Human Pancreatic Cancer Tissues Research Articles

Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

Background and AimsEfforts to decode pancreatic ductal adenocarcinoma (PDAC) heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in PDAC progression. MethodsGlobal DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss- and gain-of function studies, and in vivo xenograft and the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in three different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. ResultsWe identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients, indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. ConclusionsHNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.

The DDX6/KIFC1 signaling axis, as regulated by YY1, contributes to the malignant behavior of pancreatic cancer.

Human DEAD/H box RNA helicase DDX6 acts as an oncogene in several different types of cancer, where it participates in RNA processing. Nevertheless, the role of DDX6 in pancreatic cancer (PC), together with the underlying mechanism, has yet to be fully elucidated. In the present study, compared with adjacent tissues, the level of DDX6 was abnormally increased in human PC tissues, and this increased level of expression was associated with poor prognosis. Furthermore, the role of DDX6 in PC was investigated by overexpressing or silencing the DDX6 in the PC cell lines, SW1990 and PaTu-8988t. A xenograft model was established by injecting nude mice with either DDX6-overexpressing or DDX6-silenced SW1990 cells. DDX6 overexpression promoted the proliferation and cell cycle transition, inhibited the cell apoptosis of PC cells, and accelerated tumor formation, whereas DDX6 knockdown elicited the opposite effects. DDX6 exerted positive effects on PC. RNA immunoprecipitation assay showed that DDX6 bound to kinesin family member C1 (KIFC1) mRNA, which was further confirmed by RNA pull-down assay. These results suggested that DDX6 positively regulated the expression of KIFC1. KIFC1 overexpression enhanced the proliferative capability of PC cells with DDX6 knockdown and inhibited their apoptosis. By contrast, DDX6 overexpression reversed the inhibitory effect of KIFC1 silencing on tumor proliferation. Subsequently, the transcription factor Yin Yang 1 (YY1) was shown to negatively regulate DDX6 at both the mRNA and protein levels. Dual-luciferase reporter assay verified that YY1 targeted the promoter of DDX6 and inhibited its transcription. High expression levels of YY1 decreased the proliferation of PC cells and promoted cell apoptosis, although these effects were reversed by DDX6 overexpression. Taken together, YY1 may target the DDX6/KIFC1 axis, thereby negatively regulating its expression, leading to an inhibitory effect on pancreatic tumor.

Identification of FOXP3+ epithelial cells contributing to pancreatic proliferation and angiogenesis.

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues.NEW & NOTEWORTHY In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology.

Stereoselective Synthesis of Sialyl Lewisa Antigen and the Effective Anticancer Activity of Its Bacteriophage Qβ Conjugate as an Anticancer Vaccine

AbstractSialyl Lewisa (sLea), also known as cancer antigen 19‐9 (CA19‐9), is a tumor‐associated carbohydrate antigen. The overexpression of sLea on the surface of a variety of cancer cells makes it an attractive target for anticancer immunotherapy. However, sLea‐based anticancer vaccines have been under‐explored. To develop a new vaccine, efficient stereoselective synthesis of sLea with an amine‐bearing linker was achieved, which was subsequently conjugated with a powerful carrier bacteriophage, Qβ. Mouse immunization with the Qβ‐sLea conjugate generated strong and long‐lasting anti‐sLea IgG antibody responses, which were superior to those induced by the corresponding conjugate of sLea with the benchmark carrier keyhole limpet hemocyanin. Antibodies elicited by Qβ‐sLea were highly selective toward the sLea structure, could bind strongly with sLea‐expressing cancer cells and human pancreatic cancer tissues, and kill tumor cells through complement‐mediated cytotoxicity. Furthermore, vaccination with Qβ‐sLea significantly reduced tumor development in a metastatic cancer model in mice, demonstrating tumor protection for the first time by a sLea‐based vaccine, thus highlighting the significant potential of sLea as a promising cancer antigen.

Stereoselective Synthesis of Sialyl Lewisa Antigen and the Effective Anticancer Activity of Its Bacteriophage Qβ Conjugate as an Anticancer Vaccine.

Sialyl Lewisa (sLea ), also known as cancer antigen 19-9 (CA19-9), is a tumor-associated carbohydrate antigen. The overexpression of sLea on the surface of a variety of cancer cells makes it an attractive target for anticancer immunotherapy. However, sLea -based anticancer vaccines have been under-explored. To develop a new vaccine, efficient stereoselective synthesis of sLea with an amine-bearing linker was achieved, which was subsequently conjugated with a powerful carrier bacteriophage, Qβ. Mouse immunization with the Qβ-sLea conjugate generated strong and long-lasting anti-sLea IgG antibody responses, which were superior to those induced by the corresponding conjugate of sLea with the benchmark carrier keyhole limpet hemocyanin. Antibodies elicited by Qβ-sLea were highly selective toward the sLea structure, could bind strongly with sLea -expressing cancer cells and human pancreatic cancer tissues, and kill tumor cells through complement-mediated cytotoxicity. Furthermore, vaccination with Qβ-sLea significantly reduced tumor development in a metastatic cancer model in mice, demonstrating tumor protection for the first time by a sLea -based vaccine, thus highlighting the significant potential of sLea as a promising cancer antigen.

Collagen XVII regulates tumor growth in pancreatic cancer through interaction with the tumor microenvironment.

Expression of the gene for collagen XVII (COL17A1) in tumor tissue is positively or negatively associated with patient survival depending on cancer type. High COL17A1 expression is thus a favorable prognostic marker for breast cancer but unfavorable for pancreatic cancer. This study explored the effects of COL17A1 expression on pancreatic tumor growth and their underlying mechanisms. Analysis of published single-cell RNA-sequencing data for human pancreatic cancer tissue revealed that COL17A1 was expressed predominantly in cancer cells rather than surrounding stromal cells. Forced expression of COL17A1 did not substantially affect the proliferation rate of the mouse pancreatic cancer cell lines KPC and AK4.4 in vitro. However, in mouse homograft tumor models in which KPC or AK4.4 cells were injected into syngeneic C57BL/6 or FVB mice, respectively, COL17A1 expression promoted or suppressed tumor growth, respectively, suggesting that the effect of COL17A1 on tumor growth was influenced by the tumor microenvironment. RNA-sequencing analysis of tumor tissue revealed effects of COL17A1 on gene expression profiles (including the expression of genes related to cell proliferation, the immune response, Wnt signaling, and Hippo signaling) that differed between C57BL/6-KPC and FVB-AK4.4 tumors. Our data thus suggest that COL17A1 promotes or suppresses cancer progression in a manner dependent on the interaction of tumor cells with the tumor microenvironment.

Similarity of oncogenic protein expression in KRASG12D gene delivery-based rat pancreatic cancer model to that of human pancreatic cancer

BackgroundThe development of effective therapies and biomarkers for pancreatic cancer is an unmet clinical need. To address this, we have developed an easy-to-use pancreatic cancer rat animal model via pancreas-targeted hydrodynamic gene delivery of human pancreatic cancer-related genes. Our study aimed to determine the molecular similarity between the pancreatic tumor in the rat model and human pancreatic cancer. MethodsKRASG12D gene-expressing plasmid was delivered to the pancreas of wild type rats via pancreas-targeted hydrodynamic gene delivery as previously reported. Tissue samples were collected at 5 weeks after the first gene delivery. The tumors developed in the rats were assessed for the expression of oncogenic proteins that are involved in human pancreatic cancer development. ResultsThe development of a tumor mimicking pancreatic ductal adenocarcinoma was confirmed. The expression levels of Cyclin D1, c-Jun, IL-33, and Zip4 proteins in the tumor were immunohistochemically assessed and the correlation of the proteins was confirmed. The expression pattern showed similarity to that of surgically resected human pancreatic cancer tissues. ConclusionsOur study findings showing a similar pattern of oncogenic protein expression in novel KRASG12D gene-induced rat pancreatic cancer model and human pancreatic cancer will be useful for establishing novel tumor markers and therapeutic options for pancreatic cancer.

B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with β-catenin

Abnormal activation of Wnt/β-catenin-mediated transcription is closely associated with the malignancy of pancreatic cancer. Family with sequence similarity 83 member A (FAM83A) was shown recently to have oncogenic effects in a variety of cancer types, but the biological roles and molecular mechanisms of FAM83A in pancreatic cancer need further investigation. Here, we newly discovered that FAM83A binds directly to β-catenin and inhibits the assembly of the cytoplasmic destruction complex thus inhibiting the subsequent phosphorylation and degradation. FAM83A is mainly phosphorylated by the SRC non-receptor kinase family member BLK (B-lymphoid tyrosine kinase) at tyrosine 138 residue within the DUF1669 domain that mediates the FAM83A-β-catenin interaction. Moreover, FAM83A tyrosine 138 phosphorylation enhances oncogenic Wnt/β-catenin-mediated transcription through promoting β-catenin-TCF4 interaction and showed an elevated nucleus translocation, which inhibits the recruitment of histone deacetylases by TCF4. We also showed that FAM83A is a direct downstream target of Wnt/β-catenin signaling and correlates with the levels of Wnt target genes in human clinical pancreatic cancer tissues. Notably, the inhibitory peptides that target the FAM83A-β-catenin interaction significantly suppressed pancreatic cancer growth and metastasis in vitro and in vivo. Our results revealed that blocking the FAM83A cascade signaling defines a therapeutic target in human pancreatic cancer.

Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.

Identification of EMT-related alternative splicing event of TMC7 to promote invasion and migration of pancreatic cancer.

Epithelial-to-mesenchymal transition (EMT) is tightly associated with the invasion and metastasis of pancreatic cancer with rapid progression and poor prognosis. Notably, gene alternative splicing (AS) event plays a critical role in regulating the progression of pancreatic cancer. Therefore, this study aims to identify the EMT-related AS event in pancreatic cancer. The EMT-related gene sets, transcriptomes, and matched clinical data were obtained from the MSigDB, The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO) databases. Key gene AS events associated with liver metastasis were identified by prognostic analysis, gene set variation analysis (GSVA), and correlation analysis in pancreatic cancer. The cell line and organoid model was constructed to evaluate these key gene AS events in regulating pancreatic cancer in vitro. Furthermore, we established an EMT-related gene set consisting of 13 genes by prognostic analysis, the role of which was validated in two other databases. Finally, the human pancreatic cancer tissue and organoid model was used to evaluate the correlation between the enrichment of this gene set and liver metastasis. Prognostic analysis and correlation analysis revealed that eight AS events were closely associated with the prognosis of pancreatic cancer. Furthermore, the expression of TMC7 and CHECK1 AS events was increased in the metastatic lesions of the human tissue and organoid model. Additionally, the knockdown of exon 17 of TMC7 significantly inhibited the proliferation, invasion, and migration of pancreatic cancer cells in 2D and 3D cell experiments. Finally, the expression of exon 17 of TMC17 exhibited a significant correlation with the poor prognosis in pancreatic ductal adenocarcinoma (PDAC). The AS events of TMC7 and CHECK1 were associated with liver metastasis in pancreatic cancer. Moreover, exon 17 of TMC7 could be a potential therapeutic target in pancreatic cancer.

Hypoxia activated HGF expression in pancreatic stellate cells confers resistance of pancreatic cancer cells to EGFR inhibition.

Epidermal growth factor receptor (EGFR) is an essential target for cancer treatment. However, EGFR inhibitor erlotinib showed limited clinical benefit in pancreatic cancer therapy. Here, we showed the underlying mechanism of tumor microenvironment suppressing the sensitivity of EGFR inhibitor through the pancreatic stellate cell (PSC). The expression of alpha-smooth muscle actin (α-SMA) and hypoxia marker in human pancreatic cancer tissues were detected by immunohistochemistry, and their correlation with overall survival was evaluated. Human immortalized PSC was constructed and used to investigate the potential effect on pancreatic cancer cell lines in hypoxia and normoxia. Luciferase reporter assay and Chromatin immunoprecipitation were performed to explore the potential mechanisms invitro. The combined inhibition of EGFR and Met was evaluated in an orthotopic xenograft mouse model of pancreatic cancer. We found that high expression levels of α-SMA and hypoxia markers are associated with poor prognosis of pancreatic cancer patients. Mechanistically, we demonstrated that hypoxia induced the expression and secretion of HGF in PSC via transcription factor HIF-1α. PSC-derived HGF activates Met, the HGF receptor, suppressing the sensitivity of pancreatic cancer cells to EGFR inhibitor in a KRAS-independent manner by activating the PI3K-AKT pathway. Furthermore, we found that the combination of EGFR inhibitor and Met inhibitor significantly suppressed tumor growth in an orthotopic xenograft mouse model. Our study revealed a previously uncharacterized HIF1α-HGF-Met-PI3K-AKT signaling axis between PSC and cancer cells and indicated that EGFR inhibition plus Met inhibition might be a promising strategy for pancreatic cancer treatment. This study was supported by The National Natural Science Foundation of China.

Characterization of antibody clones that bind exclusively to insoluble fibrin.

Previously, we established an antibody, termed 102-10, which recognizes insoluble fibrin exclusively, unlike the previously established anti-insoluble fibrin antibodies that also cross-reacted with fibrinogen. We established that the epitope of this antibody is on the β chain that lines an indented structure that becomes exposed only when insoluble fibrin is formed. The amino acid sequence of the epitope is completely conserved from mouse to humans. This study attempted to determine the most suitable insoluble fibrin clone for future diagnostic and therapeutic development. Binding kinetics and properties of antibodies were evaluated by the surface plasmon resonance analysis (SPR) and ELISA among 1101, 99, 443, and 102-10. Immunohistochemical staining for mouse and human pancreatic cancer tissues were also performed. For frozen sections, visually appropriate staining results were observed at an antibody concentration of 1-10 μg/ml, while for paraffin sections, 10 μg/ml was required. From immunohistochemistry and ELISA analyses, clone 99 and clone 1101 showed almost no nonspecific binding in normal pancreatic tissues. Hybridoma production for 1101 yielded more antibodies than that of 99 and demonstrated good long-term stability. It was, therefore, concluded that clone 1101 would be useful for future clinical development as well as basic research.

RHBDL2 promotes the proliferation, migration, and invasion of pancreatic cancer by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway

Pancreatic cancer (PC) is one of the most malignant types of cancer, and is characterized by early metastasis, limited response to chemotherapeutics, and poor prognosis. Therefore, there is an urgent need to explore new therapeutic strategies for PC treatment. Human rhomboid-like 2 (RHBDL2) is differentially expressed in cervical and breast cancer. However, the correlation between RHBDL2 and PC remains unclear. We found that RHBDL2 is highly expressed in human PC cells and tissues and is significantly associated with distant metastasis and poor survival of patients with PC. Gain- and loss-of-function assays indicated that RHBDL2 could accelerate PC cell proliferation and mobility in vitro and in vivo. The RNA-Seq results suggest that RHBDL2 may be involved in the activation of Notch signaling pathway. IMR-1 could restore the proliferation and metastatic capacity of PC cells mediated by RHBDL2. RHBDL2 interacted with and cleaved Notch1, resulting in the release of N1ICD. RHBDL2 decreased the ubiquitination level of N1ICD and collaborated with Ovarian tumor domain-containing 7B (OTUD7B) to stabilize N1ICD via the ubiquitin-proteasome pathway. RHBDL2 facilitated PC cell proliferation and mobility by stabilizing the N1ICD via the OTUD7B and activating the Notch signaling pathway. Thus, targeting this novel pathway may be a potential therapeutic strategy for PC.

Increased expression of METTL3 in pancreatic cancer tissues associates with poor survival of the patients

BackgroundMethyltransferase-like 3 (METTL3) expression could be found in various normal and cancerous tissues. As of now, the clinical significance of METTL3 expression in human pancreatic cancer (PC) tissues still remains to be understood. Our present study aims to investigate the prognostic value and clinical implications of METTL3 expression in PC tissues.MethodsThe TCGA, GTEx, and GEO public databases were used to study the mRNA expression level of the m6A family members and its relationship among PC tissues and normal pancreatic tissue. The immunohistochemistry was used to analyze the difference of METTL3 expression between cancer tissues and adjacent normal tissues. The prognostic value was evaluated by using the Log-rank survival analysis and Cox model analysis. PAAD samples from TCGA and GEO databases were used to perform the immune infiltration analysis and gene set enrichment analysis based on the genes that were highly correlated with METTL3.ResultsBased on the analysis of TCGA, GTEx, and GEO public database, we found that the m6A family members showed a higher correlation in PC tissues compared to normal pancreatic tissues, and the mRNA expression level of the m6A family members showed a significant difference between PC tissues and adjacent normal tissues. Moreover, scRNA-seq data indicated that METTL3 showed a higher expression level in malignant epithelial cells. Our immunohistochemistry results also confirmed that the intensity of METTL3 immunostaining in PC tissues was significantly higher than that in adjacent normal tissues (P = 0.015). The overall survival (OS) of PC patients with high expression of METTL3 protein were significantly poorer than those with low expression of METTL3 protein (HR = 1.788, 95% CI 1.071–2.984, P = 0.026). Further analysis of PC data from the database showed that METTL3 expression was associated with a variety of tumor-infiltrating immune cells and was involved in m6A modification and metabolism in PC tissues.ConclusionIncreased METTL3 expression at the protein level could be found in PC tissues, suggesting that the METTL3 expression was involved in the progression of PC and could serve as an important marker for prognostic prediction of this malignancy.

Label-free differentiation of human pancreatic cancer, pancreatitis, and normal pancreatic tissue by molecular spectroscopy.

.Significance: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths with a best median survival of only 40 to 50 months for localized disease despite multimodal treatment. The standard tissue differentiation method continues to be pathology with histological staining analysis. Microscopic discrimination between inflammatory pancreatitis and malignancies is demanding.Aim: We aim to accurately distinguish native pancreatic tissue using infrared (IR) spectroscopy in a fast and label-free manner.Approach: Twenty cryopreserved human pancreatic tissue samples were collected from surgical resections. In total, more than 980,000 IR spectra were collected and analyzed using a MATLAB package. For differentiation of PDAC, pancreatitis, and normal tissue, a three-class training set for supervised classification was created with 25,000 spectra and the principal component analysis (PCA) score values for each cohort. Cross-validation was performed using the leave-one-out method. Validation of the algorithm was accomplished with 13 independent test samples.Results: Reclassification of the training set and the independent test samples revealed an overall accuracy of more than 90% using a discrimination algorithm.Conclusion: IR spectroscopy in combination with PCA and supervised classification is an efficient analytical method to reliably distinguish between benign and malignant pancreatic tissues. It opens up a wide research field for oncological and surgical applications.

YOD1 serves as a potential prognostic biomarker for pancreatic cancer

BackgroundUbiquitination is a basic post-translational modification of intracellular proteins and can be reversed enzymatically by DUBs (deubiquitinating enzymes). More than 90 DUBs have been identified. Among them, the deubiquitinating enzyme YOD1, a member of the ovarian tumor domain protease (OTUs) subfamily, is involved in the regulation of endoplasmic reticulum (ER)-related degradation pathways. In fact, it is reported that YOD1 is an important proliferation and metastasis-inducing gene, which can stimulate the characteristics of cancer stem cells and maintain circulating tumor cells (CTC). However, the expression level, prognostic effect and biological functional mechanism of YOD1 in pancreatic cancer are still unclear.ResultsIn the GEO and TCGA databases, YOD1 mRNA expression is significantly up regulated in a variety of human pancreatic cancer tissues. Survival analysis showed that the up regulation of YOD1 can predict poor prognosis of pancreatic cancer. Cox analysis showed that high YOD1 expression is an independent prognostic factor of pancreatic cancer. ROC analysis shows that YOD1 has significant diagnostic value. The immunohistochemistry (IHC) results showed that the protein expression level of YOD1 in pancreatic cancer tissue was higher than that in neighboring non-pancreatic cancer tissues (P < 0.001). In addition, we found that YOD1 expression is negatively correlated with the infiltration level of CD8 + T cells, macrophages, neutrophils and dendritic cells (DC) in pancreatic cancer. The expression of YOD1 has a strong correlation with the different immune marker sets in PAAD. Co-expression network and functional enrichment analysis indicate that YOD1 may participate in the development of pancreatic cancer through cell adhesion molecules, p53, Hippo, TGF-β and other pathways. The experimental results of EDU, Transwell, Immunohistochemistry (IHC), Western blot and Flow Cytometry indicate that YOD1 is highly expressed in pancreatic cancer cells and pancreatic cancer tissues, and its overexpression can promote the proliferation and metastasis of pancreatic cancer cells and affect the immune microenvironment.ConclusionOur results indicate that YOD1 may be a useful biomarker for the prognosis of human pancreatic cancer, and it may also be a potential molecular target for the diagnosis and treatment of pancreatic cancer.

Transmembrane Protein 170B is a Prognostic Biomarker and Associated With Immune Infiltrates in Pancreatic Adenocarcinoma.

Background: Pancreatic adenocarcinoma (PAAD) is among the most common types of cancer with a poor prognosis. Transmembrane protein 170B (TMEM170B) has been reported to suppress breast cancer proliferation, metastasis, and tumorigenesis and is related to prognosis. However, its role in PAAD and the underlying molecular mechanisms are yet to be investigated. Patients and methods: We performed a comprehensive analysis of RNA sequencing data obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases to determine TMEM170B expression. Immunostaining and real-time polymerase chain reaction (RT-PCR) were done to determine TMEM170B expression in human pancreatic cancer cell lines and tissue specimens. Furthermore, the correlation of TMEM170B with clinicopathological features and PAAD prognosis was investigated, and the mechanisms were explored through enrichment analysis and immune cell infiltration analysis. Results: TCGA and GEO dataset analysis revealed that TMEM170B expression in PAAD tissue samples was significantly lower than that in non-tumorous tissues, which was further confirmed by immunohistochemistry and RT-PCR. Low TMEM170B expression was associated with poor differentiation (p = 0.014). Multivariate analysis identified that TMEM170B is an independent indicator for overall survival [hazard ratio (HR) = 0.116, 95% confidence interval (CI) = 0.014–0.995; p = 0.049] and disease-free survival (HR = 0.19, 95% CI = 0.04–0.910; p = 0.038) in patients with PAAD. Additionally, TMEM170B was involved in immune-related gene sets, including those related to chemokine signaling pathways and innate and adaptive immunity. High TMEM170B expression was linked to antitumor immune microenvironment with a high infiltration of B cells, T cells, dendritic cells, monocytes, M1 macrophages, neutrophil, and natural killer cells and a low infiltration of Tregs and myeloid-derived suppressor cells (all p < 0.05). Plain Language Summary: There is an urgent need to identify clinical prognostic biomarkers and targeted drugs for pancreatic cancer treatment. In this study, the expression status and prognostic value of transmembrane protein 170B (TMEM170B) in pancreatic adenocarcinoma were elucidated. Furthermore, TMEM170B, as a tumor suppressor gene, induced antitumor immune effects, including increased tumor infiltration of immune effector cells and reduced levels of inhibitory immune molecules and regulatory cells. Therefore, TMEM170B could be regarded as a novel target in preventing the progression of pancreatic cancer. Conclusion: The findings suggest that low TMEM170B expression is remarkably correlated with poor PAAD prognosis, which might provide a therapeutic target for PAAD.

GAS41 mediates proliferation and GEM chemoresistance via H2A.Z.2 and Notch1 in pancreatic cancer.

GAS41 is a YEATS domain protein that binds to acetylated histone H3 to promote the chromatin deposition of H2A.Z in non-small cell lung cancer. The role of GAS41 in pancreatic cancer is still unknown. Here, we aimed to reveal this role. GAS41 expression in pancreatic cancer tissues and cell lines was examined using qRT-PCR, Western blotting and immunohistochemistry. MTT, colony formation, spheroid formation and in vivo tumorigenesis assays were performed to assess the proliferation, tumorigenesis, stemness and gemcitabine (GEM) resistance of pancreatic cancer cells. Mechanistically, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) assays were used to evaluate the roles of GAS41, H2A.Z.2 and Notch1 in pancreatic cancer. We found that GAS41 is overexpressed in human pancreatic cancer tissues and cell lines, and that its expression increases following the acquisition of GEM resistance. We also found that GAS41 up-regulates Notch, as well as pancreatic cancer cell stemness and GEM resistance in vitro and in vivo. We show that GAS41 binds to H2A.Z.2 and activates Notch and its downstream mediators, thereby regulating stemness and drug resistance. Depletion of GAS41 or H2A.Z.2 was found to down-regulate Notch and to sensitize pancreatic cancer cells to GEM. Our data indicate that GAS41 mediates proliferation and GEM resistance in pancreatic cancer cells via H2A.Z.2 and Notch1.

Prognostic values of B7-H3, B7-H4, and HHLA2 expression in human pancreatic cancer tissues based on mIHC and spatial distribution analysis

BackgroundPancreatic cancer (PC) is one of the most malignant solid tumors and its 5-year survival rate remains poor. Although immunotherapy has achieved certain therapeutic efficacy in some clinical trials, such treatment still shows low responses and overall remission rate. Therefore, it is urgently necessary to dissect the tumor microenvironment and optimize the immunotherapeutic strategies against this malignancy. MethodsUsing the multi-color immunohistochemistry assay, we investigated the expressions of B7-H3, B7-H4, HHLA2, CD8, and CD68 in 63 cases of PC tissues in a tissue microarray. Moreover, we analyzed immunolocalization features, clinical associations and prognostic values of these molecules. ResultsThe expressions of B7-H3, B7-H4, and HHLA2 could be detected in cytokeratin staining positive (CK+) cancer epithelial cells, CD68+tumor-associated macrophages (TAMs), and even other cells defined as CK-CD8-CD68-. Higher expression of B7-H3 in tumor cells could predict a better survival of the PC patients. A positive correlation was found between the expressions of B7-H3 and HHLA2 in tumor cells, while there was a negative correlation between the expressions of B7-H4 and HHLA2 in tumor cells. A positive correlation was found between the expressions of B7-H3 and B7-H4 or HHLA2 in CD68+TAMs, but not B7-H4 and HHLA2. Tumor-infiltrating CD8+T cells in combination with CD68+TAMs could serve as an important predictor for the postoperative prognosis of PC patients. Higher expression of B7-H3, or HHLA2 in CD68+TAMs could serve as an important predictor for poorer prognosis of PC patients. Patients with B7-H3lowB7-H4low, B7-H3lowHHLA2low, or B7-H4lowHHLA2low on CD68+TAMs could have a better postoperative prognosis compared with the other sub-populations in the combinational analysis. ConclusionsTaken together, our study indicated variable expressions and prognostic values of B7-H3, B7-H4, and HHLA2, in human PC tissues, and demonstrated that these co-stimulator molecules expressed by CD68+TAMs could be used as important bio-markers for the prognostic prediction of PC patients. Moreover, these results supported that the evaluation of these markers could be used as essential candidate targets for immunotherapy against PC.

The effect and mechanism of miR-30e-5p targeting SNAI1 to regulate epithelial-mesenchymal transition on pancreatic cancer

ABSTRACT Accumulating evidence indicates that abnormally expressed microRNAs (miRNAs, miRs) contribute to cancer progression. Nonetheless, the role of miR-30e-5p in pancreatic cancer (PCa) remains unclear. In this study, using quantitative real-time polymerase chain reaction analysis, we found that miR-30e-5p expression was downregulated in human PCa tissues compared with that in normal para-cancerous tissues. After transfecting with miR-30e-5p inhibitors, miR-30e-5p mimics, or empty vectors in the BxPC-3 and PANC-1 cells, respectively, the experiments revealed that the upregulation of miR-30e-5p expression inhibited cell growth, invasion, migration and epithelial-mesenchymal transition (EMT), and promoted apoptosis, while miR-30e-5p downregulation had the opposite effects. RNA sequencing of miR-30e-5p inhibitor-, miR-30e-5p mimic-, and the negative control (NC)-treated groups revealed that miR-30e-5p may affect epithelial cell differentiation, cell growth and death. Next, the snail family transcriptional repressor 1 (SNAI1) was predicted and verified as the target gene of miR-30e-5p using bioinformatics analysis and luciferase assays. SNAI1 expression levels were decreased in the PCa cells transfected with miR-30e-5p mimics, whereas the opposite was observed in the cells transfected with miR-30e-5p inhibitors. Subsequently, PCa cells were transfected with a vector overexpressing SNAI1 (OE-SNAI1) and miR-30e-5p mimics, miR-30e-5p inhibitors, or empty vectors. Compared with that in the OE-SNAI1 + miR-30e-5p NC group, transfection with OE-SNAI1 + miR-30e-5p mimics inhibited the PCa cell growth, migration, and increased apoptosis, whereas transfection with OE-SNAI1 + miR-30e-5p inhibitors had the opposite effects. In conclusion, miR-30e-5p potentially inhibits PCa cell proliferation, migration, and invasion via the SNAI1/EMT axis.