Promoter regulation heterogeneity and stratification of colorectal cancer organoids

Developing tumours are composed of a mixture of clonal subpopulations, containing distinct patterns of mutations leading to distinct transcriptome features. In this respect they are not akin to developing embryos, which are also characterized by lineage-specific transcriptomes. The tumourigenic potential of these mixed populations of cancer forming stem cells is driven by Darwinian selection permitting a high level plasticity, allowing the tumour to evade treatment. Clonal architecture analysis of a tumour together with transcriptomic analysis and mathematical modeling can help in stratifying tumors and to address the significance of heterogeneity in contribution to the ontogeny and treatment resistance of tumour formation.

In this PhD studentship we shall test the hypothesis, that transcriptome heterogeneity of tumors modeled by organoids will allow informative stratification of tumors, which will eventually lead to improved biomarkers and intervention strategy designs. One of the important signaling pathways, which define tumour progression is the mTOR pathway, which has been reported to be upregulated in various tumors and as a result has become an appealing therapeutic target. Differential regulation of gene expression by the mTOR pathway has been described in colon cancers and in their model organoids and have been suggested to respond differentially to radiation therapy. The mTOR pathway responds to nutrient and metabolic stress and is activated by growth factors and thus regulates translation pathways via a specialized transcriptional promoter. A hallmarks of this activity is the upregulation of a non-canonical transcription initiation pathway. An understanding of the mechanisms by which tumour cells receive and integrate these signals, which influence their growth and metabolism, is essential to developing a well-targeted therapy. We have recently shown by promoter genomics technology (CAGE-sequencing) that the transcription initiation mechanisms representative for mTOR signaling targets are far more pervasively used in development than previously reported. In this project, transcription mechanisms associated with tumor progression will be analysed to the analogy of our previous work on spatio-temporal heterogeneity during development and will be used to stratify colon cancer organoid models.

The supervisory team representing expertise in developmental genomics (Mueller), cancer genomics (Beggs) and computational biology (Stekel) will build on their complementing expertise to train in in vivo organoid imaging, in promoter transcriptomics and computational biology. We shall utilise a combination of genetic lineage and cell cycle tracing tools (such as FUCCI and Brainbow) introduced into organoids by transgenesis. To monitor tumour heterogeneity in vivo we propose 4D high resolution, light sheet microscopy imaging of colon cancer cell organoids preliminary data has been generated the supervisory team, which in vivo lineage tracing will be applied. The genetic lineage tools will allow sorting tumour cell lineages and selectively analysed for their transcriptomes. The genetic lineage tracing expressed from combinations of reporters in evolving cell lineages of the organoids. For transcriptome and particularly promoterome analysis we shall use CAGE sequencing an improved approach, which works with small cell numbers called SLIC CAGE to selectively detect mTOR signaling responsive transcripts as well as conventional RNA sequencing for global transcriptome characterization in irradiated and control organoids. The transriptome datasets will be computationally analysed for identification of signatures of heterogeneity as well as for stratification of irradiation responsive and non-responsive organoids (Beggs, unpublished data).
The candidate will develop advanced research skills, including next generation sequencing data mapping and computational analysis, as well as organoid imaging and image processing techniques. The student will train in transcription regulation and various aspects of molecular biology and cancer genomics, thus offering the candidate a highly desirable set of skills attractive to prospective academic and non-academic employers.

The two training environments at Birmingham (Birmingham Centre for Genome Biology and Institute of Cancer and Genomic Sciences) and the Sutton Bonnington Campus of the University of Nottingham offer internationally outstanding environment for high quality research including the recent £10M investment into COMPARE, a unique, shared in vivo imaging centre of the two participating institutions.

Person Specification
Applicants should have a strong background in molecular biology or molecular genetics, and ideally a background in in vivo microscopy. They should have a commitment to research in cancer genetics and hold or realistically expect to obtain at least an Upper Second Class Honours Degree in a relevant subject.