Genetic mutations associated with lung cancer metastasis to the brain.

Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. As a barrier to metastasis, cells normally undergo an apoptotic process known as 'anoikis', in circulation. The recent technological advances in the isolation and characterisation of rare circulating tumour cells (CTCs) will allow a better understanding of anoikis resistance. Detailed molecular and functional analyses of anoikis-resistant cells may provide insight into the biology of cancer metastasis and help identify novel targets for prevention of cancer dissemination. To uncover the molecular changes that govern the transition from a primary lung tumour to a secondary metastasis and specifically the mechanisms by which CTCs survive in circulation, we carried out whole genome sequencing (WGS) of normal lung, primary tumours and the corresponding brain metastases from five patients with progressive metastatic non-small-cell lung carcinoma. We also isolated CTCs from patients with metastatic cancer and subjected them to whole genome amplification and Sanger sequencing of genes of interest. While the primary tumours showed mutations in genes associated with cell adhesion and motility, brain metastases acquired mutations in adaptive, cytoprotective genes involved in response to cellular stress such as Keap-1, Nrf2 and P300, which are key players of the Keap1-Nrf2-ARE survival pathway. Nrf2 is a transcriptional factor that upon stress translocates into the nucleus, binds to the anti-oxidant response elements (ARE) and drives the expression of anti-oxidant genes. The identified mutations affect regulatory domains in all three proteins, suggesting a functional role in providing a survival advantage to CTCs in the peripheral blood allowing their dissemination to distant organs.

Whole genome sequencing of glioblastoma multiforme identifies multiple structural variations involved in EGFR activation.

Next generation sequencing has become a powerful tool in dissecting and identifying mutations and genomic structural variants that accompany tumourigenesis. Sequence analysis of glioblastoma multiforme (GBM) illustrates the ability to rapidly identify mutations that may affect phenotype. Approximately 50% of human GBMs overexpress epidermal growth factor receptor (EGFR) which renders the EGFR protein a compelling therapeutic target. In brain tumours, attempts to target EGFR as a cancer therapeutic, however, have achieved little or no benefit. The mechanisms that drive therapeutic resistance to EGFR inhibitors in brain tumours are not well defined, and drug resistance contributes to the deadly and aggressive nature of the disease. Whole genome sequencing of four primary GBMs revealed multiple pathways by which EGFR protein abundance becomes deregulated in these tumours and will guide the development of new strategies for treating EGFR overexpressing tumours. Each of the four tumours displayed a different mechanism leading to increased EGFR protein levels. One mechanism is mediated by gene amplification and tandem duplication of the kinase domain. A second involves an intragenic deletion that generates a constitutively active form of the protein. A third combines the loss of a gene which encodes a protein that regulates EGFR abundance as well as an miRNA that modulates EGFR expression. A fourth mechanism entails loss of an ubiquitin ligase docking site in the C-terminal part of the protein whose absence inhibits turnover of the receptor.

Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma.

BACKGROUND: Findings based on recent advances in next-generation sequence analysis suggest that, in some tumors, a single catastrophic event, termed chromothripsis, results in several simultaneous tumorigenic alterations. Previous studies have suggested that glioblastoma (GBM) may exhibit chromothripsis at a higher rate (39%) than other tumors (9%). Primary glioblastoma is an aggressive form of brain cancer that typically appears suddenly in older adults. With aggressive treatment, the median survival time is only 15 months. Their acute onset and widespread genomic instability indicates that chromothripsis may play a key role in their initiation and progression. GBMs are often characterized by EGFR amplification, CDKN2A and PTEN deletion, although approximately 20% of GBMs harbor additional amplifications in MDM2 or MDM4 with CDK4. METHODS: We used the chromothripsis prediction tool, Shatterproof, in conjunction with a custom whole genome sequence analysis pipeline in order to generate putative regions of chromothripsis. The data derived from this study was further expanded on using fluorescence in situ hybridization (FISH) analysis and susceptibility studies with colony formation assays. RESULTS: We show that primary GBMs are associated with higher chromothripsis scores and establish a link between chromothripsis and gene amplification of receptor tyrosine kinases (RTKs), as well as modulators of the TP53 and RB1 pathways. CONCLUSIONS: Utilizing a newly introduced bioinformatic tool, we provide evidence that chromothripsis is associated with the formation of amplicons containing several oncogenes involved in key pathways that are likely essential for post-chromothriptic cell survival.

Targeting DNA repair mechanisms in cancer.

Preservation of genomic integrity is an essential process for cell homeostasis. DNA-damage response (DDR) promotes faithful transmission of genomes in dividing cells by reversing the extrinsic and intrinsic DNA damage, and is required for cell survival during replication. Radiation and genotoxic drugs have been widely used in the clinic for years to treat cancer but DNA repair mechanisms are often associated with chemo- and radio-resistance. To increase the efficacy of these treatments, inhibitors of the major components of the DDR such as ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), DNA-PK (DNA-dependent protein kinase, catalytic subunit), Chk1 (checkpoint protein 1) and Chk2 (checkpoint protein 2) have been used to confer radio- and/or chemosensitivity upon cancer cells. The elucidation of the molecular mechanisms of DNA repair and the discovery that tumors are frequently repair-deficient provide a therapeutic opportunity to selectively target this deficiency. Genetic mutations in the DNA repair genes constitute not only the initiating event of the cancer cell but also its weakness since the mutated gene is often needed by the cancer cell to maintain its own survival. This weakness has been exploited to specifically kill the tumor cells while sparing the normal ones, a concept known as 'synthetic lethality'. Recent efforts in the design of cancer therapies are directed towards exploiting synthetic lethal interactions with cancer-associated mutations in the DDR. In this review, we will discuss the latest concepts in targeting DNA repair mechanisms in cancer and the novel and promising compounds currently in clinical trials.