BackgroundPancreatic cancer (PC) is a common malignancy of the digestive system and is characterized by poor prognosis and early metastasis. Tumor immune escape plays an important role in PC progression. Programmed death 1 (PD1) blockade therapy is a promising treatment for patients with PC, but is yet to achieve significant clinical effects so far. Interferon gamma (IFN-γ) is a soluble dimeric cytokine that is closely associated with tumor immune surveillance and cytotoxicity. IFN-γ suppresses a variety of tumor-derived cytokines in PC, such as CXCL8. In the present study, we investigated the therapeutic efficacy of combined anti-PD1 and IFN-γ treatment in PC.MethodsBxPC-3 and Panc-1 human PC cell lines were used to construct a murine PC model. Blood samples (n=44) and surgical resection specimens (n=36) from human patients with PC were also collected. χ2test, two-tailed unpaired t-test or Kaplan-Meier survival analysis was used to calculate p values.ResultsPD1/PD-L1 signaling was overexpressed in PC tumor-bearing mice. Anti-PD1 prevented tumor growth if initiated early after tumor inoculation; however, delayed anti-PD1 treatment showed limited benefit. Murine PC model had a preferential expansion of CXCR2+CD68+macrophages, and these cells showed an immunosuppressive nature (M2 polarization). PC tumors overexpressed CXCL8 and tumor-derived CXCL8 deficiency prohibited the trafficking of CXCR2+CD68+macrophages. IFN-γ suppressed the expression of tumor-derived CXCL8, and combined with IFN-γ treatment, delayed anti-PD1 treatment showed significant antitumor effects. Thus, we conclude that murine CXCR2+CD68+macrophages traffic to PC tumors by tumor-derived CXCL8 and mediate local immunosuppression, which limits the efficacy of PD1 blockade therapy. IFN-γ suppresses tumor-derived CXCL8 and inhibits the tumor trafficking of CXCR2+CD68+macrophages by blocking the CXCL8–CXCR2 axis to enhance anti-PD1 efficacy. Human PC also produces high levels of CXCL8. Patients with PC present elevated CXCR2 expression on peripheral and tumor-infiltrating CD68+macrophages, which are associated with advanced tumor stage and poor prognosis.ConclusionOur findings suggest that IFN-γ is a translatable, therapeutic option to improve the efficacy of PD1 blockade therapy by preventing trafficking of CXCR2+CD68+macrophages via blocking the CXCL8–CXCR2 axis.

Pancreatic cancer is one of the most aggressive tumors and patients have poor survival rates. Fisetin, a natural flavonoid, was recently reported to have antitumor effects in various cancer models. Autophagy is a conserved catabolic process that maintains cellular homoeostasis in response to stress, and together with apoptosis, determines cell fate. Herein, we examined the effect of fisetin on pancreatic cancer. We reveal that fisetin inhibits PANC-1 cell proliferation using a real-time cell analysis system. Moreover, the in vivo antitumor effect of fisetin was verified in pancreatic cancer using a luciferase-expressing murine xenograft pancreatic cancer model. We found that the AMPK/mTOR signaling pathway was enhanced after fisetin treatment; however, autophagy was not diminished by adding the AMPK inhibitor compound C. Thus, we hypothesized that an another autophagy regulating pathway existed. RNA-seq analysis revealed that the unfolded protein response pathway, which is activated by ER stress, was enriched. We also found that the stress-induced transcription factor p8 was increased in fisetin-treated PANC-1 cells, and that fisetin-induced autophagy was blocked by silencing p8. We revealed that p8-dependent autophagy was AMPK-independent, and that p8 regulated ATF6, ATF4, and PERK in response to ER stress via p53/PKC-α-mediated signaling. Furthermore, mitophagy was associated with Parkin and PINK1 in response to mitochondrial stress. Interestingly, ATF4 and ATF6 were increased in cells treated with fisetin and compound C. Moreover, inhibiting the AMPK/mTOR pathway with compound C may upregulate p8-dependent autophagy. Thus, there may be crosstalk between the AMPK/mTOR and p8-dependent pathways.

Pancreatic cancer is the fourth most lethal malignancy and is characterized by poor immunogenicity. Pancreatic cancer cells have various strategies to suppress host immune response, evade immune defenses, and facilitate tumor growth and development. As a mode of long‐range intercellular communication, cancer‐derived exosomes contribute to impairment of the immune system. However, the mechanisms that induce changes in the activities of signal transduction pathways in immune cells, which are influenced by tumor‐derived exosomes, are poorly understood. We (1) treated peripheral T lymphocytes with pancreatic cancer‐derived exosomes, tagged CD63 with tdTomato, to trace exosome transfer from pancreatic cancer cells to T lymphocytes; (2) carried out a cytotoxicity assay of exosome‐treated T lymphocytes using the Real Time Cellular Analysis system; (3) performed RNA sequencing and gene set enrichment analysis to explore the pivotal signaling pathway that mediates apoptosis in exosome‐treated T lymphocytes; and (4) demonstrated the role of p38 mitogen‐activated protein kinase (MAPK) and endoplasmic reticulum (ER) stress in exosome‐induced T‐lymphocyte apoptosis. In conclusion, these results indicate that pancreatic cancer cells secrete exosomes, which are taken up by T lymphocytes to activate p38 MAPK, and then induce ER stress‐mediated apoptosis, ultimately causing immunosuppression.

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BackgroundMyelodysplastic syndromes (MDS) are a group of heterogeneous diseases with variable clinical course. Predicting disease progression is difficult due to lack of specific molecular marker(s). SALL4 plays important roles in normal hematopoiesis and leukemogenesis. SALL4 transgenic mice develop MDS prior to acute myeloid leukemia (AML) transformation. However, the role of SALL4 in human MDS has not been extensively investigated. In this study, we evaluate the diagnostic/prognostic value of SALL4 in MDS by examining its expression levels in a cohort of MDS patients.MethodsFifty-five newly diagnosed MDS, twenty MDS-AML, and sixteen post-treatment MDS patients were selected for our study along with ten healthy donors.ResultsWe demonstrated that SALL4 was over-expressed in MDS patients and proportionally increased in MDS patients with high grade/IPSS scores. This expression pattern was similar to that of Bmi-1, an important marker in predicting MDS/AML progression. In addition, the level of SALL4 was positively correlated with increased blast counts, high-risk keryotypes and increased significantly in MDS-AML transformation. Furthermore, higher level of SALL4 expression was associated with worse survival rates and SALL4 level decreased following effective therapy.ConclusionsTo the best of our knowledge, this is the largest series and the first to report the expression pattern of SALL4 in detail in various subtypes of MDS in comparison to that of Bmi-1. We conclude that SALL4 is a potential molecular marker in predicting the prognosis of MDS.

ObjectiveTo date, there are no studies regarding the lactylation profile and its role in critically ill patients. Thus, we aimed to examine expression of histone H3 lysine 18 (H3K18) lactylation and its role in patients with septic shock.MethodsThirteen healthy volunteers and 35 critically ill patients from the Department of Surgical Intensive Care Medicine, Beijing Hospital were enrolled in our study. Baseline information and clinical outcomes were obtained prospectively. Lactylation levels of all proteins and H3K18 from peripheral blood mononuclear (PBMC) were determined by western blotting and serum levels of inflammatory cytokines by flow cytometry. Arginase-1 (Arg1) and Krüppel-like factor-4 (Klf4) mRNA expression was evaluated by quantitative real-time PCR (qRT-PCR).ResultsLactylation was found to be an all-protein post-translational modification and was detected in PBMCs from both healthy volunteers and critically ill patients, with a significantly higher relative density in shock patients (t=2.172, P=0.045). H3K18la was expressed in all subjects, including healthy volunteers, with the highest level in septic shock patients (compared with non-septic shock patients, critically ill without shock patients and healthy volunteers P=0.033, 0.000 and 0.000, respectively). Furthermore, H3K18la protein expression correlated positively with APACHE II scores, SOFA scores on day 1, ICU stay, mechanical ventilation time and serum lactate (ρ=0.42, 0.63, 0.39, 0.51 and 0.48, respectively, ρ=0.012, 0.000, 0.019, 0.003 and 0.003, respectively). When we matched patients with septic shock and with non-septic shock according to severity, we found higher H3K18la levels in the former group (t=-2.208, P =0.040). Moreover, H3K18la exhibited a close correlation with procalcitonin levels (ρ=0.71, P=0.010). Patients with high H3K18la expression showed higher IL-2, IL-5, IL-6, IL-8, IL-10, IL-17, IFN-α levels (ρ=0.33, 0.37, 0.62, 0.55, 0.65, 0.49 and 0.374 respectively, P=0.024, 0.011, 0.000, 0.000, 0.000 and 0.000 respectively). H3K18la expression also displayed a positive correlation with the level of Arg1 mRNA (ρ=0.561, P=0.005).ConclusionsLactylation is an all-protein post-translational modification occurring in both healthy subjects and critically ill patients. H3K18la may reflect the severity of critical illness and the presence of infection. H3K18la might mediate inflammatory cytokine expression and Arg1 overexpression and stimulate the anti-inflammatory function of macrophages in sepsis.

Pancreatic adenocarcinoma (PDAC) is an extremely malignant tumor that is associated with low survival rates. Fisetin is a natural flavonoid that shows diverse antitumor effects, including DNA damage, in various cancers. Increasing studies have demonstrated that epigenetic modifications play critical roles in DNA-damage response. However, the epigenetic regulation mechanism of fisetin in cancers is hardly studied. RFXAP is a critical transcription factor for MHC II molecules, however, its transcriptional role in PDAC is poorly understood. The anti-PDAC effect of fisetin was measured by CCK-8, flow cytometry, xenograft tumor nude mice model. DNA-damage levels were examined by immunofluorescence. Bioinformatics analysis was used to examine the expression of RFXAP and other genes involved in DNA-damage response. ChIP sequencing was used to explore the transcriptional role of RFXAP. The expression of target gene KDM4A was measured by qRT-PCR and western blots. KDM4A promoter activity was analyzed using dual-luciferase reporter assay. RFXAP overexpressing or silencing of PDAC cells was used to explore the effect of RFXAP in DNA damage induced by fisetin. We found that fisetin inhibited cell proliferation and induced DNA damage and S-phase arrest in PDAC. Expression of RFXAP and other DNA-damage response genes were upregulated by fisetin. We revealed that RFXAP expression was relatively low in PDAC and correlated with tumor stage and poor prognosis. Then we explored the transcriptional role of RFXAP and found that RFXAP targeted KDM4A, a special demethylase specific for tri- and dimethylated histone H3K36. We found that overexpression of RFXAP upregulated KDM4A and attenuated methylation of H3K36, thereby impairing DNA repair and enhancing the DNA damage induced by fisetin, while RFXAP silencing showed the opposite effect. We also found the function of fisetin in enhancing the effect of chemotherapy on pancreatic cancer cells. Our findings revealed that fisetin induced DNA damage via RFXAP/KDM4A-dependent histone H3K36 demethylation, thus causing inhibition of proliferation in PDAC.

Colorectal cancer (CRC) is the third leading cause of cancer-associated mortality worldwide. Ginsenoside Rh1 (Rh1) is a traditional medicine monomer with antitumor activity; however, the effects of Rh1 in CRC remain to be determined. In the present study, SW620 cells were treated with different concentrations of Rh1. Cell Counting Kit-8, wound healing and Transwell assays were performed to measure cell viability and proliferation, migration and invasion, respectively. Subsequently, the mRNA expression levels of matrix metallopeptidase (MMP)1, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were detected by reverse transcription-quantitative PCR analysis. In addition, the protein expression levels of MMP1, MMP3, TIMP3, and total or phosphorylated (p-)ERK1/2, P38, JNK were detected by western blotting. Furthermore, tumor growth was examined in a nude mouse xenograft model. The results of the present study indicated that Rh1 was not toxic to CRC cells at various concentrations (0, 50 or 100 µM) and treatment durations (24 or 48 h). However, cell proliferation was suppressed by Rh1 in a dose-dependent manner. Rh1 (100 µM) significantly inhibited cell migration and invasion in vitro. Additionally, Rh1 suppressed the mRNA and protein expression of MMP1 and MMP3, and promoted TIMP3 expression. Rh1 decreased the ratios of p-P38/P38, p-ERK1/2/ERK1-2 and p-JNK/JNK in vitro and in vivo, which suggested that Rh1 inactivated the mitogen-activated protein kinase (MAPK) signaling pathway. Notably, Rh1 markedly decreased tumor volume and weight in vivo. In conclusion, the present study demonstrated that Rh1 inhibited the proliferation, migration and invasion of CRC cells in vitro and tumor growth in vivo. This inhibition was at least partially due to the inhibition of MMP1 and MMP3 expression, the increase in TIMP3 expression level and the MAPK signaling pathway inactivation. Therefore, Rh1 may effectively inhibit the development of CRC as an anticancer drug, and may have a supporting effect during CRC treatment.