Stem cells in human oral squamous cell carcinomas
Lead Research Organisation:
CANCER RESEARCH UK
Department Name: Cambridge Research Institute
Abstract
Squamous cell carcinomas (SCCs) are the most common type of oral cancer. There were 4,400 new cases diagnosed in the United Kingdom (UK) in 2001, accounting for 1.6% of all new cancers. The disease is on the increase, particularly in men in their 40‘s and 50‘s. In 2003 there were 1,592 deaths from oral cancer in the UK. Within normal human oral mucosa (skin lining the mouth) there are ‘stem cells‘, which allow the tissue to regenerate and grow. These are also thought to be the driving force in SCCs. As cancers develop, stem cells continue to promote further growth of the tumour, therefore, any potential treatment must target these cells in order to effectively destroy the cancer. Without removing all of these cancerous stem cells there remains the potential for the tumour to regenerate. Different cell types within skin and oral mucosa will have differing patterns of cell marker molecules allowing them to communicate with each other and other cells in the body. The aim of this project would be to identify cell surface markers, which are unique to SCC stem cells, allowing us to then target them in the treatment and hopefully cure oral cancers.
Technical Summary
Squamous cell carcinomas of the head and neck (HNSCC) represent an enormous burden to a patient, the clinician and the health service. Surgery and adjuvant therapies are mutilating and have a devastating effect on normal oral function as well as the general health and mental wellbeing of the patient. It has been proposed that tumour stem cells are responsible for tumour re-growth following conventional treatment. Treatments that are specific for cancer stem cells may be more effective and less damaging to the patient.
The goal of my project is to evaluate potential markers of SCC tumour stem cells using both cell lines derived from human HNSCC and specimens of fresh tumours. Previous work from my host lab has identified a putative cytoskeletal regulatory protein, FERM4D, as a candidate stem cell marker in SCC lines. My first goal will be to examine FERM4D expression in HNSCC. I will use radioactive in situ hybridisation (ISH) to screen for FERM4D expression in normal oral mucosa and a panel of 20-30 HNSCC. I will investigate where within the tumour FERM4D is expressed and whether high expression correlates with the differentiated state of the tumour and prognosis. For further studies I will raise anti-peptide antibodies to the N- and C-terminals (which are highly conserved). I will screen the antisera by immunofluorescence staining and Western blotting.
If I am able to confirm that FERM4D is upregulated in HNSCC I will perform functional studies in normal keratinocytes and SCC cell lines. I will generate retroviral vectors to overexpress FERM4D and I will also knockdown expression using RNAi. I will then investigate whether FERM4D increases clonal growth of normal and SCC keratinocytes, whether it reduces differentiation, and whether it affects cell-cell or cell-extracellular matrix adhesion. To validate and extend my in vitro observations, I will determine whether knockdown or overexpression of FERM4D affects the ability of SCC cells to form tumours in nude mice.
The final goal of my project will be to use FERM4D in combination with other potential tumour stem cell markers, such as CD44 and MCSP, to obtain clues as to the location of putative cancer stem cells in HNSCC sections. If I am able to define a subpopulation of cells in this way, I will use laser capture microdissection to isolate RNA from the cells for microarray analysis in order to obtain a full list of markers that define this subpopulation of tumour cells.
The goal of my project is to evaluate potential markers of SCC tumour stem cells using both cell lines derived from human HNSCC and specimens of fresh tumours. Previous work from my host lab has identified a putative cytoskeletal regulatory protein, FERM4D, as a candidate stem cell marker in SCC lines. My first goal will be to examine FERM4D expression in HNSCC. I will use radioactive in situ hybridisation (ISH) to screen for FERM4D expression in normal oral mucosa and a panel of 20-30 HNSCC. I will investigate where within the tumour FERM4D is expressed and whether high expression correlates with the differentiated state of the tumour and prognosis. For further studies I will raise anti-peptide antibodies to the N- and C-terminals (which are highly conserved). I will screen the antisera by immunofluorescence staining and Western blotting.
If I am able to confirm that FERM4D is upregulated in HNSCC I will perform functional studies in normal keratinocytes and SCC cell lines. I will generate retroviral vectors to overexpress FERM4D and I will also knockdown expression using RNAi. I will then investigate whether FERM4D increases clonal growth of normal and SCC keratinocytes, whether it reduces differentiation, and whether it affects cell-cell or cell-extracellular matrix adhesion. To validate and extend my in vitro observations, I will determine whether knockdown or overexpression of FERM4D affects the ability of SCC cells to form tumours in nude mice.
The final goal of my project will be to use FERM4D in combination with other potential tumour stem cell markers, such as CD44 and MCSP, to obtain clues as to the location of putative cancer stem cells in HNSCC sections. If I am able to define a subpopulation of cells in this way, I will use laser capture microdissection to isolate RNA from the cells for microarray analysis in order to obtain a full list of markers that define this subpopulation of tumour cells.