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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.

Glioblastoma multiforme is a common malignant brain tumor that portends extremely poor patient survival. Recent studies reveal that glioma stem-like cells (GSC) are responsible for glioblastoma multiforme escape from chemo-radiotherapy and mediators of tumor relapse. Previous studies suggest that AEG-1 (MTDH), an oncogene upregulated in most types of cancers, including glioblastoma multiforme, plays a focal role linking multiple signaling pathways in tumorigenesis. We now report a crucial role of AEG-1 in glioma stem cell biology. Primary glioblastoma multiforme cells were isolated from tumor specimens and cultured as neurospheres. Using the surface marker CD133, negative and positive cells were separated as nonstem and stem populations by cell sorting. Tissue samples and low passage cells were characterized and compared with normal controls. Functional biological assays were performed to measure stemness, self-renewal, differentiation, adhesion, protein–protein interactions, and cell signaling. AEG-1 was upregulated in all glioblastoma multiforme neurospheres compared with normal neural stem cells. Expression of AEG-1 was strongly associated with stem cell markers CD133 and SOX2. AEG-1 facilitated β-catenin translocation into the nucleus by forming a complex with LEF1 and β-catenin, subsequently activating Wnt signaling downstream genes. Through an AEG-1/Akt/GSK3β signaling axis, AEG-1 controlled phosphorylation levels of β-catenin that stabilized the protein.Implications: This study discovers a previously unrecognized role of AEG-1 in GSC biology and supports the significance of this gene as a potential therapeutic target for glioblastoma multiforme. Mol Cancer Res; 15(2); 225–33. ©2016 AACR.

S1. Primary GBM neurospheres have higher AEG-1 expression than neuronal stem cells. S2. Primary GBM neurospheres sorted with the CD133 GSC cell surface marker. S3. Isolated GSCs show enhanced expression of the stem cell marker SOX2 and are capable of differentiating into astrocytes and neurons. Supplemental Table 1. Comparison of normalized expression levels of each protein for Western blots in Figure 2B.

Legends for supplemental figures, table and antibody list

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.

PDF file - 215K, Supplementary Figure S8: An association between increased level of MDA-9 mRNA expression and the development of therapeutic resistance and stable UCC was also evident in datasets available at TCGA database (TCGA_BLCA_SDCBP)

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.

PDF file - 64K, Table S3. Expression level of EGFR, c-Src (Y418), CTNNB1 (Y333), AKT (S473) and E-cadherin in the primary UCC tumors

PDF file - 142K, Supplementary Figure S1: A: Expression of MDA-9/Syntenin in non-tumorigenic HUC-1 cells is low or undetectable compared to the tumorigenic and aggressive T24 cells. B: Exogenously overexpressed MYC-tagged MDA-9/Syntenin expression was detectable in different clones. beta-actin was used as a loading control

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.

PDF file - 133K, Table S1. Clinical characteristics and expression level of MDA-9/Syntenin in primary UCC tumors

PDF file - 1469K, Supplementary Figure S3: Membranous (yellow arrow) and cytoplasmic (white arrow) co-localization of MDA-9/Syntenin and EGFR in T24 cells

PDF file - 105K, Table S2. Association between MDA-9/Syntenin, EGFR, c-Src, AKT, CTNNB1 and E-cadherin expression and different clinical characteristics

MDA-9/Syntenin activates IGF-1R

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7–induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal–regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH2-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7–induced phosphorylation of protein kinase R–like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R–like endoplasmic reticulum kinase that correlated with reduced c-jun NH2-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal–regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.[Mol Cancer Ther 2009;8(5):OF1–12]

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.

PDF file - 621K, Supplementary Figure S2: Immunofluorescence analysis of EGFR and C-Src (Y418) expression in MDA-9/syntenin-depleted T24 cells

Purpose: Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression.Experimental Design: Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knockdown was examined in multiple cells lines and key findings were validated in primary tumors.Results: Significantly higher (P = 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28 of 44) of primary tumors and an association was evident with stage (P = 0.01), grade (P = 0.03), and invasion status (P = 0.02). MDA-9/Syntenin overexpression in nontumorigenic HUC-1 cells increased proliferation (P = 0.0012), invasion (P = 0.0001), and EGF receptor (EGFR), AKT, phosphoinositide 3-kinase (PI3K), and c-Src expression. Alteration of β-catenin, E-cadherin, vimentin, claudin-1, ZO-1, and T-cell factor-4 (TCF4) expression was also observed. MDA-9/Syntenin knockdown in three UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and colocalization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin:AKT expressions with stage (P = 0.04, EGFR; P = 0.01, AKT). A correlation between MDA-9/Syntenin:β-catenin coexpression with stage (P = 0.03) and invasion (P = 0.04) was also evident.Conclusions: Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring, and therapeutic strategies for managing UCC. Clin Cancer Res; 19(17); 4621–33. ©2013 AACR.

Figure legends for supplementary figures 1 to 8

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

PDF file - 120K, Supplementary Figure S4: Map of the wild type and mutant MDA-9/Syntenin construct lacking N and C terminal domains with a C terminal HA tag

Supplementary Fig. from MDA-7/IL-24–induced cell killing in malignant renal carcinoma cells occurs by a ceramide/CD95/PERK–dependent mechanism

PDF file - 154K, Supplementary Figure S7: Progressive increase of MDA-9 mRNA from normal urothelium to CIS to muscle-invasive cancer was evident in datasets available in Gene Expression Omnibus database (GEO # GSE 3167)

PDF file - 1070K, Supplementary Figure S6: A steady-state level of MDA-9/Syntenin expression was evident in the in vivo lung metastasis of T24 cells transfected with an empty vector. Magnification X 200

PDF file - 6610K, Supplementary Figure S5: Co-expression signature of MDA-9/Syntenin, EGFR, AKT, �-catenin, E-cadherin and c-Src in primary UCC tumors

Legends for Supplementary Tables S1-S7 and Supplementary Figures S1-S5.

Supplementary Figure Legends 1-9

Supplemental Fig. 6. Effect of over-expression of AKT on [pIC]PEI-induced apoptosis

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Supplemental figure legend for figure S1 through S8

Subtraction hybridization combined with induction of cancer cell terminal differentiation in human melanoma cells identified melanoma differentiation–associated gene-7/interleukin-24 (mda-7/IL-24) and SARI (suppressor of AP-1, induced by IFN) that display potent antitumor activity. These genes are not constitutively expressed in cancer cells and forced expression of mda-7/IL-24 (Ad.mda-7) or SARI (Ad.SARI) promotes cancer-specific cell death. Ectopic expression of mda-7/IL-24 induces SARI mRNA and protein in a panel of different cancer cells, leading to cell death, without harming corresponding normal cells. Simultaneous inhibition of K-ras downstream extracellular signal-regulated kinase 1/2 signaling in pancreatic cancer cells reverses the translational block of MDA-7/IL-24 and induces SARI expression and cell death. Using SARI-antisense-based approaches, we demonstrate that SARI expression is necessary for mda-7/IL-24 antitumor effects. Secreted MDA-7/IL-24 protein induces antitumor “bystander” effects by promoting its own expression. Recombinant MDA-7/IL-24 (His-MDA-7) induces SARI expression, supporting the involvement of SARI in the MDA-7/IL-24-driven autocrine loop, culminating in antitumor effects. Moreover, His-MDA-7, after binding to its cognate receptors (IL-20R1/IL-20R2 or IL-22R/IL-20R2), induces intracellular signaling by phosphorylation of p38 MAPK, leading to transcription of a family of growth arrest and DNA damage inducible (GADD) genes, culminating in apoptosis. Inhibition of p38 MAPK fails to induce SARI following Ad.mda-7 infection. These findings reveal the significance of the mda-7/IL-24-SARI axis in cancer-specific killing and provide a potential strategy for treating both local and metastatic disease. Cancer Res; 74(2); 563–74. ©2013 AACR.

Differential count of bone marrow cells (% of total cell count).

Supplementary Figures 1-9. Sabutoclax has greater efficacy than ABT-737. (S1) Sabutoclax causes a G1-S phase cell cycle arrest. (S2) Pancreatic cancer cells exhibit varying levels of sensitivity to Minocycline. (S3) Sabutoclax and Minocycline induce cytotoxicity in PANC-1 cells that is reversed with zVAD. (S4) Sabutoclax and Minocycline show synergy. Combination index (CI) values for the combination of Sabutoclax and Minocycline in MIA PaCa-2 cells. (S5) The cytotoxicity induced by Sabutoclax and Minocycline is caspase-dependent and dependent upon loss of Stat3 activation. (S6) Sabutoclax and Minocycline reduce tumor growth in a subcutaneous xenograft model of pancreatic cancer. (S7) Tumors from Pdx-1-Cre/K-rasLSL-G12D/p53flox/wt and Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox mice overexpress Mcl-1 and show sensitivity to Sabutoclax and Minocycline. (S8) Tumors from KPC (Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox )mice treated with Sabutoclax and Minocycline show decreased Stat3 activation. (S9)

Supplemental Materials. Supplemental methods and figure legend

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Supplemental Fig. 3. [pIC]PEI does not change XIAP and survivin at the mRNA level

Generation of AEG-1KO mouse (S1); Analysis of spleen cells of adult WT and AEG-1KO mice (S2); F4/80 staining for macrophages in spleen sections of aged (16 m) WT and AEG-1KO mouse. Magnification 400X (S3); Graphical representation of quantification of F4/80 staining for macrophages in liver sections of WT and AEG-1KO mice, either untreated (naïve) or treated with DEN or DEN/PB (S4); Stellate cells are activated in the tumor region of WT liver (S5).

PDF file - 155K, IL-8 neutralizing antibody blocks C8161.9-induced HuVEC proliferation, migration and tube formation.

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