Lactate promoting cancer stem cell phenotype and inducing epithelial-mesenchymal transition

Purpose: Cancer stem cells were considered to be the genesis of cancer and account for cancer initiation, progression, and recurrence. Studies have highlighted a role for Hexokinase 2 (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 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.

Materials and Methods: The endogenous HK2 in human bladder cancer (TCCSUP, J82 and TSGH8301) and normal (SVHUC) cells was examined by immunoblot and immunofluoscence. Effects of lactate exposure on cell proliferation, morphologic change and cancer stem cell phenotype were analyzed in human bladder cancer cells. Stable HK2-overexpression and –knockdown clones were also examined for their effects on EMT, lactate secretion, 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 cells. 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-overexpression cancers compared with mock counterparts (survival, P = 0.034). In animal models, HK2-overexpression cancers also induced morphologic change and CD133 activity.

Conclusion: 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.