The Gain-of-Function p53 R248W Mutant Promotes Migration by STAT3 Deregulation in Human Pancreatic Cancer Cells.

Luisa Klemke,Shiv K Singh,Ramona Schulz-Heddergott,Nadine Winkler,Ute M Moll,Clara F Fehlau,Felicia Toboll

doi:10.3389/fonc.2021.642603

Abstract

Missense p53 mutations (mutp53) occur in approx. 70% of pancreatic ductal adenocarcinomas (PDAC). Typically, mutp53 proteins are aberrantly stabilized by Hsp90/Hsp70/Hsp40 chaperone complexes. Notably, stabilization is a precondition for specific mutp53 alleles to acquire powerful neomorphic oncogenic gain-of-functions (GOFs) that promote tumor progression in solid cancers mainly by increasing invasion and metastasis. In colorectal cancer (CRC), we recently established that the common hotspot mutants mutp53R248Q and mutp53R248W exert GOF activities by constitutively binding to and hyperactivating STAT3. This results in increased proliferation and invasion in an autochthonous CRC mouse model and correlates with poor survival in patients. Comparing a panel of p53 missense mutations in a series of homozygous human PDAC cell lines, we show here that, similar to CRC, the mutp53R248W protein again undergoes a strong Hsp90-mediated stabilization and selectively promotes migration. Highly stabilized mutp53 is degradable by the Hsp90 inhibitors Onalespib and Ganetespib, and correlates with growth suppression, possibly suggesting therapeutic vulnerabilities to target GOF mutp53 proteins in PDAC. In response to mutp53 depletion, only mutp53R248W harboring PDAC cells show STAT3 de-phosphorylation and reduced migration, again suggesting an allele-specific GOF in this cancer entity, similar to CRC. Moreover, mutp53R248W also exhibits the strongest constitutive complex formation with phosphorylated STAT3. The selective mutp53R248W GOF signals through enhancing the STAT3 axis, which was confirmed since targeting STAT3 by knockdown or pharmacological inhibition phenocopied mutp53 depletion and reduced cell viability and migration preferentially in mutp53R248W-containing PDAC cells. Our results confirm that mutp53 GOF activities are allele specific and can span across tumor entities.

Highlights

Already in the early 1990s, the tumor suppressor p53 was coined as ‘guardian of the genome’ [1, 2] and it was known that mutation of the TP53 gene is an essential step in human tumor development [1, 3]
To stimulate the STAT3 pathway, cells were seeded in 6-well plates (Sarstedt) and treated with Interleukin-6 (IL-6) or Oncostatin M (OSM 209a.a.) or solvent control for 24 h and analyzed by immunoblots
We analyze a panel of p53 missense mutants in a series of homozygous human pancreatic ductal adenocarcinoma (PDAC) cell lines and compare the impact of various mutants on protein properties and functions

Summary

INTRODUCTION

Already in the early 1990s, the tumor suppressor p53 was coined as ‘guardian of the genome’ [1, 2] and it was known that mutation of the TP53 gene (tumor protein p53, HGNC:11998) is an essential step in human tumor development [1, 3]. 70% are missense mutations leading to amino acid substitutions mostly in the DNA binding domain, some alleles are selected and occur at a high frequency, termed hotspots. Recent results from our group highlight the GOF hotspot mutp53R248Q/W specificity in mouse and human colorectal cancer (CRC). 70% of PDAC patients, TP53 undergoes mainly missense mutations (www.cbioportal.org) as a late genetic event at the transition from high grade PanIN dysplasia to invasiveness during pancreatic cancer progression [35, 36]. We show in a panel of common human PDAC cell lines harboring different homozygous missense p53 mutants that mutp variants differ in their protein stability, with mutp53R248W again accumulating the highest protein levels in the pancreatic cell context. Our results support a GOF function of mutp53R248W in pancreatic cancer cell lines, justifying future investigations in this tumor entity in vivo

MATERIAL AND METHODS

RESULTS

Findings

DISCUSSION