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Dr J. Timms' UCL Institute for Women's Health Cancer Proteomics Group aims to advance the understanding of the molecular basis of cancer, with particular focus on signalling mechanisms in cancer and normal cells. Our aim is also to identify clinically useful biomarkers of ovarian, breast and other cancers through the application of proteomic techniques to clinical samples (tissue, serum, urine) and cellular models.

2D gel- and LC-MS-based platforms have been developed for high-throughput quantitative protein and peptide profiling of in vitro cell models and clinical samples. These including 2D-DIGE/MS profiling, automated peptide extraction linked to MALDI-TOF profiling, serum immunoaffinity depletion strategies, phosphopeptide enrichment and 2D-LC-MS/MS profiling which incorporate stable isotope labelling of amino acids in culture (SILAC) and isobaric peptide mass tagging approaches (TMT, iTRAQ) for quantification. These separation, identification and quantification methods have been combined into effective workflows and linked to downstream data analysis and bioinformatics tools.

Research Projects
Analysis of the human serum proteome for screening, diagnosis and cancer biomarker discovery
Our work aims to establish the potential of the human serum proteome for screening and diagnosis of disease in large populations and uses samples collected for the UKCTOCS and UKOPS studies. In an MRC-funded project, we are using robotic liquid-handling for magnetic bead-based extraction of serum polypeptides linked to automated MALDI-TOF MS for the high-throughput profiling of the serum peptidome. Advanced biostatistics and machine learning methods are being applied to find discriminatory peptide peaks generated by the action of tumour-specific proteases which are being identified by MS/MS. High-throughput sample fractionation and SELDI-TOF MS profiling is also being applied to find novel biomarkers of ovarian cancer and to validate previously reported serum biomarkers of ovarian cancer. By comparing profiles of serum from healthy volunteers, patients with benign and malignant ovarian cancer and samples that predate diagnosis, we hope to identify early biomarkers for differential diagnosis and screening. Promising markers are being further validated in independent sample sets using immune-based assays.

serum biomarker

Automated profiling of case control serum samples using magnetic bead extraction and MALDI-TOF MS profiling.

serum

MALDI-TOF MS profiling of serum polypeptides from QC serum and healthy, benign and malignant ovarian cancer samples from the UKOPS. The figure shows 852 aligned spectra.



Characterisation of ErbB2/HER2 signalling
Research is aimed at identifying targets of ErbB2 overexpression in cancer and the growth factors which activate the ErbB receptor family of tyrosine kinases. Parallel differential mRNA and protein expression analyses are being applied to compare gene and protein expression in model cell lines to understand how target genes are dysregulated by ErbB2 overexpression in breast cancer. Targets of interest are being validated and characterised as biomarkers of cancer progression using conventional biochemical and cell-based assays, including the application of siRNA-mediated knockdown. Further work, applying more directed and in-depth proteomic methods for improved detection and quantitation of low-abundance proteins and their post-translational modifications, is aimed at a better understanding of ErbB signalling and regulated gene expression and will establish improved strategies for the proteomic analysis of intractable proteins. Methods include SILAC and multi-dimensional phospho-peptide enrichment.

dige_1

2D-DIGE profiling of cell lysates from parental human mammary luminal epithelial cell line HB4a (red) and a derivative cell line overexpressing ErbB2/HER2 (blue).



Identification and characterisation of ErbB2-dependent and metastatic markers of breast cancer
2D-DIGE and MS-based protein identification have been applied to assess protein expression differences between sets of non-metastatic and metastatic primary breast tumour samples with different levels of ErbB2 expression. Expression of selected candidates has been validated by immunoblotting and immuno-histochemical staining of tissue microarrays and in model cell lines triggered with growth factors. The role of selected targets in proliferation and migration is being further tested in cell-based assays using siRNA-mediated knockdown of gene expression.

dige_2

2D-DIGE profiling of extracted protein from ErbB2-positive (blue) and ErbB2-negative (red) breast tumour tissues.



bc_1



bc_2

Immunohistochemical validation of markers of breast cancer found from tumour tissue proteomic profiling.



Proteomic analysis of ovarian cancer cell models
Microcell-mediated chromosome transfer (MMCT) has been used to transfer normal chromosomal material into ovarian cancer cell lines resulting in hybrids with suppressed tumorigenic phenotype. A combination of quantitative 2D-DIGE and 2D-LC-MS/MS with iTRAQ labelling is being applied to whole cell lysates, the secreted proteome and cell surface proteome of these cell models to identify protein expression signatures of tumour suppression. The work is also aimed at identifying potential biomarkers of ovarian cancer.

mmct_1

2D-DIGE/MS profiling of proteins secreted from TOV112D & TOV21G OC cell lines and MMCT hybrids harbouring normal Chr18 material.




mmct_2

Tandem mass tagging for relative peptide quantitation



Characterisation of molecular stress responses in model systems
This basic research is focused on deciphering cellular responses to environmental stresses in model systems. Work in fission yeast and now in primary human fibroblast cells combines quantitative 2D-DIGE, transcriptional and metabolomic analyses to elucidate responses to oxidative stress and exposure to electromagnetic fields. Novel cysteine-labelling 2D-DIGE reagents and affinity reagents have been used as probes to identify protein targets of oxidative damage in mammary luminal epithelial cells and to assess the effects of ErbB2 overexpression on these responses. Finally, phospho-proteomic affinity methods and cell-based assays are being applied to study the signalling events associated with oxidative stress in mammary luminal epithelial cells and to relate the phosphorylation of specific target proteins with the cellular response to oxidative stress.

kinase

Known interactors of Src kinase identified by affinity purification of tyrosine phosphorylated proteins from epithelial cells under oxidative stress.



Collaborative projects
Other collaborative projects include the application of proteomic methods for characterising novel 3D models of ovarian and endometrial cancer and for the discovery of biomarkers of polycystic ovary syndrome, pelvic inflammatory disease, pancreatic cancer and neuropathic pain.

Links:
Cancer Proteomics Laboratory
Dr J. Timms


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