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Bile acids (BAs), well-defined signaling molecules with diverse metabolic functions, play important roles in cellular processes associated with many cancers. As one of the most common BAs, deoxycholic acid (DCA) is originally synthesized in the liver, stored in the gallbladder, and processed in the gut. DCA plays crucial roles in various tumors; however, functions and molecular mechanisms of DCA in gallbladder cancer (GBC) still remain poorly characterized. Here, we analyzed human GBC samples and found that DCA was significantly downregulated in GBC, and reduced levels of DCA was associated with poor clinical outcome in patients with GBC. DCA treatment impeded tumor progression by halting cell proliferation. DCA decreased miR-92b-3p expression in an m6A-dependent posttranscriptional modification manner by facilitating dissociation of METTL3 from METTL3–METTL14–WTAP complex, which increased the protein level of the phosphatase and tensin homolog, a newly identified target of miR-92b-3p, and subsequently inactivated the PI3K/AKT signaling pathway. Our findings revealed that DCA might function as a tumor suppressive factor in GBC at least by interfering with miR-92b-3p maturation, and suggested that DCA treatment could provide a new therapeutic strategy for GBC.

Gemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. Here, we utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNA-modifying enzymes. Mechanistically, loss of ELP5 impairs the integrity and stability of the Elongator complex to abrogate wobble U34 tRNA modification, and directly impedes the wobble U34 modification-dependent translation of hnRNPQ mRNA, a validated P53 internal ribosomal entry site (IRES) trans-acting factor. Downregulated hnRNPQ is unable to drive P53 IRES-dependent translation, but rescuing a U34 modification-independent hnRNPQ mutant could restore P53 translation and gemcitabine sensitivity in ELP5-depleted GBC cells. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy. These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells., Gemcitabine is used to treat gallbaldder cancer but patient responses are variable. Here, the authors use a genome-wide CRISPR screen and identify the translational elongator protein ELP5 as a protein that is important for mediating gemcitabine-induced apoptosis.

Gallbladder cancer (GBC) is the leading malignancy of biliary system showing refractory chemoresistance to current first‐line drugs. Growing epidemiological evidences have established that the incidence of GBC exhibits significant gender predominance with females two‐threefold higher than males, suggesting oestrogen/oestrogen receptors (ERs) signalling might be a critical driver of tumorigenesis in gallbladder. This study aims to evaluate the antitumour activity of tamoxifen (TAM), a major agent of hormonal therapy for breast cancer, in preclinical GBC model. Quantitative real‐time PCR was used to investigate mRNA levels. Protein expression was measured by immunohistochemistry and Western blot. Glycolytic levels were measured by glucose consumption and lactic acid measurement. The antitumour activity of TAM alone or with cisplatin was examined with CCK8 assay, colony formation, flow cytometry and in vivo models. The results revealed that ERɑ expression was higher in GBC tissues and predicted poor clinical outcomes. TAM was showed effective against a variety of GBC cell lines. Mechanical investigations revealed that TAM enabled potent reactive oxygen species (ROS) production by reduced nuclear factor Nrf2 expression and its target genes, leading to the activation of AMPK, which subsequently induced impaired glycolysis and survival advantages. Notably, TAM was demonstrated to sensitize GBC cells to cisplatin (CDDP) both in vitro and in vivo. In agreement with these findings, elimination of oestrogens by ovariectomy in nude mice prevented CDDP resistance. In summary, these results provide basis for TAM treatment for GBC and shed novel light on the potential application of endocrine therapy for patients with GBC.

SPARC is a secreted glycoprotein that plays a complex and multifaceted role in tumour formation and progression. However, whether SPARC is an oncogene or a tumour suppressor is still unclear. Moreover, SPARC demonstrates potential in clinical pancreatic adenocarcinoma (PAAD) treatment, although it has been identified as an oncogene in some studies and a tumor suppressor in others. In the present study, a pan-cancer analysis of SPARC was carried out using The Cancer genome Atlas data, which demonstrated that SPARC was an oncogene in most cancer types and a cancer suppressor in others. In addition, SPARC expression was significantly upregulated in PAAD and associated with poor prognosis. SPARC also promoted the proliferation and migration of PANC-1 and SW1990 cell lines in vitro. SPARC was detected in the culture supernatant of PAAD cells and pancreatic acinar AR42J cells. SPARC regulated PAAD cell proliferation only when secreted into the extracellular milieu, thus explaining why the prognosis of patients with PAAD is correlated with the SPARC expression of both tumour cells and stromal cells. Collectively, the present findings demonstrated that the function of SPARC was associated with tumour type and that SPARC may represent an important oncogene in PAAD that merits further study.

Bile acids (BAs), well-defined signaling molecules with diverse metabolic functions, play important roles in cellular processes associated with many cancers. As one of the most common BAs, deoxycholic acid (DCA) is originally synthesized in the liver, stored in the gallbladder, and processed in the gut. DCA plays crucial roles in various tumors; however, functions and molecular mechanisms of DCA in gallbladder cancer (GBC) still remain poorly characterized. Here, we analyzed human GBC samples and found that DCA was significantly downregulated in GBC, and reduced levels of DCA was associated with poor clinical outcome in patients with GBC. DCA treatment impeded tumor progression by halting cell proliferation. DCA decreased miR-92b-3p expression in an m6A-dependent posttranscriptional modification manner by facilitating dissociation of METTL3 from METTL3–METTL14–WTAP complex, which increased the protein level of the phosphatase and tensin homolog, a newly identified target of miR-92b-3p, and subsequently inactivated the PI3K/AKT signaling pathway. Our findings revealed that DCA might function as a tumor suppressive factor in GBC at least by interfering with miR-92b-3p maturation, and suggested that DCA treatment could provide a new therapeutic strategy for GBC.

Gallbladder cancer (GBC) is a malignant cancer with very poor prognosis. Although promyelocytic leukemia zinc-finger protein (PLZF) was reported to be deregulated in numerous cancers and also relevant to clinical prognosis, its role in GBC progression has been little known. In this study, we found PLZF expression was decreased in GBC, correlating to advanced TNM stage, distant metastasis, and shorter overall survival. Moreover, ectopic PLZF expression in GBC cells (NOZ and GBC-SD) significantly reduced the cell proliferation, migration, and invasion. Consistently, overexpression of PLZF in xenograft mice model could suppress tumor growth and liver metastasis. Mechanical investigations verified PLZF could regulate the expression of cell cycle arrest-associated gene p21 and epithelial–mesenchymal transition (EMT)-related genes (E-cadherin and N-cadherin) in GBC cell lines. Importantly, PLZF remarkably increased the mRNA transcription of interferon-induced protein with tetratricopeptide repeat 2 (IFIT2) by increasing STAT1 protein level, a known factor involved in tumor progression. Furthermore, ablation of IFIT2 in PLZF overexpression cells abrogated the tumor-suppressive function of PLZF, at least partially, leading to impaired tumor growth and EMT program. These studies indicated PLZF inhibited the proliferation and metastasis via regulation of IFIT2. In conclusion, our study demonstrated PLZF could be a promising tumor biomarker for GBC, and also be a potential therapeutic target.

Brusatol, isolated from brucea, has been proved to exhibit anticancer influence on various kind of human malignancies. However, the role that brusatol plays in pancreatic cancer is seldom known by the public. Through researches brusatol was proved to inhibit growth and induce apoptosis in both PATU-8988 and PANC-1 cells by decreasing the expression level of Bcl-2 and increasing the expression levels of Bax, Cleaved Caspase-3. Then we found the activation of the JNK, p38 MAPK and inactivation of the NF-κb, Stat3 are related with the potential pro-apoptotic signaling pathways. However, SP600125 could not only abrogated the JNK activation caused by brusatol, but also reverse the p38 activation and the decrease of Bcl-2 as SB203580 did. Besides, SP600125 and SB203580 also reversed the inactivation of NF-κb and Stat3. Furthermore, BAY 11-7082 and S3I-201 indeed had the similar effect as brusatol had on the expression of Phospho-Stat3 and Bcl-2. To sum up, we came to a conclusion that in pancreatic cancer, brusatol do inhibit growth and induce apoptosis. And we inferred that brusatol illustrates anticancer attribution via JNK/p38 MAPK/NF-κb/Stat3/Bcl-2 signaling pathway.

Background CircRNAs are found to affect initiation and progression of several cancer types. However, whether circRNAs are implicated in gallbladder cancer (GBC) progression remains obscure. Methods We perform RNA sequencing in 10 pairs of GBC and para-cancer tissues. CCK8 and clone formation assays are used to evaluate proliferation ability of GBC cells. qPCR and Western blot are used to determine expression of RNAs and proteins, respectively. CircRNA-protein interaction is confirmed by RNA pulldown, RNA immunoprecipitation, and fluorescence in situ hybridization. Results We find that circRNA expression pattern is tremendously changed during GBC development. Among dozens of significantly changed circRNAs, a circRNA generated from the oncogene ERBB2, named as circERBB2, is one of the most significant changes. CircERBB2 promotes GBC proliferation, in vitro and in vivo. Other than being a miRNA sponge, circERBB2 accumulates in the nucleoli and regulates ribosomal DNA transcription, which is one of the rate-limiting steps of ribosome synthesis and cellular proliferation. CircERBB2 regulates nucleolar localization of PA2G4, thereby forming a circERBB2-PA2G4-TIFIA regulatory axis to modulate ribosomal DNA transcription and GBC proliferation. Increased expression of circERBB2 is associated with worse prognosis of GBC patients. Conclusions Our findings demonstrate that circERBB2 serves as an important regulator of cancer cell proliferation and shows the potential to be a new therapeutic target of GBC.

Background Gallbladder cancer (GBC) is an extremely malignant tumor with a high mortality rate. Little is known about its invasion and metastasis mechanism so far. Methods To identify the driver genes in GBC metastasis, we performed a mRNA microarray of metastatic GBC and paired non-tumor samples, and found PLEK2 was markedly upregulated in GBC tissues. Next, the expression of PLEK2 in GBC were examined in a larger cohort of patients by qRT-PCR, western blot and IHC staining. The clinicopathologic correlation of PLEK2 was determined by statistical analyses. The biological involvement of PLEK2 in GBC metastasis and the underlying mechanisms were investigated. Results In this study, we found that PLEK2 had higher expression in GBC tumor tissues compared to non-cancerous adjacent tissues and cholecystolithiasis tissues. The clinicopathologic analyses showed PLEK2 expression was positively correlated with tumor TNM stage, distant metastasis and PLEK2 was an independent predictor of overall survival (OS) in GBC patients. The cellular function assays showed PLEK2 promoted GBC cells migration, invasion and liver metastasis in mouse model via the regulation of epithelial-mesenchymal transition (EMT) process. Our mass spectrum and co-immunoprecipitation (co-IP) assays demonstrated that PLEK2 could interact with the kinase domain of EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, RNA-sequencing and qRT-PCR results demonstrated chemokine (C-C motif) ligand 2 (CCL2), a target gene downstream of PLEK2/EGFR signaling, mediated the motility-promoting function of PLEK2. Conclusions On the basis of these collective data, we propose that PLEK2 promotes the invasion and metastasis of GBC by EGFR/CCL2 pathway and PLEK2 can serve as a potential therapeutic target for GBC treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1250-8) contains supplementary material, which is available to authorized users.

Figure S4B. EGFR mRNA levels of NOZ and GBC-SD cells with stable PLEK2 knockdown and overexpression were detected by qRT-PCR. (PDF 118 kb)

Figure S5. Representative images of H&E staining of mouse model. Figure S5A and S5B were the representative images of H&E staining of metastatic focuses in livers, Figure S5C was a representative image of the H&E staining of subcutaneous xenografts. (PDF 1697 kb)

Figure S2A. Cells construction of PLEK2 down-regulation NOZ and GBC-SD cells (NOZ-shPLEK2, GBC-SD-shPLEK2, respectively), also PLEK2 overexpression NOZ and GBC-SD cells (NOZ-PLEK2, GBC-SD-PLEK2, respectively). (PDF 95 kb)

Figure S4C. GBC cells were treated with 100 μM Chloroquine for 8 h, followed by 50 ng/ml EGF stimulation for 5 m. Alterations of EGFR expression in PLEK2 knockdown cells were detected by western blot. (PDF 139 kb)

Figure S4D. Protein levels of EGFR in PLEK2 overexpression cells with increasing ectopic c-CBL expression were detected by western blot. (PDF 93 kb)

Figure S1A. Analysis the gene expression differences and its distribution in human cancer cells by bioinformatics data ( http://www.broadinstitute.o.rg ). (PDF 145 kb)

Figure S2C. EMT markers of NOZ and GBC-SD cells with stable PLEK2 knockdown and overexpression were detected by qRT-PCR. (PDF 186 kb)

Tingting Ye,1 Jiadong Su,1 Chaohao Huang,1 Dinglai Yu,1 Shengjie Dai,1 Xince Huang,1 Bicheng Chen,1,2 Mengtao Zhou1 1Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, 2Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou Key Laboratory of Surgery, Wenzhou, Zhejiang Province, People’s Republic of China Abstract: Isoorientin (or homoorientin) is a flavone, which is a chemical flavonoid-like compound, and a 6-C-glucoside of luteolin. Isoorientin has been demonstrated to have anti-cancer activities against various tumors, but its effects on pancreatic cancer (PC) have not been studied in detail. In this study, we aim to investigate whether isoorientin has potential anti-PC effects and its underlying mechanism. In PC, isoorientin strongly inhibited the survival of the cells, induced cell apoptosis, and decreased its malignancy by reversing the expression of epithelial–mesenchymal transition and matrix metalloproteinase and decreased vascular endothelial growth factor expression. Meanwhile, we investigated the activity of the AMP-activated protein kinase (AMPK) signaling pathway after isoorientin treatment, which was forcefully activated by isoorientin, as expected. In addition, in the PC cells that were transfected with lentivirus to interfere with the expression of the gene PRKAA1, there were no differences in the apoptosis rate and the expression of malignancy biomarkers in the tumors of the isoorientin-treated and untreated groups. Thus, we demonstrated that isoorientin has potential antitumor effects via the AMPK signaling pathway, and isoorientin merits further investigation. Keywords: pancreatic cancer, AMPK, isoorientin, apoptosis, invasiveness, VEGF

Purpose: Gallbladder cancer (GBC) is the leading malignancy of biliary system showing refractory chemoresistance to current first-line drugs. Growing epidemiological evidences have established that the incidence of GBC exhibits significant gender predominance with females 2-3 fold higher than males, suggesting estrogen/estrogen receptors (ERs) signaling might be a critical driver of tumorigenesis in gallbladder. This study aims to evaluate the antitumor activity of tamoxifen (TAM), a major agent of hormonal therapy for breast cancer, in preclinical GBC model. Experimental Design: The antitumor activity of TAM alone or with cisplatin was examined using cell viability, apoptosis and in vivo models. Results: Increased ERɑ expression was found in GBC tissues and predicted poor clinical outcomes. TAM was showed effective against a variety of GBC cell lines. Mechanical investigations revealed that TAM enabled potent reactive oxygen species (ROS) production by reduced nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression and its target genes, leading to the activation of Adenosine Monophosphate Activated Protein Kinase (AMPK), which subsequently induced impaired glycolysis and survival advantages. Moreover, the inhibitory effect of TAM for GBC cells was remarkably attenuated by N-acetyl-L-cysteine (NAC) and AMPK inhibitor, suggesting ROS/AMPK axis was required for TAM tumor-suppressive function. Notably, TAM was demonstrated to sensitize GBC cells to cisplatin (CDDP) both in vitro and in vivo. In agreement with these findings, elimination of estrogens by ovariectomy in nude mice prevented CDDP resistance. Conclusions: These results provide basis for TAM-mediated chemosensitivity for GBC and shed novel light on the potential application of endocrine therapy for patients with GBC. Funding Statement: This work was supported by National Science Foundation of China (81472240，81272748 and 81072011)，Science and Technology Commission of Shanghai Municipality (16411952700, 10411955400, and 09411960800), Program for Outstanding Academic Leader of Shanghai (2016, JW) Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The collection and analysis of patient samples were approved by the Ethical Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine, and written informed consent was obtained from all patients. All methods and experiments were carried out in accordance with the approved guidelines and regulations. Animal maintenance and experimental procedures were strictly performed following the guidelines of the Animal Care and Use Committee of Shanghai Jiao Tong University and approved by IACUC committee of Shanghai Jiao Tong University

Luteolin, a flavone, has been demonstrated to have anti‑cancer properties. In the current study, the effects of luteolin on certain carcinogenesis‑associated changes induced by pancreatitis, which are significant risk factors for pancreatic cancer, were investigated. Male six‑week‑old C57BL6 mice used in the current study were divided into three groups; the control group, acute pancreatitis group and luteolin group. Intra‑peritoneal injection of cearulein was performed in the acute pancreatitis group and luteolin group to induce acute pancreatitis whereas the luteolin group received intra‑peritoneal injection of luteolin. The control group received intra‑peritoneal injection of normal saline. Then, the expression of SOX9, phosphorylated (p‑) STAT3, p‑EGFR, cytokeratin‑19, Ki67 and N‑cadherin were determined by immunohistochemistry. Morphological changes of acinar cells were determined by hematoxylin and eosin staining. The mRNA expression of the epithelial‑mesenchymal transition markers CDH1, CDH2, Slug, Zeb1, EpCAM, ZO1, Vimentin, Snail and Twist was determined by reverse transcription‑quantitative polymerase chain reaction. It was identified that luteolin inhibits the formation of tubular complexes and ectopic expression of cytokeratin‑19 and luteolin also decreased proteins of SOX9, p‑STAT3 and p‑EGFR. In addition, luteolin inhibits proliferation and epithelial‑mesenchymal transition of acinar cells induced by acute pancreatitis. As tubular complex formation and ectopic expression of cytokeratin‑19 were two prominent characters of acinar‑ductal metaplasia, it was concluded that luteolin inhibits acinar‑ductal metaplasia induced by pancreatitis and also inhibits pancreatitis‑induced proliferation and epithelial‑mesenchymal transition of acinar cells. Acinar‑ductal metaplasia and proliferation have close associations with pancreatic carcinogenesis. It is suggested that luteolin has potential anti‑pancreatic carcinogenesis effects and merits further investigation.

The aim of the present study was to investigate the pancreatic exocrine function in a canine model and to analyze the changes in organelles of pancreatic acinar cells during the early stage of acute pancreatitis (AP). AP was induced by retrograde injection of 5% sodium taurocholate (0.5 ml/kg) into the main pancreatic duct of dogs. The induction of AP resulted in serum hyperamylasemia and a marked reduction of amylase activity in the pancreatic fluid (PF). The pancreatic exocrine function was markedly decreased in subjects with AP compared with the control group. After the induction of AP, histological examination showed acinar cell edema, cytoplasmic vacuolization, fibroblasts infiltration, and inflammatory cell infiltration in the interstitium. Electron micrographs after the induction of AP revealed that most of the rough endoplasmic reticulum (RER) were dilated and that some of the ribosomes were no longer located on the RER. The mitochondria were swollen, with shortened and broken cristae. The present study demonstrated, in a canine model, a reduced volume of PF secretion with decreased enzyme secretion during the early stage of AP. Injury of mitochondria and dilatation and degranulation of RER may be responsible for the reduced exocrine function in AP. Furthermore, the present model and results may be useful for researching novel therapeutic measures in AP.

Xince Huang,1 Shengjie Dai,1 Juji Dai,1 Yuwu Xiao,1 Yongyu Bai,1 Bicheng Chen,1,2 Mengtao Zhou1 1Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of China; 2Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou Key Laboratory of Surgery, Wenzhou, Zhejiang Province, People’s Republic of China Abstract: Luteolin, a flavone, has been shown to exhibit anticancer properties. Here, we investigated whether luteolin affects epithelial–mesenchymal transition (EMT) and invasiveness of pancreatic cancer cell lines and their underlying mechanism. Pancreatic cancer cell lines PANC-1 and SW1990 were used in our study, and their EMT characters, matrix metalloproteinase (MMP) expression level, invasiveness, and signal transducer and activator of transcription 3 (STAT3) activity were determined after luteolin treatment. We also treated pancreatic cancer cells with interleukin-6 (IL-6) to see whether IL-6-induced activation of STAT3, EMT, and MMP secretion was affected by luteolin. We found that luteolin inhibits EMT and MMP2, MMP7, and MMP9 expression in a dose-dependent manner, similar to STAT3 signaling. Through Transwell assay, we found that invasiveness of pancreatic cancer cells was inhibited by luteolin. EMT characters and MMP secretion increase with STAT3 activity after IL-6 treatment and these effects, caused by IL-6, were inhibited by luteolin. We concluded that luteolin inhibits invasiveness of pancreatic cancer cells, and we speculated that luteolin inhibits EMT and MMP secretion likely through deactivation of STAT3 signaling. Luteolin has potential antitumor effects and merits further investigation. Keywords: epithelial–mesenchymal transition, matrix metalloproteinase, luteolin, STAT3

Dexamethasone (Dex)-induced osteoporosis has been described as the most severe side effect in long-term glucocorticoid therapy. The decreased bone mass and the increased marrow fat suggest that Dex possibly shifts the differentiation of bone marrow stromal cells (BMSCs) to favor adipocyte over osteoblast, but the underlying mechanisms are still unknown. In this paper, we established a Dex-induced osteoporotic mouse model, and found that BMSCs from Dex-treated mice are more likely to differentiate into adipocyte than those from control mice, even under the induction of bone morphogenetic protein-2 (BMP2). We also discovered both in vitro and in vivo that the expression level of adipocyte regulator CCAAT/enhancer-binding protein alpha (C/EBPalpha) is significantly upregulated in Dex-induced osteoporotic BMSCs during osteoblastogenesis by a mechanism that involves inhibited DNA hypermethylation of its promoter. Knockdown of C/EBPalpha in Dex-induced osteoporotic cells rescues their differentiation potential, suggesting that Dex shifts BMSC differentiation by inhibiting C/EBPalpha promoter methylation and upregulating its expression level. We further found that the Wnt/beta-catenin pathway is involved in Dex-induced osteoporosis and C/EBPalpha promoter methylation, and its activation by LiCl rescues the effect of Dex on C/EBPalpha promoter methylation and osteoblast/adipocyte balance. This study revealed the C/EBPalpha promoter methylation mechanism and evaluated the function of Wnt/beta-catenin pathway in Dex-induced osteoporosis, providing a useful therapeutic target for this type of osteoporosis.