The PKCepsilon-regulated oncogenic and tumor suppressor roles of ATF2 in melanoma

PKCepsilon 调节 ATF2 在黑色素瘤中的致癌和抑癌作用

• 批准号: 9330795
• 负责人: Eric Kirk Lau
• 金额: $ 23.94万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-18 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9330795
• 关键词: ATF2 gene; Accounting; Affect; Apoptotic; Automobile Driving; Award; BCL-2 Protein; BCL2 gene; BRAF gene; Binding; Biochemistry; Biological; Biology; Cancer Biology; Cell Death; Cell Proliferation; Cells; Cellular biology; Cessation of life; Clinical; Complex; Critical Pathways; Cutaneous Melanoma; Cytosol; DNA Damage; Data; Development; Diagnosis; Extravasation; Future; Gene Chips; Gene Cluster; Gene Expression Microarray Analysis; Genes; Genetic Transcription; Genotoxic Stress; Grant; Immunotherapy; Institution; Interferon-beta1; Investigation; Lesion; Light; Malignant Neoplasms; Mediating; Medical Research; Melanoma Cell; Mentors; Metabolic; Metabolism; Microarray Analysis; Mitochondria; Modality; Modeling; Molecular; Molecular Profiling; Mus; Non-Malignant; Nuclear; Oncogenes; Oncogenic; Outcome; Outer Mitochondrial Membrane; Output; Pathway interactions; Phase; Phosphorylation; Phosphotransferases; Physiological; Play; Protein Family; Protein Isoforms; Protein Kinase C; Proteins; Regulation; Reporting; Research; Research Institute; Resistance; Resistance development; Respiration; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Skin Cancer; Stress; Technical Expertise; Threonine; Transcription Factor AP-1; Transcriptional Activation; Transcriptional Regulation; Tumor Suppressor Proteins; United States; Voltage-Dependent Anion Channel; Xenograft procedure; arm; base; cancer cell; chemotherapy; cohort; faculty research; hexokinase; in vivo; inhibitor/antagonist; melanoma; metabolomics; mitochondrial membrane; mouse model; mutant; novel; novel therapeutics; nutrient deprivation; outcome forecast; overexpression; prevent; protein kinase C epsilon; response; targeted treatment; therapeutic target; therapy resistant; transcription factor; tumor

DESCRIPTION (provided by applicant): Accounting for an estimated ~70,000 new diagnoses and ~8,800 deaths in 2011, malignant melanoma is the most lethal skin cancer, representing ~8% of total cancers cases in the United States. Elucidation of the molecular mechanisms that drive its development, progression and therapeutic resistance is urgently needed. Activating Transcription Factor 2 (ATF2) is an AP1 transcription factor that functions divergently as an oncogene in melanoma and as a tumor suppressor in nonmalignant skin cancers. How ATF2 plays both functions has remained unclear. I recently found that Protein Kinase C, isoform epsilon (PKCepsilon) phosphorylates ATF2 on a novel phosphoacceptor site (T52), promoting its nuclear localization and transcriptional activation, conferring resistance to genotoxic stress. Thi phosphorylation blocks the ability of ATF2 to translocate to the mitochondria during genotoxic stress. At the mitochondrial outer membrane, ATF2 induces mitochondrial membrane leakage by perturbing hexokinase1 and voltage-dependent anion channel 1-(HK1:VDAC1) containing complexes, and activating pro-apoptotic Bcl2 protein, Bax. Specifically how PKCepsilon phosphorylation affects the transcriptional and non-transcriptional DNA damage response functions of ATF2 is not known. Precisely how ATF2 activates Bax and alters HK1:VDAC1 complexes is not clear. Furthermore, I have observed ATF2 in the cytosol during contexts other than genotoxic stress. Its precise function and biological ramifications (e.g., mitochondrial/metabolic changes) during such contexts is not known. In Aim 1, I propose to determine how phosphorylation by PKCepsilon modulates ATF2 transcriptional activity by modulating its interaction with transcriptional regulators and AP1 partners, as well as its non-transcriptiona function in DNA damage response. I will also investigate how PKCepsilon affects ATF2 transcriptional output programming by investigating hits identified in gene expression microarray analyses that I have now performed on ATF2 mutants that mimic phosphorylation by PKCepsilon in the presence or absence of genotoxic stress. I have found that PKCepsilon phosphorylated ATF2 represses the expression of Interferon Beta 1 (IFNB1) and related downstream targets. IFNB1related signaling is known to suppress cellular proliferation, and its administration can sensitize cancer cells to chemotherapeutics. My preliminary data suggests that melanomas might develop resistance to genotoxic stress by suppressing IFNB1 expression through PKCepsilon ATF2mediated signaling. This raises the exciting notion that the therapeutic targeting of ATF2 in melanomas might derepress IFNB1 expression, rendering the cells sensitive to genotoxic stress (such as that exerted by frontline chemotherapeutic melanoma treatments). I will further investigate how PKCepsilon ATF2 suppresses IFNB1 expression during genotoxic stress. Detailed mechanistic studies of such pathways identified from my expression profiling studies will identify functional clusters of PKCepsilon ATF2regulated genes that are critical for melanoma development and/or therapeutic resistance. I will develop a syngeneic xenograft mouse melanoma model to assess how PKCepsilon phosphoregulation of ATF2 drives melanoma. In Aim 2, I propose to determine how specifically ATF2 activates Bcl2 proteins and modulates HK1:VDAC1 complexes to promote mitochondrial membrane leakage. I will also identify and characterize non-genotoxic stress, physiological conditions, where we have observed ATF2 mitochondrial localization without cell death. I will characterize the resulting biological consequences, such as altered mitochondrial respiration or metabolism, and begin investigation of the molecular mechanism(s) underlying those changes that are mediated by ATF2. By determining how PKCepsilon alters ATF2 transcriptional activity and control of specific downstream genes that are critical for melanoma development and resistance, investigation from Aim 1 may be able to identify novel therapeutic modalities for melanoma. My proposed studies in Aim 2 will provide a better understanding of how ATF2 regulates of the integrity of the mitochondrial outer membrane, both during stress and physiological conditions. The K99/R00 award would allow for me to conduct my proposed studies under the continued guidance of my current mentor, Dr. Ze'ev Ronai, together with a panel of co-mentors who are renowned experts in melanoma and skin cancer biology, transcriptional regulation, PKCmediated signaling, and mitochondrial dynamics and cell death. During the mentored phase, I will complete several arms of Aims 1 and 2 at the Sanford-Burnham Medical Research Institute. During the R00 independent phase, I will independently continue my research at another academic or nonprofit research institution. My R00 phase investigation will further our understanding of how PKCepsilon regulated ATF2 transcription drives melanoma progression and resistance via regulation of IFNB1 (Aim 1 subaim iv and Aim 2, which will begin in the K99 phase and be completed within the R00 phase). I will then focus on other functional gene clusters identified from the expression array analyses in Aim 1. I will further investigate the metabolic ramifications of mitochondrial ATF2 identified in Aim 2. I believe that with my extensive biochemistry and cell biology background, being awarded a K99/R00 grant will promote and expedite the further development of my scientific and technical expertise, and my transition into a successful, independent research faculty in transcriptional control, protein signal transduction, melanoma and cancer.

描述（由申请人提供）：恶性黑色素瘤是最致命的皮肤癌，在2011年估计有约70，000例新诊断和约8，800例死亡，占美国总癌症病例的约8%。迫切需要阐明其发展、进展和耐药性的分子机制。 转录激活因子2（ATF 2）是一种AP 1转录因子，在黑色素瘤中作为癌基因发挥不同的功能，在非恶性皮肤癌中作为肿瘤抑制因子发挥作用。ATF 2如何发挥这两种功能尚不清楚。我最近发现，蛋白激酶C亚型（PKC β）在一个新的磷酸受体位点（T52）磷酸化ATF 2，促进其核定位和转录激活，赋予对遗传毒性应激的抗性。这种磷酸化阻断了ATF 2在遗传毒性应激期间易位至线粒体的能力。在线粒体外膜，ATF 2通过干扰含己糖激酶1和电压依赖性阴离子通道1-（HK 1：VDAC 1）的复合物并激活促凋亡Bcl 2蛋白Bax诱导线粒体膜渗漏。 具体而言，PKC β磷酸化如何影响ATF 2的转录和非转录DNA损伤反应功能尚不清楚。ATF 2如何激活Bax并改变HK 1：VDAC 1复合物的确切机制尚不清楚。此外，我观察到ATF 2在细胞质中的背景下，而不是遗传毒性的压力。它的确切功能和生物学分支（例如，线粒体/代谢变化）的情况是未知的。在目的1中，我建议确定如何磷酸化的PKC β调节ATF 2的转录活性，通过调节其与转录调控因子和AP 1的合作伙伴，以及它的nontranscriptiona在DNA损伤反应的功能。我还将研究如何PKC 3影响ATF 2的转录输出编程，通过研究在基因表达微阵列分析中发现的命中，我现在已经对ATF 2突变体进行了模拟PKC 3在存在或不存在遗传毒性应激的情况下磷酸化。我发现PKC β磷酸化的ATF 2抑制干扰素β 1（IFNB 1）和相关下游靶点的表达。已知IFN 1相关信号传导抑制细胞增殖，并且其施用可使癌细胞对化疗药物敏感。我的初步数据表明，黑色素瘤可能通过抑制IFN-β 1的表达，通过PKC α-ATF 2介导的信号传导，对遗传毒性应激产生抗性。这提出了一个令人兴奋的概念，即在黑色素瘤中靶向治疗ATF 2可能会抑制IFNB 1的表达，使细胞对遗传毒性应激敏感（如一线化疗黑色素瘤治疗所产生的应激）。我将进一步研究如何PKCCR 2 ATF 2抑制IFNB 1的表达在遗传毒性应激。通过对这些途径的详细机制研究，我的表达谱研究将确定对黑色素瘤发展和/或治疗耐药性至关重要的PKC β 2调节基因的功能簇。我将开发一个同基因异种移植小鼠黑色素瘤模型，以评估PKC β磷酸化调节ATF 2如何驱动黑色素瘤。在目标2中，我建议确定如何具体ATF 2激活Bcl 2蛋白和调节HK 1：VDAC 1复合物，以促进线粒体膜渗漏。我还将鉴定和表征非遗传毒性应激、生理条件，在这些条件下，我们观察到ATF 2线粒体定位而没有细胞死亡。我将描述由此产生的生物学后果，如线粒体呼吸或代谢的改变，并开始研究ATF 2介导的这些变化背后的分子机制。通过确定PKC β如何改变ATF 2转录活性和控制对黑色素瘤发展和耐药性至关重要的特定下游基因，Aim 1的研究可能能够确定黑色素瘤的新治疗方式。我在Aim 2中提出的研究将更好地了解ATF 2如何在应激和生理条件下调节线粒体外膜的完整性。 K99/R 00奖将允许我在我目前的导师Ze'ev Ronai博士的持续指导下进行我的研究，以及一组共同导师，他们是黑色素瘤和皮肤癌生物学，转录调控，PKC介导的信号传导，线粒体动力学和细胞死亡方面的知名专家。在指导阶段，我将在Sanford-Burnham医学研究所完成目标1和2的几个方面。在R 00独立阶段，我将在另一个学术或非营利研究机构独立继续我的研究。我的R 00期研究将进一步了解PKC β调节的ATF 2转录如何通过调节IFNB 1（Aim 1 subaim iv和Aim 2，将开始于K99期并在R 00期内完成）驱动黑色素瘤进展和耐药性。然后，我将集中在其他功能基因簇确定的表达阵列分析目标1。我将进一步研究目标2中确定的线粒体ATF 2的代谢分支。我相信，凭借我广泛的生物化学和细胞生物学背景，被授予K99/R 00赠款将促进和加快我的科学和技术专长的进一步发展，以及我在转录控制，蛋白质信号转导，黑色素瘤和癌症方面成为一名成功的独立研究人员的过渡。

项目成果

Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3.

• DOI: 10.3390/cells10061310
• 发表时间: 2021-05-25
• 期刊: Cells
• 影响因子: 6
• 作者: Watson G;Lester D;Ren H;Forsyth CM;Medina E;Gonzalez Perez D;Darville L;Yao J;Luca V;Koomen J;Cen L;Lau E
• 通讯作者: Lau E