Catherine Vaughan

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.

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 p53s 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.

Upregulation of the mitochondrial transport protein, Tim50, by mutant p53 contributes to cell growth and chemoresistance.

The p53 gene is one of the most frequently mutated genes in human cancer. Some p53 mutations impart additional functions that promote oncogenesis. To investigate how these p53 mutants function, a proteomic analysis was performed. The protein, translocator of the inner mitochondrial membrane 50 (Tim50), was upregulated in a non-small cell lung carcinoma cell line (H1299) that expressed the p53 mutants R175H and R273H compared to cells lacking p53. Tim50 was also elevated in the breast cancer cell lines MDA-MB-468 and SK-BR-3, that endogenously express the p53 mutants R175H and R273H, respectively, compared to MCF-10A. The p53 mutants R175H and R273H, but not WT p53, upregulated the expression of a Tim50 promoter construct and chromatin immunoprecipitation (ChIP) analysis indicated increased histone acetylation and increased interaction of the transcription factors Ets-1, CREB and CREB-binding protein (CBP) with the Tim50 promoter in the presence of mutant p53. Finally, reduction of Tim50 expression reduced the growth rate and chemoresistance of cells harboring mutant p53 but had no effect upon cells lacking p53. Taken together, these findings identify the Tim50 gene as a transcriptional target of mutant p53 and suggest a novel mechanism by which p53 mutants enhance cell growth and chemoresistance.

Human oncoprotein MDM2 activates the Akt signaling pathway through an interaction with the repressor element-1 silencing transcription factor conferring a survival advantage to cancer cells

The current paradigm states that the Akt signaling pathway phosphorylates the human oncoprotein mouse double minute 2 (MDM2), leading to its nuclear translocation and degradation of the tumor suppressor p53. Here we report a novel Akt signaling pathway elicited by MDM2. Upregulation of endogenous MDM2 promotes, whereas its downregulation diminishes, Akt phosphorylation irrespective of p53 status. MDM2 requires phosphatidylinositol (PI)3-kinase activity for enhancing Akt phosphorylation and upregulates this activity by repressing transcription of the regulatory subunit p85 of PI3-kinase. MDM2 interacts with the repressor element-1 silencing transcription factor (REST), a tumor suppressor that functions by downregulating PI3-kinase activity and Akt phosphorylation, prevents localization of REST on the p85 promoter and represses p85 expression. The deletion mutant of MDM2 capable of upregulating Akt phosphorylation represses p85 expression and interferes with localization of REST on the p85 promoter, whereas the deletion mutant of MDM2 that does not increase Akt phosphorylation cannot perform these functions. Silencing of REST abrogates the ability of MDM2 to upregulate Akt phosphorylation and downregulate p85 expression, implicating the ability of MDM2 to interact with REST in its ability to inhibit p85 expression and activate Akt phosphorylation. Inhibition of MDM2-mediated Akt phosphorylation with an Akt-phosphorylation-specific inhibitor abrogates its ability to improve cell survival. Consistently, the Akt phosphorylation function of MDM2 was required for its ability to improve cell survival after treatment with a chemotherapeutic drug. Our report not only unravels a novel signaling pathway that contributes to cell survival but also implicates a p53-independent transcription regulatory function of MDM2 in Akt signaling.

p53: Its Mutations and Their Impact on Transcription

p53 is a tumor suppressor protein whose key function is to maintain the integrity of the cell. Mutations in p53 have been found in up to 50 % of all human cancers and cause an increase in oncogenic phenotypes such as proliferation and tumorigenicity. Both wild-type and mutant p53 have been shown to transactivate their target genes, either through directly binding to DNA, or indirectly through protein-protein interactions. This review discusses possible mechanisms behind both wild-type and mutant p53-mediated transactivation and touches on the concept of addiction to mutant p53 of cancer cells and how that may be used for future therapies.

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing.

Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.

Isolation and Characterization of Murine Multipotent Lung Stem Cells

Cancer stem cells possess the ability to self-renew and differentiate into specific cells found in tumor types, a characteristic feature of normal multipotent stem cells. These cells harbor within the bulk of tumors and if the tumor suppressor p53 is mutated in these cells, can be more likely to cause relapse and metastasis by giving rise to new tumors. This new paradigm of oncogenesis has been observed in various cancers, including lung cancer. Determining the interaction of critical cellular pathways in the ontogeny of lung tumors is expected to lead to identification of molecular targets for effective therapeutic strategies. To achieve this, it is important to characterize and dissect the differences between the cancer cells with aberrant stem cell like properties and normal multipotent stem cells that contribute to regeneration. This could be accomplished by using cell surface markers unique for certain cell types by employing techniques such as flow cytometry and magnetic bead isolation. This chapter summarizes the isolation process of the resident stem cell Sca1 (+ve), CD-45 (-ve), and CD-31 (-ve) populations for its potential use in assessing correlations between specific p53 gain of function phenotypes in different murine lung cancer models.

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.

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.

Human Oncoprotein MDM2 Up-regulates Expression of NF-κB2 Precursor p100 Conferring a Survival Advantage to Lung Cells

The current model predicts that MDM2 is primarily overexpressed in cancers with wild-type (WT) p53 and contributes to oncogenesis by degrading p53. Following a correlated expression of MDM2 and NF-κB2 transcripts in human lung tumors, we have identified a novel transactivation function of MDM2. Here, we report that in human lung tumors, overexpression of MDM2 was found in approximately 30% of cases irrespective of their p53 status, and expression of MDM2 and NF-κB2 transcripts showed a highly significant statistical correlation in tumors with WT p53. We investigated the significance of this correlated expression in terms of mechanism and biological function. Increase in MDM2 expression from its own promoter in transgenic mice remarkably enhanced expression of NF-κB2 compared with its non-transgenic littermates. Knockdown or elimination of endogenous MDM2 expression in cultured non-transformed or lung tumor cells drastically reduced expression of NF-κB2 transcripts, suggesting a normal physiological role of MDM2 in regulating NF-κB2 transcription. MDM2 could up-regulate expression of NF-κB2 transcripts when its p53-interaction domain was blocked with Nutlin-3, indicating that the MDM2-p53 interaction is dispensable for up-regulation of NF-κB2 expression. Consistently, analysis of functional domains of MDM2 indicated that although the p53-interaction domain of MDM2 contributes to the up-regulation of the NFκB2 promoter, MDM2 does not require direct interactions with p53 for this function. Accordingly, MDM2 overexpression in non-transformed or lung cancer cells devoid of p53 also generated a significant increase in the expression of NF-κB2 transcript and its targets CXCL-1 and CXCL-10, whereas elimination of MDM2 expression had the opposite effects. MDM2-mediated increase in p100/NF-κB2 expression reduced cell death mediated by paclitaxel. Furthermore, knockdown of NF-κB2 expression retarded cell proliferation. Based on these data, we propose that MDM2-mediated NF-κB2 up-regulation is a combined effect of p53-dependent and independent mechanisms and that it confers a survival advantage to lung cancer cells.