Semaphorin7A Promotion of Tumoral Growth and Metastasis in Human Oral Cancer by Regulation of G1 Cell Cycle and Matrix Metalloproteases: Possible Contribution to Tumoral Angiogenesis

<div><p>Background</p><p>Semaphorins (SEMAs) consist of a large family of secreted and membrane-anchored proteins that are important in neuronal pathfinding and axon guidance in selected areas of the developing nervous system. Of them, SEMA7A has been reported to have a chemotactic activity in neurogenesis and to be an immunomodulator; however, little is known about the relevance of SEMA7A in the behaviors of oral squamous cell carcinoma (OSCC).</p><p>Methods</p><p>We evaluated SEMA7A expression in OSCC-derived cell lines and primary OSCC samples using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and semiquantitative immunohistochemistry (sq-IHC). In addition, SEMA7A knockdown cells (shSEMA7A cells) were used for functional experiments, including cellular proliferation, invasiveness, and migration assays. We also analyzed the clinical correlation between SEMA7A status and clinical behaviors in patients with OSCC.</p><p>Results</p><p>SEMA7A mRNA and protein were up-regulated significantly (P<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. The shSEMA7A cells showed decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors (p21^Cip1 and p27^Kip1) and down-regulation of cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6); and decreased invasiveness and migration activities by reduced secretion of matrix metalloproteases (MMPs) (MMP-2, proMMP-2, pro-MMP-9), and expression of membrane type 1- MMP (MT1-MMP). We also found inactivation of the extracellular regulated kinase 1/2 and AKT pathways, an upstream molecule of cell-cycle arrest at the G1 phase, and reduced secretion of MMPs in shSEMA7A cells. sq-IHC showed that SEMA7A expression in the primary OSCCs was significantly (P = 0.001) greater than that in normal counterparts and was correlated with primary tumoral size (P = 0.0254) and regional lymph node metastasis (P = 0.0002).</p><p>Conclusion</p><p>Our data provide evidence for an essential role of SEMA7A in tumoral growth and metastasis in OSCC and indicated that SEMA7A may play a potential diagnostic/therapeutic target for use in patients with OSCC.</p></div

Inactivation of the ERK1/2 and AKT pathways in SEMA7A knockdown cells.

<p>(<b>A, B</b>) Immunoblotting analysis shows that SEMA7A knockdown results in decreased levels of pERK1/2 and pAKT compared with the shMock cells (SAS and KOSC-2-derived transfectants). Densitometric pERK1/2, ERK1/2, pAKT, and AKT protein data are normalized to GAPDH protein levels.</p

Evaluation of SEMA7A expression in OSCC-derived cell lines.

<p>(<b>A</b>) Quantification of <i>SEMA7A</i> mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Significant (^✽P<0.05, Student’s t-test) up-regulation of <i>SEMA7A</i> mRNA is seen in seven OSCC-derived cell lines compared with the HNOKs. Data are expressed as the mean ± SEM of triplicate results. (<b>B</b>) Immunoblotting analysis of SEMA7A protein in OSCC-derived cell lines and HNOKs. SEMA7A protein expression is up-regulated in OSCC-derived cell lines compared with that in the HNOKs. Densitometric SEMA7A protein data are normalized to the GAPDH protein levels. The values are expressed as a percentage of the HNOKs.</p

Cell-cycle analysis of SEMA7A knockdown cells.

<p>(<b>A</b>) Flow cytometric analysis was performed to investigate cell-cycle progression in the shSEMA7A and shMock cells (SAS and KOSC-2-derived transfectants) after synchronization at the G2/M phase to using nocodazole. The percentage of cells at the G1 phase in the shSEMA7A cells is increased markedly compared with the shMock cells. (<b>B</b>) Immunobloting analysis shows up-regulation of p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 in the shSEMA7Acells (SAS and KOSC-2-derived transfectants) compared with the shMock cells.</p

Establishment of SEMA7A knockdown cells.

<p>(<b>A</b>) Expression of <i>SEMA7A</i> mRNA in shMock and shSEMA7A cells (SAS and KOSC-2-derived transfectants). <i>SEMA7A</i> mRNA expression in shSEMA7A cells is significantly (^✽P<0.05, Student’s t-test) lower than in the shMock cells. (<b>B</b>) Immunoblotting analysis shows that the SEMA7A protein levels in the shSEMA7A cells also are decreased markedly compared with the shMock cells.</p

Functional analyses of SEMA7A knockdown cells.

<p>(<b>A</b>) Cellular proliferation assay of shMock and shSEMA7A cells (SAS and KOSC-2-derived transfectants). To determine the effect of shSEMA7A on cellular proliferation, shMock and shSEMA7A cells were seeded in 6-cm dishes at a density of 1×10⁴ viable cells/well. The cellular growth of shSEMA7A cells is inhibited significantly compared with the shMock cells after 4 days (96 hours). The results are expressed as the means ± SEM of values from three assays (^✽P<0.05, Student’s t-test). (<b>B</b>) Invasiveness assay of shMock and shSEMA7A cells (SAS and KOSC-2-derived transfectants). To evaluate the effect of SEMA7A knockdown on invasiveness, we seeded 2.5×10⁵ cells in the serum-free medium of Matrigel^®-coated Transwell^® inserts (8 μm pores) and added serum-supplemented medium in the lower chamber as a chemoattractant. After incubation at 37°C for 48 hours, cells that penetrated through the pores were fixed, stained, and counted using a light microscope at ×100 magnification. The number of shSEMA7A cells penetrating through the pores is decreased significantly (^✽P<0.05, Student’s t-test) compared with the shMock cells. The mean value was calculated from data obtained from three separate chambers. (<b>C</b>) Migration assay of shMock and shSEMA7A cells (SAS and KOSC-2-derived transfectants). To evaluate the effect of SEMA7A knockdown on migration, uniform wounds were made in confluent cultures of the shSEMA7A and shMock cells and the extent of closure was monitored visually every 6 hours for 12 hours. The mean value was calculated from data obtained from three separate chambers. The wound area is decreased significantly (^✽P<0.05, Student’s t-test) in the culture of shMock cells after 12 hours, whereas a gap remained in the shSEMA7A cells.</p

Evaluation of SEMA7A expression in primary OSCCs.

<p>(<b>A, B</b>) Representative sq-IHC results of SEMA7A in primary OSCCs and normal oral tissues. Original magnification, ×200. Scale bars, 50 μm. (<b>C</b>) The status of SEMA7A protein expression in primary OSCCs (n = 150) and normal counterparts based on the sq-IHC scoring system. The SEMA7A sq-IHC scores for OSCCs and normal oral tissues range from 36.7 to 203.8 (median, 109.3) and 13.5 to 87.5 (median, 37.9), respectively. SEMA7A protein expression levels in OSCCs are significantly higher than in normal oral tissues (*P<0.05, Student’s t-test). (<b>D</b>) ROC curve analysis revealed that the optimal cutoff point is 69.5 (AUC, 0.91; 95% CI, 0.8749–0.9378; P<0.05). (<b>E</b>) Youden index analysis shows that the optimal cutoff point is 69.5 (sensitivity, 70.5%; specificity, 96.7%, P<0.05).</p

SEMA7A expression in SEMA7A knockdown cells after TGF-β₁ treatment.

<p>The mRNA levels of <i>SEMA7A</i> in TGF-β₁ incubated shSEMA7A cells (SAS and KOSC-2) are increased significantly (SAS, *P = 0.0022, **P = 0.0021, †P = 0.0001, ††P = 0.0005, Student’s t-test) (KOSC-2, *P = 0.0003, **P = 0.0003, †P = 0.0029, ††P = 0.0045, Student’s t-test) compared with that in TGF-β₁ unincubated shSEMA7A cells.</p

Phosphorylation levels of ERK1/2 and AKT in SEMA7A knockdown cells with/without TGF-β₁.

<p>Immunoblot analysis of the phosphorylation levels of SEMA7A, ERK1/2, and AKT. SEMA7A knockdown results in decreased levels of pERK1/2 and pAKT compared with the shMock cells (SAS and KOSC-2-derived transfectants). TGF-β₁ treated shSEMA7A cells show increased SEMA7A level compared with TGF-β₁ non-treated shSEMA7A cells. The pERK1/2 and pAKT level also show increased in TGF-β₁ treated shSEMA7A cells.</p

Reduced expression of MT1-MMP in SEMA7A knockdown cells.

<p>(<b>A</b>) Expression of <i>MT1-MMP</i> mRNA in shMock and shSEMA7A cells (SAS and KOSC-2-derived transfectants). <i>MT1-MMP</i> mRNA expression in shSEMA7A cells are significantly (^✽P<0.05, Student’s t-test) lower than in shMock cells. (<b>B</b>) Immunoblotting analysis shows that the MT1-MMP protein levels in the shSEMA7A cells are also decreased markedly compared with the shMock cells. CHX treated shMock cells also inihibited the expression of MT1-MMP expression. Densitometric MT1-MMP protein data are normalized to the GAPDH protein levels.</p