Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.
Department of Pathology, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, 200030, China.
J Exp Clin Cancer Res. 2019 Apr 11;38(1):154. doi: 10.1186/s13046-019-1131-1.
Metabolic reprogramming is one of the hallmarks of cancer cells. The pentose phosphate pathway (PPP), a branch of glycolysis, is an important metabolic pathway for the survival and biosynthesis of cancer cells. Transketolase (TKT) is a key enzyme in the non-oxidative phase of PPP. The mechanistic details of TKT in hepatocellular carcinoma (HCC) development remain unclear.
TKT level and subcellular location were examined in HCC cell lines and tissue samples. We established the TKT overexpression and knocking-down stable cells in HCC cell lines. Proliferation, migration, viability and enzyme activity assays in vitro, tumor growth and metastasis assays in vivo were employed to test the effects of TKT on HCC development. GFP-tagged TKT truncations and mutants were used to locate the nuclear localization sequence (NLSs) of TKT. Cross-linking co-IP/MS was applied to identify the interaction proteins of nuclear TKT.
We showed that TKT increased the proliferation and migration of HCC cells, as well as the viability under oxidative stress in vitro and accelerated the growth and metastasis of HCC cells in vivo. We found as a key enzyme of PPP, TKT could promote the proliferation, cell cycle, migration and viability by regulating the metabolic flux. Moreover, it was firstly reported that unlike other key enzymes in PPP, TKT showed a strong nuclear localization in HCC cells. We found not only high TKT expression, but also its nuclear localization was a prediction for poor prognosis of HCC patients. We further identified the nuclear localization sequences (NLS) for TKT and demonstrated the NLS mutations decreased the pro-tumor function of TKT independent of the enzyme activity. Cross-linking Co-IP/MS showed that nuclear TKT interacted with kinases and transcriptional coregulators such as EGFR and MAPK3, which are associated with cell activation or stress response processes. EGF treatment significantly increased the viability and proliferation of HCC cells in the enzyme-inactivating mutation TKT-D155A overexpression cells but not in the NLS-D155A double mutant group, which could be blocked by EGFR inhibitor erlotinib treatment.
Our research suggests that in addition to the metabolic manner, TKT can promote the development of HCC in a non-metabolic manner via its nuclear localization and EGFR pathway.
代谢重编程是癌细胞的特征之一。戊糖磷酸途径（PPP）是糖酵解的一个分支，是癌细胞存活和生物合成的重要代谢途径。转酮醇酶（TKT）是 PPP 非氧化阶段的关键酶。TKT 在肝细胞癌（HCC）发展中的机制细节尚不清楚。
在 HCC 细胞系和组织样本中检查 TKT 水平和亚细胞定位。我们在 HCC 细胞系中建立了 TKT 过表达和敲低稳定细胞。体外增殖、迁移、活力和酶活性测定、体内肿瘤生长和转移测定用于测试 TKT 对 HCC 发展的影响。使用 GFP 标记的 TKT 截断体和突变体来定位 TKT 的核定位序列（NLS）。交联共免疫沉淀/质谱法用于鉴定核 TKT 的相互作用蛋白。
我们表明，TKT 增加了 HCC 细胞的增殖和迁移，以及体外氧化应激下的活力，并加速了 HCC 细胞在体内的生长和转移。作为 PPP 的关键酶，TKT 可以通过调节代谢通量来促进增殖、细胞周期、迁移和活力。此外，我们首次报道 TKT 在 HCC 细胞中表现出强烈的核定位，而 PPP 的其他关键酶则没有。我们不仅发现 TKT 表达水平高，而且其核定位也是 HCC 患者预后不良的预测因素。我们进一步确定了 TKT 的核定位序列（NLS），并证明 NLS 突变降低了 TKT 的促肿瘤功能，而不依赖于酶活性。交联共免疫沉淀/质谱显示，核 TKT 与 EGFR 和 MAPK3 等激酶和转录共调节剂相互作用，这些激酶和转录共调节剂与细胞激活或应激反应过程有关。EGF 处理显著增加了酶失活突变 TKT-D155A 过表达细胞中的 HCC 细胞活力和增殖，但在 NLS-D155A 双突变体组中没有，这可以被 EGFR 抑制剂厄洛替尼治疗阻断。
我们的研究表明，除了代谢方式外，TKT 还可以通过核定位和 EGFR 途径以非代谢方式促进 HCC 的发展。
