MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

Bone metastasis is a frequent and incurable consequence of advanced prostate cancer (PC). An interplay between disseminated tumor cells and heterogeneous bone resident cells in the metastatic niche initiates this process. Melanoma differentiation associated gene-9 (mda-9/Syntenin/syndecan binding protein) is a prometastatic gene expressed in multiple organs, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), under both physiological and pathological conditions. We demonstrate that PDGF-AA secreted by tumor cells induces CXCL5 expression in BM-MSCs by suppressing MDA-9-dependent YAP/MST signaling. CXCL5-derived tumor cell proliferation and immune suppression are consequences of the MDA-9/CXCL5 signaling axis, promoting PC disease progression. mda-9 knockout tumor cells express less PDGF-AA and do not develop bone metastases. Our data document a previously undefined role of MDA-9/Syntenin in the tumor and microenvironment in regulating PC bone metastasis. This study provides a framework for translational strategies to ameliorate health complications and morbidity associated with advanced PC.

Noninvasive therapy of brain cancer using a unique systemic delivery methodology with a cancer terminator virus.

Primary, glioblastoma, and secondary brain tumors, from metastases outside the brain, are among the most aggressive and therapeutically resistant cancers. A physiological barrier protecting the brain, the blood-brain barrier (BBB), functions as a deterrent to effective therapies. To enhance cancer therapy, we developed a cancer terminator virus (CTV), a unique tropism-modified adenovirus consisting of serotype 3 fiber knob on an otherwise Ad5 capsid that replicates in a cancer-selective manner and simultaneously produces a potent therapeutic cytokine, melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24). A limitation of the CTV and most other viruses, including adenoviruses, is an inability to deliver systemically to treat brain tumors because of the BBB, nonspecific virus trapping, and immune clearance. These obstacles to effective viral therapy of brain cancer have now been overcome using focused ultrasound with a dual microbubble treatment, the focused ultrasound-double microbubble (FUS-DMB) approach. Proof-of-principle is now provided indicating that the BBB can be safely and transiently opened, and the CTV can then be administered in a second set of complement-treated microbubbles and released in the brain using focused ultrasound. Moreover, the FUS-DMB can be used to deliver the CTV multiple times in animals with glioblastoma  growing in their brain thereby resulting in a further enhancement in survival. This strategy permits efficient therapy of primary and secondary brain tumors enhancing animal survival without promoting harmful toxic or behavioral side effects. Additionally, when combined with a standard of care therapy, Temozolomide, a further increase in survival is achieved. The FUS-DMB approach with the CTV highlights a noninvasive strategy to treat brain cancers without surgery. This innovative delivery scheme combined with the therapeutic efficacy of the CTV provides a novel potential translational therapeutic approach for brain cancers.

Pharmacological inhibition of MDA-9/Syntenin blocks breast cancer metastasis through suppression of IL-1ß.

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)-1ß 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.

MDA-7/IL-24 regulates the miRNA processing enzyme DICER through downregulation of MITF.

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.

Recent insights into apoptosis and toxic autophagy: The roles of MDA-7/IL-24, a multidimensional anti-cancer therapeutic.

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.

MDA-9/Syntenin/SDCBP: new insights into a unique multifunctional scaffold protein.

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.

MDA-9/Syntenin regulates protective autophagy in anoikis-resistant glioma stem cells.

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.

Insights into the Mechanisms of Action of MDA-7/IL-24: A Ubiquitous Cancer-Suppressing Protein.

Melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24), a secreted protein of the IL-10 family, was first identified more than two decades ago as a novel gene differentially expressed in terminally differentiating human metastatic melanoma cells. MDA-7/IL-24 functions as a potent tumor suppressor exerting a diverse array of functions including the inhibition of tumor growth, invasion, angiogenesis, and metastasis, and induction of potent "bystander" antitumor activity and synergy with conventional cancer therapeutics. MDA-7/IL-24 induces cancer-specific cell death through apoptosis or toxic autophagy, which was initially established in vitro and in preclinical animal models in vivo and later in a Phase I clinical trial in patients with advanced cancers. This review summarizes the history and our current understanding of the molecular/biological mechanisms of MDA-7/IL-24 action rendering it a potent cancer suppressor.

MDA-9/Syntenin (SDCBP): Novel gene and therapeutic target for cancer 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.

Comprehensive meta-analysis of QTL and gene expression studies identify candidate genes associated with Aspergillus flavus resistance in maize.

Aflatoxin (AF) contamination, caused by Aspergillus flavus, compromises the food safety and marketability of commodities, such as maize, cotton, peanuts, and tree nuts. Multigenic inheritance of AF resistance impedes conventional introgression of resistance traits into high-yielding commercial maize varieties. Several AF resistance-associated quantitative trait loci (QTLs) and markers have been reported from multiple biparental mapping and genome-wide association studies (GWAS) in maize. However, QTLs with large confidence intervals (CI) explaining inconsistent phenotypic variance limit their use in marker-assisted selection. Meta-analysis of published QTLs can identify significant meta-QTLs (MQTLs) with a narrower CI for reliable identification of genes and linked markers for AF resistance. Using 276 out of 356 reported QTLs controlling resistance to A. flavus infection and AF contamination in maize, we identified 58 MQTLs on all 10 chromosomes with a 66.5% reduction in the average CI. Similarly, a meta-analysis of maize genes differentially expressed in response to (a)biotic stresses from the to-date published literature identified 591 genes putatively responding to only A. flavus infection, of which 14 were significantly differentially expressed (-1.0 ≤ Log2Fc ≥ 1.0; p ≤ 0.05). Eight MQTLs were validated by their colocalization with 14 A. flavus resistance-associated SNPs identified from GWAS in maize. A total of 15 genes were physically close between the MQTL intervals and SNPs. Assessment of 12 MQTL-linked SSR markers identified three markers that could discriminate 14 and eight cultivars with resistance and susceptible responses, respectively. A comprehensive meta-analysis of QTLs and differentially expressed genes led to the identification of genes and makers for their potential application in marker-assisted breeding of A. flavus-resistant maize varieties.

A novel computational predictive biological approach distinguishes Integrin ß1 as a salient biomarker for breast cancer chemoresistance.

Chemoresistance is a primary cause of breast cancer treatment failure, and protein-protein interactions significantly contribute to chemoresistance during different stages of breast cancer progression. In pursuit of novel biomarkers and relevant protein-protein interactions occurring during the emergence of breast cancer chemoresistance, we used a computational predictive biological (CPB) approach. CPB identified associations of adhesion molecules with proteins connected with different breast cancer proteins associated with chemoresistance. This approach identified an association of Integrin ß1 (ITGB1) with chemoresistance and breast cancer stem cell markers. ITGB1 activated the Focal Adhesion Kinase (FAK) pathway promoting invasion, migration, and chemoresistance in breast cancer by upregulating Erk phosphorylation. FAK also activated Wnt/Sox2 signaling, which enhanced self-renewal in breast cancer. Activation of the FAK pathway by ITGB1 represents a novel mechanism linked to breast cancer chemoresistance, which may lead to novel therapies capable of blocking breast cancer progression by intervening in ITGB1-regulated signaling pathways.

Cytoplasmic-delivery of polyinosine-polycytidylic acid inhibits pancreatic cancer progression increasing survival by activating Stat1-CCL2-mediated immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective therapies and with poor prognosis, causing 7% of all cancer-related fatalities in the USA. Considering the lack of effective therapies for this aggressive cancer, there is an urgent need to define newer and more effective therapeutic strategies. Polyinosine-polycytidylic acid (pIC) is a synthetic double-stranded RNA (dsRNA) which directly activates dendritic cells and natural killer cells inhibiting tumor growth. When pIC is delivered into the cytoplasm using polyethyleneimine (PEI), pIC-PEI, programmed-cell death is induced in PDAC. Transfection of [pIC]PEI into PDAC cells inhibits growth, promotes toxic autophagy and also induces apoptosis in vitro and in vivo in animal models. METHODS: The KPC transgenic mouse model that recapitulates PDAC development in patients was used to interrogate the role of an intact immune system in vivo in PDAC in response to [pIC]PEI. Antitumor efficacy and survival were monitored endpoints. Comprehensive analysis of the tumor microenvironment (TME) and immune cells, cytokines and chemokines in the spleen, and macrophage polarization were analyzed. RESULTS: Cytosolic delivery of [pIC]PEI induces apoptosis and provokes strong antitumor immunity in vivo in immune competent mice with PDAC. The mechanism underlying the immune stimulatory properties of [pIC]PEI involves Stat1 activation resulting in CCL2 and MMP13 stimulation thereby provoking macrophage polarization. [pIC]PEI induces apoptosis via the AKT-XIAP pathway, as well as macrophage differentiation and T-cell activation via the IFNγ-Stat1-CCL2 signaling pathways in PDAC. In transgenic tumor mouse models, [pIC]PEI promotes robust and profound antitumor activity implying that stimulating the immune system contributes to biological activity. The [pIC]PEI anti-PDAC effects are enhanced when used in combination with a standard of care (SOC) treatment, that is, gemcitabine. CONCLUSIONS: In summary, [pIC]PEI treatment is non-toxic toward normal pancreatic cells while displaying strong cytotoxic and potent immune activating activities in PDAC, making it an attractive therapeutic when used alone or in conjunction with SOC therapeutic agents, potentially providing a safe and effective treatment protocol with translational potential for the effective therapy of PDAC.

Dual Targeting of the PDZ1 and PDZ2 Domains of MDA-9/Syntenin Inhibits Melanoma Metastasis.

Genome-wide gene expression analysis and animal modeling indicate that melanoma differentiation associated gene-9 (mda-9, Syntenin, Syndecan binding protein, referred to as MDA-9/Syntenin) positively regulates melanoma metastasis. The MDA-9/Syntenin protein contains two tandem PDZ domains serving as a nexus for interactions with multiple proteins that initiate transcription of metastasis-associated genes. Although targeting either PDZ domain abrogates signaling and prometastatic phenotypes, the integrity of both domains is critical for full biological function. 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 disruption of MDA-9/Syntenin signaling, an inhibitor has now been developed that simultaneously binds and blocks activity of both PDZ domains. PDZ1i was joined to the second PDZ binding peptide (TNYYFV) with a PEG linker, resulting in PDZ1i/2i (IVMT-Rx-3) that engages both PDZ domains of MDA-9/Syntenin. IVMT-Rx-3 blocks MDA-9/Syntenin interaction with Src, reduces NF-κB activation, and inhibits MMP-2/MMP-9 expression, culminating in repression of melanoma metastasis. The in vivo antimetastatic properties of IVMT-Rx-3 are enhanced when combined with an immune-checkpoint inhibitor. Collectively, our results support the feasibility of engineering MDA-9 dual-PDZ inhibitors with enhanced antimetastatic activities and applications of IVMT-Rx-3 for developing novel therapeutic strategies effectively targeting melanoma and in principle, a broad spectrum of human cancers that also overexpress MDA-9/Syntenin.

Conversion of a Non-Cancer-Selective Promoter into a Cancer-Selective Promoter.

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.

Enhanced Cancer Therapy Using an Engineered Designer Cytokine Alone and in Combination With an Immune Checkpoint Inhibitor.

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.

Theranostic Tripartite Cancer Terminator Virus for Cancer Therapy and Imaging.

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.

Engineering T Cells to Express Tumoricidal MDA-7/IL24 Enhances Cancer Immunotherapy.

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.

MDA-9/Syntenin (SDCBP) Is a Critical Regulator of Chemoresistance, Survival and Stemness in Prostate Cancer Stem Cells.

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.

Mechanism of internalization of MDA-7/IL-24 protein and its cognate receptors following ligand-receptor docking.

Melanoma differentiation associated gene-7 (mda-7/IL-24) is a member of the IL-10 family of cytokines, with ubiquitous direct and "bystander" tumor-selective killing properties. MDA-7/IL-24 protein binds distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2, IL-22R1/IL-20R1 and IL-22R1/IL-20R2. Recombinant MDA-7/IL-24 protein induces endogenous mda-7/IL-24 expression in a receptor-dependent manner; since A549 cells that lack a complete set of cognate receptors are not responsive to exogenous protein. The mechanism of MDA-7/IL-24 ligand-receptor biology is not well understood. We explored the interaction of MDA-7/IL-24 with its' receptors and the consequences of ligand-receptor docking. Using both pharmacological and genetic approaches we demonstrate that MDA-7/IL-24 internalization employs the clathrin-mediated endocytic pathway leading to degradation of receptors via the lysosomal/ubiquitin proteosomal pathway. This clathrin-mediated endocytosis is dynamin-dependent. This study resolves a novel mechanism of MDA-7/IL-24 protein "bystander" function, which involves receptor/protein-mediated internalization and receptor degradation.

Regulation of neuroblastoma migration, invasion, and in vivo metastasis by genetic and pharmacological manipulation of MDA-9/Syntenin.

Despite multi-modality treatments, prognosis for advanced stage neuroblastoma (NB) remains challenging with residual long-term disabilities in survivors. Advanced stage NB is metastatic, which is a principal cause of cancer-related deaths. We presently document a primary role of MDA-9 in NB progression and define the molecular mechanisms by which MDA-9 promotes transformed phenotypes. NB cell lines and clinical samples display elevated MDA-9 expression and bioinformatic analysis supports an association between elevated MDA-9 and bone metastasis and poor prognosis. Genetic (shmda-9, mda-9 siRNA) or pharmacological (small molecule inhibitor of protein-protein interactions; PDZ1i) blockade of MDA-9 decreases NB migration, invasion, and metastasis. Blocking mda-9 expression or disrupting MDA-9 partner protein interactions downregulates integrin α6 and ß4, diminishing Src activity and suppressing Rho-Rac-Cdc42 activity. These signaling changes inhibit cofilin and matrix metalloproteinases reducing in vitro and in vivo NB cell migration. Overexpression of integrin α6 and ß4 rescues the invasion phenotype and increases Src activity, supporting integrins as essential regulators of MDA-9-mediated NB migration and invasion. We identify MDA-9 as a key contributor to NB pathogenesis and show that genetic or pharmacological inhibition suppresses NB pathogenesis by an integrin-mediated Src-disruption pathway.