Andre R. Jordan

The Role of CD44 in Disease Pathophysiology and Targeted Treatment

The cell-surface glycoprotein CD44 is involved in a multitude of important physiological functions including cell proliferation, adhesion, migration, hematopoiesis, and lymphocyte activation. The diverse physiological activity of CD44 is manifested in the pathology of a number of diseases including cancer, arthritis, bacterial and viral infections, interstitial lung disease, vascular disease, and wound healing. This diversity in biological activity is conferred by both a variety of distinct CD44 isoforms generated through complex alternative splicing, posttranslational modifications (e.g., N- and O-glycosylation), interactions with a number of different ligands, and the abundance and spatial distribution of CD44 on the cell surface. The extracellular matrix glycosaminoglycan hyaluronic acid (HA) is the principle ligand of CD44. This review focuses both CD44-hyaluronan dependent and independent CD44 signaling and the role of CD44–HA interaction in various pathophysiologies. The review also discusses recent advances in novel treatment strategies that exploit the CD44–HA interaction either for direct targeting or for drug delivery.

Targeting Hyaluronic Acid Family for Cancer Chemoprevention and Therapy

Hyaluronic acid or hyaluronan (HA) is perhaps one of the most uncomplicated large polymers that regulates several normal physiological processes and, at the same time, contributes to the manifestation of a variety of chronic and acute diseases, including cancer. Members of the HA signaling pathway (HA synthases, HA receptors, and HYAL-1 hyaluronidase) have been experimentally shown to promote tumor growth, metastasis, and angiogenesis, and hence each of them is a potential target for cancer therapy. Furthermore, as these members are also overexpressed in a variety of carcinomas, targeting of the HA family is clinically relevant. A variety of targeted approaches have been developed to target various HA family members, including small-molecule inhibitors and antibody and vaccine therapies. These treatment approaches inhibit HA-mediated intracellular signaling that promotes tumor cell proliferation, motility, and invasion, as well as induction of endothelial cell functions. Being nontoxic, nonimmunogenic, and versatile for modifications, HA has been used in nanoparticle preparations for the targeted delivery of chemotherapy drugs and other anticancer compounds to tumor cells through interaction with cell-surface HA receptors. This review discusses basic and clinical translational aspects of targeting each HA family member and respective treatment approaches that have been described in the literature.

Dietary Supplement 4-Methylumbelliferone: An Effective Chemopreventive and Therapeutic Agent for Prostate Cancer

BACKGROUND Prevention and treatment of advanced prostate cancer (PCa) by a nontoxic agent can improve outcome, while maintaining quality of life. 4-methylumbelliferone (4-MU) is a dietary supplement that inhibits hyaluronic acid (HA) synthesis. We evaluated the chemopreventive and therapeutic efficacy and mechanism of action of 4-MU. METHODS TRAMP mice (7-28 per group) were gavaged with 4-MU (450mg/kg/day) in a stage-specific treatment design (8-28, 12-28, 22-28 weeks). Efficacy of 4-MU (200-450mg/kg/day) was also evaluated in the PC3-ML/Luc(+) intracardiac injection and DU145 subcutaneous models. PCa cells and tissues were analyzed for HA and Phosphoinositide 3-kinase (PI-3K)/Akt signaling and apoptosis effectors. HA add-back and myristoylated Akt (mAkt) overexpression studies evaluated the mechanism of action of 4-MU. Data were analyzed with one-way analysis of variance and unpaired t test or Tukeys multiple comparison test. All statistical tests were two-sided. RESULTS While vehicle-treated transgenic adenocarcinoma of the prostate (TRAMP) mice developed prostate tumors and metastases at 28 weeks, both were abrogated in treatment groups, without serum/organ toxicity or weight loss; no tumors developed at one year, even after stopping the treatment at 28 weeks. 4-MU did not alter the transgene or neuroendocrine marker expression but downregulated HA levels. However, 4-MU decreased microvessel density and proliferative index (P < .0001,). 4-MU completely prevented/inhibited skeletal metastasis in the PC3-ML/Luc(+) model and DU145-tumor growth (85-90% inhibition, P = .002). 4-MU also statistically significantly downregulated HA receptors, PI-3K/CD44 complex and activity, Akt signaling, and β-catenin levels/activation, but upregulated GSK-3 function, E-cadherin, and apoptosis effectors (P < .001); HA addition or mAkt overexpression rescued these effects. CONCLUSION 4-MU is an effective nontoxic, oral chemopreventive, and therapeutic agent that targets PCa development, growth, and metastasis by abrogating HA signaling.

Antitumor activity of sulfated hyaluronic acid fragments in pre-clinical models of bladder cancer

Tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into angiogenic fragments (AGF: 10-12 disaccharides). AGF support tumor growth and progression. Urine and tissue HAase/HYAL-1 levels are sensitive markers for high-grade bladder cancer (BCa) and its metastasis. In preclinical models of BCa, we evaluated whether o-sulfated AGF (sHA-F) inhibits HAase activity and has antitumor activity. At IC50 for HAase activity inhibition (5-20 μg/ml [0.4-1.7 μM]), sHA-F significantly inhibited proliferation, motility and invasion of HYAL-1 expressing BCa cells (253J-Lung, HT1376, UMUC-3), P<0.001. sHA-F did not affect the growth of HYAL-1 non-expressing BCa (5637, RT4, T24, TCCSUP) and normal urothelial (Urotsa, SV-HUC1) cells. sHA-F treatment induced apoptosis by death receptor pathway. sHA-F downregulated transcript and/or protein levels of HA receptors (CD44, RHAMM), p-AKT, β-catenin, pβ-Catenin(S552), Snail and Twist but increased levels of pβ-Catenin(T41/S45), pGSK-3α/β(S21/S9) and E-cadherin. sHA-F also inhibited CD44/Phosphoinositide 3-kinase (PI-3K) complex formation and PI-3K activity. AGF addition or myristoylated-AKT overexpression attenuated sHA-F effects. Contrarily, HYAL-1 expression sensitized RT4 cells to sHA-F treatment. In the 253J-L and HT1376 xenograft models, sHA-F treatment significantly inhibited tumor growth (P<0.001), plausibly by inhibiting angiogenesis and HA receptor-PI-3K/AKT signaling. This study delineates that sHA-F targets tumor-associated HA-HAase system and could be potentially useful in BCa treatment.

Hyaluronic acid family in bladder cancer: potential prognostic biomarkers and therapeutic targets

Background:Molecular markers of clinical outcome may aid in designing targeted treatments for bladder cancer. However, only a few bladder cancer biomarkers have been examined as therapeutic targets.Methods:Data from The Cancer Genome Atlas (TCGA) and bladder specimens were evaluated to determine the biomarker potential of the hyaluronic acid (HA) family of molecules – HA synthases, HA receptors and hyaluronidase. The therapeutic efficacy of 4-methylumbelliferone (4MU), a HA synthesis inhibitor, was evaluated in vitro and in xenograft models.Results:In clinical specimens and TCGA data sets, HA synthases and hyaluronidase-1 levels significantly predicted metastasis and poor survival. 4-Methylumbelliferone inhibited proliferation and motility/invasion and induced apoptosis in bladder cancer cells. Oral administration of 4MU both prevented and inhibited tumour growth, without dose-related toxicity. Effects of 4MU were mediated through the inhibition of CD44/RHAMM and phosphatidylinositol 3-kinase/AKT axis, and of epithelial–mesenchymal transition determinants. These were attenuated by HA, suggesting that 4MU targets oncogenic HA signalling. In tumour specimens and the TCGA data set, HA family expression correlated positively with β-catenin, Twist and Snail expression, but negatively with E-cadherin expression.Conclusions:This study demonstrates that the HA family can be exploited for developing a biomarker-driven, targeted treatment for bladder cancer, and 4MU, a non-toxic oral HA synthesis inhibitor, is one such candidate.

MP39-15 SDCT2 AS A FUNCTIONAL BIOMARKER OF RENAL CELL CARCINOMA

patients were stained for iron and TfR1 protein using Prussian Blue and immunohistochemistry, respectively. 178 tissue cores from 14 different body sites of non-cancer patients were included as controls. Staining was scored by a clinically blinded genitourinary pathologist based on the product of intensity and tissue percentage (Z-score), and tested for association with clinicopathologic features and survival using a MannWhitney U test, Kruskal Wallis test and Cox regression model. RESULTS: Renal epithelium from non-cancer patients had low iron content (mean Z-score,MZS1⁄4 0.1), but by far the highest TfR1 levels of any tissue site in the body (MZS1⁄4 153). Compared to non-cancer patients, iron content in RCC patients increased mildly (4-fold) in benign renal epithelium (MZS1⁄4 0.6) and dramatically (>100 fold) in primary tumors (MZS1⁄4 21, p< 0.001). Higher tumor iron content was accompanied by moderate TfR1 downregulation (MZS1⁄4 21, p< 0.001) and associated with clear cell and papillary histologies, male gender and tobacco usage (p< 0.05 each). Opposite to changes observed with tumorigenesis, iron and TfR1 levels decreased and increased, respectively, with progression in tumor size, grade, pT stage andmetastatic stage (all p< 0.05). Iron loss andTfR1upregulationweremost apparent inmetastatic lesions (MZS1⁄45 and 111, respectively) and each associated with patient anemia and worse RCC-specific survival (all p< 0.05). CONCLUSIONS: Benign renal epithelium has uniquely high levels of the iron import protein, TfR1, potentially priming these cells for dysregulated iron uptake and large intracellular iron increases (>100 fold) during tumorigenesis. Reduction in iron content during RCC progression to metastasis, despite TfR1 increases, may reflect lower systemically available iron in advanced RCC patients and raises the possibility that these cancers might have increased susceptibility to iron deprivation as a novel therapeutic strategy.

Abstract 2647: Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells

INTRODUCTION AND OBJECTIVE: Tumor cell-derived hyaluronidase HYAL1, which degrades hyaluronic acid (HA) into angiogenic fragments, promotes tumor growth and metastasis. HYAL-1 is an independent prognostic marker for predicting metastasis. Small molecular mass sulfated hyaluronic acid (sHA) derivatives (sHA8k) inhibit HYAL1 activity through a mixed inhibition mechanism. Antitumor activity of sHA8k and mechanism of action was evaluated in BCa models.. METHODS: Effect of sHA8k (0-40 ug/ml) on cell proliferation and apoptosis was examined in BCa cells (253J-Lung, HT1376, UMUC-3, T24, RT4) by cell counting and Cell Death ELISA kit. Matrigel invasion and Boyden chamber assays were used to test the effect of sHA8k on invasive activity. Effect of sHA on signaling, apoptosis cascade, HA receptor (CD44, RHAMM), EMT markers (β-catenin, E-cadherin, Snail, Twist) levels, was evaluated by Q-pCR and immunoblotting assays. Angiogenic fragment addition and mAkt transfection were performed to elucidate mechanism of action. Athymic mice bearing 253J-Lung xenografts were treated with sHA (25 and 50 mg/kg) by i.p. injection. RESULTS: sHA8k inhibited proliferation, motility and invasion in BCa cells that expressed HYAL-1. At IC50 for HAase activity inhibition (∼ 20-μg/ml), sHA induced > 3-fold apoptosis and inhibited invasive activity of BCa cells. sHA8k induced caspase-3, -8, -9 activation, up-regulation of Fas, Fas-L, FADD, DR4, DR5 and E-cadherin. sHA8k downregulated CD44, RHAMM, bcl-2, phospho(p)-Akt, pGSK3β, pβcatenin(ser552), snail and Twist expression. Effect of sHA8k were attenuated by angiogenic HA fragments or overexpression of m-Akt and downregulation of CD44 and RHAMM mimicked sHA8k effects. sHA significantly inhibited 253J-Lung xenograft growth. The majority of the animals did not form palpable tumors at 50-mg/kg dose. No weight loss or serum and organ toxicity was observed in sHA treated animals. Biochemical analysis of tumors also showed the same alterations in Akt and EMT pathways as observed cell culture. CONCLUSION: This is the first study that shows sHA8k, a small molecular mass HYAL-1 inhibitor has potent antitumor activity. Support: R01 CA 72821-14 (VBL) Citation Format: Andre R. Jordan, Juan Chipollini, Luis Lopez, Travis Yates, Vinata Lokeshwar. Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2015-2647