Validation of a Potential Biomarker for the Diagnosis/Treatment of Metastatic Breast Cancer within Omani Females

Breast cancer is the most common malignancy within the Omani females. The alarming fact is that the disease was found to affect Omani females at younger ages than their counterparts in the west and to have lower survival rates with advanced stages of the disease. The etiology of this malignancy within the Omani population is not yet well established and further investigation is needed. In the present proposal we will investigate the potential role of a promising molecule Neuropilin-1 (NRP-1) to be used as a biomarker for breast cancer metastasis and progression. Neuropilin-1 is a multifunctional protein which is intensively studied by many different scientists around the globe. Its high expression was correlated with advanced stages of several types of cancer including breast cancer. However, the mechanism by which it induces breast cancer malignancy is not yet well defined. In this proposal and up to our knowledge we are the first to hypothesize that andldquo;Neuropilin-1 enhances breast cancer progression via Transforming Growth factor andbeta; mediated increase in the activity of the Epithelial to Mesenchymal Transition (EMT) pathwayandrdquo;. Therefore the proposal will deal with two main aspects: the investigation of the molecular mechanism by which NRP-1 induces more tumorogenic behavior in vitro by knocking down and over expressing it in a regulated fashion in several breast cancer cell lines. The in vitro experiments will be done in a way to mimic the clinical investigation by which we will establish 2D and 3D cultures and measure the soluble levels of NRP-1 as well as all the related inducers of the EMT pathway such as the epithelial marker E cadherin and its main repressors Snail-1, Snail2 (Slug), and Twist. As well, the TGF-andbeta; signaling pathway including Smad-2 and Smad-3 as the main transducers for the EMT pathway will be examined. The levels of the former proteins including NRP-1 will be measured in the cell lysates and in conditioned media treated with or without chemotherapy agents after 24 hours and at different time points depending on the cellular survival in culture. The second part of the proposal will focus on the clinical aspect of NRP-1 expression and measure its levels in clinical samples beside the other EMT related proteins using the proximity ligation assay PLA system. We will measure its soluble form in the plasma of non breast cancer control individuals, patients with other types of cancer (non reproductive related) such as colon cancer, and breast cancer patients. We will compare NRP-1 levels before and after their treatment with chemotherapy; the time of sample collection will be after the third week of treatment and at the end of the assigned treatment. We will also quantify NRP-1expression and study its tissue localization in at least 70 breast cancer tissue samples and by using sophisticated statistical analysis we will be able to determine if it is positively or negatively correlated with the other EMT markers such as E-cadherin, Slug, Snail, Twist, N-cadherin and vimentin. All the results will be then compared to the stage and clinico-pathological background data of each case. Knowing the molecular involvement of NRP-1 with the EMT process will enable us to identify and validate this novel biomarker for the prediction/prognosis of breast cancer. The outcome of this proposed project will improve our understanding about the relationship between NRP-1 and the progression of breast cancer in general and within Omani females in particular. Finally, these studies will lead us to better diagnose and treat this deadly disease using NRP-1 as a predictive/prognosis marker.