Exploiting Metabolic Changes in HCC for Cancer Therapy

项目总结： 肝细胞癌(HCC)是美国第十大常见癌症，但也是癌症相关死亡的第四大原因。到目前为止，唯一获得FDA批准的分子疗法索拉非尼尚未有效。这项拨款旨在确定己糖激酶2(HK2)在肝癌发生发展中的作用，以及HK2是否为治疗肝癌的可行药物靶点。正常的健康成年肝细胞表达葡糖激酶，这是一种低亲和力的己糖激酶。然而，在向肝硬变和肝细胞癌的病理转变过程中，转化的肝细胞沉默了葡糖激酶并诱导了HK2。我们已经在人类肝细胞癌细胞系和来自153名患者的312个样本的肿瘤微阵列中显示了这种异构体转变。这一转变也代表了一种更普遍的代谢变化，即癌细胞在氧气充足的情况下增加了葡萄糖的利用和乳酸的分泌，也被称为华宝效应。HK2是一种“线粒体”己糖激酶，这意味着它能够与线粒体上的电压依赖性离子通道结合，我们有数据表明这种相互作用对于肿瘤的发生和有效的葡萄糖利用都是必不可少的。这笔赠款的一部分将确定HK2的线粒体相互作用通过什么机制促进肿瘤发生，以及更广泛地说，为什么线粒体结合是完全糖酵解能力所必需的。我们已经证明，超过70%的HK2基因敲除对肝癌细胞是致命的，事实上，另一个小组已经使用CRISPR/Cas9基因敲除筛查表明，HK1或HK2对于癌细胞的生存是必不可少的。由于肝癌细胞只表达HK2，我们认为HK2是治疗肝癌的理想药物靶点。我们将使用已建立的小鼠肝细胞癌模型来确定这种治疗策略的可行性，以确定HK2在肝细胞癌的发生和发展中的细胞自主必要性。我们将在裸鼠皮下移植后，在人肝癌细胞中使用多西环素诱导的靶向HK2的shRNA来模拟药物治疗。我们还将确定靶向线粒体谷氨酰胺代谢是否与HK2抑制协同作用。初步数据表明，NADPH的产生完全依赖于肝癌细胞中苹果酸酶1和谷氨酰胺的代谢。NADPH是癌细胞中脂肪酸合成和氧化还原平衡的重要电子来源。我们将利用谷氨酰胺酶-1抑制剂BPTES或复合体-1抑制剂二甲双胍与HK2抑制相结合来利用这种依赖性。HK2的缺失将阻止磷酸戊糖途径的代偿激活和糖酵解丙酮酸进入线粒体的代偿性通量以获得TCA-通量，从而与BPTES或二甲双胍产生协同作用。

Project Summary: Hepatocellular carcinoma (HCC) is the 10th most common cancer but the fourth leading cause of cancer-related death in the United States. To date, the only FDA-approved molecular therapy, sorafenib, has not been effective. This grant is aimed at determining the role of hexokinase 2 (HK2) in HCC development and progression, and whether or not HK2 is a viable drug target for HCC treatment. Normal healthy adult hepatocytes express glucokinase, a low affinity hexokinase. However, during pathological transition to cirrhosis and HCC, transformed hepatocytes silence glucokinase and induce HK2. We have shown this isoform transition in both human HCC cell lines and in a tumor microarray of 312 samples from 153 patients. This transition also represents a more general metabolic alteration whereby cancer cells increase glucose utilization and lactate secretion despite ample oxygen being present, also known as the Warburg Effect. HK2 is a “mitochondrial” hexokinase, meaning it is capable of binding to the voltage dependent ion channel on the mitochondria, and we have data suggesting this interaction is essential for both tumorigenesis and efficient glucose utilization. Part of this grant will determine by what mechanism the mitochondrial interaction of HK2 promotes tumorigenesis and more generally why mitochondrial binding is necessary for full glycolytic capacity. We have shown that knockdown of HK2 beyond 70% is lethal to HCC cells, and indeed another group has shown that having either HK1 or HK2 is essential for cancer cell viability using a CRISPR/Cas9 knockout screen. Since HCC cells only express HK2, we believe HK2 is an ideal drug target for HCC. We will determine the feasibility of this therapeutic strategy using established mouse models of HCC to determine the cell autonomous necessity of HK2 in HCC initiation and progression. We will emulate drug therapy by using a doxycycline-inducible shRNA targeting HK2 in human HCC cells following subcutaneous implantation in nude mice. We will also determine if targeting mitochondrial glutamine metabolism synergizes with HK2 inhibition. Preliminary data suggests that production of NADPH, an important electron source for fatty acid synthesis and redox balance in cancer cells, is completely dependent on malic enzyme 1 and glutamine metabolism in HCC cells. We will exploit this dependency with a glutaminase-1 inhibitor, BPTES, or a complex-1 inhibitor, metformin in concert with HK2 inhibition. HK2 deletion should prevent compensatory activation of the pentose phosphate pathway and compensatory flux of glycolytic pyruvate into the mitochondria for TCA-flux, creating synergism with BPTES or metformin.