Predictive gene lists for breast cancer prognosis: a topographic visualisation study

Background: The controversy surrounding the non-uniqueness of predictive gene lists (PGL) of small selected subsets of genes from very large potential candidates as available in DNA microarray experiments is now widely acknowledged 1. Many of these studies have focused on constructing discriminative semi-parametric models and as such are also subject to the issue of random correlations of sparse model selection in high dimensional spaces. In this work we outline a different approach based around an unsupervised patient-specific nonlinear topographic projection in predictive gene lists. Methods: We construct nonlinear topographic projection maps based on inter-patient gene-list relative dissimilarities. The Neuroscale, the Stochastic Neighbor Embedding(SNE) and the Locally Linear Embedding(LLE) techniques have been used to construct two-dimensional projective visualisation plots of 70 dimensional PGLs per patient, classifiers are also constructed to identify the prognosis indicator of each patient using the resulting projections from those visualisation techniques and investigate whether a-posteriori two prognosis groups are separable on the evidence of the gene lists. A literature-proposed predictive gene list for breast cancer is benchmarked against a separate gene list using the above methods. Generalisation ability is investigated by using the mapping capability of Neuroscale to visualise the follow-up study, but based on the projections derived from the original dataset. Results: The results indicate that small subsets of patient-specific PGLs have insufficient prognostic dissimilarity to permit a distinction between two prognosis patients. Uncertainty and diversity across multiple gene expressions prevents unambiguous or even confident patient grouping. Comparative projections across different PGLs provide similar results. Conclusion: The random correlation effect to an arbitrary outcome induced by small subset selection from very high dimensional interrelated gene expression profiles leads to an outcome with associated uncertainty. This continuum and uncertainty precludes any attempts at constructing discriminative classifiers. However a patient's gene expression profile could possibly be used in treatment planning, based on knowledge of other patients' responses. We conclude that many of the patients involved in such medical studies are intrinsically unclassifiable on the basis of provided PGL evidence. This additional category of 'unclassifiable' should be accommodated within medical decision support systems if serious errors and unnecessary adjuvant therapy are to be avoided.

Breast clinic and life style study BLLISS

Background: Independent, strong and unequivocal evidence suggests that life style factors such as obesity and lack of physical activity along with certain reproductive choices can increase the risk of breast cancer. There are no studies measuring the effectiveness of guidelines from the Department of Health regarding life style choices made by women presenting to breast clinics. The aim of this audit was to study the prevalence of obesity, physical activity and reproductive factors in women referred to breast clinic. Patients and methods: All patients attending the Breast clinic as new referrals were invited to complete a life style questionnaire. The data was analysed for prevalence of various risk factors for breast cancer. Three hundred and 73 patients completed the questionnaire. Results: Final analyses of 373 patients demonstrated that 42% of women performed no exercise and only 24% of patients met Department of Health guideline of 30 minutes of exercise for 5 days a week. Overall 50% of patients were either obese or overweight and 22% of patients had BMI of > 30 kg/m. The median age of menarche was 13 and 18% of women started their period below the age 12. Twenty one percent of women were nulliparous and 14% had their first live birth after the age of 30. Fourteen percent of patients were on the hormone replacement therapy of which 57% have used hormones for more than 5 years. Twenty two percent of women smoked and 9% of women consumed alcohol 5 days a week of which 13% had more than 4 glasses of alcohol in a day. Conclusion: There is preponderance of high risk life style choices in women attending breast clinic. If these life style options are not modified, there could potentially be a significant rise in the number of breast cancer in West Midlands.

Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

Cellular and molecular consequences of S100A4-induced motility in rat breast tumour Rama 37 cells

Since the first discovery of S100 members in 1965, their expressions have been affiliated with numerous biological functions in all cells of the body. However, in the recent years, S100A4, a member of this superfamily has emerged as the central target in generating new avenue for cancer therapy as its overexpression has been correlated with cancer patients’ mortality as well as established roles as motility and metastasis promoter. As it has no catalytic activity, S100A4 has to interact with its target proteins to regulate such effects. Up to date, more than 10 S100A4 target proteins have been identified but the mechanical process regulated by S100A4 to induce motility remains vague. In this work, we demonstrated that S100A4 overexpression resulted in actin filaments disorganisation, reduction in focal adhesions, instability of filopodia as well as exhibiting polarised morphology. However, such effects were not observed in truncated versions of S100A4 possibly highlighting the importance of C terminus of S100A4 target recognition. In order to assess some of the intracellular mechanisms that may be involved in promoting migrations, different strategies were used, including active pharmaceutical agents, inhibitors and knockdown experiments. Treatment of S100A4 overexpressing cells with blebbistatin and Y-27632, non muscle myosin IIA (NMMIIA) inhibitors, as well as knockdown of NMMIIA, resulted in motility enhancement and focal adhesions reduction proposing that NMMIIA assisted S100A4 in regulating cell motility but its presence is not essential. Further work done using Cos 7 cell lines, naturally lacking NMMIIA, further demonstrated that S100A4 is capable of regulating cell motility independent of NMMIIA, possibly through poor maturation of focal adhesion. Given that all these experiments highlighted the independency of NMMIIA towards migration, a protein that has been put at the forefront of S100A4-induced motility, we aimed to gather further understanding regarding the other molecular mechanisms that may be at play for motility. Using high throughput imaging (HCI), 3 compounds were identified to be capable of inhibiting S100A4-mediated migration. Although we have yet to investigate the underlying mechanism for their effects, these compounds have been shown to target membrane proteins and the externalisation of S100 proteins, for at least one of the compounds, leading us to speculate that preventing externalisation of S100A4 could potentially regulate cell motility.