Protein Kinase C Signaling Mechanisms in Cancer

描述(由申请人提供)：我们的长期目标是阐明导致癌症的蛋白激酶C(PKC)信号机制，并将这些机制见解转化为更好的预后和治疗策略。在之前的资助期间，我们发现PKCι是美国癌症死亡的主要原因之一的非小细胞肺癌的癌基因，阐明了主要的致癌PKCι信号通路，并开发了一种针对致癌PKCι信号的治疗剂，目前正在临床上进行评估。在本资助期间，我们发现：1)PKCι在非小细胞肺癌细胞胞浆中形成致癌的PKCι/PAR6信号复合体，对细胞的体外增殖、侵袭和体内肿瘤的形成是必需的；2)鸟嘌呤核苷酸交换因子ECT2结合PKCι/PAR6复合体并激活该复合体的关键下游效应因子RAC1；3)PKCι通过直接结合和磷酸化来调节ECT2的细胞内定位和致癌活性；4)基质金属蛋白酶10(MMP10)是PKCι/ECT2/PAR6/RAC1信号轴的下游效应因子，在非小细胞肺癌细胞体外增殖、侵袭和Kras介导的体内肺肿瘤发生中起重要作用；5)Kras介导的细支气管肺泡干细胞(BASCs)体内转化需要基质金属蛋白酶ι和MMP10。我们的初步研究表明：1)PKCι/ECT2/PAR6/RAC1/MMP10信号轴在非小细胞肺癌细胞中维持肿瘤起始细胞的表型，其特征是干细胞样行为和增强的致瘤性；2)大量的细胞ECT2以PKCι依赖的方式定位在核仁上，在那里它调节核糖体ι(RRNA)的转录；3)PKCι转录激活非小细胞肺癌肿瘤起始细胞中的细胞自主刺激性(HH)信号；以及4)PKC RNA2调节干细胞多能因子Sox2到编码Hedgehog Acyl Transferase(HHAT)基因的启动子区域的招募，HHAT是一种催化HH产生和连接的关键步骤的酶。基于这些数据，我们假设：1)PKCι介导的转化涉及ECC2核仁定位和核糖体前核糖体合成的调节；2)ECC2信号对于Kras介导的基底细胞转化和体内肺肿瘤的发生是必需的；3)PKCι至少部分地通过SOX2介导的诱导HHAT转录和激活细胞自主HH信号轴来维持肺肿瘤起始细胞的表型；以及4)HHAT是PKCι依赖的转录靶点，在体内肺肿瘤起始活性中发挥关键作用。这些假说将通过四个相互关联的特定目标的完成来检验：1)确定PKCι和ECT2调控核糖体ι转录的机制；2)评估ECC2在Kras介导的肺肿瘤发生中的作用；3)确定PKC RNARNA调控刺猬酰基转移酶(HHAT)表达的机制；以及4)评估HHAT在肺肿瘤发生中的作用。

DESCRIPTION (provided by applicant): Our long-term goals are to elucidate protein kinase C (PKC) signaling mechanisms that contribute to cancer and translate these mechanistic insights into better prognostic and treatment strategies. In previous funding periods, we discovered that PKCι is an oncogene in non-small cell lung cancer (NSCLC) the leading cause of cancer death in the United States, elucidated a major oncogenic PKCι signaling pathway, and developed a therapeutic agent that targets oncogenic PKCι signaling that is currently being evaluated in the clinic. During the current funding period we showed that: 1) PKCι forms an oncogenic PKCι/Par6 signaling complex in the cytoplasm of NSCLC cells that is necessary for cell proliferation and invasion in vitro, and tumor formation in vivo; 2) the guanine nucleotide exchange factor (GEF) Ect2 binds the PKCι/Par6 complex and activates Rac1, a key downstream effector of this complex; 3) PKCι regulates the intracellular location and oncogenic activity of Ect2 through direct binding and phosphorylation; 4) matrix metalloproteinase 10 (Mmp10) is a critical downstream effector of the PKCι/Ect2/Par6/Rac1 signaling axis that is required for NSCLC cell proliferation and invasion in vitro, and Kras-mediated lung tumorigenesis in vivo; and 5) both PKCι and Mmp10 are required for Kras-mediated transformation of bronchio-alveolar stem cells (BASCs), putative lung tumorinitiating cells (TICs) in vivo. Our preliminary studies indicate that: 1) the PKCι/Ect2/Par6/Rac1/Mmp10 signaling axis maintains a tumor-initiating cell phenotype in NSCLC cells characterized by stem-like behavior and enhanced tumorigenicity; 2) a significant pool of cellular Ect2 localizes to the nucleolus in a PKCι- dependent manner where it regulates ribosomal RNA (rRNA) transcription; 3) PKCι transcriptionally activates cell autonomous hedgehog (Hh) signaling in NSCLC tumor-initiating cells; and 4) PKCι regulates recruitment of the stem cell pluripotency factor Sox2 to th promoter region of the gene encoding Hedgehog Acyl Transferase (HHAT), an enzyme that catalyzes a key step in the production of Hh ligand. Based on these data, we hypothesize that: 1) PKCι-mediated transformation involves regulation of Ect2 nucleolar localization and pre-ribosomal RNA synthesis; 2) Ect2 signaling is required for Kras-mediated BASC transformation and lung tumorigenesis in vivo; 3) PKCι maintains a lung tumor-initiating cell phenotype, at least in part, through Sox2-mediated induction of HHAT transcription and activation of a cell autonomous Hh signaling axis; and 4) HHAT, a PKCι-dependent transcriptional target, plays a key role in lung tumor-initiating activity in vivo. These hypotheses will be tested through completion of four interrelated specific aims to: 1) determine the mechanism by which PKCι and Ect2 regulate ribosomal RNA transcription; 2) assess the role of Ect2 in Kras-mediated lung tumorigenesis; 3) determine the mechanism by which PKCι regulates hedgehog acyl-transferase (HHAT) expression; and 4) assess the role of HHAT in lung tumorigenesis.