Classical models of cancer development involve the accrual of activating mutations in oncogenes and inactivating mutations in tumour suppressor genes, which result in a large survival or proliferative advantage. This is proposed to result in a ‘selective sweep’, such that the affected cell population becomes much the most prevalent tumour cell population. It is proposed that a limited number of such mutations are required to drive the typical human cancer, perhaps 5 or 6 for solid tumours and a smaller number for early onset haematological malignancy.
More recently, accruing data from large-scale DNA-sequencing programs and pan-genomic analyses of signalling pathways in cancer has revealed a number of findings that are difficult to accommodate under this classical ‘multiple discreet step’ model. For most cancers, the mutational spectrum is much more constrained than might have been predicted. Thus, precise mutations of specific amino acid residues (which are not necessarily the most activating or the most inactivating) are associated with specific cancer types. Mutations in pathways that operate in all cells are often associated with highly tissue-specific cancer predisposition. Specific combinations and even ordering of mutations are observed. All these findings suggest that the oncogenic drive must be quantitatively precise and context specific.
The laboratory is interested in these findings from the perspective of hypoxia signalling pathways, which are genetically activated in clear cell renal cancer (RCC) by mutations of the von Hippel-Lindau tumour suppressor (pVHL) which normally acts to promote proteolytic destruction of the key hypoxia inducible transcription factor (HIF) in the presence of oxygen. In VHL-associated RCC, the impaired function of VHL leads to constitutive activation of the HIF transcriptional cascade. Pan-genomic analysis of this (and many other) biological pathways has revealed massively greater complexity than was foreseen before the advent of high throughput sequencing technology. Following oncogenic activation, all these pathway connections will be entrained, irrespective of whether they support, or hinder oncogenic drive, or are neutral.
Such a process potentially explains the above observations from cancer genetics, and suggests a different model of cancer evolution, which will be studied in this project. In this model it is nett balance of pathway effects that is important. This balance is likely very precise and highly context (e.g. tissue) specific, explaining the highly constrained and tissue specific patterns of mutation.
The new model has important implications for cancer evolution. It suggests first that an ‘oncogenic switch’ may only be tolerated in a small number of cells, and/or may place the relevant cell under strong selective pressure to modify the pathway to more oncogenic output. It is possible that many mutations or copy number variations acting in cis or epigenetic regulators acting in trans are involved in this process.
The main difficulty in proof/disproof of the model is that it is not easy to observe and monitor the earliest events in cancer when these processes are proposed to be operational. Most mouse models using Cre-recombinase permit timed oncogenic mutations to be created, but in general result in a large ‘field change’ in the relevant tissue. This doesn’t mimic cancer, and also creates an abnormal context which might obscure relevant events.
The aim of the project is to use precise lineage tracing technology to observe and monitor the earliest events following inactivation of two kidney tissue specific tumour suppressors in the mouse (von Hippel-Lindau and fumarate hydratase). Inactivation of these genes is directly coupled to the activation of the marker, tdTomato. In this way small numbers of genetic events can be detected and monitored. Relevant cells can be retrieved for single or oligo cellular analyses. Outcomes in cancer prone and cancer resistant organs will be compared and contrasted. Mechanisms of ‘accommodation’ will be studied in surviving and proliferating cells.
The project will provide a training in a range of state-of-the-art technologies that are central to cancer research, including mouse models, lineage tracing, cell biology, pan-genomic pathway analyses and informatics.
Project reference number: 1031
|Professor Sir Peter J Ratcliffe FRS||Target Discovery Institute||Oxford University, NDM Research Building||GBRemail@example.com|
|Dr Julie Adam||Nuffield Department of Medicine||University of Oxford||GBR|
|Professor David R Mole FRCP||Target Discovery Institute||Oxford University, NDM Research Building||GBRfirstname.lastname@example.org|
The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors. Hide abstract
General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumor suppressor. However HIF-1α and HIF-2α have contrasting effects on experimental tumor progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1α and HIF-2α and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of canonical and non-canonical HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1α-specific binding, and genes associated with favorable prognosis and between HIF-2α-specific binding and adverse prognosis. However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-α isoform contributes a highly complex mix of pro- and anti-tumorigenic effects. Hide abstract
Un-physiological activation of hypoxia inducible factor (HIF) is an early event in most renal cell cancers (RCC) following inactivation of the von Hippel-Lindau tumor suppressor. Despite intense study, how this impinges on cancer development is incompletely understood. To test for the impact of genetic signals on this pathway, we aligned human RCC-susceptibility polymorphisms with genome-wide assays of HIF-binding and observed highly significant overlap. Allele-specific assays of HIF binding, chromatin conformation and gene expression together with eQTL analyses in human tumors were applied to mechanistic analysis of one such overlapping site at chromosome 12p12.1. This defined a novel stage-specific mechanism in which the risk polymorphism, rs12814794, directly creates a new HIF-binding site that mediates HIF-1α isoform specific upregulation of its target BHLHE41. The alignment of multiple sites in the HIF cis-acting apparatus with RCC-susceptibility polymorphisms strongly supports a causal model in which minor variation in this pathway exerts significant effects on RCC development. Hide abstract
Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrecognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippel-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control. Hide abstract
Both tumor hypoxia and dysregulated metabolism are classical features of cancer. Recent analyses have revealed complex interconnections between oncogenic activation, hypoxia signaling systems and metabolic pathways that are dysregulated in cancer. These studies have demonstrated that rather than responding simply to error signals arising from energy depletion or tumor hypoxia, metabolic and hypoxia signaling pathways are also directly connected to oncogenic signaling mechanisms at many points. This review will summarize current understanding of the role of hypoxia inducible factor (HIF) in these networks. It will also discuss the role of these interconnected pathways in generating the cancer phenotype; in particular, the implications of switching massive pathways that are physiologically 'hard-wired' to oncogenic mechanisms driving cancer. Hide abstract