Fengming Gong

Comparative Proteomic Approach Identifies Pkm2 and Cofilin-1 as Potential Diagnostic, Prognostic and Therapeutic Targets for Pulmonary Adenocarcinoma

Lung cancer is the leading cause of cancer-related death in the world. Non-small cell lung carcinomas (Non-SCLC) account for almost 80% of lung cancers, of which 40% were adenocarcinomas. For a better understanding of the molecular mechanisms behind the development and progression of lung cancer, particularly lung adenocarcinoma, we have used proteomics technology to search for candidate prognostic and therapeutic targets in pulmonary adenocarcinoma. The protein profile changes between human pulmonary adenocarcinoma tissue and paired surrounding normal tissue were analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE) based approach. Differentially expressed protein-spots were identified with ESI-Q-TOF MS/MS instruments. As a result, thirty two differentially expressed proteins (over 2-fold, p<0.05) were identified in pulmonary adenocarcinoma compared to normal tissues. Among them, two proteins (PKM2 and cofilin-1), significantly up-regulated in adenocarcinoma, were selected for detailed analysis. Immunohistochemical examination indicated that enhanced expression of PKM2 and cofilin-1 were correlated with the severity of epithelial dysplasia, as well as a relatively poor prognosis. Knockdown of PKM2 expression by RNA interference led to a significant suppression of cell growth and induction of apoptosis in pulmonary adenocarcinoma SPC-A1 cells in vitro, and tumor growth inhibition in vivo xenograft model (P<0.05). In addition, the shRNA expressing plasmid targeting cofilin-1 significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. While additional works are needed to elucidate the biological significance and molecular mechanisms of these altered proteins identified in this study, PKM2 and cofilin-1 may serve as potential diagnostic and prognostic biomarkers, as well as therapeutic targets for pulmonary adenocarcinoma.

Quantitative protein expression profiling of 14-3-3 isoforms in human renal carcinoma shows 14-3-3 epsilon is involved in limitedly increasing renal cell proliferation.

14‐3‐3 proteins regulate many cellular processes that are implicated in cancer development, and the seven 14‐3‐3 isoforms have different expression level and isoform‐specific roles in different tumors. However, the biological functions of 14‐3‐3 proteins and their correlations with renal carcinoma have not been investigated so far. In our study, the expression profiles and functional characterization of 14‐3‐3 proteins were discovered by a sensitive stable isotope labeling with amino acids in cell culture based quantitative proteomics analysis in human renal carcinoma tissues. We found that 14‐3‐3ε was up‐regulated with 1.44‐fold changes in renal cancerous tissues compared with that in counterpart kidney tissues, and 14‐3‐3σ was almost not detected in both tissues due to its DNA highly methylated in our previous reports. The other five isoforms almost have similar expression level in two states of renal tissues. The following RT‐PCR, Western blot and immunohistochemistry analysis for specific 14‐3‐3 isoform expression were all consistent with the quantitative proteomic data. Furthermore, the overexpression of 14‐3‐3ε in vitro can limitedly prompt the abnormal growth of renal tumor cells.

Isoform-specific expression and characterization of 14-3-3 proteins in human glioma tissues discovered by stable isotope labeling with amino acids in cell culture-based proteomic analysis.

Human 14‐3‐3 proteins have isoform‐specific expression and functions in different kinds of normal or tumor cells and tissues. However, the expression profiling of 14‐3‐3 proteins and isoform‐specific biological functions are unclear in human glioma so far. In our study, the expression levels and characterization of 14‐3‐3 isoforms in human glioma tissues were investigated by a sensitive, accurate stable isotope labeling with amino acids in cell culture‐based quantitative proteomic strategy. As a result, except unexpressed 14‐3‐3σ, the other six isoforms, with different expression levels, were existed in glioma tissues and para‐cancerous brain tissues (PBTs). 14‐3‐3β and η were upregulated, whereas 14‐3‐3ζ was downregulated in glioma tissues compared with that in PBTs. And the other three isoforms 14‐3‐3ε, θ, and γ had similar expression levels in human glioma tissues and PBTs. Western blot and immunohistochemistry analysis were both consistent with the quantitative proteomic data. The loss of expression of 14‐3‐3σ was further discovered due to DNA high methylation in its coding region in glioma by methylation‐specific PCR analysis. These results indicated that the four isoforms, including 14‐3‐3β, η, ζ, and σ, may play important roles in tumorigenesis of human glioma, which is probably used as potential biomarkers for diagnosis and targets for treatment of human gliomas in future.

Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts☆

Ovarian cancers highly overexpress folate receptor α (FRα) and claudin3 (CLDN3), both of which are associated with tumor progression and poor prognosis of patients. Downregulation of FRα and CLDN3 in ovarian cancer may suppress tumor growth and promote benign differentiation of tumor. In this study, F-P-LP/CLDN3, a FRα targeted liposome loading with short hairpin RNA (shRNA) targeting CLDN3 was prepared and the pharmaceutical properties were characterized. Then, the antitumor effect of F-P-LP/CLDN3 was studied in an in vivo model of advanced ovarian cancer. Compared with Control, F-P-LP/CLDN3 promoted benign differentiation of tumor and achieved about 90% tumor growth inhibition. In the meantime, malignant ascites production was completely inhibited, and tumor nodule number and tumor weight were significantly reduced (p<0.001). FRα and CLDN3 were downregulated together in tumor tissues treated by F-P-LP/CLDN3. The antitumor mechanisms were achieved by promoting tumor cell apoptosis, inhibiting tumor cell proliferation and reducing microvessel density. Finally, safety evaluation indicated that F-P-LP/CLDN3 was a safe formulation in intraperitoneally administered cancer therapy. We come to a conclusion that F-P-LP/CLDN3 is a potential targeting formulation for ovarian cancer gene therapy.

Cathepsin B as a potential prognostic and therapeutic marker for human lung squamous cell carcinoma

BackgroundThe lung squamous cell carcinoma survival rate is very poor despite multimodal treatment. It is urgent to discover novel candidate biomarkers for prognostic assessment and therapeutic targets to lung squamous cell carcinoma (SCC).ResultsHerein a two-dimensional gel electrophoresis and ESI-Q-TOF MS/MS-based proteomic approach was used to identify differentially expressed proteins between lung SCC and adjacent normal tissues. 31 proteins with significant alteration were identified. These proteins were mainly involved in metabolism, calcium ion binding, signal transduction and so on. Cathepsin B (CTSB) was one of the most significantly altered proteins and was confirmed by western blotting. Immunohistochemistry showed the correlation between higher CTSB expression and lower survival rate. No statistically significant difference between CTSB-shRNA treated group and the controls was observed in tumor volume, tumor weight, proliferation and apoptosis. However, the CTSB-shRNA significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. Moreover, CTSB, Shh and Ptch were up-regulated in patients with metastatic lung SCC, suggesting that hedgehog signaling might be activated in metastatic lung SCC which could affect the expression of CTSB that influence the invasive activity of lung SCC.ConclusionsThese data suggested that CTSB might serve as a prognostic and therapeutic marker for lung SCC.

Gene expression and methylation status of 14‐3‐3σ in human renal carcinoma tissues

Loss of 14‐3‐3σ expression mainly by methylation‐mediated silencing has been reported in several human cancers, but the methylation status of 14‐3‐3σ in human renal carcinoma is rarely studied so far. In this report, 14‐3‐3σ expression was first examined by RT‐PCR and immunohistochemistry, and further we investigated the methylation status by methylation‐specific PCR and the correlation between 14‐3‐3σ expression and its methylation. We found 14‐3‐3σ expression was lost in 27 of 31 renal tissues including 16 renal carcinoma tissues, eight para‐cancerous kidney tissues and seven normal kidney tissues. Among 16 renal carcinoma tissues, 14 cases had complete hypermethylation of 14‐3‐3σ. Eight para‐cancerous kidney tissues were almost completely methylated except one case had both methylation and unmethylation. Among seven normal kidney tissues, five cases had partial methylation, and the other two cases were completely methylated. In addition, 14‐3‐3σ mRNA had weak expression in OS‐RC‐2 cells, but it increased with gradual demethylation after treatment by a demethylation agent, 5‐aza‐2′‐deoxycytidine. In general, 14‐3‐3σ mRNA was mostly unexpressed, and its DNA frequently hypermethylated within 14‐3‐3σ coding region was closely associated with the gene silencing in cancerous and para‐cancerous kidney tissues. 14‐3‐3σ was also frequently methylated and almost silencing in normal kidney tissues. However, the methylation frequency was gradually reinforced with the extent of malignancy from normal to para‐cancerous and cancerous kidney tissues.

Application of the SILAC (stable isotope labelling with amino acids in cell culture) technique in quantitative comparisons for tissue proteome expression

Stable isotope labelling has recently become a popular tool for the quantitative profiling of the proteome, especially the emergence and development of the SILAC (stable isotope labelling with amino acids in cell culture) technique. Here we have expanded the application of SILAC to comparison of the relative protein expression levels between two different states of tissues based on cultured cells with [2H]leucine labelling as an internal standard in mass spectra. The SILAC ratio of tissue proteins versus labelled cells was determined by the calculation of peak intensity of the pair of labelled and unlabelled peptide fragment ions from the mass spectra, and the relative expression level of proteins in two groups of tissues was estimated by calculating the ratio of their SILAC ratio. To validate our [2H]leucine‐based differential proteome analysis for tissues, we successfully compared two known proteins, one up‐regulated vimentin and one down‐regulated enoyl‐CoA hydratase in human renal cancerous tissues versus human normal kidney tissues, which was previously confirmed by other groups using conventional two‐dimensional PAGE analysis. Furthermore, we identified a previously unknown down‐regulated protein, COX4I1 (cytochrome c oxidase subunit 4 isoform 1), in renal carcinoma tissues by this [2H]leucine‐based quantitative proteomics method, which was also validated by immunohistochemistry and Western‐blot analysis. In conclusion, the application of the [2H]leucine‐based quantitative technique can be effectively expanded to comparison of the expression levels for the tissue proteome at different states, which would help us to identify new candidate biomarkers for tumours.

Prokaryotic expression, purification of a new tumor-relative protein FAM92A1-289 and its characterization in renal cell carcinoma

In order to study the characterization of a new tumor-relative FAM92A1-289 protein, we first constructed plasmid FAM92A1-pQE30 for fusion expression in Escherichia coli. The recombinant protein FAM92A1-289 was affinity-purified by Ni2+-charged resin and separated by HPLC chromatography with high purity, and it was further identified by electrospray ionization-mass spectrometry. Furthermore, the expression and cell localization of FAM92A1-289 by immunohistochemistry using our self-prepared polyclonal antibody showed it was expressed in cytoplasm of renal carcinoma. FAM92A1-289 mRNA was expressed in 2 of 10 kidney tissues and in 6 of 12 primary renal tumors. FAM92A1-289 can promote cell growth in vitro and in vivo by colony formation and mouse xenograft assay. Our present data indicated FAM92A1-289 is a new tumor-related gene with oncogenic potentials to probably play roles in renal carcinogenesis.

Ammonia Drives Dendritic Cells into Dysfunction

Ammonia levels are often elevated in patients with cirrhosis or tumors. Patients with these diseases are immunocompromised. In this study, we investigated the effects of ammonia on a member of the immune cell family, the dendritic cells (DCs). Our results demonstrated that ammonia diminished cell count, phagocytosis, and lymphocyte stimulation of DCs. Ammonia also induced DC swelling, excessive reactive oxygen species production, and mitochondrial damage, which may constitute the underlying mechanism of ammonia-induced DC dysfunction. In ammonium chloride (NH4Cl)–loaded mice, DCs exhibited lowered phagocytosis and a weakened immune response to the chicken OVA vaccine. DCs from patients with cirrhosis or ammonia-treated healthy human blood both exhibited diminished phagocytosis. Moreover, tumor cell conditioned medium drove DCs into dysfunction, which could be reversed by ammonia elimination. In a murine colon carcinoma model, we found that ammonia could regulate tumor growth involving DCs and their related immune response. These findings reveal that ammonia could drive DCs into dysfunction, which contributes to the immunocompromised state of patients with cirrhosis or tumors.