Autophagy, mTOR inhibitors and phenformin in hepatocellular carcinoma

 描述（由申请人提供）：2012年，肝癌是全球癌症死亡的第二大常见原因。肝细胞癌（HCC）的发病率正在增加，特别是在西方社会，它与肥胖的日益流行有关。尽管索拉非尼等靶向药物最近取得了进展，但晚期肝癌患者的预后仍然很差，这突出了对新疗法的迫切需求。哺乳动物雷帕霉素靶蛋白激酶（mTOR）是HCC中一个有前途的靶点，因为mTOR途径已显示在约50%的HCC中上调，并且mTOR变构抑制剂雷帕霉素已显示抑制肿瘤生长。然而，雷帕霉素在HCC中的临床应用取得了有限的成功，这与我们在HCC小鼠模型中使用雷帕霉素RAD 001的临床前数据一致。在同一研究中，我们发现RAD 001与双重PI 3 K/mTOR抑制剂BEZ 235组合协同抑制mTOR信号传导和HCC肿瘤的生长，这与增加的线粒体自噬相关。这一发现与遗传数据一致，表明肝脏中的自噬可能作为肿瘤抑制因子。然而，最近报道mTOR抑制剂促进CD 133 + HCC干细胞样细胞（HSC）的存活，推测是通过增加自噬。尽管有这种观察结果，双胍类药物苯丙氨酸和二甲双胍激活AMPK，一种上游调节unc-51样激酶（ULK 1）和自噬，据报道，选择性抑制癌症干细胞。在初步研究中，我们着手比较用RAD 001/BEZ 235处理的细胞与用苯丙氨酸处理的细胞中自噬诱导的程度，以及两种处理对HSC存活的影响。出乎意料的是，我们发现苯丙氨酸不仅不能单独诱导自噬，而且还能抑制RAD 001/BEZ 235诱导的自噬。与早期报道一致，RAD 001/BEZ 235处理促进了HSC在源自肝肿瘤的类器官中的存活，这被苯丙氨酸逆转。这些发现使我们假设用组合的变构和ATP位点竞争性抑制剂（mTOR配对抑制剂）治疗HCC有效地防止了大量肿瘤生长，但促进了HSC的自噬依赖性存活，HSC通过抑制自噬对苯丙氨酸敏感，我们将在两个目标中测试。在目标1中，我们将确定苯丙氨酸抑制自噬的机制以及自噬与细胞能量状态之间的关系。我们将确定的步骤（S）的自噬抑制phenylene和AMPK在调节这种反应的作用。在目标2中，我们将确定当同时或顺序给予时，在HCC中将苯丙氨酸与mTOR配对抑制剂组合的治疗优势。我们将用mTOR配对抑制剂在有或没有苯丙氨酸的情况下处理人HSC和小鼠HSC衍生的多群体类器官，以测试它们的治疗响应性。将离体结果与HCC荷瘤小鼠的治疗进行比较，目的是利用人HCC衍生的类器官用于未来的治疗应用。

 DESCRIPTION (provided by applicant): In 2012, liver cancer was the second most common cause of cancer deaths worldwide. The incidence of hepatocellular cancer (HCC) is increasing, particularly in western societies where it is linked to the growing epidemic of obesity. Despite recent advances with targeted drugs like sorafenib, the prognosis for HCC patients with advanced disease remains poor, highlighting the urgent need for new therapies. The mammalian target of rapamycin (mTOR) protein kinase is a promising target in HCC, as the mTOR pathway has been shown to be upregulated in ~50% of HCCs, and the mTOR allosteric inhibitor rapamycin has been shown to inhibit tumor growth. However, the clinical use of rapamycin in HCC has had limited success, in agreement with our preclinical data using the rapamycin RAD001 in a mouse model of HCC. In the same study we found that RAD001 in combination with the dual PI3K/mTOR inhibitor BEZ235 synergistically inhibited mTOR signaling and growth of HCC tumors, which correlated with increased mitophagy. This finding is consistent with genetic data showing that autophagy in liver may act as a tumor suppressor. However, it was recently reported that mTOR inhibitors promote the survival of CD133+ HCC stem-like cells (HSCs), presumably through increased autophagy. Despite this observation, the biguanides phenformin and metformin, which activate AMPK, an upstream regulator of unc-51 like kinase (ULK1) and autophagy, are reported to selectively suppress cancer stem cells. In preliminary studies, we set out to compare the extent of autophagy induction in cells treated with RAD001/BEZ235 to those treated with phenformin, and the effects of the two treatments on the survival of HSCs. Unexpectedly, we found that phenformin not only failed to induce autophagy on its own, but inhibited RAD001/BEZ235 induced autophagy. Consistent with earlier reports, RAD001/BEZ235 treatment promoted the survival of HSCs in organoids derived from liver tumors, which was reversed by phenformin. These findings have led us to hypothesize that treatment of HCC with combined allosteric and ATP-site competitive inhibitors (mTOR paired inhibitors) effectively prevents bulk tumor growth, but promotes the autophagic-dependent survival of HSCs, which are sensitive to phenformin through the inhibition of autophagy, which we will test in two aims. In Aim 1, we will determine the mechanism by which phenformin inhibits autophagy and the relationship between autophagy and cellular energy status. We will determine the step(s) of autophagy inhibited by phenformin and the role of AMPK in regulating this response. In Aim 2, we will determine the therapeutic advantage of combining phenformin with mTOR paired inhibitors in HCC, when given together or sequentially. We will treat human HSCs and mouse HSC derived multi-population organoids with mTOR paired inhibitors with or without phenformin to test their therapeutic responsiveness. The ex vivo results will be compared to treatment of HCC tumor-bearing mice, with the goal of utilizing human HCC-derived organoids for future therapeutic applications.