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）： 2011 年，恶性黑色素瘤估计有约 70,000 例新诊断病例和约 8,800 例死亡病例，是最致命的皮肤癌，约占美国癌症病例总数的 8%。迫切需要阐明驱动其发生、进展和治疗耐药的分子机制。 激活转录因子 2 (ATF2) 是一种 AP1 转录因子，在黑色素瘤中作为癌基因，在非恶性皮肤癌中作为肿瘤抑制因子发挥不同的作用。 ATF2 如何发挥这两种功能仍不清楚。我最近发现蛋白激酶 C，同种型 epsilon (PKCepsilon) 在一个新的磷酸受体位点 (T52) 上磷酸化 ATF2，促进其核定位和转录激活，从而赋予对基因毒性应激的抵抗力。 Thi 磷酸化可阻断 ATF2 在基因毒性应激期间易位至线粒体的能力。在线粒体外膜，ATF2 通过扰乱己糖激酶 1 和电压依赖性阴离子通道 1-(HK1:VDAC1) 复合物并激活促凋亡 Bcl2 蛋白 Bax 来诱导线粒体膜渗漏。 具体来说，PKCepsilon 磷酸化如何影响 ATF2 的转录和非转录 DNA 损伤反应功能尚不清楚。 ATF2 究竟如何激活 Bax 并改变 HK1:VDAC1 复合物尚不清楚。此外，我在基因毒性应激以外的环境下观察到细胞质中存在 ATF2。在这种情况下，其精确功能和生物学后果（例如线粒体/代谢变化）尚不清楚。在目标 1 中，我建议确定 PKCepsilon 的磷酸化如何通过调节 ATF2 与转录调节因子和 AP1 伴侣的相互作用以及其在 DNA 损伤反应中的非转录功能来调节 ATF2 转录活性。我还将通过研究基因表达微阵列分析中确定的命中来研究 PKCepsilon 如何影响 ATF2 转录输出编程，我现在对 ATF2 突变体进行了基因表达微阵列分析，这些突变体在存在或不存在基因毒性应激的情况下模拟 PKCepsilon 的磷酸化。我发现 PKCepsilon 磷酸化 ATF2 会抑制干扰素 Beta 1 (IFNB1) 和相关下游靶标的表达。已知 IFNB1 相关信号传导可抑制细胞增殖，其给药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 PKCepsilon ATF2 介导的信号传导抑制 IFNB1 表达，从而产生对基因毒性应激的抵抗力。这提出了一个令人兴奋的想法，即黑色素瘤中 ATF2 的治疗靶向可能会抑制 IFNB1 的表达，使细胞对基因毒性应激（例如一线化疗黑色素瘤治疗所施加的压力）敏感。我将进一步研究 PKCepsilon ATF2 在基因毒性应激期间如何抑制 IFNB1 表达。对我的表达谱研究中确定的此类途径的详细机制研究将确定对黑色素瘤发展和/或治疗耐药至关重要的 PKCepsilon ATF2 调节基因的功能簇。我将开发同基因异种移植小鼠黑色素瘤模型来评估 PKCepsilon 磷酸化 ATF2 如何驱动黑色素瘤。在目标 2 中，我建议确定 ATF2 如何特异性激活 Bcl2 蛋白并调节 HK1:VDAC1 复合物以促进线粒体膜渗漏。我还将识别和描述非基因毒性应激、生理条件，我们在这些条件下观察到 ATF2 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，例如线粒体呼吸或代谢的改变，并开始研究 ATF2 介导的这些变化背后的分子机制。通过确定 PKCepsilon 如何改变 ATF2 转录活性以及对黑色素瘤发展和耐药性至关重要的特定下游基因的控制，目标 1 的研究可能能够确定黑色素瘤的新治疗方式。我在目标 2 中提出的研究将有助于更好地理解 ATF2 在压力和生理条件下如何调节线粒体外膜的完整性。 K99/R00 奖将使我能够在我现任导师 Ze'ev Ronai 博士以及一组共同导师的持续指导下进行我拟议的研究，他们是黑色素瘤和皮肤癌生物学、转录学领域的知名专家。调节、PKC 介导的信号传导、线粒体动力学和细胞死亡。在指导阶段，我将在桑福德-伯纳姆医学研究所完成目标 1 和 2 的几个部分。在R00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 R00 阶段研究将进一步了解 PKCepsilon 调节 ATF2 转录如何通过 IFNB1 调节驱动黑色素瘤进展和耐药性（目标 1 subaim iv 和目标 2，将在 K99 阶段开始并在 R00 阶段完成）。然后，我将重点关注从目标 1 中的表达阵列分析中鉴定出的其他功能基因簇。我将进一步研究目标 2 中鉴定的线粒体 ATF2 的代谢影响。我相信，凭借我广泛的生物化学和细胞生物学背景，获得 K99 /R00 资助将促进和加速我的科学和技术专业知识的进一步发展，以及我向转录控制、蛋白质信号转导、黑色素瘤和癌症领域成功的独立研究人员的转变。

项目成果

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

岩藻糖基化蛋白质组分析可鉴定核糖体蛋白 S3 的岩藻糖基化、非核糖体、应激反应性物种。

• 发表时间: 2021
• 影响因子: 6
• 作者: Watson, Gregory;Lester, Daniel;Ren, Hui;Forsyth, Connor M;Medina, Elliot;Gonzalez Perez, David;Darville, Lancia;Yao, Jiqiang;Luca, Vince;Koomen, John;Cen, Ling;Lau, Eric
• 通讯作者: Lau, Eric