| Objective:Head and neck squamous cell carcinoma （HNSCC） is a devastating disease, which accounts for3%f all malignancies and is the sixth most common cancer worldwide. It is a disease primarily affecting the oral cavity, hypopharynx, oropharynx, larynx, and salivary glands. More than80%f HNSCC are oral squamous cell carcinoma （OSCC）, which is one of the undertreated and understudied cancer types and remains a lethal disease in over50%of the cases diagnosed annually. Although we made appreciable advances in the treatment and understanding of the underlying molecular pathogenesis of OSCC, survival rates have not improved significantly over the last several decades. Therefore, there is increasing concern about determining novel molecular targets for the treatment of OSCC. Cellular activities, such as cell survival, proliferation, and differentiation, are tightly controlled by intracellular signaling processes initiated by extracellular signals, in which protein phosphorylation （conducted by protein kinases） and dephosphorylation （conducted by protein phosphatases） are necessary events. Src homology phosphotyrosyl phosphatase2（Shp2） encoded by the PTPN11gene in humans, is a ubiquitously expressed protein tyrosine phosphatase （PTP） with two N-terminal Src homology2（SH2） domains （N-SH2, C-SH2, respectively） and a catalytic （PTP） domain that acts as an important transducer of the MAPK pathway by growth factors, cytokines, and hormones. Autosomal-dominant mutations in the human gene PTPN11have been detected in nearly50%of patients with Noonan syndrome who have higher risk of suffering juvenile myelomonocytic leukemia （JMML）, and somatic mutations constitutively activating Shp2have also been detected in several types of leukemias. Meanwhile, high level of Shp2expression has been reported in several types of cancers, such as breast cancer, gastric cancer, cervical cancer, prostate cancer, and melanomas, which indicated that Shp2functions as a proto-oncogene. Interestingly, the opposite effect of Shp2on tumorigenesis was found in hepatocellular carcinoma, implying that Shp2acted as a tumor suppressor. However, so far there is a lack of information concerning the expression and clinical significance of Shp2in OSCC. Therefore, this study investigated the clinical significance of Shp2protein expression in OSCC and elucidated the effect of Shp2gene on biological behaviours of in vitro. The correlation between the expression level and clinical parameters in OSCC tissues was analyzed. The biological significance in OSCC cells was elucidated preliminarily.Methods:1. Seventy paraffin-embedded specimens were retrospectively recruited and immunohistochemical method was used to detect the expression of Shp2in OSCC. Based on the score of immunochemistry, the correlation between the expression of Shp2and various clinicopathologic parameters （age, sex, tumor size, tumor location, clinical stage, histological grade and lymphatic metastasis） was investigated.2. Eighteen cases of cancerous and normal tissues were obtained from patients with OSCC. These samples were histologically confirmed by frozen sections to have OSCC and used for Western blot analysis of Shp2expression.3. To knockdown Shp2expression in cell lines, we designed and synthesized Shp2shRNAs according to Shp2cDNA sequences （Gen-Bank accession NM002834） from Shanghai Genomeditech Co. Ltd. These oligonucleotides were then subcloned into a lentiviral vector （pGMLV-SB1RNAi） and after estimating a multiplicity of infection （MOI） using a standard procedure, these viruses were used to infect SCC-4cells using an oligofectamine transfection reagent. Thereafter, the cells were observed under a fluorescence microscope and harvested on the4th,7th and10th day after infection. The efficiency of Shp2knockdown in SCC-4cells was confirmed by using real time PCR and Western blot.4. The proliferation rate of SCC-4cells at24h,48h,72h and96h after Shp2-shRNA3lentivirus vector and empty vector infection was investigated by MTT dection.5. Cells were collected after lentivirus vector and empty vector infection for72h and then5μl PE Annexin V and5μl7-ADD were added into cell solutions and incubated in the dark for30min. The apoptotic rate was detected by a flow cytometer. P53, Bax and Bcl-2, were detected for further demonstrating the mechanism involved in apoptosis.6. SCC-4cells were transfected with Shp2lentiviral vector and empty vector for investigating the effect of Shp2on cell invasion ability by tumor cell transwell invasion assay.Results:1. Immunohistochemistry results showed that in the adjacent normal epithelia, Shp2protein was either absent or weakly expressed, whereas the average total score of Shp2protein levels in OSCC tissues was significantly higher than that in adjacent normal epithelia. Furthermore, there was significant association of Shp2expression with tumor clinical stage and lymph node metastasis.2. The expression of Shp2protein in the fresh OSCC tissues was higher than that in the adjacent non-tumor tissue, except in one sample.3. We produced lentiviruses carrying Shp2shRNA to knockdown Shp2expression in SCC-4cells. Real-time PCR data showed that the levels of Shp2expression in Shp2shRNA1,2, and3subgroups were remarkably reduced by39%,47%, and75%, respectively. Western blot analysis revealed that the levels of Shp2protein expression in these three subgroups were also significantly reduced.4. The effect of Shp2knockdown on regulation of SCC-4cell viability was determined by using MTT assay. The result showed that the Shp2shRNA3lentivirus-infected cell had a reduction in cell viability compared with the control cells （p<0.05）.5. Annexin V/7-ADD staining by flow cytometry showed that the reduced cell viability was due to induction of apoptosis （p<0.05）. The western blot results showed that the expression of p53protein was increased upon Shp2knockdown in SCC-4cells. Meanwhile, Shp2knockdown was able to induce increase in expression of Bax protein, but decreased the expression of Bcl-2protein.6. Knockdown of Shp2expression on SCC-4cells significantly reduced tumor cell invasive capacity compared to the control cells （p<0.05）.Conclusions:Expression of Shp2protein was significantly upregulated in OSCC tissues compared to the normal tissues and Shp2overexpression was associated with advanced tumor clinical stages and lymph node metastasis in vivo. Knockdown of Shp2expression in vitro inhibited OSCC cell viability and invasion but induced apoptosis by regulating expression of the apoptosis-related proteins. In summary, these results demonstrated that Shp2may be an oncogene and promote OSCC lymph node metastasis. These data also suggest that Shp2might be further evaluated as a biomarker for prediction of OSCC progression and that target of Shp2may be a novel therapeutic strategy for clinical control of OSCC in future. |
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