Novel Tumor-Specific Antigens for Immunotherapy Identified From Multi-omics Profiling in Thymic Carcinomas.

Thymic carcinoma (TC) is the most aggressive thymic epithelial neoplasm. TC patients with microsatellite instability, whole-genome doubling, or alternative tumor-specific antigens from gene fusion are most likely to benefit from immunotherapies. However, due to the rarity of this disease, how to prioritize the putative biomarkers and what constitutes an optimal treatment regimen remains largely unknown. Therefore, we integrated genomic and transcriptomic analyses from TC patients and revealed that frameshift indels in KMT2C and CYLD frequently produce neoantigens. Moreover, a median of 3 fusion-derived neoantigens was predicted across affected patients, especially the CATSPERB-TC2N neoantigens that were recurrently predicted in TC patients. Lastly, potentially actionable alterations with early levels of evidence were uncovered and could be used for designing clinical trials. In summary, this study shed light on our understanding of tumorigenesis and presented new avenues for molecular characterization and immunotherapy in TC.

Using proteomic profiling to characterize protein signatures of different thymoma subtypes.

BACKGROUND: Histology is a traditional way to classify subtypes of thymoma, because of low cost and convenience. Yet, due to the diverse morphology of thymoma, this method increases the complexity of histopathologic classification, and requires experienced experts to perform correct diagnosis. Therefore, in this study, we developed an alternative method by identifying protein biomarkers in order to assist clinical practitioners to make right classification of thymoma subtypes. METHODS: In total, 204 differentially expressed proteins in three subtypes of thymoma, AB, B2, and B3, were identified using mass spectrometry. Pathway analysis showed that the differentially expressed proteins in the three subtypes were involved in activation-related, signaling transduction-related and complement system-related pathways. To predict the subtypes of thymoma using the identified protein signatures, a support vector machine algorithm was used. Leave-one-out cross validation methods and receiver operating characteristic analysis were used to evaluate the predictive performance. RESULTS: The mean accuracy rates were > 80% and areas under the curve were ≧0.93 across these three subtypes. Especially, subtype B3 had the highest accuracy rate (96%) and subtype AB had the greatest area under the curve (0.99). One of the differentially expressed proteins COL17A2 was further validated using immunohistochemistry. CONCLUSIONS: In summary, we identified specific protein signatures for accurately classifying subtypes of thymoma, which could facilitate accurate diagnosis of thymoma patients.

Direct repression of the oncogene CDK4 by the tumor suppressor miR-486-5p in non-small cell lung cancer.

MicroRNAs are a class of non-coding single-stranded RNA, 20-23 nucleotide in length, which can be involved in the regulation of gene expression. Through binding with 3'-untranslated regions (3'-UTR), microRNAs can cause degradation of target mRNAs or inhibition of translation, and thus regulating the expression of genes at the post-transcriptional level. In this study, we found that miR-486-5p was significantly downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, suggesting that miR-486-5p might function as a tumor suppressor in lung cancer. Additionally, we showed that CDK4, an oncogene that plays an important role in cell cycle G1/S phase progression, was directly targeted by miR-486-5p. Furthermore, our data reveals that knockdown of CDK4 by siRNA can inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression. In epigenetics, the upstream promoter of miR-486-5p was strongly regulated by methylation in NSCLC. Collectively, our results suggest that miR-486-5p could not only inhibit NSCLC by downregulating the expression of CDK4, but also be as a promising and potent therapy in the near future.

Proteome analysis and tissue array for profiling protein markers associated with type B thymoma subclassification.

BACKGROUND: The prognostic relevance of World Health Organization (WHO) subtypes within type B thymomas is still controversial. Understanding of the molecular characteristics of the different histologic types of thymomas will provide meaningful information for diagnosis and therapeutic management in type B thymoma. METHODS: Proteins extracted from twelve type B thymoma tissue specimens (six type B1 and six type B2) were analyzed by two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF-MS. Differentially expressed proteins were then assayed in sixty-nine type B thymoma tissues (including B1, B2 and B3) by tissue array analysis with immunohistochemistry staining. The relationship of their expression with clinicopathological parameters, such as tumor stage or WHO classification, was estimated by Spearman's Rank Correlation Test. RESULTS: Sixteen differentially expressed proteins between type B1 and B2 thymoma tissues were identified. The differential levels of ezrin and glutathione S-transferase pi (GSTP1) were validated using immunohistochemistry staining. A statistically significant difference was observed in the positive rate of ezrin expression between type B1 thymoma and type B3 thymoma (Z = -2.963, P < 0.01). Ezrin showed a tendency to be expressed in higher classification tumors from type B1 to B3. A statistical analysis demonstrated that type B2 and B3 tumors had significantly higher positive expression of GSTP1 than the B1 group (type B2 vs. B1: Z = -2.582, P = 0.01; type B3 vs. B1: Z = -4.012, P ≤ 0.001). The results also showed a strong correlation between GSTP1 and WHO type staging of B1 to B3 tumors (Spearman's correlation coefficient: 0.633, P ≤ 0.001). Statistical analysis showed that there was close correlation between GSTP1 and ezrin expression with the clinical stage (Spearman's correlation coefficients, ezrin: 0.481, P < 0.05; GSTP1: 0.484, P < 0.01). CONCLUSIONS: Differentially expressed proteins between type B1 and B2 thymoma tissues were analyzed by comparative proteomic analysis. The techniques of proteomic analysis and tissue array provide a potential tool for screening of key molecules in type B thymoma histological sub-classifications. The statistical analysis of ezrin and GSTP1 expression by immunohistochemistry, especially GSTP1, may be a useful approach for type B thymoma classification.

Comparative proteomic analysis of paclitaxel sensitive A549 lung adenocarcinoma cell line and its resistant counterpart A549-Taxol.

PURPOSE: Paclitaxel is used as the first-line chemotherapy for Non-Small Cell Lung Cancer (NSCLC), but acquired resistance becomes a critical problem. Several mechanisms have been proposed in paclitaxel resistance, but they are not sufficient to exhaustively explain this resistance emergence. To better investigate molecular resistance mechanisms, a comparative proteomic approach was carried out to identify differentially expressed proteins between human lung adenocarcinoma A549 cell line (paclitaxel sensitive) and A549-Taxol cell line (acquired resistant). METHODS: A paclitaxel-resistant subline (A549-Taxol) derived from the parental-sensitive cell line A549 was established by stepwise selection by paclitaxel. Total proteins in the two cell lines were separated by fluorescent differential gel electrophoresis (DIGE). Image analysis was carried out with the DeCyder 2D 6.5 software. Proteins associated with chemoresistance process were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Some key molecules were valuated by Western blot. RESULTS: Thirty proteins were identified and grouped into eight main functional classes according to the biological processes in which they are likely to participate, i.e. signal transduction, cytoskeleton, redox reaction, energy and metabolism, and so on. Alterations of these processes might be involved in paclitaxel resistance. Most of the proteins showed mitochondrial and cytoplasm location. The up-regulation of CK8, CK18, ALDH1, CAST and ANX I in A549-Taxol cell line was verified by Western blot, in coincidence with the data obtained from proteomic analysis. CONCLUSION: For the first time, differentially expressed proteins between paclitaxel-sensitive cell line and paclitaxel-resistant one were explored by comparative proteomic approach in human lung adenocarcinoma. It may be useful for further studying of resistance mechanisms and screening of resistance biomarkers, so as to develop tailored therapeutic strategies.

A glycoproteome database of normal human liver tissue.

PURPOSE: To extensively investigate the glycoproteins of normal human liver tissue, constructing the glycoprotein profile and database of the normal human liver tissue. METHODS: The total proteins were extracted from the normal human liver tissue and then subjected to two-dimensional electrophoresis (2-DE). Finally, 2-DE gels were stained according to the methods of multiplexed proteomics (MP) technology. Glycoprotein spots were excised from 2-DE gel and then characterized by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: The PDQuest software detected 1,011 glycoprotein spots and 1,923 total protein spots in the 2-DE gels of sample from the normal human liver tissue. Furthermore, 116 species of glycoproteins were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and annotated to our databases. In addition, we also applied bioinformatics softwares to predict N- or O-glycosylation sites of identified glycoproteins. CONCLUSION: This study demonstrates the feasibility of a novel technological platform to contruct glycoprotein databases. These results lay the foundation for future physiological and pathological studies of the human liver.

[Cloning and expression of hTRF1 in Escherichia coli and preparation of polyclonal antibody].

Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.