Invasion Of Pancreatic Ductal Adenocarcinoma Cells Research Articles

Loss of Heterozygosity for KrasG12D Promotes Malignant Phenotype of Pancreatic Ductal Adenocarcinoma by Activating HIF-2α-c-Myc-Regulated Glutamine Metabolism.

Loss of heterozygosity (LOH) for KRAS, in which a wild-type KRAS allele is progressively lost, promotes invasive and migratory abilities of pancreatic ductal adenocarcinoma (PDAC) cells and tissues. Moreover, the occurrence of KrasG12D-LOH activates nonclassical glutamine metabolism, which is related to the malignant behavior of PDAC cells. Herein, we aim to demonstrate the regulatory link between hypoxia-inducible factor-2α (HIF-2α) and glutamine metabolism that mediates malignant phenotypes in KrasG12D-LOH PDAC cells. HIF-2α-shRNA knockdown lentivirus transfection and metabolite analysis were performed in KrasG12D-LOH and KrasG12D cell lines, respectively. Cell proliferation, migration, and invasion were examined using Cell Counting Kit-8, colony formation, and Transwell assays. Cell cycle phase and apoptosis were determined using flow cytometry. Western blotting and real-time quantitative PCR were also performed. Additionally, a subcutaneous xenograft mouse model was established. LOH stimulated HIF-2α activity and transactivated c-Myc, which has a central regulatory effect on glutamine metabolism independent of hypoxia. Meanwhile, HIF-2α silencing repressed KrasG12D-LOH PDAC cell proliferation, invasion, and migration. HIF-2α knockdown inhibited glutamine uptake and GOT1 expression via a c-Myc-dependent pathway. Collectively, KrasG12D-LOH can activate HIF-2α to regulate c-Myc-mediated glutamine metabolism and promote malignant phenotypes. Moreover, targeting HIF-2α-c-Myc regulated nonclassical glutamine metabolism, providing a new therapeutic perspective for KrasG12D-LOH PDAC.

Overexpression of laminin-5 gamma-2 promotes tumorigenesis of pancreatic ductal adenocarcinoma through EGFR/ERK1/2/AKT/mTOR cascade.

Pancreatic ductal adenocarcinoma (PDAC) is correlated with poor outcomes because of limited therapeutic options. Laminin-5 gamma-2 (LAMC2) plays a critical role in key biological processes. However, the detailed molecular mechanism and potential roles of LAMC2 in PDAC stay unexplored. The present study examines the essential role and molecular mechanisms of LAMC2 in the tumorigenesis of PDAC. Here, we identified that LAMC2 is significantly upregulated in microarray cohorts and TCGA RNA sequencing data of PDAC patients compared to non-cancerous/normal tissues. Patients with higher transcript levels of LAMC2 were correlated with clinical stages; dismal overall, as well as, disease-free survival. Additionally, we confirmed significant upregulation of LAMC2 in a panel of PDAC cell lines and PDAC tumor specimens in contrast to normal pancreatic tissues and cells. Inhibition of LAMC2 significantly decreased cell growth, clonogenic ability, migration and invasion of PDAC cells, and tumor growth in the PDAC xenograft model. Mechanistically, silencing of LAMC2 suppressed expression of ZEB1, SNAIL, N-cadherin (CDH2), vimentin (VIM), and induced E-cadherin (CDH1) expression leading to a reversal of mesenchymal to an epithelial phenotype. Interestingly, co-immunoprecipitation experiments demonstrated LAMC2 interaction with epidermal growth factor receptor (EGFR). Further, stable knockdown of LAMC2 inhibited phosphorylation of EGFR, ERK1/2, AKT, mTOR, and P70S6 kinase signaling cascade in PDAC cells. Altogether, our findings suggest that silencing of LAMC2 inhibited PDAC tumorigenesis and metastasis through repression of epithelial-mesenchymal transition and modulation of EGFR/ERK1/2/AKT/mTOR axis and could be a potential diagnostic, prognostic, and therapeutic target for PDAC.

PTPN3 Could Βe a Therapeutic Target of Pancreatic Cancer.

Recently, protein tyrosine phosphatase non-receptor type 3 (PTPN3) has gained attention. However, the role of PTPN3 in cancer has not been fully elucidated. In the present study, we analyzed the role of PTPN3 in pancreatic cancer and investigated whether PTPN3 could be a new therapeutic target for pancreatic cancer. Two pancreatic ductal adenocarcinoma (PDAC) cell lines were used as target cells. Cell proliferation was investigated using cell counting and a xenograft mouse model. Migration and invasion were analyzed using Transwell inserts. Activation-related signaling molecules were examined by western blotting. PTPN3 contributes to the proliferation, migration, and invasion of PDAC cells in vitro. PTPN3 promotes tumor growth in a mouse xenograft model, an action mediated partially through the MAPK pathway. PTPN3 could be a new therapeutic target for pancreatic cancer.

Oncogenic Kras-Mediated Cytokine CCL15 Regulates Pancreatic Cancer Cell Migration and Invasion through ROS.

Simple SummaryOncogenic KrasG12D and tumor inflammation are critical components of the development and dissemination of pancreatic ductal adenocarcinoma (PDAC). The aim of this study is to investigate a lesser-known cytokine, CCL15, that functions as a new downstream target of KrasG12D with the purpose of regulating PDAC cell migration and invasion. We showed increased levels of CCL15 as well as the presence of its receptors, including CCR1 and CCR3, in PDAC tissues and cell lines. The knockdown of CCL15 diminished metastatic Panc-1 cell migration, whereas the treatment of CCL15 in non-metastatic BxPC-3 cells promoted BxPC-3 cell motility. Similar results were verified using murine metastatic PDAC KP-2 cells. Furthermore, we demonstrated that CCL15-modulated PDAC cell migration through the upregulation of cellular reactive oxygen species (ROS) levels and the knockdown of KrasG12D resulted in a decrease in CCL15. Altogether, our data unveiled a new mechanism of oncogenic KrasG12D in modulating PDAC inflammation and spreading.Pancreatic ductal adenocarcinoma (PDAC) is well known for its high death rate due to prompt cancer metastasis caused by cancer cell migration and invasion within the early stages of its development. Here, we reveal a new function of cytokine CCL15, namely the upregulation of PDAC cell migration and invasion. We showed increased levels of CCL15 transcripts and protein expressions in human PDAC tissue samples, as well as in cultured cell lines. Furthermore, PDAC cells also expressed CCL15 receptors, including CCR1 and CCR3. Murine PDAC cell lines and tissues strengthened this finding. The manipulation of CCL15 in metastatic Panc-1 cells through CCL15 knockdown or CCL15 neutralization decreased Panc-1 cell motility and invasiveness. In addition, treating non-metastatic BxPC-3 cells with recombinant CCL15 accelerated the cell migration of BxPC-3. A reduction in the levels of reactive oxygen species (ROS) by either N-Acetyl-L-Cysteine treatment or p22phox knockdown led to a decrease in Panc-1 cell migration and a reversed effect on recombinant CCL15-promoted BxPC-3 cell movement. Importantly, the knockdown of oncogenic Kras in Panc-1 cells abolished CCL15 protein expression and impeded cell migration without affecting PDAC cell growth. Altogether, our work elucidates an additional molecular pathway of oncogenic Kras to promote PDAC metastasis through the upregulation of cell migration and invasion by the Kras downstream CCL15, a lesser-known cytokine within the cancer research field.

Cytotoxic Nitrobenzoyl Sesquiterpenoids from an Antarctica Sponge-Derived Aspergillus insulicola.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases of the pancreas with fatal proliferation and metastasis and no medicine available for treatment. From an Antarctica sponge-derived fungus, Aspergillus insulicola HDN151418, four new nitrobenzoyl sesquiterpenoids, namely, insulicolides D-G (1-4), were isolated. Compounds 3 and 4 exhibited selective inhibition against human PDAC cell lines. Further studies indicated that compound 4 could significantly suppress cell proliferation to induce apoptosis and blocked migration and invasion of PDAC cells. Compound 4 could also avoid resistance and improved the therapeutic effect of the chemotherapy drug gemcitabine. A preliminary mechanism study showed that compound 4 can significantly inhibit the expression of EGFR and XIAP in PDAC cells. Altogether, 4 is a potential lead compound for anti-PDAC drug research.

HnRNPC Promotes Malignancy in Pancreatic Cancer through Stabilization of IQGAP3

Due to challenges in early-stage detection, aggressive behavior, and poor response to systemic therapy, pancreatic cancer is one of the most fatal cancer types globally. The role of RNA-binding protein (RBP) transcription and translation of cancer cells has been well demonstrated, although their roles in pancreatic cancer is less well understood. In this study, we found that heterogeneous nuclear ribonucleoprotein C (hnRNPC), a RBP, is highly expressed in pancreatic ductal adenocarcinoma (PDAC) tissues and cells. In addition, we discovered that overexpression of hnRNPC in PDAC cells in vitro increased cell proliferation, migration, invasion, and metastasis. The presence of hnRNPC promoted tumorigenesis of pancreatic cells in metastatic in vivo models, which was also validated. In silico analyses revealed that hnRNPC is a strong positive regulator of IQ Motif Containing GTPase Activating Protein 3 (IQGAP3) activity. The experimental confirmation of this association revealed a direct interaction of IQGAP3 and hnRNPC to induce cell growth and invasion in PDAC cells by activating the epithelial-mesenchymal transition. In light of the findings that hnRNPC accelerates PDAC progression by interfering with IQGAP3, it appears that this technique for diagnosis and treatment of PDAC may have promise.

Circular RNA circ_0047744 suppresses the metastasis of pancreatic ductal adenocarcinoma by regulating the miR-21/SOCS5 axis

There is increasing evidence that circular RNAs (circRNAs) can serve as microRNA (miRNA) sponges to regulate metastasis of multiple tumors, including pancreatic ductal adenocarcinoma (PDAC). However, the role of the circRNA/miRNA regulatory network in metastasis of PDAC has not been elucidated. The purpose of this study is to explore the role of circ_0047744/miR-21/SOCS5 in the metastasis of PDAC. We found that circRNA_0047744 was weakly expressed in PDAC tissues and cell lines. The expression of circ_0047744 was negatively correlated with lymph node metastasis and positively correlated with overall survival in PDAC patients. Functionally, the overexpression of circ_0047744 suppressed cell migration and invasion in vitro and in vivo. Mechanistically, circ_0047744 could regulate SOCS5 expression by acting as a sponge of miR-21 to inhibit migration and invasion of PDAC cells. Our study demonstrates that circ_0047744 acts as an anti-oncogene to inhibit PDAC metastasis by regulating the miR-21/SOCS5 axis, indicating that circ_0047744 may be a potential novel therapeutic target for PDAC patients.

COL11A1-Driven Epithelial-Mesenchymal Transition and Stemness of Pancreatic Cancer Cells Induce Cell Migration and Invasion by Modulating the AKT/GSK-3β/Snail Pathway.

Background: Collagen type XI α1 (COL11A1) is associated with tumorigenesis and development in many human malignancies. Previous reports indicate that COL11A1 may be a significant diagnostic marker for pancreatic ductal adenocarcinoma (PDAC); however, its biological role in PDAC progression remains unclear. In this study, we investigated the influence of COL11A1 on the invasion and migration abilities of pancreatic cancer cells and explored its potential molecular mechanisms. Methods: Cell migration and invasion were assessed using Transwell assays in pancreatic cancer cells transfected with siCOL11A1 and pCNV3-COL11A1 plasmids. The protein and mRNA expression levels of N-cadherin, E-cadherin, Vimentin, cluster of differentiation (CD)-24, CD44, serine–threonine kinase (AKT), glycogen synthase kinase (GSK)-3β, phospho (p)-AKTSer473, p-GSK-3βSer9, and Snail were analyzed using Western blotting and real-time polymerase chain reaction (PCR). The effect of COL11A1 on cell stemness was tested using flow cytometry and clone formation assays. Results: These results demonstrated that COL11A1 significantly promoted the invasion and migration abilities of PDAC cells. Furthermore, COL11A1 facilitated the occurrence of epithelial–mesenchymal transition (EMT) and cell stemness by upregulating the expression levels of p-AKTSer473, p-GSK-3βSer9, and Snail. Conclusions: This study suggests that the activation of the AKT/GSK-3β/Snail signaling pathway induced by COL11A1 plays a major role in the progression of PDAC. Therefore, COL11A1 could serve as a potential target for PDAC treatment.

Identification of DDX31 as a Potential Oncogene of Invasive Metastasis and Proliferation in PDAC.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignant tumors worldwide and has poor prognosis. DEAD box proteins31 (DDX31) participate in cellular processes involving RNA secondary structure changes. However, the functions of DDX31 in PDAC remain to be elucidated. Methods: The key gene DDX31 was identified using a combination of a risk model and weighted gene co-expression network analysis (WGCNA) with R software. The biological functions of DDX31 in PDAC were investigated through bioinformatics analysis and in vitro experiments. Results: Combining with WGCNA and risk model, DDX31 was identified as a potential factor of the invasive metastasis properties of PDAC, and its expression was closely related to the malignant differentiation of PDAC. The results of gene set enrichment analysis (GSEA) showed that DDX31 was correlated with cell invasive metastasis and proliferation by activating MAPK signaling pathway. The inhibition of DDX31 inhibited the invasion and migration of PDAC cells. Survival analysis showed that DDX31 expression was negatively associated with the poor prognosis in patients with PDAC. Interpretation: DDX31 may be a potential factor for PDAC. The inhibition of DDX31 may be a potential way to treat PDAC.

Circ_0072008, an oncogene in pancreatic ductal adenocarcinoma, contributes to tumour cell malignant progression and glycolysis by regulating miR-545-3p/SLC7A11 axis

Background The hsa_circRNA_103809 (circ_0072088) has been an emerging tumour regulator in human cancers, and is identified as one most aberrantly expressed circRNA in patients with pancreatic ductal adenocarcinoma (PDAC). However, the role of circ_0072088 remains unclear in PDAC cells. Methods Expression of circ_0072088, microRNA (miR)-545-3p and solute carrier family 7 member 11 (SLC7A11) was detected by real-time quantitative PCR and western blotting. Cell progression was measured by cell counting kit (CCK)-8 assay, transwell assays and flow cytometry, as well as xenograft tumour models. Glycolysis was evaluated by commercial assay kits. The interaction among circ_0072088, miR-545-3p and SLC7A11 was confirmed by dual-luciferase reporter assay. Results Circ_0072088 was upregulated in PDAC tumours and cells; besides, high circ_0072088 level was associated with high tumour-node-metastasis (TNM) stage. The circ_0072088 siRNA suppressed cell viability, migration, invasion, extracellular acidification rate (ECAR), lactate production, glucose uptake, and ATP generation, but promoted apoptosis rate and oxygen consumption rate (OCR) in SW1990 and PANC-1 cells. In vivo, circ_0072088 knockdown retarded tumour growth of PANC-1 cells. Overexpressing miR-545-3p mimicked circ_0072088 siRNA-induced actions, and inhibited cell progression and glycolysis of SW1990 and PANC-1 cells. Moreover, SLC7A11 downregulation could be mediated by both circ_0072008 siRNA and miR-545-3p mimic, and participating in suppressive role in cell progression and glycolysis of SW1990 and PANC-1 cells. In mechanism, miR-545-3p was targeted by circ_0072008, and SLC7A11 was target of miR-545-3p. Conclusion Circ_0072088 elicited oncogenic role in malignant cell progression and glycolysis of PDAC cells through circ_0072088/miR-545-3p/SLC7A11 pathway. Highlights Circ_0072088 was upregulated in PDAC tumours and was associated with high tumour burden. Blocking circ_0072088 suppressed cell proliferation, migration, invasion, and glycolysis in PDAC cells. Circ_0072088 could directly regulate miR-545-3p, and SLC7A11 was a target of miR-545-3p. Restoring miR-545-3p mimicked the effects of circ_0072088 knockdown in PDAC cell in vitro.

The CTCF/LncRNA-PACERR complex recruits E1A binding protein p300 to induce pro-tumour macrophages in pancreatic ductal adenocarcinoma via directly regulating PTGS2 expression.

BackgroundTumour‐associated macrophages (TAMs) play an important role in promoting the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to study the epigenetic mechanisms in regulating pro‐tumour M2‐polarised TAMs in the PDAC tumour microenvironment.MethodsThis study was conducted based on ex vivo TAMs isolated from PDAC tissues and in vitro THP1‐derived TAM model. RNA‐sequencing (RNA‐seq), assay for transposase‐accessible chromatin with sequencing and chromatin immunoprecipitation sequencing were performed to investigate gene expression, chromatin accessibility, transcription factor binding sites and histone modifications. Gene knockdown in THP1‐derived TAMs was performed with lentivirus, and the impact of THP1‐derived TAMs on invasion and metastasis ability of PDAC cells were investigated with in vitro and in vivo functional assays. RNA‐chromatin interaction was analysed by chromatin isolation through RNA purification with sequencing. RNA‐protein interaction was studied by RNA immunoprecipitation and RNA pull‐down.ResultsOur data showed that the transcription factor CTCF (CCCTC‐binding factor) was highly expressed in TAMs and predicted to be significantly enriched in hyper‐accessible chromatin regions when compared to monocytes. High infiltration of CTCF+ TAMs was significantly associated with poor prognosis in PDAC patients. Knockdown of CTCF in THP1‐derived TAMs led to the down‐regulation of specific markers for M2‐polarised TAMs, including CD206 and CD163. When THP1‐derived TAMs with CTCF knockdown, they showed a decreased ability of invasion and metastasis. Further integrative analysis of multi‐omics data revealed that prostaglandin‐endoperoxide synthase 2 (PTGS2) and PTGS2 antisense NF‐κB1 complex‐mediated expression regulator RNA (PACERR) were critical downstream targets of CTCF and positively correlated with each other, which are closely situated on a chromosome. Knockdown of PACERR exhibited a similar phenotype as observed in CTCF knockdown THP1‐derived TAMs. Moreover, PACERR could directly bind to CTCF and recruit histone acetyltransferase E1A binding protein p300 to the promoter regions of PACERR and PTGS2, thereby enhancing histone acetylation and gene transcription, promoting the M2 polarization of TAMs in PDAC.ConclusionsOur study demonstrated a novel epigenetic regulation mechanism of promoting pro‐tumour M2‐polarised TAMs in the PDAC tumour microenvironment.

HDAC4 Mediates Smoking-Induced Pancreatic Cancer Metastasis.

Cigarette smoking is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). In this project, we investigated the effect of smoking and the role of histone deacetylase 4 (HDAC4) in PDAC invasion and metastasis. Cells were treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and cigarette smoke extract and the mRNA levels of HDACs were measured by real-time polymerase chain reaction. Invasion was measured using the Matrigel Invasion Assay. Syngeneic PDAC mice were treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and metastasis measured. Human PDAC primary and metastatic tissues were analyzed by immunohistochemistry. Levels of HDAC4 mRNA were increased by smoking. Smoking compounds significantly promoted invasion of cancer cells and promoted metastasis of PDAC cells to different organs, including the liver and the lung, whereas inhibition of HDAC4 prevented this effect. The effect of HDAC4 inhibition on preventing smoking-induced metastasis was greater in the liver compared with the lung. We found that HDAC4 is highly expressed in primary and metastatic PDAC tumors. We found that HDAC4 is the only HDAC induced by smoking among all HDACs analyzed. We found that smoking promotes invasion and metastasis of PDAC cells through a mechanism that involves HDAC4 and that HDAC4 is a promising target for preventing PDAC metastasis.

Oxysterol-Binding Protein 2 Promotes Pancreatic Ductal Adenocarcinoma Progression Through Epithelial-Mesenchymal Transition.

Oxysterol-binding protein 2 (OSBP2) is crucial for promoting the growth and development of cancers; however, its effects on pancreatic ductal adenocarcinoma (PDAC) are still unclear. Here, we report that OSBP2 is an efficient tumor-associated protein to lead to extremely malignant characteristics in PDAC. We discovered that increased OSBP2 expression in primary tumors was associated with shorter survival in PDAC patients. Therefore, we used immunohistochemistry (IHC) to analyze the levels of OSBP2 expression in PDAC tissues and adjacent paracancerous tissues. We used wound healing and Transwell assays to evaluate the effects of OSBP2 on PDAC cell (ASPC-1 and BXPC-3) migration and invasion, respectively, and CCK-8 and Annexin V/PI double staining to evaluate the effects of OSBP2 on PDAC cell proliferation and apoptosis, respectively. Western blotting was used to analyze the effect of OSBP2 on the PDAC cell phenotype. We also explored the effect of OSBP2 on chemosensitivity to gemcitabine (GEM) and 5-fluorouracil (5-FU). We validated these findings in an in vivo mouse model. The data show that OSBP2 overexpression promoted PDAC cell migration, invasion, proliferation and chemotherapy resistance, and decreased apoptosis. OSBP2 overexpression downregulated E-cadherin expression and upregulated N-cadherin, vimentin, Snail, Slug, ZEB1, and β-catenin expression. Taken together, our findings indicated that OSBP2 was overexpressed in PDAC and that upregulation of OSBP2 may promote PDAC progression. Therefore, OSBP2 may have potential diagnostic and therapeutic value in PDAC.

LncRNA LINC01857 drives pancreatic adenocarcinoma progression via modulating miR-19a-3p/SMOC2

ObjectivesEmerging evidence has demonstrated that LINC01857 exerts a pivotal function in many cancers. However, its function in Pancreatic Ductal Adenocarcinoma (PDAC) still remains unclear. This study was designed to investigate the regulatory character of LINC01857 in PDAC. MethodsBioinformatic tools and databases were used to seek potential miRNAs and mRNAs. Gene expression was evaluated by Reverse Transcription quantitative real-time Polymerase Chain Reaction (RT-qPCR), and western blot was used for protein level detection. A subcellular fraction assay was done to ascertain the location of LINC01857 in PANC-1 and BxPC-3 human pancreatic cancer cells. CCK-8, EdU, wound healing and Transwell assays were performed to inquire into the influence of LINC01857, and SPARC -related Modular Calcium-binding protein-2 (SMOC2) on cell viability, proliferation, migration, and invasion, respectively. The interaction between LINC01857 and its downstream genes was explored by RNA immunoprecipitation and luciferase reporter assays. ResultsLINC01857 levels were significantly elevated in PDAC. Knockdown of LINC01857 significantly restrained the proliferation, migration, invasion, and Epithelial-Mesenchymal Transition (EMT) process of PDAC cells. MiR-19a-3p was a downstream target of LINC01857, and miR-19a-3p levels were significantly decreased in PDAC cells. In addition, SMOC2 expression had a negative correlation with that of miR-19a-3p, and SMOC2 was a downstream target of miR-19a-3p. Furthermore, SMOC2 upregulation partially abolished the inhibitive influence of LINC01857 downregulation on cell proliferation, migration, invasion, and the EMT process. ConclusionLINC01857 promotes malignant phenotypes of PDAC cells via upregulation of SMOC2 by interacting with miR-19a-3p.

Rosmarinic Acid Decreases the Malignancy of Pancreatic Cancer Through Inhibiting Gli1 Signaling

BackgroundRosmarinic acid (RA) has been shown to exert anti-tumor effects on various types of cancer. However, its roles in the treatment of pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanisms remain elusive. PurposeThe present study aimed to investigate the therapeutic effects of RA on PDAC as well as the underlying mechanisms. Study designEvaluation of the effects of RA on PDAC malignancy both in vitro and in vivo. MethodsCell counting kit 8 (CCK8) assay, colony formation assay, 5-Ethynyl-2′-deoxyuridine (EDU) incorporation assay, cell cycle analysis, and apoptosis assay were conducted to assess the inhibitory effect of RA on PDAC cell proliferation. Meanwhile, western blotting and RT-qPCR assay were performed to detect the target gene expression at protein and mRNA levels, respectively. Moreover, the in vivo anti-tumor activities of RA were assayed in an xenograft mouse model of PDAC. ResultsRA dramatically down-regulated Gli1 and its downstream targets. Further studies showed that RA prevents the nuclear translocation of Gli1, while promoting the degradation of cytosolic Gli1 via the proteasome pathway. Moreover, we observed that RA induced G1/S cell cycle arrest and apoptosis in the PDAC cells through regulating the expression of P21, P27, CDK2, Cyclin E, Bax, and Bcl-2, it inhibited the PDAC cell migration and invasion via E-cadherin and MMP-9. Notably, Gli1 overexpression markedly reversed the above RA-induced effects on PDAC cells, whereas Gli1 knockdown enhanced the effects. Additionally, the in vivo assays demonstrated that RA suppresses the tumor growth of PDAC presumably by inhibiting Gli1. ConclusionWe provided evidence that RA restrained the nuclear translocation of Gli1 and facilitates Gli1 degradation via proteasome pathway, reducing the malignancy of PDAC cells. These findings implicated RA as a therapeutic agent for PDAC.

HNRNPC impedes m6A-dependent anti-metastatic alternative splicing events in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with poor prognosis due to early metastasis. The aberrant N6-methyladenosine (m6A) RNA modification has emerged as an important mechanism in cancer progression and metastasis, but its role in PDAC remained largely unknown. Here, we demonstrated that an m6A regulator, heterogeneous nuclear ribonucleoprotein C (HNRNPC), modulated alternative splicing events to promote PDAC metastasis. In clinical PDAC tissues, high expression of HNRNPC was correlated with metastasis, resulting in poor prognosis in PDAC patients. Knockdown of HNRNPC significantly reduced PDAC cell invasion in vitro and metastasis in vivo. In contrast, overexpression of HNRNPC provoked malignant phenotypes of PDAC cells. Mechanistically, HNRNPC antagonized the anti-metastatic isoform of TAF8 (TAF8L) but increased the pro-metastatic alternative splicing isoform of TAF8 (TAF8S). Mutation of the m6A-site of TAF8 attenuated the interaction between HNRNPC and TAF8 transcript, leading to the decrease of TAF8S. Furthermore, experimental manipulation of the anti-metastasis splicing isoform TAF8L revealed that splice isoform switching of TAF8 is crucial for PDAC metastasis. In conclusion, our findings demonstrate the essentiality of HNRNPC-mediated alternative splicing events that impinges on metastatic PDAC.

NR2F1-AS1 Promotes Pancreatic Ductal Adenocarcinoma Progression Through Competing Endogenous RNA Regulatory Network Constructed by Sponging miRNA-146a-5p/miRNA-877-5p.

The role of NR2F1-AS1 in pancreatic ductal adenocarcinoma (PDAC) remains unknown. Therefore, we aimed to investigate the biological mechanism of NR2F1-AS1 in PDAC. The expression of NR2F1-AS1 was measured by using microarray data and real-time PCR. The effects of NR2F1-AS1 knockdown on proliferation, cell cycle progression, invasion in vitro and tumorigenesis in vivo were investigated. The mechanism of competitive endogenous RNAs was determined from bioinformatics analyses and validated by a dual-luciferase reporter gene assay. Potential target mRNAs from TargetScan 7.2 were selected for subsequent bioinformatics analysis. Key target mRNAs were further identified by screening hub genes and coexpressed protein-coding genes (CEGs) of NR2F1-AS1. NR2F1-AS1 was highly expressed in PDAC, and the overexpression of NR2F1-AS1 was associated with overall survival and disease-free survival. The knockdown of NR2F1-AS1 impaired PDAC cell proliferation, migration, invasion and tumorigenesis. NR2F1-AS1 competitively sponged miR-146a-5p and miR-877-5p, and low expression of the two miRNAs was associated with a poor prognosis. An integrative expression and survival analysis of the hub genes and CEGs demonstrated that the NR2F1-AS1–miR-146a-5p/miR-877-5p–GALNT10/ZNF532/SLC39A1/PGK1/LCO3A1/NRP2/LPCAT2/PSMA4 and CLTC ceRNA networks were linked to the prognosis of PDAC. In conclusion, NR2F1-AS1 overexpression was significantly associated with poor prognosis. NR2F1-AS1 functions as an endogenous RNA to construct a novel ceRNA network by competitively binding to miR-146a-5p/miR-877-5p, which may contribute to PDAC pathogenesis and could represent a promising diagnostic biomarker or potential novel therapeutic target in PDAC.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

Background & AimsInactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development.MethodsWe performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis.ResultsLoss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial–mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata.ConclusionsLoss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

The long non-coding RNA DKFZp434J0226 regulates the alternative splicing process through phosphorylation of SF3B6 in PDAC

BackgroundLong noncoding RNAs (lncRNAs), a type of pervasive genes that regulates various biological processes, are differentially expressed in different types of malignant tumors. The role of lncRNAs in the carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we investigated the role of the lncRNA DKFZp434J0226 in PDAC.MethodsAberrantly expressed mRNAs and lncRNAs among six PDAC and paired non-tumorous tissues were profiled using microarray analysis. Quantitative real-time polymerase chain reaction was used to evaluate DKFZp434J0226 expression in PDAC tissues. CCK-8 assay, wound-healing assay, soft agar colony formation assay, and transwell assay were performed to assess the invasiveness and proliferation of PDAC cells. Furthermore, RNA pull-down, immunofluorescence, RNA immunoprecipitation, and western blotting assays were performed to investigate the association between DKFZp434J0226 and SF3B6. Tumor xenografts in mice were used to test for tumor formation in vivo.ResultsIn our study, 222 mRNAs and 128 lncRNAs were aberrantly expressed (≥ twofold change). Of these, 66 mRNAs and 53 lncRNAs were upregulated, while 75 lncRNAs and 156 mRNAs were downregulated. KEGG pathway analysis and the Gene ontology category indicated that these genes were associated with the regulation of mRNA alternative splicing and metabolic balance. Clinical analyses revealed that overexpression of DKFZp434J0226 was associated with worse tumor grading, frequent perineural invasion, advanced tumor-node-metastasis stage, and decreased overall survival and time to progression. Functional assays demonstrated that DKFZp434J0226 promoted PDAC cell migration, invasion, and growth in vitro and accelerated tumor proliferation in vivo. Mechanistically, DKFZp434J0226 interacted with the splicing factor SF3B6 and promoted its phosphorylation, which further regulated the alternative splicing of pre-mRNA.ConclusionsThis study indicates that DKFZp434J0226 regulates alternative splicing through phosphorylation of SF3B6 in PDAC and leads to an oncogenic phenotype in PDAC.

ELK3 Mediated by ZEB1 Facilitates the Growth and Metastasis of Pancreatic Carcinoma by Activating the Wnt/β-Catenin Pathway.

Rapid progression and metastasis are the major causes of death in patients with pancreatic ductal adenocarcinoma (PDAC). ELK3, a member of the ternary complex factor (TCF), has been associated with the initiation and progression of various cancers. However, the role of ELK3 in PDAC is not yet fully understood. Online databases and immunohistochemistry were used to analyze the ELK3 levels in PDAC tissues. The function of ELK3 was confirmed by a series of in vivo and in vitro studies. Western blotting and immunofluorescence were used to detect the molecular mechanisms of PDAC. ChIP-qPCR was used to study the mechanism responsible for the elevation of ELK3 expression in PDAC. The ELK3 levels were higher in PDAC tissues than in adjacent normal tissues. Functionally, we demonstrated that ELK3 acted as an oncogene to promote PDAC tumorigenesis and metastasis. Further study suggested that ELK3 promoted PDAC cell migration and invasion by activating the Wnt/β-catenin pathway, and proved that ZEB1 could directly bind to the promoter of ELK3 to increase its transcription. Finally, both were associated with the patients’ clinicopathological features and worse overall survival. Conclusively, our findings enrich the role of ELK3 in PDAC, and provide potential avenues for exploring more effective biomarkers and therapeutic strategies for the treatment of PDAC.