About the Project
As part of the successful CRUK accelerator award studying the microenvironment in hepatocellular carcinoma (HCC), we are looking for a graduate student to be jointly supervised by Dr Matthew Hoare at the Cancer Research UK Cambridge Institute and Dr Meritxell Huch at the Gurdon Institute.
Introduction
Cellular senescence is a highly conserved tumour suppressor mechanism, whereby cells undergo permanent cell cycle arrest in response to diverse stressors such as replicative exhaustion, oxidative stress and unrestricted oncogene-activation (1). Senescent cells accumulate in the diseased human liver (2), are enriched in areas of dysplasia (3) and their presence predicts the subsequent development of HCC (4). Mouse models of hepatocyte replicative senescence develop features of liver failure (5).
Despite being non-proliferative senescent cells are both metabolically active and highly secretory, driving non-autonomous effects that can be both tumour suppressive and pro-oncogenic dependent on context (6). Through signalling to immune cells they trigger their own immune-mediated clearance, termed senescence surveillance (7). When this process is interrupted senescent cells persist and are chronically oncogenic. The mechanism for this pro-oncogenic effect, though, remains elusive.
We have demonstrated that RAS-senescent hepatocytes drive signalling in adjacent hepatocytes (8), but the functional effect of non-autonomous signalling from persistent senescent cells to surrounding ‘normal’ hepatocytes is unknown. Also, whether persistent intrahepatic senescent cells drive pro-oncogenic signals or inhibit the normal function of adjacent cells is unknown. Novel organoid culture systems that allow long-term expansion of liver progenitors that retain the capacity to differentiate in to hepatocytes in vitro represent an ideal model to study the interactions of two or more cell populations in a more physiological context (9).
Hypotheses
We hypothesize that senescent hepatocytes have significant non-autonomous effects upon:
1. the transcriptional programme and chromatin architecture of adjacent normal hepatocytes;
2. the functionality of adjacent normal hepatocytes.
Aims and experimental design
We aim to utilise liver organoid-based models to study non-autonomous signalling from senescent hepatocytes to adjacent normal cells and utilise these models to understand the functional effects of senescence in the pre-cancerous liver microenvironment.
1) Model establishment. To understand non-autonomous senescence signalling upon key pathways in adjacent hepatocytes, we will develop in vitro models using hepatocyte-derived organoids. We will use cells from two mouse strains with distinct tags allowing the temporal induction of oncogene-induced senescence in the signal-sending hepatocyte population with subsequent fractionation and analysis of downstream signalling in the signal-receiving hepatocyte population.
2) Systematic profiling of non-autonomous effects of senescence upon hepatocytes. We will profile the transcriptional landscape and chromatin accessibility of signal-receiving hepatocytes. (A) We will utilise single-cell RNA-sequencing of fractionated signal-receiving hepatocytes organoids to understand the transcriptional programmes that are induced non-autonomously in hepatocytes by senescence in the microenvironment. This will allow us to understand not only the programmes that are induced, but also the variability of these phenotypes in the signal-receiving population. (B) We will utilise ATAC-sequencing (10) of fractionated signal-receiving hepatocytes to understand whether chromatin accessibility in adjacent hepatocytes is also non-autonomously regulated.
3) Non-autonomous senescence signalling in the human liver in vitro and in vivo. We will extend these concepts to human liver tissue: (A) we will derive hepatocyte lines from resected human liver for use as signal-receiving cells in our organoid cultures to confirm whether signalling pathways are dysregulated in adjacent primary human hepatocytes and whether this differs in normal or chronically diseased hepatocytes. (B) Utilising these same organoids we will analyse whether non-autonomous senescence signaling impairs the normal functionality of hepatocytes.
Preferred skills/knowledge
The ideal candidate will have a solid knowledge of cell or cancer biology and experience in one or more of the areas mentioned above. He or she must be highly motivated to drive an independent research project.
Funding Notes
Funding
This project is funded by a Cancer Research UK studentship that includes full funding for University and College fees and a stipend of £19,000 per annum.
Eligibility
The studentship provides a maintenance grant and tuition fees at the UK/EU rate. Owing to funding restrictions the studentship is not available to non-EU nationals.
Applications are invited from recent graduates or final year undergraduates who hold or expect to gain a first/upper second class degree (or equivalent) in a relevant subject from any recognised university worldwide.
References
1. Salama, R., Sadaie, M., Hoare, M. & Narita, M. Cellular senescence and its effector programs. Genes & Development 28, 99–114 (2014).
2. Marshall, A. et al. Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection. Gastroenterology 128, 33–42 (2005).
3. Ikeda, H. et al. Large cell change of hepatocytes in chronic viral hepatitis represents a senescent-related lesion. Human Pathology 40, 1774–1782 (2009).
4. Rey, S. et al. Liver damage and senescence increases in patients developing hepatocellular carcinoma. Journal of Gastroenterology and Hepatology 32, 1480–1486 (2017).
5. Rudolph, K. L., Chang, S., Millard, M., Schreiber-Agus, N. & DePinho, R. A. Inhibition of experimental liver cirrhosis in mice by telomerase gene delivery. Science 287, 1253–1258 (2000).
6. Coppé, J.-P., Desprez, P.-Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. Mech. Dis. 5, 99–118 (2010).
7. Kang, T.-W. et al. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479, 547–551 (2011).
8. Hoare, M. et al. NOTCH1 mediates a switch between two distinct secretomes during senescence. Nature Cell Biology 18, 979–992 (2016).
9. Broutier, L. et al. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nature Medicine 23, 1424–1435 (2017).
10. Hänsel-Hertsch, R. et al. G-quadruplex structures mark human regulatory chromatin. Nature Genetics 48, 1267–1272 (2016).