Presentation Authors: Soum Lokeshwar*, Miami, FL, Daley Morera, Sarrah Lahorewala, Zachary Klaassen, Augusta, GA, Bruce Kava, Miami, FL, Vinata Lokeshwar, Augusta, GA
Introduction: Muscle invasive bladder cancer (MIBC) causes the majority of morbidity and mortality in BCa patients. Prognosis of MIBCa patients may be improved by identification of novel prognostic biomarkers and therapeutic targets. Chondroitin sulfate proteoglycans are known to promote tumor growth and metastasis. However, a Chondroitinase (Chase) that degrades chondroitin sulfate has not been identified. HYAL4, a member of the glycosaminoglycan (GAG) degrading enzyme-family, potentially has Chase activity; however, HYAL4 (HY4) has not be studied in any biological system, normal or disease. In this study, we evaluated the expression of all six members of the GAG-degrading enzyme family in bladder cancer (BCa) specimens and evaluated HY4 functions in preclinical models of BCa.
Methods: Cohort 1: 79 bladder tissues (normal (NBL) = 31; tumor (TBL) = 52); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment for metastatic disease with chemotherapy. Gene expression was measured by q-PCR in both cohorts. HY4 was either overexpressed (HY4) or knockdown in immortalized normal urothelial (Urotsa) and 3 BCa cell lines. Transfectants were assayed for Chase activity, anchorage independent growth, motility, invasion, molecular signaling. Tumor growth and metastasis in intravesical examined in HT1376 bladder orthotopic model.
Results: In cohort 1 HYAL1, HYAL4 mRNA levels were significantly (6-13-fold) elevated in TBL tissues when compared to NBL tissues (P < 0.001). HY4 levels were 7-fold elevated in MIBC. HY4 levels were an independent predictor of metastasis and death due to BCa (chi-sq: 6.9; P=0.0087; chi-sq: 7.59; P=0.006). In cohort 2, high HY4 levels significantly correlated with G+C treatment failure (ï£2 = 8.7; P=0.003) with > 80% accuracy. HY4-expressing transfectants secreted HY4 and Chase activity in their conditioned media. Overexpression of HYAL-4 in normal urothelial and BCa cells significantly increased, while its knockdown abrogated, anchorage-independent growth, invasion, chemotactic motility (> 3-fold). HY4 expression induced Gemcitabine resistance (IC50: vector: 3.1 nM; HY4: 126 nM). HY4 expression upregulated a stem cell signature. Urothelial cells expressing HY4 formed tumors in NOD/SCID mice. HY4-expressing BCa cells metastasized to lung and spleen in an orthotopic BCa model.
Conclusions: This first study on HY4 shows that it is a novel molecular determinant of MIBCa and Gemcitabine resistance and a potential marker for clinical outcome.
Source of Funding: 1R01CA227277-01A1 (VBL); Department of Defense-PRCRP (W81XWH1810277 (CA170470; PRCRP)