| Background and purpose Esophageal cancer is the fourth most common malignancies in China and more than 90% patients are squamous cell carcinoma. Owing to the advanced stage when patients are examined, the 5-year survival rate is still low although they might receive comprehensive treatment including operation. Recently, abnormal expression of tyrosine kinase signaling pathway has been found in many tumors by some researchers, however, tyrosine phosphorylation is at low abundance in tissues. Hence there was no tyrosine phospho-proteomics research on esophageal cancer yet. Here, we investigated differential protein tyrosine phosphorylation between esophageal cancer and para-esophageal cancer normal tissues (simply, para-cancer tissues) with a new tyrosine phospho-proteomics method. This study would provide basic proof for understanding the role of tyrosine kinase signaling in the occurrence and development of esophageal cancer, and also offer new insights into the treatment and early diagnosis of esophageal cancer.Methods In the first chapter, we collected 28 cases of esophageal squamous cancer and corresponding para-cancer tissues. Tyrosine phosphorylated peptides were enriched by immunoaffinity purification, and analyzed by LTQ Orbitrap mass spectrometer. Differential phosphorylated proteins were analyzed by using Genepattern software. MYH9 (encoding NMⅡA) was the top phosphorylated protein between the cancer and para-cancer group and was closely correlated with DDR1.In the second chapter, we extracted genomic DNA from ten cancer tissues with highest MYH9 phosphorylation and sequenced PCR products to find any mutations of MYH9. Western blot was adopted to validate NMⅡA expression in the 28 pair specimen. Another 50 cancer specimen and 30 para-cancer normal specimen were chosen to investigate the expression of NMⅡA and DDR1 using immunohistochemistry (IHC) and correlations between their expressions and patients'clinical data were also analyzed.In chapter three, we cultured and profiled three esophageal squamous cancer cell lines(KYSE-140,KYSE-150,KYSE-510) by mass spectrometer. Comparison of primary human esophageal cancer and cancer cell lines was made and suggested that cell lines can be used as models to validate targets identified in primary tumors.In the fourth chapter, we silenced MYH9 with small interfering RNA; qRT-PCR and western blot were used to study the silence effect. We counted cell number to find any effect of decreased NMⅡA on cell proliferation. Cell adhesion assay was used to analyze the effect of decreased NMⅡA on cell adhesion ability. Wound closure assay and transwell assay were used to assess the effect of decreased NMⅡA on cell migration ability.Results1. In this study, we identified 1037 tyrosine phosphorylated proteins. 477 of them were enriched in cancers while 560 were enriched in para-cancer tissues. The top 5 tyrosine phosphorylated proteins were MYH9, palladin, APP, DDR1 and K10 in the cancer group and K23, K20, Sciellin, envoplakin and EphA1 in para-cancer group. No significant difference of tyrosine phosphorylated protein clusters was found between the two groups. The tyrosine phosophorylated sites of NMⅡA were Tyr754 and Tyr1408. Correlation analysis showed that MYH9 was closely related with DDR1. 2. There was no mutation in exon 1,10,16,24-26,30-32 and 35-39 of MYH9. Western blot analysis found that the expression of NMⅡA was higher in cancer tissues than para-cancer tissues in the 28 pair tissues. In IHC analysis, NMⅡA was expressed in all cancer tissues, and were significantly higher than the expression in para-cancer tissues (100% VS 26.7%, P=0.000), strong positive expression of NMⅡA was found in 25 cancer specimen(50%) and was correlated with metastatic lymph nodes and the differentiation of the cancer but not with cancer invasive depth. Corresponding increased strong positive NMⅡA expression was correlated with the increasing metastatic lymph nodes (P=0.015<0.05) and the poorer differentiation (P=0.018<0.05). The expression of DDR1 was significantly higher in cancer tissues than para-cancer tissues (86% VS 30%, P=0.000). DDR1 expression was only correlated with the invasive depth (P=0.027<0.05), but not with metastatic lymph nodes or the differentiation of the cancer.3. Significant differences of tyrosine phosphorylated protein levels and clusters were found between the esophageal cancer tissues and cell lines. In three cell lines, phosphorylation of the motor/contractile protein was only found in KYSE-510 cell line, and there were only three phosphorylated proteins:KLC2, MYH9 and dnnactin2. The expression of NMⅡA protein was found in all three cancer cell lines with a relative lower expression in KYSE-140 cell line.4. The silence efficacy was highest when the ratio of SiRNA/transfection reagent was 75ng/1.5ul at 72h post-transfection with western blot analysis. MYH9 mRNA was silenced up to 89.9% demonstrated by qRT-PCR. When the expression of NMⅡA protein was almost eliminated by targeted MYH9 SiRNA, KYSE-510 cells exhibited decreased proliferation rate, planer and 3-D migration rate while with higher adhesion ability compared with cells transfected with negative SiRNA (P＜0.01).Conclusions1. PhosphoScan technology was successfully applicated in proteomic analysis of tyrosine phosphorylation in esophageal squamous cancer and para-cancer tissues; a number of over-expressed tyrosine phosphorylated proteins were screened in esophageal cancer tissues, of which the most obvious were the motor/contractile protein NMⅡA and the receptor tyrosine kinase DDR1;2. Increased tyrosine phosphorylation of NMⅡA might not be induced by genetic mutation of MYH9 gene, but might be caused by elevated total NMⅡA protein expression;3. NMⅡA expression correlated with esophageal squamous cancer differentiation and metastasis in cancer tissues; DDR1 expression only correlated with the cancer invasion depth.4. KYSE-510 cell line expressed NMⅡA protein and tyrosine phosphorylated NMⅡA protein;5. Decreased NMⅡA expression by targeted MYH9 SiRNA inhibited the migration and proliferation ability of KYSE-510 cell line but enhanced the adhesive ability. |
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