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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.

Saito T, Kasamatsu A, Ogawara K, Miyamoto I, Saito K, Iyoda M, Suzuki T, Endo-Sakamoto Y, Shiiba M, Tanzawa H, Uzawa K - PLoS ONE (2015)

Bottom Line: 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 (p21Cip1 and p27Kip1) 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

ABSTRACT

Background: 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).

Methods: 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.

Results: 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 (p21Cip1 and p27Kip1) 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).

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Cell-cycle analysis of SEMA7A knockdown cells.(A) 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) Immunobloting analysis shows up-regulation of p21Cip1 and p27Kip1 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.
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pone.0137923.g004: Cell-cycle analysis of SEMA7A knockdown cells.(A) 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) Immunobloting analysis shows up-regulation of p21Cip1 and p27Kip1 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.

Mentions: Since the cellular growth of the SEMA7A knockdown cells decreased (Fig 3A), we investigated the cell-cycle distributions using flow cytometry. The percentage of the cells in the G1 phase in shSEMA7A cells was significantly (P<0.05) higher than that in the shMock cells (Fig 4A).We also assessed the expression levels of the G1 arrest-related proteins, such as CDKIs (p21Cip1 and p27Kip1), cyclins, and CDKs. As expected, the CDKIs were up-regulated, and cyclin D1, cyclin E, CDK2, CDK4, and CDK6 were down-regulated significantly (P<0.05) in the shSEMA7A cells (Fig 4B). These results indicated that shSEMA7A cells inhibited cellular proliferation by cell-cycle arrest at the G1 phase.


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.

Saito T, Kasamatsu A, Ogawara K, Miyamoto I, Saito K, Iyoda M, Suzuki T, Endo-Sakamoto Y, Shiiba M, Tanzawa H, Uzawa K - PLoS ONE (2015)

Cell-cycle analysis of SEMA7A knockdown cells.(A) 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) Immunobloting analysis shows up-regulation of p21Cip1 and p27Kip1 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4574527&req=5

pone.0137923.g004: Cell-cycle analysis of SEMA7A knockdown cells.(A) 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) Immunobloting analysis shows up-regulation of p21Cip1 and p27Kip1 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.
Mentions: Since the cellular growth of the SEMA7A knockdown cells decreased (Fig 3A), we investigated the cell-cycle distributions using flow cytometry. The percentage of the cells in the G1 phase in shSEMA7A cells was significantly (P<0.05) higher than that in the shMock cells (Fig 4A).We also assessed the expression levels of the G1 arrest-related proteins, such as CDKIs (p21Cip1 and p27Kip1), cyclins, and CDKs. As expected, the CDKIs were up-regulated, and cyclin D1, cyclin E, CDK2, CDK4, and CDK6 were down-regulated significantly (P<0.05) in the shSEMA7A cells (Fig 4B). These results indicated that shSEMA7A cells inhibited cellular proliferation by cell-cycle arrest at the G1 phase.

Bottom Line: 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 (p21Cip1 and p27Kip1) 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.

ABSTRACT

Background: 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).

Methods: 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.

Results: 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 (p21Cip1 and p27Kip1) 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).

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus