HYAL4-V1/Chondroitinase (Chase) Drives Gemcitabine Resistance and Predicts Chemotherapy Failure in Patients with Bladder Cancer.

PURPOSE: Gemcitabine-based chemotherapy regimens are first-line for several advanced cancers. Because of better tolerability, gemcitabine + cisplatin is a preferred neoadjuvant, adjuvant, and/or palliative chemotherapy regimen for advanced bladder cancer. Nevertheless, predicting treatment failure and overcoming resistance remain 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 bladder cancer and drives a malignant phenotype. In this study, we investigated whether V1 drives chemotherapy resistance. EXPERIMENTAL DESIGN: V1 expression was measured in muscle-invasive bladder cancer (MIBC) specimens by qRT-PCR and IHC. HYAL-4 wild-type (Wt) and V1 were stably expressed or silenced in normal urothelial and three bladder cancer 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 bladder cancer xenograft growth, V1-expressing tumors were resistant. Low-dose combination of gemcitabine and tetrahydrouridine abrogated the growth of V1 tumors with minimal toxicity. CONCLUSIONS: V1/Chase drives gemcitabine resistance and potentially predicts gemcitabine + cisplatin failure. CDA inhibition resensitizes V1-expressing tumors to gemcitabine. Because several chemotherapy regimens include gemcitabine, our study could have broad significance.

A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Patients with Bladder Cancer.

PURPOSE: Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer 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 bladder cancer. EXPERIMENTAL DESIGN: In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer 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 bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival 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 bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically. CONCLUSIONS: 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 bladder cancer and potential predictor of outcome in patients with bladder cancer.

Impairment of fetal hematopoietic stem cell function in the absence of Fancd2.

Fanconi anemia (FA) results from mutations in the 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 that 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 embryonic day 14.5 (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-HSC and ST-HSC, respectively) 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 that these defects are intrinsic to the fetal liver and are 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 mimics key phenotypes from adult Fancd2 KO bone marrow independently of aging-accrued DNA damage.

CD44 induced enhancement of phosphatase activity and calcium influx: Modifications of EGR-1 expression and cell proliferation.

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.

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.

Chronic psychological stress suppresses contact hypersensitivity: potential roles of dysregulated cell trafficking and decreased IFN-γ production.

Increasing evidence shows that psychological stress can have dramatic impacts on the immune system, particularly the cutaneous immune response in dermatological disorders. While there have been many studies examining the impact of acute psychological stress on contact hypersensitivity there are relatively few studies concerning the impact of chronic psychological stress. Furthermore, the local immunological mechanisms by which chronic psychological stress impacts contact hypersensitivity still remain to be explored. Here we show that restraint-induced chronic psychological stress stimulates activation of the hypothalamus-pituitary-adrenal axis and delays weight gain in female BALB/c mice. We observed that chronic psychological stress reduces the cutaneous immune response as evidence by reduced ear swelling. This correlated with a significant decrease in the inflammatory cell infiltrate. On the other hand, chronic psychological stress does not influence T cell proliferation, activation, or sensitivity to corticosterone but does increase CD4(+) and CD8(+) T cell percentages in draining lymph nodes during a contact hypersensitivity reaction. Chronic psychological stress induces a decrease in overall circulating white blood cells, lymphocytes, and monocytes during a contact hypersensitivity reaction suggesting extravasation from the circulation. Finally, we found markedly reduced local IFN-γ production in chronically stressed animals. Based on these findings we propose that chronic psychological stress reduces contact hypersensitivity due to dysregulated cell trafficking and reduced production of IFN-γ.