The Role of p53-R249S’s GOF in HCC development

项目摘要 这项应用旨在确定特定的p53突变体R249S(p53-RS)的生物学作用，其 Arg 249被Ser取代，在肝细胞癌的发生和发展中起作用 揭示其功能获得(GOF)的分子机制，GOF对其致癌作用至关重要。值得注意的是， P53-RS与经常接触饮食黄曲霉毒素B1(AFB1)和 在亚洲和中非感染乙肝病毒(乙肝)，因为这是唯一发现的P53突变热点 在肝细胞癌患者中。由于小鼠R246S(相当于人类R249S)在没有任何 致癌挑战仅显示其对癌症的功能丧失(LOF)和显性负效应(DN) 没有任何GOF活性的发育，如果P53-RS具有重要的GOF活性，它仍然是难以捉摸的 在肝细胞癌的增殖、侵袭和肿瘤发生中起重要作用。如果是这样的话，背后的机制是什么 GoF活动。我们最近的研究揭示了细胞周期调节的激酶CDK4，Cyclin的独特联系 D_1(CycD_1)、肽基-脯氨基顺式-反式异构酶NIMA相互作用1(Pin1)和癌蛋白c-Myc。 P53-RS的GOF活性。这四种癌蛋白，CDK4，CycD1，Pin1和c-Myc在 细胞周期进程和癌细胞的增殖和生长，并在各种类型的 人类癌症，包括肝细胞癌。我们的初步研究和已发表的研究表明，CDK4/CycD1可以 在其肝癌来源的Ser249上与P53-RS特异性结合并磷酸化，这种磷酸化促进 P53-RS的Pin1结合及随后的核定位。在细胞核中，P53-RS与c-Ras结合并稳定c- MYC通过阻断FBW7介导的降解，从而导致c-Myc激活和增加 核糖体蛋白编码转录本的合成。通过这些动作，P53-RS执行其GOF活性 对肝癌细胞的增殖和存活至关重要。根据我们的初步结果，我们假设一个独特的 这个突变体的GOF机制，即用Ser替换Arg249使这个突变体变成一个新的 细胞周期中CDK4/CycD1的磷酸化底物，该残基的磷酸化使 P53-RS更容易与Pin1结合；因此，Pin1促进了这个突变的p53向 通过与c-Myc结合并激活c-Myc促进肝癌增殖的细胞核，促进肝癌的发展 和进步。我们将通过解决两个具体目标来检验这一假设：1)进一步破译 P53-RS基因GOF在肝细胞癌中的生化机制2)确定P53-RS基因的GOF是否起作用 在与乙肝病毒相关的肝细胞癌的发展中起作用。完成这些全面的研究将获得至关重要的 更好地了解肝癌中P53-RS‘s GOF的独特信号通路 CDK4/CycD1、Pin1和c-Myc作为分子靶点的研究进展 开发一种更有效的联合疗法治疗携带p53-RS的肝癌。

Project Summary This application is proposed to determine the biological role of a specific p53 mutant, R249S (p53-RS), whose Arg 249 is substituted by Ser, in development and progression of hepatocellular carcinoma (HCC) and to divulge molecular mechanisms underlying its gain of function (GOF) crucial for its oncogenic role. Remarkably, p53-RS is highly associated with HCC patients who are often exposed to dietary aflatoxin B1 (AFB1) and infected with Hepatitis Virus B (HBV) in Asia and central Africa, as it is the only hotspot p53 mutation identified among HCC patients. Since genetic knockin of mouse R246S (equivalent to human R249S) without any oncogenic challenges only showed its loss of function (LOF) and dominant negative (DN) effect on cancer development without any GOF activity, it has still remained elusive if p53-RS possesses GOF activity important for proliferation, invasion and tumorigenesis of HCC. If so, what would be the underlying mechanism for this GOF activity. Our recent studies uncovered the unique link of a cell cycle-regulated kinase, CDK4, Cyclin D1(CycD1), a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), and an oncoprotein c-Myc with the GOF activity of p53-RS. All of these four oncoproteins, CDK4, CycD1, PIN1, and c-Myc play critical roles in the cell cycle progression and cancer cell proliferation and growth, and are highly expressed in various types of human cancers, including HCC. Our preliminary and published studies showed that CDK4/CycD1 can specifically bind to and phosphorylate p53-RS at its HCC-derived Ser249, and this phosphorylation facilitates p53-RS's PIN1 binding and subsequent nuclear localization. In the nucleus, p53-RS binds to and stabilizes c- Myc by blocking FBW7-mediated degradation, consequently leading to c-Myc activation and increase of synthesis of ribosomal proteins-encoded transcripts. Through these actions, p53-RS executes its GOFs activity crucial for HCC cell proliferation and survival. In light of our preliminary results, we hypothesize a unique mechanism for this p53 mutant's GOF, i.e., substitution of Arg249 with Ser renders this mutant to a new phospohorylation substrate for CDK4/CycD1 during the cell cycle, and phosphorylation at this residue makes p53-RS more accessible for PIN1-binding; As a result, PIN1 facilitates the transport of this mutant p53 to the nucleus where it promotes HCC proliferation by binding to and activating c-Myc, promoting HCC development and progression. We will test this hypothesis by addressing two specific aims: 1) To further decipher biochemical mechanisms underlying the GOF of p53-RS in HCC; 2) To determine if p53-RS's GOF plays a role in HBV-associated HCC development. Completing these comprehensive studies would gain critical information for our better understanding of the unique signaling pathway underlying p53-RS's GOF in HCC development and progression, and also unveil CDK4/CycD1, PIN1 and c-Myc as molecular targets for developing a more effective combinatorial therapy for HCCs that harbor p53-RS.