Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1‐p21/ZEB‐1 pathway

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol‐3‐phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan‐2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB‐1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB‐1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1‐p21/ZEB‐1 pathway, which provides new gene target therapy for PC.     

GDP Induces PANC-1 Human Pancreatic Cancer Cell Death Preferentially under Nutrient Starvation by Inhibiting PI3K/Akt/mTOR/Autophagy Signaling Pathway

Pancreatic tumors are hypovascular, which leads to a poor nutrient supply to support the aggressively proliferating tumor cells. However, human pancreatic cancer cells have extreme resistance to nutrition starvation, which enables them to survive under severe metabolic stress conditions within the tumor microenvironment, a phenomenon known as “austerity” in cancer biology. Discovering agents which can preferentially inhibit the cancer cells’ ability to tolerate starvation conditions represents a new generation of anticancer agents. In this study, geranyl 2,4-dihydroxy-6-phenethylbenzoate (GDP), isolated from Boesenbergia pandurata rhizomes, exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition starvation conditions. GDP also possessed PANC-1 cell migration and colony formation inhibitory activities under normal nutrient-rich conditions. Mechanistically, GDP inhibited PI3K/Akt/mTOR/autophagy survival signaling pathway, leading to selective PANC-1 cancer cell death under the nutrition starvation condition. Therefore, GDP is a promising anti-austerity agent for drug development against pancreatic cancer.     

GATA1通过上调NANOG促进胰腺癌肿瘤干细胞形成

目的:探究GATA1在胰腺癌肿瘤干细胞形成中的功能和作用机制。方法:通过流式细胞术检测GATA1对胰腺癌肿瘤干细胞形成的影响;通过实时荧光定量PCR和Western印迹筛选和验证GATA1下游的干性基因;通过双萤光素酶报告基因实验和染色质免疫共沉淀明确GATA1的调控机制。结果:GATA1过表达细胞株中肿瘤干细胞含量增加;GATA1上调NANOG的mRNA和蛋白表达水平;GATA1可以增强NANOG启动子的活性;GATA1结合在NANOG启动子-527^-524bp处的GATA序列。结论:GATA1可以通过结合在NANOG启动子上激活其转录,促进胰腺癌肿瘤干细胞的形成。     

Long noncoding RNA FEZF1-AS1 predicts poor prognosis and modulates pancreatic cancer cell proliferation and invasion through miR-142/HIF-1α and miR-133a/EGFR upon hypoxia/normoxia

Nowadays, pancreatic cancer (PC) remains the most lethal tumor, partially due to the invasive and treatment-resistant phenotype induced by the extent of hypoxic stress within the tumor tissue. According to previous studies, miR-142/HIF-1α and miR-133a/EGFR could modulate PC cell proliferation under hypoxic and normoxic conditions, respectively. In the present study, FEZF1-AS1, a recently described oncogenic long noncoding RNA, was predicted to target both miR-142 and miR-133a; thus, we hypothesized that FEZF1-AS1 might affect PC cell proliferation through these two axes under hypoxic or normoxic conditions. In PC cell lines, FEZF1-AS1 acted as an oncogene via promoting PC cell proliferation and invasion through miR-142/HIF-1α axis under hypoxic condition; however, FEZF1-AS1 failed to affect the protein levels of HIF-1α and VEGF under the normoxic condition, suggesting the existence of another signaling pathway under normoxic condition. As predicted by an online tool, FEZF1-AS1 could target miR-133a to inhibit its expression; under the normoxic condition, FEZF1-AS1 exerted its effect on PC cell lines through miR-133a/EGFR axis. Taken together, FEZF1-AS1 might be a promising target in controlling the aberrant proliferation and invasion of PC cell lines.     

Itraconazole inhibits proliferation of pancreatic cancer cells through activation of Bak-1

Itraconazole is an FDA-approved antifungal agent, which has been reported to possess promising anticancer activities in recent years. This study investigates the antiproliferative effects of itraconazole on pancreatic cancer cells and the molecular mechanism of its apoptosis-inducing effects. In this study, our results showed that itraconazole inhibited the growth of pancreatic cancer cells in vitro, and it also significantly inhibited the tumor growth of CFPAC-1 xenografts in vivo. Itraconazole induced apoptosis through ROS generation and mitochondrial membrane depolarization. A Bak-1 activation dependent apoptosis was identified in CFPAC-1 cells. These data suggested that itraconazole exhibited antiproliferative effects in pancreatic cancer cells by inducing apoptosis through Bak-1 activation.     

炎症评分与晚期胰腺癌患者预后相关性研究

目的:评估炎症评分对接受放化疗(CRT)的局部晚期胰腺癌患者的无进展生存期(PFS)和总生存期(OS)的预测价值。方法:选取235例晚期胰腺癌患者,接受治疗前,所有患者均进行临床评估、实验室检查和影像学检查。比较两组患者的PFS和OS;评估与患者预后差、肿瘤减少率、6个月内肿瘤转移相关的预测因子。结果:患者平均PFS和OS分别为10.2个月和18.8个月。格拉斯哥预后评分(GPS)2、血浆纤维蛋白原(FIB)≥400 mg/dL为PFS和OS较差的的独立预测因素;小野寺预后营养指数(OPNI)是CRT后肿瘤减少率提高的预测因子(P0.05);GPS 2、FIB≥400 mg/dL的患者的早期转移发生率显著提高(P0.05)。结论:格拉斯哥预后评分、纤维蛋白原、小野寺预后营养指数是评估接受放化疗治疗的局部晚期胰腺癌患者有效的治疗和预后预测因子。     

Palbociclib,a selective CDK4/6 inhibitor,restricts cell survival and epithelial-mesenchymal transition in Panc-1 and MiaPaCa-2 pancreatic cancer cells

The mortality rate of pancreatic cancer has close parallels to its incidence rate because of limited therapeutics and lack of effective prognosis. Despite various novel chemotherapeutics combinations, the 5-year survival rate is still under 5%. In the current study, we aimed to modulate the aberrantly activated PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) signaling with the treatment of CDK4/6 inhibitor PD-0332991 (palbociclib) in Panc-1 and MiaPaCa-2 pancreatic cancer cells. It was found that PD-0332991 effectively reduced cell viability and proliferation dose-dependently within 24 hours. In addition, PD-0332991 induced cell cycle arrest at the G1 phase by downregulation of aberrant expression of CDK4/6 through the dephosphorylation of Rb in each cell lines. Although PD-0332991 treatment increased epithelial markers and decreased mesenchymal markers, the nuclear translocation of β-catenin was not prevented by PD-0332991 treatment, especially in MiaPaCa-2 cells. Effects of PD-0332991 on the regulation of PI3K/AKT signaling and its downstream targets such as GSK-3 were cell type-dependent. Although the activity of AKT was inhibited in both cell lines, the phosphorylation of GSK-3β at Ser9 increased only in Panc-1. In conclusion, PD-0332991 induced cell cycle arrest and reduced the cell viability of Panc-1 and MiaPaCa-2 cells. However, PD-0332991 differentially affects the regulation of the PI3K/AKT pathway and EMT process in cells due to its distinct influence on Rb and GSK-3/β-catenin signaling. Understanding the effect of PD-0332991 on the aberrantly activated signaling axis may put forward a new therapeutic strategy to reduce the cell viability and metastatic process of pancreatic cancer.     

Targeting Wnt/tenascin C-mediated cross talk between pancreatic cancer cells and stellate cells via activation of the metastasis suppressor NDRG1

A major barrier to successful pancreatic cancer (PC) treatment is the surrounding stroma, which secretes growth factors/cytokines that promote PC progression. Wnt and tenascin C (TnC) are key ligands secreted by stromal pancreatic stellate cells (PSCs) that then act on PC cells in a paracrine manner to activate the oncogenic β-catenin and YAP/TAZ signaling pathways. Therefore, therapies targeting oncogenic Wnt/TnC cross talk between PC cells and PSCs constitute a promising new therapeutic approach for PC treatment. The metastasis suppressor N-myc downstream-regulated gene-1 (NDRG1) inhibits tumor progression and metastasis in numerous cancers, including PC. We demonstrate herein that targeting NDRG1 using the clinically trialed anticancer agent di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibited Wnt/TnC-mediated interactions between PC cells and the surrounding PSCs. Mechanistically, NDRG1 and DpC markedly inhibit secretion of Wnt3a and TnC by PSCs, while also attenuating Wnt/β-catenin and YAP/TAZ activation and downstream signaling in PC cells. This antioncogenic activity was mediated by direct inhibition of β-catenin and YAP/TAZ nuclear localization and by increasing the Wnt inhibitor, DKK1. Expression of NDRG1 also inhibited transforming growth factor (TGF)-β secretion by PC cells, a key mechanism by which PC cells activate PSCs. Using an in vivo orthotopic PC mouse model, we show DpC downregulated β-catenin, TnC, and YAP/TAZ, while potently increasing NDRG1 expression in PC tumors. We conclude that NDRG1 and DpC inhibit Wnt/TnC-mediated interactions between PC cells and PSCs. These results further illuminate the antioncogenic mechanism of NDRG1 and the potential of targeting this metastasis suppressor to overcome the oncogenic effects of the PC-PSC interaction.     

NGF from pancreatic stellate cells induces pancreatic cancer proliferation and invasion by PI3K/AKT/GSK signal pathway

Pancreatic cancer (PC) is a continuously high lethal disease, and the tumour microenvironment plays a pivotal role during PC progression. Herein, we focus on that the Nerve growth factor (NGF)/Tropomyosin-related kinase A (TrkA), in pancreatic stellate cells-pancreatic cancer cells (PSCs-PC cells) co-culture system, influences PC proliferation and invasion. The model of PC cells and PSCs was directly co-cultured in a no-touch manner, using the Transwell as the co-culture system. NGF and TrkA expression was measured in cultured system by real-time PCR, immunofluorescence, Western blotting analysis or ELISA. Small interfering RNA transfection was used to regulate the expression of TrkA in PC cells. The promotion of cancer invasion was investigated using Matrigel Transwell assay. In our study, NGF/TrkA is overexpressed in PSCs-PC cells co-culture system and promotes the invasion and proliferation of PC cells. And the epithelial-mesenchymal transition-related genes are influenced by si-TrkA. What's more, NGF/TrkA regulates the PC cell proliferation and invasion via activation of PI3K/AKT/GSK signalling. The present study demonstrated NGF/TrkA promoted the PC cell proliferation and invasion in the co-culture system by the activation of the PI3K/AKT/GSK signal cascade, providing a potential therapeutic target for PC patients.     

Metabonomic models of human pancreatic cancer using 1D proton NMR spectra of lipids in plasma

In this study, we hypothesized that the altered insulin and glucose levels in male pancreatic cancer patients reported in a recent JAMA article would result in an altered lipid profile in the blood of pancreatic cancer patients when compared to controls (Stolzenberg-Solomon et al., 2005). Proton nuclear magnetic resonance (NMR) spectra of human lipophilic plasma extracts were used in order to build partial least squares discriminant function (PLS-DF) models that classified samples as belonging to the pancreatic control group or to the pancreatic cancer group. The sensitivity, specificity, and overall accuracy of the PLS-DF models based on 4 bins were 96%, 88%, and 92%, respectively. The sensitivity, specificity, and overall accuracy of the PLS-DF models based on 5 bins were 98%, 94%, and 96%, respectively. The sensitivity, specificity and overall accuracy of both the 4-bin and 5-bin PLS-DF models dropped only 1–2% during leave-25%-out cross-validation testing. Mass spectrometric profiling of phospholipids in plasma found three phosphatidylinositols that were significantly lower in pancreatic cancer patients than in healthy controls. The cancer models are based upon changes in lipid profiles that may provide a more sensitive and accurate diagnosis of pancreatic cancer than current methods that are based upon a single biomarker.     

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.     

TMEM158 promotes pancreatic cancer aggressiveness by activation of TGFβ1 and PI3K/AKT signaling pathway

Pancreatic cancer (PC) is one of the most deadly digestive cancers world-wide, with a dismal five-year survival rate of <8%. Upregulation of transmembrane protein 158 (TMEM158) is known to facilitate the progression of several carcinomas. However, little is known concerning the potential roles of TMEM158 in PC. Herein, we first found that TMEM158 was significantly upregulated in PC samples as well as PC cell lines. The overexpression of TMEM158 was significantly correlated with advanced clinicopathologic features (including tumor size, TNM stage, and blood vessel invasion) and poorer prognosis of patients with PC in clinic. Evidenced based on a series of loss- and gain-of-function assays uncovered that TMEM158 enhanced PC cell proliferation, migration, and invasion by stimulating the progression of cell cycle, epithelial–mesenchymal transition, and MMP-2/9 production. Furthermore, mechanism-related investigations disclosed that activation of TGFβ1 and PI3K/AKT signal might be responsible for TMEM158-triggered PC aggressiveness. Collectively, TMEM158 was upregulated in PC and promoted PC cell proliferation, migration, and invasion through the activation of TGFβ1 and PI3K/AKT signaling pathways, highlighting its potential as a tumor promoter and a therapeutic target for PC.     

LyP-1-fMWNTs enhanced targeted delivery of MBD1siRNA to pancreatic cancer cells

Functionalized multi-walled carbon nanotubes have been extensively gained popularity in pancreatic cancer gene therapy. LyP-1, a peptide, has been proved to specifically bind pancreatic cancer cells. The potential therapeutic effect of LyP-1–conjugated functionalized multi-walled carbon nanotubes in treating pancreatic cancer is still unknown. In this study, LyP-1–conjugated functionalized multi-walled carbon nanotubes were successfully synthesized, characterized and showed satisfactory size distribution and zeta potential. Compared with functionalized multi-walled carbon nanotubes, cellular uptake of LyP-1–functionalized multi-walled carbon nanotubes was shown to be increased. Compound of LyP-1–functionalized multi-walled carbon nanotubes and MBD1siRNA showed superior gene transfection efficiency. Moreover, LyP-1-fMWNTs/MBD1siRNA complex could significantly decrease the viability and proliferation and promoted apoptosis of pancreatic cancer cells in vitro. Further xenograft assays revealed that the tumour burden in the nude mice injected with LyP-1–functionalized multi-walled carbon nanotubes/MBD1siRNA was significantly relieved. The study demonstrated that LyP-1–functionalized multi-walled carbon nanotubes/MBD1siRNA could be a promising candidate for tumour active targeting therapy in pancreatic cancer.     

Cell-specific transgene expression from a widely transcribed promoter using Cre/lox in mice

Mice carrying two or more transgenes are used frequently to evaluate oncogene interactions during carcinogenesis. However, neoplastic transformation typically results in reduced expression both of differentiation-specific genes and of transgenes that use their promoters. In contrast, the more widely expressed metallothionein (MT) gene remains expressed at a high level in certain neoplasms, including those developing in pancreas. We have developed a system to maintain high-level, tissue-specific transgene expression during pancreatic carcinogenesis that uses Cre recombinase and a lox site-containing target transgene. Cre was expressed in pancreatic acinar cells under control of the elastase promoter (EL). Cre-mediated target transgene recombination placed a previously silent open-reading frame, encoding rat transforming growth factor alpha (TGFalpha), under control of the MT gene promoter. As long as DNA rearrangement does not occur in other cell types that express MT, TGFalpha expression will be restricted to acinar cells. Development of an effective target transgenic mouse required evaluation of multiple lineages to identify one with sufficient TGFalpha expression to induce pancreatic lesions after transgene rearrangement.     

Chromatin modifying protein 1A (Chmp1A) of the endosomal sorting complex required for transport (ESCRT)-III family activates ataxia telangiectasia mutated (ATM) for PanC-1 cell growth inhibition

Chromatin modifying protein 1A (Chmp1A) is a member of the endosormal sorting complex required for transport (ESCRT)-III family whose overexpression induces growth inhibition, chromatin condensation and p53 phosphorylation. p53 is a substrate for ataxia telangiectasia mutated (ATM), which can be activated upon chromatin condensation. Thus, we propose that Chmp1A regulates ATM, and the nuclear localization signal (NLS) is required for ATM activation. Our data demonstrated that overexpression of full-length Chmp1A induced an increase in active, phosphorylated ATM in the nucleus, where they co-localized. It also induced an increase in phospho-p53 in the nucleus, and in vitro ATM kinase and p53 reporter activities. The intensity of phospho-p53 closely followed that of ectopically induced full-length Chmp1A, suggesting a tight correlation between Chmp1A overexpression and p53 phosphorylation. On the other hand, Chmp1A depletion (reported to promote cell growth) had minor effects on phospho-ATM and p53 expression compared with control, which had very little expression of these proteins. NLS-deleted cells showed uniform cytoplasmic-Chmp1A expression and acted like shRNA-expressing cells (cell growth promotion and minimal effect on ATM), demonstrating the significance of NLS on ATM activation and growth inhibition. C-deleted Chmp1A, detected in the cytoplasm at the enlarged vesicles, increased phospho-ATM and p53, and inhibited growth; yet it had no effect on in vitro ATM kinase or p53 reporter activities, suggesting that the C-domain is not required for ATM activation. Finally, ATM inactivation considerably reduced Chmp1A mediated growth inhibition and phosphorylation of p53, showing that Chmp1A regulates tumor growth partly through ATM signaling.     

Cold Shock Domain Containing E1 (CSDE1) Protein is Overexpressed and Can be Targeted to Inhibit Invasiveness in Pancreatic Cancer Cells

Pancreatic cancer has a dismal prognosis and to date there are no targeted therapies for this malignancy. Using shotgun proteomics, the mRNA binding protein cold shock domain containing E1 (CSDE1), also called upstream‐of‐N‐Ras, is detected in pancreatic cancer cell lines but not in normal pancreatic epithelial cells. The expression of CSDE1 in pancreatic cancer cells is confirmed by Western blotting and immunohistochemistry of human pancreatic tumors. In vitro functional assays show that siRNA downregulation of CSDE1 or gene knockout using CRISPR‐Cas9 significantly reduce the invasiveness of pancreatic cancer cells. Together, this study reveals that CSDE1 is overexpressed in pancreatic cancer and is a potential therapeutic target to inhibit pancreatic cancer cell invasion.