Mutations In Human Cancers Research Articles

CTCF Haploinsufficiency Destabilizes DNA Methylation and Predisposes to Cancer

Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

Prevalence of inactivating protein kinase C mutations in human cancers (1055.2)

Protein kinase C (PKC) levels are aberrant in diverse cancers and over 400 mutations in PKC have been identified in human cancers. Here we characterized PKC mutations identified in human cancers to investigate whether PKCs function as oncogenes or tumor suppressors or whether their function is isozyme and/or context dependent. Live‐cell imaging was used to determine how these mutations affect PKC translocation, subcellular localization, and activity. Cell‐based assays were used to determine how PKC mutations alter proliferation, apoptosis, migration, and anchorage‐independent growth. Of 37 mutations we examined thus far, 24 of them reduced or completely abolished PKC activity, while only 1 increased it. These mutations reside within the diacylglycerol‐binding C1 domain, the Ca2+‐binding C2 domain, or the kinase domain of conventional, novel, or atypical PKCs. The inactivating mutations ablate activity by either impeding Ca2+ or diacylglycerol binding, or by yielding a misfolded kinase. Our results suggest that PKC can act as a tumor suppressor in certain cancers. Understanding whether mutations are activating or inactivating in specific cancers would allow us to determine whether therapeutic strategies should target ways to inactivate or activate PKC in the respective cancers.Grant Funding Source: This work was supported by GM43154 to ACN and DGE1144086 and T32 GM007752 to CEA.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity

Mutation of p53 is the most common genetic change in human cancer, causing complex effects including not only loss of wild-type function but also gain of novel oncogenic functions (GOF). It is increasingly likely that p53-hotspot mutations may confer different types and magnitudes of GOF, but the evidences are mainly supported by cellular and transgenic animal models. Here we combine large-scale cancer genomic data to characterize the prognostic significance of different p53 mutations in human cancers. Unexpectedly, only mutations on the Arg248 and Arg282 positions displayed significant association with shorter patient survival, but such association was not evident for other hotspot GOF mutations. Gene set enrichment analysis on these mutations revealed higher activity of drug-metabolizing enzymes, including the CYP3A4 cytochrome P450. Ectopic expression of p53 mutant R282W in H1299 and SaOS2 cells significantly upregulated CYP3A4 mRNA and protein levels, and cancer cell lines bearing mortality-associated p53 mutations display higher CYP3A4 expression and resistance to several CYP3A4-metabolized chemotherapeutic drugs. Our results suggest that p53 mutations have unequal GOF activities in human cancers, and future evaluation of p53 as a cancer biomarker should consider which mutation is present in the tumor, rather than having comparison between wild-type and mutant genotypes.

Synonymous Mutations Frequently Act as Driver Mutations in Human Cancers

Synonymous mutations change the sequence of a gene without directly altering the sequence of the encoded protein. Here, we present evidence that these "silent" mutations frequently contribute to human cancer. Selection on synonymous mutations in oncogenes is cancer-type specific, and although the functional consequences of cancer-associated synonymous mutations may be diverse, they recurrently alter exonic motifs that regulate splicing and are associated with changes in oncogene splicing in tumors. The p53 tumor suppressor (TP53) also has recurrent synonymous mutations, but, in contrast to those in oncogenes, these are adjacent to splice sites and inactivate them. We estimate that between one in two and one in five silent mutations in oncogenes have been selected, equating to ~6%- 8% of all selected single-nucleotide changes in these genes. In addition, our analyses suggest that dosage-sensitive oncogenes have selected mutations in their 3' UTRs.

A1.47 BRAF (v raf murine sarcoma viral oncogene homologue B1) mutations in rheumatoid arthritis patients

BackgroundBRAF (v raf murine sarcoma viral oncogene homologue B1) is an attractive new autoantigen with the ability to identify rheumatoid arthritis (RA) patients who do not have the classical ACPAs...

Mutational patterns in the breast cancer mitochondrial genome, with clinical correlates

The presence of mitochondrial DNA (mtDNA) mutations in human cancer has long been recognized, but their functional significance has remained obscure. Debate persists as to whether the mutations help drive the tumor, or are bystander events. Here, we analyze next-generation mtDNA sequence data from 99 breast cancer patients. High depth coverage enables detection of even low-level heteroplasmic variants, and data from matched normal tissue allow us to distinguish between shifts in heteroplasmy and acquired mutations. Somatic mtDNA mutations are found in 73 (73.7%) of patient tumors, and dramatic shifts from the initial germline allele proportions are observed for many heteroplasmies. Clustering of somatic mutations in promoter and replication regions, and also in genes coding for electron transport chain complex I, suggest selection for mutations affecting critical mitochondrial processes. Furthermore, statistical tests for Darwinian selection reveal evidence for positive and relaxed negative selection for somatic missense mutations. We also observe a dramatic decrease in per-cell mtDNA content in tumor tissues, as well as a surprising positive correlation between somatic mtDNA mutational burden and patient survival. Taken together, our results support the view that somatic mtDNA mutations are not solely bystander events, but have significance in cancer from both biological and clinical perspectives. We also anticipate that the catalog of heteroplasmies and somatic mutations presented here will serve as a reference for future studies of cancer mitochondrial genomes.

NGAL Can Alternately Mediate Sunitinib Resistance in Renal Cell Carcinoma

Serum NGAL is highly expressed in patients with advanced renal cancer treated with sunitinib. We investigated the role of NGAL in sunitinib resistance in renal cell carcinoma to identify potential tactics to overcome it. NGAL expression was correlated with sunitinib sensitivity. Vascular endothelial growth factor related upstream Ras, Erk1/2 and STAT1 phosphorylation activity in Caki-1 and NGAL transfected Caki-1 cells after sunitinib treatment was analyzed using Western blot. NGAL and vascular endothelial growth factor-A interaction with sunitinib therapeutic efficacy was monitored in renal cell carcinoma tumor xenografted mice by tumor growth inhibition, serum NGAL and vascular endothelial growth factor-a levels, and microscopic examination of tumor microvascular density. Sunitinib cytotoxicity in various renal cell carcinoma cell lines was reversibly related to NGAL expression. Sunitinib showed the lowest 50% inhibitory concentration (5.53 μM) in Caki-1 cells, which had the lowest NGAL expression of these renal cell carcinoma cell lines. After sunitinib treatment adding NGAL inhibited Ras and Erk1/2 phosphorylation but activated STAT1α phosphorylation in Caki-1 cells and Caki-1 cells transfected with NGAL. In a xenograft mouse model sunitinib significantly inhibited tumor growth in Caki-1 mice. NGAL transfected Caki-1 mice had higher serum NGAL and lower vascular endothelial growth factor-A than Caki-1 mice. Microvascular density was decreased in Caki-1 mice with sunitinib treatment. NGAL in tumor cells may show crosstalk with vascular endothelial growth factor-a and alternative activation in stimulating tumor growth during sunitinib treatment. It may become a therapeutic target to reverse sunitinib resistance in renal cell carcinoma.

When defense turns into attack

The APOBEC3 cytidine deaminases play an important role in innate immunity but have also emerged as mediators of somatic mutations in human cancer. We recently reported a high incidence of APOBEC-mediated driver mutations in human papillomavirus-associated cancer, suggesting a key role for these enzymes in the development of such tumors.

Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer

Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer

High throughput RNAi screening identifies ID1 as a synthetic sick/lethal gene interacting with the common TP53 mutation R175H

The TP53 mutation (R175H) is one of the most common mutations in human cancer. It is a highly attractive strategy for cancer therapy to find the genes that lead the R175H-expressing cancer cells. The aim of this study was to identify the synthetic sick/lethal gene interacting with R175H. Using lentiviral bar-coded comprehensive shRNA library and a tetracycline-inducible R175H expressed in the SF126 human glioblastoma cell line (SF126-tet-R175H), we conducted high-throughput screening to identify the candidate genes that induce synthetic sickness/lethality in R175H-expressing cells. We identified 906 candidate gene suppressions that may lead to accelerated cell growth inhibition in the presence of R175H. Inhibitor of differentiation 1 (ID1) was one of the candidate genes, and its suppression by siRNA resulted in the acceleration of growth inhibition in cell lines both transiently and endogenously expressing R175H but not in TP53-null cell lines or other common p53 mutants (such as R273H). Flow cytometry analysis showed that ID1 suppression resulted in G1 arrest, and the arrest was accelerated by the expression of R175H. ID1 is a synthetic sick/lethal gene that interacts with R175H and is considered to be a novel molecular target for cancer therapy in R175H-expressing cells.

The mitochondrial C16069T polymorphism, not mitochondrial D310 (D-loop) mononucleotide sequence variations, is associated with bladder cancer

BackgroundBladder cancer is a relatively common and potentially life-threatening neoplasm that ranks ninth in terms of worldwide cancer incidence. The aim of this study was to determine deletions and sequence variations in the mitochondrial displacement loop (D-loop) region from the blood specimens and tumoral tissues of patients with bladder cancer, compared to adjacent non-tumoral tissues.MethodsThe DNA from blood, tumoral tissues and adjacent non-tumoral tissues of twenty-six patients with bladder cancer and DNA from blood of 504 healthy controls from different ethnicities were investigated to determine sequence variation in the mitochondrial D-loop region using multiplex polymerase chain reaction (PCR), DNA sequencing and southern blotting analysis.ResultsFrom a total of 110 variations, 48 were reported as new mutations. No deletions were detected in tumoral tissues, adjacent non-tumoral tissues and blood samples from patients. Although the polymorphisms at loci 16189, 16261 and 16311 were not significantly correlated with bladder cancer, the C16069T variation was significantly present in patient samples compared to control samples (p < 0.05). Interestingly, there was no significant difference (p > 0.05) of C variations, including C7TC6, C8TC6, C9TC6 and C10TC6, in D310 mitochondrial DNA between patients and control samples.ConclusionOur study suggests that 16069 mitochondrial DNA D-Loop mutations may play a significant role in the etiology of bladder cancer and facilitate the definition of carcinogenesis-related mutations in human cancer.

Abstract B179: Targeted next generation sequencing for somatic mutations in human cancer.

Abstract Objective: We have replaced single gene PCR-based methods for the detection of BRAF, KRAS and EGFR variants in routine clinical practice with next generation sequencing assays for the Ion Torrent AmpliSeq Cancer Hotspot Panel v2 (50 genes) and the Illumina TruSeq Amplicon Cancer panel (48 genes). Implementation of these panels requires constant optimization of technical and analytical procedures as reagents and software continue to be developed. In this study we describe somatic mutation findings from our clinical laboratory service. Methods: DNA was extracted from 90 formalin fixed, paraffin embedded tissue specimens that included 50 lung cancer, 24 colon cancer and 16 other cancers. Barcoded libraries were prepared from up to 10ng of extracted DNA and multiplexed on single 318 chips. Data analysis was performed using Golden Helix SVS for the Ion Torrent panel and Variant Studio for the Illumina panel. Variants that remained after the analysis pipeline were individually interrogated using the Integrative Genomics Viewer (IGV). Results: Of the 90 cancer cases sequenced, each panel returned on average 20 variants. Of these, there was 100% concordance between the two panels with respect to the clinically actionable mutations identified. Conclusions: We have implemented next generation sequencing technologies for the detection of somatic mutations in human cancers. Two platforms, the Ion Torrent PGM and the Illumina MiSeq, have been used for the routine analysis of FFPE cancer tissues in a clinical setting with actionable and non-actionable variants reported and archived. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B179. Citation Format: Jason D. Peterson, Francine de Abreu, Torrey L. Gallagher, Paul R. Burchard, Christopher I. Amos, Wendy A. Wells, J. Marc Pipas, Marc S. Ernstoff, Camilo E. Fadul, James R. Rigas, Gregory J. Tsongalis. Targeted next generation sequencing for somatic mutations in human cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B179.

A comparative survey of functional footprints of EGFR pathway mutations in human cancers

Genes functioning in epidermal growth factor receptor (EGFR) signaling pathways are among the most frequently activated oncogenes in human cancers. We have conducted a comparative analysis of functional footprints (that is, effect on signaling and transcriptional landscapes in cells) associated with oncogenic and tumor suppressor mutations in EGFR pathway genes in human cancers. We have found that mutations in the EGFR pathway differentially have an impact on signaling and metabolic pathways in cancer cells in a mutation- and tissue-selective manner. For example, although signaling and metabolic profiles of breast tumors with PIK3CA or AKT1 mutations are, as expected, highly similar, they display markedly different, sometimes even opposite, profiles to those with ERBB2 or EGFR amplifications. On the other hand, although low-grade gliomas and glioblastomas, both brain cancers, driven by EGFR amplifications are highly functionally similar, their functional footprints are significantly different from lung and breast tumors driven by EGFR or ERBB2. Overall, these observations argue that, contrary to expectations, the mechanisms of tumorigenicity associated with mutations in different genes along the same pathway, or in the same gene across different tissues, may be highly different. We present evidence that oncogenic functional footprints in cancer cell lines have significantly diverged from those in tumor tissues, which potentially explains the discrepancy of our findings with the current knowledge. Nevertheless, our analyses reveal a common inflammatory response signature in EGFR-driven human cancers of different tissue origins. Our results may have implications in the design of therapeutic strategies in cancers driven by these oncogenes.

A conserved domain in exon 2 coding for the human and murine ARF tumor suppressor protein is required for autophagy induction

The ARF tumor suppressor, encoded by the CDKN2A gene, has a well-defined role regulating TP53 stability; this activity maps to exon 1β of CDKN2A. In contrast, little is known about the function(s) of exon 2 of ARF, which contains the majority of mutations in human cancer. In addition to controlling TP53 stability, ARF also has a role in the induction of autophagy. However, whether the principal molecule involved is full-length ARF, or a small molecular weight variant called smARF, has been controversial. Additionally, whether tumor-derived mutations in exon 2 of CDKN2A affect ARF’s autophagy function is unknown. Finally, whereas it is known that silencing or inhibiting TP53 induces autophagy, the contribution of ARF to this induction is unknown. In this report we used multiple autophagy assays to map a region located in the highly conserved 5′ end of exon 2 of CDKN2A that is necessary for autophagy induction by both human and murine ARF. We showed that mutations in exon 2 of CDKN2A that affect the coding potential of ARF, but not p16INK4a, all impair the ability of ARF to induce autophagy. We showed that whereas full-length ARF can induce autophagy, our combined data suggest that smARF instead induces mitophagy (selective autophagy of mitochondria), thus potentially resolving some confusion regarding the role of these variants. Finally, we showed that silencing Tp53 induces autophagy in an ARF-dependent manner. Our data indicated that a conserved domain in ARF mediates autophagy, and for the first time they implicate autophagy in ARF’s tumor suppressor function.

Metabolic alteration in tumorigenesis

Altered metabolism in cancer was first discovered by Otto Warburg early last century. Although the Warburg Effect has been widely used in tumor detection, relatively little progress had been made in mechanistic understanding of cancer metabolism in the subsequent eight decades. Genetic studies have recently identified mutations in human cancer targeting multiple enzymes involved in intermediate metabolism. One emerging mechanism common to these mutant enzymes is the accumulation of a metabolite that alters the epigenetic control.

Abstract IA6: Murine models of nonserous ovarian cancer: Effects of mutant Trp53, Pik3ca, and Arida1a in a model of endometrioid carcinoma based on Wnt and PI3K/Akt pathway defects

Abstract A dualistic pathway model of ovarian carcinoma (OvCA) pathogenesis has been proposed in which type I OvCAs are low grade, genetically stable, and relatively more indolent than type II OvCAs, most of which are high-grade serous carcinomas. Endometrioid OvCA (EOC) is a prototypical type I tumor, often harboring mutations that affect the Wnt and phosphatidylinositol 3-kinase/AKT/mTOR signaling pathways. Molecular and histopathologic analyses indicate type I and II OvCAs share overlapping features, and a subset of EOCs may undergo type I→type II progression accompanied by acquisition of somatic TP53 or PIK3CA mutations. We used a murine model of EOC initiated by conditional inactivation of the Apc and Pten tumor suppressor genes to investigate mutant Trp53 or Pik3ca alleles as key drivers of type I→type II OvCA progression. In the mouse EOC model, the presence of somatic Trp53 or Pik3ca mutations resulted in shortened survival and more widespread metastasis. In contrast, bi-allelic inactivation of Arid1a in addition to Apc and Pten actually prolonged survival and tumors with Arid1a inactivation showed more epithelial differentiation than their counterparts with intact Arid1a alleles. Our findings indicate that the adverse prognosis associated with TP53 and PIK3CA mutations in human cancers can be functionally replicated in mouse models of type I→type II OvCA progression. Moreover, the models should represent a robust platform for assessment of the contributions of Trp53, Pik3ca, and Arid1a mutations to the response of EOCs to conventional and targeted drugs. Citation Format: Kathleen R. Cho, Rong Wu, Yali Zhai, Isao Sakaguchi. Murine models of nonserous ovarian cancer: Effects of mutant Trp53, Pik3ca, and Arida1a in a model of endometrioid carcinoma based on Wnt and PI3K/Akt pathway defects. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA6.

A short tutorial in analyzing NGS data of cancer genomes for somatic mutation calling

Background Somatic mutation is the key element of tumorigenesis as these changes in nucleotide sequence of the cancer genome in somatic cells acquired throughout life can lead to protein alteration, cellular damage and thus cause cancer. The advent of next generation sequencing has significantly improved our ability to identify somatic mutations in cancer genomes paving the way for the comprehensive online catalogue for somatic mutation in human cancer (COSMIC) 1 which contains more than 820,000 mutations so far. Nevertheless, there are still many challenges in detecting somatic mutation in cancer especially for low frequency mutation due to either tumor heterogeneity or contamination with normal cells. Here, in this short tutorial, I will present a recent somatic mutation caller tool developed by the Broad Institute called Mutect 2 as part of the GATK (Genome Analysis Toolkit). I will use my own NGS dataset to demonstrate the tools and address some issues of troubleshooting input data and interpreting output.

E2F-7 couples DNA damage-dependent transcription with the DNA repair process

The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.

Frequency of TERT promoter mutations in human cancers

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.