Exploring the Role of Neuropilin Receptors and Semaphorin-3F in Oral Squamous Cell Carcinoma
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CitationAlmazyad, Asma. 2020. Exploring the Role of Neuropilin Receptors and Semaphorin-3F in Oral Squamous Cell Carcinoma. Doctoral dissertation, Harvard School of Dental Medicine.
AbstractOral squamous cell carcinoma (OSCC) is the most common cancer in the oral cavity with a 5-year survival rate of only 38% in metastatic cases. OSCC dissemination is correlated with enhanced tumor angiogenesis and lymphangiogenesis. Neuropilin 1 and 2 are membrane receptors in endothelial cells (EC) that bind to angiogenic factors (VEGF-A) and lymphangiogenic factors (VEGF-C and VEGF-D), respectfully, and complex with VEGFR1-3 to stimulate the sprouting of blood and lymphatic vessels during developmental angiogenesis and lymphangiogenesis. In addition to EC, tumor cells also express Neuropilin (NRP) receptors but whether NRPs can signal via VEGF ligands in tumor cells lacking VEGFRs and what role either NRP1 or NRP2 plays in the tumorigenesis process is unclear. I hypothesize that NRP1 expression in oral epithelial cells promotes survival in physiologic conditions and tumor initiation or tumor growth (either directly or indirectly by enhancing angiogenesis) during pathologic (carcinogenic) conditions. I hypothesize that NRP2 expression promotes carcinoma progression either directly by increasing tumor cell motility or indirectly by increasing microvessel density in the tumor microenvironment thereby increasing the chances of dissemination. Our laboratory has reported NRP1 upregulation in human and mouse oral dysplasia and OSCC samples. One of my aims is to expand our analysis to examine NRP2 expression in human and mouse normal oral mucosa, oral dysplasia and OSCC samples. NRP2 has an alternative ligand called Semaphorin-3F (SEMA3F) that competes with VEGF binding and inhibits angiogenesis, lymphangiogenesis, and cell motility. Another aim will examine the expression of SEMA3F in human and mouse normal oral mucosa and OSCC samples with the prediction that SEMA3F will be lost during tumor progression. In order to characterize the necessity of the Nrp1, Nrp2, or Sema3F genes in keratinocytes during the tumorigenesis process, Cre-Lox technology will be used to conditionally delete each gene in the oral mucosa followed by chemical (4-NQO) induction of carcinogenesis in mice. Lastly, novel preclinical trials using SEMA3F protein for the first time will be examined in human and mouse OSCC models. Taken together, my results demonstrate that normal oral epithelium expresses both NRP1 receptor and SEMA3F ligand in the suprabasal layers and lacks expression of NRP2. During dysplasia and OSCC, NRP1 and NRP2 are both upregulated and SEMA3F expression is downregulated. In vivo carcinogenesis experiments in specific knockout mice revealed striking findings and demonstrated that Nrp1 expression was essential to cancer initiation in the oral epithelium as K14Nrp1iKO mice failed to develop any dysplasia or OSCC in the 4-NQO model. Nrp2 expression appeared to contribute more to tumor growth and progression as K14iNrp2KO mice developed fewer and smaller OSCC than control groups. My results surrounding the effects of SEMA3F suggest that its bioactivity may be more complex than initially anticipated. In vitro, exogenous SEMA3F inhibited the migration and invasion of Nrp2-expressing mouse OSCC cells (WT3). In vivo, slow-release mini osmotic pumps releasing exogenous SEMA3F systemically (0.3 mg/kg/day) inhibited human OSCC tumor-associated angiogenesis and lymphangiogenesis compared to control treated tumors. Over the course of just 2 weeks, systemic exogenous SEMA3F protein therapy induced complete regression in immunocompetent mice in the majority of mice with established mouse OSCC. In contrast, SEMA3F therapy did not induce regression of OSCC in immunodeficient mice, but did slow OSCC growth and progression. Results examining the role of endogenous Sema3F in keratinocytes were more confounding. Whereas oral epithelial cells in wildtype mice undergoing carcinogenesis were found to downregulate endogenous Sema3F expression during dysplasia, mice depleted in Sema3F fail to develop OSCC tumors after 4-NQO treatment. These results suggest that endogenous Sema3F expression actually promotes carcinogenesis, likely via an indirect paracrine effect on another cell type in expressing Nrp2 in the oral mucosa such as melanocytes, dendritic cells, or T cells. Overall, I have identified NRP2 as a novel target in OSCC and its associated vasculature. Our 4-NQO models in transgenic mice showed both NRPs and SEMA3F to play key roles in OSCC initiation and progression. SEMA3F is a promising anti-tumor therapy specifically targeting the NRP2 axis in carcinoma cells and stromal cells, but future studies will be needed to uncover its role in tumor immunity.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365594