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WISP-1 a novel angiogenic regulator of the CCN family promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression.

Chuang JY, Chen PC, Tsao CW, Chang AC, Lein MY, Lin CC, Wang SW, Lin CW, Tang CH - Oncotarget (2015)

Bottom Line: In vitro results indicated that WISP-1 induced VEGF-A expression via the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signaling pathway in OSCC.Our in vivo data revealed that tumor-secreted WISP-1 promoted the angiogenesis through VRGF expression and increased angiogenesis-related tumor growth.Our study offers new information that highlights WISP-1 as a potential novel therapeutic target for OSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan.

ABSTRACT
Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by poor prognosis and a low survival rate. VEGF-A is the most established angiogenic factor involved in the angiogenic-regulated tumor progression. WISP-1/CCN4 is an extracellular matrix-related protein that belongs to the Cyr61, CTGF, Nov (CCN) family and regulates many biological functions, such as angiogenesis. Previous studies indicated the role of WISP-1 in tumor progression. However, the angiogenic property of WISP-1 in the cancer microenvironment has never been discussed. Here, we provide novel insights regarding the role of WISP-1 in the angiogenesis through promoting VEGF-A expression. In this study, the correlation of WISP-1 and VEGF-A was confirmed by IHC staining of specimens from patients with OSCC. In vitro results indicated that WISP-1 induced VEGF-A expression via the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signaling pathway in OSCC. This pathway in turn induces the recruitment of endothelial progenitor cells and triggers the neovascularization in the tumor microenvironment. Our in vivo data revealed that tumor-secreted WISP-1 promoted the angiogenesis through VRGF expression and increased angiogenesis-related tumor growth. Our study offers new information that highlights WISP-1 as a potential novel therapeutic target for OSCC.

No MeSH data available.


Related in: MedlinePlus

WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo(A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (103) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM *P < 0.05 compared to control shRNA/SCC4.
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Figure 6: WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo(A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (103) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM *P < 0.05 compared to control shRNA/SCC4.

Mentions: Our results indicated that WISP-1 promoted VEGF-A expression and enhanced angiogenesis by recruiting EPCs and increasing tube formation in the microenvironment. Thus, we investigated the role of WISP-1 in vivo. To confirm its regulatory role in VEGF-A expression, we utilized OSCC cells stably expressing a WISP-1 shRNA. Knockdown of WISP-1 expression in OSCC cells didn’t affect cell proliferation (Figure 6A). Results showed that WISP-1 and VEGF-A expression levels were decreased in WISP-1 shRNA transfected OSCC cells (Figure 6B and 6C). CM collected from OSCC cells stably expressing a control-shRNA promoted EPC cell migration and tube formation, while CM collected from OSCC cells stably expressing WISP-1 shRNA decreased EPC cell migration and tube formation (Figure 6D and 6E). Finally, the role of WISP-1, in vivo, was examined by chick embryo chorioallantoic membrane (CAM) assay. As expected, CM collected from OSCC cells stably expressing a control shRNA enhanced CAM angiogenesis, while CM from WISP-1 shRNA cells completely reduced angiogenesis in CAMs (Figure 6F). The results of the in vivo Matrigel plug formation assay by subcutaneous implantation in mice showed that Matrigel mixed with CM from control-shRNA transfected SCC4 cells increased blood vessel growth, while CM from WISP-1 shRNA transfected SCC4 cells reduced neovascularization (Figure 6G, upper panel). CD31 IHC and hemoglobin content assay indicated a decline in vascular formation in Matrigel (Figure 6G, lower panel). These results indicate that WISP-1 promotes the angiogenesis in vivo.


WISP-1 a novel angiogenic regulator of the CCN family promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression.

Chuang JY, Chen PC, Tsao CW, Chang AC, Lein MY, Lin CC, Wang SW, Lin CW, Tang CH - Oncotarget (2015)

WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo(A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (103) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM *P < 0.05 compared to control shRNA/SCC4.
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Figure 6: WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo(A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (103) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM *P < 0.05 compared to control shRNA/SCC4.
Mentions: Our results indicated that WISP-1 promoted VEGF-A expression and enhanced angiogenesis by recruiting EPCs and increasing tube formation in the microenvironment. Thus, we investigated the role of WISP-1 in vivo. To confirm its regulatory role in VEGF-A expression, we utilized OSCC cells stably expressing a WISP-1 shRNA. Knockdown of WISP-1 expression in OSCC cells didn’t affect cell proliferation (Figure 6A). Results showed that WISP-1 and VEGF-A expression levels were decreased in WISP-1 shRNA transfected OSCC cells (Figure 6B and 6C). CM collected from OSCC cells stably expressing a control-shRNA promoted EPC cell migration and tube formation, while CM collected from OSCC cells stably expressing WISP-1 shRNA decreased EPC cell migration and tube formation (Figure 6D and 6E). Finally, the role of WISP-1, in vivo, was examined by chick embryo chorioallantoic membrane (CAM) assay. As expected, CM collected from OSCC cells stably expressing a control shRNA enhanced CAM angiogenesis, while CM from WISP-1 shRNA cells completely reduced angiogenesis in CAMs (Figure 6F). The results of the in vivo Matrigel plug formation assay by subcutaneous implantation in mice showed that Matrigel mixed with CM from control-shRNA transfected SCC4 cells increased blood vessel growth, while CM from WISP-1 shRNA transfected SCC4 cells reduced neovascularization (Figure 6G, upper panel). CD31 IHC and hemoglobin content assay indicated a decline in vascular formation in Matrigel (Figure 6G, lower panel). These results indicate that WISP-1 promotes the angiogenesis in vivo.

Bottom Line: In vitro results indicated that WISP-1 induced VEGF-A expression via the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signaling pathway in OSCC.Our in vivo data revealed that tumor-secreted WISP-1 promoted the angiogenesis through VRGF expression and increased angiogenesis-related tumor growth.Our study offers new information that highlights WISP-1 as a potential novel therapeutic target for OSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan.

ABSTRACT
Oral squamous cell carcinoma (OSCC), which accounts for nearly 90% of head and neck cancers, is characterized by poor prognosis and a low survival rate. VEGF-A is the most established angiogenic factor involved in the angiogenic-regulated tumor progression. WISP-1/CCN4 is an extracellular matrix-related protein that belongs to the Cyr61, CTGF, Nov (CCN) family and regulates many biological functions, such as angiogenesis. Previous studies indicated the role of WISP-1 in tumor progression. However, the angiogenic property of WISP-1 in the cancer microenvironment has never been discussed. Here, we provide novel insights regarding the role of WISP-1 in the angiogenesis through promoting VEGF-A expression. In this study, the correlation of WISP-1 and VEGF-A was confirmed by IHC staining of specimens from patients with OSCC. In vitro results indicated that WISP-1 induced VEGF-A expression via the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signaling pathway in OSCC. This pathway in turn induces the recruitment of endothelial progenitor cells and triggers the neovascularization in the tumor microenvironment. Our in vivo data revealed that tumor-secreted WISP-1 promoted the angiogenesis through VRGF expression and increased angiogenesis-related tumor growth. Our study offers new information that highlights WISP-1 as a potential novel therapeutic target for OSCC.

No MeSH data available.


Related in: MedlinePlus