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

描述(申请人提供)：恶性黑色素瘤是最致命的皮肤癌，约占美国癌症总数的8%，2011年估计有约70,000例新诊断和约8,800例死亡。迫切需要阐明其发生、发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发挥不同的功能，在非恶性皮肤癌中也作为肿瘤抑制因子发挥作用。ATF2如何发挥这两种功能尚不清楚。我最近发现，蛋白激酶C，异构体epsilon(PKCepsilon)使ATF2在一个新的磷酸受体位点(T52)上磷酸化，促进其核定位和转录激活，从而对遗传毒性应激产生抗性。THI的磷酸化抑制了ATF2在遗传毒性应激过程中移位到线粒体的能力。在线粒体外膜，ATF2通过干扰己酮酸酶1和含有电压依赖性阴离子通道1-(HK1：VDAC1)的复合体，并激活促凋亡的Bcl2蛋白Bax来诱导线粒体膜渗漏。 具体地说，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如何激活Bax并改变HK1：VDAC1复合体尚不清楚。此外，在基因毒性应激以外的环境中，我还观察到胞浆中的ATF2。在这种情况下，其确切的功能和生物学分支(例如，线粒体/代谢变化)尚不清楚。在目标1中，我建议确定PKCepsilon如何通过调节ATF2与转录调控因子和AP1伙伴的相互作用来调节ATF2的转录活性，以及它在DNA损伤反应中的非转录功能。我还将通过调查在基因表达微阵列分析中确定的命中来调查PKCepsilon如何影响ATF2转录输出编程，这些分析是我现在对ATF2突变体进行的，这些突变体在存在或不存在遗传毒性应激的情况下模仿PKCepsilon的磷酸化。我发现PKCepsilon磷酸化的ATF2抑制干扰素β1(IFNB1)的表达和相关的下游靶点。已知IFNB1相关信号可抑制细胞增殖，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介导的信号抑制IFNB1的表达而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ATF2在黑色素瘤中的治疗性靶向可能会降低IFNB1的表达，使细胞对遗传毒性应激(如一线化疗黑色素瘤治疗施加的应激)敏感。我将进一步研究PKCepsilon ATF2在基因毒性应激过程中如何抑制IFNB1的表达。从我的表达谱研究中确定的这种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/或治疗耐药至关重要。我将建立一个同基因异种移植小鼠黑色素瘤模型，以评估PKCepsilon对ATF2的磷酸化调节如何驱动黑色素瘤。在目标2中，我建议确定ATF2如何特异性地激活Bcl2蛋白并调节HK1：VDAC1复合体以促进线粒体膜渗漏。我还将确定和描述非遗传毒性应激的生理条件，在这些条件下，我们观察到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，如线粒体呼吸或新陈代谢的改变，并开始研究ATF2介导的这些变化背后的分子机制(S)。通过确定PKCepsilon如何改变ATF2转录活性和对黑色素瘤发生和耐药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上提出的研究将更好地理解ATF2如何在应激和生理条件下调节线粒体外膜的完整性。 K99/R00奖将允许我在我目前的导师Ze‘ev Ronai博士的持续指导下，以及一个共同导师小组的持续指导下，开展我提议的研究，他们是黑色素瘤和皮肤癌生物学、转录调控、PKC介导的信号以及线粒体动力学和细胞死亡方面的知名专家。在指导阶段，我将在桑福德-伯纳姆医学研究所完成AIMS 1和AIMS 2的几个分支。在R00独立阶段，我将在另一家学术或非营利性研究机构独立继续我的研究。我的R00期研究将进一步加深我们对PKCepsilon调控ATF2转录如何通过调控IFNB1(Aim 1亚Aim IV和Aim 2，将在K99期开始并在R00期完成)调控黑色素瘤进展和耐药的理解。然后，我将专注于从Aim 1的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中确定的线粒体ATF2的代谢分支。我相信，以我广泛的生物化学和细胞生物学背景，获得K99/R00奖学金将促进和加速我的科学和技术专长的进一步发展，并使我过渡到转录控制、蛋白质信号转导、黑色素瘤和癌症方面的成功的独立研究人员。

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.