Enormous efforts have been made to target metabolic dependencies of cancer cells for developing new therapies. However, the therapeutic efficacy of glycolysis inhibitors is limited due to their inability to elicit cell death. Hexokinase 2 (HK2), via its mitochondrial localization, functions as a central nexus integrating glycolysis activation and apoptosis resilience. Here we identify that K63-linked ubiquitination by HectH9 regulates the mitochondrial localization and function of HK2. Through stable isotope tracer approach and functional metabolic analyses, we show that HectH9 deficiency impedes tumor glucose metabolism and growth by HK2 inhibition. The HectH9/HK2 pathway regulates cancer stem cell (CSC) expansion and CSC-associated chemoresistance. Histological analyses show that HectH9 expression is upregulated and correlated with disease progression in prostate cancer. This work uncovers that HectH9 is a novel regulator of HK2 and cancer metabolism. Targeting HectH9 represents an effective strategy to achieve long-term tumor remission by concomitantly disrupting glycolysis and inducing apoptosis.

为了开发新的疗法，已经做出了巨大的努力来靶向癌细胞的代谢依赖性。然而，由于糖酵解抑制剂不能引起细胞死亡，因此其治疗功效受到限制。己糖激酶2（HK2）通过线粒体定位，充当整合糖酵解激活和凋亡弹性的中央联系。在这里，我们确定由HectH9与K63连接的泛素化调节HK2的线粒体定位和功能。通过稳定的同位素示踪方法和功能性代谢分析，我们表明，HectH9缺乏症通过HK2抑制作用阻碍肿瘤葡萄糖的代谢和生长。HectH9 / HK2通路调节癌症干细胞（CSC）的扩张和与CSC相关的化学抗药性。组织学分析表明，HectH9表达上调并与前列腺癌的疾病进展相关。这项工作揭示了HectH9是HK2和癌症代谢的新型调节剂。靶向HectH9代表一种有效的策略，可通过同时破坏糖酵解和诱导细胞凋亡来实现长期肿瘤缓解。