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.