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THE ONCOGENIC ROLE OF TISSUE INHIBITOR METALLOPROTEINASE-1, TIMP-1, THROUGH REGULATION OF YAP IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

Background: Head and neck squamous cell carcinoma (HNSCC) affects more 
than 600,000 patients per year in the world. Despite the treatment, about half of 
all patients will die of this disease. More advanced diagnosis and treatment are 
required. Tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the 
degradation of extracellular matrix regulated by matrix metalloproteinases 
(MMP). Although this function is unfavorable for cancer cells to invade or 
migrate using MMP, TIMP-1 is controversially overexpressed in various types of 
cancer including HNSCC. Therefore, an oncogenic aspect of TIMP-1 has been 
featured, which can resolve the discrepancy. Actually, TIMP-1 binds to its 
receptor CD63 and activate integrinβ1, leading to aberrant cell proliferation. 
However, a true role and its mechanism of TIMP-1 in HNSCC is still unclear. 
Yes-Associated Protein (YAP), a transcription co-activator, enhances 
transcription of specific genes related to cell proliferation, such as CTGF or 
Cyr61. YAP is inactivated due to its phosphorylation inducing localization in 
cytoplasm and degradation, which is regulated by Hippo-signaling pathway, 
composed of MST1/2 and LATS1/2, or Hippo-independent signaling pathway 
like actin dynamics by GPCR or integrinβ1. YAP is dysregulated and contributes 
to aberrant cell proliferation in a variety of cancer including HNSCC. When I 
searched TIMP-1 expression by Oncomine data base analysis, I found that TIMP- 
1 was highly overexpressed in association with YAP-associated genes including 
CTGF and Cyr61 in HNSCC. Then, I hypothesized TIMP-1 might regulate YAP 
in HNSCC. 
Purpose: To reveal an oncogenic role of TIMP-1 through regulation of novel 
downstream YAP in HNSCC. 
Method: HNSCC cell lines (HSC-2, 3, 4 and KOSCC33A) and human embryonic 
kidneycells (HEK293) were used. Expressing or shRNA plasmids were 
transfected and stable clones were analyzed by western blotting, RT-PCR, Real 
time-PCR and proliferation assay. Data were evaluated by student’s t-test with 
significant level set at P＜0.05. 
Results: TIMP-1, YAP and CTGF expression were positively correlated among 
HNSCC cell lines. Stable overexpression or recombinant TIMP-1 induced YAP 
activation (dephosphorylation), CTGF expression and accelerated cell 
proliferation. In contrast, stable TIMP-1 knockdown inactivated (phosphorylated) 
YAP and inhibited proliferation. Furthermore, stable CD63 knockdown 
inactivated YAP and disturbed activation triggered by TIMP-1. 
Conclusion: I revealed TIMP-1 with its receptor CD63 play an oncogenic role in 
HNSCC through regulation of YAP. This finding suggests that targeting of TIMP- 
1 and/or its new downstream YAP will be beneficial for the improvement of 
diagnosis and treatment for the patients with HNSCC.