The Role and Regulation of Semaphorin 3B in Breast Cancer Progression

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Saudi Digital Library
Abstract
Semaphorin 3B (SEMA3B) is an 83 kDa secreted protein of the semaphorin family which has recently been shown to play an important role in angiogenesis and tumour progression and is thought to act as a tumour suppressor. SEMA3B is located at a site of frequent allele loss in the early pathogenesis of breast cancer, and the full-length form of SEMA3B is thought to be cleaved by furin-like pro-protein convertases (PPCs), into an inactive 51 kDa fragment. However, the role and expression of SEMA3B in breast cancer remain unclear. This project, therefore, tests the hypothesis that PPCs cleavage of SEMA3B results in inactivation of SEMA3B in invasive breast cancer. In order to test the hypothesis the project used a series of breast cell-lines consisting of normal breast epithelial cells, pre-malignant, pre-invasive, invasive and metastatic cell-lines, as a model of breast cancer progression. SEMA3B and PPCs mRNA and protein expression were investigated in these cell-lines using qPCR and Western blotting, respectively. Immunohistochemistry was then performed on tissue microarray slides in a panel representing different stages of breast disease. In addition, the effect of full-length recombinant SEMA3B was assessed in cell based functional assays. Finally, two PPC inhibitors, namely decRVKR-CMK and alpha-1 antitrypsin Portland variant (α1-PDX) were used to inhibit PPC activity and potentially restore the active full-length SEMA3B in breast cancer cells. The data in this thesis show that SEMA3B gene and protein expression was detected in all cell-lines tested, with the highest level of gene expression seen in the normal MCF-10A cells. These cells were the only ones to express the full-length SEMA3B with all other cells only expressing cleaved SEMA3B, which is likely to be inactive. Treatment with full-length recombinant SEMA3B showed that this protein inhibited breast cancer cell growth, migration and invasion. PPCs analysis did not show a significant relationship with increasing malignancy of cell-line for either mRNA or protein expression, although all cell-lines expressed at least some PPCs. Histological analysis showed that SEMA3B expression was significantly reduced with increasing malignancy of lesion and correlated closely with the expression of many of the PPCs. Although the CMK inhibitor did not prevent cleavage of SEMA3B, the α1-PDX inhibitor partially restored full-length SEMA3B. These data suggest that full-length SEMA3B is a tumour suppressor in breast cancer, and that the PPCs may be involved in cleavage of SEMA3B to allow breast cancer progression. Further work is required to confirm the hypothesis and to see whether α1-PDX may be a potential future therapy for breast cancer.
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