ABSTRACT
Vascular endothelial growth factor A (VEGF) is one of the major regulators of angiogenesis. It plays an important role during the process of physiological and pathological neovascularization. Variant VEGF isoforms are generated as a result of alternative pre-mRNA splicing. To determine the expression of VEGF isoforms in angioma and head and neck squamous cell carcinoma (HNSCC), both being dependent on pathological neovascularization, we included 11 HNSCC cell lines, 4 hemangiomas and 5 vascular malformations (VMs) in the study. Tonsil mucosa served as normal control. Using reverse transcription polymerase chain reaction (RT-PCR) sequencing, the VEGF isoforms VEGF(189), VEGF(165) and VEGF(121) were regularly detected in all tested samples. VEGF(121) was the most abundant isoform in all tested tissues, whereas VEGF(165) exhibited lower levels, and VEGF(189) only very small amounts of transcript. Interestingly, VMs expressed significantly higher (p=0.0286) amounts of VEGF(121) compared with hemangiomas, which had levels similar to normal control mucosa. HNSCC cell lines demonstrated on-average higher levels of all three isoforms compared with the controls. Consistent with the clinical staging, a trend for VEGF overexpression was observed in tumor cells derived from N+ tumors compared to those derived from N0 tumors. One drawback of this study was the small number of specimens available, particularly since VMs and hemangiomas are relatively rare diseases. Future studies need to follow-up on these observations and further evaluate the potential role of specific VEGF isoforms in the pathogenesis of hemangioma and VM.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Head and Neck Neoplasms/metabolism , Hemangioma/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Head and Neck Neoplasms/blood supply , Head and Neck Neoplasms/genetics , Hemangioma/blood supply , Hemangioma/genetics , Humans , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Protein Isoforms , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Head and neck squamous cell carcinomas (HNSCCs) are the most frequent malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis concludes that CSCs constitute the dangerous tumor cell population due to their ability of self-renewal and being associated with relapse of tumor disease, invasiveness and resistance to chemo(radio)therapy. The aim of this study was to look for CSC candidates and expression of MMP-9 that previously was implicated in HNSCC invasiveness. Immunohistochemical, immunofluorescence and Western blot analysis were performed on HNSCC tumor specimens using antibodies specific for MMP-9, CD44, ALDH1 and CK14. Gelatinolytic activity was assessed by zymography. Pearson correlation analysis was used for statistical comparison. Immunohistochemical analysis found CD44 and MMP-9 to co-localize in tumor cells at the invasive front. Western blot analysis demonstrated a significant correlation (p=0.0047) between CD44 and MMP-9 in the tested tissues. In addition gelatinolytic activity of HNSCC tissues was found to significantly correlate (p=0.0010) with MMP-9 expression. The CD44(+) invasive front of the tumor was also positive for ALDH1 and CK14, all of them being typically expressed by cells in the basal cell layer of normal stratified squamous epithelia that also harbors the epithelial stem cells. The observations point to a role of a MMP-9 positive basal-cell-like cell layer in the process of HNSCC invasiveness. This compartment likely contains CSCs since it is expressing the putative CSC markers CD44, ALDH1 and CK14. This cell layer therefore should be considered a major therapeutic target in the treatment of head and neck cancer.
