P53 Gain Of Function Research Articles

The Diverse Functions of Mutant 53, Its Family Members and Isoforms in Cancer.

The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53′s inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics.

CAF hierarchy driven by pancreatic cancer cell p53-status creates a pro-metastatic and chemoresistant environment via perlecan

Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.

Abstract LB-311: Exosomal gain-of-function p53 protein promotes cancer-associated phenotype

Abstract TP53 is the most commonly mutated gene in cancer. Certain mutations can produce gain-of-function (GOF) phenotypes, resulting in pathogenic effects beyond the loss of p53 function. Recent study suggested that tumor cells could affect the p53 status of stromal cells while the mechanisms remains unclear. Exosomes have emerged as an important mode of extracellular communication and a major player in cancer pathogenesis, by promoting pro-tumor microenvironment. We hypothesized that packaging of mutated p53 protein into exosomes and dissemination via exosomes could impact a variety of stromal cell processes, producing a pro-tumorigenic microenvironment. In this study, we carried out a series of in vitro (immunoblot analysis, Q-PCR, Gene array, Co-Immunoprecipitation and lentiviral transfection) and in vivo (subcutaneous mouse models) experiments to determine the presence of mutant p53 in exosomes and its biological significance in tumor microenvironment. We demonstrated that gain-of-function (GOF) p53 protein is packaged into p53 mutant cancer cell-derived exosomes and transferred to fibroblasts. Further studies showed that this process is facilitated by the enhanced stability and consequent intracellular concentration of GOF p53. The packaging of GOF p53 into exosomes is selectively bound by HSP90 chaperone proteins. Inhibition of HSP90 blocks the packaging of GOF p53, but not wild-type into exosomes. Uptake of GOF p53-containing exosomes by fibroblasts results in conversion to a cancer-associated phenotype. In vivo study have shown that GOF p53 exosomes-induced cancer-associated fibroblasts promote tumor growth in a colon cancer cells (HT29 cells) xenograft mouse model. These findings reveal a crucial role of exosomes in modifying tumor microenvironment and hold great consequence in broadening our understanding of tumor biology. Citation Format: Shaolin Ma, Michael H. McGuire, Elaine Stur, Emine Bayraktar, Alejandro Villar-Prados, Sherry Y. Wu, Akira Yokoi, Santosh K. Dasari, Cristian Rodriguez-Aguayo, Jinsong Liu, Gabriel Lopez-Berestein, Anil K. Sood. Exosomal gain-of-function p53 protein promotes cancer-associated phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-311.

Abstract 5289: Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5

Abstract As the guardian of genome, p53 plays important roles in maintaining the genome integrity, and TP53 mutations thereby often lead to genomic instability in cancers. In addition to the loss-of-function, many TP53 mutations can lead to the gain-of-functions (GOF) that often promote tumor development and tumor progression. As an effort to identify the mechanisms involved in mutant p53 GOF activities in head and neck squamous cell carcinoma (HNSCC), we performed an unbiased proteomic screen to identify the mutant p53 G245D interactome in HNSCC cells by immunoprecipitation, and quantitative proteomics using stable isotope labeling with amino acids in cell culture and liquid chromatography coupled with tandem mass spectrometry. Our results identified that MCM5, a member of replication licensing heterohexameric origin recognition complex minichromosome maintenance 2-7 (MCM2-7), interacts with mutant p53s, which suggests a possible role of mutant p53 in regulation of DNA replication. Consistent with this, we show that overexpression of mutant p53 in p53-depelted oral keratinocytes and HNSCC cells predisposes cells susceptibility to replication stress by inhibiting the dormant origin firing, leading to increased chromosomal instability (CIN) under replication stress. Moreover, by overexpressing and downregulating MCM5, we demonstrate that MCM5 modulates GOF mutant p53-mediated susceptibility to replication stress and CIN. Given the importance of MCM2-7 in replication licensing, initiation, and elongation, our discovery of the relationship between mutant p53 and MCM5 suggests that the MCM2-7 complex is one of the key downstream effectors, through which GOF mutant p53s regulate DNA replication and genomic instability in HNSCC. Citation Format: Mei Zhao, Tianxiao Wang, Chen Zhen, Jing Wang, Curtis R. Pickering, Junjie Chen, Jeffrey N. Myers, Ge Zhou. Gain-of-function mutant p53 predisposes head and neck keratinocytes and squamous cell carcinoma cells to replicative stress and genomic instability through minichromosome maintenance complex component 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5289.

Diverse and cancer type‑specific roles of the p53 R248Q gain‑of‑function mutation in cancer migration and invasiveness.

Gain-of-function (GOF) mutations in the TP53 gene lead to acquisition of new functions by the mutated tumor suppressor p53 protein. A number of the over-represented ‘hot spot’ mutations, including the ones in codons 175, 248 or 273, convey GOF phenotypes. Such phenotypes may include resistance to chemotherapeutics or changes in motility and invasiveness. Whereas the prevalent notion is that the acquisition of the p53 GOF phenotype translates into poorer prognosis for the patient, the analysis of a human somatic p53 mutations dataset demonstrated earlier tumor onset, but decreased frequency and altered location of metastases in patients with the p53-R248Q allele. Therefore, the GOF activities of p53-R248Q and p53-D281G were analyzed in triple negative breast cancer MDA-MB-231 and lung adenocarcinoma H1299 cell lines with regard to invasive and metastatic traits. The expression of p53-D281G increased the motility and invasiveness of the lung cancer cells, but not those of the breast cancer cells. In contrast, the expression of p53-R248Q decreased the motility and invasiveness of the breast and lung cancer cells in a p53 transactivation-dependent manner. The intravenous xenotransplantation of MDA-MB-231 cells expressing p53-R248Q into zebrafish embryos resulted in an alteration of the distribution of cancer cells in the body of the fish. In p53-R248Q-expressing H1299 cells a decrease in the expression of TCF8/ZEB1 and N-cadherin was observed, suggesting partial mesenchymal-to-epithelial transition. In the two cell lines expressing p53-R248Q a decrease was noted in the expression of myosin light chain 2, a protein involved in actomyosin-based motility. To the best of our knowledge, the present study is one of only few reports demonstrating the mutated p53 GOF activity resulting in a decrease of a malignant trait in human cancer.

EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis.

In light of the increasing number of identified cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target several mutants, rather than developing strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation-sequencing (RIP-seq), we identified the Polycomb-group histone methyltransferase EZH2 as a p53 mRNA-binding protein. EZH2 bound to an internal ribosome entry site (IRES) in the 5'UTR of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing protein levels of mutant p53. EZH2 overexpression was associated with worsened outcome selectively in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.

The p53 Pathway in Glioblastoma.

The tumor suppressor and transcription factor p53 plays critical roles in tumor prevention by orchestrating a wide variety of cellular responses, including damaged cell apoptosis, maintenance of genomic stability, inhibition of angiogenesis, and regulation of cell metabolism and tumor microenvironment. TP53 is one of the most commonly deregulated genes in cancer. The p53-ARF-MDM2 pathway is deregulated in 84% of glioblastoma (GBM) patients and 94% of GBM cell lines. Deregulated p53 pathway components have been implicated in GBM cell invasion, migration, proliferation, evasion of apoptosis, and cancer cell stemness. These pathway components are also regulated by various microRNAs and long non-coding RNAs. TP53 mutations in GBM are mostly point mutations that lead to a high expression of a gain of function (GOF) oncogenic variants of the p53 protein. These relatively understudied GOF p53 mutants promote GBM malignancy, possibly by acting as transcription factors on a set of genes other than those regulated by wild type p53. Their expression correlates with worse prognosis, highlighting their potential importance as markers and targets for GBM therapy. Understanding mutant p53 functions led to the development of novel approaches to restore p53 activity or promote mutant p53 degradation for future GBM therapies.

Abstract 556: Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants

Abstract The tumor suppressor p53 protein is mutated in over 50% of cancers. The most frequent mutations in p53, termed “hotspot” mutations, occur in the DNA binding domain of the p53 protein. These mutations not only remove normal p53 function but also convey additional oncogenic properties greater than loss of tumor suppressor activities associated with loss of wild-type (WT) p53 activities. With newly acquired oncogenic properties, mutations in the DNA binding domain of p53 are classified as gain of function (GOF) mutations. GOF p53 mutants can be subdivided into conformational mutations (e.g. R175H, H193Y) involving residues necessary for maintenance of structure and contact mutations (e.g. R248Q, R273H) involving resides necessary for DNA interaction. Cancer cells bearing a GOF p53 mutation increase metastasis with application of histone deacetylase inhibitors (HDACi). This uncharacterized pro-oncogenic GOF p53 property is of clinical relevance as HDACi are FDA approved for hematological cancers but have failed in clinical trials for solid tumors. To explore this hypothesis, metastatic capacity and proliferation rates of common GOF p53 mutations were assessed with and without HDACi treatment and in the presence or absence of specific inhibitors of target GOF p53. The p53-null, H1299 non-small cell lung carcinoma line was transfected with GOF p53 mutants and WT p53 using a tetracycline inducible expression system. Inducible expression of stable subclones was varified by immunofluorescence and western blot. Proliferation rate was measured by incorporation of EdU and metastatic capacity was assessed by measuring MMP activity and migration assays with and without HDACi treatment. Specific GOF p53 mutations possess a subset of the oncogenic properties of the entire GOF p53 class. Data suggest that the GOF p53 mutants display differing rates of proliferation with the R175H mutation exhibiting the highest rate. Application of NSC319726 significantly decreases the proliferation rate and appears to successfully refold the R175H mutant to a WT conformation. The R248Q mutation exhibited morphological change suggestive of the epithelial to mesenchymal transition associated with increased metastatic capacity. Preliminary data suggest that HDACi affect among these cell lines in conjunction with NSC 319726 and SCH 529074 treatment. In conclusion, HDACi treatment may be contraindicated while treatment with HDACi may have great benefit in tumors which are WT or null for p53 protein expression. Citation Format: Brianna Flores, Elizabeth E. Hull. Oncogenic properties and response to HDAC inhibitor treatment of H1299 cells expressing GOF p53 mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 556.

Abstract 5462: Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma

Abstract One of the most frequently mutated genes reported in human cancers is TP53. Esophageal squamous cell carcinoma (ESCC) has an extremely high frequency of p53 mutations of 60-90% in ESCC patients. Evidence of gain-of-function (GOF) p53 mutations that exert wild-type (WT) function-independent oncogenic effects are emerging. We aim to determine the functional role of several candidate GOF p53 mutations in ESCC and explore their clinical utility for prognostication and as potential treatment targets. In our recent study of Hong Kong ESCC specimens, we detected TP53 alterations in over 90% of ESCC tumors, ~36% of which are candidate GOF mutations verified in other cancers. By analyzing published ESCC sequencing data, we showed that patients from the high-risk ESCC region show frequent candidate GOF p53 mutants. We applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to functionally knockout (fKO) mutant p53 protein expression in various ESCC cell lines with different endogenous p53 mutants and subject them to in vivo and in vitro functional assays. Interestingly, only KYSE150 cells with an endogenous p53R248Q mutation show reduced tumor growth in nude mice after p53-fKO, suggesting that there is a specific mutant p53 that confers oncogenic features in ESCC. Consistently, only KYSE150 p53-fKO cells show reduced colony formation in three-dimension in vitro culture. Tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) is significantly reduced in KYSE150 p53-fKO cells, suggesting that regulation of the STAT3 pathway contributed to the oncogenic feature of the GOF p53 mutant. p53R248Q exhibits concentrated subcellular localization to the membrane fraction (including ER, mitochondria, etc.) and shows increased resistance to HSP90/HDAC inhibition-induced protein degradation, as compared to other p53 mutants. Together, these results suggest a GOF role of certain mutant p53 proteins with specific characteristics. Targeting such mutants and/or related oncogenic pathways may be of therapeutic benefit. Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for General Research Fund support (17109515 to M.L.L). Citation Format: Valen Z. Yu, Maria L. Lung. Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5462.

Abstract 562: HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy

Abstract The purpose of this study is to evaluate the expression and possible upregulation of the salvage pathway enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) in prostate cancer cells to determine if it could serve as a biomarker for prostate cancer diagnosis and treatment. Prostate cancer is the second most lethal cancer in men, and an estimated 26,730 men will die from the disease in 2017. We chose to evaluate the HPRT enzyme due to its involvement in nucleotide synthesis and cell cycle progression. Two prostate cancer cell lines were used for this analysis (PC3 and DU145) along with malignant tissue from 35 patients with prostate carcinoma. The surface localization of HPRT was determined utilizing flow cytometry and scanning electron microscopy, while upregulation within tissue was assessed using immunohistochemistry. Additionally, RNA-seq data from TCGA was used to evaluate general HPRT upregulation in patients with prostate cancer (n = 502) when compared to healthy individuals (n = 52). Throughout our investigation, we found a significant association between HPRT and the plasma membrane of DU145 cells, but found no presence on PC3 cells. Flow cytometry showed insignificant (p = 0.14) changes in fluorescence when PC3 cells were exposed to HPRT antibodies, while there was a significant increase in fluorescence when DU145 cells were treated with fluorescent HPRT antibody (p = 0.0004). To determine the distribution of HPRT across the membrane and ensure the observed expression was not due to cytoplasmic HPRT, gold conjugated antibodies were used for analysis with an electron microscope. The distribution of the gold on the cell surface showed random HPRT binding across the membrane with no patterns of localization. This analysis aided in confirming HPRT surface presence as the gold weight % of DU145 cells increased significantly when exposed to HPRT antibodies (p < 0.0001). In addition to being presented on the surface of DU145 cells, tissue samples revealed variable HPRT expression as approximately 53% of prostate cancer patients evaluated had elevated levels of HPRT compared to normal controls, while 47% of patients had no upregulation. TCGA data revealed a significant (p = 1.53x10-4) increase in HPRT levels upon malignancy. While some patients had levels consistent with healthy controls, there was a significant number of patients with increased protein expression upon cancer development. The control of HPRT expression within these cancer cells has been linked preliminarily to p53 functionality. While PC3 cells are null for p53, DU145 cells have a gain of function (GOF) p53 mutation. GOF p53 mutations are known to influence salvage pathway enzyme expression and is an influential gene in HPRT expression in these cancer cells. These results strongly indicate a unique relationship between prostate cancer cells and HPRT and suggest HPRT as a possible biomarker for the detection and treatment of patients with prostate cancer. Citation Format: Michelle H. Townsend, Abigail M. Felsted, Taylor P. Cox, Zachary E. Ence, Stephen R. Piccolo, Richard A. Robison, Kim O'Neill. HPRT surface localization on prostate cancer cells as a biomarker for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 562.

Abstract 2988: Loss of Rbm38 cooperates with mutant p53 to promote lymphomagenesis through downregulation of Pten

Abstract The tumor suppressor TP53 is the most commonly mutated gene in human cancers and mutant p53 has a gain of function by promoting tumor progression, metastasis, and resistance to anticancer therapy. Interestingly, the RNA binding protein Rbm38, a critical regulator of mutant p53 translation, is frequently lost in tumors with mutant p53. To determine the role of the Rbm38-mutant p53 axis in tumorigenesis, we showed that loss of Rbm38 significantly alters the cancer susceptibility of mutant p53 knockin mice by shortening the lifespan, altering the tumor incidence, and promoting T-cell lymphomagenesis. To understand how Rbm38 deficiency promotes mutant p53 gain of function, we showed that loss of Rbm38 not only enhances mutant p53 expression but also decreases expression of tumor suppressor Pten, a critical regulator of T-cell development. Furthermore, we showed that Rbm38 is required for Pten expression by stabilizing Pten mRNA through an AU-rich element in its 3'UTR. Thus, we have uncovered a novel mechanism by which Pten is regulated by Rbm38 via mRNA stability. Our data also suggest that Rbm38 controls T-cell lymphomagenesis by concomitantly modulating mutant p53 and Pten, which may be explored as a therapeutic strategy for the treatment of T-cell malignancies. Citation Format: Jin Zhang, Heejung yang, Yanhong Zhang, Mingyi Chen, Xinbin Chen. Loss of Rbm38 cooperates with mutant p53 to promote lymphomagenesis through downregulation of Pten [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2988.

Abstract A28: Understanding the role of the tumor suppressor p53 in pancreatic cancer development

Abstract In the next decade, pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer-related death. This high rate of mortality is attributed to late-stage diagnosis and lack of effective therapeutic strategies. The tumor-suppressor gene TP53 is mutated in ~75% of PDAC patients. The most frequently observed TP53 mutations in PDAC occur within the DNA binding domain at codons 175 and 273. TP53 mutations may induce loss of wild-type p53 function and gain of novel, oncogenic functions. We aim to use different mouse models of PDAC to identify the molecular pathways deregulated when wild-type p53 is lost and mutant p53 is expressed, to elucidate the molecular mechanisms driving PDAC development. While previous studies have suggested that point mutant p53 drives gain-of-function phenotypes, our studies of aging Pdx1-Cre;KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato mice with conditional p53 knockout (p53Flox/Flox or p53Flox/Null) or p53 mutation (p53LSL-R172H/Flox or p53LSL-R172H/Null) did not demonstrate a clear mutant p53 gain-of-function phenotype in tumor latency. We hypothesize that tumor latency is too rapid in these models to manifest a clear gain-of-function phenotype, requiring the establishment of different model systems. Indeed, our preliminary studies using tumor-derived p53R172H cell lines and isogenic p53 null cell lines suggest a role for mutant p53 gain of function in later-stage tumorigenesis, specifically in metastatic seeding. Furthermore, we are evaluating the role of the putative PDAC cells of origin, pancreatic acinar and ductal cells, in PDAC development. Using both tumor-derived mouse cell lines and pancreatic lineage-specific genetically engineered mouse models of PDAC, we can evaluate the consequences of loss of wild-type p53 expression and gain of mutant p53 expression in different pancreatic compartments. Ultimately, using these systems we aim to elucidate the target genes and molecular pathways controlled by wild-type p53 or by mutant p53 to regulate PDAC initiation. Understanding the molecular mechanisms driving PDAC initiation is critical for the development of new therapeutic strategies for early detection and effective intervention in PDAC patients. Citation Format: Brittany M. Flowers, Patty B. Garcia, Barbara M. Grüner, Monte M. Winslow, Laura D. Attardi. Understanding the role of the tumor suppressor p53 in pancreatic cancer development [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A28.

Biallelic TP53 gain of function mutations in rapidly progressing solid tumors

Recent studies are discovering TP53 mutations with gain of function (GOF) properties that promote tumorigenesis via a variety of mechanisms. To our knowledge, all reported compound mutations are allelic. We identified two patients with biallelic GOF TP53 mutations in their tumors and a third with allelic compound variants. The correlation with p53 expression was also examined. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and mutational analysis was performed using Ion AmpliSeq™Cancer HotSpot Panel V2. Biallelic GOF mutations (p.R273H and p.R273C) were identified in a 19-year-old male with glioblastoma (allele frequencies 94% and 48%) and a 54-year-old with pT3 penile squamous cell carcinoma (allele frequencies 19% and 27%). Immunohistochemistry showed nuclear accumulation of p53. The third patient, a 62-year-old female with metastatic lung adenocarcinoma, had allelic p.P278S (GOF) and p.R283L (non-GOF) variants at frequencies of 61% but with null staining for p53. Germline testing for Patient 1 confirmed wildtype TP53. No other variants were discovered among the genes tested in these cases. All patients succumbed within two years of diagnosis despite aggressive treatment. In conclusion, implementation of TP53 mutation analysis in clinical practice may predict patient outcome, and inhibition of GOF p53 could represent an attractive target for therapy.

Improved potency of F10 relative to 5-fluorouracil in colorectal cancer cells with p53 mutations.

Aim:Resistance to fluoropyrimidine drugs (FPs) is a major cause of mortality in colorectal cancer (CRC). We assessed the potency advantage of the polymeric FP F10 relative to 5-fluorouracil (5FU) in four human CRC cell lines that differ only in TP53 mutational status to determine how p53 mutations affect drug response and whether F10 is likely to improve outcomes.Methods:HCT-116 human CRC cells (p53+/+) and three isogenic variants (p53−/−, R248W/+, R248W/−) were assessed for drug response. Resistance factors were derived from cell viability data and used to establish the relative potency advantage for F10. Rescue studies with exogenous uridine/thymidine determined if cytotoxicity resulted from DNA-directed processes.Results:Significant resistance to 5-FU resulted from p53-loss or from gain-of-function (GOF) mutation (R248W) and was greatest when GOF mutation was coupled with loss of wild-type p53. F10 is much more potent than 5-FU (137–314-fold depending on TP53 mutational status). F10 and 5-FU induce apoptosis by DNA- and RNA-directed mechanisms, respectively, and only F10 shows a modest enhancement in cytotoxicity upon co-treatment with leucovorin.Conclusion:TP53 mutational status affects inherent sensitivity to FPs, with p53 GOF mutations most deleterious. F10 is much more effective than 5-FU regardless of TP53 mutations and has potential to be effective to CRC that is resistant to 5-FU due, in part, to TP53 mutations.6,7

Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cells by targeting the transcription factors FOXO3a and FOXM1

Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that promote cancer cell invasive growth and metastasis, yet the mechanisms mediating these functions still largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances the invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in in vitro three-dimensional culture and in an in vivo orthotopic nude mouse model of HNSCC through a novel transcription-independent mechanism. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the AMP-activated protein kinase (AMPK)-mediated phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. This downregulation of FOXO3a’s activity, in turn, leads to de-repression of FOXM1 expression. Importantly, we show that either overexpression of FOXO3a or downregulation of FOXM1 impairs both GOF mutant p53-mediated cell invasion in vitro and pulmonary metastases of UM-SCC-1 cells in vivo. Finally, not only do oral cancer patients with p53 mutations exhibit higher levels of FOXM1 expression than patients with wild-type p53, but also HNSCC patients with TP53 mutations and high levels of FOXM1 expression have the poorest survival outcomes. Given our prior demonstration that GOF mutant p53s inhibit AMPK, our current study, establishes and demonstrates a novel transcription-independent GOF mutant p53-AMPK-FOXO3a-FOXM1 signaling cascade that plays an important role in mediating mutant p53s’ gain-of-function activities in HNSCCs.

Abstract 73: Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1

Abstract Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties in promoting cancer cell invasive growth and metastasis, but the mechanisms mediating these functions largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in vitro 3D culture and in vivo orthotopic tumor mouse model. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s in UM-SCC-1 cells and tumors. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the inhibitory phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. Consistent with the observation that FOXO3a negatively regulates FOXM1 expression, we demonstrate that overexpression FOXO3a decreases GOF mutant p53-mediated FOXM1 expression, whereas downregulation of FOXO3a partially restores decreased FOXM1 expression due to loss of GOF mutant p53. In support of the roles of FOXO3a-FOXM1 signaling axis in mutant p53's GOF signaling, we further show that overexpression of FOXO3a or downregulation of FOXM1 impairs GOF mutant p53-mediated cell invasion, whereas downregulation of FOXO3a, in part, rescues impaired cell invasion due to downregulated GOF mutant p53. Finally, given that AMP-activated protein kinase (AMPK) directly phosphorylates and promotes FOXO3a function, and that our prior demonstration that GOF mutant p53s inhibit AMPK, our current study establishes that GOF mutant p53-AMPK-FOX3a-FOXM1 signaling is one of important mechanisms through which mutant p53s gain oncogenic functions in HNSCCs. Citation Format: Noriaki Tanaka, Mei Zhao, Jiexin Zhang, Jing Wang, Ge Zhou, Jeffrey N. Myers. Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 73.

Abstract IA08: Personalizing HNSCC treatment based on TP53 mutational status

Abstract Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer deaths worldwide, and resistance to standard treatments with radiation/chemotherapy accounts for the majority of HNSCC deaths. Mutation of TP53 occurs in 60-85% of non-HPV-associated HNSCC cases, and some mutant p53s display oncogenic properties, termed “gain-of-function (GOF),” which are independent of wild-type (WT) p53 functions. These GOF p53 mutant proteins can enhance cell transformation, increase tumor formation, and confer cellular resistance to chemo- and/or radio-therapy. Therefore, we are interested in the development of innovative therapeutic approaches to overcome this resistance. Pre-clinical studies both in vitro and in vivo have shown the promise of some novel targeted agents including Chk-1, Wee-1, and PARP inhibitors used alone or in combination with HDAC inhibition. In addition, COTI-2 is a novel drug with striking single-agent and radio-/chemo-sensitizing activity in pre-clinical models of HNSCC bearing TP53 mutations. Our preliminary data demonstrate that COTI-2 markedly decreases cell growth in vitro and induces p21, noxa, and puma expression in HNSCC cells. COTI-2 also negatively modulates the PI3K/AKT/mTOR survival pathway, which is constitutively activated in many types of cancer including HNSCC. In vitro studies in our laboratory have shown that COTI-2 has a nanomolar (IC50: 1.3-13.2 nm) activity in nine human HNSCC cell lines. Additionally, GOF mutant p53-bearing HNSCC cells are significantly delayed in outgrowth in an orthotopic nude mouse model of tongue cancer when treated with COTI-2. Current clinical studies are evaluating the safety and efficacy of the combination of COTI-2 alone and in combination with cisplatin in phase I/II studies of patients with refractory HNSCC and whether response is associated with TP53 mutational status of the tumor determined from sequencing of circulating cell free DNA. If this combination is found to safely provide sensitization of HNSCC to cisplatin-based chemotherapy in the majority of human patients who have TP 53 mutant tumors, this should make a major impact on the survival of individuals suffering from HNSCC, and potentially other p53 driven epithelial malignancies. Citation Format: Jeffrey Myers. Personalizing HNSCC treatment based on TP53 mutational status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA08.

Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.

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.

Gain-of-function mutant p53 activates small GTPase Rac1 through SUMOylation to promote tumor progression.

Tumor suppressor p53 is frequently mutated in human cancer. Mutant p53 often promotes tumor progression through gain-of-function (GOF) mechanisms. However, the mechanisms underlying mutant p53 GOF are not well understood. In this study, we found that mutant p53 activates small GTPase Rac1 as a critical mechanism for mutant p53 GOF to promote tumor progression. Mechanistically, mutant p53 interacts with Rac1 and inhibits its interaction with SUMO-specific protease 1 (SENP1), which in turn inhibits SENP1-mediated de-SUMOylation of Rac1 to activate Rac1. Targeting Rac1 signaling by RNAi, expression of the dominant-negative Rac1 (Rac1 DN), or the specific Rac1 inhibitor NSC23766 greatly inhibits mutant p53 GOF in promoting tumor growth and metastasis. Furthermore, mutant p53 expression is associated with enhanced Rac1 activity in clinical tumor samples. These results uncover a new mechanism for Rac1 activation in tumors and, most importantly, reveal that activation of Rac1 is an unidentified and critical mechanism for mutant p53 GOF in tumorigenesis, which could be targeted for therapy in tumors containing mutant p53.

Abstract LB-263: P53 promotes replication genome stability by epigenetics-enabled MRE11 restart

Abstract DNA damage activates p53, the most frequently mutated tumor suppressor, to guard the genome and control gene transcription, epigenetics and apoptosis. Yet, how p53 suppresses genome instability remains ill-defined and is clouded by its genome-wide alterations. Here, enabled by our kPOND technology for single-cell nascent DNA/protein interactions and single-molecule DNA spreading, we define p53’s role in promoting genomic stability by enabling replication restart of stalled replication forks. Mechanistically, p53 enables MRE11 nuclease recruitment for stalled replication fork restart via control of epigenetic factor. We find an apoptosis-proficient and tumor promoting p53 mutant allele to be replication restart defective, while an apoptosis defective and poorly tumor-promoting murine p53 allele remains mostly restart proficient, revealing an improved correlation of restart functions with tumorigenesis compared to apoptosis functions. Importantly, both null and p53 gain-of-function (GOF) mutant cells show restart defects resulting in replication dependent genomic instability, uncovering a common function defect aside gene transcription. Both mutant and p53 null cells show increased recruitment of error-prone repair factors and DNA polymerases, providing a mechanism for genomic instability observed in these cells by pathway homeostasis imbalance. Collective results unveil a common transcription-independent pathway defect for both p53 null and GOF mutants laying groundwork for new targeting strategies of p53 defects in cancer. Note: This abstract was not presented at the meeting. Citation Format: Katharina Schlacher, Sunetra Roy, Karl-Heinz Tomaszowski. P53 promotes replication genome stability by epigenetics-enabled MRE11 restart [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2017-LB-263