謝嘉興^a、楊文宏^d、蕭璦莉^c、吳昭良^b

衛生福利部 台南醫院 泌尿科^a, 國立成功大學醫學院 泌尿部^d, 國立成功大學醫學院 生化所^b，國立成功大學醫學院 微免所^c

Shieh G-S.^a, Yang W-H.^d, Wu C-L.^b, Shiau A-L.^c

Tainan Hospital, Dept. of Urology, Tainan, Taiwan^a, National Cheng Kung University Hospital, Dept. of Urology, Tainan, Taiwan^d, National Cheng Kung University Hospital, Dept. of Biochemistry and Molecular Biology, Tainan, Taiwan^b, National Cheng Kung University Hospital, Dept. of Microbiology and Immunology, Tainan, Taiwan^c

 

Purpose: Cancer stem cells were considered to be the genesis of cancer and account for cancer initiation, progression, and recurrence. Hexokinase 2 (HK2) catalyzed the first irreversible step of glycolysis and was associated with poor outcomes in cancer patients. Studies have highlighted a role for HK2 in facilitating tumor growth and lactate production, which is downstream product of HK2 reaction in cancer cells. Tumor cells can extrude and shuttle lactate to neighboring cancer cells, adjacent stromal cells, and vascular endothelial cells to induce signaling molecular change. However, in tumor microenvironment, the molecular mechanisms underlying this association of tumor lactate shuttle, HK2 activity and cancer metastases were not well established. In this study, we assessed the contribution of HK2 expression in human bladder cancer samples and the association of survival data, and explored the role of lactate shuttle induced by HK2 in cancer stem cell formation and epithelial-mesenchymal transition (EMT) between bladder cancer cells in vitro and in vivo.

Methods: We examined HK2 expression by immunohistochemistry in tumors (n=151) and normal specimens (n=20) from bladder cancer patients. The association of HK2 with survival and pathological characteristics was assessed using Kaplan-Meier survival curves and two-sided chi-square test. Endogenous HK2 in human bladder cancer (TCCSUP, J82 and TSGH8301) and normal (SVHUC) cells was examined by immunoblot and immunofluoscence. Effects of HK2 overexpression on cell proliferation, morphologic change and cancer stem cell phenotype were analyzed in human bladder cancer TSGH8301 and TCCSUP cells. Stable HK2-overexpression clones were also examined for their effects on EMT, NF-κB phosphorylation and CD133 activity in vitro and mouse models. The animal survival and lung metastasis were assessed in a mouse subcutaneous model using TSGH8301 cells with HK2-overexpression clones. All statistical tests were two-sided.

Results: The HK2 expression was significantly higher in bladder cancers compared with normal tissues. In human bladder cancer samples, 54.1% and 45.9% showed positive and negative HK2 expression, respectively. The urine lactate detection was higher in human bladder cancers than in non-cancer subjects (204.9 vs 54.79 μM; P < 0.001). Stable HK2 overexpression induced cell proliferation, and showed morphologic changes with gain of cancer stem cell markers. HK2 Knockdown also reduced lactate extrusion in vitro. In response to lactate exposure, nuclear translocation of NF-κB phosphorylation and Twist1 as well as mesenchymal markers was promoted in human bladder cancer cells. In addition, lactate exposure enhanced CD133 activity in vitro. In mice bearing subcutaneous tumors, increased tumor growth and lung metastasis were observed in stable HK2-expression cancers compared with mock counterparts (survival, P= 0.034). In animal models, HK2-overexpression cancers also induced morphologic change and CD133 activity.

Conclusions: High HK2 expression in bladder cancers induced oversecretion of lactate, which was associated with metastatic behaviors through the cancer stem cell formation, EMT promotion and nuclear translocation of phosphorylated NF-κB and Twist1. HK2 may be a novel oncoprotein and play as target for bladder cancer therapy.