• Expression of MT1-MMP in Head and Neck Squamous Cell Carcinomas (HNSCCs) and Endothelial Cells is Regulated by Hypoxia and Semaphorin 4D (Sema4D)

      Bugshan, Amr; Basile, John R.; 0000-0001-7920-4556 (2016)
      Membrane type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane protein that is important in tumor growth, migration, and invasion. It has the ability to degrade ECM, non-matrix proteins such as CD44 and integrin, and activate MMP2. Semaphorin 4D (Sema4D), a membrane-bound semaphorin, is highly expressed in malignancies such as head and neck squamous cell carcinoma (HNSCC) and is known to be pro-angiogenic, promoting the growth of blood vessels into a developing tumor by acting as a chemoattractant when bound to its receptor, Plexin-B1 (PB1), on endothelial cells. Our central hypothesis is that tumor hypoxia causes an increase in Sema4D, which acts in an autocrine and paracrine manner on tumor cells to induce the overexpression of MT1-MMP, which, in turn, cleaves Sema4D and increases availability to the tumor microenvironment to promote tumor-induced angiogenesis and invasion. Using immunoblots and flow cytometry, we demonstrate that MT1-MMP increases in HNSCC cells in a Sema4D and Plexin-B1-dependent manner in hypoxia. Also, we show that RhoA and NF-?B (downstream effectors of Plexin-B1) are important in the regulation of cell surface MT1-MMP expression under hypoxic conditions. Consequently, tumor-induced invasion and angiogenesis are enhanced. Soluble Sema4D diffuses out from the tumor and acts as a chemoattractant for endothelial cells, which also upregulate MT1-MMP on their surface to facilitate migration through the extracellular matrix. We conclude that Sema4D controls its own availability and, therefore, its own pro-angiogenic potential through autocrine/paracrine regulation of MT1-MMP.
    • A novel role for Semaphorin 4D in tumor metastasis

      Binmadi, Nada Omar; Basile, John R. (2011)
      The semaphorin proteins were originally identified as axonal guidance factors expressed during neuronal development. In addition to this function, several semaphorins are now known to play diverse roles outside of the nervous system. Semaphorin 4D (Sema4D/CD100), a transmembrane molecule that shares structural homology with the scatter factors, exerts important biological effects on a variety of cells, including neural, epithelial and immune cells. Interaction between Sema4D and its receptor, Plexin-B1, has proven to be important in many facets of tumor progression such as tumor angiogenesis, regulation of tumor-associated macrophages and control of invasive growth. Invasion and metastasis are key components of cancer progression. A distinct and largely forgotten path for tumor spread is perineural invasion (PNI), defined as the presence of cancer cells in the perineural space. PNI is frequently used by many human carcinomas, in particular by pancreatic, prostate, and oral squamous cell carcinoma (OSCC), and is associated with tumor recurrence and pain in patients with advanced disease. The work presented in this thesis shows that Sema4D and Plexin-B1 are involved in PNI in tumors and identifies Sema4D as a new player in the complex interaction between tumor cells, nerve cells and the tumor microenvironment. We have previously found that Sema4D is able to promote angiogenesis in several in vitro assays and tumor growth and vascularity of head and neck squamous cell carcinoma xenografts in vivo, but the intracellular signaling pathways engaged and the mechanisms of regulation of this pro-angiogenic function remained unclear. Results presented in the second part of this thesis demonstrate that ligation of Plexin-B1 by Sema4D activates a RhoA-dependent pro-angiogenic phenotype in endothelial cells that signals through phosphatidylinositol 4-phosphate 5-kinase (PI(4)P5K) and generates lipid second messengers. We also discuss the essential role of hypoxia inducible factor (HIF) in endothelial cell migration and tumor vascularity. In conclusion, deciphering the regulatory and signaling mechanisms of Sema4D could lead to the identification of new therapeutic targets for anti-angiogenic and anti-metastatic cancer therapy.
    • The Role of Semaphorin 4D (Sema4D) in Bone Metastasis

      Buhamrah, Asma; Basile, John R. (2014)
      The Role of Semaphorin 4D (Sema4D) in Bone Metastasis Background: Bone metastasis is a catastrophic endpoint of many neoplastic diseases, but especially for patients with advanced breast cancer. Despite the continuous advances in pharmacological and cancer research, bone loss and subsequent bone complications are seen in 70% of females diagnosed with breast cancer. Semaphorin 4D (Sema4D), a protein originally described to regulate the immune response, is now known to have a novel role in bone regulation. Sema4D is also found to be highly expressed by many tumor cells including those of breast cancer. In this study we focus on the role of Sema4D produced by tumor cells on their ability to metastasize to bone. Materials and methods: The osteoblast cell line MC3T3 was treated under different osteogenic conditions to examine the effects of Sema4D on bone differentiation in vitro. We also used tumor cells with silenced Sema4D to investigate the effects of tumor-derived Sema4D on their ability to metastasize to bone in vivo. Results: Sema4D produced by the breast cancer cell line MDA-231 inhibited bone matrix formation and mineralization in vitro. In vivo, however, MDA-231 tend to spread to bone only when Sema4D was highly expressed by these cells and not when it was silenced. Conclusion: Over-expression of Sema4D by breast cancer cells inhibits bone formation in vitro and tends to increase the ability of these cells to metastasize to bone in vivo and establish osteolytic lesions characterized by this tumor type. Our findings may serve as a solid starting point to investigate the role of anti-Sema4D therapy in tumor metastasis. Further in vivo studies are strongly encouraged to clinically determine their effects.