ZC3H13-mediated N6-methyladenosine modification of PHF10 is impaired by fisetin which inhibits the DNA damage response in pancreatic cancer.

DNA damage repair is a major barrier for chemotherapy efficacy of pancreatic ductal adenocarcinoma (PDAC), including the efficacy of platinum-based and gemcitabine/nab-paclitaxel treatments. N6-methyladenosine modifications (m6A) have recently been reported to play a role in homologous recombination (HR) repair of DNA double strand breaks (DSBs); however, the mechanism of action remains unknown. Our previous work indicated that fisetin may be a promising anti-tumour agent that induces DNA damage. In this study, we reported that fisetin induced DSBs and suppressed HR repair through m6A modification in PDAC cells. The m6A writer ZC3H13 and PHF10, which is a subunit of the PBAF chromatin remodelling complex, were identified as the main molecules affected by fisetin treatment. To our knowledge, it's the first time that PHF10 was found and involved in the DNA damage response. PHF10 loss-of-function resulted in elevated recruitment of γH2AX, RAD51, and 53BP1 to DSB sites and decreased HR repair efficiency. Moreover, ZC3H13 knockdown downregulated the m6A methylation of PHF10 and decreased PHF10 translation in a YTHDF1-dependent manner. In conclusion, our study demonstrates that fisetin enhanced DSBs via ZC3Hl3-mediated m6A modification of PHF10, which may provide insight into novel therapeutic approaches for PDAC.

Acute hemorrhagic necrotizing enterocolitis caused by non-O1/non-O139 Vibrio cholerae infection: A case report.

RATIONALE: Acute hemorrhagic necrotizing enterocolitis (AHNE) is a rapidly progressive and extremely dangerous disease. Here we report a rare case caused by Vibrio cholerae (V cholerae). PATIENT CONCERNS: A 70-year-old man was admitted to our emergency department because of a sudden loss of consciousness. DIAGNOSES: On admission with severe toxic shock, the patient presented with elevated body temperature, decreased blood pressure, abdominal tenderness and rebound pain, predominantly on the right side. Computed tomography showed swelling and thickening of the right colon and peritoneal effusion. Necrosis was found in the hepatic flexure of the colon. On the basis of these results, the patient was diagnosed with AHNE. INTERVENTIONS AND OUTCOMES: After fluid resuscitation, an exploratory laparotomy was performed immediately. The procedure was successful. Despite antibiotic therapy, the patient's clinical condition progressively deteriorated and he died of multi-organ failure on day 3 after admission. LESSONS: AHNE is a rapidly progressive and extremely dangerous disease. Here we report a case of AHNE caused by non-O1/non-O139 V cholerae infection. The clinical features, phenotypic analyses and the presence of a panel of known virulence genes in the isolated strain are described. To the best of our knowledge, this is the first report of V cholerae causing severe AHNE, which is of profound pedagogical significance.

BxPC-3-Derived Small Extracellular Vesicles Induce FOXP3+ Treg through ATM-AMPK-Sirtuins-Mediated FOXOs Nuclear Translocations.

Immunotherapy in pancreatic ductal adenocarcinoma (PDAC) treatment faces serious challenges, due particularly to the poor immunogenicity. Cancer cell-derived small extracellular vesicles (sEVs) play important roles in damaging the immune system. However, the effects of pancreatic cancer-derived sEVs on T lymphocytes are unknown. Here we investigated changes in phenotypes and signal transduction pathways in sEVs-treated T lymphocytes. We identified the overexpression of immune checkpoint proteins PD-1, PD-L1, CTLA4, and Tim-3 and the enrichment of FOXP3+ Treg cluster in sEVs-treated T lymphocytes by CyTOF. Gene set enrichment analysis revealed that DNA damage response and metabolic pathways might be involved in sEVs-induced Tregs. ATM, AMPK, SIRT1, SIRT2, and SIRT6 were activated sequentially in sEVs-treated T lymphocytes and essential for sEVs-upregulated expressions of FOXO1A, FOXO3A, and FOXP3. Our study reveals the impact and mechanism of pancreatic cancer cell-derived sEVs on T lymphocytes and may provide insights into developing immunotherapy strategies for PDAC treatment.

Down-regulation of miR-30a-5p is Associated with Poor Prognosis and Promotes Chemoresistance of Gemcitabine in Pancreatic Ductal Adenocarcinoma.

MicroRNA-30a-5p (miR-30a-5p) plays an important role in many biological and pathological processes, and therefore has been studied extensively. However, its expression and function in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Furthermore, whether miR-30a-5p affects sensitivity of PDAC cells to gemcitabine (GEM) is worthy of further exploration. The results showed that miR-30a-5p expression in pancreatic cancer was decreased and the down-regulated expression correlated with poor prognosis, while up-regulating miR-30a-5p suppressed tumor cell proliferation, cell cycle and increased apoptosis. MiRNA expression profiles between gemcitabine-resistant pancreatic cancer cells and parental pancreatic cancer cells showed significant change of miR-30a-5p expression. Besides, up-regulating miR-30a-5p in PDAC significantly increased the chemosensitivity of gemcitabine. Furthermore, FOXD1 is a direct target of miR-30a-5p and the miR-30a-5p/FOXD1/ERK axis may play an important role in the development of gemcitabine resistance in pancreatic cancer. In summary, our study showed that miR-30a-5p increases the sensitivity of pancreatic cancer to gemcitabine, and it may be a potential therapeutic target to overcome gemcitabine resistance.

The distinct role of CD73 in the progression of pancreatic cancer.

Recent studies have shown that the non-enzymatic function of CD73 plays a key role in tumor progression, but this function of CD73 in pancreatic cancer cells has not been studied. Furthermore, little is known about the mechanism involved in CD73 regulation in tumors. Here, we found that CD73 expression was upregulated in pancreatic ductal adenocarcinoma (PDAC) and that its expression correlated with poor prognosis. CD73 knockdown inhibited cell growth and induced G1 phase arrest via the AKT/ERK/cyclin D signaling pathway. We also found that tumor necrosis factor receptor (TNFR) 2 was involved in CD73-induced AKT and ERK signaling pathway activation in PDAC. Further, miR-30a-5p overexpression significantly increased the cytotoxic effect of gemcitabine in pancreatic cancer by directly targeting CD73 messenger RNA (mRNA), suggesting that regulation of the miR-30a-5p/CD73 axis may play an important role in the development of gemcitabine resistance in pancreatic cancer. In summary, this regulatory network of CD73 appears to represent a new molecular mechanism underlying PDAC progression, and the mechanistic interaction between miR-30a-5p, CD73, and TNFR2 may provide new insights into therapeutic strategies for pancreatic cancer. KEY MESSAGES: CD73 was upregulated in PDAC and correlated with poor prognosis. CD73 knockdown inhibited cell growth and induced G1 phase arrest. TNFR2 was involved in CD73-induced AKT and ERK signaling pathway. miR-30a-5p targeted CD73 and increased the sensitivity to gemcitabine.

Interferon Gamma Inhibits CXCL8-Induced Proliferation and Migration of Pancreatic Cancer BxPC-3 Cell Line via a RhoGDI2/Rac1/NF-κB Signaling Pathway.

Interferon gamma (IFN-γ) is a dimeric soluble cytokine and the only type II interferon. Accumulated evidence suggests that IFN-γ inhibits tumor progression. This study investigated the effects of IFN-γ on the proliferation and migration of pancreatic cancer (PC) cells and the underlying mechanism. IFN-γ treatment decreased the expression and secretion of CXCL8 in BxPC-3 PC cells, suppressed the proliferation and migration of these cells, and enhanced their apoptosis, as determined by increased levels of cleaved Caspase-8 and Bax together with reduced expression of Bcl-2. These effects were abolished by overexpression of CXCL8. Moreover, IFN-γ treatment downregulated RhoGDI2 expression. Depletion of RhoGDI2 and Rac1 by using small interfering RNAs and inhibition of NF-κB by BMS-345541 (an IκB kinase [IKK] inhibitor) suppressed expression of CXCL8. Our results indicate that IFN-γ inhibits the proliferation and migration of PC cells by suppressing CXCL8 expression via a RhoGDI2/Rac1/NF-κB signaling pathway.

High Expression of P38α and Preoperative Carbohydrate Antigen 19-9 Indicate Poor Prognosis in Patients with Pancreatic Ductal Adenocarcinoma.

Background: P38α is a ubiquitous protein kinase, which plays diverse roles in cancers. Surprisingly, P38α functions vary markedly in different cancers (e.g., cancer suppressor vs cancer promoter). However, there is no report on the expression of P38α, the family's most important member, in pancreatic ductal adenocarcinoma (PDAC) and its association with clinicoathological parameters and patients' prognosis. Materials and methods: We retrospectively analyzed 152 patients who underwent surgery and were pathologically diagnosed with PDAC from September 2013 to September 2015. We used immunohistochemistry to detect P38α expression in tumor and adjacent normal tissues. The significance of the association between P38α and clinicopathological parameters was evaluated using the χ² test and t tests. The Kaplan-Meier method was used to assess the association between P38α expression and preoperative carbohydrate antigen 19-9 (CA19-9) levels and patients' overall survival. The Cox regression model was used to analyze the association between clinicopathological parameters, P38α and preoperative CA19-9 levels, and prognosis. Statistical significance was defined as P < 0.05. Results: P38α was expressed in 63.16% tumor tissues of PDAC, which was significantly higher compared with the adjacent normal tissues (26.32%, P < 0.001). High expression of P38α was associated with patients' histological grade (P = 0.013), lymphatic metastasis (P = 0.025) and TNM stage (P = 0.048). The median survival time of the P38α-high group was 9.2 months, which was shorter compared with that of the P38α-low group (17.3 months, P = 0.011). The median survival time of the CA19-9 > 43.63 group was 11.1 months shorter than that of the CA19-9 < 43.63 group (24.8 months, P < 0.001). The Cox regression model revealed that age (P = 0.003), lymphatic invasion (P = 0.015), TNM stage (P = 0.003), histological grade (P < 0.001), preoperative CA19-9 (P = 0.049), and P38α expression (P = 0.008) were statistically significant independent risk factors affecting prognosis. Specifically, overall survival was 28.4 months in the P38α-low and CA19-9 < 43.63 groups, 16.3 months in the P38α-high or CA19-9 > 43.63 groups, and 9.7 months in the P38α-high and CA19-9 > 43.63 groups (P < 0.001). Conclusions: High expression of P38α was significantly associated with histological grade, lymphatic metastasis, TNM stage and prognosis in patients with PDAC. P38α and preoperative CA19-9 levels were independent risk factors affecting the prognosis of PDAC patients. High expression of p38α and preoperative carbohydrate antigen 19-9 indicate poor prognosis in patients with PDAC.

Down-expression of CD36 in pancreatic adenocarcinoma and its correlation with clinicopathological features and prognosis.

Recent studies show that CD36 plays a key role in the occurrence and development of tumors, especially in the metastasis of tumors. However, the expression and role of CD36 has not been reported in pancreatic cancer. This study is aimed to explore the expression of CD36 in pancreatic cancer and corresponding non-tumor normal tissues, and its correlation with clinicopathological features and prognosis of pancreatic cancer patients. By analyzing the chip results of database GSE16515, we found that there was significant differential expression of CD36 in pancreatic cancer and corresponding non-tumor normal tissues. In this study, western blot and immunohistochemistry were used to show that the expression of CD36 in pancreatic cancer cells and tissues is significantly lower than that in corresponding non-tumor normal tissues. By statistically analyzing clinical and pathological data, we found that low expression of CD36 predicts lower TNM staging and CA19-9 levels, but larger tumor size and poor survival prognosis. These findings indicated that CD36 can be used as a predictor of clinicopathological features and prognosis, but the contradiction is worthy of our further study.

IFN-γ induces the upregulation of RFXAP via inhibition of miR-212-3p in pancreatic cancer cells: A novel mechanism for IFN-γ response.

Previous studies have demonstrated that pancreatic cancer-derived microRNA (miR)-212-3p can inhibit the expression of regulatory factor X-associated protein (RFXAP), an important transcription factor for major histocompatibility complex (MHC) class II, and thereby lead to downregulation of MHC class II in dendritic cells. It has also been established that interferon (IFN)-γ can increase the expression of MHC class II in immune cells. It was therefore hypothesized that IFN-γ can inhibit miR-212-3p expression in pancreatic cancer, leading to the upregulation of RFXAP and MHC class II expression. This may represent a novel molecular mechanism underlying the use of IFN-γ in immunotherapy. Data from the present study revealed that miR-212-3p was inhibited by IFN-γ in a dose and time-dependent manner in the pancreatic ductal adenocarcinoma cell line PANC-1. RFXAP and MHC class II expression were increased following IFN-γ stimulation. A luciferase assay was performed to validate RFXAP as a target gene of miR-212-3p. The expression levels of RFXAP and MHC class II were decreased by miR-212-3p mimics and increased by miR-212-3p inhibitors. In PANC-1 cells transfected with miR-212-3p mimics, IFN-γ stimulation could not increase the RFXAP and MHC class II. The results from the present study suggest that IFN-γ increases RFXAP and MHC class II expression by inhibiting miR-212-3p. To the best of our knowledge, this is the first report of this novel molecular mechanism underlying the effects of IFN-γ on pancreatic cancer, which may aid with the development of immunotherapies for patients with pancreatic cancer.

Prognostic value of programmed cell death protein 1 expression on CD8+ T lymphocytes in pancreatic cancer.

Pancreatic cancer is one of the most aggressive malignancies and has a highly immunosuppressive tumour microenvironment. Immune checkpoint blockade has led to remarkable and durable objective responses in a number of malignancies and antibody-based strategies targeting programmed cell death protein 1 (PD-1) are showing promise where traditional modalities of surgery, radiotherapy, and chemotherapy have failed. In this study, we examined the clinical value of PD-1 protein expression by CD8+ peripheral T lymphocytes or tumour-infiltrating T lymphocytes (TILs) in pancreatic ductal adenocarcinoma (PDAC). Expression of PD-1 protein on CD8+ TILs correlated with overall survival and clinicopathological characteristics such as clinical stage, N classification, and M classification. Similar findings were observed for the expression of PD-1 protein on peripheral CD8+ T cells, whereas its expression on peripheral CD4+ T cells showed no significance. Comparison of the levels of PD-1 protein expressed by peripheral CD8+ T cells before and 4 weeks after surgery indicated that preoperative and postoperative status of peripheral PD-1 expression was unchanged. Our findings showed that PD-1 protein expressed by peripheral or tumour-infiltrated CD8+ T cells was a promising biomarker for diagnosis and prognosis in PDAC and might help guide future immunotherapies.

Overexpressions of miR-212 are associated with poor prognosis of patients with pancreatic ductal adenocarcinoma.

BACKGROUND: The miR-212 was among the top differentially expressed miRNAs in pancreatic ductal adenocarcinoma (PDAC). OBJECTIVE: The aim of this study was to investigate the expression of miR-212 in PDAC and evaluate its correlation with major clinicopathologic features and patients' survival. METHODS: Fluorescence in situ hybridization (FISH) was adopted to examine miRNA expression in 45 pancreatic cancer and 20 normal pancreatic tissues. The relationship of miR-212 expression with clinicopathologic parameters and clinical outcome was evaluated. RESULTS: miR-212 was confirmed to have significantly higher expression in PDAC compared with normal pancreatic tissues (51.1% vs 10%, p< 0.01). High expression of miR-212 was significantly associated with tumor size (p = 0.048) and tumor stage (p = 0.023). Moreover, in univariant analysis, patients with high expression of miR-212 demonstrate significantly poorer overall survival (p= 0.02). CONCLUSIONS: High expression of miR-212 in PDAC is associated with shorter overall survival. It may be not only a potential prognostic marker, but also a possible therapeutic target in PDAC.

Pancreatic cancer-derived exosomes transfer miRNAs to dendritic cells and inhibit RFXAP expression via miR-212-3p.

It has been reported tumor-derived exosomes can transfer miRNAs to recipient cells in the tumor microenvironment, promoting tumor invasion and metastasis. The present research aimed to explore how pancreatic cancer (PC) derived exosomal miRNAs inhibited mRNA expression of dendritic cells and induced immune tolerance. Our study revealed that 9 PC-related miRNAs were increased and 208 mRNAs were inhibited in exosome-stimulated dendritic cells (exo-iDCs) compared to immature dendritic cells (iDCs). A target prediction between the 9 miRNAs and 208 mRNAs was performed by bioinformatics database analysis. From the target prediction, it was predicted and validated that regulatory factor X-associated protein (RFXAP), an important transcription factor for MHC II, was inhibited by miR-212-3p transferred from PC-secreted exosomes, resulting in decreased MHC II expression. Moreover, a clinical study showed a negative correlation between miR-212-3p and RFXAP in PC tissue. From these data, we concluded that PC-related miRNAs can be transferred to dendritic cells via exosome and inhibit target mRNA expression. More importantly, PC-derived exosomes inhibit RFXAP expression via miR-212-3p, which decrease MHC II expression and induce immune tolerance of dendritic cells. RFXAP deficiency has never been reported in solid tumors. The functions and mechanisms of RFXAP in tumors deserve future explorations.

Overexpressions of DLL4 and CD105 are Associated with Poor Prognosis of Patients with Pancreatic Ductal Adenocarcinoma.

The aim of this study was to investigate the expression of Delta-like ligand 4(DLL4) and Endoglin(CD105) labeled microvessel density(MVD) in pancreatic ductal adenocarcinoma (PDAC) and evaluate their correlation with major clinicopathologic features and patients' survival. Forty-two pancreatic cancer and 20 normal pancreatic tissues were included in the study. Immunohistochemical staining was employed to assess the expression level of DLL4 both in tumor cells and stromal vascular endothelial cells, as well as CD105 which was used to determine MVD. The relationships of DLL4 and CD105 expression with clinicopathologic parameters and clinical outcome were evaluated. Both DLL4 and CD105-labeled microvessel were observed highly immunostained in PDAC cases, and high expression of DLL4 was positively correlated with MVD. Moreover, the high expression of DLL4 was significantly associated with histological grade, node stage and TNM stage in not only the cancer cells but also stroma; while high expression of CD105 was associated with histological grade, TNM stage, node stage and distant metastasis. In univariant analysis, patients with high expression of DLL4 and CD105 tended to significantly poorer overall survival. Both DLL4 and CD105 were overexpressed in a large proportion of patients with PDAC. The expression of DLL4 was positively correlated with CD105-labeled MVD, indicating DLL4 may involved in angiogenesis. In addition, high DLL4 and CD105 expression correlated with the poor clinical outcome and overall survival in patients with PDAC.

Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203.

MicroRNAs (miRNAs) are aberrant in many human tumors which can be transferred to immune cells by tumor-derived exosomes. Dendritic cells (DCs) play an important role in activation of immune response. However, the effect of tumor-derived exosomes on toll-like receptor (TLR) in DCs remains unclear. We investigated the influence of pancreatic cancer derived exosomes on TLR4, and downstream cytokines via miR-203. Our results showed that miR-203 expressed in panc-1 cells and exosomes, and upregulated in exosomes-treated DCs. TLR4 decreased after treatment of exosomes and miR-203 mimics, while increased in exosomes-treated DCs by miR-203 inhibitors. But the mRNA level of TLR4 was not significantly different between DCs and exosomes-treated DCs. Tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) also decreased under treatment of exosomes and miR-203 mimics, both of which increased in exosomes-treated DCs by miR-203 inhibitors. Collectively, pancreatic cancer derived exosomes downregulate TLR4 and downstream cytokines in DCs via miR-203.