Rebecca Frum

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.

The Growth Arrest Function of the Human Oncoprotein Mouse Double Minute-2 Is Disabled by Downstream Mutation in Cancer Cells

We have reported earlier that ectopic expression of mouse double minute-2 (MDM2) induces G1 arrest in normal cells. To explain occasional overexpression of MDM2 in cancer cells, we searched for deletion or substitution mutation in the growth suppressor domains of MDM2 in several breast cancer cell lines that overexpress the oncoprotein. Our results suggest the absence of alteration (deletion or substitution) in the open reading frame of MDM2 transcripts in such cells. Because the breast cancer cell line MCF-7 overexpresses MDM2, we isolated the full-length MDM2 transcript from this cell line. The MDM2 cDNA synthesized from transcripts isolated from MCF-7 cells induced inhibition of G1 to S phase transition in normal human diploid cells such as WI38, suggesting that the genetic alterations in breast cancer cells that overexpress MDM2 disable the growth arrest function of the oncoprotein. Consistently, overexpression of full-length MDM2 in MCF-7 cells over its high endogenous level did not inhibit G1-S transition efficiently. Although MDM2 overexpression was accompanied by CDK4 overexpression or absence of cdk4 inhibitor p16 in most breast cancer cells, we found remarkably high levels of cyclin A rather than cyclin E in these cells. Ectopic expression of cyclin A released MDM2-mediated inhibition of G1-S transition in normal human diploid WI38 cells. We propose that cancer cells expressing high levels of cyclin A escape MDM2-mediated G1 arrest, which may account for a selective growth advantage over normal cells.

MDM2 controls the timely expression of cyclin A to regulate the cell cycle.

Overexpression of MDM2 has been related to oncogenesis. In this communication, we present evidence to show that MDM2 controls the cell cycle–dependent expression of cyclin A by using a pathway that ensures its timely expression. MDM2 does not inhibit cyclin D or E expression. Silencing of endogenous MDM2 expression elevates cyclin A expression. The p53-binding domain of MDM2 harbors a SWIB region homologous to a conserved domain of a chromosome remodeling factor BRG1-associated protein. The SWIB domain of MDM2 inhibits cyclin A expression in a p53- and BRG1-dependent fashion, suggesting that MDM2 interferes with p53 binding of the BRG1 complex freeing it to repress cyclin A expression. Silencing of cyclin-dependent kinase (cdk) inhibitor p16 prevents MDM2-mediated inhibition of cyclin A expression, implicating its role in the process. MDM2-mediated repression of cyclin A expression induces G1-S arrest, which can be rescued by ectopic expression of cyclin A. Cancer cells lacking p53, p16, or BRG1 escape MDM2-mediated repression of cyclin A expression and growth arrest. Our data propose a novel mechanism by which MDM2 controls the cell cycle in normal cells and how cancer cells may escape this important safety barrier. (Mol Cancer Res 2009;7(8):1253–67)

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.

Constitutive Activation of DNA Damage Checkpoint Signaling Contributes to Mutant p53 Accumulation via Modulation of p53 Ubiquitination

Many mutant p53 proteins exhibit an abnormally long half-life and overall increased abundance compared with wild-type p53 in tumors, contributing to mutant p53s gain-of-function oncogenic properties. Here, a novel mechanism is revealed for the maintenance of mutant p53 abundance in cancer that is dependent on DNA damage checkpoint activation. High-level mutant p53 expression in lung cancer cells was associated with preferential p53 monoubiquitination versus polyubiquitination, suggesting a role for the ubiquitin/proteasome system in regulation of mutant p53 abundance in cancer cells. Interestingly, mutant p53 ubiquitination status was regulated by ataxia–telangectasia mutated (ATM) activation and downstream phosphorylation of mutant p53 (serine 15), both in resting and in genotoxin-treated lung cancer cells. Specifically, either inhibition of ATM with caffeine or mutation of p53 (serine 15 to alanine) restored MDM2-dependent polyubiquitination of otherwise monoubiquitinated mutant p53. Caffeine treatment rescued MDM2-dependent proteasome degradation of mutant p53 in cells exhibiting active DNA damage signaling, and ATM knockdown phenocopied the caffeine effect. Importantly, in cells analyzed individually by flow cytometry, p53 levels were highest in cells exhibiting the greatest levels of DNA damage response, and interference with DNA damage signaling preferentially decreased the relative percentage of cells in a population with the highest levels of mutant p53. These data demonstrate that active DNA damage signaling contributes to high levels of mutant p53 via modulation of ubiquitin/proteasome activity toward p53. Implication: The ability of DNA damage checkpoint signaling to mediate accumulation of mutant p53 suggests that targeting this signaling pathway may provide therapeutic gain. Mol Cancer Res; 14(5); 423–36. ©2016 AACR.

Flow cytometric analysis of MDM2-mediated growth arrest

Although MDM2, the product of mouse double minute-2 (mdm2) gene, or its human homologue possesses the potential to confer tumorigenic properties, it induces G1/S arrest in nontransformed cells. Flow cytometry provides a way to determine the effects of MDM2 on the cell cycle by expressing the protein ectopically, immunostaining cells expressing MDM2 and analyzing their DNA content. The DNA histograms of MDM2-transfected and untransfected cells can then be used to visualize the effect of ectopically expressed MDM2 on the cell cycle. Fluorescence-activated cell sorter (FACS) analysis following bromodeoxyuridine (BrdU) incorporation can be used to determine whether MDM2-expressing cells are synthesizing DNA. Incorporation of BrdU during DNA synthesis or repair can be detected in partially denatured DNA with a BrdU-specific fluorescent antibody. Subsequent staining of transfected MDM2 with a different fluorochrome provides information about whether transfected cells make significant progression through S phase. Further analysis of the growth-regulatory properties of MDM2 will elucidate both its normal function and the ways in which its deregulation leads to tumorigenesis.

Use of the DNA Fiber Spreading Technique to Detect the Effects of Mutant p53 on DNA Replication

DNA replication involves a coordinated progression through S phase, and disruption of these regulated steps may cause gene abnormalities, which may lead to cancer. Different stages of DNA replication can be detected immunofluorescently that would indicate how replication is progressing in a cell population or under specific conditions. We describe a method for labeling replicating DNA with two nucleotide analogs, and then detecting the sequential patterns of incorporation using fluorescently labeled antibodies on DNA spread onto a glass slide. Quantification of the different types of replication patterns produced by this method reveals how replication is achieved under different conditions by the predominance and lengths of elongating replication forks progressing from single or clustered origins, as well as the sites of termination from two converging forks.

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.

Transactivation and transrepression studies with p53.

The methods outlined in this chapter are designed to facilitate the study of the transactivation and transrepression properties of p53 (as well as p63 and p73). Once a gene of interest is identified, its presumptive promoter region can be cloned upstream of a luciferase gene in a plasmid. The most common reason for transfection experiments is to study gene expression patterns in the presence or absence of a particular gene product (e.g., p53). Three methods of transfection are outlined in this chapter: (i) cationic lipofection; (ii) calcium phosphate precipitation; and (iii) BES precipitation. The first method is ideal for the study of transactivation and transrepression properties of p53 (or other transcription factors). The last two are more suited for experiments where larger numbers of transfected cells are needed. Several examples of transfections and their respective results are provided.