Hexokinase 2透過Lactate/NF-κB/Twist1/EMT路徑促進乳酸分泌去誘導膀胱癌轉移和增強hTERT驅動之溶瘤病毒療法

謝宜岑¹、蕭璦莉²、吳昭良³、蘇秉驊⁴、謝嘉興⁵

¹國立成功大學醫學院附設醫院 教學中心；²國立成功大學 微生物及免疫學研究所，³生物化學暨分子生物學研究所；⁴臺北醫學大學 呼吸治療學系；⁵衛生福利部臺南醫院 泌尿科

Hexokinase 2 drives lactate secretion to induce bladder cancer metastasis via the lactate/NF-κB/Twist1/EMT pathway and enhance hTERT-driven oncolytic virotherapy

Yi-Tsen Hsieh¹, Ai-Li Shiau², Chao-Liang Wu³, Bing‐Hua Su⁴, Gia-Shing Shieh⁵

¹Education Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan Taiwan; ²Department of Microbiology and Immunology and ³Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; ⁴School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan; ⁵Department of Urology, Tainan Hospital, Ministry of Health and Welfare, Executive Yuan, Tainan, Taiwan

Purpose: We investigate the role of HK2-induced lactate in the tumor microenvironment in promoting metastasis and epithelial–mesenchymal transition (EMT), as well as in enhancing hTERT-driven oncolytic virotherapy (Ad5GS1) in bladder cancer.

Materials and Methods: We analyzed survival and pathological correlations with HK2 in 144 bladder cancer patients. 14 patients underwent pre-surgery [18F] fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) scans to correlate HK2 expression with uptake values. Urine lactate levels were quantified using ELISA, and the effects of shRNA-mediated HK2 knockdown on cell proliferation, expression of EMT markers, and Twist1 activity were investigated in vitro. Mice bearing HK2-knockdown human bladder tumors were used to examine the survival and cancer metastasis. For oncolytic virotherapy experiments, intratumoral administration of Ad5GS1 was performed to evaluate its tumor-selective oncolytic effect in immunocompetent mice bearing bladder tumors.

Results: Expression of HK2 in bladder cancer was positively correlated with advanced tumor stages and poorer survival. Elevated HK2 levels were associated with higher FDG-PET/CT standard uptake values (SUVs) and urine lactate levels. Urine lactate levels were correlated with tumor stage. Knockdown of HK2 reduced cancer cell proliferation, invasion, lactate extrusion, NF-κB phosphorylation (p-NF-κB), and Twist1 activity as well as promoted a shift to epithelial characteristics in vitro. In response to lactate exposure, nuclear translocation of p-NF-κB and Twist1 as well as mesenchymal markers were promoted in bladder cancer cells. HK2-knockdown bladder tumors reduced growth and metastasis in mice. HK2-knockdown cancers also diminished mesenchymal markers and nuclear translocation of p-NF-κB and Twist1. As p-NF-κB upregulation, which was induced via microenvironment lactate, played the activator in hTERT promoter, intratumoral administration of Ad5GS1 significantly inhibited tumor growth and prolonged the survival time in mice bearing bladder tumors.

Conclusion: HK2 expression in bladder cancer increases lactate production and promotes metastasis through EMT and invasion pathways mediated by p-NF-κB and Twist1. Urine lactate levels and SUVs on FDG-PET/CT, reflecting HK2 activity, can serve as diagnostic and prognostic tools. Targeting HK2/lactate shuttling/p-NF-κB/Twist1/EMT pathways may offer new therapeutic avenues. Moreover, as lactate/p-NF-κB is activated, the oncolytic effect of Ad5GS1 provides multiple safety features for tumor-selective killing and yields a therapeutic benefit in the syngeneic murine tumor model.