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.

Purpose: Poor prognosis of patients with muscle-invasive bladder cancer (BC) that often metastasizes drives the need for discovery of molecular determinants of BC progression.Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in BC. Experimental design:In clinical specimens HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, immunohistochemistry and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder.Results: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In BC tissues, V1 levels associated with metastasis and cancer-specificsurvival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing BC cells formed tumors at low-density and formed metastatic bladder tumors when implanted orthotopically. Conclusion:Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant BC and potential predictor of outcome in BC patients.

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Fanconi Anemia (FA), results from mutations in genes necessary for DNA damage repair and often leads to progressive bone marrow failure. Although the exhaustion of the bone marrow leads to cytopenias in FA patients as they age, evidence from human FA and mouse model fetal livers suggests hematopoietic defects originate in utero which may lead to deficient seeding of the bone marrow. To address this possibility, we examined the consequences of loss of Fancd2, a central component of the FA pathway. Examination of E14.5 Fancd2 knockout (KO) fetal livers showed a decrease in total cellularity and specific declines in long-term and short-term hematopoietic stem cell (LT- and ST-HSC) numbers. Fancd2 KO fetal liver cells display similar functional defects to Fancd2 adult bone marrow cells including reduced colony forming units, increased mitomycin C sensitivity, increased LT-HSC apoptosis and heavily impaired competitive repopulation, implying these defects are intrinsic to the fetal liver and not dependent on the accumulation of DNA damage during aging. Telomere shortening, an aging-related mechanism proposed to contribute to HSC apoptosis and bone marrow failure in FA, was not observed in Fancd2 KO fetal livers. In summary, loss of Fancd2 yields significant defects to fetal liver hematopoiesis, particularly the HSC population, which mimic key phenotypes from adult Fancd2 KO bone marrow independent of aging-accrued DNA damage.

Gemcitabine-based chemotherapy regimens are first-line for several advanced cancers.Due to better tolerability, Gemcitabine+Cisplatin is a preferred neoadjuvant, adjuvant, and/or palliative chemotherapy regimen for advanced bladder cancer (BC). Nevertheless, predicting treatment failure and overcoming resistance remain as unmet clinical needs. We discovered that splice variant (V1) of HYAL-4 is a first-in-class eukaryotic chondroitinase (Chase), and CD44 is its major substrate. V1 is upregulated in BC and drives a malignant phenotype. In this study, we investigated whether V1 drives chemotherapy resistance. Experimental design: V1 expression was measured in muscle-invasive BC (MIBC) specimens by RT-qPCR and immunohistochemistry. HYAL-4 wildtype (Wt) and V1 were stably expressed or silenced in normal urothelial and three BC cell lines. Transfectants were analyzed for chemoresistance and associated mechanism in preclinical models. Results: V1 levels in MIBC specimens of patients who developed metastasis, predicted response to Gemcitabine+Cisplatin adjuvant/salvage treatment and disease-specific mortality. V1-expressing bladder cells were resistant to Gemcitabine but not to Cisplatin. V1 expression neither affected Gemcitabine influx nor the drug-efflux transporters. Instead, V1 increased Gemcitabine metabolism and subsequent efflux of difluorodeoxyuridine, by upregulating cytidine deaminase (CDA) expression through increased CD44-JAK2/STAT3 signaling. CDA inhibitor Tetrahydrouridine resensitized V1-expressing cells to Gemcitabine. While Gemcitabine (25-50-mg/kg) inhibited BC xenograft growth, V1-expressing tumors were resistant. Low-dose combination of Gemcitabine and Tetrahydrouridine abrogated the growth of V1 tumors with minimal toxicity. Conclusion:V1/Chase drives Gemcitabine resistance and potentially predicts Gemcitabine+Cisplatin failure. CDA inhibition re-sensitizes V1-expressing tumors to Gemcitabine.Since several chemotherapy regimens include Gemcitabine, our study could have broad significance.Research.

The purpose of this study was to investigate how CD44 impaired Akt phosphorylation, EGR-1 expression and cell proliferation. E6.1 Jurkat cells, which lack endogenous CD44 expression, were engineered to express CD44. Previously we showed that Akt is hypophosphorylated, EGR-1 expression is reduced and proliferation is impaired in CD44 expressing E6.1 Jurkat cells. The cell cycle was studied using flow cytometry and the role of calcium (Ca2+) in Akt phosphorylation and EGR-1 expression was investigated using Western blotting. Phosphatase activity was assessed using a commercially available kit. CD44 expressing cells showed disruption at the G1 to S transition. Chelation of Ca2+ from the culture media impaired Akt phosphorylation and EGR-1 expression in both CD44 expressing cells and the open vector control. Moreover, Ni2+ disrupted cell proliferation in both cell types suggesting Ca2+ import through calcium release activated calcium channels (CRAC). Staining of cells with fura-2 AM showed significantly higher Ca2+ in CD44 expressing cells as compared with the vehicle control. Finally, non-calcium mediated phosphatase activity was significantly greater in CD44 expressing cells. We propose that the enhanced phosphatase activity in the CD44 cells increased the dephosphorylation rate of Akt; at the same time, the increased intracellular concentration of Ca2+ in the CD44 cells ensured that the phosphorylation of Akt remains intact albeit at lower concentrations as compared with the vector control. Reduced Akt phosphorylation resulted in lowered expression of EGR-1 and hence, reduced the cell proliferation rate.

<p>Four Supplementary Figures and Seven Supplementary Tables</p>