The role of PTEN and AKT2 in the malignant transformation of liver progenitor cel

描述(申请人提供)：本项目的长期目标是阐明肝癌发生的分子机制。为此，我们开发并鉴定了一种携带肿瘤抑制基因缺失的小鼠模型，该肿瘤抑制基因在人类肝癌中经常发生突变(高达44%)。这些小鼠缺乏肿瘤抑制基因PTEN(10号染色体上缺失的磷酸酶和紧张素同源物)，表现出从脂肪肝到纤维化再到肝癌和肺转移的进展，这在大多数人类患者中都能观察到。我们的初步数据显示，在Pten Null模型中，具有祖细胞特性的细胞在肿瘤发展之前就发生了大规模的扩张。我们在癌前阶段分离了这些祖细胞，并使用异种移植模型发现它们是肿瘤起始细胞(TICS)。除了分离这些细胞外，我们还开始研究导致它们生长和转化的机制。AKT是PTEN信号通路中最具代表性的下游效应分子。我们意外地发现，AKT2的缺失抑制了Pten缺失小鼠肿瘤的发展。AKT2以其在代谢调节中的作用而不是AKT1，而AKT1以其在细胞生长/生存中的作用而闻名。当Akt2与Pten同时缺失时，祖细胞激活形态的急剧减少伴随着肝癌发展的显着延迟。我们的分析表明，脂肪肝的损伤是Pten基因缺失小鼠肿瘤发展所必需的，而AKT2可以减轻脂肪肝和损伤。在这个提案中，我们计划研究PTEN缺失/AKT2激活诱导肿瘤发生的分子和细胞机制。在第一个目的中，我们将分析AKT2调节的脂肪生成在肿瘤发生和TIC细胞激活中的作用。我们将使用饮食方法来控制肝脏脂肪变性的发展，并解决当PTEN或PTEN/AKT2丢失时脂肪生成在肿瘤发生中的作用。在第二个目标中，我们将确定AKT2对祖细胞转化的影响。我们将在我们建立的独特的Pten零和Pten/Akt2双零肝祖细胞系中过表达MYR-AKT2和击倒AKT2，并确定这些细胞系的影响 对这些细胞形成克隆、移植肿瘤和增殖能力的操纵。我们还将确定可能支持这种表型的分子机制(典型的PI3K/AKT信号和由AKT2调控的独特的代谢信号)。在最后一个目标中，我们将确定AKT2在Pten缺失观察到的Wnt/？-catenin信号上调中的作用。我们发现，在Pten基因缺失的小鼠中，已知的调节祖细胞活动的途径Wnt/？-catenin被激活。我们推测AKT2可能通过控制Wnt信号来拮抗PTEN对祖细胞激活的影响。这可以通过建立脂肪肝/损伤生态位来诱导Wnt的表达，也可以通过直接调节抽搐中的b-catenin活性来实现。我们将使用TIC培养和体内培养的方法来解决这一目标。综上所述，这三个目标将建立AKT2激活在体内和体外对肝癌发生的因果作用以及这种作用的潜在机制。

DESCRIPTION (provided by applicant): The long term goal of this project is to elucidate the molecular mechanism for liver cancer development. Toward this end, we developed and characterized a mouse model that carries deletion of a tumor suppressor that is often mutated (up to 44%) in human liver cancer. These mice, lacking the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), displayed the progression from fatty liver disease to fibrosis to liver cancer and lung metastasis that has been observed in most human patients. Our preliminary data showed that a massive expansion of cells with progenitor cell properties occurs prior to tumor development in the Pten null model. We isolated these progenitor cells at the pre-malignant stage and found that they are tumor initiating cells (TICs) using xenograft models. In addition to isolating these cells, we have also begun to investigate the mechanism that lead to their growth and transformation. AKT is the best characterized downstream effector molecule of PTEN signaling. We found unexpectedly that loss of AKT2, known for its role in metabolic regulation but not AKT1, known for its role in cell growth/survival inhibits the development of tumors in the Pten null mice. A dramatic reduction in the progenitor cell activation morphology accompanied a significant delay in liver cancer development when Akt2 is simultaneously deleted with Pten. Our analysis shows that injury occurring with fatty liver is necessary for tumor development in the Pten null mice, whereas AKT2 attenuates fatty liver and injury. In this proposal, we plan to investigate the molecular and cellular mechanisms underlying the PTEN loss/AKT2 activation induced tumorigenesis. In the first aim, we will analyze the role of the AKT2 regulated lipogenesis on tumorigenesis and TIC cell activation. We will use dietary means to manipulate liver steatosis development and address the role of lipogenesis in tumorigenesis when PTEN or PTEN/AKT2 is lost. In the second aim, we will determine the effects of AKT2 on the transformation of the progenitor cells. We will overexpress myr-AKT2 and knockdown AKT2 in the unique Pten null and Pten/Akt2 double null liver progenitor cell lines that we have established and determine the effects of these manipulations on the abilities of these cells to form colonies, graft tumors and proliferate. We will additionally determine the molecular mechanisms (both canonical PI3K/AKT signaling and the unique metabolic signaling regulated by AKT2) that may underlie such a phenotype. In the last aim, we will determine the role of AKT2 in the upregulation of Wnt/ß-catenin signaling observed with Pten deletion. We have found that Wnt/ß-catenin, a pathway known to regulate progenitor cell activity, is activated in the Pten null mice. We hypothesize that AKT2 may antagonize the effect of PTEN on progenitor cell activation by controlling Wnt signaling. This may occur either through establishing a fatty liver/injury niche to induce Wnt expression or through directly regulating b-catenin activity within TICs. We will use both in TIC culture and in vivo approaches to address this aim. Together, these three aims will establish the causal effect of AKT2 activation on liver carcinogenesis in vivo and in vitro and the underlying mechanisms for such effect.