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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

FAK/Src signaling pathway is involved in WISP-1-promoted VEGF-A expression and contributing to angiogenesis(A–B) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM) for 30 min or transfected with FAK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. (C–D) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM), followed by WISP-1 (20 ng/mL) stimulation for 24 h. CM was collected. EPCs were incubated with CM for 6 h and capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h and cell migration was examined by transwell assay (D) (E–H) SCC4 cells were treated with a Src inhibitor (pp2; 1 μM) for 30 min or transfected with Src siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. Assay procedure was performed as in (A–D) (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times, and FAK and c-Src phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with an integrin αvβ3 antibody or FAKi for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and FAK (J) and c-Src (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM *P < 0.05 compared with control; #P < 0.05 compared with the WISP-1-treated group.
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Figure 3: FAK/Src signaling pathway is involved in WISP-1-promoted VEGF-A expression and contributing to angiogenesis(A–B) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM) for 30 min or transfected with FAK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. (C–D) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM), followed by WISP-1 (20 ng/mL) stimulation for 24 h. CM was collected. EPCs were incubated with CM for 6 h and capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h and cell migration was examined by transwell assay (D) (E–H) SCC4 cells were treated with a Src inhibitor (pp2; 1 μM) for 30 min or transfected with Src siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. Assay procedure was performed as in (A–D) (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times, and FAK and c-Src phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with an integrin αvβ3 antibody or FAKi for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and FAK (J) and c-Src (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM *P < 0.05 compared with control; #P < 0.05 compared with the WISP-1-treated group.

Mentions: Our data indicate that WISP-1 elicits signal transduction through integrin αvβ3. We therefore analyzed signal pathways that are known to be regulated by integrin receptors in OSCC cells. FAK/c-Src dual kinase complex, the common signal modulator downstream of integrin, functions to promote cell motility, cell cycle, and cell survival. It has recently been implicated in tumor progression processes such as angiogenesis and metastasis [27]. Pretreatment with FAK inhibitor or FAK siRNA transfection reversed the WISP-1-induced VEGF-A expression in OSCC cells (Figure 3A and 3B). Furthermore, EPC migration and tube formation, which were induced by WISP-1-treated OSCC cells CM, were also abolished (Figure 3C and 3D). Similarly, pretreatment of OSCC cells with c-Src inhibitor or cSrc siRNA transfection confirmed the involvement of c-Src in WISP-1-induced VEGF-A expression and subsequent angiogenesis function in EPCs (Figure 3E–3H). Finally, pretreatment of OSCC cells with WISP-1 increased the phosphorylation of FAK and c-Src signaling proteins (Figure 3I). We also demonstrated the role of the integrin αvβ3/FAK/c-Src axis in OSCC cells using an integrin αvβ3 antibody and FAK inhibitor. Pretreatment with integrin αvβ3 antibody or FAKi block downstream signal proteins FAK or c-Src activation respectively (Figure 3J–3K). These results indicate that WISP-1 regulates VEGF-A expression in OSCC cells and contributing to angiogenesis through the FAK/c-Src signaling pathway.


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)

FAK/Src signaling pathway is involved in WISP-1-promoted VEGF-A expression and contributing to angiogenesis(A–B) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM) for 30 min or transfected with FAK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. (C–D) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM), followed by WISP-1 (20 ng/mL) stimulation for 24 h. CM was collected. EPCs were incubated with CM for 6 h and capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h and cell migration was examined by transwell assay (D) (E–H) SCC4 cells were treated with a Src inhibitor (pp2; 1 μM) for 30 min or transfected with Src siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. Assay procedure was performed as in (A–D) (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times, and FAK and c-Src phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with an integrin αvβ3 antibody or FAKi for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and FAK (J) and c-Src (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM *P < 0.05 compared with control; #P < 0.05 compared with the WISP-1-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414186&req=5

Figure 3: FAK/Src signaling pathway is involved in WISP-1-promoted VEGF-A expression and contributing to angiogenesis(A–B) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM) for 30 min or transfected with FAK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. (C–D) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM), followed by WISP-1 (20 ng/mL) stimulation for 24 h. CM was collected. EPCs were incubated with CM for 6 h and capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h and cell migration was examined by transwell assay (D) (E–H) SCC4 cells were treated with a Src inhibitor (pp2; 1 μM) for 30 min or transfected with Src siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. Assay procedure was performed as in (A–D) (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times, and FAK and c-Src phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with an integrin αvβ3 antibody or FAKi for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and FAK (J) and c-Src (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM *P < 0.05 compared with control; #P < 0.05 compared with the WISP-1-treated group.
Mentions: Our data indicate that WISP-1 elicits signal transduction through integrin αvβ3. We therefore analyzed signal pathways that are known to be regulated by integrin receptors in OSCC cells. FAK/c-Src dual kinase complex, the common signal modulator downstream of integrin, functions to promote cell motility, cell cycle, and cell survival. It has recently been implicated in tumor progression processes such as angiogenesis and metastasis [27]. Pretreatment with FAK inhibitor or FAK siRNA transfection reversed the WISP-1-induced VEGF-A expression in OSCC cells (Figure 3A and 3B). Furthermore, EPC migration and tube formation, which were induced by WISP-1-treated OSCC cells CM, were also abolished (Figure 3C and 3D). Similarly, pretreatment of OSCC cells with c-Src inhibitor or cSrc siRNA transfection confirmed the involvement of c-Src in WISP-1-induced VEGF-A expression and subsequent angiogenesis function in EPCs (Figure 3E–3H). Finally, pretreatment of OSCC cells with WISP-1 increased the phosphorylation of FAK and c-Src signaling proteins (Figure 3I). We also demonstrated the role of the integrin αvβ3/FAK/c-Src axis in OSCC cells using an integrin αvβ3 antibody and FAK inhibitor. Pretreatment with integrin αvβ3 antibody or FAKi block downstream signal proteins FAK or c-Src activation respectively (Figure 3J–3K). These results indicate that WISP-1 regulates VEGF-A expression in OSCC cells and contributing to angiogenesis through the FAK/c-Src signaling pathway.

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