In silico identification and biological evaluation of a selective MAP4K4 inhibitor against pancreatic cancer.

Inhibiting a specific target in cancer cells and reducing unwanted side effects has become a promising strategy in pancreatic cancer treatment. MAP4K4 is associated with pancreatic cancer development and correlates with poor clinical outcomes. By phosphorylating MKK4, proteins associated with cell apoptosis and survival are translated. Therefore, inhibiting MAP4K4 activity in pancreatic tumours is a new therapeutic strategy. Herein, we performed a structure-based virtual screening to identify MAP4K4 inhibitors and discovered the compound F389-0746 with a potent inhibition (IC50 120.7 nM). The results of kinase profiling revealed that F389-0746 was highly selective to MAP4K4 and less likely to cause side effects. Results of in vitro experiments showed that F389-0746 significantly suppressed cancer cell growth and viability. Results of in vivo experiments showed that F389-0746 displayed comparable tumour growth inhibition with the group treated with gemcitabine. These findings suggest that F389-0746 has promising potential to be further developed as a novel pancreatic cancer treatment.

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.

RNA N6-methyladenosine demethylase FTO promotes pancreatic cancer progression by inducing the autocrine activity of PDGFC in an m6A-YTHDF2-dependent manner.

RNA N6-methyladenosine (m6A) is an emerging regulator of mRNA modifications and represents a novel player in tumorigenesis. Although it has functional significance in both pathological and physiological processes, the role of m6A modification in pancreatic ductal cancer (PDAC) remains elusive. Here, we showed that high fat mass and obesity-associated gene (FTO) expression was associated with a poor prognosis in PDAC patients and that suppression of FTO expression inhibited cell proliferation. Here, m6A sequencing (m6A-seq) was performed to screen genes targeted by FTO. The effects of FTO stimulation on the biological characteristics of pancreatic cancer cells, including proliferation and colony formation, were investigated in vitro and in vivo. The results indicate that FTO directly targets platelet-derived growth factor C (PDGFC) and stabilizes its mRNA expression in an m6A-YTHDF2-dependent manner. m6A-methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR), RNA immunoprecipitation (RIP), and luciferase reporter assays were employed to validate the specific binding of FTO to PDGFC. PDGFC upregulation led to reactivation of the Akt signaling pathway, promoting cell growth. Overall, our study reveals that FTO downregulation leads to increased m6A modifications in the 3' UTR of PDGFC and then modulates the degradation of its transcriptional level in an m6A-YTHDF2-dependent manner, highlighting a potential therapeutic target for PDAC treatment and prognostic prediction.

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 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.

Carboxypeptidase A1 (CPA1) Immunohistochemistry Is Highly Sensitive and Specific for Acinar Cell Carcinoma (ACC) of the Pancreas.

Carboxypeptidase A1 (CPA1) is a zinc metalloprotease that is produced in pancreatic acinar cells and plays a role in cleaving C-terminal branched-chain and aromatic amino acids from dietary proteins. This study assessed the utility of immunohistochemical CPA1 staining for diagnosing pancreatic acinar cell carcinoma (ACC). A total of 12,274 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types were interpretable by immunohistochemistry in a tissue microarray format. CPA1 was strongly expressed in acinar cells of all normal pancreas samples but not in any other normal tissues. CPA1 immunostaining was detected in 100% of 11 pancreatic ACCs and 1 mixed acinar endocrine carcinoma, but absent in 449 pancreatic ductal adenocarcinomas, 75 adenocarcinomas of the ampulla Vateri, and 11,739 other evaluable cancers from 128 different tumor entities. A weak to moderate diffuse staining of epithelial and stromal cells of cancer tissues immediately adjacent to non-neoplastic pancreatic acinar cells often occurred and was considered to be caused by the diffusion of the highly abundant CPA1 from normal acinar cells that may have suffered some autolytic cell damage. In conclusion, our data show that CPA1 is a highly sensitive and largely specific marker for normal and neoplastic pancreatic acinar cells. CPA1 immunohistochemistry greatly facilitates the otherwise often difficult diagnosis of pancreatic ACC.

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.

Pancreatic medullary carcinoma developed on a pancreatic intraductal papillary mucinous neoplasm with loss of MSH2 and MSH6 expression: a case report.

BACKGROUND: Pancreatic medullary carcinoma (PMC) is a rare pancreatic tumor, usually showing the presence of microsatellite instability, mostly MLH1 silencing, and a wild-type KRAS mutation status. We report here a PMC arising from a Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN), both having KRAS and TP53 mutations. CASE PRESENTATION: We report the case of a 73-year-old woman presenting with right iliac fossa pain. MRI revealed a 16 mm diameter mass in the pancreas, leading to a pancreatic duct stricture and upstream a dilatation of the distal pancreatic duct of Wirsung. A fine needle aspiration was performed, and pathology analysis revealed malignant glandular cells. The patient underwent distal pancreatectomy. Gross examination revealed an12 mm indurated white lesion, adjacent to a cystic lesion extending into the rest of the pancreatic body. Microscopically, the cystic area represented a mixed (gastric-type and pancreatobiliary-type) IPMN, involving the main and secondary pancreatic ducts with low-grade and high-grade dysplasia. In the periphery of this IPMN, a 14mm associated invasive carcinoma was observed, characterized by focal gland formation and by poorly differentiated cells with a syncytial appearance, associated with a dense lymphoplasmocytic and neutrophilic infiltrate. Immunohistochemical analyses showed loss of MSH2 and MSH6 expression. Microsatellite instability was confirmed by molecular test. Molecular analysis was performed both on the invasive carcinoma and on the high-grade dysplasia IPMN, revealing the same mutation profile with KRAS and TP53 mutations. The proposed diagnosis was mixed IPMN with associated invasive medullary carcinoma that presented loss of MSH2 and MSH6 expression. CONCLUSIONS: The present case reports for the first time, at the best of our knowledge, the coexistence of IPMN lesions and PMC, both having the same molecular alterations. It also describes the second case of PMC with microsatellite instability, MSH2 and MSH6 silenced.

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.

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer.

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.

UGT1A Gene Family Members Serve as Potential Targets and Prognostic Biomarkers for Pancreatic Cancer.

BACKGROUND: Pancreatic cancer (PC) is one of the most common cancers worldwide, with high mortality. The UGT1A gene family plays important roles in pharmacology and toxicology, contributing to interindividual differences in drug disposition. However, mRNA expression and prognostic value of the UGT1A gene family in PC have not been identified. METHODS: Oncomine, GEPIA2, DAVID 6.8, Metascape, Kaplan-Meier plotter, cBioPortal, GeneMANIA, TRRUST v2, TIMER, and R software were used in our study. RESULTS: The transcriptional levels of UGT1A1/3/6/8/9/10 in PC tissues were significantly higher than those in normal tissues. These results were further validated using five pairs of PC tumor tissues and adjacent nontumor tissues. A significant correlation was found between the expression of UGT1A1/6/10 and the pathological stage of PC. PC patients with lower transcriptional levels of UGT1A1/4/5/6/10 were associated with a better prognosis. The differentially expressed UGT1A gene family functions were primarily related to the glucuronidation pathway, cytokine-cytokine receptor interactions, and the ILK signaling pathway. Our data suggest that HNF1A, AHR, and CDX2 are key transcription factors for the UGT1A gene family. Furthermore, the expression levels of UGT1A1/3/8/9/10 were positively correlated with the activities of tumor-infiltrating immune cells, especially B cells. The expression levels of UGT1A6/9 were negatively correlated with macrophage infiltration levels. CONCLUSIONS: These results suggest that the UGT1A gene family could serve as a potential prognostic biomarker and target for PC. However, future studies are required to validate our findings and promote the clinical utility of the UGT1A gene family in PC.

MK8722, an AMPK activator, inhibiting carcinoma proliferation, invasion and migration in human pancreatic cancer cells.

BACKGROUND: MK8722 is a potent and systemic pan-AMPK activator. It is an effective, direct, allosteric activator of AMPK complex in many mammals. This study tried to explore the underlying anti-cancer molecular mechanism of MK8722 in human pancreatic cancer cells (PCCs). METHODS: The anti-proliferation, invasion and migration functions of MK8722 in human pancreatic cancer analyzed by real time cellular analysis, colony formation assay, cell migration assay, transwell assay and flow cytometery analysis. Moreover, the potential targeted signaling pathway was tested via RNA-seq and pathway enrichment analysis. RESULTS: In the present study, we investigated the anti-PCCs effects of MK8722 on two different human pancreatic cancer cell lines (PANC-1 and Patu8988). The results showed that MK8722 significantly inhibited human tumor cells proliferation and migration/invasion in a dose-dependent manner. Additionally, the influence of MK8722 was examined by analyzing the expression of potential key genes and pathways, which may provide novel insights to the mechanism of MK8722. CONCLUSION: The inhibition of pancreatic cancer by MK8722 through a number of pathways that inhibit carcinoma proliferation, invasion and migration. The potential effect of MK8722 might be determined by regulating the expression of AL162151, IER2, REPIN1, KRT80 to inhibit cycle arrest and migration.

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.

LncRNA RGMB-AS1 facilitates pancreatic cancer cell proliferation and migration but inhibits cell apoptosis via miR-574-3p/PIM3 axis.

Pancreatic cancer (PC) is among the most notorious malignancies worldwide. Long noncoding RNA (lncRNA) repulsive guidance molecule bone morphogenetic protein (BMP) coreceptor b antisense RNA 1 (RGMB-AS1) was an oncogene in glioma. However, the RGMB-AS1 function in PC remains largely unknown. Herein, quantitative real-time polymerase chain reaction was performed to analyze the expression of RGMB-AS1. We determined RGMB-AS1 influence on PC cell malignant behaviors via functional assays. Besides, we applied subcellular fractionation and fluorescence in situ hybridization (FISH) assays to confirm the cellular distribution of RGMB-AS1 in PC cells. We used mechanism assays to detect the regulatory axis of RGMB-AS1 in PC cells. Briefly, the level of RGMB-AS1 expression in PC cells was abnormally high. RGMB-AS1 knockdown impeded PC cell proliferation and migration, but induced cell apoptosis, and RGMB-AS1 overexpression led the opposite consequences. RGMB-AS1 acted as a competing endogenous RNA (ceRNA) to sequester miR-574-3p and thereby regulated Pim-3 proto-oncogene, serine/threonine kinase (PIM3) expression. Conclusively, our work revealed the cancer-promoting function of RGMB-AS1 in PC and that the regulatory mechanism of the RGMB-AS1/miR-574-3p/PIM3 axis might contribute to novel biomarker development in PC treatment.NEW & NOTEWORTHY RGMB-AS1 promotes PC cell proliferation, elevates PC cell migration capacity, inhibits PC cell apoptosis, and promotes PC cell proliferation and migration but inhibits cell apoptosis via targeting miR-574-3p. PIM3 is directly targeted by miR-574-3p.

Evaluation of Micro Satellite Instability and Mismatch Repair Status in Different Solid Tumors: A Multicenter Analysis in a Real World Setting.

Immune-checkpoint inhibitors (ICIs) play a key role in the treatment of advanced stage colorectal cancer (CRC) patients featuring a deficient DNA mismatch repair (dMMR) system or a high microsatellite instability (MSI-H) profile. However, beyond the established role in CRC patients, ICIs have highly proven efficacy in other solid tumors featuring MSI-H/dMMR status represented by endometrial, gastric, ovarian, prostatic, and pancreatic carcinomas (EC, GC, OC, PrC, and PaC). Our aim was to compare the concordance rates among the Idylla™ MSI test, TapeStation 4200, and immunohistochemical (IHC) analysis in assessing MSI-H/dMMR status in EC, GC, OC, PrC, and PaC patients. The Sanger sequencing-based Titano MSI test was used in discordant cases. One hundred and eighty-five cases (n = 40 PrC, n = 39 GC, n = 38 OC, n = 35 PaC, and n = 33 EC) were retrospectively selected. MMR protein expression was evaluated by IHC. After DNA quality and quantity evaluations, the IdyllaTM and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Remarkably, compared to IHC, the Idylla™ platform achieved a global concordance rate of 94.5% (154/163) for the microsatellite stable (MSS)/proficient MMR (pMMR) cases and 77.3% (17/22) for the MSI-H/dMMR cases. Similarly, a global concordance rate of 91.4% (149/163) and 68.2% (15/22) for MSS/pMMR and MSI-H/dMMR cases was also identified between IHC and the TapeStation 4200 microfluidic system. In addition, a global concordance of 93.1% (148/159) and 69.2% (18/26) for MSS/pMMR and MSI-H/dMMR cases was observed between the Idylla™ and TapeStation 4200 platforms. Discordant cases were analyzed using the Titano MSI kit. Overall, our data pinpointed a central role for molecular techniques in the diagnostic evaluation of dMMR/MSI-H status not only in CRC patients but also in other types of solid tumors.

The enhancement of Tetrandrine to gemcitabine-resistant PANC-1 cytochemical sensitivity involves the promotion of PI3K/Akt/mTOR-mediated apoptosis and AMPK-regulated autophagy.

BACKGROUND: In the process of tumor development, the resistance of pancreatic cancer cells to gemcitabine (GEM) is mainly due to the suppression and dysregulation of apoptosis signals to a large extent. Therefore, it is very necessary to develop pro-apoptotic drugs for combined treatment of pancreatic cancer to increase the activity of GEM and improve the prognosis of pancreatic cancer. METHODS AND RESULTS: GEM-resistant PANC-1 cells were treated with increasing doses of GEM. The effects of GEM and TET on apoptosis were evaluated by flow cytometry and Hoechst 33258 staining. We also evaluated the expression of survivin by real-time PCR, and the expression levels of proteins involved in apoptosis, autophagy, and PI3K/Akt/mTOR signaling were detected by western blotting. The results showed that TET downregulated expression of survivin by inhibiting the PI3K/Akt/mTOR signaling pathway to promote pancreatic cancer cell apoptosis, thereby enhancing pancreatic cancer cell sensitivity to GEM. Moreover, TET enhanced cytotoxic and autophagy-dependent cell death by upregulating the AMPK-autophagy axis, and this effect was reversed by inhibition of AMPK. CONCLUSIONS: TET promotes apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway and promotes autophagy via up-regulating the AMPK signaling pathway to play an anti-tumor effect in GEM-resistant pancreatic cancer cells, which represents a new therapeutic strategy for the treatment of GEM-resistant pancreatic cancer.

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.

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

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.