Aurora kinase a inhibitor MLN8237 suppresses pancreatic cancer growth.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for high mortality due to limited options of appropriate chemotherapy drugs. Here we report that Aurora kinase-A expression is elevated in both human and mouse PDAC samples. MLN8237, an inhibitor of Aurora kinase-A, efficiently reduced the proliferation and motility of PDAC cells in vitro as well as tumor growth in orthotropic xenograft model and genetic pancreatic cancer animal models (p53/LSL/Pdx-Cre mice) in vivo. MLN8237 exhibited tumor inhibitory effect through inhibiting proliferation and migration, and inducing apoptosis and senescence. These results provide the molecular basis for a novel chemotherapy strategy for PDAC patients.

The edited UPF1 is correlated with elevated asparagine synthetase in pancreatic ductal adenocarcinomas.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) is a malignant disorder and is the most common pancreatic cancer type. The malignant cells depend on the uptake of asparagine (Asn) for growth. The synthesis of Asn occurs through the enzyme asparagine synthetase (ASNS). Interestingly, ASNS is known as is direct target of nonsense-mediated RNA decay (NMD). We have previously reported that NMD major factor UPF1 mutations in the pancreatic tumors. However, the relationship between NMD and the level of ASNS is unknown. METHOD: We constructed point mutations by site-specific mutagenesis. To evaluate NMD magnitude, we assessed the expression ratio of an exogenously expressed wild-type and mutated ß-globin mRNA with N39 allele, and five known NMD targets. Then, reverse transcription-polymerase chain reaction (RT-PCR), RT-qPCR and western bolt to determine RNA or protein levels, after knockdown of endogenous UPF1 by small RNA interference in the cells. RESULTS: An RNA editing event (c.3101 A > G) at UPF1 transcripts resulting in an Asparagine (p.1034) changed to a Serine is found in one primary PDAC patient. The edited UPF1 increases the ability of degrading of NMD provoking transcripts, such as ß-globin mRNA with N39 allele and 5 out of 5 known endogenous NMD substrate mRNAs, including ASNS. In addition, ASNS mRNA is subjected to NMD degradation by virtue of its possessing uORFs at the 5'UTR. A reduction of endogenous ASNS RNA and the increased protein expression level is found either in the PDAC patient or in the cells with edited UPF1 at c.3101 A > G relative to the controls. CONCLUSIONS: This edited UPF1 found in the PDAC results in hyperactivated NMD, which is tightly correlation to elevated expression level of ASNS. The targeting of knockdown of ASNS may improve the antitumor potency in PDACs.

The Complement C3a-C3a Receptor Axis Regulates Epithelial-to-Mesenchymal Transition by Activating the ERK Pathway in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: We aimed to clarify the role of complement C3a and its receptor C3aR in progression of pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: We evaluated the serum levels of C3 and C3a in patients with PDAC. C3aR expression in tissue was assessed using a tissue microarray. To confirm the protumoral effects of C3a in PDAC, we conducted in vitro experiments using PDAC cell lines (Panc-1 and MiaPaca-2) that exhibit high C3aR expression. RESULTS: Serum levels of both C3 and C3a were higher in 26 patients with PDAC than in 28 nontumor-bearing controls. In the tissue microarray, we observed increased expression of C3aR in PDAC cells, especially in cases with metastatic lesions. In vitro experiments showed that C3a facilitated tumor cell proliferation, migration and invasion by activating the extracellular-regulated kinase signaling pathway and inducing epithelial-to-mesenchymal transition. Inhibition of the C3a-C3aR axis by pharmacological blockade and short-hairpin RNA-mediated knockdown of C3aR alleviated its protumoral effect. CONCLUSION: These findings provide a new approach for the development of treatments targeting the C3a-C3aR axis.

The effect of danusertib, an Aurora kinase inhibitor, onto the cytotoxicity, cell cycle and apoptosis in pancreatic ductal adenocarcinoma cells.

BACKGROUND: Pancreatic cancer is the second type of cancer that causes the most death among the digestive system cancers. Difficulties in early diagnosis and rapidly progressing to advanced stages are most common in high mortality rate of pancreatic carcinoma. The mutation of Bcr-Abl tyrosine kinase and mitotic kinases (such as Aurora kinases), which are involved in the cell cycle, plays an important role in the progression of cancer. Enzymes belonging to Aurora kinase family (-A, -B, -C) have been reported to play a major role in cancer progression, invasion and metastasis. Therefore, the purpose of this study, investigate of the effect of danusertib, an Aurora kinase inhibitor, onto cytotoxicity, apoptosis and cell cycle in human pancreatic carcinoma CFPAC-1 cells. MATERIALS AND METHODS: For determining the IC50 value, the 20,000 cells were seeded in E-plate 16 wells in a real-time cell analyzer and various concentrations of danusertib (1-10,000 nM) were applied onto CFPAC-1 cells incubated in IMDM medium. Cell index demonstrated that the proliferation of fraction cells was measured in real time. On the other hand, cell apoptosis and cell cycle arrest test were stained with Annexin V-APC/PI and DNA-cell cycle PI staining respectively by using flow cytometry. RESULTS: The IC50 value was found to be approximately 400 nM. Danusertib at this concentration induced apoptosis in CFPAC-1 cells (%14,8 at 24 hours; %21,3 at 48 hours). Furthermore, in the cells treated with danusertib, 31.77% and 11.05% were arrested in the S and G2 phases, respectively. CONCLUSIONS: Aurora kinase inhibitor danusertib induced a significant effect of cytotoxic, apoptotic and cell cycle arrest in CFPAC-1 ductal adenocarcinoma cells. Therefore, it may be a potential alternative to the treatment of pancreatic cancers.

The KRAS-regulated kinome identifies WEE1 and ERK coinhibition as a potential therapeutic strategy in KRAS-mutant pancreatic cancer.

Oncogenic KRAS drives cancer growth by activating diverse signaling networks, not all of which have been fully delineated. We set out to establish a system-wide profile of the KRAS-regulated kinase signaling network (kinome) in KRAS-mutant pancreatic ductal adenocarcinoma (PDAC). We knocked down KRAS expression in a panel of six cell lines and then applied multiplexed inhibitor bead/MS to monitor changes in kinase activity and/or expression. We hypothesized that depletion of KRAS would result in downregulation of kinases required for KRAS-mediated transformation and in upregulation of other kinases that could potentially compensate for the deleterious consequences of the loss of KRAS. We identified 15 upregulated and 13 downregulated kinases in common across the panel of cell lines. In agreement with our hypothesis, all 15 of the upregulated kinases have established roles as cancer drivers (e.g., SRC, TGF-ß1, ILK), and pharmacological inhibition of one of these upregulated kinases, DDR1, suppressed PDAC growth. Interestingly, 11 of the 13 downregulated kinases have established driver roles in cell cycle progression, particularly in mitosis (e.g., WEE1, Aurora A, PLK1). Consistent with a crucial role for the downregulated kinases in promoting KRAS-driven proliferation, we found that pharmacological inhibition of WEE1 also suppressed PDAC growth. The unexpected paradoxical activation of ERK upon WEE1 inhibition led us to inhibit both WEE1 and ERK concurrently, which caused further potent growth suppression and enhanced apoptotic death compared with WEE1 inhibition alone. We conclude that system-wide delineation of the KRAS-regulated kinome can identify potential therapeutic targets for KRAS-mutant pancreatic cancer.

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-Induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was used for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in patients with PDAC. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited antitumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased noncanonic use of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC.

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.

AGR2-Dependent Nuclear Import of RNA Polymerase II Constitutes a Specific Target of Pancreatic Ductal Adenocarcinoma in the Context of Wild-Type p53.

BACKGROUND & AIMS: Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2-RNAPII complex. RESULTS: We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation in vivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents in vivo. Of note, this therapeutic effect is efficient across various cancer types. CONCLUSIONS: Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

NADK is activated by oncogenic signaling to sustain pancreatic ductal adenocarcinoma.

Metabolic adaptations and the signaling events that control them promote the survival of pancreatic ductal adenocarcinoma (PDAC) at the fibrotic tumor site, overcoming stresses associated with nutrient and oxygen deprivation. Recently, rewiring of NADPH production has been shown to play a key role in this process. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells. However, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). In this study, we show that oncogenic KRAS promotes protein kinase C (PKC)-mediated NADK phosphorylation, leading to its hyperactivation, thus sustaining both NADP+ and NADPH levels in PDAC cells. Together, our data show that increased NADK activity is an important adaptation driven by oncogenic signaling. Our findings indicate that NADK could serve as a much-needed therapeutic target for PDAC.

Novel circular RNA circSLIT2 facilitates the aerobic glycolysis of pancreatic ductal adenocarcinoma via miR-510-5p/c-Myc/LDHA axis.

Increasing evidence has indicated the great diagnostic and therapeutic potentials of circular RNAs (circRNAs) in human cancers. Although the biological roles of circRNAs in pancreatic ductal adenocarcinoma (PDAC) have been partially annotated, the potential regulatory mechanism of circRNAs in PDAC tumorigenesis remains poorly understood. Here, our study found that the novel circRNA circSLIT2 was significantly upregulated in PDAC tissues and cells. Clinically, ectopic high-expression of circSLIT2 was correlated with unfavorable prognosis of PDAC patients. Functional experiments demonstrated that circSLIT2 promoted the aerobic glycolysis and proliferation of PDAC cells in vitro, and circSLIT2 knockdown inhibited tumor growth in vivo. Mechanistically, circSLIT2 acted as miRNA sponge to target miR-510-5p/c-Myc axis. Furthermore, c-Myc bound with the promoter region of lactate dehydrogenase A (LDHA) to activate the transcription. Collectively, present findings reveal that circSLIT2/miR-510-5p/c-Myc/LDHA axis participates in the aerobic glycolysis and carcinogenesis of PDAC, and may act as a promising therapeutic target.

SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells.

Glutaminase regulates glutaminolysis to promote cancer cell proliferation. However, the mechanism underlying glutaminase activity regulation is largely unknown. Here, we demonstrate that kidney-type glutaminase (GLS) is highly expressed in human pancreatic ductal adenocarcinoma (PDAC) specimens with correspondingly upregulated glutamine dependence for PDAC cell proliferation. Upon oxidative stress, the succinyl-coenzyme A (CoA) synthetase ADP-forming subunit ß (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 dissociates from GLS, resulting in enhanced GLS K311 succinylation, oligomerization, and activity. Activated GLS increases glutaminolysis and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby counteracting oxidative stress and promoting tumor cell survival and tumor growth in mice. In addition, the levels of SUCLA2 pS79 and GLS K311 succinylation, which were mutually correlated, were positively associated with advanced stages of PDAC and poor prognosis for patients. Our findings reveal critical regulation of GLS by SUCLA2-coupled GLS succinylation regulation and underscore the regulatory role of metabolites in glutaminolysis and PDAC development.

TET2 Drives 5hmc Marking of GATA6 and Epigenetically Defines Pancreatic Ductal Adenocarcinoma Transcriptional Subtypes.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples. METHODS: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo. RESULTS: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. CONCLUSIONS: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.

PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis.

Although induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, has emerged as an anticancer strategy, the metabolic basis of ferroptotic death remains poorly elucidated. Here, we show that glucose determines the sensitivity of human pancreatic ductal carcinoma cells to ferroptosis induced by pharmacologically inhibiting system xc-. Mechanistically, SLC2A1-mediated glucose uptake promotes glycolysis and, thus, facilitates pyruvate oxidation, fuels the tricyclic acid cycle, and stimulates fatty acid synthesis, which finally facilitates lipid peroxidation-dependent ferroptotic death. Screening of a small interfering RNA (siRNA) library targeting metabolic enzymes leads to identification of pyruvate dehydrogenase kinase 4 (PDK4) as the top gene responsible for ferroptosis resistance. PDK4 inhibits ferroptosis by blocking pyruvate dehydrogenase-dependent pyruvate oxidation. Inhibiting PDK4 enhances the anticancer activity of system xc- inhibitors in vitro and in suitable preclinical mouse models (e.g., a high-fat diet diabetes model). These findings reveal metabolic reprogramming as a potential target for overcoming ferroptosis resistance.

Selective killing of cancer cells harboring mutant RAS by concomitant inhibition of NADPH oxidase and glutathione biosynthesis.

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.

Inhibition of Stearoyl-CoA Desaturase Induces the Unfolded Protein Response in Pancreatic Tumors and Suppresses Their Growth.

OBJECTIVE: Pancreatic ductal adenocarcinoma is the fourth-leading cause of cancer death in the United States, and there is an urgent need for effective therapies. Stearoyl-CoA desaturase (SCD) is an enzyme localized in the endoplasmic reticulum and generates monounsaturated fatty acid from saturated fatty acid. In this study, we examined the role of SCD in pancreatic cancer. METHODS: We isolated epithelial cell adhesion molecule-positive pancreatic tumors from the Pdx1Cre;LSL-KrasG12D mouse and formed organoids in Matrigel. Using a SCD inhibitor, A939572, we tested its effects on growth and cell death in tumor organoids, tumors developed in the Pdx1Cre;LSL-KrasG12D mouse, and a human pancreatic ductal adenocarcinoma cell line, PANC-1. RESULTS: A939572 treatment rapidly induced degeneration of mouse tumor organoids and activated the unfolded protein response (UPR). Cotreatment of oleic acid, but not stearic acid, reduced the UPR in the organoids and rescued the inhibitory effect of the SCD inhibitor on their growth. Administration of A939572 to Pdx1Cre;LSL-KrasG12D mice caused cell death in early pancreatic tumors, but not in acini or islets. The SCD inhibitor induced the UPR in PANC-1 and suppressed their growth but did not induce cell death. CONCLUSIONS: The inhibition of the SCD enzyme causes an UPR and cell death in early pancreatic tumors.

Ubiquitin-specific protease 14 (USP14) promotes proliferation and metastasis in pancreatic ductal adenocarcinoma.

Previous studies have shown aberrant expression of ubiquitin-specific protease 14 (USP14) in multiple malignancies, suggesting an important role of USP14 in tumorigenesis. However, the functional role of USP14 in pancreatic ductal adenocarcinoma (PDAC) has never been elucidated. In this study, we found that USP14 was remarkably upregulated in PDAC tissues compared with normal pancreatic tissues. Notably, Kaplan-Meier curves showed that high expression of USP14 predicted significantly worse prognosis in PDAC patients than low expression of USP14. To determine whether USP14 could regulate the proliferation, apoptosis and metastasis of PDAC cells, we knocked down endogenous USP14 or overexpressed exogenous USP14 in Panc-1 and BxPC-3 cells. Using MTT assays, colony formation analyses, flow cytometry assays, and cell invasion and migration assays, we found that knockdown of USP14 attenuated proliferation, induced apoptosis and restrained invasion and migration of PDAC cells. Overexpression of USP14 could enhance proliferation, prevent apoptosis and promote invasion and migration of PDAC cells. In addition, USP14 could regulate the expression of cyclin D1, PCNA and E-cadherin, three important carcinogenic factors, in PDAC cells. These findings suggest that USP14 might play an important role in promoting the tumorigenesis of PDAC and thus be a promising therapeutic target to prevent PDAC progression.

mTORC1 and mTORC2 Converge on the Arp2/3 Complex to Promote KrasG12D-Induced Acinar-to-Ductal Metaplasia and Early Pancreatic Carcinogenesis.

BACKGROUND & AIMS: Oncogenic KrasG12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM), an actin-based morphogenetic process, and drives pancreatic ductal adenocarcinoma (PDAC). mTOR (mechanistic target of rapamycin kinase) complex 1 (mTORC1) and 2 (mTORC2) contain Rptor and Rictor, respectively, and are activated downstream of KrasG12D, thereby contributing to PDAC. Yet, whether and how mTORC1 and mTORC2 impact on ADM and the identity of the actin nucleator(s) mediating such actin rearrangements remain unknown. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven early pancreatic carcinogenesis was used. Rptor, Rictor, and Arpc4 (actin-related protein 2/3 complex subunit 4) were conditionally ablated in acinar cells to deactivate the function of mTORC1, mTORC2 and the actin-related protein (Arp) 2/3 complex, respectively. RESULTS: We found that mTORC1 and mTORC2 are markedly activated in human and mouse ADM lesions, and cooperate to promote KrasG12D-driven ADM in mice and in vitro. They use the Arp2/3 complex as a common downstream effector to induce the remodeling the actin cytoskeleton leading to ADM. In particular, mTORC1 regulates the translation of Rac1 (Rac family small GTPase 1) and the Arp2/3-complex subunit Arp3, whereas mTORC2 activates the Arp2/3 complex by promoting Akt/Rac1 signaling. Consistently, genetic ablation of the Arp2/3 complex prevents KrasG12D-driven ADM in vivo. In acinar cells, the Arp2/3 complex and its actin-nucleation activity mediated the formation of a basolateral actin cortex, which is indispensable for ADM and pre-neoplastic transformation. CONCLUSIONS: Here, we show that mTORC1 and mTORC2 attain a dual, yet nonredundant regulatory role in ADM and early pancreatic carcinogenesis by promoting Arp2/3 complex function. The role of Arp2/3 complex as a common effector of mTORC1 and mTORC2 fills the gap between oncogenic signals and actin dynamics underlying PDAC initiation.

Prognostic impact of CD73 expression and its relationship to PD-L1 in patients with radically treated pancreatic cancer.

Immune suppressing molecule CD73 is overexpressed in various cancers and associated with poor survival. Little is so far known about the predictive value of CD73 in pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the prognostic significance of CD73 in PDAC. The study material consisted of 110 radically treated patients for PDAC. Tissue microarray blocks were constructed and stained immunohistochemically using CD73 antibody. Staining intensity and numbers of stained tumour cells, inflammatory cells, stroma, and blood vessels were assessed. High-level CD73 expression in tumour cells was positively associated with PD-L1 expression, perineural invasion, and histopathological grade. CD73 positivity in tumour-infiltrating lymphocytes was significantly associated with lymph node metastasis. Lymphocytic CD73 positivity was also associated with staining positivity in both stroma and vascular structures. In addition, CD73 positivity in vascular structures and stroma were associated with each other. There were no significant associations between CD73 positive tumour cells and CD73 positivity in any other cell types. PD-L1 expression was associated with CD73 staining positivity in stroma (p = 0.007) and also with histopathological grade (p = 0.033) and T class (p = 0.016) of the primary tumour. CD73 positivity in tumour cells was significantly associated with poor disease-specific (p = 0.021) and overall survival (p = 0.016). In multivariate analysis, CD73 positivity in tumour cells was an independent negative prognostic factor together with histopathological grade, TNM stage, and low immune cell score. In conclusion, high CD73 expression in tumour cells is associated with poor survival in PDAC independently of the number of tumour-infiltrating lymphocytes or TNM stage.

FAK inhibition radiosensitizes pancreatic ductal adenocarcinoma cells in vitro.

INTRODUCTION: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase protein frequently overexpressed in cancer and has been linked to an increase in the stem cell population of tumors, resistance to therapy, and metastatic spread. Pharmacological FAK inhibition in pancreatic cancer has received increased attention over the last few years, either alone or in combination with other therapeutics including chemotherapy and immunotherapy. However, its prognostic value and its role in radioresistance of pancreatic ducal adenocarcinoma (PDAC) is unknown. METHODS AND MATERIALS: Using the TCGA and GTEx databases, we investigated the genetic alterations and mRNA expression levels of PTK2 (the encoding-gene for FAK) in normal pancreatic tissue and pancreatic cancer and its impact on patient survival. Furthermore, we evaluated the expression of FAK and its tyrosine domain Ty-397 in three pancreatic cancer cell lines. We went further and evaluated the role of a commercial FAK tyrosine kinase inhibitor VS-4718 on the viability and radiosensitization of the pancreatic cell lines as well as its effect on the extracellular matrix (ECM) production from the pancreatic stellate cells. Furthermore, we tested the effect of combining radiation with VS-4718 in a three-dimensional (3D) multicellular pancreatic tumor spheroid model. RESULTS: A database analysis revealed a relevant increase in genetic alterations and mRNA expression of the PTK2 in PDAC, which were associated with lower progression-free survival. In vitro, there was only variation in the basal phosphorylation level of FAK in cell lines. VS-4718 radiosensitized pancreatic cell lines only in the presence of ECM-producing pancreatic stellate cells and markedly reduced the ECM production in the stromal cells. Finally, using a 3D multicellular tumor model, the combination of VS-4718 and radiotherapy significantly reduced the growth of tumor aggregates. CONCLUSION: Pharmacological inhibition of FAK in pancreatic cancer could be a novel therapeutic strategy as our results show a radiosensitization effect of VS-4718 in vitro in a multicellular 2D- and in a 3D-model of pancreatic cancer.

A new prognosis prediction model combining TNM stage with MAP2K4 and JNK in postoperative pancreatic cancer patients.

Mitogen-activated protein kinase kinase 4 (MAP2K4) is a tumor suppressor in many cancers. However, its roles and action mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Here, we analyzed MAP2K4 and its downstream kinases (c-Jun N-terminal kinase (JNK) and p38) using immunohistochemical staining and their prognostic significances using univariate and multivariate Cox proportional hazards regression analysis in our PDAC cohort. Then, we validated MAP2K4/JNK/p38 mRNA levels and prognostic significances using The Cancer Genome Atlas (TCGA) database. Finally, we evaluated the effects of MAP2K4 on the proliferation and invasion of PDAC cells. MAP2K4, JNK, and p38 proteins were expressed in 97.3 % (72/74), 95.6 % (65/68), and 88.6 % (62/70) of the samples, respectively, and their levels in tumor tissues were significantly higher than those in normal ducts. MAP2K4 protein expression was lower in male patients (p = 0.028). In our PDAC cohort, advanced TNM stage, low MAP2K4, and high JNK protein levels were significant prognostic factors for poor overall survival (OS) based on a univariate survival analysis (p = 0.006, p < 0.001, and p = 0.004, respectively). N stage and MAP2K4 and JNK protein levels were independent prognostic factors for OS based on multivariate analysis. We then built a prognosis prediction nomogram combining the standard TNM staging system with MAP2K4 and JNK expression that had a Harrell's C-index of 0.645. The new prognosis prediction model effectively stratified the resected patients with PDAC, from both our cohort and TCGA database, into low- and high-risk groups. Finally, MAP2K4 overexpression inhibited pancreatic cancer cell proliferation and migration in vitro. This study shows that reduced protein and mRNA levels of MAP2K4 found in PDAC patients, coupled to in vitro effects observed, support the tumor suppressor role of MAP2K4 in PDAC. Importantly, combining MAP2K4 and JNK expression with the TNM staging system results in a better prediction of postoperative survival of patients with PDAC.