Feature-based classifiers for somatic mutation detection in tumour–normal paired sequencing data

Motivation: The study of cancer genomes now routinely involves using next-generation sequencing technology (NGS) to profile tumours for single nucleotide variant (SNV) somatic mutations. However, surprisingly few published bioinformatics methods exist for the specific purpose of identifying somatic mutations from NGS data and existing tools are often inaccurate, yielding intolerably high false prediction rates. As such, the computational problem of accurately inferring somatic mutations from paired tumour/normal NGS data remains an unsolved challenge

The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes

The genomic landscape of breast cancer is complex, and inter- and intra-tumour heterogeneity are important challenges in treating the disease. In this study, we sequence 173 genes in 2,433 primary breast tumours that have copy number aberration (CNA), gene expression and long-term clinical follow-up data. We identify 40 mutation-driver (Mut-driver) genes, and determine associations between mutations, driver CNA profiles, clinical-pathological parameters and survival. We assess the clonal states of Mut-driver mutations, and estimate levels of intra-tumour heterogeneity using mutant-allele fractions. Associations between PIK3CA mutations and reduced survival are identified in three subgroups of ER-positive cancer (defined by amplification of 17q23, 11q13-14 or 8q24). High levels of intra-tumour heterogeneity are in general associated with a worse outcome, but highly aggressive tumours with 11q13-14 amplification have low levels of intra-tumour heterogeneity. These results emphasize the importance of genome-based stratification of breast cancer, and have important implications for designing therapeutic strategies.The METABRIC project was funded by Cancer Research UK, the British Columbia Cancer Foundation and Canadian Breast Cancer Foundation BC/Yukon. This sequencing project was funded by CRUK grant C507/A16278 and Illumina UK performed all the sequencing. The authors also acknowledge the support of the University of Cambridge, Hutchinson Whampoa, the NIHR Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre, the Centre for Translational Genomics (CTAG) Vancouver and the BCCA Breast Cancer Outcomes Unit. We thank the Genomics, Histopathology, and Biorepository Core Facilities at the Cancer Research UK Cambridge Institute, and the Addenbrooke’s Human Research Tissue Bank (supported by the National Institute for Health Research Cambridge Biomedical Research Centre).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1147

Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis.

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barrett's esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barrett's esophagus (NDBE; n=66) and high-grade dysplasia (HGD; n=43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barrett's esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies

Cell-cell contact induced resistance to etoposide

Many tumour cell lines grown in close three dimensional cell-cell contact either as multicell spheroids or tumours in mice exhibit a form of multicellular drug and radiation resistance that has been called the "contact effect". This resistance is often associated with agents that produce DNA double-strand breaks such as ionizing radiation and the anticancer drug and topoisomerase II inhibitor, etoposide. The hypothesis was that growth in three dimensional contact results in changes in gene expression that act, directly or indirectly, to increase resistance to etoposide. The objectives were to 1) determine the mechanism for etoposide resistance of spheroids, 2) identify genes that are differentially expressed in monolayers and spheroids, and 3) based on these results, examine the importance of intracellular free calcium levels as mediators of contact resistance. Cycling cells from Chinese hamster V79 spheroids are about 10 times more resistant than monolayers to cell killing by etoposide. Previous results indicated that the outer cells of spheroids and monolayers contained the same total amount and activity of the target enzyme, topo lice, and grew at the same rate. Using immunoblotting and immunohistochemistry, topo Hoc was found to be localized primarily in the cytoplasm of the proliferating outer cells of V79, SiHa and C6 spheroids, while nuclear localization was observed in their corresponding monolayers. Conversely, only WiDr cells, which did not show an increase in resistance to etoposide when grown as spheroids, demonstrated a predominantly nuclear localization of topo Ila. This difference in localization pattern was subsequently explained by the 10-fold decrease in phosphorylation of topo Ila in spheroids relative to monolayers, since phosphorylation is apparently required for nuclear translocation of this enzyme. Cells sorted from xenograft tumours grown in immunodeficient mice resembled the spheroid pattern both in terms of sensitivity to etoposide and location of topo Hoc. When the outer cells of V79 spheroids were returned to monolayer growth, the rate of redistribution of topo Hot to the nucleus occurred with the same kinetics as the increase in sensitivity to the cytotoxic effects of etoposide. Thus, close 3-dimensional cell-cell contact can lead to a change in posttranslational modification of topo Hoc that results in resistance to etoposide. A more direct approach was taken to identify changes in gene expression that occur when cells are grown as spheroids. Using the technique of differential display, 8 reproducible genes were found to be differentially expressed in the outer layer of V79 spheroids compared to monolayers. Up-regulation of 3 genes (cytochrome oxidase c, mtsl and calretinin) was associated with an increase in calcium binding capacity in outer cycling cell of spheroids, suggesting a possible role for calcium for the development of a contact effect. Consistent with this hypothesis, a 2-fold lower concentration of intracellular calcium was found in spheroids compared to monolayers using fluo-3 as a calcium indicator dye. Exposure of monolayers and outer spheroid cells to non-cytotoxic concentrations of BAPTA-AM, a calcium chelating agent, eliminated the difference in etoposide sensitivity between V79 monolayers and spheroids. Calcium depletion has been previously shown to protect against etoposide-induced damage by affecting both the phosphoryaltion of topo Ila and by stablizing the cleavable complex. To determine whether over-expression of a calcium binding protein would increase resistance to etoposide, V79, SiHa and C6 monolayers were transduced with metastasin (mtsl). Expression of this transgene did not reduce killing by etoposide, however because the ultimate goal of reduction in intracellular free Ca²⁺ was also not achieved with this method, the importance of calcium regulation in etoposide resistance cannot be ruled out. In conclusion, growth of cells in 3-dimensional contact as spheroids or as solid tumours induces resistance to etoposide. The basis for this resistance could lie in a change in intracellular Ca²⁺ that alters cleavable complex formation and affects phosphorylation of topo Ila, both of which can cause resistance to the anti-cancer drug etoposide.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Regulation of E-cadherin expression and β-catenin/Tcf transcriptional activity by the integrin-linked kinase

AbstractIntegrin-linked kinase (ILK) is a serine/threonine protein kinase which interacts with the cytoplasmic domains of β1 and β3 integrins. ILK structure and its localization at the focal adhesion allows it not only to interact with different structural proteins, but also to mediate many different signalling pathways. Extracellular matrices (ECM) and growth factors each stimulate ILK signalling. Constitutive activation of ILK in epithelial cells results in oncogenic phenotypes such as disruption of cell extracellular matrix and cell to cell interactions, suppression of suspension-induced apoptosis, and induction of anchorage independent cell growth and cell cycle progression. More specifically, pathological overexpression of ILK results in down-regulation of E-cadherin expression, and nuclear accumulation of β-catenin, leading to the subsequent activation of the β-catenin/Tcf transcription complex, the downstream components of the Wnt signalling pathway. Here we review the data implicating ILK in the regulation of these two signalling pathways, and discuss recent novel insights into the molecular basis and requirement of ILK in the process of epithelial to mesenchymal transformation (EMT)

Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival

An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH2- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser473 phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser473P-Akt were detected in ILK immunoprecipitates and small interfering RNA-mediated depletion of rictor, but not mTOR, inhibited the amount of Ser473P-Akt in the ILK complex. Expression of the NH2-terminal (1-398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser473 phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa