Target For Pancreatic Ductal Adenocarcinoma Research Articles

H2A.Z overexpression suppresses senescence and chemosensitivity in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-β-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.

KLHDC7B-DT aggravates pancreatic ductal adenocarcinoma development via inducing cross-talk between cancer cells and macrophages.

Tumor microenvironment (TME) exerts key roles in pancreatic ductal adenocarcinoma (PDAC) development. However, the factors regulating the cross-talk between PDAC cells and TME are largely unknown. In the present study, we identified a long noncoding RNA (lncRNA) KLHDC7B divergent transcript (KLHDC7B-DT), which was up-regulated in PDAC and correlated with poor survival of PDAC patients. Functional assays demonstrated that KLHDC7B-DT enhanced PDAC cell proliferation, migration, and invasion. Mechanistically, KLHDC7B-DT was found to directly bind IL-6 promoter, induce open chromatin structure at IL-6 promoter region, activate IL-6 transcription, and up-regulate IL-6 expression and secretion. The expression of KLHDC7B-DT was positively correlated with IL-6 in PDAC tissues. Via inducing IL-6 secretion, KLHDC7B-DT activated STAT3 signaling in PDAC cells in an autocrine manner. Furthermore, KLHDC7B-DT also activated STAT3 signaling in macrophages in a paracrine manner, which induced macrophage M2 polarization. KLHDC7B-DT overexpressed PDAC cells-primed macrophages promoted PDAC cell proliferation, migration, and invasion. Blocking IL-6/STAT3 signaling reversed the effects of KLHDC7B-DT on macrophage M2 polarization and PDAC cell proliferation, migration, and invasion. In conclusion, KLHDC7B-DT enhanced malignant behaviors of PDAC cells via IL-6-induced macrophage M2 polarization and IL-6-activated STAT3 signaling in PDAC cells. The cross-talk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.

Mutations in key driver genes of pancreatic cancer: molecularly targeted therapies and other clinical implications.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a minimal difference between its incidence rate and mortality rate. Advances in oncology over the past several decades have dramatically improved the overall survival of patients with multiple cancers due to the implementation of new techniques in early diagnosis, therapeutic drugs, and personalized therapy. However, pancreatic cancers remain recalcitrant, with a 5-year relative survival rate of <9%. The lack of measures for early diagnosis, strong resistance to chemotherapy, ineffective adjuvant chemotherapy and the unavailability of molecularly targeted therapy are responsible for the high mortality rate of this notorious disease. Genetically, PDAC progresses as a complex result of the activation of oncogenes and inactivation of tumor suppressors. Although next-generation sequencing has identified numerous new genetic alterations, their clinical implications remain unknown. Classically, oncogenic mutations in genes such as KRAS and loss-of-function mutations in tumor suppressors, such as TP53, CDNK2A, DPC4/SMAD4, and BRCA2, are frequently observed in PDAC. Currently, research on these key driver genes is still the main focus. Therefore, studies assessing the functions of these genes and their potential clinical implications are of paramount importance. In this review, we summarize the biological function of key driver genes and pharmaceutical targets in PDAC. In addition, we conclude the results of molecularly targeted therapies in clinical trials and discuss how to utilize these genetic alterations in further clinical practice.

Comprehensive genomic profiling (CGP) utilizing cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

421 Background: PDAC has a propensity for early systemic dissemination and most pts with primary tumors radiographically confined to the pancreas harbor micro-metastases. cfDNA based CGP offers a non-invasive mechanism to identify actionable genomic targets in PDAC and account for serial clonal evolution in response to therapeutic selection pressure. We interrogated the Foundation Medicine database to characterize cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of pts with PDAC who underwent cfDNA based CGP between May 2016 and February 2020. cfDNA based CGP was done utilizing either the FoundationOne Liquid (F1-Liquid) or the Foundation-ACT (F-ACT) testing platform. Samples were interrogated for exonic and select intronic regions of cancer-related genes for both F1-Liquid (Exons: 70 genes; Select Introns: 7 genes) and F-ACT (Exons: 59 genes; Select Introns: 6 genes). Variant zygosity and somatic/germline status (SGZ) for short variant mutations was computationally predicted without matched normal tissue using an investigational method. A Gaussian Mixture Model (GMM) was used to identify latent molecular classes in samples harboring somatic, pathogenic variants (n=613); the Bayesian Information Criteria (BIC) was used for model selection. Results: We identified 1,009 pts with cfDNA based CGP - median age was 65, 55% were male, and median cfDNA concentration was 24 ng/µL. cfDNA yield had an influence on the detection of somatic alterations (p &lt;0.001). 613/1009 (60.8%) of pts had at least one somatic alteration detected on cfDNA based CGP. TP53 (53.7%), KRAS (40.3%) and CDKN2A (6.5%) were the most frequently altered genes (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [somatic and/or germline BRCA2, ATM, and CHEK2 alterations], were detected in 12.3% (124/1009) pts – 11.4% (46/403) in KRAS mutated pts and 12.9% (78/606) in KRAS wildtype (WT) pts. Fusions were detected only in KRAS WT pts (1.5%, 6/403). Pts with cfDNA based CGP could be classified into 4 latent classes – class 1 ( KRAS - 100%, TP53 - 100% and CDKN2A – 33.3%), class 2 ( KRAS - 65.7%), class 3 ( KRAS – 68.2%, CDKN2A/B – 7.7% ) and class 4 ( KRAS – 38.5%, TP53 - 26.7%). Conclusions: The putative role of cfDNA based CGP in monitoring therapeutic response and clonal evolution in pts with PDAC may be predicated on cfDNA yield. In clinical scenarios where pre-treatment tissue availability is limited, cfDNA based CGP may be have utility in leveraging the predictive value of somatic and/or germline HR-DDR gene alterations. The prognostic and/or predictive relevance of the four latent classes needs validation in clinically annotated data sets. [Table: see text]

CircRNA circ_102049 Implicates in Pancreatic Ductal Adenocarcinoma Progression through Activating CD80 by Targeting miR-455-3p.

Emerging evidence has shown that circular RNAs (circRNAs) and DNA methylation play important roles in the causation and progression of cancers. However, the roles of circRNAs and abnormal methylation genes in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) are still largely unknown. Expression profiles of circRNA, gene methylation, and mRNA were downloaded from the GEO database, and differentially expressed genes were obtained via GEO2R, and a ceRNA network was constructed based on circRNA-miRNA pairs and miRNA-mRNA pairs. Inflammation-associated genes were collected from the GeneCards database. Then, functional enrichment analysis and protein-protein interaction (PPI) networks of inflammation-associated methylated expressed genes were investigated using Metascape and STRING databases, respectively, and visualized in Cytoscape. Hub genes of PPI networks were identified using the NetworkAnalyzer plugin. Also, we analyzed the methylation, protein expression levels, and prognostic value of hub genes in PDAC patients through the UALCAN, Human Protein Atlas (HPA), and Kaplan-Meier plotter databases, respectively. The circRNA_102049/miR-455-3p/CD80 axis was identified by the ceRNA network and hub genes. In vitro and in vivo experiments were performed to evaluate the functions of circRNA_102049. The regulatory mechanisms of circRNA_102049 and miR-455-3p were explored by RT-PCR, western blot, and dual-luciferase assays. In the present study, twelve hub genes (STAT1, CCND1, KRAS, CD80, ICAM1, ESR1, RAF1, RPS6KA2, KDM6B, TNRC6A, FOSB, and DNM1) were determined from the PPI networks. Additionally, the circRNA_102049 was upregulated in PDAC cell lines. Functionally, the knockdown of circRNA_102049 by siRNAs inhibited cell growth, inflammatory factors, and migratory and invasive potential and promoted cell apoptosis. Mechanistically, circRNA_102049 functioned as a sponge of miR-455-3p and partially reversed the effect of miR-455-3p and consequently upregulated CD80 expression. Our findings showed that circRNA_102049 and methylated hub genes play an important role in the proliferation, apoptosis, migration, invasion, and inflammatory response of PDAC, which might be selected as a promising prognostic marker and therapeutic target for PDAC.

Hypoxic microenvironment induced spatial transcriptome changes in pancreatic cancer.

Objective:Hypoxia is a significant feature of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). It is associated with tumor invasion, metastasis, and drug resistance. However, the spatial distribution of hypoxia-related heterogeneity in PDAC remains unclear.Methods:Spatial transcriptomics (STs), a new technique, was used to investigate the ST features of engrafted human PDAC in the ischemic hind limbs of nude mice. Transcriptomes from ST spots in the hypoxic tumor and the control were clustered using differentially-expressed genes. These data were compared to determine the spatial organization of hypoxia-induced heterogeneity in PDAC. Clinical relevance was validated using the Tumor Cancer Genome Atlas and KM-plotter databases. The CMAP website was used to identify molecules that may serve as therapeutic targets for PDAC.Results:ST showed that the tumor cell subgroups decreased to 7 subgroups in the hypoxia group, compared to 9 subgroups in the control group. Different subgroups showed positional characteristics and different gene signatures. Subgroup 6 located at the invasive front showed a higher proliferative ability under hypoxia. Subgroup 6 had active functions including cell proliferation, invasion, and response to stress. Expressions of hypoxia-related genes, LDHA, TPI1, and ENO1, induced changes. CMAP analysis indicated that ADZ-6482, a PI3K inhibitor, was targeted by the invasive subgroup in hypoxic tumors.Conclusions:This study is the first to describe hypoxic microenvironment-induced spatial transcriptome changes in PDAC, and to identify potential treatment targets for PDAC. These data will provide the basis for further investigations of the prognoses and treatments of hypoxic tumors.

The COL11A1/Akt/CREB signaling axis enables mitochondrial-mediated apoptotic evasion to promote chemoresistance in pancreatic cancer cells through modulating BAX/BCL-2 function.

Collagen XI, a member of the collagen family, is present in the extracellular matrix (ECM), and high collagen XI/αI (COL11A1) expression in tumor tissue is reportedly correlated with the clinicopathological parameters of pancreatic ductal adenocarcinoma (PDAC). However, the function of COL11A1 in the development of pancreatic cancer cells remains unclear. In the current study, we assessed mRNA expression of COL11A1 and its receptors and created a testing-model of both a COL11A1-overexpressing tumor microenvironment and/or altered-COL11A1 expression in pancreatic cancer cell lines. Next, we investigated the mechanism by which COL11A1 affects growth, gemcitabine (GEM) resistance and apoptosis in pancreatic cancer cells. We demonstrated that COL11A1 phosphorylated AktSer473, promoting proliferation of cancer cells and inhibiting their apoptosis. Additionally, our data showed that COL11A1/Akt/CREB altered the balance between BCL-2 and BAX and mediated their mitochondrial translocation in pancreatic cancer cells. The COL11A1/Akt axis induced disruption of mitochondrial transmembrane function, enabling mitochondria-mediated apoptotic evasion to promote chemoresistance. We also explored the regulatory effect of COL11A1/Akt on molecular signaling in the mitochondria-mediated apoptotic program. COL11A1/Akt disturbed the BCL-2/BAX balance, inhibiting cytochrome c (Cyt-C) release and binding of Apaf-1/procaspase-9/Cyt-C, which suppressed the apoptotic program and induced GEM resistance in pancreatic cancer cells. In conclusion, COL11A1 modulates apoptotic inhibition and chemoresistance in pancreatic cancer cells by activating the Akt/CREB/BCL-2/BAX signaling pathway. COL11A1 may represent a distinct prognostic indicator and may be an attractive therapeutic target for PDAC.

NEAT1/miR-101-dependent Up-regulation of DNA-PKcs Enhances Malignant Behaviors of Pancreatic Ductal Adenocarcinoma Cells.

Background: Although we previously revealed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and important for gemcitabine resistance, the role of DNA-PKcs in the progression and metastasis of PDAC remain unclear. To date, the upstream signaling events stimulating DNA-PKcs overexpression in PDAC are still not well characterized.Methods: Expression of DNA-PKcs was measured by western blot. The levels of miRNA-101 and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) were detected by real-time PCR. Cell viability was determined by CCK-8. Cell migration and cell invasion were measured by transwell assay. The regulatory relationship between NEAT1 and miR-101 was determined by a luciferase assay.Results: DNA-PKcs expression was significantly elevated in human PDAC tissues and cells. DNA-PKcs overexpression was correlated with TNM stage and lymph node metastasis. Higher expression of DNA-PKcs was closely correlated with patients of worse overall survival (OS). DNA-PKcs knockdown suppresses malignant behaviors of PDAC cells. Further study showed that miRNA-101 level was decreased in PDAC tissues and cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in PDAC cells. Moreover, NEAT1 functions as an oncogene influencing cell proliferation, migration and invasion in part by serving as a competing endogenous RNA (ceRNAs) modulating miR-101 expression, leading to up-regulation of DNA-PKcs.Conclusion: These findings suggest that NEAT1/miR-101-dependent up-regulation of DNA-PKcs promotes the malignant behaviors of PDAC cells. The NEAT1/miR-101/DNA-PKcs axis may serve as a viable prognostic marker and therapeutic target for PDAC.

Carbohydrate antigen 19-9 - tumor marker: Past, present, and future.

Carbohydrate antigen 19-9 (CA 19-9) is a cell surface glycoprotein complex most commonly associated with pancreatic ductal adenocarcinoma (PDAC). Koprowski first described it in 1979 using a mouse monoclonal antibody in a colorectal carcinoma cell line. Historically, it is one of the most commonly used tumor markers for diagnosing, managing, and prognosticating PDAC. Additionally, elevated CA 19-9 levels are used as an indication for surgery in suspected benign pancreatic conditions. Another common application of CA 19-9 in the biliary tract includes its use as an adjunct in diagnosing cholangiocarcinoma. However, its clinical value is not limited to the hepatopancreatobiliary system. The reality is that the advancing literature has broadened the clinical value of CA 19-9. The potential value of CA 19-9 in patients' workup extends its reach to gastrointestinal cancers – such as colorectal and oesophageal cancer – and further beyond the gastrointestinal tract - including urological, gynecological, pulmonary, and thyroid pathologies. Apart from its role in investigations, CA 19-9 presents a potential therapeutic target in PDAC and acute pancreatitis. In a bid to consolidate its broad utility, we appraised and reviewed the biomarker’s current utility and limitations in investigations and management, while discussing the potential applications for CA 19-9 in the works for the future.

The RNA-binding protein MEX3A is a prognostic factor and regulator of resistance to gemcitabine in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA-binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression-free survival, we focused on MEX3A because it was recently shown to mark an intestinal stem cell population that is refractory to chemotherapeutic treatments, a typical feature of PDAC. Increased expression of MEX3A was correlated with higher disease stage in PDAC patients and with tumor development in a mouse model of PDAC. Depletion of MEX3A in PDAC cells enhanced sensitivity to chemotherapeutic treatment with gemcitabine, whereas its expression was increased in PDAC cells selected upon chronic exposure to the drug. RNA-sequencing analyses highlighted hundreds of genes whose expression is sensitive to MEX3A expression, with significant enrichment in cell cycle genes. MEX3A binds to its target mRNAs, like cyclin-dependent kinase 6 (CDK6), and promotes their stability. Accordingly, knockdown of MEX3A caused a significant reduction in PDAC cell proliferation and in progression to the S phase of the cell cycle. These findings uncover a novel role for MEX3A in the acquisition and maintenance of chemoresistance by PDAC cells, suggesting that it may represent a novel therapeutic target for PDAC.

X-aptamers targeting Thy-1 membrane glycoprotein in pancreatic ductal adenocarcinoma

Modified DNA aptamers incorporated with amino-acid like side chains or drug-like ligands can offer unique advantages and enhance specificity as affinity ligands. Thy-1 membrane glycoprotein (THY1 or CD90) was previously identified as a biomarker candidate of neovasculature in pancreatic ductal adenocarcinoma (PDAC). The current study developed and evaluated modified DNA X-aptamers targeting THY1 in PDAC. The expression and glycosylation of THY1 in PDAC tumor tissues were assessed using immunohistochemistry and quantitative proteomics. Bead-based X-aptamer library that contains 108 different sequences was used to screen for high affinity THY1 X-aptamers. The sequences of the X-aptamers were analyzed with the next-generation sequencing. The affinities of the selected X-aptamers to THY1 were quantitatively evaluated with flow cytometry. Three high affinity THY1 X-aptamers, including XA-B217, XA-B216 and XA-A9, were selected after library screening and affinity binding evaluation. These three X-aptamers demonstrated a high binding affinity and specificity to THY1 protein and the THY1 expressing cell lines, using THY1 antibody as a comparison. The development of these X-aptamers provides highly specific and non-immunogenic affinity ligands for THY1 binding in the context of biomarker development and clinical applications. They could be further exploited to assist molecular imaging of PDAC targeting THY1.

Emerging Kinase Therapeutic Targets in Pancreatic Ductal Adenocarcinoma and Pancreatic Cancer Desmoplasia.

Kinase drug discovery represents an active area of therapeutic research, with previous pharmaceutical success improving patient outcomes across a wide variety of human diseases. In pancreatic ductal adenocarcinoma (PDAC), innovative pharmaceutical strategies such as kinase targeting have been unable to appreciably increase patient survival. This may be due, in part, to unchecked desmoplastic reactions to pancreatic tumors. Desmoplastic stroma enhances tumor development and progression while simultaneously restricting drug delivery to the tumor cells it protects. Emerging evidence indicates that many of the pathologic fibrotic processes directly or indirectly supporting desmoplasia may be driven by targetable protein tyrosine kinases such as Fyn-related kinase (FRK); B lymphoid kinase (BLK); hemopoietic cell kinase (HCK); ABL proto-oncogene 2 kinase (ABL2); discoidin domain receptor 1 kinase (DDR1); Lck/Yes-related novel kinase (LYN); ephrin receptor A8 kinase (EPHA8); FYN proto-oncogene kinase (FYN); lymphocyte cell-specific kinase (LCK); tec protein kinase (TEC). Herein, we review literature related to these kinases and posit signaling networks, mechanisms, and biochemical relationships by which this group may contribute to PDAC tumor growth and desmoplasia.

Hepatocyte nuclear factor 1 alpha influences pancreatic cancer growth and metastasis

Hepatocyte nuclear factor 1 homeobox alpha (HNF1α) is a transcription factor involved in endodermal organogenesis and pancreatic precursor cell differentiation and development. Earlier studies have reported a role for HNF1α in pancreatic ductal adenocarcinoma (PDAC) but it is controversial. The mechanism by which it impacts PDAC is yet to be explored in depth. In this study, using the online databases we observed that HNF1α is upregulated in PDAC, which was also confirmed by our immunohistochemical analysis of PDAC tissue microarray. Silencing HNF1α reduced the proliferative, migratory, invasive and colony forming capabilities of pancreatic cancer cells. Key markers involved in these processes (pPI3K, pAKT, pERK, Bcl2, Zeb, Snail, Slug) were significantly changed in response to alterations in HNF1α expression. On the other hand, overexpression of HNF1α did not induce any significant change in the aggressiveness of pancreatic cancer cells. Our results demonstrate that reduced expression of HNF1α leads to inhibition of pancreatic cancer growth and progression, which indicates that it could be a potential oncogene and target for PDAC.

Abstract PO-004: Identification of MLL4 as a novel therapeutic target of pancreatic ductal adenocarcinoma by in vivo genome-wide RNAi screening

Abstract The prognosis of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to the lack of effective therapeutics. It is urgent to identify novel therapeutic target for PDAC. We performed in vivo genome-wide RNAi screening to identify cancer-associated candidate genes with critical roles in the tumorigenesis of PDAC. We identify mixed-lineage leukaemia 4 (MLL4 also known as KMT2B), which belongs to histone H3 lysine 4 (H3K4) methyltransferase of MLL family, as one of the cancer-associated gene to PDAC tumorigenesis. Here, we firstly report that MLL4 expression was frequently upregulated in PDAC cell lines as well as human PDAC tissue, depicting its potential role in PDAC tumorigenesis. Lysine methylation of different position of Histone is performed by SET (suppressor of variegation) domain of MLL family, aberrant expression of SET domain results in disruption of epigenetic gene regulation and leads to cancer. To characterize the in vitro tumorigenic role of SET domain of MLL4 in the development of PDAC, gain-of-function and loss-of-function approaches of SET domain of MLL4 and MLL4 were performed in PDAC cells, respectively. Ectopic SET expression promotes cell proliferation, colony formation, cell migration as well as invasion, demonstrating its oncogenic role in PDAC cells. Moreover, MLL4-depleted PDAC cells exhibited significantly reduction of tumor growth in nude mice xenograft model. Putative MLL4 activating and repressive targets were identified using gene expression microarray analysis in PDAC cell lines Capan2 and Panc-1. Expression alteration of these genes by regulated by MLL4 contribute to PDAC development and progression. To further delineate whether MLL4 regulates expression of the activating and repressing targets via H3K4 methylation, activity of H3K4 methylation was investigated in PDAC cells with MLL4 knockdown. Suppression of MLL4 reduced the H3K4Me2 and H3K4Me3 occupancies at promoter of MLL4 target genes, suggesting that MLL4 regulates downstream genes expression through H3K4 methylation. Taken together, our findings demonstrate that aberrant overexpression of MLL4 in PDAC cells alters its downstream oncogene and tumor suppressor genes, contributing to PDAC tumorigenesis. Thus, targeting MLL4 may serve as a potential therapeutic modality in the management of PDAC. Citation Format: Frederic K. C. Fung, Yinxin Zhu, Chi Hin Wong, Yangchao Chen. Identification of MLL4 as a novel therapeutic target of pancreatic ductal adenocarcinoma by in vivo genome-wide RNAi screening [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-004.

Abstract PO-009: Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system

Abstract Introduction: Exosomes are emerging as promising nanocarriers for genes and drugs; however, major challenges exist including the absence of active tumor targeting and a high proportion of clearance by mononuclear phagocytic system (MPS) in liver and spleen. To overcome these obstacles currently preventing the effective application of nanodelivery technology in the field of cancer biology, we generate a novel exosome that recognizes both pancreatic ductal adenocarcinoma (PDAC) specific cell surface marker avb3 and the signal regulatory protein (SIRPa) on macrophages. Using this novel exosome, we have achieved active PDAC targeting and MPS evasion. Methods: We engineered the CD9, an exosome marker, to display a HA tagged RGD peptide at the E174 site of CD9 (CD9-HA-RGD). We overexpressed CD9-HA-RGD and a Flag tagged CD47 (CD47-Flag) in HEK 293 cells. CD47, a “don’t eat me” protein signal, mediates inhibition of phagocytosis through interacting with SIRPa on macrophages. Exosomes overexpressing CD9-HA-RGD and CD47-Flag were harvested from HEK 293 cell culture media by ultracentrifugation followed by purification with size exclusion chromatography (SEC). Exosomes displaying RGD and CD47 (Exo-RGD/CD47) were characterized in vitro using ELISA, western blot (WB), flow cytometry (FCM), PDAC cell uptake, and macrophage phagocytosis assays. Exo-RGD/CD47 was also evaluated in vivo in patient-derived PDAC cell line (PDCL5) orthotopic xenograft mouse model. Results: RGD and CD47 were demonstrated to be successfully displayed on exosome surface by analysis of HA and Flag expression using ELISA and FCM. Engineered Exo-RGD/CD47 exosomes achieved enhanced cell binding and increased uptake capacities in an avb3 expression dependent manner in PDAC cells. This was confirmed using excess RDG peptide in a competition assay. Control exosomes demonstrated low uptake efficiency by PDAC cells. Exo-RGD/CD47 resulted in 71.5% reduction of macrophage phagocytosis when compared to control exosomes as evaluated by THP1 cells phagocytosis assay. Using DiR stained exosomes, we demonstrated that Exo-RGD/CD47 systemic delivery significantly reduced liver accumulation up to 80.6% and 65.8% after 1h and 6h after injection, respectively, as compared to control group (p&amp;lt;0.05). Similarly, Exo-RGD/CD47 delivery resulted in 70.1% reduction of splenic accumulation as compared to control at 6h after application (p&amp;lt;0.05). Exo-RGD/CD47 systemic delivery also showed significant tumor accumulation in the PDCL5 xenograft tumor model as compared to control (p&amp;lt;0.05). Conclusions: These data demonstrate that CD9 is an editable exosome biomarker by which peptides can be effectively displayed on the surface of an exosome. Displaying RGD and CD47 on the exosome surface results in a novel exosome that enables active PDAC targeting and evasion of phagocytosis by MPS. These findings suggest the newly designed exosome may have a better translational potential in PDAC diagnosis and therapy than traditional exosomes. Citation Format: Shi-He Liu, Justin Creeden, F. Charles Brunicardi, JianTing Zhang. Genetically engineered exosomes enable active pancreatic cancer targeting and evading mononuclear phagocytic system [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-009.

CEP164 Deficiency Causes Hyperproliferation of Pancreatic Cancer Cells.

Primary cilia are hair-like projections that protrude from most mammalian cells and mediate various extracellular signaling pathways. Pancreatic ductal adenocarcinoma (PDAC) cells are known to lose their primary cilia, but the relevance of this phenomenon remains unclear. In this study, we generated PDAC-originated Panc1 cells devoid of primary cilia by mutating a centriolar protein, centrosomal protein 164 (CEP164), which is required for ciliogenesis. CEP164 depletion enhanced the clonogenicity of Panc1 cells, along with chemically induced elimination of primary cilia, suggesting that a lack of these organelles promotes PDAC cells proliferation. In addition, the loss of CEP164 altered the cell cycle progression irrespective of absence of primary cilia. We found that CEP164 was co-localized with the GLI2 transcription factor at the mother centriole and controlled its activation, thus inducing Cyclin D-CDK6 expression. Furthermore, CEP164-mutated Panc1 cells were significantly tolerant to KRAS depletion-dependent growth inhibition. This study suggests that CEP164 deficiency is advantageous for PDAC cells proliferation due to not only lack of ciliation but also cilia-independent GLI2-Cyclin D/CDK6 activation, and that CEP164 is a potential therapeutic target for PDAC.

KIF23 enhances cell proliferation in pancreatic ductal adenocarcinoma and is a potent therapeutic target.

BackgroundIn recent research, high expression of kinesin family member 23 (KIF23), one of the kinesin motor proteins involved in the regulation of cytokinesis, has been shown to be related to poor prognosis in glioma and paclitaxel-resistant gastric cancer, as a results of the enhancement of proliferation, migration, and invasion. In this study, we analyzed the role of KIF23 in the progression of pancreatic ductal adenocarcinoma.MethodsA bioinformatic method was used to analyze the KIF23 mRNA level in pancreatic tumor tissues compared with normal pancreatic tissues and to analyze the connection between high KIF23 expression and prognosis. We examined the expression of KIF23 using immunohistochemistry and analyzed the connection between the expression of KIF23 and clinicopathological features in pancreatic ductal adenocarcinoma patients. In addition, a colony formation assay, MTT assay, and western blot assay were performed in vitro, along with a mouse xenograft model in vivo, to analyze the effect of KIF23 on proliferation. Further, the correlation between KIF23 and CDCA8 was analyzed by TCGA and immunohistochemical data.ResultsBioinformatic results showed that KIF23 mRNA expression was higher in pancreatic tumor tissues than in normal pancreatic tissues and a poor prognosis has been linked to the high expression of KIF23. Immunohistochemistry revealed that KIF23 was highly expressed at the protein level and high expression of KIF23 correlated with adverse clinicopathological features. Our experimental results demonstrated that knockdown of KIF23 could inhibit the proliferation of pancreatic cells. Further, a positive correlation between KIF23 and CDCA8 expression existed, and KIF23 might promote pancreatic cancer proliferation by affecting CDCA8 expression.ConclusionsOur data showed that high expression of KIF23 is associated with a poor prognosis, and KIF23 might be a potential therapeutic target for pancreatic ductal adenocarcinoma.

Human Positive Coactivator 4 Affects the Progression and Prognosis of Pancreatic Ductal Adenocarcinoma via the mTOR/P70s6k Signaling Pathway.

IntroductionPancreatic cancer is one of the deadliest cancers in the world, and pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of all cases. Human positive coactivator 4 (PC4) is a transcriptional coactivator that has been associated with the development and progression of several tumors. However, no studies investigated the potential role of PC4 in PDAC.MethodsWe investigated PC4 expression in 81 PDAC tissue samples using immunohistochemistry and studied the impact of PC4 expression and the molecular mechanisms of this altered expression on PDAC tumorigenesis and proliferation both in vitro and in vivo.ResultsPC4 overexpression was correlated with a poor outcome in PDAC patients. The RNAi-mediated knockdown of PC4 expression in CFPAC-1 and AsPC-1 cell lines reduced cell proliferation and tumor growth. The loss of PC4 in PDAC inhibits cell growth by inducing cell cycle arrest at the G1/S transition and suppressing the mTOR/p70s6k pathway.Discussion/ConclusionOur findings reveal for the first time that PC4 exerts oncogenic functions by activating mTOR/p70s6k signaling pathway-mediated cell proliferation, implying that PC4 is a promising therapeutic target for PDAC.

Integrated transcriptome meta-analysis of pancreatic ductal adenocarcinoma and matched adjacent pancreatic tissues.

A comprehensive meta-analysis of publicly available gene expression microarray data obtained from human-derived pancreatic ductal adenocarcinoma (PDAC) tissues and their histologically matched adjacent tissue samples was performed to provide diagnostic and prognostic biomarkers, and molecular targets for PDAC. An integrative meta-analysis of four submissions (GSE62452, GSE15471, GSE62165, and GSE56560) containing 105 eligible tumor-adjacent tissue pairs revealed 344 differentially over-expressed and 168 repressed genes in PDAC compared to the adjacent-to-tumor samples. The validation analysis using TCGA combined GTEx data confirmed 98.24% of the identified up-regulated and 73.88% of the down-regulated protein-coding genes in PDAC. Pathway enrichment analysis showed that “ECM-receptor interaction”, “PI3K-Akt signaling pathway”, and “focal adhesion” are the most enriched KEGG pathways in PDAC. Protein-protein interaction analysis identified FN1, TIMP1, and MSLN as the most highly ranked hub genes among the DEGs. Transcription factor enrichment analysis revealed that TCF7, CTNNB1, SMAD3, and JUN are significantly activated in PDAC, while SMAD7 is inhibited. The prognostic significance of the identified and validated differentially expressed genes in PDAC was evaluated via survival analysis of TCGA Pan-Cancer pancreatic ductal adenocarcinoma data. The identified candidate prognostic biomarkers were then validated in four external validation datasets (GSE21501, GSE50827, GSE57495, and GSE71729) to further improve reliability. A total of 28 up-regulated genes were found to be significantly correlated with worse overall survival in patients with PDAC. Twenty-one of the identified prognostic genes (ITGB6, LAMC2, KRT7, SERPINB5, IGF2BP3, IL1RN, MPZL2, SFTA2, MET, LAMA3, ARNTL2, SLC2A1, LAMB3, COL17A1, EPSTI1, IL1RAP, AK4, ANXA2, S100A16, KRT19, and GPRC5A) were also found to be significantly correlated with the pathological stages of the disease. The results of this study provided promising prognostic biomarkers that have the potential to differentiate PDAC from both healthy and adjacent-to-tumor pancreatic tissues. Several novel dysregulated genes merit further study as potentially promising candidates for the development of more effective treatment strategies for PDAC.

GOLPH3 Promotes Cancer Growth by Interacting With STIP1 and Regulating Telomerase Activity in Pancreatic Ductal Adenocarcinoma.

Overexpression of Golgi phosphoprotein 3 (GOLPH3) predicts poor prognosis and is a potential therapeutic target in pancreatic ductal adenocarcinoma (PDAC). However, its role and underlying molecular mechanisms in the progression of PDAC remain unknown. In the present study, using high-throughput bimolecular fluorescence complementation (BiFC) analysis, we identified that stress-inducible protein-1 (STIP1) interacts with GOLPH3 and confirmed the interaction using co-localization and co-immunoprecipitation. The levels of GOLPH3 and STIP1 in PDAC tissues and adjacent non-cancerous pancreatic tissues were determined using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR. Real-time Quantitative-telomere repeat amplification (Q-TRAP) was applied to detect relative telomerase activity, and cell proliferation was measured when small interfering RNAs targeting GOLPH3 or STIP1 were transfected into PDAC cell lines. BALB/c nude mice were used to assess tumor growth inhibition of BXPC3 cells stably transfected with GOLPH3 short hairpin RNA. In summary, GOLPH3 was found to interact with STIP1 and both proteins were overexpressed and co-localized in PDAC tissues and cell lines. Moreover, suppression of GOLPH3 expression using shRNAs in PANC1 and BXPC3 cells inhibited tumor cell proliferation both in vitro and in vivo. Mechanistically, GOLPH3 interacts with STIP1 to activate telomerase reverse transcriptase (hTERT) and telomerase activity by c-Myc, and then upregulates cell cycle-related signaling proteins, including cyclin D1, to promote tumor cell growth, suggesting that disrupting the interaction between STIP1 and GOLPH3 would be a promising new strategy to treat PDAC.