SST-DNL treatment. In xenograft model, immunohistochemistry of Ki-67 and CD-31 indicated that SST-DNL improved the anti-proliferative and anti-angiogenic impacts of Diacerein. In vivo pharmacokinetic studies in rats showed enhanced circulation time in the DNL or SST-DNL treated groups as compared to free DN. Considering all of these findings, we conclude that SST-DNL provides a novel strategy with better efficacy for breast cancer therapy.