Gain-of-function mutant p53 upregulates CXC chemokines and enhances cell migration.

The role of dominant transforming p53 in carcinogenesis is poorly understood. Our previous data suggested that aberrant p53 proteins can enhance tumorigenesis and metastasis. Here, we examined potential mechanisms through which gain-of-function (GOF) p53 proteins can induce motility. Cells expressing GOF p53 -R175H, -R273H and -D281G showed enhanced migration, which was reversed by RNA interference (RNAi) or transactivation-deficient mutants. In cells with engineered or endogenous p53 mutants, enhanced migration was reduced by downregulation of nuclear factor-kappaB2, a GOF p53 target. We found that GOF p53 proteins upregulate CXC-chemokine expression, the inflammatory mediators that contribute to multiple aspects of tumorigenesis. Elevated expression of CXCL5, CXCL8 and CXCL12 was found in cells expressing oncogenic p53. Transcription was elevated as CXCL5 and CXCL8 promoter activity was higher in cells expressing GOF p53, whereas wild-type p53 repressed promoter activity. Chromatin immunoprecipitation assays revealed enhanced presence of acetylated histone H3 on the CXCL5 promoter in H1299/R273H cells, in agreement with increased transcriptional activity of the promoter, whereas RNAi-mediated repression of CXCL5 inhibited cell migration. Consistent with this, knockdown of the endogenous mutant p53 in lung cancer or melanoma cells reduced CXCL5 expression and cell migration. Furthermore, short hairpin RNA knockdown of mutant p53 in MDA-MB-231 cells reduced expression of a number of key targets, including several chemokines and other inflammatory mediators. Finally, CXCL5 expression was also elevated in lung tumor samples containing GOF p53, indicating relevance to human cancer. The data suggest a mechanistic link between GOF p53 proteins and chemokines in enhanced cell motility.

Allele specific gain-of-function activity of p53 mutants in lung cancer cells.

p53 mutations are mostly single amino acid changes resulting in expression of a stable mutant protein with "gain of function" (GOF) activity having a dominant oncogenic role rather than simple loss of function of wild-type p53. Knock-down of mutant p53 in human lung cancer cell lines with different endogenous p53 mutants results in loss of GOF activity as shown by lowering of cell growth rate. Two lung cancer cell lines, ABC1 and H1437, carrying endogenous mutants p53-P278S and -R267P, show reduction in growth rate on knock-down on p53 levels. However, whereas reduction of the p53 level induces loss of tumorigenicity in nude mice for ABC1 cells, it escalates tumorigenicity for H1437 cells. We have tested their transactivation potential on p53 target gene promoters by performing transient transcriptional assays in the p53-null H1299 lung cancer cell line. Interestingly, while the mutant p53 target promoter Axl was activated by both the mutants, the p21 promoter was activated by p53-R267P and wild-type p53 but not by p53-P278S; showing a clear difference in transcriptional activity between the two mutants. Our results demonstrate allele specificity between GOF p53 mutants and attempt to show that the specificity is dependent on the transactivation property of GOF p53; it also suggests importance of p21 activation in tumor suppression by p53.

p53 mutants induce transcription of NF-κB2 in H1299 cells through CBP and STAT binding on the NF-κB2 promoter and gain of function activity.

Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 and show "gain of function" (GOF) phenotypes such as increased growth rate, enhanced resistance to chemotherapeutic drugs, increased cell motility and tumorigenicity; although the mechanism for this function remains unknown. In this communication we report that p53-mediated NF-κB2 up-regulation significantly contributes to the aggressive oncogenic behavior of cancer cells. Lowering the level of mutant p53 in a number of cancer cell lines resulted in a loss of GOF phenotypes directly implicating p53 mutants in the process. RNAi against NF-κB2 in naturally occurring cancer cell lines also lowers GOF activities. In H1299 cells expressing mutant p53, chromatin immunoprecipitation (ChIP) assays indicate that mutant p53 induces histone acetylation at specific sites on the regulatory regions of its target genes. ChIP assays using antibodies against transcription factors putatively capable of interacting with the NF-κB2 promoter show increased interaction of CBP and STAT2 in the presence of mutant p53. Thus, we propose that in H1299 cells, mutant p53 elevates expression of genes capable of enhancing cell proliferation, motility, and tumorigenicity by inducing acetylation of histones via recruitment of CBP and STAT2 on the promoters causing CBP-mediated histone acetylation.

The oncogenicity of tumor-derived mutant p53 is enhanced by the recruitment of PLK3.

p53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.

DNA replication in progenitor cells and epithelial regeneration after lung injury requires the oncoprotein MDM2.

Depletion of epithelial cells after lung injury prompts proliferation and epithelial mesenchymal transition (EMT) of progenitor cells, and this repopulates the lost epithelial layer. To investigate the cell proliferative function of human oncoprotein MDM2, we generated mouse models targeting human MDM2 expression in either lung Club or alveolar cells after doxycycline treatment. We report that MDM2 expression in lung Club or alveolar cells activates DNA replication specifically in lung progenitor cells only after chemical- or radiation-induced lung injury, irrespective of their p53 status. Activation of DNA replication by MDM2 triggered by injury leads to proliferation of lung progenitor cells and restoration of the lost epithelial layers. Mouse lung with no Mdm2 allele loses its ability to replicate DNA, whereas loss of 1 Mdm2 allele compromises this function, demonstrating the requirement of endogenous MDM2. We show that the p53-independent ability of MDM2 to activate Akt signaling is essential for initiating DNA replication in lung progenitor cells. Furthermore, MDM2 activates the Notch signaling pathway and expression of EMT markers, indicative of epithelial regeneration. This is the first report to our knowledge demonstrating a direct p53-independent participation of MDM2 in progenitor cell proliferation and epithelial repair after lung injury, distinct from a p53-degrading antiapoptotic effect preventing injury.

Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication.

Gain-of-function (GOF) p53 mutations are observed frequently in most intractable human cancers and establish dependency for tumor maintenance and progression. While some of the genes induced by GOF p53 have been implicated in more rapid cell proliferation compared with p53-null cancer cells, the mechanism for dependency of tumor growth on mutant p53 is unknown. This report reveals a therapeutically targetable mechanism for GOF p53 dependency. We have shown that GOF p53 increases DNA replication origin firing, stabilizes replication forks, and promotes micronuclei formation, thus facilitating the proliferation of cells with genomic abnormalities. In contrast, absence or depletion of GOF p53 leads to decreased origin firing and a higher frequency of fork collapse in isogenic cells, explaining their poorer proliferation rate. Following genome-wide analyses utilizing ChIP-Seq and RNA-Seq, GOF p53-induced origin firing, micronuclei formation, and fork protection were traced to the ability of GOF p53 to transactivate cyclin A and CHK1. Highlighting the therapeutic potential of CHK1's role in GOF p53 dependency, experiments in cell culture and mouse xenografts demonstrated that inhibition of CHK1 selectively blocked proliferation of cells and tumors expressing GOF p53. Our data suggest the possibility that checkpoint inhibitors could efficiently and selectively target cancers expressing GOF p53 alleles.

Gain-of-function p53 activates multiple signaling pathways to induce oncogenicity in lung cancer cells.

Gain-of-function (GOF) mutants of p53 upregulate genes implicated in cell proliferation and oncogenesis. Here, we report that GOF p53 induces tumorigenicity through simultaneous activation of key oncogenic pathways including those controlling putative tumor-initiating cell functions. We determined that in cells expressing p53-R273H, GOF p53 simultaneously upregulates genes from multiple signaling pathways by recognizing promoters containing distinct transcription factor (TF) binding sites. Our analytical data support a model in which GOF p53 complexes with two TFs on the promoter-a mediator protein, Med17, and a histone acetyl transferase, activating histone acetylation-and enhances gene expression to signal cell proliferation and oncogenesis. Thus, therapeutic inhibition of one GOF p53-induced pathway would be insufficient to prevent tumor growth as the oncoprotein activates a multitude of parallel pathways. This discovery suggests enormous selection advantage for cancer cells with GOF p53 to induce oncogenic growth, highlighting the problems of cancer therapy.

Addiction of lung cancer cells to GOF p53 is promoted by up-regulation of epidermal growth factor receptor through multiple contacts with p53 transactivation domain and promoter.

Human lung cancers harboring gain-of-function (GOF) p53 alleles express higher levels of the epidermal growth factor receptor (EGFR). We demonstrate that a number of GOF p53 alleles directly upregulate EGFR. Knock-down of p53 in lung cancer cells lowers EGFR expression and reduces tumorigenicity and other GOF p53 properties. However, addiction of lung cancer cells to GOF p53 can be compensated by overexpressing EGFR, suggesting that EGFR plays a critical role in addiction. Chromatin immunoprecipitation (ChIP) using lung cancer cells expressing GOF p53 alleles showed that GOF p53 localized to the EGFR promoter. The sequence where GOF p53 is found to interact by ChIP seq can act as a GOF p53 response element. The presence of GOF p53 on the EGFR promoter increased histone H3 acetylation, indicating a mechanism whereby GOF p53 enhances chromatin opening for improved access to transcription factors (TFs). ChIP and ChIP-re-ChIP with p53, Sp1 and CBP histone acetylase (HAT) antibodies revealed docking of GOF p53 on Sp1, leading to increased binding of Sp1 and CBP to the EGFR promoter. Up-regulation of EGFR can occur via GOF p53 contact at other novel sites in the EGFR promoter even when TAD-I is inactivated; these sites are used by both intact and TAD-I mutated GOF p53 and might reflect redundancy in GOF p53 mechanisms for EGFR transactivation. Thus, the oncogenic action of GOF p53 in lung cancer is highly dependent on transactivation of the EGFR promoter via a novel transcriptional mechanism involving coordinated interactions of TFs, HATs and GOF p53.

Preferred binding of gain-of-function mutant p53 to bidirectional promoters with coordinated binding of ETS1 and GABPA to multiple binding sites.

Gain-of-function mutant p53 is thought to induce gene expression in part by binding transcription factors bound to promoters for genes that mediate oncogenesis. We investigated the mechanism of mutant p53 binding by mapping the human genomic binding sites for p53 R273H using ChIP-Seq and showed them to localize to ETS DNA sequence motifs and locations with ETS1 and GABPA binding, both within promoters and distal to promoters. Strikingly, p53 R273H showed statistically significant and substantial binding to bidirectional promoters, which are enriched for inverted repeated ETS DNA sequence motifs. p53 R273H exhibited an exponential increase in probability of binding promoters with a higher number of ETS motifs. Both ETS1 and GABPA also showed an increase in the probability of binding to promoters with a higher number of ETS motifs. However, despite this increase in probability of binding by p53 R273H and ETS1, there was no increase in the binding signal, suggesting that the number of ETS1 and p53 R273H proteins bound per promoter is being limited. In contrast, GABPA did exhibit an increase in binding signal with higher numbers of ETS motifs per promoter. Analysis of the distance between inverted pairs of ETS motifs within promoters and binding by p53 R273H, ETS1 and GABPA, showed a novel coordination of binding for the three proteins. Both ETS1 and p53 R273H exhibited preference for binding promoters with distantly spaced ETS motifs in face-to-face and back-to-back orientations, and low binding preference to promoters with closely spaced ETS motifs. GABPA exhibited the inverse pattern of binding by preferring to bind promoters with closely spaced ETS motifs. Analysis of the helical phase between ETS motifs showed that ETS1 and p53 R273H exhibited a low preference for binding promoters with ETS motifs on the same face of the DNA helix. We propose a model for the binding of ETS1 and p53 R273H in which two inverted ETS motifs on a looped DNA helix are juxtaposed for ETS1 binding as a homodimer, with p53 R273H bound to ETS1. We propose that the formation of this DNA loop and protein-bound complex prevents additional binding of ETS1 and p53 R273H proteins to other proximal binding sites.

Gain-of-Function Activity of Mutant p53 in Lung Cancer through Up-Regulation of Receptor Protein Tyrosine Kinase Axl.

p53 mutations are present in up to 70% of lung cancer. Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 or no p53. Expression of tumor-derived mutant p53 in cells leads to up-regulated expression of genes that may affect cell growth and oncogenesis. In our study of this aggressive phenotype, we have investigated the receptor protein tyrosine kinase Axl, which is up-regulated by p53 mutants at both RNA and protein levels in H1299 lung cancer cells expressing mutants p53-R175H, -R273H, and -D281G. Knockdown of endogenous mutant p53 levels in human lung cancer cells H1048 (p53-R273C) and H1437 (p53-R267P) led to a reduction in the level of Axl as well. This effect on Axl expression is refractory to the mutations at positions 22 and 23 of p53, suggesting that p53's transactivation domain may not play a critical role in the up-regulation of Axl gene expression. Chromatin immunoprecipitation (ChIP) assays carried out with acetylated histone antibodies demonstrated induced histone acetylation on the Axl promoter region by mutant p53. Direct mutant p53 nucleation on the Axl promoter was demonstrated by ChIP assays using antibodies against p53. The Axl promoter has a p53/p63 binding site, which however is not required for mutant p53-mediated transactivation. Knockdown of Axl by Axl-specific RNAi caused a reduction of gain-of-function (GOF) activities, reducing the cell growth rate and motility rate in lung cancer cells expressing mutant p53. This indicates that for lung cancer cell lines with mutant p53, GOF activities are mediated in part through Axl.