A new high-throughput screening methodology for the discovery of cancer-testis antigen using multi-omics data.

BACKGROUND: Cancer/testis antigens (CTAs), also known as tumor-specific antigens (TSAs) are specifically expressed in cancer cells and exhibit high immunogenicity, making them promising targets for immunotherapy and cancer vaccines. METHODS: A new integrated high-throughput screening methodology for CTAs was proposed in this study through combining DNA methylation and RNA sequencing data. Briefly, the genes with increased transcript level and decreased DNA methylation were identified by multi-omics analysis. RNA sequencing studies in cell lines exposed to DNA methyltransferase (DNMT) inhibitors were performed to validate the inherent causal relationship between DNA hypomethylation and gene expression upregulation. RESULTS: We proposed a new integrated high-throughput screening methodology for identification of CTAs using multi-omics analysis. In addition, we tested the feasibility of this method using gastric cancer (GC) as an example. In GC, we identified over 2000 primary candidate CTAs and ultimately identified 20 CTAs with significant tissue-specificity, including a testis-specific serine protease TESSP1/PRSS41. Integrated analysis confirmed that PRSS41 expression was reactivated in gastrointestinal cancers by promoter DNA hypomethylation at the CpG site (cg08104780). Additionally, DNA hypomethylation of PRSS41 predicted a poor prognosis in GC. CONCLUSION: We propose a new high-throughput screening method for the identification of CTAs in cancer and validate its effectiveness. Our work emphasizes that serine protease PRSS41 is a novel TSA that is reactivated in GC due to promoter DNA hypomethylation.

Serine protease PRSS56, a novel cancer-testis antigen activated by DNA hypomethylation, promotes colorectal and gastric cancer progression via PI3K/AKT axis.

BACKGROUND: Cancer/testis (CT) antigens/genes are usually overexpressed in cancers and exhibit high immunogenicity, making them promising targets for immunotherapy and cancer vaccines. The role of serine protease PRSS56 in cancers remains unknown to date. METHODS: RNA sequencing studies were performed to screen CT genes in gastric cancer (GC) and colorectal cancer (CRC) cells exposed to DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AZA-CdR). Bioinformatics analysis was conducted to analyze the correlation between PRSS56 expression and DNA methylation. Functional experiments were performed to explore the biological function of PRSS56 in GC and CRC. RESULTS: In this study, we identified the testis-specific serine proteases PRSS56 as a novel CT antigen. PRSS56 was frequently overexpressed in various cancers, especially in gastrointestinal cancer. PRSS56 expression was negatively associated with promoter DNA methylation level, and positively associated with gene body methylation level. PRSS56 expression was significantly activated in colorectal and gastric cancer cells exposed to DNA methyltransferase inhibitors. Importantly, our finding highlights that the decreased methylation level of the CpG site cg10242318 in the PRSS56 promoter region resulted in its overexpression in GC and CRC. Additionally, functional assays verified that PRSS56 overexpression activated PI3K-AKT signaling in GC and CRC. CONCLUSION: Serine protease PRSS56 is a novel CT antigen that is reactivated in cancers by promoter DNA hypomethylation. PRSS56 functions oncogenic roles in GC and CRC by activating of PI3K/AKT axis. Our results presented here represent the first data on the function of the serine protease PRSS56 in cancers.

Heterogeneity and plasticity of epithelial-mesenchymal transition (EMT) in cancer metastasis: Focusing on partial EMT and regulatory mechanisms.

Epithelial-mesenchymal transition (EMT) or mesenchymal-epithelial transition (MET) plays critical roles in cancer metastasis. Recent studies, especially those based on single-cell sequencing, have revealed that EMT is not a binary process, but a heterogeneous and dynamic disposition with intermediary or partial EMT states. Multiple double-negative feedback loops involved by EMT-related transcription factors (EMT-TFs) have been identified. These feedback loops between EMT drivers and MET drivers finely regulate the EMT transition state of the cell. In this review, the general characteristics, biomarkers and molecular mechanisms of different EMT transition states were summarized. We additionally discussed the direct and indirect roles of EMT transition state in tumour metastasis. More importantly, this article provides direct evidence that the heterogeneity of EMT is closely related to the poor prognosis in gastric cancer. Notably, a seesaw model was proposed to explain how tumour cells regulate themselves to remain in specific EMT transition states, including epithelial state, hybrid/intermediate state and mesenchymal state. Additionally, this article also provides a review of the current status, limitations and future perspectives of EMT signalling in clinical applications.

Cancer-associated fibroblast-secreted IGFBP7 promotes gastric cancer by enhancing tumor associated macrophage infiltration via FGF2/FGFR1/PI3K/AKT axis.

We previously reported that IGFBP7 plays a role in maintaining mRNA stability of oncogenic lncRNA UBE2CP3 by RNA-RNA interaction in gastric cancer (GC). Clinical cohort studies had implied an oncogenic role of IGFBP7 in GC. However, the molecular mechanism of IGFBP7 in GC progression remains unknown. In this study, clinical analysis based on two independent cohorts showed that IGFBP7 was positively associated with poor prognosis and macrophage infiltration in GC. Loss-of-function studies confirmed the oncogenic properties of IGFBP7 in regulating GC cell proliferation and invasion. Mechanismly, IGFBP7 was highly expressed in cancer-associated fibroblasts (CAF) and mesenchymal cells, and was induced by epithelial-to-mesenchymal transition (EMT) signaling, since its expression was increased by TGF-beta treatment and reduced by overexpression of OVOL2 in GC. RNA sequencing, qRT-PCR, ELISA assay showed that IGFBP7 positively regulated FGF2 expression and secretion in GC. Transcriptome analysis revealed that FGFR1 was downregulated in M1 polarization but upregulated in M2 polarization. Exogenous recombinant IGFBP7 treatment in macrophages and GC cells further identified that IGFBP7 promotes tumor associated macrophage (TAM) polarization via FGF2/FGFR1/PI3K/AKT axis. Our finding here represented the first evidence that IGFBP7 promotes GC by enhancing TAM/M2 macrophage polarization through FGF2/FGFR1/PI3K/AKT axis.

Identification of cellular heterogeneity and key signaling pathways associated with vascular remodeling and calcification in young and old primate aortas based on single-cell analysis.

Aging of the vascular system is the main cause of many cardiovascular diseases. The structure and function of the blood vessel wall change with aging. To prevent age-related cardiovascular diseases, it is essential to understand the cellular heterogeneity of vascular wall and changes of cellular communication among cell subpopulations during aging. Here, using published single-cell RNA sequencing datasets of young and old monkey aortas, we analyzed the heterogeneity of vascular endothelial cells and smooth muscle cells in detail and identified a distinct endothelial cell subpopulation that involved in vascular remodeling and calcification. Moreover, cellular communication that changed with aging was analyzed and we identified a number of signaling pathways that associated with vascular aging. We found that EGF signaling pathway play an essential role in vascular remodeling and calcification of aged aortas. This work provided a better understanding of vascular aging and laid the foundation for prevention of age-related vascular pathologies.

Serine protease PRSS23 drives gastric cancer by enhancing tumor associated macrophage infiltration via FGF2.

Serine proteases has been considered to be closely associated with the inflammatory response and tumor progression. As a novel serine protease, the biological function of PRSS23 is rarely studied in cancers. In this study, the prognostic significance of PRSS23 was analyzed in two-independent gastric cancer (GC) cohorts. PRSS23 overexpression was clinically correlated with poor prognosis and macrophage infiltration of GC patients. Loss-of-function study verified that PRSS23 plays oncogenic role in GC. RNA-seq, qRT-PCR, western blotting and ELISA assay confirmed that serine protease PRSS23 positively regulated FGF2 expression and secretion. Single-cell analysis and gene expression correlation analysis showed that PRSS23 and FGF2 were high expressed in fibroblasts, and highly co-expressed with the biomarkers of tumor associated macrophages (TAMs), cancer-associated fibroblasts (CAFs) and mesenchymal cells. Functional analysis confirmed PRSS23/FGF2 was required for TAM infiltration. Rescue assay further verified that PRSS23 promotes GC progression and TAM infiltration through FGF2. Survival analysis showed that high infiltration of M1-macrophage predicted favorable prognosis, while high infiltration level of M2-macrophage predicted poor prognosis in GC. Our finding highlights that PRSS23 promotes TAM infiltration through regulating FGF2 expression and secretion, thereby resulting in a poor prognosis.

The EMT-induced lncRNA NR2F1-AS1 positively modulates NR2F1 expression and drives gastric cancer via miR-29a-3p/VAMP7 axis.

Deregulated lncRNAs play critical roles in tumorigenesis and tumor progression. NR2F1-AS1 is an antisense lncRNA of NR2F1. However, the biological function of NR2F1-AS1 in gastric cancer (GC) remains largely unclear. In this study, we revealed that NR2F1-AS1 and NR2F1 were both positively correlated with the degree of malignancy and predicted poor prognosis in two independent GC cohorts. Besides, NR2F1-AS1 and NR2F1 can respond to Epithelial-to-mesenchymal transition (EMT) signaling in GC, since their expression was increased by TGF-beta treatment and decreased after stable overexpression of OVOL2 in GC cell lines. NR2F1-AS1 and NR2F1 were highly co-expressed in pan-tissues and pan-cancers. Depletion of NR2F1-AS1 compromised the expression level of NR2F1 in GC cells. Furthermore, NR2F1-AS1 knockdown inhibited the proliferation, migration, invasion and G1/S transition of GC cells. More importantly, transcriptome sequencing revealed a novel ceRNA network composed of NR2F1-AS1, miR-29a-3p, and VAMP7 in GC. The overexpression of VAMP7 predicted poor prognosis in GC. Rescue assay confirmed that NR2F1-AS1 promotes GC progression through miR-29a-3p/VAMP7 axis. Our finding highlights that the aberrant expression of NR2F1-AS1 is probably due to the abnormal EMT signaling in GC. LncRNA NR2F1-AS1 plays crucial roles in GC progression by modulating miR-29a-3p/VAMP7 axis, suggesting that NR2F1-AS1 may serve as a potential therapeutic target in GC.

Genome-wide association studies identify miRNA-194 as a prognostic biomarker for gastrointestinal cancer by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5.

Background: The dysregulated genes and miRNAs in tumor progression can be used as biomarkers for tumor diagnosis and prognosis. However, the biomarkers for predicting the clinical outcome of gastrointestinal cancer (GIC) are still scarce. Methods: Genome-wide association studies were performed to screen optimal prognostic miRNA biomarkers. RNA-seq, Ago-HITS-CLIP-seq, western blotting and qRT-PCR assays were conducted to identify target genes of miR-194. Genome-wide CRISPR-cas9 proliferation screening analysis were conducted to distinguish passenger gene and driver gene. Results: A total of 9 prognostic miRNAs for GIC were identified by global microRNA expression analysis. Among them, miR-194 was the only one miRNA that significantly associated with overall survival, disease-specific survival and progress-free interval in both gastric, colorectal and liver cancers, indicating miR-194 was an optimal prognostic biomarker for GIC. RNA-seq analysis confirmed 18 conservative target genes of miR-194. Four of them, including ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5, were directly targeted by miR-194 and required for cell proliferation. Cell proliferation assay validated that miR-194 inhibits cell proliferation by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5 in GIC. Conclusion: In summary, miR-194 is an optimal biomarker for predicting the outcome of GIC. Our finding highlights that miR-194 exerts a tumor-suppressive role in digestive system cancers by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5.