Mutant p53 Prolongs NF-κB Activation and Promotes Chronic Inflammation and Inflammation-Associated Colorectal Cancer

Mutant p53 Prolongs NF-kB Activation and Promotes Chronic Inflammation and Inflammation-Associated Colorectal Cancer

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

Tumor cells, including SW480 carcinoma cells that carry a mutant p53, are addicted to the mutant for their survival and resistance to growth suppression by chemotherapeutic agents. Here, we investigated whether various classes of p53 mutants share a common property and functional domains necessary for mutant p53 gain of function. To test this, we generated SW480 cell lines in which endogenous mutant R273H/P309S can be inducibly or stably knocked down, whereas a small interfering RNA-resistant mutant p53 along with a mutated functional domain can be inducibly or stably expressed. We found that both contact-site (R248W and R273H) and conformation (G245S and R249S) mutants are able to maintain the transformed phenotypes of SW480 cells conferred by endogenous mutant p53. We also found that activation domains 1-2 and the proline-rich domain are required for mutant p53 gain of function. Interestingly, we showed that the C-terminal basic domain, which is required for wild-type p53 activity, is an inhibitory domain for mutant p53. Furthermore, we showed that deletion of the basic domain enhances, whereas a mutation in activation domains 1-2 and deletion of the proline-rich domain abolish mutant p53 to regulate Gro1 and Id2, both of which are regulated by and mediate endogenous mutant p53 gain of function. These results indicate that both conformation and contact-site mutants share a property for cell transformation, and the domains critical for wild-type p53 tumor suppression are also required for mutant p53 tumor promotion. Thus, the inhibitory basic domain and the common property for p53 mutants can be explored for targeting tumors with mutant p53.

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the β1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to β1 integrin. Function-blocking antibodies to α2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in α2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of α2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type α2 integrin or a non-signaling chimeric α2 integrin. Overexpression of wild-type α2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric α2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor α2β1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

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.

DNA Hypermethylation Contributes to Incomplete Synthesis of Carbohydrate Determinants in Gastrointestinal Cancer

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.

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.

Tumor suppressor p53 is frequently mutated in human cancers. Many tumor-associated mutant p53 (mutp53) proteins gain new functions in promoting tumorigenesis, defined as gain of function (GOF). The mechanisms for mutp53 GOF are not well-understood. Mutp53 has been reported to interact with some proteins and it has been suggested that some interactions of mutp53 with its binding partners are important for mutp53 GOF through regulating the levels or functions of mutp53 and/or its binding proteins. To understand the molecular mechanisms for mutp53 GOF, we searched for mutp53 binding proteins in thymic lymphomas of R172H mutp53 knock-in (p53R172H/R172H) mice with significant mutp53 protein accumulation by employing an unbiased immunoprecipitation (IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach. Normal tissues of p53R172H/R172H mice with low mutp53 levels and tumors of p53-/- mice were used as controls. Using this approach, we identified Pontin, a highly conserved AAA+ ATPase important for various cellular functions, as a new mutp53 binding protein. This Pontin-mutp53 is conserved in both humans and mice. Importantly, this Pontin-mutp53 interaction promotes mutp53 GOF in invasion, migration, and anchorage-independent growth of tumor cells. Furthermore, we found that the C-terminus of Pontin is required for Pontin-mutp53 interaction and mutp53 GOF. The ATPase domain in the C-terminus of Pontin is crucial for its promoting effect on mutp53 GOF; blocking the ATPase activity of Pontin by a Pontin specific ATPase inhibitor or an ATPase deficient dominant-negative Pontin expression vector greatly diminished mutp53 GOF. We found that Pontin was largely co-localized with mutp53 in the nucleus and promotes mutp53 GOF through regulation of mutp53 transcriptional activity; knock-down of Pontin abolished the transcriptional regulation of mutp53 towards a group of genes involved in many important signaling pathways, including gap junction signaling, IGF-1 signaling, EGF signaling etc. Furthermore, overexpression of Pontin is associated with poor survival in cancer patients, especially those containing mutp53 in tumors. This work demonstrates that Pontin promotes mutp53 GOF in tumorigenesis in an ATPase-dependent manner, and raises interesting and intriguing possibilities to block mutp53 GOF in tumors through targeting Pontin or its ATPase activity. In summary, our results highlight an important role and mechanism for Pontin, a new p53 partner, in promoting mutp53 GOF in tumorigenesis. Citation Format: Yuhan Zhao, Cen Zhang, Xuetian Yue, Xiaoyan Li, Juan Liu, Haiyang Yu, Qifeng Yang, Zhaohui Feng, Wenwei Hu. Pontin, a new mutant p53 binding protein, promotes gain-of-function of mutant p53. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1221. doi:10.1158/1538-7445.AM2015-1221

IntroductionMost cancers harbor mutations in the TP53 gene (encoding for the p53 tumor suppressor protein). Furthermore, gain-of-function (GOF) mutation in p53 imparts an aggressive traits to the cells when compared to the cancer cells harboring inactivating mutations or wild-type (WT)-p53. Notably, multiple studies have delineated the presence of GOF-mutant p53 protein in untransformed cells or in stromal compartments of tumor microenvironment (TME). In recent years, the involvement of extracellular vesicles (EVs) in cell-to-cell communication has emerged as a major route by which cancer cells can interact and educate immune, and non-immune cells in TME to become tumor supportive. To this end, we hypothesize that mutant p53 protein can be shuttled via EVs to TME cells thus shedding light on a novel non-cell autonomous role of mutant p53 cancers.MethodsEVs were isolated from various cancer cell lines (pancreas, lung, colon) differing by their p53 status and the effect on neighboring cancer cells and TME cells was studied in vitro and in-vivo. We also utilized the human colorectal Colo-320DM cancer cell xenograft model, which expresses the R248W p53 mutant. FFPE sections of subcutaneous tumors derived from the Colo-320DM xenografts were stained for p53 using the DO-1 antibody that specifically recognizes human p53.ResultsOur data demonstrated that mutant p53 protein can be selectively sorted into EVs; that mutant p53 in EVs can be taken up by neighboring cancer cells and macrophages that do not harbor mutant p53 protein. Evident of macrophage education was seen with the increased expression and secretion of pro-inflammatory cytokines. Notably, mutant p53 expression was also found in non-tumor cells in both human cancers, and in non-human tissues in human xenografts.SummaryCancer cells harboring GOF p53 mutants, can package mutant p53 proteins in EVs, and deliver them to neighboring cancer cells and to the TME. Citation Format: Bibek Bhatta, Ishai Luz, Christian Krueger, Fanny Xueting Teo, David P. Lane, Kanaga Sabapathy, Tomer Cooks. Oncogenic mutant p53 proteins are selectively shuttled by cancer extracellular vesicles toeducate the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1312.

Mutations in the K-ras gene are frequent in human cancer. ras activation in primary cells results in a cellular senescence phenotype that is precluded by inactivation of p16. At the clinical level, this may imply a differential behavior for tumors with alternative or cooperative activation of K-ras function and impairment of p16 pathways. We have determined the presence of mutations in the K-ras gene and the methylation status of p16 promoter in a series of 119 prospectively collected colorectal carcinomas. p53 mutations and p14 alternative reading frame methylation status were also assessed. Associations with survival were investigated. K-ras mutations were present in 44 (38%) of 115 cases, and p16 methylation was present in 42 (37%) of 113 cases. p53 mutations were detected in 50% (56 of 115) and p14 methylation in 29% (32 of 112) of cases. K-ras and p16 alterations were independent genetic events. Presence of K-ras or p16 genetic alterations (analyzed independently) was associated with shorter survival, although differences were not statistically significant. Cox analysis of the two variables combined showed a diminished survival as the results of an interaction between p16 and K-ras. Alternative alteration of K-ras and p16 genes was an independent prognostic factor in human colorectal cancer in univariate and multivariate analysis. Differences were maintained when cases undergoing radical surgery and without distant metastases were considered. These results suggest that the combined K-ras and p16 analyses may be of prognostic use in human colorectal cancer.

Prostaglandin E2 Promotes Colorectal Cancer Stem Cell Expansion and Metastasis in Mice

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

We conducted quantitative cellular respiration analysis on samples taken from human breast cancer (HBC) and human colorectal cancer (HCC) patients. Respiratory capacity is not lost as a result of tumor formation and even though, functionally, complex I in HCC was found to be suppressed, it was not evident on the protein level. Additionally, metabolic control analysis was used to quantify the role of components of mitochondrial interactosome. The main rate-controlling steps in HBC are complex IV and adenine nucleotide transporter, but in HCC, complexes I and III. Our kinetic measurements confirmed previous studies that respiratory chain complexes I and III in HBC and HCC can be assembled into supercomplexes with a possible partial addition from the complex IV pool. Therefore, the kinetic method can be a useful addition in studying supercomplexes in cell lines or human samples. In addition, when results from culture cells were compared to those from clinical samples, clear differences were present, but we also detected two different types of mitochondria within clinical HBC samples, possibly linked to two-compartment metabolism. Taken together, our data show that mitochondrial respiration and regulation of mitochondrial membrane permeability have substantial differences between these two cancer types when compared to each other to their adjacent healthy tissue or to respective cell cultures.

Two of the most common events in human tumors are mutation of the tumor suppressor gene TP53 and development of aneuploidy. In addition to losing their wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mechanistic understanding of mutant p53 GOF activities is complicated by the diversity and context-specific nature of reported GOF phenotypes. The study of mutant p53 GOF activities is especially challenging because mutations in p53 are positively correlated with the development of aneuploidy, which can increase heterogeneity through diverse chromosomal alterations and itself contributes to tumorigenesis. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic epithelial cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Additionally, the use of clonally-derived cell lines originating from the same near diploid parental genetic background allows for assessment of the genomic alterations and resulting molecular heterogeneity following mutation of TP53. Through genomic, transcriptomic, and cellular based assays we have validated our cell line models and found that missense mutant and p53 null cells display loss of p53 function. Through functional genomics analyses comparing isogenic epithelial cells, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and aneuploidy and assessed whether our clonal cell lines display several previously reported mutant p53 GOF phenotypes such as altered gene expression, proliferation, metabolism, drug sensitivity, and migration. Further, using lentiviral mediated knockdown of p53 protein we evaluated the dependency of these phenotypes on expression of mutant p53 protein. Finally, data from The Cancer Genome Atlas (TCGA) was used for the analysis of manifestations of clinical mutant p53 GOF phenotypes. The dissection of mutant p53 GOF phenotypes will improve the current understanding of the role of mutant p53 in tumorigenesis. The results generated from these studies have the potential for clinical translation in major types of human cancer that have high-frequency p53 mutation. Citation Format: Lindsay N. Redman-Rivera, Timothy M. Shaver, Hailing Jin, Johanna M. Schafer, Quanhu Sheng, Rachel A. Hongo, Kathryn E. Beckermann, Brian D. Lehmann, Ferrin C. Wheeler, Jennifer A. Pietenpol. A functional genomics approach to determine mutant p53 gain-of-function mechanisms and phenotypes in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2489.