Pancreatic Ductal Epithelial Cells Research Articles

Expression of ZKSCAN3 protein suppresses proliferation, migration, and invasion of pancreatic cancer through autophagy.

Elevated autophagy activity enhances the malignancy of pancreatic cancer (PaCa), and autophagy is recognized as a novel therapeutic target. Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) is a transcription factor that suppresses autophagy, but its association with PaCa is unknown. We analyzed the function of ZKSCAN3 in PaCa and investigated whether autophagy regulation through ZKSCAN3 could become a new therapeutic target for PaCa. Using reverse transcription-quantitative polymerase chain reaction and western blotting, we observed that ZKSCAN3 expression was upregulated in several PaCa cell lines compared with normal pancreatic ductal epithelial cells. Additionally, comparing ZKSCAN3 expression with the prognosis of PaCa patients using web databases, we found that higher ZKSCAN3 expression in PaCa was associated with extended overall survival. Knocking down ZKSCAN3 promoted the proliferation of PaCa cells. Moreover, following ZKSCAN3 knockdown, PaCa cells exhibited significantly enhanced migratory and invasive properties. Conversely, overexpression of ZKSCAN3 significantly suppressed the proliferation, migration and invasion of PaCa cells. Additionally, the knockdown of ZKSCAN3 increased the expression of LC3-II, a marker of autophagy, whereas ZKSCAN3 overexpression decreased LC3-II expression. In a xenograft mouse model, tumors formed by MIA PaCa-2 cells in which ZKSCAN3 was knocked down significantly increased in size compared with the control group. In conclusion, ZKSCAN3 expression was upregulated in several pancreatic cancer cells. Additionally, it was revealed that ZKSCAN3 is negatively correlated with the malignancy of PaCa through autophagy. These results suggest that autophagy regulation via ZKSCAN3 may be a new therapeutic target for PaCa.

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.

Abstract 1900: TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer

Abstract Cadherin 17 (CDH17) is a cell-to-cell adhesion protein that is a member of the cadherin superfamily. In normal tissues, expression of this single pass transmembrane protein is restricted to the lateral surfaces of intestinal and pancreatic ductal epithelial cells. However, in cancer, CDH17 is frequently overexpressed in tumors of the colon, stomach, and pancreas. The selective expression of this cell surface protein in these hard-to-treat cancer makes it an attractive target for the development of antibody-based therapeutics. Here, we describe the preclinical development of TORL-3-600, a novel CDH17-targeting antibody drug conjugate (ADC). CDH17 specific monoclonal antibodies were generated using traditional hybridoma technology and splenocytes isolated from mice immunized with cocktails of NIH3T3 cells overexpressing full length hCHD17 plus purified mammalian expressed hCDH17 extracellular domain protein (aa 23-787). Selective mAb binding was confirmed by flow cytometry and mAb internalization rate was assessed by immunofluorescence (IF). TORL-3-600 was generated from a fully humanized CDH17 mAb by MMAE conjugation with a cleavable linker. Cell membrane staining of CDH17 in patient tumor microarrays (TMAs), cell line (CDX) and patient derived xenografts (PDX) was evaluated by IHC assay. Selective binding of the TORL-3-600 to CDH17 was confirmed in human cancer cell lines and cells engineered to overexpress CDH17. Binding of TORL-3-600 to cell surface CDH17 induced Internalization and translocation to the lysosome of the protein-ADC complex for release of the MMAE payload. Treatment with TORL-3-600 induced significant regressions and tumor growth inhibition (TGI) in four CDH17-positive (CDH17+) human colorectal cancer (CRC) CDXs (103.6 - 140.4% TGI) and three CDH17+ CRC PDXs (63.3 - 102.2% TGI). Responses in these models were sustained for up to nine-weeks following cessation of treatment. Sustained inhibition of xenograft tumor progression was also observed in a cell line model of CDH17+ pancreatic cancer (85.8% TGI). In contrast, greatly reduced responses (37.3 - 58.2% TGI) to TORL-3-600 were observed in the CDH17- human colon cancer CDX and PDX models. Each of the doses tested in this study were well tolerated in mice with no dose-limiting toxicities observed. Analyses of large human patient TMAs by CDH17 IHC assay demonstrated detectable expression of CDH17 in 90.1% (173/193) of CRC, 51.8% (86/166) of gastric and 20.4% (69/331) of pancreatic cancers. These numbers suggest that an ADC directed against CDH17 could provide benefit to a significant number of patients diagnosed with these cancers. These data support the clinical development of TORL-3-600 for the treatment of CDH17+ cancers. TORL-3-600 has completed IND enabling toxicity studies with acceptable PK and toxicity profiles and is now in phase 1 clinical testing (NCT05948826). Citation Format: Neil A. O'Brien, Martina SJ McDermott, Jun Zhang, Ming Lu, Ke Wei Gong, Benjamin Hoffstrom, Wei Ping Jia, Tong Luo, Athena M. Madrid, Min Liang, John A. Glaspy, Dennis J. Slamon. TORL-3-600, a novel antibody drug conjugate directed against cadherin 17 (CDH17), has preclinical efficacy in colorectal, gastric, and pancreatic cancer [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 1900.

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.

EGR1 mediates MDR1 transcriptional activity regulating gemcitabine resistance in pancreatic cancer

BackgroundGemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells.MethodsThe following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry.FindingsWe revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group.ConclusionsOur study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.

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.

Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Mitochondrial calcium uniporter promotes mitophagy by regulating the PINK1/Parkin pathway in caerulein‑treated pancreatic ductal epithelial cells invitro.

The mitochondrial calcium uniporter (MCU) is a major protein for the uptake of mitochondrial calcium to regulate intracellular energy metabolism, including processes such as mitophagy. The present study investigated the effect of the MCU on mitophagy in pancreatic ductal epithelial cells (PDECs) in acute pancreatitis (AP) in vitro. The normal human PDECs (HPDE6-C7) were treated with caerulein (CAE) to induce AP-like changes, with or without ruthenium red to inhibit the MCU. The mitochondrial membrane potentials (MMPs) and mitochondrial Ca2+ levels were analyzed by fluorescence. The expression levels of MCU, LC3, p62, and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20), putative kinase 1 (PINK1), and Parkin were measured by western blotting and immunofluorescence. Mitophagy was observed by confocal fluorescence microscopy and transmission electron microscopy. The results showed that CAE increased the MCU protein expression, mitochondrial Ca2+ levels, MMP depolarization and the protein expression of mitophagy markers including the LC3II/I ratio, PINK1, and Parkin. CAE decreased the protein expression of p62 and TOMM20, and promoted the formation of mitophagosomes in HPDE6-C7 cells. Notably, changes in these markers were reversed by inhibiting the MCU. In conclusion, an activated MCU may promote mitophagy by regulating the PINK1/Parkin pathway in PDECs in AP.

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.

Circular RNA hsa_circ_0001846 facilitates the malignant behaviors of pancreatic cancer by sponging miR-204-3p and upregulating KRAS expression

Pancreatic cancer (PC) is mainly derived from the exocrine pancreatic ductal epithelial cells, and it is strongly aggressive malignant tumor. Due to its insidious onset and the lack of effective diagnostic biomarkers, PC currently remains one of the main causes of cancer-related mortality worldwide. Recent studies have found that hsa_circ_0001846 is involved in the progression of multiple cancers and has the potential to become biomarkers, but its function and mechanism in PC remains unclear. We found by qRT-PCR experiments that hsa_circ_0001846 was upregulated in PC cells and tissues, while circBase, Sanger sequencing, agarose gel electrophoresis and FISH experiments identified the splicing site, ring structure and cellular localization of hsa_circ_0001846. Various functional experiments by using the construction of small interfering RNA targeting hsa_circ_0001846 and overexpression plasmid demonstrated that hsa_circ_0001846 promoted the proliferation, migration and invasion of PC cells. Moreover, the tumor weight and volume of nude mice were significantly reduced after the stable knockdown of hsa_circ_0001846. In the mechanism exploration, RNA pull-down experiments and dual-luciferase experiments helped us to determine that hsa_circ_0001846 regulated the KRAS expression by sponging miR-204-3p in PC, thus playing a pro-cancer role. In this study, the effect of miR-204-3p on PC was also explored for the first time, and we found that knockdown of miR-204-3p reversed the tumor suppressive effect caused by silencing hsa_circ_0001846, and silencing KRAS also rescued the pro-cancer effect caused by overexpression of hsa_circ_0001846. In conclusion, our study revealed the pro-cancer role of hsa_circ_0001846 in PC, and for the first time identified the mechanism that hsa_circ_0001846 regulated KRAS by sponging miR-204-3p to promote PC progression and had the potential to become a cancer biomarker.

Transcriptome of pancreatic ductal epithelial cell line suggests the role of unexplored calcium activated chloride channel BEST 4 in chronic pancreatitis

Transcriptome of pancreatic ductal epithelial cell line suggests the role of unexplored calcium activated chloride channel BEST 4 in chronic pancreatitis

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.

Hydrogen Peroxide Mediates Pharmacological Ascorbate Induced Radio-Sensitization of Pancreatic Cancer Cells by Enhancing G2-accumulation and Reducing Cyclin B1 Protein Levels.

Pharmacological ascorbate (P-AscH-, high dose, intravenous vitamin C) preferentially sensitizes human pancreas ductal adenocarcinoma (PDAC) cells to radiation-induced toxicity compared to non-tumorigenic epithelial cells. Radiation-induced G2-checkpoint activation contributes to the resistance of cancer cells to DNA damage induced toxicity. We hypothesized that P-AscH- induced radio-sensitization of PDAC cells is mediated by perturbations in the radiation induced activation of the G2-checkpoint pathway. Both non-tumorigenic pancreatic ductal epithelial and PDAC cells display decreased clonogenic survival and increased doubling times after radiation treatment. In contrast, the addition of P-AscH- to radiation increases clonogenic survival and decreases the doubling time of non-tumorigenic epithelial cells but decreasing clonogenic survival and increasing the doubling time of PDAC cells. Results from the mitotic index and propidium iodide assays showed that while the P-AscH- treatments did not affect radiation-induced G2-checkpoint activation, it enhanced G2-accumulation. The addition of catalase reverses the increases in G2-accumulation, indicating a peroxide-mediated mechanism. In addition, P-AscH- treatment of PDAC cells suppresses radiation-induced accumulation of cyclin B1 protein levels. Both translational and post-translational pathways appear to regulate cyclin B1 protein levels after the combination treatment of PDAC cells with P-AscH- and radiation. The protein changes seen are reversed by the addition of catalase suggesting that hydrogen peroxide mediates P-AscH- induced radiation sensitization of PDAC cells by enhancing G2-accumulation and reducing cyclin B1 protein levels.

Engineered Extracellular Vesicles from Human Skin Cells Induce Pro‐β‐Cell Conversions in Pancreatic Ductal Cells

Direct nuclear reprogramming has the potential to enable the development of β cell replacement therapies for diabetes that do not require the use of progenitor/stem cell populations. However, despite their promise, current approaches to β cell‐directed reprogramming rely heavily on the use of viral vectors. Herein, the use of extracellular vesicles (EVs) derived from human dermal fibroblasts (HDFs) is explored as novel nonviral carriers of endocrine cell‐patterning transcription factors, to transfect and transdifferentiate pancreatic ductal epithelial cells (PDCs) into hormone‐expressing cells. Electrotransfection of HDFs with expression plasmids for Pdx1, Ngn3, and MafA (PNM) leads to the release of EVs loaded with PNM at the gene, mRNA, and protein levels. Exposing PDC cultures to PNM‐loaded EVs leads to successful transfection and increases PNM expression in PDCs, which ultimately result in endocrine cell‐directed conversions based on the expression of insulin/c‐peptide, glucagon, and glucose transporter 2 (Glut2). These findings are further corroborated in vivo in a mouse model following intraductal injection of PNM‐ versus sham‐loaded EVs. Collectively, these findings suggest that dermal fibroblast‐derived EVs can potentially serve as a powerful platform technology for the development and deployment of nonviral reprogramming‐based cell therapies for insulin‐dependent diabetes.

ECM Composition Differentially Regulates Intracellular and Extracellular pH in Normal and Cancer Pancreatic Duct Epithelial Cells

Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid–base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na+/H+ exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid–base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO3, especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO3. We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO3 gradients similar to that expected in the tumor.

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.

Pharmacological inhibition of human EZH2 can influence a regenerative β-like cell capacity with in vitro insulin release in pancreatic ductal cells

BackgroundTherapeutic replacement of pancreatic endocrine β-cells is key to improving hyperglycaemia caused by insulin-dependent diabetes . Whilst the pool of ductal progenitors, which give rise to the endocrine cells, are active during development, neogenesis of islets is repressed in the human adult. Recent human donor studies have demonstrated the role of EZH2 inhibition in surgically isolated exocrine cells showing reactivation of insulin expression and the influence on the H3K27me3 barrier to β-cell regeneration. However, those studies fall short on defining the cell type active in transcriptional reactivation events. This study examines the role of the regenerative capacity of human pancreatic ductal cells when stimulated with pharmacological inhibitors of the EZH2 methyltransferase.ResultsHuman pancreatic ductal epithelial cells were stimulated with the EZH2 inhibitors GSK-126, EPZ6438, and triptolide using a 2- and 7-day protocol to determine their influence on the expression of core endocrine development marker NGN3, as well as β-cell markers insulin, MAFA, and PDX1. Chromatin immunoprecipitation studies show a close correspondence of pharmacological EZH2 inhibition with reduced H3K27me3 content of the core genes, NGN3, MAFA and PDX1. Consistent with the reduction of H3K27me3 by pharmacological inhibition of EZH2, we observe measurable immunofluorescence staining of insulin protein and glucose-sensitive insulin response.ConclusionThe results of this study serve as a proof of concept for a probable source of β-cell induction from pancreatic ductal cells that are capable of influencing insulin expression. Whilst pharmacological inhibition of EZH2 can stimulate secretion of detectable insulin from ductal progenitor cells, further studies are required to address mechanism and the identity of ductal progenitor cell targets to improve likely methods designed to reduce the burden of insulin-dependent diabetes.