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有助于癌症的蛋白激酶C（PKC）信号传导机制，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PKC i是非小细胞肺癌（NSCLC）中的致癌基因，非小细胞肺癌（NSCLC）是美国癌症死亡的主要原因，阐明了主要致癌PKC i信号传导途径，并开发了一种靶向致癌PKC i信号传导的治疗剂，目前正在临床上进行评估。在当前的资助期内，我们显示：1）PKC 1在NSCLC细胞的细胞质中形成致癌的PKC 1/Par 6信号传导复合物，其是体外细胞增殖和侵袭以及体内肿瘤形成所必需的; 2）鸟嘌呤核苷酸交换因子（GEF）Ect 2结合PKC 1/Par 6复合物并激活Rac 1，该复合物的关键下游效应物; 3）PKC 1通过直接结合和磷酸化调节Ect 2的细胞内定位和致癌活性; 4）基质金属蛋白酶10（Mmp 10）是NSCLC细胞体外增殖和侵袭所需的PKC 1/Ect 2/Par 6/Rac 1信号传导轴的关键下游效应物，和Kras介导的肺肿瘤发生;和5）PKC 1和Mmp 10两者都是Kras介导的支气管肺泡干细胞（BASC）（推定的肺肿瘤起始细胞（TIC））体内转化所需的。我们的初步研究表明：1）PKC 1/Ect 2/Par 6/Rac 1/Mmp 10信号传导轴在NSCLC细胞中维持肿瘤起始细胞表型，其特征在于干细胞样行为和增强的致瘤性; 2）细胞Ect 2的显著库以PKC 1依赖性方式定位于核仁，其中其调节核糖体RNA（rRNA）转录; 3）PKC i转录激活NSCLC肿瘤起始细胞中的细胞自主性刺猬（Hh）信号传导;和4）PKC 1调节干细胞多能性因子Sox 2向编码Hedgehog酰基转移酶（HHAT）的基因的启动子区的募集，一种催化Hh配体产生的关键步骤的酶。基于这些数据，我们假设：1）PKC 1介导的转化涉及调节Ect 2核仁定位和前核糖体RNA合成; 2）Ect 2信号传导是体内Kras介导的BASC转化和肺肿瘤发生所需的; 3）PKC 1至少部分地维持肺肿瘤起始细胞表型，通过Sox 2介导的HHAT转录诱导和细胞自主Hh信号传导轴的激活;和4）HHAT，一种PKC 1依赖性转录靶标，在体内肺肿瘤起始活性中起关键作用。这些假设将通过完成四个相互关联的具体目标来测试：1）确定PKC 1和Ect 2调节核糖体RNA转录的机制; 2）评估Ect 2在Kras介导的肺肿瘤发生中的作用; 3）确定PKC 1调节刺猬酰基转移酶（HHAT）表达的机制;和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.