Autophagy, mTOR inhibitors and phenformin in hepatocellular carcinoma

 描述(申请人提供)：2012年，肝癌是全球癌症死亡的第二大常见原因。肝细胞癌的发病率正在增加，特别是在西方社会，在那里它与日益流行的肥胖症有关。尽管索拉非尼等靶向药物最近取得了进展，但患有晚期疾病的肝细胞癌患者的预后仍然很差，这突显了对新疗法的迫切需要。哺乳动物靶标雷帕霉素(MTOR)蛋白激酶是肝癌治疗的一个有前景的靶点，因为mTOR通路在~50%的肝癌细胞中上调，mTOR变构抑制剂雷帕霉素被证明能抑制肿瘤生长。然而，雷帕霉素在肝癌的临床应用取得了有限的成功，这与我们在小鼠肝癌模型中使用雷帕霉素RAD001的临床前数据是一致的。在同一项研究中，我们发现RAD001与双重PI3K/mTOR抑制剂BEZ235协同抑制mTOR信号转导和肝癌肿瘤的生长，这与增加有丝分裂相关。这一发现与表明肝脏自噬可能起到肿瘤抑制作用的遗传数据是一致的。然而，最近有报道称，mTOR抑制剂可能通过增加自噬来促进CD133+肝癌干细胞样细胞(HSCs)的存活。尽管有这样的观察，但据报道，双胍类苯福明和二甲双胍激活了UNC-51样激酶(ULK1)的上游调节因子AMPK和自噬，有选择性地抑制癌症干细胞。在初步研究中，我们开始比较RAD001/BEZ235和苯福明处理的细胞自噬诱导的程度，以及这两种处理对HSCs存活的影响。出乎意料的是，我们发现苯乙双胍不仅不能单独诱导自噬，而且还能抑制RAD001/BEZ235诱导的自噬。与早先的报道一致，RAD001/BEZ235治疗促进了来源于肝脏肿瘤的器官中HSCs的存活，这一作用可被苯福明逆转。这些发现使我们假设，联合使用变构和ATP位点竞争抑制剂(mTOR配对抑制剂)治疗肝癌有效地防止了实体肿瘤的生长，但促进了HSC的自噬依赖性存活，HSC通过抑制自噬对苯福明敏感，我们将在两个目标中进行测试。在目标1中，我们将确定苯福明抑制自噬的机制以及自噬与细胞能量状态之间的关系。我们将确定苯福明抑制自噬的步骤(S)以及AMPK在调节这一反应中的作用。在目标2中，我们将确定联合使用苯福明和mTOR成对抑制剂在肝癌中的治疗优势，无论是联合给药还是顺序给药。我们将用含或不含苯福明的mTOR成对抑制剂处理人HSC和小鼠HSC衍生的多种群有机化合物，以测试它们的治疗反应性。体外结果将与对肝癌荷瘤小鼠的治疗进行比较，目标是将人类肝癌衍生的有机化合物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.