Bin Tean Teh

Patterns of somatic mutation in human cancer genomes

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be ‘passengers’ that do not contribute to oncogenesis. However, there was evidence for ‘driver’ mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.

Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma

The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ∼3,500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (also known as KDM6A), JARID1C (also known as KDM5C) and SETD2 (ref. 2). These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodelling complex gene PBRM1 (ref. 4) as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology.

Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes

Clear cell renal cell carcinoma (ccRCC) is the most common form of adult kidney cancer, characterized by the presence of inactivating mutations in the VHL gene in most cases, and by infrequent somatic mutations in known cancer genes. To determine further the genetics of ccRCC, we have sequenced 101 cases through 3,544 protein-coding genes. Here we report the identification of inactivating mutations in two genes encoding enzymes involved in histone modification—SETD2, a histone H3 lysine 36 methyltransferase, and JARID1C (also known as KDM5C), a histone H3 lysine 4 demethylase—as well as mutations in the histone H3 lysine 27 demethylase, UTX (KMD6A), that we recently reported. The results highlight the role of mutations in components of the chromatin modification machinery in human cancer. Furthermore, NF2 mutations were found in non-VHL mutated ccRCC, and several other probable cancer genes were identified. These results indicate that substantial genetic heterogeneity exists in a cancer type dominated by mutations in a single gene, and that systematic screens will be key to fully determining the somatic genetic architecture of cancer.

Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome.

We report here the identification of a gene associated with the hyperparathyroidism–jaw tumor (HPT–JT) syndrome. A single locus associated with HPT–JT (HRPT2) was previously mapped to chromosomal region 1q25–q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT–JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT–JT and in development of some sporadic parathyroid tumors.

Signatures of mutation and selection in the cancer genome

The cancer genome is moulded by the dual processes of somatic mutation and selection. Homozygous deletions in cancer genomes occur over recessive cancer genes, where they can confer selective growth advantage, and over fragile sites, where they are thought to reflect an increased local rate of DNA breakage. However, most homozygous deletions in cancer genomes are unexplained. Here we identified 2,428 somatic homozygous deletions in 746 cancer cell lines. These overlie 11% of protein-coding genes that, therefore, are not mandatory for survival of human cells. We derived structural signatures that distinguish between homozygous deletions over recessive cancer genes and fragile sites. Application to clusters of unexplained homozygous deletions suggests that many are in regions of inherent fragility, whereas a small subset overlies recessive cancer genes. The results illustrate how structural signatures can be used to distinguish between the influences of mutation and selection in cancer genomes. The extensive copy number, genotyping, sequence and expression data available for this large series of publicly available cancer cell lines renders them informative reagents for future studies of cancer biology and drug discovery.

Gene expression profiling of clear cell renal cell carcinoma: Gene identification and prognostic classification

To better understand the molecular mechanisms that underlie the tumorigenesis and progression of clear cell renal cell carcinoma (ccRCC), we studied the gene expression profiles of 29 ccRCC tumors obtained from patients with diverse clinical outcomes by using 21,632 cDNA microarrays. We identified gene expression alterations that were both common to most of the ccRCC studied and unique to clinical subsets. There was a significant distinction in gene expression profile between patients with a relatively nonaggressive form of the disease [100% survival after 5 years with the majority (15/17 or 88%) having no clinical evidence of metastasis] versus patients with a relatively aggressive form of the disease (average survival time 25.4 months with a 0% 5-year survival rate). Approximately 40 genes most accurately make this distinction, some of which have previously been implicated in tumorigenesis and metastasis. To test the robustness and potential clinical usefulness of this molecular distinction, we simulated its use as a prognostic tool in the clinical setting. In 96% of the ccRCC cases tested, the prediction was compatible with the clinical outcome, exceeding the accuracy of prediction by staging. These results suggest that two molecularly distinct forms of ccRCC exist and that the integration of expression profile data with clinical parameters could serve to enhance the diagnosis and prognosis of ccRCC. Moreover, the identified genes provide insight into the molecular mechanisms of aggressive ccRCC and suggest intervention strategies.

Interleukin-8 Mediates Resistance to Antiangiogenic Agent Sunitinib in Renal Cell Carcinoma

The broad spectrum kinase inhibitor sunitinib is a first-line therapy for advanced clear cell renal cell carcinoma (ccRCC), a deadly form of kidney cancer. Unfortunately, most patients develop sunitinib resistance and progressive disease after about 1 year of treatment. In this study, we evaluated the mechanisms of resistance to sunitinib to identify the potential tactics to overcome it. Xenograft models were generated that mimicked clinical resistance to sunitinib. Higher microvessel density was found in sunitinib-resistant tumors, indicating that an escape from antiangiogenesis occurred. Notably, escape coincided with increased secretion of interleukin-8 (IL-8) from tumors into the plasma, and coadministration of an IL-8 neutralizing antibody resensitized tumors to sunitinib treatment. In patients who were refractory to sunitinib treatment, IL-8 expression was elevated in ccRCC tumors, supporting the concept that IL-8 levels might predict clinical response to sunitinib. Our results reveal IL-8 as an important contributor to sunitinib resistance in ccRCC and a candidate therapeutic target to reverse acquired or intrinsic resistance to sunitinib in this malignancy.

HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours

Background: Hyperparathyroidism is a common endocrinopathy characterised by the formation of parathyroid tumours. In this study, we determine the role of the recently identified gene, HRPT2, in parathyroid tumorigenesis. Methods: Mutation analysis of HRPT2 was undertaken in 60 parathyroid tumours: five HPT-JT, three FIHP, three MEN 1, one MEN 2A, 25 sporadic adenomas, 17 hyperplastic glands, two lithium associated tumours, and four sporadic carcinomas. Loss of heterozygosity at 1q24-32 was performed on a subset of these tumours. Results:HRPT2 somatic mutations were detected in four of four sporadic parathyroid carcinoma samples, and germline mutations were found in five of five HPT-JT parathyroid tumours (two families) and two parathyroid tumours from one FIHP family. One HPT-JT tumour with germline mutation also harboured a somatic mutation. In total, seven novel and one previously reported mutation were identified. “Two-hits” (double mutations or one mutation and loss of heterozygosity at 1q24-32) affecting HRPT2 were found in two sporadic carcinomas, two HPT-JT-related and two FIHP related tumours. Conclusions: The results in this study support the role of HRPT2 as a tumour suppressor gene in sporadic parathyroid carcinoma, and provide further evidence for HRPT2 as the causative gene in HPT-JT, and a subset of FIHP. In light of the strong association between mutations of HRPT2 and sporadic parathyroid carcinoma demonstrated in this study, it is hypothesised that HRPT2 mutation is an early event that may lead to parathyroid malignancy and suggest intragenic mutation of HRPT2 as a marker of malignant potential in both familial and sporadic parathyroid tumours.

A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.