Semaphorin 3A Is A Novel Tumor Suppressor In Head And Neck Squamous Cell Carcinoma

Background:Head and neck squamous cell carcinoma (HNSCC) is the eighth most common malignancy and accounts for approximately 4.8% of all cancer incidence.HNSCC is characterized by a propensity for early metastasis and lymph-node metastasis and a poor prognosis. Epithelial-to-mesenchymal transition (EMT), which has been associated with tumor invasion and metastasis, is frequently observed in HNSCC. The transcription factors Snail and Zeb are representative EMT regulators. They have previously been implicated as inducers of EMT and potent repressors of E-cadherin expression during tumor progression. In addition, nuclear factor-kappa B (NF-κB) plays an essential role in both the induction and maintenance of EMT and tumor progression. The Semaphorins (SEMAs) were originally described as a large family of conserved axon guidance factors that are crucial for the formation of the nervous system. SEMAs were recently recognized to play a role in various developmental processes, particularly as regulators of cell migration, the immune response, angiogenesis and cancer progression. Class-3 Semaphorins, especially Semaphorin3A (SEMA3 A), is involved in the suppression of tumor progression in various types of cancers through binding to its receptor neuropilins (NRPs),acting as a tumor suppressor because of its distinct antiangiogenic effects. However, the expression pattern of SEMA3A and its associated molecular mechanisms in HNSCC have not been explored. In addition, the therapeutic potential of SEMA3A as a new option for HNSCC treatment remains to be clarifiedObjective1. To test the expression level of SEMA3A of HNSCC samples, analyse the relationship between the level of expression of SEMA3A and pathologic and clinical features of HNSCC.2. To investigate whether SEMA3A overexpression or silence would effect EMT and biological behaviors of HNSCC in vitro;3. To detect SEMA3A’s fundamental function in the tumor progression in HNSCC xenograft model;4. To explore the molecular mechanism of SEMA3A-induced anticancer effects in HNSCC cell lines and xenograft tumors.Methods1. Immunohistochemical staining was used to investigate the expression of SEMA3A in 100 HNSCC and 20 normal tissue samples. The correlation of the SEMA3A level with patients’clinicopathological parameters and prognosis was analyzed.2. A xenograft tumor model was established to further determine if the over-expression of SEMA3A has an effect on tumor growth in vivo. The adenovirus (Ad-SEMA3A or Ad-Con) packaged and purified from vector pCMV-SEMA3A-MCS-EGFP, encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP was injected several points within the tumor, twice a week for 7 weeks. The volume of tumors was measured and recorded.3. The SEMA3A over-expression adenovirus pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were transfected into HNSCC cells to obtain experiment group cells which express high level of SEMA3A and control group cells with low SEMA3 A expression. The variations in gene and protein expression of the epithelial and mesenchymal markers of HNSCC cell lines were evaluated by Real-time RT-PCR and western blotting. Wound healing, transwell invasion, colony-formation CCK-8 assays and TUNEL assays were performed to measure the biological properties of the SEMA3 A overexpression or silenced cells and xenograft tumor samples.4. Western blotting and RT-PCR were conducted to assess the alteration of the components of the NF-κB pathway and Zeb family, and Snail family, which plays an important role in EMT regulation. We then detected whether caspase family protein has an essential role in SEMA3A-induced apoptosis.Results1. SEMA3A expression is reduced in HNSCC specimens and is associated with poor post-operative overall survival.High expression of SEMA3A was observed in 18 of the 20 normal tissues, while among the 100 HNSCC cases, only 43 of the 100 HNSCC specimens exhibited high SEMA3A expression (P<0.001). A significant correlation was observed between SEMA3A expression levels and pathological stage (P=0.002), lymph node metastasis (P=0.017) and tumor T-stage (P=0.016). Consistent with the data for lymph node metastasis and tumor stage, patients with HNSCC whose tumors had a low level of SEMA3A staining had a poorer prognosis than those whose tumors had a high level of SEMA3A staining. Multivariate analysis showed that SEMA3A expression were independently associated with overall survival (P=0.025)2. SEMA3A over-expression inhibits tumor growth and induces apoptosis in vivo.In the Control group (n=5), all mice developed large tumors with an average size of 1100mm3 by week 13. By contrast, the mice in the Ad-SEMA3A group (n=5) developed much smaller tumors with an average size of 108mm3. Apoptosis quantification results revealed that xenograft tumors injected with Ad-SEMA3A adenovirus showed enhanced apoptosis compared with the tumors injected with Ad-Con adenovirus. Increased level of SEMA3A and over-expression of cleaved caspase-5 in the Ad-SEMA3 A group were also confirmed by immunohistochemistry.3. Endogenous SEMA3A inhibits HNSCC cell proliferation.Compared with cells transfected with control vector (Ad-Con-CAL27, Ad-Con-HN6), SEMA3A-transduced cells (Ad-SEMA3A-CAL27, Ad-SEMA3A-HN6) exhibited a lower colony-formation ability. Conversely, si-SEMA3A-SCC9 and si-SEMA3A-HN4 cells exhibited higher colony-formation ability. CCK-8 assays results showed that compared with control cells (CAL27, HN6), viability and proliferation remained unchanged in Ad-Con-cells (Ad-Con-CAL27, Ad-Con-HN6), whereas significantly lower proliferation ability was observed in Ad-SEMA3A-cells. changes in the expression of cell cycle-specific proteins were analyzed by Western blot. As expected, SEMA3A over-expression resulted in the down-regulation of CDKs (2,4,6) and cyclins (E1, D1, D3), whereas the expression of P27 and P21 was increased. Cell cycle changes were further verified by flow cytometry revealed that Ad-SEMA3A cells were mostly arrested in S-phase of the cell-cycle.4. SEMA3A over-expression induces apoptosis of HNSCC cells in a caspase-dependent manner.Flow cytometric analysis evolving apoptosis was performed and the results showed that the rate of apoptosis was significantly higher in Ad-SEMA3A-CAL27 and Ad-SEMA3A-HN6 cells than Ad-Con-CAL27 and Ad-Con-HN6 cells. Caspase (caspase-3,-5,-7) was also detected by Western blot analysis. As anticipated, caspase-5 and caspase-7 were substantially activated, while caspase-3 expression was nearly unchanged. CAL27 cells also adopted an apoptosis-like phenotype after 48 h of treatment with rhSEMA3A protein as the concentration of rhSEMA3A increased.5. EMA3A inhibits cancer cell migration, invasion and induces reversion of EMT on NF-κB, SNAI2 related pathways. Gene expression levels of the classic EMT-inducers Zeb (Zebl, Zeb2) and Snail (SNAI1, SNAI2) were detected by real-time PCR. A decrease in SNAI2 gene expression was observed in Ad-SEMA3A cells, while the expression of Zebl, Zeb2 and SNAI1 was not significantly different in Ad-SEMA3A cells. Decreased SNAI2 expression was observed by Western blot analysis, whereas SEMA3A depletion reverted its effects on SNAI2. Moreover, decreased nuclear translocation of the P65 subunit of NF-κB and reduced phosphorylation of the inhibitor of NF-κB (IκB) were detected in SEMA3A-over-expressing cells by western blot analysis; SEMA3 A-depleted cells exhibited the opposite pattern of nuclear translocation and phosphorylation. in xenograft tumor sections by IHC staining. As anticipated, decreased nuclear translocation of P65 and reduced p-IκB were detected in Ad-SEMA3 A xenograft tumors, compared with Ad-Con tumors.ConclusionsIn the present study, we provide evidence that SEMA3A is abnormallydown-regulated in a large fraction of HNSCCs. Its down-regulation is associated with several aggressive characteristics and serves as an independent prognostic factor for patients with HNSCC. Moreover, adenovirus transfection and recombinant SEMA3A protein approaches were used to identify the multiple tumor suppressor roles of SEMA3A and the potential underlying mechanisms in vitro. Furthermore, the therapeutic efficacy of SEMA3A-targeted delivery against HNSCC was confirmed in a cancer xenograft model.