The C-terminus of Gain-of-Function Mutant p53 R273H Is Required for Association with PARP1 and Poly-ADP-Ribose

Abstract

The TP53 gene is mutated in 80% of triple-negative breast cancers. Cells that harbor the hot-spot p53 gene mutation R273H produce an oncogenic mutant p53 (mtp53) that enhances cell proliferative and metastatic properties. The enhanced activities of mtp53 are collectively referred to as gain-of-function (GOF), and may include transcription-independent chromatin-based activities shared with wild type p53 (wtp53) such as association with replicating DNA and DNA replication associated proteins like Poly-ADP-Ribose Polymerase 1 (PARP1). However how mtp53 up-regulates cell proliferation, is not well understood. Wild-type p53 interacts with PARP1 using a portion of its C-terminus. The wtp53 oligomerization (OD) and far C-terminal domain (CTD) located within the C-terminus constitute putative GOF-associated domains, since R273H breast cancer cells lacking both domains manifest slow proliferation phenotypes. We addressed if the C-terminal region of mtp53 R273H is important for chromatin interaction and breast cancer cell proliferation using CRISPR-Cas9 mutated MDA-MB-468 cells endogenously expressing mtp53 R273H C-terminal deleted isoforms (R273HΔ381-388 and R273HΔ347-393). The mtp53 R273HΔ347-393 lacks the CTD and a portion of the OD. We observed that cells harboring mtp53 R273HΔ347-393 (compared to mtp53 R273H full-length) manifest a significant reduction in chromatin, PARP1, Poly-ADP-Ribose (PAR), and replicating DNA binding. These cells also exhibited impaired response to hydroxyurea (HU) replicative stress, decreased sensitivity to the PARP-trapping drug combination temozolomide-talazoparib, and increased phosphorylated 53BP1 foci, suggesting reduced Okazaki fragment processing. Implications: The C-terminal region of mtp53 confers GOF activity that mediates mtp53-PARP1 and PAR interactions assisting DNA replication, thus implicating new biomarkers for PARP inhibitor therapy.

Role of p53 in breast cancer progression: An insight into p53 targeted therapy

“…This synergistic sensitivity was enhanced upon R273H overexpression [33]. Moreover, the deletion of CTD of R273H disrupts the R273H-PARP1 interaction, causing a reduced ability of cells to proceed pass the G2/M checkpoint, slower proliferation, and decreased sensitivity to the talazoparib-temolozomide combination [154]. These results highlighted the clinical benefit of PARP inhibitors against breast cancers, especially those that express R273H p53 mutation.…”

A Cancer Persistent Dna Repair Circuit Driven by Mdm2, Mdm4 (Mdmx), and Mutant P53 for Recruitment of Mdc1 and 53bp1 to Chromatin

“…The protocol for the proximity ligation assay was performed as described previously [29] using the Sigma Aldrich Duolink System TM (anti-mouse, rabbit or goat (-) or (+) secondary antibodies; either 594 (cat# DUO92008) or 647 (cat# DUO92013) detection kit) with modifications indicated below (unless indicated otherwise, all incubations were performed as specific by the manufacturer). Cells were seeded in 12-well un-coated glass-bottomed plates (MatTek) at 1x10 5 cells per well in complete media, cultured until 50% confluency and then washed 3x with ice cold PBS for 1 min/wash followed by fixation with 4% Paraformaldehyde in PBS for 15 min.…”

“…These observations suggest mtp53 works with MDM2 and MDMX to drive functions that promote tumorigenesis [26]. Examples of mtp53 GOF activities that suggest alterations in DNA metabolism promoting cell survival have been reported by many laboratories, and include mtp53-PARP1 modulation of DNA replication and repair intermediates [27–29], mtp53-Treslin modulation of replication fork assembly [30], and the observation that inhibition of PARP or Treslin or MDM-Two Binding Protein (MTBP) leads to DNA damage [31]. Both MDM2 and MDMX also participate in regulating DNA replication [32, 33].…”