SDCBP) PDZ1 Small-Molecule Inhibitor Swadesh K

Published OnlineFirst July 25, 2019; DOI: 10.1158/1535-7163.MCT-18-1019

Small Molecule Therapeutics Molecular Cancer Therapeutics Suppression of Prostate Cancer Pathogenesis Using an MDA-9/Syntenin (SDCBP) PDZ1 Small-Molecule Inhibitor Swadesh K. Das1,2,3, Timothy P. Kegelman1, Anjan K. Pradhan1, Xue-Ning Shen1, Praveen Bhoopathi1, Sarmistha Talukdar1, Santanu Maji1, Devanand Sarkar1,2,3, Luni Emdad1,2,3, and Paul B. Fisher1,2,3

Abstract

Metastasis is the primary determinant of death in patients domain-targeted small molecule (PDZ1i) was previously with diverse solid tumors and MDA-9/Syntenin (SDCBP), a developed using fragment-based drug discovery (FBDD) pro-metastatic and pro-angiogenic gene, contributes to guided by NMR spectroscopy and was found to be well- this process. Recently, we documented that by physically tolerated in vivo, had significant half-life (t1/2 ¼ 9hours)and interacting with IGF-1R, MDA-9/Syntenin activates STAT3 displayed substantial anti-prostate cancer preclinical in vivo and regulates prostate cancer pathogenesis. These observa- activity. PDZ1i blocked tumor cell invasion and migration tions firmly established MDA-9/Syntenin as a potential in vitro,andmetastasisin vivo.Hence,wedemonstrate molecular target in prostate cancer. MDA-9/Syntenin con- that PDZ1i an MDA-9/Syntenin PDZ1 target-specific tains two highly homologous PDZ domains predicted to small-molecule inhibitor displays therapeutic potential for interact with a plethora of proteins, many of which are prostate and potentially other cancers expressing elevated central to the cancerous process. An MDA-9/Syntenin PDZ1 levels of MDA-9/Syntenin.

Introduction cloned by us in 1996, has recently achieved considerable interest for its central pathogenic role in multiple diverse Despite significant success in clinically managing localized cancers (6–10). MDA-9/Syntenin expression positively corre- prostate cancer through surgical, radiation and chemotherapeutic lates with prostate cancer progression (11), suggesting that approaches, metastatic prostate cancer remains essentially incur- this gene/protein might provide a suitable therapeutic target. able (1). Numerous molecular events, including intracellular- MDA-9/Syntenin physically interacts with Insulin-like growth regulated and/or extracellular tumor microenvironment- factor receptor (IGF-1R) activating the STAT3 pathway, and mediated are driving forces in controlling prostate cancer pro- turning on a plethora of downstream effector proteins gression (1, 2). Consequently, understanding disease pathogen- that facilitate invasion and angiogenesis. The relevance of esis and developing rationally targeted therapies are mandatory to IGF-1R (12) and STAT3 (13) activation in prostate cancer develop treatments that are potentially curative for advanced progression are well established. Hence, our recent work prostate cancer. defined a novel molecular pathway whereby MDA-9/Syntenin Melanoma differentiation associated gene-9 (mda-9) Syndecan- playsadecisiveroleinIGF-1R–mediated STAT3 activation in binding protein (SDCBP; refs. 3, 4), also known as Syntenin-1 (5), the context of prostate cancer (11). Functionally, MDA-9/Syntenin is an adaptor protein that physically interacts with selective binding partners such as 1Department of Human and Molecular Genetics, Virginia Commonwealth Uni- IGF-1R (11), c-Src (14), EGFR (15), TGF-bR (16), and versity, School of Medicine, Richmond, Virginia. 2VCU Institute of Molecular TGF-b (17), thereby stimulating downstream cancer-context– Medicine, Virginia Commonwealth University, School of Medicine, Richmond, fi 3 speci c signaling pathways. These interactions result in induction Virginia. VCU Massey Cancer Center, Virginia Commonwealth University, of metastasis-related phenotypes including invasion (14, 18, 19), School of Medicine, Richmond, Virginia. migration (18), angiogenesis (20) and epithelial–mesenchymal Note: Supplementary data for this article are available at Molecular Cancer transition (16, 17). In addition to its role in cancer, the con- Therapeutics Online (http://mct.aacrjournals.org/). sequences of MDA-9/Syntenin and its' binding partners in dif- Current address for T.P. Kegelman: Department of Radiation Oncology, Univer- ferent pathological conditions is also well documented (21–23). sity of Pennsylvania, Philadelphia, PA. With few exceptions [Ubiquitin (24), TGF-bR (16)] two highly Corresponding Authors: Paul B. Fisher, Virginia Commonwealth University, conserved PDZ domains (PSD95/SAP90, DLGA, and ZO-1), 113- School of Medicine, 1101 East Marshall Street, Sanger Hall Building, Room 11-015, 275 amino acids, represent nodal hubs for binding part- Richmond, VA 23298. Phone: 804-628-3336; Fax: 804-827-1124; E-mail: ners (14, 15). In these contexts, designing small molecules that paul.fi[email protected]; and Swadesh K. Das, [email protected] specifically target the PDZ domains, thereby disrupting interac- Mol Cancer Ther 2019;18:1997–2007 tions or altering signaling, would in principle have enormous doi: 10.1158/1535-7163.MCT-18-1019 potential in blocking MDA-9/Syntenin-mediated phenotypes, 2019 American Association for Cancer Research. such as invasion, migration, and angiogenesis.

www.aacrjournals.org 1997

Das et al.

There is significant interest in targeting the PDZ class of recommended by the manufacturer. PC-3ML-Luc cells were proteins with some initial successes using small peptides, obtained from Dr. M.G. Pomper (Johns Hopkins Medical natural products, and small molecules (25). These initial Institutions, Baltimore, MD) and maintained as previously accomplishments are noteworthy and suggested that the PDZ described (28). The androgen-refractory mouse prostate domains may in fact be "druggable." However, there were cancer cell line RM1 was provided by Dr. T.C. Thompson several challenges associated with targeting PDZ domains for (Baylor College of Medicine, Houston, TX) and was maintained therapeutic purposes, because they are involved in multiple in DMEM as previously described. The metastatic capacity of protein-protein interactions, some of which may be essential stable luciferase expressing RM1 cells (RM1-Luc)hasbeen for normal cellular functions. Nonetheless, we noted that reported previously (29). although there are over 150 PDZ domains in the human genome discovered thus far, their binding surfaces are Reagents and antibodies likely distinct and with different substrate specificities (26). MDA-9/Syntenin (SDCBP) antibody was obtained from We have confirmed the hypothesis that the PDZ domains are Abonova Inc. (Taiwan). Phospho-IGF-1R (Tyr1135), IGF-1R, in fact "druggable" and by using a combination of Fragment- Phospho-Src (Tyr416), Src, Phospho-FAK (Tyr397), FAK, and NMR-based drug discovery (FBDD) approaches we Phospho-STAT3 (Tyr 705), and STAT3 antibodies were purchased derived novel initial pharmacological tools targeting MDA- from Cell Signaling Technology. b-Actin and EF-1a were from 9/Syntenin (27). These strategies enabled the identification Sigma-Aldrich and EMD Milipore, respectively, and were used as and initial optimization of a small-molecule capable of target- loading controls for the different experiments. All reagents for cell ing and antagonizing MDA-9/Syntenin in vitro,incellcultures cultures, including media and serum, were purchased from through its PDZ1 domain (PDZ1i; ref. 27). Interestingly, these Thermo Fisher Scientific. Recombinant IGFBP-2 was purchased small molecules do not target the PDZ2 domain of MDA-9/ from R & D Biosystems. Syntenin, demonstrating that despite the similar global fold, these domains tend to have fairly distinct binding sur- Gelatin Zymography fi faces (27). These ndings are intriguing and form the basis Gelatin zymography was used to determine the gelatinolytic for developing new classes of PDZ inhibitors and the activity of MMP-2 and MMP-9 in conditioned media collected approaches would be paradigm shifting and pave the way in from in vitro cell cultures. The experimental protocol is described fi the future for an entire new eld of research for targeting other in the "Supplementary Document" section. therapeutically relevant PDZs. In the present study, we evaluated the effect of inhibiting Real-Time PCR. For qPCR, total RNAs were extracted using miR- MDA-9/Syntenin functions with PDZ1i on prostate cancer cell Neasy kits (Qiagen) as recommended by the manufacturer and invasion, downstream biochemical changes, tumor angiogen- cDNA was prepared as previously described (20). Quantitative esis, and tumor cell retention in the lungs, and formation of qPCR was performed using an ABI ViiA7 fast real-time PCR system lung and bone metastases. Our studies demonstrate that and TaqMan gene expression assays according to the manufac- PDZ1i blocks key interactions between IGF-1R and MDA-9/ turer's protocol (Applied Biosystems). Syntenin thereby resulting in inhibition of STAT3 and con- comitant blocking of production of angiogenic factors and matrix degrading enzymes, culminating in an inhibition of Constructs and stable cell clones metastasis. These findings suggest that PDZ1i may represent a Various vector constructs used in this study were either cloned useful small-molecule inhibitor capable of reducing prostate by our group (mda-9,shmda-9; REF. 20) or obtained from com- cancer pathogenicity. mercial vendors. As described previously (20), cells were trans- fected with an expression vector producing luciferase and selected for neomycin resistance for approximately 2 weeks. Individual Materials and Methods colonies (clones) were picked and analyzed for luciferase Synthesis of PDZ1i expression. A detail description of synthesis protocol and properties were previously described (27). Additional details presented in the In vivo experiments "Supplementary Documents" section. All in vivo experiments were performed in accordance with IACUC approved protocols. To determine the effect of PDZ1i Human cell lines on tumor cell retention in the lungs, we inoculated cohorts of All cells except ARCaP with its metastatic variant ARCaP-M, mice (n ¼ 5, each group) via tail vein injection with either were obtained from the ATCC and maintained in culture as per vehicle (DMSO) or test compound (50 mmol/L) pre-treated ATCC recommendations. ARCaP and ARCaP-M cells were ARCaP-M-Luc (1 106 cells in 100 mL saline) metastatic obtained from Novicure Biotechnology (Birmingham, AL) and prostate cancer cells. Luciferase activity was monitored for maintained in media as recommended by the provider. Primary differential cellular clearance from the lungs between 15 min- immortal prostate epithelial cells (RWPE-1) were purchased utes and 5 hours by Bioluminescence imaging (Xenogen in vivo from the ATCC. HUVEC (Human Umbilical Vein Endothelial imaging (IVIS) system (Caliper Life Sciences, Inc.). For the lung Cells) were obtained from Lonza. All cell lines were routinely experimental metastasis model, a total of 5 105 ARCaP-M-Luc checked for Mycoplasma contamination by using commercial cells were injected (in 100 mL PBS) by intravenous tail vein kits. The majority of experiments used RWPE-1, DU-145, and injection. Treatment began 12 hours after prostate cancer cell ARCaP-M cells. All of these cell lines were purchased recently injection. PDZ1i was given at a dose of 30 mg/kg body weight (within the last 3 years) and were strictly maintained as in solution containing DMSO. Drugs were delivered every

1998 Mol Cancer Ther; 18(11) November 2019 Molecular Cancer Therapeutics

Small-Molecule Inhibitor of Prostate Cancer

alternate day for the first three weeks (total 9 injections). Mice Results were periodically observed for any signs of toxicity. Mice were PDZ1i suppresses mda-9/syntenin–mediated invasion in kept until euthanized as recommended by IACUC. In another prostate cancer and mda-9/syntenin overexpressing normal experiment, PC-3ML-Luc cells were injected by the intracardiac immortal prostate epithelial cells route to develop bone-metastases. Mice were divided into two The chemical structure of PDZ1i was presented (Fig. 1A). To groups (n ¼ 8), control and PDZ1i. Vehicle or drug were determine the effect of PDZ1i on cell growth and proliferation, administered on alternate days for 9 doses within the first long-term (3 week) colony formation (clonal) assays were per- three weeks from the day of implantation. Incidence of metas- formed using a series of prostate cancer cell lines and normal tasis was monitored using Bioluminescence imaging, as immortal prostate epithelial cells (RWPE-1). No significant tox- described previously (30). Two sets of experiments were icity was evident in the long-term clonal assay in either prostate conducted using the murine-derived prostate cancer cell line cancer or RWPE-l cells after exposure to 50 mmol/L PDZ1i RM1-Luc (29). In the first experiment, 1 105 RM1-Luc cells (Fig. 1B). Further confirmation of a lack of overt toxicity were injected by intracardiac route in C57BL/6 mice to develop or dramatic effects on short term growth of early passage lung metastases. Similar to the athymic nude mouse study, immortal prostate epithelial cells was documented using MTT experimental mice received only three doses of PDZ1i within assays after exposure to 50 mmol/L PDZ1i (Fig. 1C). In addi- the first week of treatment. Mice were euthanized on day 9 and tion, only minimal growth inhibition was evident in MTT the lungs were collected for comparing metastases. In the assays when early passage immortal prostate epithelial cells second experiment, tumor cells were implanted by intracardiac were treated with 75 or 100 mmol/L PDZ1i (Fig. 1C). route, divided into two groups, control and PDZ1i and treated Initially, to assess the direct effect of PDZ1i on mda-9/syntenin– as described above. A cohort of 20 mice were used in mediated invasion, we used genetically modified (transiently) this experiment. Mice were maintained until they required mda-9/syntenin overexpressing normal immortal prostate epithe- euthanasia. In an additional experiment, Hi-myc mice (ref. 31; lial cells (RWPE-1; referred to as RWPE-1 mda-9), which originally a prostate cancer spontaneous transgenic mouse model) were expressed minimal levels of MDA-9/Syntenin and minimal to injected intraperitoneally with PDZ1i starting at 2 months zero invasive ability (Fig. 1D). RWPE-1 mda-9 cells displayed of age and treatment continued for a subsequent 3 weeks increased invasion; however, this enhancement in invasion (total 9 injections). Mice were kept until they reached was significantly attenuated when cells were treated with 6-months of age. Prostates were removed, photographed, PDZ1i (25 and 50 mmol/L; Fig. 1D). We have also tested the weighed, and processed for paraffin sectioning. Immunohis- anti-invasive effect on prostate cancer cells, including DU-145, tochemistry was done as previously described (20) with the ARCaP and its metastatic variant ARCaP-M (Fig. 1E). ARCaP is indicated antibodies. the parental cell line from which ARCaP-M was derived, consisting of a heterogeneous cell population with both mes- Co-immunoprecipitation enchymal and epithelial phenotypes. All of these prostate Co-Immunoprecipitation was performed as described previ- cancer cell lines have similar levels of MDA-9/Syntenin (9) ously (9, 14) using a kit from Pierce (Pierce Biotechnology). and demonstrated comparable inhibition of invasion when treated with PDZ1i. Invasion assays Boyden chamber assays were used to investigate the invasive properties of cancer cells (19, 20). Briefly, cells were pretreated PDZ1i blocks MDA-9/Syntenin and IGF-1R interactions in with PDZ1i or DMSO and plated on the upper chamber. After prostate cancer cells 18 hours, invasive cells in the lower chamber were photographed Our recent work documented a physical interaction between and analyzed. MDA-9/Syntenin and IGF-1R following stimulation with exogenous IGFBP-2 and the resulting significant impact on prostate In vitro tube formation and chorioallantoic membrane assays cancer invasion (11). We also experimentally documented the Tube formation and chorioallantoic membrane (CAM) assays potential involvement of the first PDZ domain of MDA-9/Synte- were performed as described previously (20). Briefly, tumor- nin in mediating this interaction (11). On the basis of these derived conditioned media were collected after 24 hours of considerations, we explored the effect of PDZ1i on MDA-9/ treatment (either DMSO or PDZ1i) and concentrated. Equal Syntenin and IGF-1R interactions. DU-145 and ARCaP-M cells amounts of protein (50 mg) containing conditioned media were were treated with PDZ1i for 6 hours and cell lysates were subjected mixed with basal media and incubated with HuVEC cells on to co-IP analysis to determine potential physical interactions Matrigel layers. Photographs were taken after 6 hours. In CAM between MDA-9/Syntenin and IGF-1R. The same samples were assays, DMSO- or PDZ1i-treated tumor cell-derived conditioned also analyzed for MDA-9/Syntenin and Src interactions confirm- media were implanted on the CAM of 8-day-old fertilized eggs. ing that PDZ1i selectively inhibits MDA-9/Syntenin/IGF-1R, but Photographs were taken on day 12, 4 days after the addition of not MDA-9/Src interactions, which occur through the PDZ2 conditioned media. domain, providing further evidence for specificity of PDZ1i (Fig. 2A). Previous studies suggest a role for both PDZ domains Statistical analysis of MDA-9/Syntenin in Src interaction, with binding potentially Statistical significance analysis was performed using the occurring through the PDZ2 domain and then co-interaction with Student t test in comparison with corresponding controls. Prob- the PDZ1 domain (14). Both proteins, IGF-1R (red) and MDA-9/ ability values <0.05 were considered statistically significant. Sur- Syntenin (green) were co-localized (yellow spots in the merged vival curves were analyzed using Cox proportional hazards sur- image) in the plasma membrane upon stimulation with IGFBP-2. vival regression using GraphPad Prism. Treatment of PDZ1i visibly reduced this association and

www.aacrjournals.org Mol Cancer Ther; 18(11) November 2019 1999

Das et al.

A D RWPE-1 mda-9 RWPE-1 control PDZ1i PDZ1i PDZ1i (0 mmol/L) (25 mmol/L) (50 mmol/L) H N N HN NH O N N O O N N O

B 120 90

DMSO 60

(% of control) of (% Invading cells Invading 0

PDZ1i Control + – –– RWPE-1 DU-145 ARCaP-M mda-9 – +++ PDZ1i 0 0 25 50 CE 125 ns 120

100 100 80 75 60 50

(% of control) of (% (vs. control) (vs. 25 cells Invading 20 0 Percentage of growth of Percentage 00 0 50 75 100 0 25 50 0 25 50 0 25 50 m PDZ1i ( mol/L) DU-145 ARCaP ARCaP-M

Figure 1. Effect of PDZ1i on growth and invasion. A, Chemical structure of PDZ1i. B, The indicated cells were treated with DMSO or PDZ1i (50 mmol/L) and plated at low density (50 cells/6-well plate). Every third day media containing DMSO or PDZ1i were replaced. Colonies were stained after 3 weeks. Representative photomicrographs are shown. C, MTT assays were done to test the activity of PDZ1i on immortal human prostate epithelial cell (RWPE-1) growth. Data presented as the percentage of antiproliferation activity of PDZ1i compared with the DMSO-treated group. D, The designated cells were treated with DMSO or PDZ1i (25 or 50 mmol/L) and invasion was determined using a modified Boyden Chamber assay (BD Biosciences). RWPE-1 control or mda-9: control plasmid or mda-9 transiently overexpressing RWPE-1 cells. Photomicrographs were taken at 10 magnification. E, Different prostate cancer cells (25,000 cells/well) were pre-treated with either DMSO (vehicle) or PDZ1i (dose as indicated) and invasion ability was assayed using a modified Boyden Chamber according to the manufacturer's instructions. In both (D and E), quantification of the results of three independent experiments are provided in the graphs. The data are the mean S.D. , represents statistical significance from the corresponding control group.

substantiated the ability of PDZ1i to block the physical interac- mediated STAT3 activation. Additional support for this conclu- tion between MDA-9/Syntenin and IGF-1R (Fig. 2B). Because, this sion comes from experiments where IGFBP-2 and IL-6 were used interaction affected STAT3 activation, we investigated whether in combination either in the presence or absence of PDZ1i. As PDZ1i could downregulate IGF-1R and STAT3 activity in both a predicted, PDZ1i only downregulated IGFBP-2–induced STAT3 dose- and time-dependent manner (Fig. 3A and B). Exogenous activation, but not when IGFBP-2 and IL-6 were used in combi- stimulation of IGFBP-2 upregulated phosphorylated IGF-1R and nation (Fig. 3C). PDZ1i might directly or indirectly down regulate STAT3 (active state) in both prostate cancer cells without changing multiple receptor tyrosine kinases, which is currently under the total corresponding proteins, which was suppressed when investigation. Finally, consistent with our previous observations, cells were pre-treated with PDZ1i. The IGFBP-2–mediated acti- PDZ1i downregulated both MMP-2 and MMP-9 (32), at both the vation and suppression by PDZ1i was sustained for at least 2 hours protein and activity level as documented by Western blotting and in both cell lines, which is sufficient to provide signals to regulate zymography, respectively (Fig. 3D). downstream invasion-related gene(s) expression. Although PDZ1i did not affect MDA-9/Syntenin/Src interactions, Src activ- Anti-angiogenic role of PDZ1i ity was reduced (Fig. 3A and B), suggesting that IGF-1R might also MDA-9/Syntenin non-autonomously (through secretion of play a role in Src activation. several angiogenic factors, including IGFBP-2 and IL-8) regulates Previous studies indicate that IL-6 can activate STAT3 through tumor angiogenesis in melanoma cells (20). In this study, we IGF-1R in prostate cancer (13). In agreement with that study, we explored the potential impact of PDZ1i on tumor-derived angio- also observed IGF-1R activation in IL-6–treated samples (Sup- genesis in prostate cancer. The experimental strategy is outlined in plementary Fig. S1); however, this effect was not suppressed by Supplementary Fig. S2A. Data analysis from an antibody-based PDZ1i, suggesting specificity for IGFBP-2/MDA-9/IGF-1R– array highlighted a number of defined pro-angiogenic proteins

2000 Mol Cancer Ther; 18(11) November 2019 Molecular Cancer Therapeutics

Small-Molecule Inhibitor of Prostate Cancer

AB DMSO PDZ1i IP: IgG MDA-9 IgG MDA-9 - + ---+++PDZ1i MDA-9 WB:IGF-1R 110.7 0.6

Input

WB:MDA-9

IGF-1R DU-145 ARCaP-M

IP:IgG IP:MDA-9 - +++---+ PDZ1i WB: Src

Merge WB:MDA-9

DU-145 ARCaP-M DU-145 ARCaP-M

Figure 2. Effect of PDZ1i on MDA-9/Syntenin/IGF-R1 interactions. A, Cell lysates prepared from DU-145 and ARCaP-M cells, treated or untreated with PDZ1i, were subjected to IP using anti–MDA-9/Syntenin antibody and IB was performed using anti–IGF-1R antibody. Values from densitometry analysis are presented using MDA-9/Syntenin expression in the input as control. B, Representative photomicrographs of confocal images from PDZ1i pre-treated ARCaP-M cells. MDA-9/ Syntenin and IGF-1R were labeled with green and red florescent probes, respectively. "Yellow dots" represent co-localized proteins. that are downregulated by PDZ1i (Fig. 4A and B). A complete list the lungs (within 2 hours post-inoculation). This suggests that of proteins that have been tested is provided in Supplementary pre-treatment with PDZ1i might alter the adhesion ability of Fig. S2. A robust downregulation (>50%) of several pro- potentially metastatic tumor cells, which in principle, ultimately anagiogenic proteins, for example, Angiogenin, Angiopoetin-2, have a direct effect on development of metastatic lesions at Amphiregulin, FGF-basic, GM-CSF, and IL-1b, was evident in the secondary sites. Moreover, animals injected with PDZ1i-treated PDZ1i-treated samples. In addition, IGFBP-2, VEGF-A, and ARCaP-M-Luc cells survived longer than control animals treated CXCL16 also displayed considerable downregulation in PDZ1i- with vehicle without the small-molecule PDZ inhibitor (Fig. 5A). treated samples. The reduction of the anti-angiogenic factor In the second set of experiments (Fig. 5B), an equal number endostanin is surprising and unanticipated and requires further of cells was implanted into animals using an intravenous tail study. Because VEGF-A is a potent angiogenic factor and a down- vein route. Experimental groups received 30 mg/kg body weight stream target of STAT3 (33), we also scrutinized the expression of PDZ1i intraperitoneally as described in the figure legend. pattern of VEGF-A at an mRNA level in different prostate cancer As predicted, the treated groups developed significantly fewer cells, including mda-9/syntenin stably expressing RWPE-1 cells lesions as compared with control groups, as detected by biolu- (Fig. 4C). MDA-9/Syntenin mediates regulation of CXCL16, minescence imaging (BLI), ultimately resulting in prolonged TSP-1, uPA (34) and IGFBP-2 (20) and this study supports the survival (Fig. 5B). To investigate the potential role of PDZ1i in specificity of PDZ1i to MDA-9/Syntenin action. Finally, both inhibiting bone metastasis, PC-3ML-Luc cells were implanted by in vitro tube formation and in vivo CAM assays confirmed the intracardiac route in animals (n ¼ 8) and treated with vehicle or anti-angiogenic properties of PDZ1i-treated tumor cell-derived PDZ1i. Six out of eight mice from the control group developed conditioned media (Fig. 4D). metastases in the left or right femur. In contrast, no femur metastases were detected in the PDZ1i-treated group. In addition, PDZ1i inhibits prostate cancer development in vivo a lower incidence of lung (7 out of 8 vs. 5 out of 8) metastases were To assess the potential therapeutic efficacy of PDZ1i we con- evident in animals that received PDZ1i. ducted six independent sets of in vivo experiments. First, stable In the fourth and fifth set of experiments, two cohorts of luciferase expressing ARCaP-M (ARCaP-M-Luc) cells were pre- C57BL/6 mice were injected through intracardiac route with treated with PDZ1i and injected intravenously into animals to RM1-Luc cells. The first cohort consisted of 5 mice from each determine the effect of this MDA-9/Syntenin inhibitor in mod- group (vehicle and PDZ1i) and were euthanized after 9 days to ulating retention and adhesion of metastatic tumor cells in evaluate tumor burden in the lungs. The second cohort with 10 the lungs. As shown in Fig. 5A, mice inoculated with PDZ1i mice in each group were used to develop Kaplan–Meier survival pre-treated cells were present at lower concentrations than curves. Similar results as seen in the nude mice experiments were in control groups in the lungs and cleared more rapidly from evident, that is, PDZ1i significantly reduced tumor burden in the

www.aacrjournals.org Mol Cancer Ther; 18(11) November 2019 2001

Das et al.

A C hIGFBP-2 - + ++ +++- PDZ1i --10 - 20 - 10 20

pIGF-1R hIGFBP-2 - ++++-- - ++++- - IGF-1R PDZ1i ------+ ++ ++ + IL6 ---+++- - -- +++- pSTAT3 pSTAT3 STAT3 STAT3 pSrc pSrc Src Src EF-1a DU-145 ARCaP-M DU-145 ARCaP-M B D hIGFBP-2 ++++++++ ++ ++++++ 25 25 m m PDZ1i - ---++++ -- -- ++++ DMSO mol/L DMSO mol/L pIGF-1R MMP-2

IGF-1R MMP-9 pSTAT3 DU-145 ARCaP-M STAT3

pSrc mmol/L DMSO 10 mmol/L25 mmol/L DMSO 10 25 mmol/L Src MMP-2 EF-1a 30 60 90 120 30 60 90 120 30 60 90 120 30 60 90 120 min MMP-9 DU-145 ARCaP-M DU-145 ARCaP-M

Figure 3. PDZ1i inﬂuences the MDA-9/Syntenin/IGF-1R/STAT3 signaling axis. A, Cells were growth starved for 24 hours and treated with either DMSO or PDZ1i (dose indicated in mmol/L) for 6 hours. Cells were treated with human recombinant IGFBP-2 (hIGFBP-2, 10 ng/mL) for 2 hours, lysates were prepared and Western blotting analysis was conducted with speciﬁc antibodies. B, Cells were serum-starved for 24 hours and treated with either DMSO or PDZ1i (20 mmol/L) for 6 hours. Cells were treated with human recombinant IGFBP-2 (hIGFBP-2, 10 ng/mL) for different times (30 to 120 min), cell lysates were prepared and subjected to Western blotting. C, Cells were serum-starved for 24 hours and treated with either DMSO or PDZ1i (20 mmol/L) for 6 hours. Cells were treated with human recombinant IL-6 (1 ng/mL) and/or hIGFBP-2 for 2 hours, cell lysates were prepared and analyzed by Western blotting. D, The indicated cells were treated with DMSO or PDZ1i (25 mmol/L) for 24 hours. Tumor-derived conditioned media were subjected to Zymography (top) for enzymatic activity and Western blotting analysis (bottom) for the expression of MMP-2 and MMP-9.

lungs (Fig. 6A) and significantly enhanced survival (Fig. 6B). In ing potential targets that are affected by PDZ1i is presented the sixth experimental approach, 8-week-old Hi-myc male mice in Fig. 6F. were treated intraperitoneally with either PDZ1i (30 mg/kg) or vehicle 9 times for the first three weeks. Mice were then maintained until 6-months of age, when adenocarcinomas fully devel- Discussion oped in this transgenic animal model. In addition, to understand Genomic interrogation using whole-genome sequencing, RNA drug-mediated molecular changes, 48 hours before sacrifice, a sequencing and single-nucleotide polymorphism profiling of single additional PDZ1i treatment was given. Both the size and primary and metastatic prostate cancer has provided an unpar- weight of prostates collected from the PDZ1i-treated groups were alleled opportunity to identify potential genetic targets that may significantly less than the control groups (Fig. 6C). H&E staining afford "precision medicine" approaches for enhancing therapeu- of prostate sections (Fig. 6D) indicated that histologically the tic outcomes (35–37). A major component of cancer progression prostate from the control (vehicle-treated) group showed a sig- relates to tumor heterogeneity, which may be the driving force in nificant progression to adenocarcinoma, which was not evident in defining why only 10% of prostate cancer cases progress to the treated groups (representative photomicrographs are pre- lethality (38). On the basis of accumulating data, various path- sented). Immunostaining for the expression of pIGF-1R and ways are being targeted to effect beneficial outcomes in patients pSTAT3, two downstream effectors of MDA-9/Syntenin, were also with metastatic prostate cancer, including tumor vasculature, significantly reduced in PDZ1i-treated animals (Fig. 6E), validat- androgen receptors, IGF-1 and IL-6 signaling, and cytoprotective ing the in vitro observations in vivo. A schematic diagram describ- chaperones (39, 40). However, therapy of advanced prostate

2002 Mol Cancer Ther; 18(11) November 2019 Molecular Cancer Therapeutics

Small-Molecule Inhibitor of Prostate Cancer

A C DMSO PDZ1i

1.5

1.2 DMSO 0.8 1

0.4 0.5

mRNA) ( VEGF-A 0 0 Relative fold changes fold Relative mda-9 PDZ1i RWPE-1 ARCaP-M DU-145

R.S.: Reference spot D DMSO PDZ1i (25 mmol/L) B

1.2 DMSO PDZ1i

1 ARCaP-M

0.8

0.6 DU-145 0.4

0.2 Relative fold changes fold Relative

ARCaP-M

(9) IL1 (9) b

(7) HGF (7)

(14) uPA (14)

(13) TSP-1 (13)

(10) MMP-9 (10)

(12) TIMP-1 (12)

(4) CXCL16 (4)

(8) IGFBP-2 (8)

(15) VEGF-A (15)

(11) Serpin E1 Serpin (11)

(5) Endostatin (5)

(1) Angiogenin (1)

DU-145

(6) Endothelin-1 (6)

(3) Amphiregulin (3) (2) Angiopoietin-2 (2)

Figure 4. PDZ1i suppresses production of tumor-derived pro-angiogenic factors. A, Blots from antibody arrays showing differential expression levels of various angiogenic factors following treatment with conditioned media derived from vehicle- or PDZ1i-treated ARCaP-M cells. B, Graphical representation of the band intensity in A quantified by densitometry. C, Cells were treated with DMSO or PDZ1i for 24 hours and expression of VEGF-A mRNA was analyzed. D, Pro-angiogenic activity of DMSO- or PDZ1i-treated tumor cell-derived conditioned media was analyzed in both in vitro (tube formation assay) and in vivo (CAM assay) contexts as described in Methods and Materials. Representative photomicrographs are presented. cancer, particularly when metastasis to bone has occurred, still recently identified IGF-1R as a new MDA-9/Syntenin binding remains an unattainable objective. Developing effective and partner and are defining the consequences of this interaction in potentially curative strategies to treat advanced prostate cancer disease progression (11). Considering the importance of these requires a detailed understanding of molecular mechanisms of interactions in STAT3 activation, which has a decisive impact on prostate cancer pathogenesis, including causative genetic changes prostate cancer progression as shown in our and other stud- and molecular determinants of tumor heterogeneity that result in ies (13, 42, 43), we sought to identify small molecule inhibitors target-site specific metastatic clonal populations. We presently that could specifically interrupt MDA-9/Syntenin/IGF-1R inter- demonstrate that MDA-9/Syntenin (SDCBP) provides a molecu- actions thereby affecting STAT3 activation and prostate cancer lar target for developing small molecules that can intervene in the invasion. Using a fragment-based drug-discovery approach invasive and metastatic properties of prostate cancer both in vitro (FBDD) guided by NMR spectroscopy in solution, we previously and in vivo in preclinical animal models. Small molecules specif- confirmed the hypothesis that the PDZ domains of MDA-9/ ically targeting interactions between the PDZ1 domain of MDA-9/ Syntenin are "druggable" and amenable to producing selective Syntenin and IGF-1R have been developed (27, 41) and may small molecules that could bind and potentially disrupt protein provide a novel approach for managing prostate cancer. interactions required for biological activity of MDA-9/Synte- MDA-9/Syntenin (SDCBP) is an adaptor scaffold protein that nin (27). This strategy resulted in the identification of a PDZ1- elicits its diverse functions by physically interacting with subsets specific binding small molecule, PDZ1i, that in micromolar doses of unique proteins in different regions of the cell (6, 8, 10). We could modify the ability of IGF-1R to bind to MDA-9/Syntenin

www.aacrjournals.org Mol Cancer Ther; 18(11) November 2019 2003

Das et al.

A 15 m 45 m 120 m 300 m B C DMSO PDZ1i DMSO PDZ1i 300 2.5

250

2.0 DMSO

200

1.5 ×105

150

1.0

100

0.5 PDZ1i 50

DMSO PDZ1i DMSO PDZ1i 150 150 Metastatic Experimental groups 100 sites 100 Control PDZ1i P < 0.01 P < 0.01 Lung 7/8 5/8 50 50 Femur 6/8 0/8

Jaw bone 2/8 1/8 Percentage of survival of Percentage

0 survival of Percentage 0 0 50 100 150 0 50 100 150 200 Days Days

Figure 5. PDZ1i suppresses prostate cancer metastasis and tumor progression. A, Representative temporal BLI images of DMSO- or PDZ1i–pre-treated ARCaP-M-Luc cells injected intravenously into athymic nude mice. B, Effect of PDZ1i in an experimental metastasis model. Athymic nude mice were injected intravenously with (n ¼ 5, each group) ARCaP-M-Luc (1 106 cells in 100 mL saline). Mice received either vehicle or PDZ1i every alternate day (9 injections during the first three months) and maintained until they required euthanization. Representative BLI image (at day 45) is presented in the top. Kaplan–Meier survival curve was prepared using GraphPad software and is presented in the bottom. C, PC-3ML-Luc cells were injected through intracardiac route in athymic nude mice. Vehicle or PDZ1i was administered by intraperitoneal injection every alternate day (total 3 injections during the first week, total 9 injections within first 3 weeks). BLI imaging was performed at day 36 to monitor metastases. Representative BLI images from each experimental group are presented (top). The metastatic incidence in different sites is presented (bottom).

(Fig. 2). Since our approach did not generate small molecules that IGF-R1 targeting has significant potential, these strategies need specifically bound to the PDZ2 of MDA-9/Syntenin (27, 41), we to be optimized and critically evaluated in vivo due to ubiquitous anticipated and confirmed that in prostate cancer cells PDZ1i did expression and the key roles of IGF-1R in different physiological not modify MDA-9/c-Src binding. This observation, despite the contexts. As documented presently, our PDZ1i small molecule considerable homology (29% sequence identity and 48% rather than targeting IGF-1R directly, inhibits IGF-1R activity by sequence homology) between the PDZ1 and PDZ2 domains of perturbing MDA-9/Syntenin/IGF-1R interactions, which may be MDA-9/Syntenin, supports the specificity of the PDZ1i. However, restricted to its' role in cancer cells. In addition, recent studies in previous studies we demonstrated that both PDZ domains of demonstrate that MDA-9/Syntenin (SDCBP) knockout mice are MDA-9/Syntenin are critical for interacting with c-Src (14), and we viable and in these mice tumor-supporting inflammation is now show that PDZ1i downregulated Src activation, thereby inhibited and melanoma metastasis is suppressed (48). Thus, negatively influencing further downstream signaling, including the activity of MDA-9/Syntenin is dispensable for normal cellular p38 and NF-kB activation in different prostate cancer cells (Fig. 2). functions and MDA-9/Syntenin-targeted molecules are more c-Src can be activated in multiple ways (44, 45) either by specific to neoplastic cells, an important prerequisite for drug autophosphorylation or through various tyrosine kinases, includ- development. In addition, a functional role for MDA-9/Syntenin ing IGF-1R (45, 46). In prostate cancer cells, PDZ1i-mediated in mediating glioma cancer stem cell growth and survival has been downregulation of c-Src activation is partially IGF-1R dependent. demonstrated recently which further supports the involvement of Further studies are warranted to understand precisely how MDA- this gene in regulating cancer phenotypes and its potential as a 9/Syntenin chooses specific partners to complex with under target for therapeutic intervention (49). normal physiological conditions as well as in specific disease In summary, this study confirms that the PDZ1 domain of states such as cancer. MDA-9/Syntenin is "druggable" and it is possible to develop Because of the clinical significance of IGF-1R in prostate can- inhibitors that are unique to this domain of MDA-9/Syntenin cer (47), diverse therapeutic approaches have been tested that and affect its interactions with specific proteins without binding target this protein, including human monoclonal antibodies and or altering protein–protein interactions to the PDZ2 domain of small molecules to inhibit IGF-1R activity through distinct target- this protein. Although it has been 20 years since MDA-9/Syntenin ing approaches, for example, IGF-1R ligands, blocking ligand was cloned, only recently has this gene attracted major attention binding, inhibiting enzymatic activity, etc. Although direct due to its ubiquitous expression in different cancers, and through

2004 Mol Cancer Ther; 18(11) November 2019 Molecular Cancer Therapeutics

Small-Molecule Inhibitor of Prostate Cancer

A CD DMSO PDZ1i P DMSO PDZ1i Luminescence < 0.01 Luminescence 0.2 10,000 1,400

1,200 0.16 8,000 1,000 0.12 6,000 800 0.08 600 4,000

400 0.04

2,000 200 Prostate weight (g) weight Prostate 0 E pIGF-1R pSTAT3 DMSO PDZ1i P < 0.01

0.5 DMSO

DMSO 0.25

DMSO PDZ1i Lung weight Lung

PDZ1i 0 DMSO PDZ1i PDZ1i

F PDZ1i B P < 0.01 100 DMSO PDZ1i Angiogeneic factors MDA-9 50 IGF-1R Metastasis

STAT3 Matrix degrading enzymes

Percentage of survival of Percentage 0 0 5 10 15 20 Days

Figure 6. PDZ1i can efficiently inhibit tumor progression in immunocompetent mice. A and B, RM1-Luc cells were injected through intracardiac route in C57BL/6 mice. Vehicle or PDZ1i was administered intraperitoneally every alternate day [in (A), total 3 injections during the first week; in setting B, mice received vehicle/drug until they were euthanized]. BLI imaging was performed at day 7 (A, top) and the animals were sacrificed on day 14. Lungs were photographed and presented in the bottom left. Average tumor weight is provided in the bottom right. B, Kaplan–Meier survival curve was prepared using GraphPad software. C, Graphical representation of the average prostate weights from control and PDZ1i-treated Hi-Myc mice (top). Photographs of the prostates from mice receiving either vehicle (control) or PDZ1i (bottom). D and E, Photomicrographs representing the histological changes (D) or the expression of indicated proteins (E) in prostate sections obtained from 6-month-old Hi-Myc mice receiving either vehicle (control) or PDZ1i. F, MDA-9/IGF-1R/STAT3 stimulates overall metastasis by upregulating invasion and angiogenesis processes (through enhancing expression of various genes) in prostate cancer cells. The impacts of PDZ1i in these processes are presented schematically. investigations by multiple independent research laboratories actions (50). In these contexts, defining MDA-9/Syntenin inter- defining unique functions in cancer cells (6, 8, 27). In principle, acting partners that are disease-specific and generating small- with appropriate advancement this small molecule (or other molecule drugs that selectively impact on these interactions are potential inhibitors targeting either PDZ1, PDZ2 or both essential to exploit important signaling axes in developing cancer- PDZ domains of MDA-9/Syntenin) could be scaffolds for devel- selective therapies. oping drugs that would be candidates for optimizing "personal- ized medicine" approaches to treat prostate cancer and other Disclosure of Potential Conflicts of Interest cancers. The role of MDA-9/Syntenin in several physiological P.B. Fisher is a co-founder, has ownership interest and was a consultant in processes (6, 50), including exosome biogenesis and pre- Cancer Targeting Systems, Inc. Virginia Commonwealth University, Johns synaptic synapses needs to be considered critically in defining Hopkins University and Columbia University have ownership interest in molecules that will have selective cancer-specific activity. MDA-9/ CTS. P.B. Fisher is a co-founder and has ownership interest in InVaMet Therapeutics, Inc. (IVMT). Virginia Commonwealth University and the Syntenin functions principally through interactions with partner fl Sanford Burnham Prebys Medical Discovery Institute have ownership inter- proteins and these interactions are in uenced by multiple factors estinIVMT.S.K.DasistheP.I.ofanSRAprovidedbyIVMTtoVirginia including phosphorylation status of ligands, disulfide bond for- Commonwealth University. No potential conflicts of interest were disclosed mation, auto-inhibition, competitive binding and allosteric inter- by the other authors.

www.aacrjournals.org Mol Cancer Ther; 18(11) November 2019 2005

Das et al.

Authors' Contributions by funding from NIH grants P50 CA058236 (to P.B. Fisher) and NCI Conception and design: S.K. Das, P.B. Fisher Cancer Center Support Grant to VCU Massey Cancer Center P30 CA016059 Development of methodology: S.K. Das, P.B. Fisher (to P.B. Fisher and D. Sarkar), the National Foundation for Cancer Research Acquisition of data (provided animals, acquired and managed patients, (NFCR; to P.B. Fisher), the Human and Molecular Genetics Enhancement provided facilities, etc.): S.K. Das, T.P. Kegelman, A.K. Pradhan, Fund (to S.K. Das and L. Emdad), VCU Massey Cancer Center (MCC) P. Bhoopathi, S. Talukdar, S. Maji, L. Emdad developmental funds (to P.B. Fisher) and VCU Institute of Molecular Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Medicine (VIMM) developmental funds (to P.B. Fisher, S.K. Das, and computational analysis): S.K. Das, L. Emdad, P.B. Fisher L. Emdad). P.B. Fisher holds the Thelma Newmeyer Corman Chair in Cancer Writing, review, and/or revision of the manuscript: S.K. Das, D. Sarkar, Research in the MCC. D. Sarkar is the Harrison Foundation Distinguished P.B. Fisher Professor in Cancer Research in the MCC. Study supervision: P.B. Fisher The costs of publication of this article were defrayed in part by the payment of Acknowledgments page charges. This article must therefore be hereby marked advertisement in We thank Drs. Maurizio Pellecchia, University of California Riverside, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and Mehmet Kahraman, Ludwig Institute for Cancer Research, UCSD, for valuable discussions and Drs. Maurizio Pellecchia, Bainan Wu, Surya K. De, Angela Purves and Jun Wei for preparing and providing speciﬁc Received September 7, 2018; revised April 10, 2019; accepted July 15, 2019; reagents used in this study. The present research was supported in part published ﬁrst July 25, 2019.

References 1. Joshi G, Singh PK, Negi A, Rana A, Singh S, Kumar R. Growth factors transition by inhibiting caveolin-mediated TGF-beta type I receptor inter- mediated cell signalling in prostate cancer progression: implications in nalization. Oncogene 2016;35:389–401. discovery of anti-prostate cancer agents. Chem Biol Interact 2015;240: 17. Menezes ME, Shen XN, Das SK, Emdad L, Sarkar D, Fisher PB. MDA-9/ 120–33. Syntenin (SDCBP) modulates small GTPases RhoA and Cdc42 via trans- 2. Shen MM, Abate-Shen C. Molecular genetics of prostate cancer: new forming growth factor beta1 to enhance epithelial–mesenchymal transi- prospects for old challenges. Genes Dev 2010;24:1967–2000. tion in breast cancer. Oncotarget 2016;749:80175–89. 3. Lin JJ, Jiang H, Fisher PB. Melanoma differentiation associated gene-9, 18. Boukerche H, Su ZZ, Emdad L, Baril P, Balme B, Thomas L, et al. mda-9/ mda-9, is a human gamma interferon responsive gene. Gene 1998;207: Syntenin: a positive regulator of melanoma metastasis. Cancer Res 2005; 105–10. 65:10901–11. 4. Lin JJ, Jiang HP, Fisher PB. Characterization of a novel melanoma differ- 19. Das SK, Bhutia SK, Sokhi UK, Azab B, Su ZZ, Boukerche H, et al. Raf kinase entiation-associated gene, mda-9, that is down-regulated during terminal inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melano- cell differentiation. Mole Cell Differ 1996;4:317–33. ma. Cancer Res 2012;72:6217–26. 5. Grootjans JJ, Zimmermann P, Reekmans G, Smets A, Degeest G, Durr J, 20. Das SK, Bhutia SK, Azab B, Kegelman TP, Peachy L, Santhekadur PK, et al. et al. Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. MDA-9/Syntenin and IGFBP-2 promote angiogenesis in human melano- Proc Natl Acad Sci U S A 1997;94:13683–8. ma. Cancer Res 2013;73:844–54. 6. Das SK, Sarkar D, Emdad L, Fisher PB. MDA-9/Syntenin: an emerging 21. Ghossoub R, Lembo F, Rubio A, Gaillard CB, Bouchet J, Vitale N, et al. global molecular target regulating cancer invasion and metastasis. Syntenin-ALIX exosome biogenesis and budding into multivesicular bod- Adv Cancer Res 2019;144:137–91. ies are controlled by ARF6 and PLD2. Nat Commun 2014;5:3477 7. Sarkar D, Boukerche H, Su ZZ, Fisher PB. mda-9/syntenin: More than just a 22. Gordon-Alonso M, Rocha-Perugini V, Alvarez S, Moreno-Gonzalo O, Ursa simple adapter protein when it comes to cancer metastasis. Cancer Res A, Lopez-Martin S, et al. The PDZ-adaptor protein syntenin-1 regulates 2008;68:3087–93. HIV-1 entry. Mol Biol Cell 2012;23:2253–63. 8. Kegelman TP, Das SK, Emdad L, Hu B, Menezes ME, Bhoopathi P, et al. 23. Sala-ValdesM,Gordon-AlonsoM,TejeraE,IbanezA,CabreroJR,Ursa Targeting tumor invasion: the roles of MDA-9/Syntenin. Expert Opin Ther A, et al. Association of syntenin-1 with M-RIP polarizes Rac-1 activation Targets 2015;19:97–112. during chemotaxis and immune interactions. J Cell Sci 2012;125: 9. Bacolod MD, Das SK, Sokhi UK, Bradley S, Fenstermacher DA, Pellecchia 1235–46. M, et al. Examination of epigenetic and other molecular factors associated 24. Rajesh S, Bago R, Odintsova E, Muratov G, Baldwin G, Sridhar P, et al. with mda-9/syntenin dysregulation in cancer through integrated analyses Binding to syntenin-1 protein defines a new mode of ubiquitin-based of public genomic datasets. Adv Cancer Res 2015;127:49–121. interactions regulated by phosphorylation. J Biol Chem 2011;286: 10. Das SK, Bhutia SK, Kegelman TP, Peachy L, Oyesanya RA, Dasgupta S, et al. 39606–14. MDA-9/syntenin: a positive gatekeeper of melanoma metastasis. 25. Fujii N, You L, Xu Z, Uematsu K, Shan J, He B, et al. An antagonist of Front Biosci 2012;17:1–15. dishevelled protein-protein interaction suppresses beta-catenin- 11. Das SK, Pradhan AK, Bhoopathi P, Talukdar S, Shen X-N, Sarkar D, et al. dependent tumor cell growth. Cancer Res 2007;67:573–9. MDA-9/Syntenin/IGF-1R/STAT3 axis directs prostate cancer invasion. 26. Daqrouq K, Alhmouz R, Balamesh A, Memic A. Application of wavelet Cancer Res 2018;78:2852–63. transform for PDZ domain classification. PLoS ONE 2015;10:e0122873. 12. Khandwala HM, McCutcheon IE, Flyvbjerg A, Friend KE. The effects of 27. Kegelman TP, Wu B, Das SK, Talukdar S, Beckta JM, Hu B, et al. Inhibition of insulin-like growth factors on tumorigenesis and neoplastic growth. radiation-induced glioblastoma invasion by genetic and pharmacological Endocr Rev 2000;21:215–44. targeting of MDA-9/Syntenin. Proc Natl Acad Sci U S A 2017;114:370–5. 13. Rojas A, Liu G, Coleman I, Nelson PS, Zhang M, Dash R, et al. IL-6 promotes 28. Bhatnagar A, Wang Y, Mease RC, Gabrielson M, Sysa P, Minn I, et al. AEG-1 prostate tumorigenesis and progression through autocrine cross-activation promoter-mediated imaging of prostate cancer. Cancer Res 2014;74: of IGF-IR. Oncogene 2011;30:2345–55. 5772–81. 14. Boukerche H, Su ZZ, Prevot C, Sarkar D, Fisher PB. mda-9/Syntenin 29. Guo C, Yi H, Yu X, Zuo D, Qian J, Yang G, et al. In situ vaccination with promotes metastasis in human melanoma cells by activating c-Src. CD204 gene-silenced dendritic cell, not unmodified dendritic cell, Proc Natl Acad Sci U S A 2008;105:15914–9. enhances radiation therapy of prostate cancer. Mol Cancer Ther 2012; 15. Dasgupta S, Menezes ME, Das SK, Emdad L, Janjic A, Bhatia S, et al. Novel 11:2331–41. role of MDA-9/syntenin in regulating urothelial cell proliferation by 30. Pradhan AK, Bhoopathi P, Talukdar S, Shen XN, Emdad L, Das SK, et al. modulating EGFR signaling. Clin Cancer Res 2013;19:4621–33. Recombinant MDA-7/IL-24 suppresses prostate cancer bone metastasis 16. Hwangbo C, Tae N, Lee S, Kim O, Park OK, Kim J, et al. Syntenin regulates through down regulation of the Akt/Mcl-1 pathway. Mol Cancer Ther TGF-beta1-induced Smad activation and the epithelial-to-mesenchymal 2018;17:1951–1960.

2006 Mol Cancer Ther; 18(11) November 2019 Molecular Cancer Therapeutics

Small-Molecule Inhibitor of Prostate Cancer

31. Ellwood-Yen K, Graeber TG, Wongvipat J, Iruela-Arispe ML, Zhang 41. Das SK, Sarkar D, Cavenee WK, Emdad L, Fisher PB. Rethinking glioblas- J, Matusik R, et al. Myc-driven murine prostate cancer shares toma therapy: MDA-9/Syntenin targeted small molecule. ACS Chem Neu- molecular features with human prostate tumors. Cancer Cell rosci 2019;10:1121–3. 2003;4:223–38. 42. Don-Doncow N, Marginean F, Coleman I, Nelson PS, Ehrnstrom R, 32. Kegelman TP, Das SK, Hu B, Bacolod MD, Fuller CE, Menezes ME, et al. Krzyzanowska A, et al. Expression of STAT3 in prostate cancer metastases. MDA-9/syntenin is a key regulator of glioma pathogenesis. Neuro Oncol Eur Urol 2016;S0302–2838:30288–3. 2013;16:50–61. 43. Shodeinde AL, Barton BE. Potential use of STAT3 inhibitors in targeted 33. Eichten A, Su J, Adler AP, Zhang L, Ioffe E, Parveen AA, et al. Resistance to prostate cancer therapy: future prospects. Onco Targets Ther 2012;5: anti-VEGF therapy mediated by autocrine IL6/STAT3 signaling and over- 119–125. come by IL6 blockade. Cancer Res 2016;76:2327–39. 44. Sekharam M, Nasir A, Kaiser HE, Coppola D. Insulin-like growth factor 1 34. Oyesanya RA, Bhatia S, Menezes ME, Dumur CI, Singh KP, Bae S, et al. receptor activates c-SRC and modiﬁes transformation and motility of colon MDA-9/Syntenin regulates differentiation and angiogenesis programs cancer in vitro. Anticancer Res 2003;23:1517–24. in head and neck squamous cell carcinoma. Oncoscience 2014;1: 45. Sanabria-Figueroa E, Donnelly SM, Foy KC, Buss MC, Castellino RC, 725–37. Paplomata E, et al. Insulin-like growth factor-1 receptor signaling increases 35. Weischenfeldt J, Simon R, Feuerbach L, Schlangen K, Weichenhan D, the invasive potential of human epidermal growth factor receptor 2-over- Minner S, et al. Integrative genomic analyses reveal an androgen-driven expressing breast cancer cells via Src-focal adhesion kinase and forkhead somatic alteration landscape in early-onset prostate cancer. Cancer Cell box protein M1. Mol Pharmacol 2015;87:150–61. 2013;23:159–70. 46. Shin DH, Lee HJ, Min HY, Choi SP, Lee MS, Lee JW, et al. Combating 36. Grasso CS, Wu YM, Robinson DR, Cao X, Dhanasekaran SM, Khan AP, et al. resistance to anti-IGFR antibody by targeting the integrin beta3-Src path- The mutational landscape of lethal castration-resistant prostate cancer. way. J Natl Cancer Inst 2013;105:1558–70. Nature 2012;487:239–43. 47. Heidegger I, Massoner P, Sampson N, Klocker H. The insulin-like growth 37. Baca SC, Prandi D, Lawrence MS, Mosquera JM, Romanel A, Drier Y, factor (IGF) axis as an anticancer target in prostate cancer. Cancer Lett 2015; et al. Punctuated evolution of prostate cancer genomes. Cell 2013;153: 367:113–21. 666–77. 48. Das SK, Guo C, Pradhan AK, Bhoopathi P, Talukdar S, Shen XN, et al. 38. Hong MK, Macintyre G, Wedge DC, Van Loo P, Patel K, Lunke S, et al. Knockout of MDA-9/Syntenin (SDCBP) expression in the microenviron- Tracking the origins and drivers of subclonal metastatic expansion in ment dampens tumor-supporting inﬂammation and inhibits melanoma prostate cancer. Nat Commun 2015;6:6605. metastasis. Oncotarget 2016;7:13. 39. Macfarlane KNC. Research in castration-resistant prostate cancer what does 49. Talukdar S, Das SK, Pradhan AK, Emdad L, Shen XN, Windle JJ, et al. Novel the future hold. Curr Oncol 2010;(Suppl 2):S80–S86. function of MDA-9/Syntenin (SDCBP) as a regulator of survival and 40. Patel JC, Maughan BL, Agarwal AM, Batten JA, Zhang TY, Agarwal N. stemness in glioma stem cells. Oncotarget 2016;7:54102–19. Emerging molecularly targeted therapies in castration refractory prostate 50. Beekman JM, Coffer PJ. The ins and outs of syntenin, a multifunctional cancer. Prostate Cancer 2013;2013:981684. intracellular adaptor protein. J Cell Sci 2008;121:1349–55.

www.aacrjournals.org Mol Cancer Ther; 18(11) November 2019 2007

Suppression of Prostate Cancer Pathogenesis Using an MDA-9/Syntenin (SDCBP) PDZ1 Small-Molecule Inhibitor

Swadesh K. Das, Timothy P. Kegelman, Anjan K. Pradhan, et al.

Mol Cancer Ther 2019;18:1997-2007. Published OnlineFirst July 25, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-1019

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2019/07/25/1535-7163.MCT-18-1019.DC1

Cited articles This article cites 49 articles, 20 of which you can access for free at: http://mct.aacrjournals.org/content/18/11/1997.full#ref-list-1

Citing articles This article has been cited by 1 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/18/11/1997.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/18/11/1997. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.