Regulation of invasive behavior by vascular endothelial growth factor is HEF1-dependent

Abstract

We previously reported a vascular endothelial growth factor (VEGF) autocrine loop in head and neck squamous cell carcinoma (HNSCC) cell lines, supporting a role for VEGF in HNSCC tumorigenesis. Using a phosphotyrosine proteomics approach, we screened the HNSCC cell line, squamous cell carcinoma-9 for effectors of VEGFR2 signaling. A cluster of proteins involved in cell migration and invasion, including the p130Cas paralog, human enhancer of filamentation 1 (HEF1/Cas-L/Nedd9) was identified. HEF1 silencing and overexpression studies revealed a role for VEGF in regulating cell migration, invasion and matrix metalloproteinase (MMP) expression in a HEF1-dependent manner. Moreover, cells plated on extracellular matrix-coated coverslips showed enhanced invadopodia formation in response to VEGF that was HEF1-dependent. Immunolocalization revealed that HEF1 colocalized to invadopodia with MT1-MMP. Analysis of HNSCC tissue microarrays for HEF1 immunoreactivity revealed a 6.5-fold increase in the odds of having a metastasis with a high HEF1 score compared with a low HEF1 score. These findings suggest that HEF1 may be prognostic for advanced stage HNSCC. They also show for the first time that HEF1 is required for VEGF-mediated HNSCC cell migration and invasion, consistent with HEF1's recent identification as a metastatic regulator. These results support a strategy targeting VEGF:VEGFR2 in HNSCC therapeutics.

Figure 1. Screen for VEGFR2 effectors using phosphotyrosine proteomics

(A) The Venn diagram illustrates the number of proteins identified within treatment group analyzed. (B) SCC-9 cells were treated with 10 ng/ml VEGF for 10 min as indicated. Cell lysates were enriched for pY-containing proteins, subjected to proteolytic digestion and subsequent analysis by LC-MS/MS. Representative spectra were taken from the VEGF treated sample of those proteins involved in cancer cell tumorigenesis. Additional tyrosine phosphorylated proteins detected but not further characterized include valosin-containing protein, α-tubulin, HNRPF, medium-chain acyl-CoA dehydrogenase and uraDNA glycosylase (see Table 1D, SI).

Figure 2. VEGF induces tyrosine phosphorylation of proteins identified in the phosphotyrosine proteomics screen

(A) SCC-9 cells were serum starved overnight followed by treatment with VEGF (10 ng/ml) for the times shown. Cell lysates were immunoprecipitated (IP) with antibodies to the individual proteins and immunoblotted (IB) with anti-phosphotyrosine (anti-pY) as indicated. (B) HEF1 tyrosine phosphorylation in SCC-9 cells in response to VEGF (10 ng/ml; 15 min) was abrogated by the Src family kinase inhibitor PP2 (1 μM). (C) Effect of FAK inhibition on HEF1 tyrosine phosphorylation. HEF1 tyrosine phosphorylation was unaffected by the FAK-specific tyrosine kinase inhibitor PF-573,228. These experiments were performed at least three times with the same results.

Figure 3. HEF1 expression augments VEGF stimulated cell migration

(A) Immunoblot analysis of HEF1 expression in HNSCC cell lines. (UM-SCC lines were obtained from Dr. Tom Carey, University of Michigan. (B) HEF1 overexpression (pEGFP-HEF1) and silencing with siRNA (siHEF1) reciprocally alters E-cadherin expression. (C) Scratch/wound assays demonstrate that VEGF stimulated cell migration is HEF1-dependent. Scr Scrambled siRNA, pHEF1 HEF1 transfected only, V+HEF1 10ng/ml VEGF treatment and HEF1 transfection, V+siHEF1 10 ng/ml VEGF treated and HEF1 siRNA transfected.

Figure 4. VEGF enhances cell invasion and MMP expression via HEF1

(A) SCC-9 cells seeded on matrigel-coated transwell filters were treated as indicated for 24 h. Filters were collected and cells quantified as detailed in Methods. VEGF stimulated invasion was increased by HEF1 transfection and attenuated by HEF1 silencing, * p<0.05; ** p<0.005. (B) MMP-2, -9, and MT1-MMP expression were increased by HEF1 transfection and VEGF addition and blocked with HEF1 siRNA. (C) HEF1 significantly increased VEGF-induced MT1-MMP expression. (D) VEGF stimulated and bevacizumab blocked MMP-9 and MMP-2 expression. All experiments were repeated a minimum of three times.

Figure 5. VEGF stimulates invadopodia formation

(A) SCC-9 cells seeded on FITC-gelatin-coated coverslips were treated and stained as indicated. The punctate staining pattern observed in response to VEGF stimulation was lost upon co-treatment with the MMP inhibitor, GM6001. (B) The pY staining of VEGF stimulated cells exhibited a central, punctate distribution, similar to that seen for actin and HEF1. (C) xyz and xzy projection of SCC-9 cells following VEGF stimulation and HEF1 overexpression. Note: HEF1 co-localization with MT1-MMP at invadopodia tips in this xzy visualization of ventral invadopodia projecting into the matrix. (D) SCC-9 cells seeded on FITC-labeled- FN coverslips were treated for 12 h as indicated. Invadopodia formation (inset, top) were identified and quantified.

Figure 6. HEF1 staining in human tissue

(A) (Upper)Tissue sections were stained with H&E, secondary antibody alone (Negative Control), and primary HEF1 antibody with secondary antibody. (B) Representative staining from each level of positivity observed from the TMA.