Raf Kinase Inhibitory Protein (RKIP): A New Hepatocellular Carcinoma Tumor Suppre

描述（申请人提供）：肝细胞癌（HCC）占原发性肝脏肿瘤的80-90%，是世界范围内最常见、最具破坏性的恶性疾病之一。胰岛素样生长因子（IGF-I和II）通过激活细胞内信号级联反应在HCC中发挥关键作用。其中最主要的是丝裂原活化蛋白激酶（MAPK）信号通路。顶端激酶Raf-1将MAPK通路偶联到细胞外酪氨酸激酶受体。Raf激酶抑制蛋白（RKIP）是一个高度保守的蛋白家族的原型，它直接结合Raf-1和该途径中的下一个激酶MEK，破坏它们的相互作用，并拮抗整个信号级联的激活。在乳腺癌、结肠癌、肝癌和前列腺癌等癌症中，RKIP的表达已被发现减少。尤其引人注目的是这一事件在HCC中的频率：几乎90%的人类HCC标本显示RKIP蛋白表达降低。更令人激动的是，RKIP可能在转移过程中发挥作用，这意味着它是少数已知的转移抑制基因之一。这就提出了调节RKIP蛋白表达或活性可能具有治疗价值的可能性。虽然RKIP蛋白在许多晚期肿瘤中表达明显降低，但很少完全缺失，这些观察结果进一步推动了这些希望。该提案的主要目标是启动小鼠模型的开发，以研究RKIP在体内的HCC肿瘤抑制活性。为了实现这些目标，提出了两个具体目标。第一个将研究下调RKIP的影响。已经存在的RKIP基因敲除将用于确定HCC发生和进展过程中RKIP功能丧失的功能后果。RKIP敲除将与一种令人兴奋的新型小鼠HCC模型相结合：一种具有肝脏特异性表达肝炎病毒Bx转录调节因子（HBx）和胰岛素受体底物-1 （IRS-1）的双转基因模型。HBx/IRS-1动物发展为肝细胞发育不良，发展为HCC。这个模型非常有趣，因为它在小鼠身上概括了人类疾病的许多组织学和分子特征。第二个目的是研究恢复RKIP表达的效果。我们将建立一个新的转基因模型来评估RKIP上调在HCC发生发展过程中的功能影响。该方法将是制造具有肝脏特异性Tet调控RKIP转基因的小鼠。这些研究将为RKIP是否是具有治疗潜力的靶点提供关键的体内功能证据。如果是肯定的，体内数据将证明未来的研究可以详细揭示肝癌和其他癌症中调控RKIP表达和失调的机制。公共卫生相关性：肝细胞癌占肝癌的80-90%，是世界上最常见的恶性肿瘤之一。深入的研究已经发现了一个关键的细胞机制，即MAPK信号通路，在这种毁灭性疾病的发展中的重要性。MAPK通路的主要控制点由一种名为Raf的蛋白激酶调节，新开发的药物Nexavar已成为靶点，该药物在III期临床试验中显示出非凡的前景。已知Raf激酶受其他细胞调节因子的影响，Raf激酶抑制蛋白（RKIP）是其中特别重要的一个。流行病学数据已将RKIP与癌症进展联系起来，这提高了它也可能是一个有价值的治疗靶点的前景。本建议的目的是建立小鼠肝细胞癌模型，使这一假设可以直接得到验证。

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) accounts for 80-90% of primary liver tumors, and is one of the most common and devastating malignant diseases worldwide. Insulin-like growth factors (IGF-I and II) have been shown to play a key role in HCC by activating intracellular signaling cascades. Chief among these is the mitogen-activated protein kinase (MAPK) signaling pathway. Raf-1, the apical kinase, couples the MAPK pathway to extracellular tyrosine kinase receptors. Raf Kinase Inhibitory Protein (RKIP) is the prototype of a highly conserved family of proteins that bind directly to both Raf-1 and the next kinase in the pathway, MEK, disrupting their interaction, and antagonizing the activation of the entire signaling cascade. RKIP expression has been found to be reduced in breast, colon, liver and prostate cancers, among others. What is especially striking is the frequency of this event in HCC: almost 90% of human HCC specimens display reduced RKIP protein expression. Even more provocative are indications that RKIP may play a role in metastatic processes, implicating it as one of only a handful of known metastasis suppressor genes. This raises the possibility that modulation of RKIP protein expression or activity may have therapeutic value. These hopes are further fueled by observations that while RKIP protein expression is clearly reduced in many advanced stage tumors, it is seldom completely absent. The broad objective of this proposal is to initiate the development of mouse models to investigate in vivo the HCC tumor suppressor activity of RKIP. In pursuit of these goals two Specific Aims are proposed. The first will investigate the effect of downregulating RKIP. An already existing RKIP knockout will be used to determine the functional consequences of RKIP loss-of-function during the development and progression of HCC. The RKIP knockout will be combined with an exciting new mouse HCC model: a double transgenic with liver-specific expression of the hepatitis virus Bx transcriptional regulator (HBx) and insulin receptor substrate-1 (IRS-1). HBx/IRS-1 animals develop hepatocellular dysplasia that progresses to HCC. This model is of great interest because it recapitulates in the mouse many of the histological and molecular hallmarks of the human disease. The second aim will investigate the effect of restoring RKIP expression. A new transgenic model will be established to evaluate the functional consequences of upregulating RKIP during the development and progression of HCC. The approach will be to make a mouse with a liver-specific Tet- regulated RKIP transgene. These studies will provide critical in vivo functional evidence whether RKIP is a target with therapeutic potential. If affirmative, the in vivo data will justify future studies to unravel in detail the mechanisms that regulate RKIP expression and dysregulation in HCC as well as other cancers. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinoma accounts for 80-90% of liver cancers, one of the most common malignancies worldwide. Intensive research has discovered the importance of a key cellular mechanism, the MAPK signaling pathway, in the development of this devastating disease. The main control point of the MAPK pathway, regulated by a protein kinase called Raf, has been targeted by the newly developed drug Nexavar, which has shown extraordinary promise in stage III clinical trials. Raf kinase is known to be influenced by other cellular regulators, Raf Kinase Inhibitory Protein (RKIP) being an especially important one. Epidemiological data has linked RKIP to cancer progression, raising the prospect that it may also be a valuable therapeutic target. The objective of this proposal is to develop mouse models of hepatocellular carcinoma in which this hypothesis can be directly tested.