Signature construction and molecular subtype identification based on liver-specific genes for prediction of prognosis, immune activity, and anti-cancer drug sensitivity in hepatocellular carcinoma.

BACKGROUND: Liver specific genes (LSGs) are crucial for hepatocyte differentiation and maintaining normal liver function. A deep understanding of LSGs and their heterogeneity in hepatocellular carcinoma (HCC) is necessary to provide clues for HCC diagnosis, prognosis, and treatment. METHODS: The bulk and single-cell RNA-seq data of HCC were downloaded from TCGA, ICGC, and GEO databases. Through unsupervised cluster analysis, LSGs-based HCC subtypes were identified in TCGA-HCC samples. The prognostic effects of the subtypes were investigated with survival analyses. With GSVA and Wilcoxon test, the LSGs score, stemness score, aging score, immune score and stromal score of the samples were estimated and compared. The HCC subtype-specific genes were identified. The subtypes and their differences were validated in ICGC-HCC samples. LASSO regression analysis was used for key gene selection and risk model construction for HCC overall survival. The model performance was estimated and validated. The key genes were validated for their heterogeneities in HCC cell lines with quantitative real-time PCR and at single-cell level. Their dysregulations were investigated at protein level. Their correlations with HCC response to anti-cancer drugs were estimated in HCC cell lines. RESULTS: We identified three LSGs-based HCC subtypes with different prognosis, tumor stemness, and aging level. The C1 subtype with low LSGs score and high immune score presented a poor survival, while the C2 subtype with high LSGs score and immune score indicated an enduring survival. Although no significant survival difference between C2 and C3 HCCs was shown, the C2 HCCs presented higher immune score and stroma score. The HCC subtypes and their differences were confirmed in ICGC-HCC dataset. A five-gene prognostic signature for HCC survival was constructed. Its good performance was shown in both the training and validation datasets. The five genes presented significant heterogeneities in different HCC cell lines and hepatocyte subclusters. Their dysregulations were confirmed at protein level. Furthermore, their significant associations with HCC sensitivities to anti-cancer drugs were shown. CONCLUSIONS: LSGs-based HCC subtype classification and the five-gene risk model might provide useful clues not only for HCC stratification and risk prediction, but also for the development of more personalized therapies for effective HCC treatment.

CapG promoted nasopharyngeal carcinoma cell motility involving Rho motility pathway independent of ROCK.

BACKGROUND: Gelsolin-like capping actin protein (CapG) modulates actin dynamics and actin-based motility with a debatable role in tumorigenic progression. The motility-associated functions and potential molecular mechanisms of CapG in nasopharyngeal carcinoma (NPC) remain unclear. METHODS: CapG expression was detected by immunohistochemistry in a cohort of NPC tissue specimens and by Western blotting assay in a variety of NPC cell lines. Loss of function and gain of function of CapG in scratch wound-healing and transwell assays were performed. Inactivation of Rac1 and ROCK with the specific small molecular inhibitors was applied to evaluate CapG's role in NPC cell motility. GTP-bound Rac1 and phosphorylated-myosin light chain 2 (p-MLC2) were measured in the ectopic CapG overexpressing cells. Finally, CapG-related gene set enrichment analysis was conducted to figure out the significant CapG-associated pathways in NPC. RESULTS: CapG disclosed increased level in the poorly differentiated NPC tissues and highly metastatic cells. Knockdown of CapG reduced NPC cell migration and invasion in vitro, while ectopic CapG overexpression showed the opposite effect. Ectopic overexpression of CapG compensated for the cell motility loss caused by simultaneous inactivation of ROCK and Rac1 or inactivation of ROCK alone. GTP-bound Rac1 weakened, and p-MLC2 increased in the CapG overexpressing cells. Bioinformatics analysis validated a positive correlation of CapG with Rho motility signaling, while Rac1 motility pathway showed no significant relationship. CONCLUSIONS: The present findings highlight the contribution of CapG to NPC cell motility independent of ROCK and Rac1. CapG promotes NPC cell motility at least partly through MLC2 phosphorylation and contradicts with Rac1 activation.

Multiomics-based analyses of KPNA2 highlight its multiple potentials in hepatocellular carcinoma.

Dysregulation and prognostic roles of Karyopherin α2 (KPNA2) were reported in many malignancies including hepatocellular carcinoma (HCC). A multi-omics analysis of KPNA2 is needed to gain a deeper understanding of its multilevel molecular characteristics and provide novel clues for HCC diagnosis, prognosis, and target therapy. Herein multi-omic alterations of KPNA2 were analyzed at genetic, epigenetic, transcript, and protein levels with evaluation of their relevance with clinicopathological features of HCC by integrative analyses. The significant correlations of KPNA2 expression with its gene copy number variation (CNV) and methylation status were shown through Spearman correlation analyses. With Cox regression, Kaplan-Meier survival, and receiver operating characteristic (ROC) analyses, based on the factors of KPNA2 CNV, methylation, expression, and tumor stage, risk models for HCC overall survival (OS) and disease-free survival (DFS) were constructed which could discriminate the 1-year, 3-year, and 5-year OS/DFS status effectively. With Microenvironment Cell Populations-counter (MCP-counter), the immune infiltrations of HCC samples were evaluated and their associations with KPNA2 were shown. KPNA2 expression in liver was found to be influenced by low fat diet and presented significant correlations with fatty acid metabolism and fatty acid synthase activity in HCC. KPNA2 was detected lowered in HCC patient's plasma by enzyme linked immunosorbent assay (ELISA), consistent with its translocation to nuclei of HCC cells. In conclusion, KPNA2 multilevel dysregulation in HCC and its correlations with immune infiltration and the fatty acid metabolism pathway indicated its multiple roles in HCC. The clinicopathological significance of KPNA2 was highlighted through the in-depth analyses at multilevels.

Correction to: Mass spectrometry-based proteomic capture of proteins bound to the MACC1 promoter in colon cancer.

In the original publication of the article, Acknowledgements section was published incorrectly. The correct Acknowledgements is given in this Correction.

Mass spectrometry-based proteomic capture of proteins bound to the MACC1 promoter in colon cancer.

MACC1 (metastasis associated in colon cancer 1) is a key driver that induces metastasis in colon cancer. However, the mechanisms by which MACC1 expression is transcriptionally regulated and the factors enriched at the MACC1 promoter remain largely unknown. The binding of proteins to specific DNA sites in the genome is a major determinant of genomic maintenance and the regulation of specific genes. The study herein utilized two methods to study the binding proteins of the MACC1 promoter region in colon cancer. Specifically, we adopted CRISPR-based chromatin affinity purification with mass spectrometry (CRISPR-ChAP-MS) and a biotin-streptavidin pulldown assay coupled with MS to identify the specific proteome bound to the MACC1 promoter in two colon cell lines with different metastatic potential. A total of 24 proteins were identified by CRISPR-ChAP-MS as binding to the MACC1 promoter, among which c-JUN was validated by ChIP-PCR. A total of 739 binding protein candidates were identified by biotin-streptavidin pulldown assays coupled with MS, of which HNF4G and PAX6 were validated and compared for their binding to the same promoter sites in the two cell lines. Our studies suggest distinctive proteomic factors associated with the MACC1 promoter in colon cells with different metastatic potential. The dynamic regulatory factors accumulated at the promoter of MACC1 may provide novel insights into the regulatory mechanisms of MACC1 transcription.

Deciphering nasopharyngeal carcinoma pathogenesis via proteomics.

Introduction: Nasopharyngeal carcinoma (NPC) is a distinct head and neck squamous cell carcinoma in its etiological association of Epstein-Barr virus (EBV) infection, hidden anatomical location, remarkable racial and geographical distribution, and high incidence of locoregional recurrence or metastasis. Thanks to the advancements in proteomics in recent decades, more understanding of the disease etiology, carcinogenesis, and progression has been gained, potentially deciphering the molecular characteristics of the malignancy. Areas covered: In this review, we provide an overview of the proteomic aberrations that are likely involved or drive NPC development and progression, focusing on the contributions of major EBV-encoded factors, intercommunication with environment, protein features of high metastasis and therapy resistance, and protein-protein interactions that allow NPC cells to evade immune recognition and elimination. Finally, multistep carcinogenesis and subtypes of NPC from a proteomic perspective are inquired. Expert commentary: Proteomic studies have covered various aspects involved in NPC pathogenesis, yet much remains to be uncovered. Coherent study designs, optimal conditions for obtaining high-quality data, and compelling interpretation are critical in ensuring the emergence of good science out of NPC proteomics. NPC proteogenomics and proteoform analysis are two promising fields to promote the application of the proteomic findings from bench to bedside.

Quantitative proteomic profiling of tumor-associated vascular endothelial cells in colorectal cancer.

To investigate the global proteomic profiles of vascular endothelial cells (VECs) in the tumor microenvironment and antiangiogenic therapy for colorectal cancer (CRC), matched pairs of normal (NVECs) and tumor-associated VECs (TVECs) were purified from CRC tissues by laser capture microdissection and subjected to iTRAQ-based quantitative proteomics analysis. Here, 216 differentially expressed proteins (DEPs) were identified and used for bioinformatics analysis. Interestingly, these proteins were implicated in epithelial mesenchymal transition (EMT), ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, angiogenesis and HIF-1 signaling pathway, which may play important roles in CRC angiogenesis. Among these DEPs we found that Tenascin-C (TNC) was upregulated in TVECs of CRC and correlated with CRC multistage carcinogenesis and metastasis. Furthermore, the reduction of tumor-derived TNC could attenuate human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation through ITGB3/FAK/Akt signaling pathway. Based on the present work, we provided a large-scale proteomic profiling of VECs in CRC with quantitative information, a certain number of potential antiangiogenic targets and a novel vision in the angiogenesis bio-mechanism of CRC.

MiR-101-3p inhibits EMT to attenuate proliferation and metastasis in glioblastoma by targeting TRIM44.

BACKGROUND: Glioblastoma (GBM) is the most common malignant tumor originating in the brain parenchyma. The invasive and infiltrative properties of glioblastoma result in poor clinical prognosis to conventional therapies. Emerging reports on microRNAs as important regulators during the process of EMT provide new insights into treating glioblastoma through new targets. However, underlying molecular mechanism of the regulation of miR-101-3p in glioblastoma remains unclear. METHODS: Level of miR-101-3p was determined in GBM cell lines by qRT-PCR. MTT, colony formation and transwell assays were utilized to evaluate functions of overexpression of miR-101-3p/knock down of TRIM44 on proliferation, migration and invasion in GBM cells. Direct interaction between miR-101-3p and TRIM44 was validated using dual luciferase reporter system and impacts of overexpression of miR-101-3p/knock down of TRIM44 on regulation of EMT markers were assessed by Western blotting. RESULTS: MiR-101-3p was validated to be repressed expressed in glioblastoma cancer cell lines. Both overexpression of miR-101-3p and knock down of TRIM44 attenuated proliferation, migration and invasion of glioblastoma cell lines in vitro. TRIM44 was shown to promote EMT in GBM progress and reverse inhibitory function of miR-101-3p. MiR-101-3p was found to suppress the expression of TRIM44 via directly targeting its 3'UTR. CONCLUSIONS: Our findings suggested miR-101-3p regulated proliferation and migration of glioblastoma cells through attenuating TRIM44 induced EMT via direct targeting 3'UTR of TRIM44, which provided preliminary study of potential therapeutic target in future GBM treatment.

Proteomic Characterization Reveals a Molecular Portrait of Nasopharyngeal Carcinoma Differentiation.

Nasopharyngeal carcinoma (NPC) is categorized into three different differentiated subtypes by World Health Organization (WHO). Based on an earlier comparative proteomic database of the three histological subtypes, the study was to deepen our understanding of molecular mechanisms associated with NPC differentiation through bio-information mining. Among the three subtypes were 194 differentially expressed proteins (DEPs) of 725 identified proteins. Two DEPs, heat shock protein family B (small) member 1 (HSPB1) and keratin 5 (KRT5), were validated in a series of NPC tissue samples by using immunohistochemistry. Quantified protein families including keratins, S100 proteins (S100s) and heat shock proteins exhibited characteristic expression alterations. Comparisons of predicted bio-function activation states among different subtypes, including formation of cellular protrusion, metastasis, cell death, and viral infections, were conducted. Canonical pathway analysis inferred that Rho GTPases related signaling pathways regulated the motility and invasion of dedifferentiated NPC. In conclusion, the study explored the proteomic characteristics of NPC differentiation, which could deepen our knowledge of NPC tumorigenesis and allow the development of novel targets of therapeutic and prognostic value in NPC.

Identifying DCN and HSPD1 as Potential Biomarkers in Colon Cancer Using 2D-LC-MS/MS Combined with iTRAQ Technology.

Colon cancer is one of the most common types of gastrointestinal cancers and the fourth cause of cancer death worldwide. To discover novel diagnostic biomarkers for colon cancer and investigate potential mechanisms of oncogenesis, quantitative proteomic approach using iTRAQ-tagging and 2D-LC-MS/MS was performed to characterize proteins alterations in colon cancer and non-neoplastic colonic mucosa (NNCM) using laser capture microdissection-harvested from the two types of tissues, respectively. As a result, 188 DEPs were identified, and the differential expression of two DEPs (DCN and HSPD1) was further verified by Western blotting and immunohistochemistry. KEGG pathway analysis disclosed that the DEPs were related to signaling pathways associated with cancer; furthermore, DCN and HSPD1 are in the relative central hub position among protein-protein interaction subnetwork of the DEPs. The results not only shed light on the mechanism by the DEPs contributed to colonic carcinogenesis, but also showed that DCN and HSPD1 are novel potential biomarkers for the diagnosis of colon cancer.

Identification of stromal differentially expressed proteins in the colon carcinoma by quantitative proteomics.

Tumor microenvironment plays very important roles in the carcinogenesis. A variety of stromal cells in the microenvironment have been modified to support the unique needs of the malignant state. This study was to discover stromal differentially expressed proteins (DEPs) that were involved in colon carcinoma carcinogenesis. Laser capture microdissection (LCM) was captured and isolated the stromal cells from colon adenocarcinoma (CAC) and non-neoplastic colon mucosa (NNCM) tissues, respectively. Seventy DEPs were identified between the pooled LCM-enriched CAC and NNCM stroma samples by iTRAQ-based quantitative proteomics. Gene Ontology (GO) relationship analysis revealed that DEPs were hierarchically grouped into 10 clusters, and were involved in multiple biological functions that were altered during carcinogenesis, including extracellular matrix organization, cytoskeleton, transport, metabolism, inflammatory response, protein polymerization, and cell motility. Pathway network analysis revealed 6 networks and 56 network eligible proteins with Ingenuity pathway analysis. Four significant networks functioned in digestive system development and its function, inflammatory disease, and developmental disorder. Eight DEPs (DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ) were validated by Western blotting, and four DEPs (DCN, FN1, PKM2, and HSP90B1) were validated by immunohistochemical analysis. It is the first report of stromal DEPs between CAC and NNCM tissues. It will be helpful to recognize the roles of stromas in the colon carcinoma microenvironment, and improve the understanding of carcinogenesis in colon carcinoma. The present data suggest that DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ might be the potential targets for colon cancer prevention and therapy.

Mitochondrial proteomics of nasopharyngeal carcinoma metastasis.

BACKGROUND: Mitochondrial proteomic alterations of nasopharyngeal carcinoma metastasis remain unknown. Our purpose is to screen mitochondrial proteins for the elucidation of the molecular mechanisms of nasopharyngeal carcinoma metastasis and the discovery of metastasis-related biomarkers. METHODS: Mitochondria were isolated from nasopharyngeal carcinoma metastatic (5-8F) and nonmetastatic (6-10B) cell lines, respectively. After characterization of isolated mitochondria, mitochondrial differentially expressed proteins (DEPs) were quantified by two-dimensional difference in-gel electrophoresis (2D-DIGE), and identified by peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). A functional enrichment analysis and a protein-protein interaction sub-network analysis for DEPs were carried out with bioinformatics. Furthermore, siRNAs transient transfections were used to suppress expressions of some up-regulated DEPs in metastatic cells (5-8F), followed by Transwell Migration assay. RESULTS: Sixteen mitochondrial DEPs including PRDX3 and SOD2 were identified. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity. A protein-protein interaction sub-network of DEPs was generated with literature data. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 were rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis. CONCLUSIONS: Sixteen mitochondrial DEPs were identified with mass spectrometry and ten of them were rationalized in the tumor-stroma co-evolution model. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.

Cancer: a proteomic disease.

The development of cancer is a pathological process involving multiple environmental carcinogenic factors and genetic alterations. For decades, cancer researchers have focused on genomic and transcriptomic analyses. The completion of the Human Genome Project has opened the door to the post-genome era and oncoproteomics. Proteins play a critical role in tumorigenesis and influence the differences between normal cells and malignant cells. This report proposes the concept that cancer is a proteomic disease. This concept is based on examining protein expression profiles, post-translational modifications, and protein-protein interactions in carcinogenesis using recent advances in comparative, functional and structural proteomics. This approach provides a new way of viewing carcinogenesis, presents new clues in biomarker discovery for cancer diagnosis and therapy, and reveals important scientific findings and their significance to clinical applications.

Cloning and characterization of the NPCEDRG gene promoter.

NPCEDRG is a novel tumor suppressive gene that localizes to 3p21.3, a chromosomal region frequently associated with loss of heterozygosity (LOH) in a number of malignancies including nasopharyngeal carcinoma (NPC). Its transcriptional down-expression has been shown in the cell lines and primary tumor tissues of NPC. Reintroduction of NPCEDRG into CNE2, a cell line derived from NPC, was effective to induce cell differentiation, control cell growth, and regulate the cell cycle. Little is known about the transcriptional mechanisms controlling NPCEDRG gene expression. In this article, we describe the NPCEDRG gene structure and the transcriptional expression of NPCEDRG; we found that NPCEDRG was expressed weakly in most of NPC cell lines. Using 5' rapid amplification of complementary DNA ends (5'-RACEs), we found that the NPCEDRG gene has several transcription start sites (TSSs) due to the existence of alternatively spliced variants, and the specific TSS of NPCEDRG was located -25 nucleotides upstream of the translation start site. We amend that Human NPCEDRG CDS containing 516 bp but not the 510 bp reported previously. To characterize the NPCEDRG promoter, transient luciferase and/or EGFP reporter assay were carried out with the constructs including various lengths of the 5' flanking region of the NPCEDRG gene. The results demonstrated that the basal promoter is located at the region from -215 to -8 nucleotides, and the optimal promoter is located at the region from -625 to -8 nucleotides upstream of the translation start site. In silico analysis suggested that the promoter region contained potential binding sites for SP1, c-Myb, AREB6, Nkx2-5, and so on. These results provide important clues to elucidate the regulation of NPCEDRG gene expression and function. Further studies are apparently required for the identification of the transcription factors, essential for NPCEDRG expression, which would lead to better understanding of the molecular mechanism of NPCEDRG expression in nasopharyngeal epithelial cells.

Quantitative proteomic analysis of formalin-fixed and paraffin-embedded nasopharyngeal carcinoma using iTRAQ labeling, two-dimensional liquid chromatography, and tandem mass spectrometry.

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method.

Triosephosphate isomerase and peroxiredoxin 6, two novel serum markers for human lung squamous cell carcinoma.

There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up-regulation of triosephosphate isomerase were observed in lung squamous cell carcinoma, and autoantibodies against triosephosphate isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up-regulated triosephosphate isomerase and peroxiredoxin 6 were further validated by enzyme-linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both triosephosphate isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between triosephosphate isomerase and distant metastasis was found. At the cut-off point 0.221 (optical density value) on the receiver operating characteristic curve, triosephosphate isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut-off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both triosephosphate isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that triosephosphate isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma.