| BackgroundRenal cell carcinoma (RCC) is the most common malignancies in adult kidney, which accounts for 2-3% of all cancers. Clear cell RCC (ccRCC) is the most common subtype, which accounts for 70～80% of all renal masses. With the population aging and medical screening approaches improving, the incidence of RCC shows a steady growing trend recently, which has become a serious thread to human beings. Clinically, nephrectomy or partial nephrectomy remains the best option for localized RCC, which is associated with favorable prognosis (5-year survival rate ～85%). Because RCC is resistant to chemotherapy and radiotherapy, prognosis is generally poor (5-year survival rate～10%) for patients with advanced RCC (including metastatic and recurrent RCC), which are totally responsible for over 50% of patients. Until today, there is still no acknowledged molecular biomarker of screening, diagnosis, stratification and response evaluation for RCC. It is still urgent to discover biomarkers and targets for early diagnosis, prognosis evaluation, treatment stratification, and monitoring for recurrence of patients with RCC.Mass spectrometry (MS)-based proteomics is a promising alternative strategy to identify, quantify, and characterize a global set of proteins, especially discover the potential biomarkers or pathways for cancer in complex biological samples. Label-free quantitative proteomics approach, which is based on the ion intensities of identical peptides or the number of acquired spectra for each protein, has become a popular and applealing alternative to compare a global set of proteins in complex biological samples. Posttranslational modifications (PTMs) of proteins perform significant influence on the cellular activity, stability, localization, and turnover. Phosphorylation, glycosylation, acetylation, and nitration have been reported in association with the pathogenesis of cancers. Because protein acetylation at the epsilon amino group of lysine (Lys, K) neutralizes the positive charge, the reversible PTM usually regulates protein (especially histone) stability and protein-protein interactions. Recently, increasing evidences have showed non-histone acetylation was associated with changing protein functions, especially during metabolic regulation and autophagy controlling processes. Recently, proteomics has been used to identify differentially expressed proteins between RCC and normal kidney tissues, such as 60kD heat shock protein (HSPD1), catalase (CAT), alphaenolase (ENO1), which might be potential candidates as diagnostic, prognostic markers, and therapeutic targets, but few reports about PTM of specific protein in RCC were elucidated. Recent studies have also reported some pathways such as mitochondrial dysfunction, glycolysis, metabolism, and protein transport in RCC tissue or cell lines, whereas none of them is concerned with PTM. Since lysine acetylation is a prevalent modification in organism, we speculate there are also some acetylated proteins in the pathogenesis and development of RCC.ObjectivePreviously, we found that S(MeO)TLC, a novel Eg5-targeted drug, displayed potent anti-cancer efficacy in RCC in vitro and in invo. In order to find more therapeutic molecular targets and diagnostic or prognostic biomarkers, we performed label-free quantitative proteomic analysis to compare protein expression profiling and elucidate signaling pathways or biological processes between ccRCC and adjacent tissues, then selected two dysregulated proteins to confirm their expression and regulation. These results might pinpoint some potential diagnostic or prognostic biomarkers and novel therapeutic targets for RCC. Meanwhile, there was some difference of MnSOD acetylation between ccRCC and adjacent kidney tissues. When Sirt3, the deacetylase of MnSOD, was found, the MnSOD enzymatic activity and cellular function of ccRCC cell line 786-0 were observed after Sirt3 interacted and deacetylated MnSOD.Methods1) Quantitative proteomics analysis of the proteins from ccRCC and adjacent tissues. After one pair of pool samples from 12 matched pairs of ccRCC and adjacent tissues were constructed, the whole cell lysates (proteins) were processed in-solution and in-gel tryptic digestion, then liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and label-free quantitative proteomic approach (total ion intensity and spectral counts) were employed to identify and quantify the dysregulated proteins between ccRCC and adjacent tissues.2) Multiple pathway analysis tools including Ingenuity Pathway Analysis (IPA), search tool for the retrieval of interacting gene (STRING), Database for Annotation, Visualization and Integrated Discovery (David) were used to identify enriched signaling pathways or networks and diseases categories.3) Western blotting (WB) and immunohistochemistry (IHC) assays were performed on manganese superoxide dismutase (MnSOD) and acetyl-CoA acetyltransferase 1 (ACAT1) using another 6 pairs of ccRCC and adjacent tissues,4) Acetylation difference of the dysregulated proteins was compared by label-free quantitative proteomic approach. Further mass spectrometer (MS) analysis was used to identify their acetylation of the dysregulated proteins, MnSOD and AC AT1, then acetylation difference of MnSOD between ccRCC and adjacent tissues, was compared by label-free quantitative proteomic approach.5) Plasmid 5’Flag-pcDNA3.1-sod2 (Sod2) construction and 786-0 cell transfection. The plasmid Sod2 was confirmed to be constructed as expectation by PCR, enzyme digestion and sequence reaction, then plasmids Sod2, empty vector (EV), Sirt2, Sirt3 an Sirt6 were transfected into 786-0 using lipofectamin 2000, respectively.6) Western blotting (WB) and immunoprecipitation (IP) were employed to confirm MnSOD acetylation and two different primary antibodies, anti-MnSOD and anti-acetylated-lysine were used. ccRCC cell line 786-0 was used to identify endogenous and transient transfected exogenous MnSOD acetylation when 786-0 cells were transfected with different plasmids or not trasfected.7) MnSOD activity was measured by superoxide dismutase (SOD) activity Kit. Plasmids EV, Sirt2, Sirt3 and Sirt6 were transfected into 786-0 using lipofectamin 2000, respectively. After cells harvest and homogenization, MnSOD activity was measured.8) Cell proliferation and apoptosis assay. After plasmids EV, Sirt2, Sirt3 and Sirt6 were transfected into 786-0, respectively,proliferation activity was measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis activity was measured using Hoechst 33342 solution with a fluorescence microscope.Results1) By in-solution digestion, a total of 1872 and 1927 proteins were identified in ccRCC and adjacent kidney tissues, respectively. Among these proteins,1037 proteins were quantified by Progenesis LC-MS, and 213 proteins were identified as dysregulated proteins between ccRCC and adjacent tissues. By in-gel digestion,5816 and 5571 proteins were identified in ccRCC and adjacent tissues, respectively, which was the biggest protein number identified in RCC to date. Of these,3381 proteins were quantified and 208 proteins were identified as dys-regulated proteins between cancer and noncancer tissues using spectra count based quantitative proteomic approach.2) Pathway analysis using IPA, STRING and DAVID tools was performed, which demonstrated the enrichment of cancer-related signaling pathways and biological processes such as mitochondrial dysfunction, metabolic pathway, cell death, and acetylation.3) Dysregulation of two mitochondrial proteins, ACAT1 and MnSOD were selected and confirmed by western blotting and immunohistochemistry assays using another 6 pairs of ccRCC and adjacent tissues:ACAT1 up-regulated in ccRCC and MnSOD down-regulated in ccRCC.4) Further mass spectrometry analysis indicated that both ACAT1 (lysine 174) and MnSOD (lysine 68 and 130) had characterized acetylation at lysine residues, which is the first time to identify acetylation of ACAT1 and MnSOD in ccRCC. The studies also discover both the acetylated peptides (containing lysine 68 and lysine 130) increased in proportion with MnSOD protein in ccRCC.5) After the plasmid pcDNA3.0-sod2 (Sod2) was successfully constructed and Sirt3, the MnSOD deacetylase, was found, the endogenous MnSOD in ccRCC cell line 786-0 was acetylated whereas transient transfected exogenous MnSOD was not.6) Sirt3, one of the HDAC family member, can deacetylate mitonchondrial MnSOD in 786-0 cells, enhance its enzymatic activity and increase cell proliferation rate, but demonstrate no effect on cell apoptosis.Conclusion1) Through MS-based label-free quantitative proteomic analysis, we identified over one thousand proteins and selected more than two hundred dysregulated proteins, which are involved in carcinogenesis and have the potential to become diagnostic, prognostic, and stratification treatment biomarkers in ccRCC.2) Using multiple pathway analysis tools including IPA, STRING, and David, we disclosed many signaling pathways and biological processes, which were activated in ccRCC, and these dysregulated pathways or processes, like mitochondrial dysfunction and lysine acetylation, might become novel therapeutic targets for ccRCC, which might greatly improve patient prognosis and increase overall survival, especially for unresectable RCC.3) There are some differences of MnSOD expression and its acetylation between ccRCC and adjacent kidney tissues, and MnSOD acetylation affect the proliferation activity of ccRCC cell line 786-0, which indicates MnSOD might participate in the ccRCC pathogenesis, and targeted MnSOD or its acetylation might be a novel therapeutic option for RCC.Innovative points1) 5816 and 5571 proteins were identified in clear cell RCC (ccRCC) and adjacent tissues by in-gel tryptic digestion, respectively, which was the biggest protein number identified in RCC to date.2) This is the first time to identify and compare MnSOD acetylation in ccRCC.3) This is the first time to report that MnSOD acetylation can affect cellualr function of ccRCC cell line 786-0, for example, MnSOD enzymatic activity, cellular proliferation activity. |
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