Human Pancreatic Ductal Epithelial Cells Research Articles

Abstract 991: Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology

Abstract Introduction: Pancreatic cancer has the lowest 5-year survival rates among all cancers because of the challenges with early detection, its aggressive nature, and resistance to chemotherapy; this makes prevention a promising approach for reducing related mortalities. The disease is predominantly diagnosed in individuals over 60, suggesting that age-related physiological decline impacts its onset; therefore, delaying aging hallmarks may delay its onset. Nicotinamide dinucleotide (NAD+) precursors may hold a unique advantage over other geroprotectors because they target multiple aging hallmarks. Heron, we investigated how NAD+ supplementation impacts normal human pancreatic ductal epithelial cells (HPDE) under stress as a surrogate for cancer prevention in vitro. We also examined the paradoxical impact of NAD+ precursor supplementation on pancreatic cancer growth during chemotherapy. Methods: In vitro, we mimicked age-related NAD+ decline in the media. We stressed HPDE cells under these conditions and evaluated the effect of supplementation with the NAD+ precursor, Nicotinamide mononucleotide (NMN). We assessed cell viability using the PicoGreen assay, mitochondrial function, and reactive oxygen species (ROS) using the Seahorse XF Analyzer and the mitoSox kit, respectively, DNA repair and DNA damage using a western blot for Poly ADP ribose (PAR) and gamma H2AX, respectively, and SIRT3 expression was determined using a western blot. To evaluate the impact of NAD+ supplementation on pancreatic cancer cells, we treated pancreatic cancer cells with NMN and oxaliplatin. We then assessed the mitochondrial function, ROS levels, DNA damage, and repair. We implanted MiaPaCa-2 xenografts into athymic nude mice and treated them daily with 300mg/Kg of NMN and biweekly 5mg/kg of Oxaliplatin. Results: Experiments revealed that NMN is not toxic to HPDE cells, even at high concentrations. There was increased cell survival of HPDE cells under stress (nutrient deprivation and alkylating agents) following NMN supplementation. We also noted reduced ROS levels, increased SIRT3 expression, and PARylation. Pancreatic cancer cells were less sensitive to Oxaliplatin following NMN supplementation and showed an increased oxygen consumption rate, increased PARylation, and reduced ROS levels. There was reduced sensitivity to oxaliplatin treatment in the mice with MiaPaCa-2 xenografts treated with NMN. Conclusion: These findings demonstrate a cytoprotective role of NAD+ precursor supplementation in normal pancreatic ductal epithelial cells, with implications for possible cancer prevention. NAD+ supplementation also rescued cancer cells under cytotoxic stress, meaning that geroprotectors like NMN, in conjunction with chemotherapy, may unintentionally promote cancer progression. Future studies will directly test NAD+ precursor supplementation in genetically engineered pancreatic cancer models. Citation Format: Faith Nakazzi, Mehrad Zarei, Hallie J. Graor, Alexander W. Loftus, Omid Hajihassanni, Jordan M. Winter. Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 991.

MicroRNA-20a in extracellular vesicles derived from duodenal fluid is a possible biomarker for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate owing to its late diagnosis and aggression. In addition, there are relatively few minimally invasive screening methods for the early detection of PDAC, making the identification of biomarkers for this disease a critical priority. Recent studies have reported that microRNAs in extracellular vesicles (EV-miRs) from bodily fluids can be useful for the diagnosis of PDACs. Given this, we designed this study to evaluate the utility of cancer EVs extracted from duodenal fluid (DF) and their resident EV-miRs as potential biomarkers for the detection of PDAC. EV-miRs were evaluated and identified in the supernatants of various pancreatic cancer cell lines (Panc-1, SUIT2, and MIAPaca2), human pancreatic duct epithelial cells, and the DF from patients with PDAC and healthy controls. EVs were extracted using ultracentrifugation and the relative expression of EV-miR-20a was quantified. We collected a total of 34 DF samples (27 PDAC patients and seven controls) for evaluation and our data suggest that the relative expression levels of EV-miR-20a were significantly higher in patients with PDAC than in controls (p = 0.0025). In addition, EV-miR-20a expression could discriminate PDAC from control patients regardless of the location of the tumor with an area under the curve values of 0.88 and 0.88, respectively. We confirmed the presence of EVs in the DF and suggest that the expression of EV-miR-20a in these samples may act as a potential diagnostic biomarker for PDAC.

Melt-electrowriting of 3D anatomically relevant scaffolds to recreate a pancreatic acinar unit in vitro

Melt-electrowriting (MEW) belongs to the group of advanced additive manufacturing techniques and consists of computer-aided design (CAD)-assisted polymer extrusion combined with a high-voltage supply to achieve deposition of polymeric fibers with diameters in the micrometric range (1 to 20 μm) similar to the size of natural extracellular matrix fibers. In this work, we exploit MEW to design and fabricate a three-dimensional (3D) model that resembles the morphology of the exocrine pancreatic functional unit without the need of supports, mandrels, or sacrificial materials. Optimized process parameters resulted in a MEW scaffold having regular fibers (19 ± 5 μm size) and an acinar cavity showing high shape fidelity. Then, human foreskin fibroblasts (HFF1) and human pancreatic ductal epithelial cells (HPDE), wild-type HPDE, and HPDE overexpressing KRAS oncogene were allowed to colonize the entire 3D structure and the acinar cavity. Thus, a physiologically relevant 3D model was created in vitro after 24 days using a co-culture protocol (14 days of HFF1 alone plus 10 days of HPDE and HFF1 co-culture). The effect of cell crosstalk within the MEW scaffolds was also assessed by monitoring HFF1 secretion of interleukin (IL)-6, a pro-inflammatory cytokine responsible for the inflammatory cascade occurring in pancreatic cancer. High levels of IL-6 were detected only when fibroblasts were co-cultured with the HPDE overexpressing KRAS. These findings confirmed that the MEW 3D in vitro model is able to recreate the characteristic hallmark of the pathological condition where cancer oncogenes mediate fibroblast activities.

Graphene Nanocomposites in the Treatment of Pancreatic Cancer

The application value of titanium dioxide (TiO2)/graphene nanocomposites in photothermal therapy of pancreatic cancer (PC) was explored. Using scale graphite as raw material, graphene was obtained by Hummer oxidation method and hydrazine hydrate reduction method, and then TiO2/graphene nanocomposites were prepared by ultrasonic heating. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and degraded methyl orange solution were adopted to detect the surface structure, particle size, element morphology, and photocatalytic activity under different composite ratios, different sonication times, and different heating temperatures. Human normal pancreatic ductal epithelial cell line HPDE6-C7 and human metastatic PC cell AsPC-1 were adopted as research models. The cytotoxicity of TiO2/graphene nanocomposites and the killing effect of photothermal therapy based on TiO2/graphene nanocomposites were analyzed by water soluble tetrazolium salt colorimetric assay (WST-1) and methyl thiazolyl tetrazolium salt colorimetric assay (MTT). The results suggested that when the ratio of graphene to TiO2 was 50:1, the ultrasonic time was 100 min, and the heating temperature was 200 °C, TiO2 was better attached to the surface of graphene, the distribution of particles was relatively more uniform, and the concentration of methyl orange was relatively lowest. The XRD pattern showed that the diffraction peak of the doped TiO2/graphene nanocomposite was basically the same as that of the pure TiO2. When the ultrasonic time was 100 min, the diffraction peak intensity in the XRD pattern was the largest. As for AsPC-1 cells, the cell viability was obviously lower than 0.1/1/10/100 μm/mL when the concentration of TiO2/graphene nanocomposites was 500 μm/mL (P <0.05). For HPDE6-C7 cells, when the concentration of TiO2/graphene nanocomposites was 100 and 500 μm/mL, the cell viability was obviously lower than 0.1/1/10 μm/mL (P <0.05), and 500 μm/mL was the lowest. The cell killing rate in group D was clearly higher as against groups A, B, and C (P <0.05). Graphene: The optimal preparation conditions of TiO2/graphene nanocomposites are 50:1, 100 min of ultrasound time, and 200 μC of composite temperature. The photothermal therapy based on TiO2/graphene nanocomposites can effectively kill PC cells, and has a good potential in the field of hyperthermia for pancreatic tumors.

Corosolic acid enhances oxidative stress-induced apoptosis and senescence in pancreatic cancer cells by inhibiting the JAK2/STAT3 pathway.

Pancreatic cancer (PC) is a fatal human malignancy with a poor prognosis. Corosolic acid (CRA) is a triterpenoid, has been reported to have inhibitory effects on tumor growth. However, the role of CRA on PC has not been explored. Here, we aimed to uncover the molecular mechanisms of CRA in PC progression. Cell viability, lactate dehydrogenase (LDH) release, cell apoptosis and senescence were detected by cell counting kit-8 (CCK-8), LDH, flow cytometry and senescence associated-β-galactosidase (SA-β-gal) assay. Levels of relevant proteins and oxidative stress (OS) markers were evaluated by Western blot and enzyme-linked immunosorbent assay (ELISA). A xenograft tumor model was established to explore the in vivo effects of CRA on PC. We found that CRA inhibited PC cell viability and promoted LDH release in a dose-dependent manner, but had no significant effect on human normal pancreatic ductal epithelial cells HPDE6C7. CRA increased OS-induced cell apoptosis and senescence in HAPC and SW1990 cells. And CRA decreased the levels of anti-apoptotic protein Bcl-2, and elevated the expression of pro-apoptotic protein Bax and senescence-associated proteins P21 and P53. Besides, CRA decreased tumor growth in xenograft models. Furthermore, CRA inactivated the Janus kinase-2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway in HAPC and SW1990 cells. Functional experiments demonstrated that activation of the JAK2/STAT3 pathway by the JAK2 activator coumermycin A1 (C-A1) or the STAT3 activator colivelin (col) reduced the contribution effect of OS, apoptosis and senescence by CRA. Taken together, our findings indicated that CRA exerted anti-cancer effects in PC by inhibiting the JAK2/STAT3 pathway.

Abstract C065: Base excision repair pathway regulates transcription-replication conflicts in pancreatic ductal adenocarcinoma

Abstract Oncogenic mutations (such as in KRAS) can dysregulate transcription and replication leading to transcription conflicts (TRCs). Unresolved TRCs can cause lethal DNA damage. Here, we sought to investigate the causal role of oncogenic mutations in regulating TRCs in pancreatic ductal adenocarcinoma (PDAC). Further, we characterize the adaptive pathways that mitigate TRCs for PDAC survival. Human PDAC demonstrated 30-120-fold higher levels of TRC genomic signature compared to breast, colon and lung cancer (p<0.001). Gene expression analysis demonstrated high levels of known TRC resolution factors in human tumors compared to normal tissues. TRCs as measured by RNAPII-PCNA proximity ligation assay were significantly enriched in human PDAC cells (Panc-1, BxPC3, MiaPaca2) compared to immortalized normal HPNE cells (p<0.001). Similarly transcriptomic complexes were enriched in nascent DNA immunoprecipitates from Panc1 cells but not HPNE cells. Ectopic expression of oncogenic KRAS(G12D) in human pancreatic ductal epithelial (HPNE) cells enhanced TRCs, and TRC-related DNA:RNA hybrids (R-loops). Inhibition of KRAS or downstream effectors abrogated TRCs in Panc1 and MiaPaca2 cells. An siRNA screen identified several factors in the base-excision repair (BER) pathway as regulators of TRCs. Sub-lethal exposure to hydrogen peroxide enhanced TRCs, whereas N-acetyl cysteine abrogated TRCs. These findings were validated by a pharmacologic approach using inhibitors of APE1 endonuclease in BER pathway (Methoxyamine and CRT). Mechanistic studies revealed that BER pathway inhibition severely altered RNA polymerase II dynamics at nascent DNA; causing RNAPII trapping and contributing to enhanced TRCs. The ensuing DNA damage activated Chk2-ATR pathway but not Chk1-ATM pathway. Co-treatment with ATR inhibitor (VX970, 10-80nM) and BER inhibitor (methoxyamine, 6-12mM) at clinically relevant doses, synergistically enhanced DNA damage and reduced clonogenic survival in PDAC cells. The study uncovers a novel role of BER pathway defects and oxidative DNA damage in promoting TRCs. Our studies provide mechanistic insights into the regulation of TRCs in PDAC which has implications for genome instability and therapy in PDAC. Citation Format: Fan Meng, Anup K. Singh, Tiane Li, Marc Attiyeh, Fatemeh Kohram, Terence Williams, Yilun Liu, Mustafa Raoof. Base excision repair pathway regulates transcription-replication conflicts in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C065.

Myoglobin improves doxycycline sensitivity in pancreatic cancer through promoting heme oxygenase-1-mediated ferroptosis.

Ferroptosis is expected to be a therapeutic target for cancers including pancreatic cancer. We aimed to screen genes that regulate ferroptosis and doxycycline resistance in pancreatic cancer and to explore the underlying mechanisms. Bioinformatics analysis was performed to identify genes that respond to ferroptosis in two human pancreatic cancer cells with GOT1 knocked down or not. 325 and 842 genes were upregulated in MiaPaCa and Tu8902 cells in response to GOT1 knockdown, with 43 genes shared. Among the 43 genes, 14 genes were identified to interact with ferroptosis key genes. MB and HMOX1 were the genes most sensitive to Erastin and doxycycline. Moreover, MB and HMOX1 expression was higher in human normal pancreatic duct epithelial cells than in pancreatic cancer cells. MB and HMOX1 proteins physically bound and promoted each other's expression. By interacting with HMOX1, MB suppressed pancreatic cancer cell proliferation, colony formation and invasion, and promoted cell ferroptosis and sensitivity to erastin and doxycycline. Silencing HMOX1 reversed the promoting effect of MB on cell ferroptosis and sensitivity to doxycycline. A pancreatic cancer xenograft model was established by subcutaneous injection of Panc-1 cells transfected with or without Ad-MB, and doxycycline was administered intraperitoneally. Overexpression of MB enhanced the inhibitory effect of doxycycline on xenograft growth. In conclusion, MB facilitated doxycycline sensitivity in pancreatic cancer cells through promoting HMOX1-mediated ferroptosis.

Fatty acids abrogate the growth-suppressive effects induced by inhibition of cholesterol flux in pancreatic cancer cells

BackgroundDespite therapeutic advances, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. Metabolic reprogramming is increasingly recognized as a key contributor to tumor progression and therapy resistance in PDAC. One of the main metabolic changes essential for tumor growth is altered cholesterol flux. Targeting cholesterol flux appears an attractive therapeutic approach, however, the complex regulation of cholesterol balance in PDAC cells remains poorly understood.MethodsThe lipid content in human pancreatic duct epithelial (HPDE) cells and human PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) was determined. Cells exposed to eight different inhibitors targeting different regulators of lipid flux, in the presence or absence of oleic acid (OA) stimulation were assessed for changes in viability, proliferation, migration, and invasion. Intracellular content and distribution of cholesterol was assessed. Lastly, proteome profiling of PANC-1 exposed to the sterol O-acyltransferase 1 (SOAT1) inhibitor avasimibe, in presence or absence of OA, was performed.ResultsPDAC cells contain more free cholesterol but less cholesteryl esters and lipid droplets than HPDE cells. Exposure to different lipid flux inhibitors increased cell death and suppressed proliferation, with different efficiency in the tested PDAC cell lines. Avasimibe had the strongest ability to suppress proliferation across the three PDAC cell lines. All inhibitors showing cell suppressive effect disturbed intracellular cholesterol flux and increased cholesterol aggregation. OA improved overall cholesterol balance, reduced free cholesterol aggregation, and reversed cell death induced by the inhibitors. Treatment with avasimibe changed the cellular proteome substantially, mainly for proteins related to biosynthesis and metabolism of lipids and fatty acids, apoptosis, and cell adhesion. Most of these changes were restored by OA.ConclusionsThe study reveals that disturbing the cholesterol flux by inhibiting the actions of its key regulators can yield growth suppressive effects on PDAC cells. The presence of fatty acids restores intracellular cholesterol balance and abrogates the alternations induced by cholesterol flux inhibitors. Taken together, targeting cholesterol flux might be an attractive strategy to develop new therapeutics against PDAC. However, the impact of fatty acids in the tumor microenvironment must be taken into consideration.

FBXO28 promotes proliferation, invasion, and metastasis of pancreatic cancer cells through regulation of SMARCC2 ubiquitination.

The E3 ligase F-box only protein 28 (FBXO28) belongs to the F-box family of proteins that play a critical role in tumor development. However, the potential function of FBXO28 in pancreatic cancer (PC) and its molecular mechanism remain unclear. In this study, we examined FBXO28 expression in PC and its biological role and explored the mechanism of FBXO28-mediated proliferation, invasion, and metastasis of PC cells. Compared with paracancerous tissues and human normal pancreatic ductal epithelial cells, FBXO28 was highly expressed in PC tissues and cell lines. High expression of FBXO28 was negatively correlated with the survival prognosis of patients with PC. Functional assays indicated that FBXO28 promoted PC cell proliferation, invasion, and metastasis in vitro and in vivo. Furthermore, immunoprecipitation-mass spectrometry was used to identify SMARCC2 as the target of FBXO28; upregulation of SMARCC2 can reverse the effect of overexpression of FBXO28 on promoting the proliferation, invasion, and metastasis of PC cells. Mechanistically, FBXO28 inhibited SMARCC2 expression in post-translation by increasing SMARCC2 ubiquitination and protein degradation. In conclusion, FBXO28 has a potential role in PC, possibly promoting PC progression through SMARCC2 ubiquitination. Thus, FBXO28 might be a potential treatment target in PC.

RETRACTED ARTICLE: Palmitoyl transferases act as novel drug targets for pancreatic cancer

BackgroundPancreatic adenocarcinoma (PAAD) is one of the most leading causes of cancer-related death across the world with the limited efficiency and response rate of immunotherapy. Protein S-palmitoylation, a powerful post-translational lipid modification, is well-known to regulate the stability and cellular distribution of cancer-related proteins, which is mediated by a family of 23 palmitoyl transferases, namely zinc finger Asp-His-His-Cys-type (ZDHHC). However, whether palmitoyl transferases can determine tumor progression and the efficacy of immunotherapy in PAAD remains unknown.MethodsBioinformatics methods were used to identify differential ZDHHCs expression in PAAD. A systematic pan-cancer analysis was conducted to assess the immunological role of ZDHHC3 using RNA sequencing data from The Cancer Genome Atlas database. In vivo Panc 02 subcutaneous tumor model validated the anti-tumor effect of knockdown of ZDHHC3 or intraperitoneal injection of 2-bromopalmitate (2-BP), a typical broad-spectrum palmitoyl transferases inhibitor. Furthermore, we explored therapeutic strategies with combinations of 2-BP with PD-1/PD-L1-targeted immunotherapy in C57BL/6 mice bearing syngeneic Panc 02 pancreatic tumors.ResultsZDHHC enzymes were associated with distinct prognostic values of pancreatic cancer. We identified that ZDHHC3 expression promotes an immunosuppressive tumor microenvironment in PAAD. 2-BP suppressed pancreatic-tumor cell viability and tumor sphere-forming activities, as well as increased cell apoptosis in vitro, without affecting normal human pancreatic ductal epithelial cells. Furthermore, genetic inactivation of ZDHHC3 or intraperitoneal injection of 2-BP impeded tumor progression in Panc 02 pancreatic tumors with enhanced anti-tumor immunity. 2-BP treatment significantly enhanced the therapeutic efficacy of PD-1/PD-L1 inhibitors in Panc 02 pancreatic tumors.ConclusionThis study revealed some ZDHHC enzyme genes for predicting the prognosis of pancreatic cancer, and demonstrated that ZDHHC3 plays a critical oncogenic role in pancreatic cancer progression, highlighting its potential as an immunotherapeutic target of pancreatic cancer. In addition, combination therapy of 2-BP and PD-1/PD-L1 achieved synergic therapy effects in a mouse model of pancreatic cancer.

Synergistic effects of agonists and two-pore-domain potassium channels on secretory responses of human pancreatic duct cells Capan-1

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium.

Targeting FTO Suppresses Pancreatic Carcinogenesis via Regulating Stem Cell Maintenance and EMT Pathway

N6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification regulating cancer self-renewal. However, despite its functional importance and prognostic implication in tumorigenesis, the relevance of FTO, an m6A eraser, in pancreatic cancer (PC) remains elusive. Here, we establish the oncogenic role played by FTO overexpression in PC. FTO is upregulated in PC cells compared to normal human pancreatic ductal epithelial (HPDE) cells. Both RNAi depletion and CS1-mediated pharmacological inhibition of FTO caused a diminution of PC cell proliferation via cell cycle arrest in the G1 phase and p21cip1 and p27kip1 induction. While HPDE cells remain insensitive to CS1 treatment, FTO overexpression confers enhancements in growth, motility, and EMT transition, thereby inculcating tumorigenic properties in HPDE cells. Notably, shRNA-mediated FTO depletion in PC cells impairs their mobility and invasiveness, leading to EMT reversal. Mechanistically, this was associated with impaired tumorsphere formation and reduced expression of CSCs markers. Furthermore, FTO depletion in PC cells weakened their tumor-forming capabilities in nude mice; those tumors had increased apoptosis, decreased proliferation markers, and MET conversion. Collectively, our study demonstrates the functional importance of FTO in PC and the maintenance of CSCs via EMT regulation. Thus, FTO may represent an attractive therapeutic target for PC.

Abstract B008: Lipocalin-2 expression modulates ferroptosis in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the US. Lipocalin 2 (LCN2) is upregulated in PDAC patients and promotes inflammation and tumor growth. LCN2 regulates iron levels, which are dysregulated in tumors as cancer cells uptake increased iron to support growth. High iron levels can lead to ferroptosis, an iron-catalyzed form of cell death that relies on the oxidation of poly-unsaturated fatty acids (PUFAs). Ferroptosis is usually inhibited in cancer to prevent cell death and promote tumor growth. Therefore, LCN2 expression might influence ferroptosis outcomes in PDAC. Objective: To determine whether LCN2 inhibits ferroptosis and whether modulating LCN2 levels and other targets can be leveraged as treatments to induce ferroptosis in PDAC. Methods: Lcn2 expression was deleted via CRISPR in a mouse PDAC cell line (mKPC) to generate control Lcn2+/+ and Lcn2-/- mKPC cells. Gene expression changes were analyzed with RNA sequencing. Human and mouse PDAC cells were treated with a ferroptosis inducer, LCN2 blockade, and various PUFAs. Treatment effects on cell proliferation and lipid peroxidation were measured via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay and BODIPY 581/591 C11 staining respectively. Gene expression of ferroptosis markers (SLC7A11, GPX4, and SLC11A2) and LCN2 were measured via RT-qPCR. Results: Deletion of Lcn2 in mKPC cells significantly decreased the expression of genes involved in ferroptosis-inhibiting antioxidant synthesis. Ferroptosis induction upregulated Lcn2 expression in PDAC cells and decreased the viability of mKPC Lcn2-/- and human PDAC cells, but not mKPC Lcn2+/+ or human pancreatic duct epithelial cells. LCN2 blockade treatment to PDAC cells modulated the expression SLC7A11, GPX4, and SLC11A2, key enablers and inhibitors of the ferroptosis antioxidant pathway. Conclusions: Ferroptosis decreases proliferation of PDAC cells, which might be mediated by LCN2 expression. LCN2 modulation has potential to induce ferroptosis and delay PDAC growth. Future studies will determine whether a combination approach with LCN2 modulation could induce ferroptosis in PDAC. Acknowledgments: This research is supported by the OSU Pelotonia Fellowship Program and the NCI R01CA223204. Citation Format: Valentina Pita Grisanti, Andrew W. Dangel, Maciej Pietrzak, Amy Hite, Zobeida Cruz-Monserrate. Lipocalin-2 expression modulates ferroptosis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B008.

Identification of MYEOV-Associated Gene Network as a Potential Therapeutic Target in Pancreatic Cancer.

The molecular mechanism that promotes pancreatic cancer remains unclear, so it is important to find the molecular network of important genes related to pancreatic cancer. To find the key molecule of pancreatic cancer, differential gene expression analyses were analyzed by the Deseq2 package, edgeR package, and limma-voom package, respectively. Pancreatic cancer survival-related genes were analyzed by COX survival analysis. Finally, we integrated the results to obtain the significantly differentially expressed gene, MYEOV (myeloma overexpressed gene), most strongly related to survival in pancreatic cancer. Experimental verification by qRT-PCR confirmed that transcription levels of MYEOV mRNA markedly increased in pancreatic cancer cells relative to normal human pancreatic ductal epithelial cells (HPDE). Through the comprehensive analysis of multiple databases, we constructed a molecular network centered on MYEOV and found specific links between molecules in this network and tumor-associated immune cells. It was noted that MYEOV could serve as a ceRNA by producing molecular sponging effects on hsa-miR-103a-3p and hsa-miR-107, thus affecting the role of GPRC5A, SERPINB5, EGFR, KRAS, EIF4G2, and PDCD4 on pancreatic cancer progression. Besides, we also identified that infiltrated immune cells are potential mediators for the molecules in the MYEOV-related network to promote pancreatic cancer progression. It is the first report to focus on the possibility that MYEOV may act as a competing endogenous RNA (ceRNA) to form an interactive network with some pancreatic cancer-related genes such as KRAS and serve as a key therapeutic target of pancreatic cancer treatment.

Design, synthesis and biological evaluation of a new series of arylidene indanones as small molecules for targeted therapy of non-small cell lung carcinoma and prostate cancer

In an attempt to identify small molecules for targeted therapy of non-small cell lung carcinoma (NSCLC) and prostate cancer (PCa), new arylidene indanones (1–10) were synthesized via the Claisen-Schmidt condensation of 5,6-methylenedioxy-1-indanone with p-substituted benzaldehyde. Compounds 1–10 were assessed for their cytotoxic effects on human lung adenocarcinoma (A549) and human pancreatic ductal carcinoma (PANC-1) cells as well as human normal lung fibroblast (CCD-19Lu) and human normal pancreatic ductal epithelial (hTERT-HPNE) cells. Among them, compounds 2, 4 and 10 were more effective on A549 and PANC-1 cells than cisplatin. Compounds 1 and 9 also showed more potent cytotoxic activity towards PANC-1 cells than cisplatin. In vitro assays were performed to assess their effects on DNA synthesis, apoptosis, caspase-3, mitochondrial membrane potential, intracellular calcium levels, morphological changes in cancer cells. Furthermore, all compounds were investigated for their inhibitory effects on cathepsin L (CatL) and cathepsin D (CatD). Compounds 2 and 4 exerted potent anti-NSCLC action through caspase-independent apoptosis induced by an increase in intracellular calcium level and correspondingly the disruption of the ΔΨm. These compounds also caused apoptotic morphological alterations in A549 cells. Compound 4 also inhibited both cathepsins but its inhibitory potency on CatL was more significant. Based on in vitro mechanistic assays, compound 4 was identified as a promising anticancer agent for targeted therapy of NSCLC. On the other hand, the marked anti- PCa activity of compound 1 mediated by apoptotic cell death is also noteworthy, but further enzymatic assays are required to elucidate its main mechanism of action.

MiR-24-3p Regulates Epithelial–Mesenchymal Transition and the Malignant Phenotype of Pancreatic Adenocarcinoma by Regulating ASF1B Expression

Anti-silencing function protein 1 homolog B (ASF1B) has been implicated in the occurrence and development of cancers. The present work explored the functional role and the expression regulation of ASF1B in pancreatic ductal adenocarcinoma (PDAC). Based on the real-time quantitative PCR (qRT-PCR) and immunohistochemistry (IHC), ASF1B was significantly upregulated in PDAC tissues. High expression of ASF1B was associated with a poor overall survival (OS) and recurrence-free survival (DFS) in the PDAC patients. ASF1B also showed a relatively higher expression in PDAC cells (AsPC-1, PANC-1) when compared with human pancreatic ductal epithelial cells (HPDFe-6). CCK8 and clone formation assay demonstrated that silencing ASF1B impaired the proliferation in PANC-1 and AsPC-1 cells, and Annexin V-PI staining showed an increased level of apoptosis upon ASF1B silencing. ASF1B silencing also suppressed the migration and invasion in PDAC cells, as revealed by Transwell assays. We further showed that miR-24-3p was downregulated in PDAC tissues and cells, which functionally interacted with ASF1B by dual-luciferase reporter assay. miR-24-3p negatively regulated ASF1B expression to modulate the malignant phenotype of PDAC cells. ASF1B shows high expression in PDAC, which promotes the malignancy and EMT process of PDAC cells. miR-24-3p is a negative regulator of ASF1B and is downregulated in PDAC cells. Our data suggest that targeting ASF1B/miR-24-3p axis may serve as an intervention strategy for the management of PDAC.

Emodin Alleviates Sodium Taurocholate-Induced Pancreatic Ductal Cell Damage by Inhibiting the S100A9/VNN1 Signaling Pathway.

Because the pathogenesis of the disease is unclear, the treatment of patients with acute pancreatitis, especially severe acute pancreatitis, is still a major challenge for clinicians. Emodin is an anthraquinone compound extracted from rhubarb that can alleviate the damage to pancreatic ductal epithelial cells induced by adenosine triphosphate, but whether it has a similar protective effect on sodium taurocholate (STC)-stimulated pancreatic ductal cells and the underlying mechanism has not yet been reported. A model of STC-induced HPDE6-C7 human pancreatic ductal epithelial cell injury was established, and then apoptosis and the levels of reactive oxygen species (ROS), glutathione, gamma-glutamylcysteine synthetase, and inflammatory cytokines were assessed in the presence or absence of emodin pretreatment. S100 calcium binding protein A9 (S100A9) and Vanin1 (VNN1) protein expression was also measured. Emodin significantly increased HPDE6-C7 cell viability, inhibited apoptosis and ROS release, and elevated glutathione levels and gamma-glutamylcysteine synthetase activity. Furthermore, emodin downregulated S100A9 and VNN1 protein expression and inhibited the production of inflammatory factors, such as interleukin (IL)-1β, IL-6, IL-8, and IL-18. Emodin attenuates STC-induced pancreatic ductal cell injury possibly by inhibiting S100A9/VNN1-mediated ROS release. This finding provides evidence for the future development of emodin as a therapeutic agent.

Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

Most cancer cells rely on aerobic glycolysis to support uncontrolled proliferation and evade apoptosis. However, pancreatic cancer cells switch to glutamine metabolism to survive under hypoxic conditions. Activation of the Wnt/β-catenin pathway induces aerobic glycolysis by activating enzymes required for glucose metabolism and regulating the expression of glutamate transporter and glutamine synthetase. The results demonstrate that riluzole inhibits pancreatic cancer cell growth and has no effect on human pancreatic normal ductal epithelial cells. RNA-seq experiments identified the involvement of Wnt and metabolic pathways by riluzole. Inhibition of Wnt-β-catenin/TCF-LEF pathway by riluzole suppresses the expression of PDK, MCT1, cMyc, AXIN, and CyclinD1. Riluzole inhibits glucose transporter 2 expression, glucose uptake, lactate dehydrogenase A expression, and NAD + level. Furthermore, riluzole inhibits glutamate release and glutathione levels, and elevates reactive oxygen species. Riluzole disrupts mitochondrial homeostasis by inhibiting Bcl-2 and upregulating Bax expression, resulting in a drop of mitochondrial membrane potential. Finally, riluzole inhibits pancreatic cancer growth in KPC (Pdx1-Cre, LSL-Trp53R172H, and LSL-KrasG12D) mice. In conclusion, riluzole can inhibit pancreatic cancer growth by regulating glucose and glutamine metabolisms and can be used to treat pancreatic cancer.

Abstract 384: Lipid nanoparticle aided delivery of a STING agonist to treat pancreatic cancer

Abstract The tumor microenvironment (TME) of pancreatic cancer is highly immunosuppressive and characterized by a huge population of cancer-associated fibroblasts, myeloid-derived suppressor cells, regulatory T cells, and a scarce number of effector immune cells. Stimulator of interferon genes (STING) is an endoplasmic reticulum receptor that plays a critical role in immunity. Agonists of STING have been shown to inflame the TME, reduce tumor burden and confer anti-tumor activity in mouse models. 2’, 3’ cyclic guanosine monophosphate adenosine monophosphate (cGAMP) is a high-affinity endogenous ligand of STING. However, the cytosolic delivery of exogenous cGAMP to antigen-presenting cells and tumor cells remains a challenge due to poor cellular permeability, poor stability, and rapid clearance. We address these limitations by encapsulating cGAMP in a lipid nanoparticle (cGAMP-LNP) engineered for efficient cytosolic delivery. The size and morphology of the cGAMP-LNP were characterized using dynamic light scattering and TEM, respectively. The in-vitro activity of the cGAMP-LNP was determined in the THP-1 Blue࣪ ISG reporter cell line, whereas the endosomolytic activity of the LNP was evaluated by red blood cell hemolysis assay. The cGAMP-LNP activates the IRF3 pathway in-vitro and shows concentration- and pH-dependent endosomolytic activity. Moreover, the cGAMP-LNP was evaluated for cytotoxicity in-vitro and were found non-toxic to human pancreatic duct epithelial cell line at bioactive concentrations. Collectively, the cGAMP-LNP represents a promising strategy to convert immunosuppressive pancreatic cancer to immune-inflamed pancreatic cancer. Citation Format: Sherin George Shaji, Pratikkumar Patel, Kun Cheng. Lipid nanoparticle aided delivery of a STING agonist to treat pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 384.

Proteome and secretome analysis of pancreatic cancer cells.

Pancreatic cancer is a lethal malignancy and no screening biomarker or targeted therapy is currently available. Here, we performed a shotgun proteomic label-free quantification (LFQ) to define protein changes in the cellular proteome and secretome of four pancreatic cancer cell lines (PANC1, Paca44, Paca2, and BXPC3) versus normal human pancreatic ductal epithelial cells (HPDE). In the cellular proteome and secretome, 149 and 43 proteins were dysregulated in the most cancer cell lines, respectively. Using Ingenuity Pathway Analysis (IPA), the most dysregulated signaling pathways in pancreatic cancer cells included the activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular regulated kinase (ERK), and the deactivation of type-I interferon (IFN) pathways, which could promote cancer cell progression and decrease antitumor immunity. Parallel reaction monitoring (PRM) mass spectrometry was used to confirm the changes of seven regulated proteins quantified by LFQ: EGFR, growth/differentiation factor 15 (GDF15), protein-glutamine gamma-glutamyltransferase 2 (TGM2), leukemia inhibitory factor (LIF), interferon-induced GTP-binding protein Mx1 (MX1), signal transducer and activator of transcription 1 (STAT1), and serpin B5 (SERPINB5). Together, this proteomic analysis highlights protein changes associated with pancreatic cancer cells that should be further investigated as potential biomarkers or therapeutic targets.