Malignant Behaviors Of Pancreatic Cancer Cells Research Articles

PELI1 overexpression contributes to pancreatic cancer progression through upregulating ubiquitination-mediated INPP5J degradation

Inositol Polyphosphate-5-Phosphatase J (INPP5J), a 5-phosphatase, has been identified as a tumor suppressor in several types of cancer. However, its role in pancreatic cancer (PC) is unknown. We found that the INPP5J expression was markedly lower in PC tissues (n = 50) compared to paired adjacent non-tumor tissues, and the lower INPP5J expression was relevant to a worse prognosis of PC patients. We thus proposed that INPP5J might inhibit PC progression and conducted gain-of- and loss-of-function experiments to test our hypothesis. Our results showed that overexpression of INPP5J inhibited cell proliferation, invasion, migration, and xenografted tumor of PC cells. INPP5J silencing showed the opposite effect. Pellino E3 Ubiquitin Protein Ligase 1 (PELI1) is one of the ubiquitin ligases known to promote ubiquitination of its downstream targets. We found that PELI1 could interact with INPP5J and promote the ubiquitination and degradation of INPP5J. PELI1 overexpression enhanced malignant behaviors of PC cells. However, INPP5J overexpression restored the alterations caused by PELI1 overexpression. In conclusion, the results suggest that the decreased INPP5J expression, caused by PELI1 through ubiquitination, may promote PC progression. The PELI1-INPP5J axis represents a potential therapeutic targetable node for PC.

GID2 Interacts With CDKN3 and Regulates Pancreatic Cancer Growth and Apoptosis

Dysregulation of deubiquitinase or ubiquitinase-mediated protein expression contributes to various diseases, including cancer. In the present study, we identified GID2, a subunit of the glucose-induced degradation-deficient (GID) complex that functions as an E3 ubiquitin ligase, as a potential key candidate gene in pancreatic cancer (PC) progression. The functional role and potential mechanism of GID2 in PC progression were investigated. Integrated bioinformatics analysis was performed to identify differentially expressed genes in PC based on the Gene Expression Profiling Interactive Analysis data sets. We found that GID2 was upregulated in PC tissues and that a high level of GID2 expression in clinical PC samples was positively associated with tumor stage and poor survival. Functional assays elucidated that GID2 expression promoted cell growth in vitro and accelerated tumor growth in vivo. GID2 knockdown effectively attenuated the malignant behaviors of PC cells and tumor formation. Furthermore, the protein network that interacted with the GID2 protein was constructed based on the GeneMANIA website. Cyclin-dependent kinase inhibitor 3 (CDKN3), a cell cycle regulator, was identified as a potential target of the GID2 protein. We revealed that GID2 positively regulated CDKN3 expression and inhibited CDKN3 ubiquitination. Furthermore, CDKN3 downregulation reversed the promoting effects of GID2 on PC progression. Therefore, the present study demonstrated that GID2 might regulate PC progression by maintaining the stability of the CDKN3 protein. These findings highlight the potential roles of the GID2/CDKN3 axis as a potential therapeutic target in PC.

Cancer-Associated Fibroblast-Derived Exosomal miRNA-320a Promotes Macrophage M2 Polarization In Vitro by Regulating PTEN/PI3Kγ Signaling in Pancreatic Cancer.

Our previous study has indicated that cancer-associated fibroblasts (CAFs) play a crucial role in regulating gemcitabine resistance through transferring exosomal miRNA-106b to cancer cells. Tumor-associated macrophages (TAMs) are recently verified to facilitate gemcitabine resistance. However, the effect of CAFs in regulating TAMs function in pancreatic cancer (PCa) remains unclear. Here, primary CAFs were extracted from tumor tissues of PCa patients, and CAFs-derived exosomes (CAFs-Exo) were acquired and authenticated by transmission electron microscopy, qNano, and western blot analysis. The role of exosomal miRNA-320a in facilitating macrophage M2 polarization was investigated in vitro. We found that CAFs-derived conditioned medium (CM) possessed a higher potential to promote macrophage M2 polarization compared with normal fibroblasts (NFs) or PCa cell-derived CM. Furthermore, CAFs-Exo treatment polarized macrophage to M2 phenotype. miRNA-320a levels were remarkably increased in CAFs-Exo versus NFs-Exo. More important, miRNA-320a could be transferred from CAFs to macrophages through exosomes, and miRNA-320a overexpression in macrophages facilitated its M2 polarization. Functionally, miRNA-320a-overexpressed macrophages facilitated PCa cell proliferation and invasion. CAFs pretreated with miRNA-320a inhibitor reduced miRNA-320a expression in CAFs-Exo and led to decreased M2 macrophage polarization. Finally, we verified that miRNA-320a polarized macrophage to M2 phenotype by regulating PTEN/PI3Kγ signaling. Taken together, the current data demonstrated that CAFs-derived exosomal miRNA-320a facilitated macrophage M2 polarization to accelerate malignant behavior of PCa cells.

Exosome-derived FGD5-AS1 promotes tumor-associated macrophage M2 polarization-mediated pancreatic cancer cell proliferation and metastasis

Inflammatory molecules and exosomes are crucial for signal transduction between tumor-associated macrophages and tumor cells. IL-6, a key inflammatory molecule secreted by M2 macrophages after polarization, can mediate malignant progression of pancreatic cancer (PC). However, the functions and mechanisms of IL-6 and tumor-derived exosomes in tumor-associated macrophages and PC remain unclear. Transcriptome chip and quantitative reverse transcription PCR experiments indicated that FGD5-AS1 induced IL-6 and high FGD5-AS1 expression correlated with the poor prognosis in PC patients. RNA pulldown, mass spectrometry, and dual luciferase reporter assays were used to identify the mechanism of exosomal FGD5-AS1 in promoting PC progression and M2 macrophage polarization. FGD5-AS1 exerted cancer-promoting functions when co-cultured with M2 macrophages. PC-derived exosomal FGD5-AS1 stimulated M2 macrophage polarization by activating STAT3/NF-κB pathway. FGD5-AS1 interacts with p300, resulting in STAT3 acetylation, thus promoting nuclear localization and transcriptional activity of STAT3/NF-κB. These data indicated that PC cells generate FGD5-AS1-rich exosomes, which cause M2 macrophage polarization to promote the malignant behaviors of PC cells. Targeting exosomal FGD5-AS1 may provide a potential diagnosis and treatment strategy for PC.

Circular RNA hsa_circ_0006117 Facilitates Pancreatic Cancer Progression by Regulating the miR-96-5p/KRAS/MAPK Signaling Pathway

Circular RNAs (circRNAs) play key roles in many malignant tumors, including pancreatic cancer (PC); however, whether circular RNA hsa_circ_0006117, a newly identified circRNA, has a role in PC has not been investigated. Here, in order to elucidate the role and potential molecular mechanisms of circRNAs, we utilized bioinformatic tolls to screen the differentially expressed circRNAs in PC. Subsequently, circular RNA hsa_circ_0006117 was identified as being highly expressed in PC tissues in a screen of two GEO datasets, which was further verified in PC cell lines and tissues. Then, its molecular characteristics were investigated using methods such as Sanger sequencing and fluorescence in situ hybridization (FISH). Functional experiments subsequently indicated that circular RNA hsa_circ_0006117 facilitated the malignant behaviors of PC cells, prompting that it plays an oncogenic role in PC. Moreover, we found that circular RNA hsa_circ_0006117 exerts its PC-promoting effects via activating the KRAS/mitogen-activated protein kinase (MAPK) signaling pathway. Through bioinformatics exploration and dual-luciferase reporter assays, miR-96-5p was identified as a downstream target of circular RNA hsa_circ_0006117. A series of assays confirmed that circular RNA hsa_circ_0006117 acted as a miR-96-5p sponge, thereby promoting the malignant features of PC in a miR-96-5p/KRAS axis-dependent manner. Taken together, our study indicated, for the first time, that the specifically highly expressed circular RNA hsa_circ_0006117 facilitates PC progression via the modulation of the miR-96-5p/KRAS/MAPK signaling pathway and might be a hopeful therapeutic target for PC.

Identification of Circ_001569 as a Potential Biomarker in the Diagnosis and Prognosis of Pancreatic Cancer.

Emerging evidence has shown that circular RNAs (circRNAs) serve as a promising biomarker in different malignancies. Specifically, circ_001569 has been found to be upregulated in some types of human gastrointestinal cancer. In this study, we aimed to investigate clinical significances, diagnostic and prognostic values of circ_001569 in pancreatic cancer (PC), and identify its effects on the malignant behaviors of PC cells. The expression of circ_001569 was determined in 26 tissues samples and 97 plasma samples from PC patients by qRT-PCR. Relationship between circ_001569 expression and clinicopathological parameters was analyzed by Chi-square test. Diagnostic and prognostic values of circ_001569 were evaluated by ROC curves, Kaplan-Meier curves, and Cox regression analysis. The effects of circ_001569 on the proliferation, migration, invasion, and apoptosis of PC cells were assessed by MTT, wound healing, Transwell invasion assays, and flow cytometric analysis, respectively. Results showed that the expression of circ_001569 was upregulated in tissues and plasma of PC patients. High circ_001569 level was positively correlated with lymphatic metastasis, clinical stage, and venous invasion. Circ_001569 level was an independent prognostic indicator for overall survival rates of PC patients, and patients with high circ_001569 level had a poor prognosis. The AUC of circ_001569 was 0.716 (95% CI: 0.642-0.790) with a sensitivity and specificity of 62.76% and 74.29%, respectively. In vitro, circ_001569 silencing decreased cell proliferation, migration, and invasion, but promoted cell apoptosis of PC cells. Our data demonstrate that high circ_001569 level associates with tumor malignant behaviors, and may serve as a potential biomarker in the diagnosis and prognosis of PC.

Role of miR-214-5p in the migration and invasion of pancreatic cancer cells.

To analyze the role of miR-214-5p in proliferation and metastasis of pancreatic cancer (PC) cells, as well as its underlying mechanism. 30 pairs of PC tissues and adjacent normal tissues were collected in our Department. The expression level of miR-214-5p was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Biological information analysis and luciferase report gene assay were used to verify potential target genes of miR-214-5p. Cell counting kit-8 (CCK-8) and transwell methods were applied to observe the interference of miR-214-5p on invasion and migration of PC cells. Western blot (WB) assay was applied to determine the expression changes of Jagged 1 (JAG1) and epithelial-mesenchymal transition (EMT)-related genes in PC cells. QRT-PCR results showed that the expression level of miR-214-5p is significantly down-regulated in PC tissues and cells. Bioinformatics software and luciferase report gene assay identified that JAG1 is a target gene of miR-214-5p. The negative correlation between protein expressions of miR-214-5p and JAG1 was assessed by Western Blot assay. Furthermore, miR-214-5p could suppress cell proliferation, invasion and migration, and it also blocked the EMT in PC cells in vitro. Meanwhile, JAG1 overexpression reversed the inhibitory effects of miR-214-5p on proliferation, invasion and migration of PC cells. Overexpressing miR-214-5p could significantly inhibit malignant behavior of PC cells through targeted regulation of JAG1. Thus, miR-214-5p might be a potential therapeutic target for treatment of PC.

Vasohibin 2 promotes malignant behaviors of pancreatic cancer cells by inducing epithelial-mesenchymal transition via Hedgehog signaling pathway.

BackgroundBased on previous findings, we hypothesized that Vasohibin 2 (VASH2) protein may induce epithelial‐mesenchymal transition (EMT) of pancreatic cancer (PC) cells by promoting the malignant behaviors of these cells. The present study aimed to test this hypothesis and explore the possible mechanisms involved.MethodsThe expression of VASH2 in PC tissues and cell lines was detected by quantitative real‐time PCR and Western blot. PC cells with overexpression or knockdown of VASH2 were used to examine the involvement of VASH2 in EMT by detecting the expression of epithelial (E‐cadherin) and mesenchymal (vimentin) markers and EMT‐related transcription factor ZEB1/2, in gemcitabine resistance and tumor cell invasion by apoptosis and invasion assays, and in cancer stem cell‐like phenotypes by detecting the proportion of CD24+CD44+ and side population (SP) cells in PC cells with flow cytometry. The impact of VASH2 overexpression and knockdown on components of the Hedgehog signaling pathway was also assessed.ResultsWe found that VASH2 was highly expressed in PC tissues and cells. It promoted the EMT of PC cells by altering ZEB1/2 expression. VASH2 also stimulated invasion and chemotherapeutic resistance of PC cells and increased the proportion of cancer stem‐like cells in PC cells. VASH2 did so by upregulating the expression of multiple molecules in the Hedgehog signaling pathway of PC cells.ConclusionVASH2 promotes malignant behaviors of PC cells by inducing EMT via activation of the Hedgehog signaling pathway.

Abstract 1457: Development of miRNA-loaded polymeric nanoformulation for pancreatic cancer therapy

Abstract MicroRNAs (miRNAs) are small (18-22 nucleotide long) non-coding RNAs that play important roles in biological processes through post-transcriptional regulation of gene expression. Their aberrant expression and functional significance are reported in several human malignancies, including pancreatic cancer (PC). Recently, we identified miR-150 as a novel tumor suppressor microRNA in PC and demonstrated that its overexpression inhibits growth and malignant behavior of PC cells suggesting that restoration of miR-150 could serve as an effective therapeutic approach for pancreatic cancer. In the present study, we have developed a nanoparticle-based miR-150 delivery system and tested its therapeutic efficacy by in vitro assays. Using double emulsion solvent evaporation method, we developed a poly (D, L-lactide-co-glycolide) (PLGA)-based nanformulation (NF) of miR-150 (miR-150-NF). Polyethyleneimine (a cationic polymer) was incorporated in PLGA matrix to increase the encapsulation of miR-150. Physical characterization of miR-150-NF demonstrated that miR-150-loaded nanoparticles were spherical in shape, had high encapsulation efficiency (>75%), were stable at room temperature and exhibited sustained release profile. miR-150-NF efficiently delivered miR-150 mimics to PC cells and led to the downregulation of its target gene (MUC4) expression. Downregulation of MUC4 correlated with a concomitant decrease in the expression of its interacting partner, HER2, and repression of its downstream signaling. Furthermore, treatment of PC cells with miR-NF suppressed their growth, clonogenicity, motility and invasion. Together, these findings suggest that PLGA-based nanovector platform could be a potential candidate for systemic delivery of miR-150 to the pancreatic tumor cells and can be a step forward in miRNA-based cancer therapeutics. Citation Format: Sumit Arora, Suresh K. Swaminathan, Sanjeev K. Srivastava, Seema Singh, Jayanth Panyam, Ajay P. Singh. Development of miRNA-loaded polymeric nanoformulation for pancreatic cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2014-1457