Summary Missense mutations in the p53 tumor suppressor inactivate its anti-proliferative properties but can also promote metastasis through a gain-of-function activity. We show that sustained expression of mutant p53 is required to maintain the pro-metastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, non-invasive cells. Blocking PDGFRb signaling by RNA interference or small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors.

SUMMARY Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8-nucleotide 5’ sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicates that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

The CRISPR-Cas system provides many prokaryotes with acquired resistance to viruses and other mobile genetic elements. The core components of this defense system are small, host-encoded prokaryotic silencing (psi)RNAs and Cas (CRISPR-associated) proteins. Invader-derived sequences within the psiRNAs guide Cas effector proteins to recognize and silence invader nucleic acids. Critical for CRISPR-Cas defense is processing of the psiRNAs from the primary transcripts of the host CRISPR (clustered regularly interspaced short palindromic repeat) locus. Cas6, a previously identified endoribonuclease present in a wide range of prokaryotes with the CRISPR-Cas system, binds and cleaves within the repeat sequences that separate the individual invader targeting elements in the CRISPR locus transcript. In the present study, we investigated several key aspects of the mechanism of function of Cas6 in psiRNA biogenesis. RNA footprinting reveals that Pyrococcus furiosus Cas6 binds to a 7-nt (nucleotide) sequence near the 59 end of the CRISPR RNA repeat sequence, 14 nt upstream of the Cas6 cleavage site. In addition, analysis of the cleavage activity of P. furiosus Cas6 proteins with mutations at conserved residues suggests that a triad comprised of Tyr31, His46, and Lys52 plays a critical role in catalysis, consistent with a possible general acid-base RNA cleavage mechanism for Cas6. Finally, we show that P. furiosus Cas6 remains stably associated with its cleavage products, suggesting additional roles for Cas6 in psiRNA biogenesis.

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

TP53 missense mutations produce a mutant p53 protein that cannot activate the p53 tumor suppressive transcriptional response, which is the primary selective pressure for TP53 mutation. Specific codons of TP53, termed hotspot mutants, are mutated at elevated frequency. Hotspot forms of mutant p53 possess oncogenic properties in addition to being deficient in tumor suppression. Such p53 mutants accumulate to high levels in the cells they inhabit, causing transcriptional alterations that produce pro-oncogenic activities, such as increased pro-growth signaling, invasiveness, and metastases. These forms of mutant p53 very likely use features of wild-type p53, such as interactions with the transcriptional machinery, to produce oncogenic effects. In this review, we discuss commonalities between wild-type and mutant p53 proteins with an emphasis on transcriptional processes.