P53 Gain Of Function Research Articles

Abstract 3030: Mutant p53-driven metastasis explored utilizing a traceable, conditional osteosarcoma model

Abstract TP53 is the most frequently mutated gene in cancer and missense mutations in particular account for the majority of mutations. Mutant p53 proteins exert oncogenic Gain-of-Function (GOF) properties that contribute to increased migration and invasion in culture and metastasis in vivo. To elucidate how mutant p53 drives metastasis, we developed a traceable somatic osteosarcoma model that initiates with a single p53 mutation (p53R172H or p53R245W), or p53 loss specifically in osteoblasts using Osx-cre. To mark tumor cells, we utilized the mTmG allele which expresses membrane-targeted Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision so the tumor cells become GFP+ and the stromal cells remain RFP+. In this high penetrant, short latency mouse model, tumor micro-metastasis can be detected by GFP expression in the context of a normal stroma and immune system. The cohort has developed osteosarcoma with complete penetrance and an average survival of 304± 65 day. We observed metastasis in 55% of p53 mutant osteosarcomas which was significantly higher than 26% metastasis observed in p53 null tumors (p<0.01). In p53 mutant osteosarcomas, the metastasis rate was found significantly associated with low p53 copy number. Spectral karyotyping (SKY) on early passage cells derived from somatic p53 mutant tumors exhibited marked aneuploidy with modal chromosome numbers near tetraploid. To characterize the transcriptional profile associated with mutant p53 GOF, we analyzed and compared the RNA-Seq transcriptomes of eight tumors derived from p53R172H mutant mice with eight p53 null tumors. Comparative analysis of gene expression identified a signature of snRNA genes to be strongly associated with mutant p53 gain of function. Functional annotation of upregulated genes in mutant tumors was also highly enriched for chromatin modifying enzymes. These findings contribute to our understanding of the role of mutant p53 GOF in metastasis. Our long term goal is to study how tumor-stromal interactions affect tumor development and metastasis. This understanding will have broad translational significance in diagnosis and treatment of tumors with mutant p53. Citation Format: Rasoul Pourebrahimabadi, Yun Zhang, Mark J. McArthur, Guillermina Lozano. Mutant p53-driven metastasis explored utilizing a traceable, conditional osteosarcoma model [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 3030. doi:10.1158/1538-7445.AM2017-3030

Abstract 2579: Identifying pathways that regulate the turnover of gain-of-function p53 mutant proteins in ovarian cancer cells

Abstract Ovarian cancer is the fifth leading cause for cancer-associated deaths among women in the US. High-grade serous ovarian carcinoma is the most common histological subtype and is characterized by frequent (>90%) mutations in the TP53 (p53) gene. Gain-of-function (GOF) p53 mutant proteins form highly stable aggregates that accumulate in ovarian cancer cells and have been shown to have oncogenic activity. Increasing evidence indicates the stabilization of GOF p53 mutants to be important for tumor progression and drug resistance. Therefore, selective degradation of the GOF p53 mutant proteins is a highly attractive therapeutic strategy to specifically target ovarian cancer cells. We identified that a small molecule named MCB-613, originally discovered as a stimulator of steroid receptor coactivator-3 (SRC-3), causes rapid degradation of the usually stable p53R175H conformational mutant aggregates leading to catastrophic cell death in human ovarian cancer cell lines. Our results indicate this effect is independent of SRC-3 mediated gene expression, but rather by post-translational modification of p53 mutant protein. Upon MCB-613 treatment, the p53R175H mutant is rapidly exported from the nucleus, ubiquitinated and degraded through a lysosomal pathway. Interestingly, in contrast to its effect on the p53R175H mutant, MCB-613 causes a slight stabilization of wild type (WT) p53 but had less of an effect on the turnover of other frequently observed p53 mutants (R248Q and R273H) in human ovarian cancer cell lines. MCB-613 also causes a rapid decrease in MDM2 protein but not mRNA. Further, the effect of MCB-613 on p53R175H is phenocopied by multiple deubiquitinase (DUB) inhibitors, confirming the role of ubiquitination in the selective turnover of p53R175H mutant aggregates. Taken together, these results show that different pathways regulate the turnover of WT p53 and the specific GOF p53 mutant proteins. We are currently investigating the precise mechanism by which MCB-613 and the different DUB inhibitors induce selective turnover of p53R175H mutant protein. We anticipate the proposed work to significantly advance our understanding of pathways important in ovarian cancer progression and pave way for the development of new targeted therapies for ovarian cancer patients with specific p53 mutations. This work is funded by NIH-CA-181808, NIH-HD-07857 and NIH-HD-076596. Citation Format: Achuth Padmanabhan, Nicholes Candelaria, Kwong-Kwok Wong, Bryan C. Nikolai, David Lonard, Bert W. O'Malley, JoAnne S. Richards. Identifying pathways that regulate the turnover of gain-of-function p53 mutant proteins in ovarian cancer cells [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 2579. doi:10.1158/1538-7445.AM2017-2579

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.

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.

P53 loss-of-heterozygosity is a necessary prerequisite for mutant p53 stabilization and gain-of-function in vivo

Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/− tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data – while cautioning that not all genetic mouse models faithfully represent the human situation – demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.

Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5

Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5'-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.

Emerging Non-Canonical Functions and Regulation by p53: p53 and Stemness.

Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53’s functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 (mutp53) GOFs that lead to survival, drug resistance and colonization are reviewed in the context of the acquisition of advantageous transformation processes, such as differentiation and dedifferentiation, epithelial-to-mesenchymal transition (EMT) and stem cell senescence and quiescence. Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53’s effects on stemness could lead to novel therapeutic strategies in cancer research.

A novel mutant p53 binding partner BAG5 stabilizes mutant p53 and promotes mutant p53 GOFs in tumorigenesis.

Tumor suppressor p53 is the most frequently mutated gene in human tumors. Many tumor-associated mutant p53 (mutp53) proteins gain new tumor-promoting activities, including increased proliferation, metastasis and chemoresistance of tumor cells, which are defined as gain-of-functions (GOFs). Mutp53 proteins often accumulate at high levels in human tumors, which is important for mutp53 to exert their GOFs. The mechanism underlying mutp53 proteins accumulation in tumors is not fully understood. Here, we report that BAG5, a member of Bcl-2-associated athanogene (BAG) family proteins, promotes mutp53 accumulation in tumors, which in turn enhances mutp53 GOFs. Mechanistically, BAG5 interacts with mutp53 proteins to protect mutp53 from ubiquitination and degradation by E3 ubiquitin ligases MDM2 and CHIP, which in turn promotes mutp53 protein accumulation and therefore GOFs in promoting cell proliferation, tumor growth, cell migration and chemoresistance. BAG5 is frequently overexpressed in many human tumors and the overexpression of BAG5 is associated with poor prognosis of cancer patients. Altogether, this study revealed that inhibition of mutp53 degradation by BAG5 is a novel and critical mechanism underlying mutp53 protein accumulation and GOFs in cancer. Furthermore, our results also uncovered that promoting mutp53 accumulation and GOFs is a novel mechanism of BAG5 in tumorigenesis.

Abstract 3688: The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity

Abstract p53 is the most commonly mutated gene in human cancers. Unlike other tumor suppressors in which loss of protein expression is common, the vast majority of changes in p53 arise as point mutations. Tumor associated mutation hotspots are localized to the DNA binding domain, either in residues involved in direct DNA contact or those that regulate its conformation. In either case, such mutations have been shown to confer gain of function activity. This was first shown in soft agar assays in which over-expression of mutant p53 increased transformation of cells on a p53 null background. Furthermore, knock-in mouse models of mutant p53 showed alterations in tumor spectrum with the development of carcinomas not seen in p53 null mice. However, the mechanism by which mutant p53 actively aids in tumorigenesis remains unclear. Although mutant p53 causes loss of sequence specific DNA binding via the core domain, it retains the ability to interact with multiple proteins including transcriptional cofactors shown to have tumor suppressor activity such as p300 and CBP. These interactions occur through multiple protein binding domains including the C-terminal domain of p53. Our laboratory and others have shown that C-terminal truncation of wildtype p53 results in multiple deficiencies. Binding of mutant p53 to p300/CBP via the C-terminus is therefore an attractive hypothesis to explain mutant p53 gain of function. To determine the role of the C-terminus in mutant p53 gain of function activity, we have expressed constructs containing a C-terminal truncation in combination with varying hotspot mutations. Transfection of mutant p53 into a p53 null cell line was required for the formation of colonies in a soft agar assay, indicating an increase in tumorigenicity. Furthermore, C-terminal truncation of the mutant p53 abrogated this phenotype suggesting that this domain is required for mutant p53 gain of function. This was independent of expression levels as shown by immunoblot. We have also extended the findings of mutant p53 interaction with p300 by IP analysis of a panel of cell lines with varying p53 hotspot mutations. To confirm the role of the C-terminus in mutant p53 gain of function, we are developing a xenograft model to detect changes in tumorigenicity in vivo. These results will therefore explore the mechanism by which mutant p53 acts as an oncogene. Citation Format: Caleb C. Lee, James Manfredi. The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3688.

Abstract SY04-03: Improving survival by exploiting tumor dependence on stabilized mutant p53 in mouse models

Abstract Missense mutations in p53 generate aberrant proteins with abrogated tumour suppressor functions that can also acquire oncogenic gain-of-function activities that promote malignant progression, invasion, metastasis and chemoresistance (1-5). Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumours, which is the key requisite for the acquisition of gain-of- functions activities (6-8). Although currently 11 million patients worldwide live with tumours expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo. Here we use a novel mutp53 knockin mouse model expressing a conditional inactivatable R248Q hotspot mutation (called floxQ; present in human cancers) to show that tumours depend on sustained mutp53 protein expression. Upon Tamoxifen-induced mutp53 ablation, allotransplanted and autochthonous tumours curb their growth, thus extending animal survival by 37%, and advanced tumours undergo apoptosis and tumour regression or stagnation. The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared with normal tissues, is a major determinant of mutp53 stabilization (9-12). We show that long-term HSP90 inhibition significantly extends the survival of hotspot mutp53 Q/- (R248Q allele; Ref 2) and H/H (R172H allele; Ref 3) mice by 59% and 48%, respectively, but not their corresponding p53 -/- (also known as Trp53 -/-) littermates. This mutp53-dependent drug effect occurs in tumor-bearing H/H mice treated with 17DMAG/SAHA, and in H/H and Q/- mice treated with the potent Hsp90 inhibitor ganetespib (the latter given as a once-weekly monotherapy). Notably, drug activity correlates with induction of mutp53 degradation, tumour cell apoptosis and strong prevention of T-cell lymphomagenesis. Together, these findings support the notion that tumors expressing mutp53 depend on it for tumor survival and maintenance, and fundamentally differ in their oncogenic wiring from p53-null tumors. Moreover, and surprisingly, the mechanism of action of these pleiotropic Hsp90 inhibitors in this specific setting is largely via targeting mutp53. Missense mutp53 is highly expressed in ∼50% of all human tumors. One therapeutic strategy, considered for nearly two decades, is to develop small compounds capable of restoring the lost wildtype function of mutp53 proteins (13). In the face of this daunting and still elusive goal, the results presented here show that eliminating stabilized mutp53 protein has positive therapeutic effects in vivo, even in the absence of a wildtype allele, since such tumors show exploitable dependence on its gain-of-function. Moreover, targeting Hsp90 might represent the first and currently only viable clinical strategy to achieve this goal. Overall, these proof-of-principle data identify mutp53 as a potentially actionable cancer-specific drug target.

092 Merkel cell polyomavirus small T antigen co-expressed with Atoh1 drives development of intraepidermal MCC

092 Merkel cell polyomavirus small T antigen co-expressed with Atoh1 drives development of intraepidermal MCC ME Verhaegen, D Mangelberger, PW Harms, D Wilbert, J Meireles, T Saunders and AA Dlugosz 1 Dermatology, University of Michigan, Ann Arbor, MI, 2 Pathology, University of Michigan, Ann Arbor, MI and 3 Internal Medicine, University of Michigan, Ann Arbor, MI Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor that frequently expresses viral transforming antigens, sTAg and tLTAg, encoded by Merkel cell polyomavirus (MCPyV). While the cell of origin is unknown, MCCs express markers detected in normal Merkel cells, including Keratin (K) 20, K8, and synaptophysin. Most MCCs and MCC cell lines also express ATOH1, a bHLH protein that is required for Merkel cell specification, and when expressed ectopically, drives de novo development of Merkel cells from epidermis. To test the potential of MCPyV viral oncoproteins to drive MCC development, we co-expressed MCPyV TAgs and Atoh1 in epidermis of transgenic mice. Combined expression of sTAg and Atoh1 in skin of multiple independent founders induced striking alterations that mimicked the appearance of human intraepidermal MCC, including numerous foci of small blue cells with nuclear hyperchromasia, mitotic activity and epidermal clefting. These tumor-like collections of cells expressed typical Merkel cell and MCC markers, including synaptophysin, K8 and K20. In contrast to normal Merkel cells, however, K20 was present in a paranuclear dot-like pattern which is pathognomonic of MCC. Moreover, many tumor cells expressed both K8 and the proliferation maker Ki67, which is characteristic of MCC tumor cells but not Merkel cells, which are post-mitotic. Addition of tLTAg did not appreciably alter the MCC-like phenotype produced by co-expression of sTAg and Atoh1. Thus, expression of MCPyV sTAg in Atoh1expressing epidermal cells of pre-term mouse embryos leads to widespread development of MCC-like tumors, strongly supporting the concept that sTAg is the primary oncogenic driver of MCC when expressed in the appropriate progenitor population. 093 Cell fate determination in the initiation of high-risk skin carcinoma WK Ryan, J Fernandez and EC Torchia Dermatology, University of Colorado Anschutz Medical Campus, Aurora, CO There are few effective treatment options for high-risk Squamous Cell Carcinomas (SCCs). Our goal in this study was to define the early signaling pathways in “initiated” Keratinocyte (KC) progenitors that contribute to the progression of high-risk tumors in a well-characterized mouse SCC model. Here, the activation of a Gain-Of-Function (GOF) p53 mutant resulted in tumors with centrosome amplification, dysregulation of the mitotic Kinase Aurora-A (Aur-A), genomic DNA instability, and enhanced metastasis, implicating abnormal mitotic regulation in the progression to high-risk SCCs. We initially observed a marked S-phase increase in both GOF and Loss-Of-Function (LOF) p53-KCs after a short course of the tumor promoter, TPA. However, histological presentation remained similar. The genetic deletion of Aur-A (Aur-A) in GOF or LOF p53 skin led to dramatic morphological changes associated with diffused hyperplasia, cellular pleomorphism, cell karyomegaly, prominent nucleoli, and multinucleation. These effects were more pronounced in Aur-A; GOF p53 skin, but surprisingly, there was minimal induction of DNA damage or cell death. By RNA seq, subtle differences between GOF and LOF p53 skin were observed. However, co-deletion of Aur-A markedly altered gene expression profiles. Analysis by the IPA software predicted the activation of the PI3K/mTOR pathway in AurA;GOF p53 skin. By immunoblotting, phospho/ total S6 protein ratios were increased in GOF vs LOF p53 skin, but markedly elevated after Aur-A deletion, whereas Bcl-xl protein levels remained unchanged. We confirmed p-S6 levels by IF and noted its predominantly suprabasal expression in untreated controls that extended to the basal layer in Aur-A skin. Lastly, we sorted KCs from TPA treated skin and perform single cell RNA sequencing. Pathway analysis revealed the enrichment of mTOR pathway signatures in GOF p53 KCs, suggesting that the involvement of the pathway in GOF p53 skin was cell-autonomous to KCs. Our data indicate that the PI3K/mTOR pathway may have role in the initiation of high-risk SCCs.

Novel targets and interaction partners of mutant p53 Gain-Of-Function.

In many human cancers p53 expression is lost or a mutant p53 protein is expressed. Over the past 15years it has become apparent that a large number of these mutant p53 proteins have lost wild typefunction, but more importantly have gained functions that promote tumorigenesis and drive chemo-resistance, invasion and metastasis. Many researchers have investigated the underlying mechanisms of these Gain-Of-Functions (GOFs) and it has become apparent that many of these functions are the result of mutant p53 hijacking other transcription factors. In this review, we summarize the latest research on p53 GOF and categorize these in light of the hallmarks of cancer as presented by Hannahan and Weinberg.

Abstract A04: Alpha-1-Antitrypsin is a secreted protein driven by mutant p53 and associated with EMT, migration and invasion in-vivo

Abstract Half of all human tumors harbor mutations in TP53 gene. Such mutations not only lead to the expression of a protein unable to impart its normal tumor suppressor ability, but also endow the mutant p53 protein with newly acquired gain-of-function (GOF) properties that drive invasion and metastasis. Although it is widely established that mutant p53-expressing tumors have a high capacity to metastasize and are associated with poor outcomes, the key molecular mechanism utilized by mutant p53 are largely unknown. Since, p53 mutations are predominantly found in pre-invasive stages of lung adenocarcinomas, the identification of critical pathways that mediate mutant p53 gain of function (GOF) properties may uncover new targets for cancer therapy. In this study, we investigated the global impact of mutant p53 induced secretome on cancer cell proteome. A1AT is a secreted protein encoded by SERPINA1, a secreted serine protease inhibitor that neutralizes the effect of proteases, and A1AT involvement in mutant p53 driven tumorigenesis is unclear. Methodology: We have developed a panel of H1299 lung adenocarcinoma derivatives that can inducibly re-express various mutant p53 proteins into a p53 null background. Expression levels were assessed by western blot analyses and real time PCR. Using iTRAQ (isobaric tag for relative and absolute quantification) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, the proteome of conditioned media from H1299 cells expressing either induced p53 mutants (R175H and R248) or their isogenic un-induced counterparts were identified and quantified. Mutant p53 and p63 bindings sites were analyzed by chromatin immune-precipitation (ChIP) assays. In vitro migration/invasion and in vivo Chicken Chorio-allantoic membrane (CAM) invasion were performed in A1AT knockdown lung adenocarcinoma cells harboring p53 mutation. Public microarray, Kaplan-Meir datasets of human non-small cell lung carcinoma (NSCLCs) and A1AT immunohistochemistry (IHC) of stage I-III lung adenocarcinoma patients tissue microarray (TMA) samples (n=107) with matching normal were analyzed to correlate A1AT with prognosis and clinical significance. Results: Induction of mutant p53 in H1299 cells drove an invasive phenotype through the release of a pro-invasive secretome, providing a novel avenue through which mutant p53 may be driving invasion and metastasis. Through proteomic characterization of induced conditioned medium from H1299 versus un-induced counterparts, we identified A1AT as a novel secreted mediator and a putative metastasis marker of mutant p53 tumors. To determine the role for A1AT as a downstream mediator of mutant p53 oncogenic pathways, we developed a double inducible H1299 system whereby we can simultaneously (i) induce expression of EI-H1299 R248Q mutant p53 and (ii) induce knockdown of the expression of its target A1AT. Remarkably, silencing the expression of A1AT significantly attenuated mutant p53 dependent migration and invasion both in vitro and in vivo. Importantly, knockdown of A1AT did not alter the basal level of motility in the absence of induced mutant p53, suggesting that the role of A1AT is specific to the mutant p53 pathway. Knockdown of A1AT significantly altered epithelial-mesenchymal transition (EMT) markers expression and reduced the ability of p53 mutant cells to grow in an anchorage independent environment. Treatment of cells with conditioned medium containing secreted A1AT enhanced the cell invasion while A1AT knockdown elicited the opposite effect. A1AT-blocking antibody attenuated the mutant p53 driven migration and invasion, strongly suggesting that mutant p53 is using secretory gene targets to potentiate its gain-of-function. In addition, A1AT intracellular levels were directly regulated by mutant p53 through the involvement with p63, indicating that A1AT is a direct target of mutant p53 transcriptomic regulation. In public microarray dataset, A1AT mRNA expression was higher in lung adenocarcinoma (AC) and associated with shorter overall survival. Consistent with these findings, immunohistochemistry of 107 lung adenocarcinoma TMA showed upregulation of A1AT expression compared with matching normal tissues. The A1AT signal was cytoplasmic in tumor but not in the core of the tumor. A1AT overexpression in tumor cells correlated with increased tumor size (P<0.046), and strongly with increased tumor progression (advanced T stage; P<0.0006) and shorter survival (P<0.001). In addition, we detected a clear correlation between elevated p53 staining and high levels of A1AT (P<0.018). This indicates A1AT expression is predominantly driven by mutant p53 gain-of-function. Conclusion: We conclude A1AT is an essential mediator of mutant p53 driven gain-of-function properties and our results highlight crucial roles of A1AT in driving oncogenic transformations. We suggest that A1AT is a potential therapeutic target in lung adenocarcinoma patient tumors expressing mutant p53. Citation Format: Reshma Shakya, Andrew G. Turner, Wendy Cooper, Noor A. Lokman, Carmela Ricciardelli, Gerard Tarulli, Paul M. Neilsen, David F. Callen. Alpha-1-Antitrypsin is a secreted protein driven by mutant p53 and associated with EMT, migration and invasion in-vivo. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A04.

Gain-of-function miRNA signature by mutant p53 associates with poor cancer outcome.

Missense mutation of p53 not only impairs its tumor suppression function, but also causes oncogenic gain of function (GOF). The molecular underpinning of mutant p53 (mutp53) GOF is not fully understood, especially for the potential roles of non-coding genes. Here we identify the microRNA expression profile (microRNAome) of mutp53 on Arg282 by controlled microarray experiments, and clarify the prognostic significance of mutp53-regulated miRNAs in cancers. A predominant repression effect on miRNA expression was found for mutant p53, with 183 significantly downregulated and only 12 upregulated miRNAs. Mutp53 and wild-type (wtp53) commonly upregulate let-7i, and other two miRNAs were upregulated by wtp53 but repressed by mutp53 (miR-610 and miR-3065–3p). Based the mutp53-regulated miRNA signature, a non-negative matrix factorization (NMF) model classified gastric cancer (GC) cases into subgroups with significantly different Disease-free survival (Kaplan-Meier test, P = 0.013). In contrast, the NMF model based on all miRNAs did not associate with cancer outcome. The mutp53 miRNA signature associated with the outcomes of breast cancer (P = 0.024) and hepatocellular cancer (P = 0.012). The miRPath analysis revealed that mutp53-suppressed miRNAs associate with Hippo, TGF-β and stem cell signaling pathways. Taken together, our results highlight a miRNA-mediated GOF mechanism of mutant p53 on Arg282, and suggest the prognostic potential of mutp53-associated miRNA signature.

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.

Small-Molecule NSC59984 Restores p53 Pathway Signaling and Antitumor Effects against Colorectal Cancer via p73 Activation and Degradation of Mutant p53.

The tumor-suppressor p53 prevents cancer development via initiating cell-cycle arrest, cell death, repair, or antiangiogenesis processes. Over 50% of human cancers harbor cancer-causing mutant p53. p53 mutations not only abrogate its tumor-suppressor function, but also endow mutant p53 with a gain of function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression, and chemo- or radiotherapy resistance. Thus, targeting mutant p53 to restore a wild-type p53 signaling pathway provides an attractive strategy for cancer therapy. We demonstrate that small-molecule NSC59984 not only restores wild-type p53 signaling, but also depletes mutant p53 GOF. NSC59984 induces mutant p53 protein degradation via MDM2 and the ubiquitin-proteasome pathway. NSC59984 restores wild-type p53 signaling via p73 activation, specifically in mutant p53-expressing colorectal cancer cells. At therapeutic doses, NSC59984 induces p73-dependent cell death in cancer cells with minimal genotoxicity and without evident toxicity toward normal cells. NSC59984 synergizes with CPT11 to induce cell death in mutant p53-expressing colorectal cancer cells and inhibits mutant p53-associated colon tumor xenograft growth in a p73-dependent manner in vivo. We hypothesize that specific targeting of mutant p53 may be essential for anticancer strategies that involve the stimulation of p73 in order to efficiently restore tumor suppression. Taken together, our data identify NSC59984 as a promising lead compound for anticancer therapy that acts by targeting GOF-mutant p53 and stimulates p73 to restore the p53 pathway signaling.

Pontin, a novel interactor of mutant p53 that promotes mutant p53 gain of function

ABSTRACTMany tumor-associated mutant p53 proteins gain new activities to promote tumorigenesis, which is termed as gain of function of mutant p53 (GOF). Currently, the mechanisms of mutant p53 GOF remain elusive. We recently identified the AAA+ ATPase pontin as a critical regulator of mutant p53 GOF, thus providing a novel mechanism for mutant p53 GOF.

The predictive value of ERCC1 and p53 for the effect of panobinostat and cisplatin combination treatment in NSCLC.

Cisplatin is one of the most common chemotherapeutic drugs for non-small cell lung cancer (NSCLC). However, the response rate is limited because of drug resistance. Histone deacetylase inhibitors (HDACis), which can alter DNA accessibility by regulating chromatin structure and inducing apoptosis, exhibit a synergistic action with cisplatin. However, no biomarkers that can predict the efficacy of the combination of HDACis and cisplatin have been reported. Our study found that panobinostat, an HDAC inhibitor, increased the cisplatin sensitivity of several NSCLC cell lines with low ERCC1 expression but not those with high ERCC1 expression or gain-of-function (GOF) p53 mutation despite of ERCC1 expression level. ERCC1 knockdown increased the cisplatin sensitivity of NSCLC cell lines with high ERCC1 expression without GOF p53 mutations. In addition, in low ERCC1 expression NSCLC cell lines, knockdown of GOF mutant p53 enhanced cisplatin sensitivity. Further double knockdown of ERCC1 and GOF mutant p53 but not ERCC1 knockdown alone increased the cisplatin sensitivity of cells with both high ERCC1 expression and GOF p53 mutations. Therefore, this study demonstrated that ERCC1 expression combined with p53 mutation status may determine the efficacy of cisplatin and HDACi combined therapy and guide the development of future NSCLC therapies.

Abstract 84: Functional evaluation of synchronous inactivation of PTEN and P53 in a murine model of prostate cancer

Abstract The prostate-specific PTEN conditional knockout (K/O) mouse model has been extremely useful as tool to investigate the biology of the tumorigenesis process, particularly during early tumor development. However, cancer progression is gradual with a low metastatic rate. This feature poses a challenge to this model when used for survival studies and end-stage biological characterization of prostate cancer. Inactivation of P53 is a characteristic that is frequent in advanced prostate cancer. To address this issue, we generated series of double K/O mice based on either P53 loss or gain of function in combination with PTEN inactivation. Conditional inactivation of both PTEN and P53 results in the development of aggressive tumors compared to PTEN K/O mice. Homozygous PTEN/P53-mutants mice grow prostate tumors at a faster rate and experience dramatically reduced survival. Homozygous double K/O mice also developed a higher frequency and number of metastases. Furthermore, haploinsufficient P53 gain of function mutations contributed to an invasive cancer phenotype compared to P53 loss of function haploinsufficient tumors. In conclusion, our analyses demonstrate that synchronous inactivation of PTEN and P53 promotes an aggressive prostate cancer and provides rationale that supports that use of such models is better suited for studies requiring long-term survival and end-stage cancer characterization. Citation Format: Hirotsugu Uemura, Yurie Kura, Naomi Ando, Emiko Fukushima, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Marco A. De Velasco. Functional evaluation of synchronous inactivation of PTEN and P53 in a murine model of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 84. doi:10.1158/1538-7445.AM2014-84

Clinical Relevance of Gain-Of-Function Mutations of p53 in High-Grade Serous Ovarian Carcinoma

PurposeInactivation of TP53, which occurs predominantly by missense mutations in exons 4–9, is a major genetic alteration in a subset of human cancer. In spite of growing evidence that gain-of-function (GOF) mutations of p53 also have oncogenic activity, little is known about the clinical relevance of these mutations.MethodsThe clinicopathological features of high-grade serous ovarian carcinoma (HGS-OvCa) patients with GOF p53 mutations were evaluated according to a comprehensive somatic mutation profile comprised of whole exome sequencing, mRNA expression, and protein expression profiles obtained from the Cancer Genome Atlas (TCGA).ResultsPatients with a mutant p53 protein (mutp53) with a GOF mutation showed higher p53 mRNA and protein expression levels than patients with p53 mutation with no evidence of GOF (NE-GOF). GOF mutations were more likely to occur within mutational hotspots, and at CpG sites, and resulted in mutp53 with higher functional severity (FS) scores. Clinically, patients with GOF mutations showed a higher frequency of platinum resistance (22/58, 37.9%) than patients with NE-GOF mutations (12/56, 21.4%) (p=0.054). Furthermore, patients with GOF mutations were more likely to develop distant metastasis (36/55, 65.5%) than local recurrence (19/55, 34.5%), whereas patients with NE-GOF mutations showed a higher frequency of locoregional recurrence (26/47, 55.3%) than distant metastasis (21/47, 44.7%) (p=0.035). There were no differences in overall or progression-free survival between patients with GOF or NE-GOF mutp53.ConclusionThis study demonstrates that patient with GOF mutp53 is characterized by a greater likelihood of platinum treatment resistance and distant metastatic properties in HGS-OvCa.