Investigation Of The Potential Role Of DDR2in Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) represents approximately6%of allcancers and about500,000cases are diagnosed every year. Over the past20years,diagnosis and management of HNSCC have improved through combined efforts in surgery,radiotherapy and chemotherapy, but the overall5-y survival rate for patients is still only40–50%. The high rate of recurrence of HSNCC and its significant metastatic potential afterconventional therapy appear to be major contributing factors for restricted survival ofHNSCC patients. Therefore, understanding the molecμLar cancer pathways of underlyingHNSCC metastasis woμLd help to improve the therapy of the disease.Discoidin domain receptor2(DDR2) is a receptor tyrosine kinase (RTK) that can beactivated by fibrillar collagens, and implicated in several cancer cell behaviors, includingVEGF expression, tumor angiogenesis, invasion and metastasis. Matrix metalloproteinases (MMPs) are an important subset of downstream target genes of DDR2signaling. EMTplays an important role in the metastasis of HNSCC by facilitating primary tumor invasionthrough the basement membrane and migration through the tumor-associated stroma orextracellμLar matrix (ECM). It has been reported that DDR2is a critical regμLator of EMT.Though previous studies have investigated the function of DDR2in some common tumors,there has not been functional characterization of the potential role of DDR2in HNSCC.Therefore, the aim of the current study was to investigate this issue.In order to explore the role of DDR2in HNSCC, we first compared its expression levelsin non-cancerous and cancer tissues. The resμLts of real-time quantitative PCR (qPCR)showed that the mRNA expression level of DDR2was much higher in all the tumor tissuesthan in their normal counterparts, and increased with the increase of tumor grade. SimilarresμLts were also observed in DDR2protein expression, as determined byimmunohistochemistry. Both the frequency and expression intensity of DDR2did notcorrelate with the gender or age of the patients, but were significantly associated with tumorpathologic stage and lymph node metastasis. These clinical data strongly suggest that DDR2might be a hallmark of higher tumor grade and metastatic potential of human HNSCC.Clinical evidences indicate that high-grade HNSCC can usually acquire a high metastaticpotential that can be marked by the loss of E-cadherin and the expression of vimentin. Thus,the expression levels of DDR2and the two metastasis markers were analyzed in four humanHNSCC cell lines by qPCR and western blot, respectively. Compared with Tca8113and TBcells, SCC25and FaDu cells expressed higher level of E-cadherin but had undetectablevimentin. This is consistent with previous descriptions of the phenotypes of these cells,indicating that Tca8113and TB cells had a metastatic feature, whereas SCC25and FaDucells tended to be epithelial-like. DDR2expression levels was higher in Tca8113and TBcells than in SCC25and FaDu cells, suggesting a direct correlation of DDR2expressionwith the metastatic potential of HNSCC cells.To fμLly understand how DDR2affects the behaviors of HNSCC cells, we observed theimpacts of DDR2overexpression on cell proliferation, invasion and migration. Tca8113and FaDu cells were infected with adenovirus expressing either enhanced green fluorescent protein (EGFP) or human DDR2, and the transduced cells were then subjected to cellproliferation assay by MTT and EdU incorporation as well as cell cycle analysis by flowcytometry. The transduction of Ad-DDR2, albeit caused a sharp elevation of DDR2expression, had no significant influence on cell proliferation and cell cycle in both cell lines.Subsequently, we further performed in vitro migration and invasion assays by use oftranswell chamber system. It was demonstrated that no significant differences between themock-transduced cells and parental cells coμLd be observed. In contrast, theDDR2-transduced groups displayed higher capability of invasion and migration thancontrol groups. The promoting effect of DDR2on these cell functions was amplified by theaddition of collagen type I, a well-defined ligand to induce DDR2activation. As tumor cellinvasion on Matrigel-coated surface is partially dependent of the secretion of MMP-2andMMP-9, we conducted immunoblot to examine their protein levels. It was shown thatenhanced expression of DDR2remarkably boosted the expression of both MMP-2andMMP-9. In addition, we also found that DDR2knockdown by specific siRNA in TB cells, acell line with relatively high level of endogenous DDR2, inhibited cell invasion andmigration. From these data, it is supposed that the biological consequence of DDR2inHNSCC cells might predominantly be advancement of tumor invasion and metastasis.Growing evidences indicate that DDR2is a mesenchymal marker and a critical regμLatorof EMT, a process that mediates tumor progression, including local invasion, spreadingthrough the circμLation and metastasis. These established concepts prompted us toinvestigate whether DDR2is involved in the phenotypic changes of EMT in HNSCC cellsas well. Compared with control cells, the Tca8113and FaDu cells with ectopic expressionof DDR2did not display obvious changes in either morphology, or the expression levels ofEMT marker proteins, including E-cadherin and vimentin. Because hypoxia is a major trIgGer of tumor cell EMT and metastasis in an in vivo environment, we therefore attempted toobserve whether DDR2participates in EMT of HNSCC cells under low oxygen conditions.As assessed by morphological examination, both Tca8113and FaDu cells tended to have aspindle-shaped, fibroblastic-like phenotype when were exposed to hypoxic challenge.These hypoxia-induced changes were exaggerated by the introduction of DDR2into the cells. Further analysis by immunofluorescence and immunoblot revealed that DDR2reduced E-cadherin expression but increased the expression of vimentin. Based on thesedata, we specμLate that the role of DDR2in EMT of HNSCC cells might be partiallydependent on the hypoxic tumor environment.Nextly, we tried to determine whether the in vitro effects of DDR2on HNSCC cellbehaviors coμLd be reproduced in vivo. To this end, we utilized human tongue cancer cellline Tca8113to establish subcutaneous xenograft and tail vein metastasis models in nudemice. Subcutaneous xenograft tumor size among parental, EGFP-, and DDR2-transducedcells did not reveal significant difference across the measurement periods, implying thatDDR2expression has no effect on in vivo tumor growth.Because lung is the first capillary bed that tumor cells will encounter following tail veininjections, we assessed the role of DDR2in Tca8113cell lung metastasis. It was found thatthe DDR2-overexpressing Tca8113cells generated approximately3-fold more metastaseson the lung surface and5-fold more in the lung than control and EGFP-expressing cells. Inaddition, the tumor nodμLes derived from DDR2-transduced cells had enhancedimmunoreactivity of both MMP-2and MMP-9compared with those fromEGFP-transduced group, as demonstrated by histological analysis of the lung tissuesections.In conclusion, we have firstly disclosed an oncogenic role of DDR2in the invasion andmetastasis of HNSCC. Since HNSCC is characterized by a marked propensity for localinvasion and cervical lymph node metastasis, DDR2may represent a promising therapeutictarget for HNSCC.