Trp53 null and R270H mutant alleles have comparable effects in regulating invasion, metastasis, and gene expression in mouse colon tumorigenesis.
ABSTRACT: Somatic APC (adenomatous polyposis coli), TP53, KRAS mutations are present in roughly 80%, 60%, and 40%, respectively, of human colorectal cancers (CRCs). Most TP53 mutant alleles in CRCs encode missense mutant proteins with loss-of-function (LOF) of p53's transcriptional activity and dominant negative (DN) effects on wild-type p53 function. Missense mutant p53 proteins have been reported to exert gain-of-function (GOF) effects in cancer. We compared the phenotypic effects of the common human cancer-associated TP53 R273H missense mutation to p53 null status in a genetically engineered mouse CRC model. Inactivation of one allele of Apc together with activation of a Kras mutant allele in mouse colon epithelium instigated development of serrated and hyperplastic epithelium and adenomas (AK mice). Addition of a Trp53R270H or Trp53null mutant allele to the model (AKP mice) led to markedly shortened survival and increased tumor burden relative to that of AK mice, including adenocarcinomas in AKP mice. Comparable life span and tumor burden were seen in AKP mice carrying Trp53R270H or Trp53null alleles, along with similar frequencies of spontaneous metastasis to lymph nodes, lung, and liver. The fraction of adenocarcinomas with submucosa or deeper invasion was higher in AKP270/fl mice than in AKPfl/fl mice, but the incidence of adenocarcinomas per mouse did not differ significantly between AKPfl/fl and AKP270/fl mice. In line with their comparable biological behaviors, mouse primary tumors and tumor-derived organoids with the Trp53R270H or Trp53null alleles had highly similar gene expression profiles. Human CRCs with TP53 R273 missense mutant or null alleles also had essentially homogeneous gene expression patterns. Our findings indicate the R270H/R273H p53 mutant protein does not manifest definite GOF biological effects in mouse and human CRCs, suggesting possible GOF effects of mutant p53 in cancer phenotypes are likely allele-specific and/or context-dependent.
Project description:TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.
Project description:Missense mutation of tumor suppressor p53, which exhibits oncogenic gain-of-function (GOF), not only promotes tumor progression, but also diminishes therapeutic efficacies of cancer treatments. However, it remains unclear how a p53 missense mutant contributes to induced pluripotency of cancer stem cells (CSCs) in tumors exposed to chemotherapeutic agents. More importantly, it may be possible to abrogate the GOF by restoring wild-type p53 activity, thereby overcoming the deleterious effects resulting from heterotetramer formation, which often compromises the efficacies of current approaches being used to reactivate p53 function. Herewith, we report that p53 R273H missense mutant urges cancer cells to spawn CSCs. SW48/TP53 cells, which heterozygously carry the p53 R273H hot-spot mutant (R273H/+, introduced by a CRISPR/Casp9 system), were subchronically exposed to doxorubicin in cell culture and in tumor-bearing mice. We found that p53-R273H (TP53-Dox) cells were drug-resistant and exhibited epithelial-mesenchymal transition (EMT) and increased numbers of CSCs (CD44v6+/CD133+), which resulted in enhanced wound healing and tumor formation. Inhibition of glucosylceramide synthase with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) sensitized p53-R273H cancer cells and tumor xenografts to doxorubicin treatments. Intriguingly, PDMP treatments restored wild-type p53 expression in heterozygous R273H mutant cells and in tumors, decreasing CSCs and sensitizing cells and tumors to treatments. This study demonstrated that p53-R273H promotes EMT and induced pluripotency of CSCs in cancer cells exposed to doxorubicin, mainly through Zeb1 and ?-catenin transcription factors. Our results further indicate that restoration of p53 through inhibition of ceramide glycosylation might be an effective treatment approach for targeting cancers heterozygously harboring TP53 missense mutations.
Project description:Over half of colorectal cancers (CRCs) harbor TP53 missense mutations (mutp53). We show that the most common mutp53 allele R248Q (p53Q) exerts gain of function (GOF) and creates tumor dependence in mouse CRC models. mutp53 protein binds Stat3 and enhances activating Stat3 phosphorylation by displacing the phosphatase SHP2. Ablation of the p53Q allele suppressed Jak2/Stat3 signaling, growth, and invasiveness of established, mutp53-driven tumors. Treating tumor-bearing mice with an HSP90 inhibitor suppressed mutp53 levels and tumor growth. Importantly, human CRCs with stabilized mutp53 exhibit enhanced Jak2/Stat3 signaling and are associated with poorer patient survival. Cancers with TP53R248Q/W are associated with a higher patient death risk than are those having nonR248 mutp53. These findings identify GOF mutp53 as a therapeutic target in CRC.
Project description:Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF) properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC) harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR) treated SW480 cells expressing mutant p53(R273H) (GEO#: GSE77533). We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53.
Project description:Missense-type mutant p53 plays a tumor-promoting role through gain-of-function (GOF) mechanism. In addition, the loss of wild-type TP53 through loss of heterozygosity (LOH) is widely found in cancer cells. However, malignant progression induced by cooperation of TP53 GOF mutation and LOH remains poorly understood. Here, we show that mouse intestinal tumors carrying Trp53 GOF mutation with LOH (AKTPM/LOH) are enriched in metastatic lesions when heterozygous Trp53 mutant cells (AKTP+/M) are transplanted. We show that Trp53 LOH is required for dormant cell survival and clonal expansion of cancer cells. Moreover, AKTPM/LOH cells show an increased in vivo tumor-initiating ability compared with AKTPNull and AKTP+/M cells. RNAseq analyses reveal that inflammatory and growth factor/MAPK pathways are specifically activated in AKTPM/LOH cells, while the stem cell signature is upregulated in both AKTPM/LOH and AKTPNull cells. These results indicate that TP53/Trp53 LOH promotes TP53/Trp53 GOF mutation-driven metastasis through the activation of distinct pathway combination.
Project description:TP53 missense mutations significantly influence the development and progression of various human cancers via their gain of new functions (GOF) through different mechanisms. Here we report a unique mechanism underlying the GOF of p53-R249S (p53-RS), a p53 mutant frequently detected in human hepatocellular carcinoma (HCC) that is highly related to hepatitis B infection and aflatoxin B1. A CDK inhibitor blocks p53-RS's nuclear translocation in HCC, whereas CDK4 interacts with p53-RS in the G1/S phase of the cells, phosphorylates it, and enhances its nuclear localization. This is coupled with binding of a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) to p53-RS, but not the p53 form with mutations of four serines/threonines previously shown to be crucial for PIN1 binding. As a result, p53-RS interacts with c-Myc and enhances c-Myc-dependent rDNA transcription key for ribosomal biogenesis. These results unveil a CDK4-PIN1-p53-RS-c-Myc pathway as a novel mechanism for the GOF of p53-RS in HCC.
Project description:TP53 is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single p53 mutation (p53R172H) or p53 loss in osteoblasts. Our study confirmed that p53 mutant mice developed osteosarcomas with increased metastasis as compared with p53-null mice. Comprehensive transcriptome RNA sequencing (RNA-seq) analysis of 16 tumors identified a cluster of small nucleolar RNAs (snoRNAs) that are highly up-regulated in p53 mutant tumors. Regulatory element analysis of these deregulated snoRNA genes identified strong enrichment of a common Ets2 transcription factor-binding site. Homozygous deletion of Ets2 in p53 mutant mice resulted in strong down-regulation of snoRNAs and reversed the prometastatic phenotype of mutant p53 but had no effect on osteosarcoma development, which remained 100% penetrant. In summary, our studies identify Ets2 inhibition as a potential therapeutic vulnerability in p53 mutant osteosarcomas.
Project description:TP53 (which encodes p53 protein) is the most frequently mutated gene among all human cancers. Prevalent p53 missense mutations abrogate its tumour suppressive function and lead to a 'gain-of-function' (GOF) that promotes cancer. Here we show that p53 GOF mutants bind to and upregulate chromatin regulatory genes, including the methyltransferases MLL1 (also known as KMT2A), MLL2 (also known as KMT2D), and acetyltransferase MOZ (also known as KAT6A or MYST3), resulting in genome-wide increases of histone methylation and acetylation. Analysis of The Cancer Genome Atlas shows specific upregulation of MLL1, MLL2, and MOZ in p53 GOF patient-derived tumours, but not in wild-type p53 or p53 null tumours. Cancer cell proliferation is markedly lowered by genetic knockdown of MLL1 or by pharmacological inhibition of the MLL1 methyltransferase complex. Our study reveals a novel chromatin mechanism underlying the progression of tumours with GOF p53, and suggests new possibilities for designing combinatorial chromatin-based therapies for treating individual cancers driven by prevalent GOF p53 mutations.
Project description:p53 missense mutant alleles are present in nearly 40% of all human tumors. Such mutated alleles generate aberrant proteins that not only lose their tumor-suppressive functions but also frequently act as driver oncogenes, which promote malignant progression, invasion, metastasis, and chemoresistance, leading to reduced survival in patients and mice. Notably, these oncogenic gain-of-function (GOF) missense mutant p53 proteins (mutp53) are constitutively and tumor-specific stabilised. This stabilisation is one key pre-requisite for their GOF and is largely due to mutp53 protection from the E3 ubiquitin ligases Mdm2 and CHIP by the HSP90/HDAC6 chaperone machinery. Recent mouse models provide convincing evidence that tumors with highly stabilized GOF mutp53 proteins depend on them for growth, maintenance, and metastasis, thus creating exploitable tumor-specific vulnerabilities that markedly increase lifespan if intercepted. This identifies mutp53 as a promising cancer-specific drug target. This review discusses direct mutp53 protein-targeting drug strategies that are currently being developed at various preclinical levels.
Project description:Despite the propensity for gastric and esophageal adenocarcinomas to select for recurrent missense mutations in TP53, the precise functional consequence of these mutations remains unclear. Here we report that endogenous mRNA and protein levels of mutant p53 were elevated in cell lines and patients with gastric and esophageal cancer. Functional studies showed that mutant p53 was sufficient, but not necessary, for enhancing primary tumor growth in vivo. Unbiased genome-wide transcriptome analysis revealed that hypoxia signaling was induced by mutant p53 in 2 gastric cancer cell lines. Using real-time in vivo imaging, we confirmed that hypoxia reporter activity was elevated during the initiation of mutant p53 gastric cancer xenografts. Unlike HIF co-factor ARNT, HIF1? was required for primary tumor growth in mutant p53 gastric cancer. These findings elucidate the contribution of missense p53 mutations in gastroesophageal malignancy and indicate that hypoxia signaling rather than mutant p53 itself may serve as a therapeutic vulnerability in these deadly set of cancers.