Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants
Anasuya PalLaura González‐MalervaSeron EatonChenxi XuYining ZhangDustin GriefLydia SakalaLilian NwekwoJia ZengGrant ChristensenChitrak GuptaEllen StreitwieserAbhishek SingharoyJin G. ParkJoshua LaBaer
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Abstract Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.Keywords:
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Abstract Given increasing numbers of patients who are undergoing exome or genome sequencing, it is critical to establish tools and methods to interpret the impact of genetic variation. While the ability to predict deleteriousness for any given variant is limited, missense variants remain a particularly challenging class of variation to interpret, since they can have drastically different effects depending on both the precise location and specific amino acid substitution of the variant. In order to better evaluate missense variation, we leveraged the exome sequencing data of 60,706 individuals from the Exome Aggregation Consortium (ExAC) dataset to identify sub-genic regions that are depleted of missense variation. We further used this depletion as part of a novel missense deleteriousness metric named MPC. We applied MPC to de novo missense variants and identified a category of de novo missense variants with the same impact on neurodevelopmental disorders as truncating mutations in intolerant genes, supporting the value of incorporating regional missense constraint in variant interpretation.
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The GJB2 gene is the most common gene responsible for hearing loss (HL) worldwide, and missense variants are the most abundant type. GJB2 pathogenic missense variants cause nonsyndromic HL (autosomal recessive and dominant) and syndromic HL combined with skin diseases. However, the mechanism by which these different missense variants cause the different phenotypes is unknown. Over 2/3 of the GJB2 missense variants have yet to be functionally studied and are currently classified as variants of uncertain significance (VUS). Based on these functionally determined missense variants, we reviewed the clinical phenotypes and investigated the molecular mechanisms that affected hemichannel and gap junction functions, including connexin biosynthesis, trafficking, oligomerization into connexons, permeability, and interactions between other coexpressed connexins. We predict that all possible GJB2 missense variants will be described in the future by deep mutational scanning technology and optimizing computational models. Therefore, the mechanisms by which different missense variants cause different phenotypes will be fully elucidated.
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Abstract The Hippo pathway is crucial in organ size control and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include the protein kinases of MST1/2, MAP4Ks, LATS1/2, the transcription co-activators YAP/TAZ, and their DNA binding partners TEADs. LATS phosphorylates YAP/TAZ to promote cytoplasmic localization and degradation, thereby inhibiting YAP/TAZ and cell growth. The Hippo pathway is regulated by a wide range of signals, including cell density, GPCR, cellular energy levels, and mechanical cues. We will present our recent progresses on Hippo pathway regulation and its role in cancer. The emerging role of the Hippo pathway in tumorigenesis suggests the potential therapeutic value of targeting this pathway for cancer treatment. Citation Format: Kun-Liang Guan. Regulation of the Hippo pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA04.
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Abstract Approximately 15% of genetic screens for mutations in BRCA1 and BRCA2 identify Variants of Uncertain Significance (VUS). Primarily missense mutations, VUS are often difficult to interpret, leading to either uncertainty in how to properly counsel a patient or an unnecessary prophylactic surgery. Given the paucity of data for which missenses are classified as truly pathogenic, computational deleterious missense prediction (DMP) algorithms are used to predict whether a mutation is likely deleterious or neutral. Accuracy of DMPs can vary considerably and have only been calibrated on a relatively small number of missense mutations of demonstrable effect on protein function. In this study, the performance of 41 different DMPs was compared to functional data from 455 functionally characterized missense variants in BRCA1 and BRCA2. New optimized thresholds for classifying missense mutations as deleterious are presented for several existing models as well as a newly derived naïve voting method (NVM). The areas under the curve estimates for the NVM approach are between 0.889-0.922, much higher than previous methods. We estimate that the overall pathogenic potential of missense variants to be 6.8% for BRCA1 and 3.2% of BRCA2, but can be as high as 50% depending on protein location. Overall these results provide key insights into how to predict deleterious missense mutations in BRCA1 and BRCA2. Citation Format: Hart SN, Hoskin T, Shimelis H, Feng B, Lindor NM, Monteiro A, Iversen E, Goldgar DE, Suman V, Couch FJ. Optimized prediction of deleterious missense mutations in BRCA1 and BRCA2 genes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-02-03.
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The Hippo signaling pathway is gaining recognition as an important player in both organ size control and tumorigenesis, which are physiological and pathological processes that share common cellular signaling mechanisms. Upon activation by stimuli such as high cell density in cell culture, the Hippo pathway kinase cascade phosphorylates and inhibits the Yes-associated protein (YAP)/TAZ transcription coactivators representing the major signaling output of the pathway. Altered gene expression resulting from YAP/TAZ inhibition affects cell number by repressing cell proliferation and promoting apoptosis, thereby limiting organ size. Recent studies have provided new insights into the Hippo signaling pathway, elucidating novel phosphorylation-dependent and independent mechanisms of YAP/Yki inhibition by the Hippo pathway, new Hippo pathway components, novel YAP target transcription factors and target genes, and the three-dimensional structure of the YAP–TEAD complex, and providing further evidence for the involvement of YAP and the Hippo pathway in tumorigenesis.
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Objective: CENPH, centromere protein H, is one of the constitutive kinetochore proteins. High expression of CENPH has been shown in various forms of cancers; however, studies searching the effect of CENPH mutations in cancers are limited. Therefore, the aim of this study is to investigate the potential effects of the missense mutations of CENPH that have been identified in different cancers.
Materials and Methods: Missense CENPH mutations, which have been observed in cancers, were downloaded from the COSMIC v89. The effect of missense mutations was predicted by using PredictSNP1.0. The protein structure of the CENPH protein was generated with I-TASSER and missense mutations were visualized on CENPH protein with UCSF Chimera. Structural effects of selected mutations were assessed with HOPE.
Results: 34 missense mutations were observed in human cancers. Of the 34 missense mutations 18 mutations were predicted as deleterious and 16 mutations were predicted as neutral with ranging expected accuracies. Predicted missense mutations showed a scattered pattern on 3D CENPH protein. Two of the predicted deleterious missense mutations with higher expected accuracy were further analyzed and assessed according to amino acid properties.
Conclusion: This study provided a systematic analysis and evaluation of missense mutations on a CENPH protein that have been observed in different cancers.
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