Tp53-Mediated Human Cancer Susceptibility Is Defined By Epigenetic Dysregulation Of Microrna-34a

Abstract Mutations in the TP53 tumor suppressor gene are the most common genetic aberrations across all human cancers. Germline TP53 mutations are also the hallmark genetic event in Li-Fraumeni syndrome (LFS), a highly penetrant human cancer susceptibility syndrome, conferring a predisposition to developing early-onset breast cancer, leukemias, bone and soft tissue sarcomas, brain tumors of various histologies, adrenocortical carcinomas, and a wide range of other malignancies. Although the link between mutant TP53 and human cancer is unequivocal, the mechanism by which this genetic aberration predisposes an individual to cancer remains to be elucidated. To address this gap, we surveyed the epigenome and describe herein the largest systematic analysis of DNA methylation in patients harboring germline TP53 mutations and TP53 wild-type individuals. Specifically, we performed genome-wide methylation analyses of peripheral blood leukocyte DNA in germline TP53 mutation carriers (n=72) and TP53 wild-type individuals who developed histologically comparable cancers (n=111). Targeted bisulfite pyrosequencing was performed on peripheral blood DNA in a validation cohort (n=76), and candidate sites were further evaluated in primary tumors from LFS patients. The differential methylation analysis demonstrates that in 183 patients, distinct DNA methylation signatures are associated with deleterious TP53 mutations. TP53 mutation-associated DNA methylation marks occur in genomic regions harboring known p53 binding sites and within genes encoding p53 pathway proteins. Moreover, loss-of-function TP53 mutations are significantly associated with differential methylation at the locus encoding miR-34A-a key component of the p53 regulatory network (adjusted p-value=3.1X10-15)-and validated in an independent patient cohort (n=76, 1.9X10-8). Targeted sequencing demonstrates that miR-34A is inactivated by hypermethylation across many different histologic types of primary tumors from LFS patients, such as brain tumors, osteosarcomas, rhabdomyosarcomas, and adrenocortical carcinomas. miR-34A promoter hypermethylation in tumors is also associated with decreased overall survival in a cohort of 29 patients with choroid plexus carcinomas, a characteristic LFS tumor (p<0.05). The relationship between miR-34A hypermethylation and TP53 mutation was further validated in sporadic cancers, using the publicly available TCGA dataset. This demonstrates the robustness of this correlation and the applicability of these findings to other cancer contexts. This study refines the role of epigenetics in a cancer predisposition syndrome and is the first to implicate a microRNA, miR-34A, in human cancer susceptibility and provides a repository of genomic regions of deregulated methylation in the context of dysfunctional TP53. These findings suggest that deregulated DNA methylation at defined genomic loci may be an important hallmark of TP53-mediated cancer susceptibility. The most striking finding from this study is the relative miR-34A promoter hypomethylation at two adjacent CpG sites in peripheral blood from TP53 mutation carriers, confirmed in two independent cohorts and shown to cosegregate with TP53 mutations in LFS families. This result is remarkable since miR-34A is a central microRNA in the p53 network and the first microRNA identified as a direct proapoptotic target of the p53 pathway. The detection of miR-34A promoter hypomethylation in TP53 mutant cells that have not undergone malignant transformation supports a putative model whereby wild-type p53 may influence methylation patterns at this locus. In particular, in nontransformed cells that do not harbor mutations in TP53, wild-type p53 may be recruited to the miR-34A locus and sustain hypermethylation. We have performed a series of in vitro studies on primary patient-derived lymphoblastoid cell lines to corroborate this model. Conversely, in the setting of loss-of-function or deleterious mutations in TP53, mutant p53 may not able to maintain hypermethylation of the miR-34A promoter, leading to upregulation of miR-34A. Owing to the known redundant cellular roles of p53 and miR-34A, upregulation of miR-34A may be beneficial to cells harboring mutant p53 by supplementing the necessary basal tumor suppressive function that is lost when p53 is mutated. This mechanism may serve to guard against mutant p53, even when the wild-type allele remains. Accordingly, this may explain why miR-34A promoter hypermethylation is characteristic of TP53-mutant tumors that lack wild-type p53 because this microRNA serves a critical role in cell maintenance, and its loss may cooperate with other genetic and/or epigenetic events to drive malignancy. It is therefore not surprising that, akin to p53, somatic miR-34A deregulation is pervasive in human cancer and miR-34A inactivation by focal deletion or promoter hypermethylation has been reported in the literature to occur in a multitude of human malignancies. The precise mechanisms of how the miR-34A promoter undergoes somatic epimutation in tissues remains to be elucidated, and likely various pathways may converge to yield this outcome in different tissues. Given the high frequency of TP53 mutations in human malignancies, the relationship between mutant p53 and miR-34A has strong implications for the targeting of miR-34A in cancer. Encouragingly, studies have demonstrated in vivo the utility of miR-34A-based therapies in cancer, including intratumor or systemic delivery of lipid-formulated synthetic miR-34A. To further probe these intriguing findings, we conducted mechanistic studies aimed at functionally interrogating the the miR-34A-p53 axis. We utilized in vitro-based assays to modulate miR-34A levels in primary patient-derived fibroblast cell lines, and subsequently performed by RNA-sequencing of the transcriptional responses. Our results uncover a number of novel cellular roles for miR-34A in cell maintenance. Significantly, the transcriptional response to miR-34A inhibition revealed that this microRNA may be a crucial switch that can lead to numerous changes to noncoding RNA networks as well as known p53 pathways. Markedly increased expression of key components of the U12 (minor) spliceosome occurs when miR-34A expression is diminished, thereby identifying a novel putative role of miR-34A in modulating transcription of the U12 spliceosomal machinery. The majority of TP53 mutation-associated transcripts are involved in chromatin remodeling and nucleosome assembly, and are enriched for histone cluster 1 genes. These linker histones are crucial for maintaining higher-order chromatin structure and for regulating gene expression, demonstrating the interplay between genetic and epigenetic states. Lastly, miR-34A is associated with transcriptional regulation of a host of lincRNAs, including LINC-PINT, a p53-induced lincRNA. These results are the first to identify miR-34A as an important node in the transcriptional regulation of numerous noncoding RNAs and point to further study of these pathways. Taken together, these findings provide strong support for the impact of TP53 mutations on epigenetic dysregulation in human cancer susceptibility and demonstrate that miR-34A may be important in the pathogenesis of TP53-mediated cancer susceptibility. Moreover, miR-34A may be a putative novel therapeutic target and a marker for clinical prognostication. These studies also demonstrate that miR-34A is a central node in numerous p53-dependent and independent networks and provide further insight into the role of this critical tumor-suppressive microRNA. Further work aimed at refining our understanding of miR-34A-mediated pathways may yield additional molecular insight into the role of this microRNA in malignant transformation. Citation Format: Nardin Samuel, Gavin Wilson, Genevieve Deblois, Badr Id Said, Nicholas W. Fischer, Mathieu Lemire, Youliang Lou, Weili Li, Roumiana Alexandrova, Ana Novokmet, James Tran, Kim E. Nichols, Jonathan L. Finlay, Sanaa Choufani, Marc Remke, Vijay Ramaswamy, Florence M.G. Cavalli, Christine Elser, Lynn Meister, Michael D. Taylor, Uri Tabori, Meredith Irwin, Rosanna Weksberg, Jonathan D. Wasserman, Jean Gariepy, Mathieu Lupien, Daniele Merico, Andrew Paterson, Jordan R. Hansford, Maria Isabel W. Achatz, Thomas J. Hudson, David Malkin. TP53-mediated human cancer susceptibility is defined by epigenetic dysregulation of microRNA-34A [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 NG05. doi:10.1158/1538-7445.AM2017-NG05