Oral squamous cell carcinoma (OSCC) is one of the most common types of cancers in developing countries. A major contributor to the high mortality rate of OSCC is the tendency of oral cancer cells to metastasize to lymph nodes around the head and neck during the early stages of cancer development. Matrix metalloproteinase 9 (MMP-9), an endopeptidase, can degrade the extracellular matrix and basement membrane and plays a key role in tumor invasion and metastasis. In vitro, cell migration ability was conducted by scratching assays. We also investigated the interaction abilities between OSCC cells and vascular endothelial cells (ECs) by an adhesion assay and transendothelial migration assay. And we established a BALB/c nude mouse tongue-xenografted metastasis model to investigate the role of MMP-9 and explore its potential underlying mechanism in OSCC growth, lymph node metastasis, and angiogenesis in vivo. The results showed that knockdown of MMP-9 could significantly suppress OSCC cell migration, proliferation, interactions between endothelial cells, xenografted tumor growth, and angiogenesis and simultaneously markedly inhibited OSCC cell metastasis to mouse lymphonodi cervicales superficiales, axillary lymph nodes, and even distant inguinal lymph nodes. Mechanistic studies revealed that knockdown of MMP-9 also led to a decreased expression of RhoC, Src, and F-actin by RT-PCR, western blotting, and immunohistochemistry. And the bioinformatic analysis showed that MMP-9, RhoC, and Src mRNA expression was positively and linearly correlated in OSCC on TCGA database. Together, our findings indicated that MMP-9 plays a very important role in OSCC growth, migration, angiogenesis, and lymph node metastasis, and its potential mechanism may be mediated by RhoC and Src gene expression.

Destruction of extracellular matrix (ECM) is one of the basic steps of tumor invasion and metastasis. Matrix metalloproteinase (MMP) 9, a kind of zinc-ion-dependent endopeptidase, can degrade almost all protein components in the ECM, destroy the histological barrier of tumor cell invasion, and play a key role in tumor invasion and metastasis. The role of MMP-9 in tumor invasion and metastasis has attracted increasing attention and is considered the main proteolytic enzyme in this process. Although the overexpression of MMP-9 was detected in Oral squamous cell carcinoma (OSCC) tissues, further basic studies in vivo and in vitro are needed to investigate the role of MMP-9 in OSCCs and provide scientific validation. In this research, we developed a novel OSCC zebrafish xenograft model to study the role of the MMP-9 gene in oral carcinogenesis. Firstly, the MMP-9/shRNA lentiviral clone and control virus were constructed and transfected into OSCC cells. Then, the decreasing expression of MMP-9 was verified by RT-PCR and immunocytochemistry. Cell proliferation was detected by MTT assay. Colony formation was evaluated by colony formation assay. Cell invasion was evaluated using transwell invasion assay in vitro. In addition, OSCC cells with MMP-9/shRNA knockdown and control vector were injected into zebrafish and an OSCC tumor model in zebrafish was established to evaluate invasion and metastasis in vivo. Knockdown of MMP-9 gene by shRNA could inhibit OSCC cell growth and clone formation and markedly suppress cell invasion in vitro. And the knockdown of the MMP-9 gene could also significantly decrease the metastatic distance and number of metastatic tumor cells or lesions in vivo and suppress the metastasis rate in xenografted zebrafish. Taken together, these evidences indicated that the knockdown of MMP-9 might suppress OSCC cell invasion and metastasis in vivo and in vitro. The MMP-9 gene may be a promising therapeutic target for OSCCs in the future.

Ras homolog family member C (RhoC) is an important component of intracellular signal transduction and its overexpression has been reported to be involved in regulating tumor proliferation, invasion, and metastasis in various malignant tumors. However, its role and underlying mechanism in oral squamous cell carcinoma (OSCC) still remain obscure. In our study, RhoC expression, its relation with clinical stages, and survival rate in OSCC were analyzed using datasets from The Cancer Genome Atlas (TCGA). Next, a RhoC knockdown cell model was established in vitro, and the effects of RhoC knockdown in OSCC cells were detected by the MTT assay, colony formation assay, transwell invasion assay, scratch assay, and F-actin phalloidin staining. An in vivo tongue-xenografted nude mouse model was established to measure the effects of knockdown of RhoC on tumor cell growth and lymph node metastasis. A mechanism study was conducted by real-time PCR and immunocytochemistry. The results of TCGA analysis showed that RhoC was overexpressed in OSCC tumor tissues. In vitro assays indicated that knockdown of RhoC did not have much effect on OSCC cell growth but significantly suppressed cell colony formation, invasion, and migration abilities, and F-actin polymerization was also reduced. The tongue-xenografted in vivo model demonstrated that knockdown of RhoC suppressed OSCC cell growth and inhibited metastasis to the superficial cervical lymph nodes. Further mechanism studies showed that knockdown of RhoC downregulated HMGA2 expression, and HMGA2 expression was highly correlated with RhoC expression in OSCC tumor tissues via the analysis of TCGA datasets. Overall, our study showed that knockdown of RhoC inhibited OSCC cells invasion and migration in vitro and OSCC cell growth and lymph node metastasis in vivo. Moreover, the potential mechanisms involved in these activities may be related to the regulation of HMGA2 expression. The RhoC gene could serve as a promising therapeutic target for OSCCs in the future.

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Oral cancer (OC), the most common cancer in the head and neck, which has a poor prognosis, histopathologically follows a stepwise pattern of hyperplasia, dysplasia, and cancer. Blocking the progression of OC in the precancer stage could greatly improve the survival and cure rates. AKT protein plays a critical role in the signal transduction of cancer cells, and we found that AKT was overexpressed in human OC samples through analysis of TCGA database. Therefore, this study is aimed at investigating the chemopreventive effect of an AKT inhibitor (MK2206 2HCl) on OC. In vivo, we established a 4-nitroquinoline-1-oxide- (4NQO-) induced mouse tongue carcinogenesis model to investigate the potential chemopreventive effect of MK2206 2HCl on mouse OC resulting from 4NQO. The results showed that MK2206 2HCl could significantly reduce the incidence rate and growth of OC, inhibit the transformation of dysplasia to cancer in the 4NQO-induced mouse tongue carcinogenesis model, and simultaneously markedly suppress cell proliferation, angiogenesis, and mast cell (MC) infiltration in 4NQO-induced mouse tongue cancers. In vitro, our results revealed that MK2206 2HCl could also inhibit oral squamous cell carcinoma (OSCC) cell malignant biological behaviors, including cell proliferation, colony formation, cell invasion, and migration, while promoting apoptosis. Mechanistic studies revealed that MK2206 2HCl suppressed matrix metalloproteinase 9 (MMP-9) and RhoC expression and promoted autophagy gene LC3 II expression. In summary, our findings demonstrated the chemopreventive effect of MK2206 2HCl on the 4NQO-induced mouse tongue carcinogenesis model, which likely has an underlying mechanism mediated by the MMP-9/RhoC signaling pathway and autophagy.