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

描述（由申请人提供）：2011年，恶性黑色素瘤是最致命的皮肤癌，约占美国癌症病例总数的8%，估计约有70,000例新诊断和约8,800例死亡。迫切需要阐明驱动其发展，进展和治疗耐药性的分子机制。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着不同的作用。目前还不清楚ATF2是如何发挥这两种功能的。我最近发现蛋白激酶C，异构体epsilon （PKCepsilon）磷酸化ATF2在一个新的磷酸化受体位点（T52）上，促进其核定位和转录激活，赋予对遗传毒性胁迫的抗性。在基因毒性应激过程中，这种磷酸化阻断了ATF2转位到线粒体的能力。在线粒体外膜，ATF2通过干扰己糖激酶1和含电压依赖性阴离子通道1-(HK1:VDAC1)复合物，激活促凋亡Bcl2蛋白Bax，诱导线粒体膜渗漏。具体而言，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激活Bax和改变HK1:VDAC1复合物的确切机制尚不清楚。此外，除了基因毒性应激，我还观察到细胞质中存在ATF2。在这种情况下，其确切功能和生物学后果（例如，线粒体/代谢变化）尚不清楚。在Aim 1中，我建议确定PKCepsilon的磷酸化如何通过调节其与转录调节因子和AP1伴侣的相互作用来调节ATF2的转录活性，以及它在DNA损伤反应中的非转录功能。我还将研究PKCepsilon如何影响ATF2转录输出编程，通过研究基因表达微阵列分析中发现的hit，我现在对ATF2突变体进行了研究，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磷酸化的ATF2抑制干扰素β 1 （IFNB1）和相关下游靶点的表达。已知ifnb1相关信号可以抑制细胞增殖，并可使癌细胞对化疗药物敏感。我的初步数据表明，黑素瘤可能通过PKCepsilon atf2介导的信号传导抑制IFNB1的表达，从而对基因毒性应激产生抗性。这提出了一个令人兴奋的概念，即在黑色素瘤中靶向治疗ATF2可能会抑制IFNB1的表达，使细胞对基因毒性应激（如一线化疗黑色素瘤治疗所施加的应激）敏感。我将进一步研究PKCepsilon ATF2如何在基因毒性应激中抑制IFNB1的表达。从我的表达谱研究中确定的这些途径的详细机制研究将确定PKCepsilon atf2调节基因的功能簇，这些基因对黑色素瘤的发展和/或治疗耐药性至关重要。我将开发一个同基因异种移植小鼠黑色素瘤模型，以评估PKCepsilon磷酸化调节ATF2如何驱动黑色素瘤。在Aim 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，我们已经观察到的ATF2线粒体定位没有细胞死亡。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，并开始研究由ATF2介导的这些变化的分子机制。通过确定PKCepsilon如何改变ATF2转录活性和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，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ATF2在应激和生理条件下如何调节线粒体外膜的完整性。K99/R00奖将允许我在我现任导师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、转录调节、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 Medical Research Institute完成Aims 1和Aims 2的几个分支。在R00独立阶段，我将在另一个学术或非营利研究机构独立地继续我的研究。我的R00期研究将进一步加深我们对PKCepsilon如何通过调节IFNB1调控ATF2转录驱动黑色素瘤进展和耐药性的理解（Aim 1亚Aim iv和Aim 2，将在K99期开始，并在R00期完成）。然后，我将重点关注Aim 1中从表达阵列分析中确定的其他功能基因簇。我将进一步研究Aim 2中鉴定的线粒体ATF2的代谢后果。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资助将促进和加快我的科学和技术专业知识的进一步发展，并使我在转录控制、蛋白质信号转导、黑色素瘤和癌症方面成为一个成功的、独立的研究人员。

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.