Cell Fate Control by Integrated E2F, FoxO and PI3K Signaling in Retinoblastoma

描述（由申请人提供）：E2F转录因子诱导增殖，但也可以促进细胞死亡，作为一种先天抗癌机制，杀死具有自发致癌突变的细胞，否则可能会形成癌症。对于使E2F1平衡在某些情况下（正常生长）倾向于增殖，在其他情况下（致癌应激/早期癌症）倾向于凋亡的分子通路，以及介导这一决定的途径知之甚少。缺乏这方面的知识是一个重要的尚未解决的医学问题，因为没有这方面的知识，获得恢复肿瘤中潜在的E2F1介导的凋亡活性以加速肿瘤细胞死亡的治疗能力是极不可能的。我们发现在人类癌症中频繁激活的PI3K/Akt通路是E2F1诱导的细胞凋亡和凋亡靶基因表达的关键负调控因子。这些发现表明，传统上“不可药物”的靶分子（转录因子）E2F1的功能可以通过干扰PI3K活性来调节细胞凋亡。我们还发现FoxO1转录因子与E2F1物理相互作用，并可以结合到相同的启动子来调节基因表达和细胞凋亡。本研究的主要假设是，E2F1和FoxO1转录因子协调了一个可被Akt激活抑制的肿瘤抑制凋亡转录程序。Aim 1将侧重于a)确定E2F1和fox01相互作用和功能所需的结构域，b)确定其他e2f和fox01是否相互作用，以及c)采用ip -质谱策略确定可能调节E2F1/ fox01复合物功能并协调其促凋亡功能的其他E2F1结合伙伴。目的2利用qPCR、RNA-Seq、ChIP和ChIP- seq深入研究共调控E2F1和FoxO1靶基因的鉴定及其表达机制。目的3研究FoxO和Pten/PI3K/Akt通路对小鼠视网膜Rb/E2F细胞凋亡和肿瘤发生的调控作用。我们还建议使用我们的小鼠模型，发展为侵袭性双侧视网膜母细胞瘤，用于抗pi3k治疗的临床前测试，无论是否采用当前的标准护理治疗。我们发现FoxO和PI3K活性促进了E2F1凋亡和增殖平衡的控制，这表明这些研究将为正常细胞和癌细胞中这些蛋白之间的机制关系提供重要的见解。本提案中概述的实验将提供有关E2F1/FoxO复合物功能、PI3K信号对其调控的重要信息，以及在体内抑制PI3K可能是一种旨在恢复肿瘤中E2F1/FoxO凋亡刺激的治疗选择的程度。考虑到Rb/E2F和PI3K/Pten通路的功能障碍在许多人类肿瘤中都很突出，我们希望我们的发现可能适用于其他类型的肿瘤。

DESCRIPTION (provided by applicant): The E2F transcription factors induce proliferation but also can promote cell death as an innate anti-cancer mechanism to kill cells with a spontaneous oncogenic mutation that might otherwise go on to form a cancer. Little is known about the molecular circuitry that tips E2F1 balance towards proliferation in some settings (normal growth), apoptosis in others (oncogenic stress/incipient cancers), and which pathways mediate this decision. Lack of such knowledge is an important unmet medical problem, because without it, acquiring the ability to restore latent E2F1 mediated apoptotic activity therapeutically in tumors to accelerate tumor cell death is highly unlikely. We discovered that the PI3K/Akt pathway, which is frequently activated in human cancer, is a key negative regulator of E2F1 induced apoptosis and apoptotic target gene expression. These findings suggest that function of E2F1, a traditionally "undruggable" target molecule (transcription factor) can be modulated towards apoptosis by interfering with PI3K activity. We also show that the FoxO1 transcription factor physically interacts with E2F1 and can bind to the same promoters to regulate gene expression and apoptosis. The major hypothesis tested in this proposal is that E2F1 and FoxO1 transcription factors coordinate a tumor suppressive apoptotic transcriptional program that can be inhibited by Akt activation. Aim 1 will focus on a) identifying domains in E2F1 and FoxO1 required for interaction and function, b) will determine if other E2Fs and FoxOs interact, and c) employ an IP-Mass Spec strategy to determine other E2F1 binding partners that may regulate the function of the E2F1/FoxO1 complex and coordinate its pro-apoptotic function. Aim 2 delves into identification of co-regulated E2F1 and FoxO1 target genes and the mechanisms of gene expression using qPCR, RNA-Seq, ChIP, and ChIP-Seq. Aim 3 tests FoxO and Pten/PI3K/Akt pathway regulation of Rb/E2F apoptosis and tumorigenesis in vivo in the mouse retina. We also propose to use our mouse model, which develops aggressive bilateral retinoblastoma, for pre-clinical testing anti-PI3K therapy with or without current standard of care treatment. Our finding that FoxO and PI3K activity facilitate control of the E2F1 apoptotic and proliferative balance suggest that these studies will provide significant insight into the mechanistic relationship between these proteins in normal and cancerous cells. The experiments outlined in this proposal will provide important information about the function of the E2F1/FoxO complex, its regulation by PI3K signaling, and the extent that inhibiting PI3K in vivo may be a therapeutic option aiming at restoring E2F1/FoxO apoptosis stimulation in tumors. Considering that dysfunction in Rb/E2F and PI3K/Pten pathways feature prominently in numerous human tumors, we expect that our findings may be applicable to other tumor types.