| Background:Lung cancer has become the leading cause of cancer death in China. Detection of lung cancer at early stage is critical for successful clinical therapy and increased survival rate. The blood contains a treasure trove of biomarkers that could reflect the ongoing physiological state of "all tissues" including tumor tissues, allowing for early detection, monitoring therapeutic efficacy, or understanding the biology for the disease.Purpose:To develop a free protein profile associated with non-small cell lung cancers (NSCLCs); and to search for the biomarkers in the circulating plasma, which could be used for early detection and/or monitoring progression of NSCLCs.Experiment design:Based on the novel method established in this laboratory previously which can be used to detect cancer-related free proteins released into the blood, primary organ cultures were generated from the tumor tissue and matched adjacent normal bronchial epithelium derived from 9 cases of NSCLC. The conditional media were collected from the primary cultures and subsequently analyzed using a liquid chromatography coupled with linear ion trap for the proteome. Two strategies of bioinformatic analysis were adopted to explore the protein profiles dysregulated in the lung cancer. One was concerning on the identification of differential free proteins with label-free quantitative proteomic technique. The other one was emphasized the enrichment analysis of the free proteins derived from lung cancers comparing with the expression pattern of invasive placental cells. The resulting proteins were then validated in human samples of plasma and tumor tissue with ELISA, Western blot and immunohistochemical analysis, respectively.Results:In total,987 unique proteins were identified with high confidence from the conditional media from both the lung cancer and the control tissues. Quantitative protein differences between the paired samples were determined by comparing the spectral counts (label-free). A total of 143 proteins were found to exhibit at least 1.5-fold difference in protein level, including 78 proteins upregulated and 65 downregulated in the tumors. Gene Ontology term enrichment analysis revealed that the predominant functional classes of these proteins were related to the oxidoreductase activity, glycolysis, etc., and the subcellular location of them was mainly membrane-bound. Interestingly, six glycolytic enzymes were upregulated concordantly, including ENO1. Network analysis showed that ENO1 was one of the "nodes" regulatory proteins. According to the statistical analysis together with the gene annotation information, ENO1, ALCAM, CLU, DCN were validated in tissue and plasma samples. The elevated ENO1 expression and repression of CLU and DCN were confirmed in 87.5% (14/16),66.7% (8/12), and 62.5% (10/16) of lung squamous cell carcinoma tumor tissues, respectively, by Western blot analysis. Moreover, the expression level of membrane protein ALCAM was correlated with the clinical staging of lung adenocarcinoma. Consistent with the expression in tissue, the plasma level of ENO1 was significantly higher in lung cancer patients than that in patients with nonmalignant diseases in lung and healthy controls, while the ALCAM plasma level was significantly lower compared to the control subjects.There are striking similarities present between the behavior of invasive placental cells and that of invasive cancer cells. The comparison to the under-controlled developmental life phenomena may hint a simplified tumor metastasis-related pattern. A total of 197 proteins with high confidence (at least twice) identified from the conditional media of lung cancer tissues showed significant enrichment in the more invasive middle term human placentation maternal-fetal interface with Gene Set Enrichment Analysis. Among them, the MMP-1 and LGALS3 were validated in the clinical samples from lung cancer patients. The result showed that the high levels of MMP-1 in both tumor tissue and plasma were correlated with lung cancer progression and poor prognosis. The intracellular expression of LGALS3 was higher in the primary tumors with lymph node metastases than that without metastases (P<0.001). Furthermore, the LGALS3 expression in the stroma was associated with lymph node invasion of SCC in lung.Conclusions:Label-free quantification method could be a feasible and effective strategy to search potential biomarkers of cancer in the simplified conditional media system. The study from a view of systems biology combined with similar biological process at different molecular level may likewise lead to a better understanding for invasion and metastasis of cancer cells. The differentially expressed proteins reported in this study would make promise for the successful protein profile and biomarkers for the non-small cell lung cancers. |
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