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

项目摘要 本申请旨在确定特定p53突变体R249 S（p53-RS）的生物学作用，其 Arg 249被Ser取代，在肝细胞癌（HCC）的发展和进展中， 揭示其功能获得（GOF）的分子机制，这对其致癌作用至关重要。值得注意的是， p53-RS与经常暴露于饮食黄曲霉毒素B1（AFB 1）的HCC患者高度相关， 在亚洲和中非感染B肝炎病毒（HBV），因为它是唯一确定的热点p53突变 在HCC患者中。由于小鼠R246 S（相当于人R249 S）的基因敲入没有任何 致癌基因攻击仅显示其对癌症的功能丧失（LOF）和显性阴性（DN）效应 尽管p53-RS在没有任何GOF活性的情况下发育，但如果p53-RS具有重要的GOF活性， 对HCC的增殖、侵袭和肿瘤发生的影响。如果是的话，其潜在机制是什么 GOF活动我们最近的研究揭示了细胞周期调节激酶CDK 4与细胞周期蛋白Cyclin D1（CycD 1）、肽基脯氨酰顺反异构酶NIMA相互作用1（PIN 1）和癌蛋白c-Myc， p53-RS的GOF活性。所有这四种癌蛋白，CDK 4，CycD 1，PIN 1和c-Myc在肿瘤的发生中起关键作用。 细胞周期进程和癌细胞增殖和生长，并在各种类型的 人类癌症，包括HCC。我们的初步和已发表的研究表明，CDK 4/CycD 1可以 特异性结合并磷酸化p53-RS在其HCC衍生Ser 249，这种磷酸化促进 p53-RS的PIN 1结合和随后的核定位。在细胞核中，p53-RS结合并稳定c- Myc通过阻断FBW 7介导的降解，从而导致c-Myc激活和增加 核糖体蛋白质编码的转录物的合成。通过这些动作，p53-RS执行其GOFs活动 对于HCC细胞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 这种p53突变体GOF的机制，即，用Ser取代Arg 249使该突变体成为新的突变体。 细胞周期中CDK 4/CycD 1的磷酸化底物，该残基的磷酸化使 p53-RS更容易与PIN 1结合;因此，PIN 1促进该突变型p53转运至 细胞核，通过结合和激活c-Myc促进HCC增殖，促进HCC发展 和进步。我们将通过解决两个具体目标来测试这一假设：1）进一步破译 p53-RS的GOF在HCC中的生化机制; 2）确定p53-RS的GOF是否起作用 在HBV相关HCC发展中的作用。完成这些全面的研究将获得关键的 为我们更好地理解HCC中p53-RS GOF的独特信号通路提供了信息 发展和进展，并揭示CDK 4/CycD 1，PIN 1和c-Myc作为分子靶点， 为携带p53-RS的HCC开发更有效的组合疗法。

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.