Normal Pancreatic Duct Epithelial Cells Research Articles

Cultures derived from pancreatic cancer xenografts with long-term gemcitabine treatment produce chemoresistant secondary xenografts: Establishment of isogenic gemcitabine-sensitive and -resistant models

In attempts to establish sophisticated models to reproduce the process of acquired drug resistance, we transformed normal human pancreatic ductal epithelial cells by introducing genes for multiple cellular factors. We also created isogenic gemcitabine-sensitive and -resistant models by short- and long-term gemcitabine treatment, respectively. These models demonstrated differences in drug resistance in vivo, but not in vitro. Gemcitabine treatment also induced squamous transdifferentiation in xenografts in mice. The transcription factor p63 was identified as a possible resistance-determining factor but was unlikely to be solely responsible for the resistance to gemcitabine. This system would prove useful to discover novel molecular targets to overcome chemotherapy resistance, by allowing the evaluation of molecules of interest in xenograft models after in vitro genetic ablation.

Inhibitory Effects of Propolis Flavonoids on Migration and Invasion of Laryngeal Cancer Cell and Analysis of Related Signal Pathways

Laryngeal cancer (LGC) is a malignant tumor that occurs in the larynx, and it is mainly treated through chemotherapy, radiotherapy, and surgery. Nevertheless, the five-year survival rate for patients is poor. Bee propolis contains various bioactive compounds and abundant anti-tumor active ingredients. Nevertheless, research on the use of propolis extracts for the treatment of LGC is relatively limited. This research aimed to demonstrate the inhibitory effects of ethanol extracts of propolis on migration (Mig) and invasion (Inv ) of LGC cells, as well as the related signaling pathways. The effects of graded ethanol extraction of propolis on the proliferation (Pro), Inv, Mig, apoptosis (Apo), and related signaling pathways of Hep-2 cells were analyzed. Propolis was extracted using ethanol (0%, 25%, 50%, 75%, and 100%) for the graded extraction of crude propolis. The flavonoid content and yield of the extracts were determined. The effects of various concentrations of propolis flavonoids on the clearance of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, O2- radicals, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals were evaluated, as well as their effects on the Pro inhibition of normal human pancreatic ductal epithelial (hTERT-HPNE) cells. Hep-2 cells of LGC were cultured using media containing 0, 25, 50, and 100 μmol/L propolis flavonoids. The cell Pro activity, Inv, Mig, Apo, and expression of PI3K/Akt pathway-related proteins were evaluated using CCK-8 assay, Transwell chamber assay, acridine orange/ethidium bromide (AO/EB) double staining method, and Western blotting, respectively. It was revealed that extraction with 50% ethanol solution yielded a higher content and yield of flavonoids, which were 51.20% and 7.42%, respectively. As the concentration of propolis flavonoids increased, the clearance rates of DPPH, O2-, and ABTS radicals, as well as the inhibition of hTERT-HPNE Pro, gradually increased. The maximum clearance rates were 84.1%, 26.6%, and 92.3%, respectively, while the maximum cell Pro inhibition rate was only 8.6%. Relative to the 0 μmol/L propolis flavonoid treatment group, the Hep-2 cells treated with 25, 50, and 100 μmol/L propolis flavonoids exhibited decreased cell Pro activity, reduced number of invasive and migratory cells, increased Apo rate, decreased PI3K and p-Akt proteins, and demonstrated a concentration-dependent effect (P < 0.05). In summary, the extraction with 50% ethanol solution resulted in a higher yield of flavonoids. Propolis flavonoids demonstrated marked antioxidant activity and did not cause damage to normal hTERT-HPNE cells. They exhibited inhibitory effects on the Pro, Inv, and Mig of Hep-2 cells in LGC, and promoted cell Apo. These effects may be associated with PI3K/Akt signaling inhibition.

Transcription factor DDIT3 is a potential driver in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and aggressive tumor that affects the digestive tract, leading to high mortality and poor survival rates. The purpose of the present study was to evaluate the expression levels of DNA damage-inducible transcript 3 (DDIT3) in pancreatic cancer and to investigate its effects in in vitro and in vivo experiments. Bioinformatics analysis indicated that DDIT3 expression was higher in pancreatic cancer tumor tissues and associated with a poor prognosis. Positive or strong positive DDIT3 expression was observed in PDAC, and no or weak expression was observed in normal pancreatic tissues. It was also highly expressed in PDAC cells, while being expressed at lower levels in normal pancreatic ductal epithelial cells. Transfection of short hairpin RNA targeting the DDIT3 gene reduced the proliferation, migration and invasion of PANC-1 cells. In vivo, in an in situ implantation tumor model with Pan02 cells, the size and weight of the tumors were reduced in the DDIT3 knockdown Pan02 cell-implanted group. These data suggested that DDIT3 represents a novel predictive biomarker for the potential treatment of patients presenting with PDAC.

Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways.

Pancreatic cancer (PC) is associated with some of the worst prognoses of all major cancers. Thymoquinone (TQ) has a long history in traditional medical practice and is known for its anti-cancer, anti-inflammatory, anti-fibrosis and antioxidant pharmacological activities. Recent studies on hypoxia-inducible factor-1α (HIF-1α) and PC have shown that HIF-1α affects the occurrence and development of PC in many aspects. In addition, TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α. Therefore, we speculate whether TQ affects HIF-1α expression in PC cells and explore the mechanism. To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression. Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation. TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation. The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway.

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.

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.

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.

Uncovering the role of FXYD3 as a potential oncogene and early biomarker in pancreatic cancer.

Pancreatic cancer is an aggressive cancer with silent symptoms and high mortality with less than 11% of the 5-year survival rate. Until now, the significance of genes as clinical biomarkers in the early stages of pancreatic cancer has not been fully understood. Hence, this study aims to reveal the significant genes in the early stages of pancreatic cancer using bioinformatic analysis and in vitro experiments, and to serve as clinical biomarkers for early detection. We used Cancer RNA-Seq Nexus database and identified one tumor suppressor gene (NAGK), and five oncogenes (FXYD3, ACTR1A, B3GNT3, SIGIRR, and EXOC1) that are significant in the early stages of pancreatic cancer. The expression of NAGK, FXYD3, ACTR1A, B3GNT3, SIGIRR, and EXOC1 were determined from the GEPIA, UALCAN, and HPA database. It has been shown that pancreatic cancer tumor dissemination is an event that can occur in early lesions, rather than being solely restricted in the developed primary tumor. Thus, the six hub genes that were differentially expressed between stage I and stage II of primary pancreatic cancer tumors were compared to metastasis-related genes (1938 genes) in the human cancer metastasis database (HCMDB), yielding two overlapped genes (B3GNT3 and FXYD3). To establish the expression correlation between these two specific genes with metastatic characteristics of the early stage of pancreatic cancer and migratory ability in pancreatic cancer cell lines, the expression patterns of B3GNT3 and FXYD3 were examined in four different migratory abilities of pancreatic cancer cell lines, including HPAC, BxPC-3, AsPC-1, and PANC-1, as well as the normal pancreatic duct epithelial cell line HPDE6-C7. The results displayed that the expression of the FXYD3 gene was dramatically increased with the migratory ability enhanced of four pancreatic cancer cell lines. Thus, in the follow-up study, we will demonstrate the functional role of FXYD3 in pancreatic cancer tumorigenesis. This study revealed that the FXYD3 may act as a significant oncogene in the early stage of pancreatic cancer.

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.

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.

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.

DNA methylation and miR-92a-3p-mediated repression of HIP1R promotes pancreatic cancer progression by activating the PI3K/AKT pathway.

Pancreatic cancer (PAAD) is a highly malignant tumour characterized of high mortality and poor prognosis. Huntingtin-interacting protein 1-related (HIP1R) has been recognized as a tumour suppressor in gastric cancer, while its biological function in PAAD remains to be elucidated. In this study, we reported the downregulation of HIP1R in PAAD tissues and cell lines, and the overexpression of HIP1R suppressed the proliferation, migration and invasion of PAAD cells, while silencing HIP1R showed the opposite effects. DNA methylation analysis revealed that the promoter region of HIP1R was heavily methylated in PAAD cell lines when compared to the normal pancreatic duct epithelial cells. A DNA methylation inhibitor 5-AZA increased the expression of HIP1R in PAAD cells. 5-AZA treatment also inhibited the proliferation, migration and invasion, and induced apoptosis in PAAD cell lines, which could be attenuated by HIP1R silencing. We further demonstrated that HIP1R was negatively regulated by miR-92a-3p, which modulates the malignant phenotype of PAAD cells in vitro and the tumorigenesis in vivo. The miR-92a-3p/HIP1R axis could regulate PI3K/AKT pathway in PAAD cells. Taken together, our data suggest that targeting DNA methylation and miR-92a-3p-mediated repression of HIP1R could serve as novel therapeutic strategies for PAAD treatment.

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 B035: Targeting ribosome biogenesis addition is a novel strategy for pancreatic cancer therapy

Abstract Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the United States with limited therapeutic options available. Gemcitabine (GEM), a deoxycytidine nucleoside analog is currently considered the most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. PanCa cells are addicted to ribosome biogenesis (RiBi) which supports their high rate of growth and proliferation. Therefore, strategically targeting the process of RiBi could be one of the ideal strategies for the prevention and treatment of PanCa. In this study, we for the first-time report that RPA194, a catalytic subunit of RNA Pol I is differentially expressed in normal pancreatic ductal epithelial (NPDE) and cancer cells. We also observed differential expression of RPA194 in various grades of pancreatic tumor tissues and its level was increased in high-grade pancreatic tumor tissues. We tested our hypothesis that targeting RPA194 with a non-toxic pharmacological inhibitor (BMH-21) will inhibit the growth of advanced PanCa. We observed that BMH-21 inhibits the growth and induced apoptosis of PanCa cells in a dose-dependent manner and this inhibition was correlated with the expression of RPA194. We observed proteasomal mediated degradation of RPA194 in PanCa cells. BMH-21 treatment inhibited RPA194 occupancy on rDNA but did not affect other nucleolar proteins (UBF, fibrillarin, nucleolin, and nucleophosmin). Two times the effective concentration of BMH-21 (5 µM) did not show any cytotoxic effect in NPDE cells. However, BMH-21 degraded RPA 194 protein in these cells in a similar manner as in PanCa cells. Therefore, we speculate that other proteins are involved in the selective toxicity of BMH-21 in PanCa cells. These findings were translated to in vivo model systems. We observed that BMH-21 significantly (P<0.01) inhibited the growth of pancreatic tumors in an orthotopic xenograft model. This pancreatic tumor growth inhibition was correlated with degradation of RPA194 protein in BMH-21 treated mice tumor tissues followed by inhibition of cell proliferation markers (ki67 and PCNA). Taken together, our results strongly suggest that targeting the RiBi process is a novel approach and BMH-21 is a non-toxic potential targeting agent for the prevention and treatment of advanced PaCa. Citation Format: Mudassier Ahmad, Carlos Perez, Andrew Massey, Vivek Kashyap, Neeraj Chauhan, Haider Ahsan, Jasmine Jasmine, Manish Tripathi, Subhash Chauhan, Bilal Hafeez. Targeting ribosome biogenesis addition is a novel strategy for pancreatic cancer therapy [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 B035.

Abstract B046: Restricted cellular plasticity of human pancreatic ductal cells into a pluripotent state

Abstract Cancer cells undergo plasticity against stress for the survival of the fittest. Pancreatic ductal adenocarcinoma (PDAC) also shows intratumoral transcriptional heterogeneity with cell plasticity, which correlates with phenotypical aggressiveness and therapeutic resistance. Cell fates are determined by master transcription factors (TFs, also called “pioneer factors”) and their co-factors by opening up chromatin and establishing competence for gene expression specific to each cell type during normal development. Force expression of various combinations of pioneer factors can switch cell fate into other lineages, including induced pluripotent stem cells (“iPSC”). Since pluripotent stem cells do not have the cancer hallmarks of uncontrolled cell growth and migration, hypothetically, a fully reprogrammed cancer iPSC should regain gene programs to control these hallmarks of cancer. Thus, we and others have reprogrammed cancer cells into pluripotent stem cell-like cells by introducing pluripotent pioneer factors. However, intriguingly, despite the nature of the high plasticity of cancer, complete reprogramming of cancers to a pluripotent state is impeded in most cases, enabling us to study and model cancer progression. Our further studies confirm that, upon reprogramming, most PDAC cells lose the original PDAC phenotypes to some extent but remain in plasticity states during reprogramming. Herein, we further update on our efforts to optimize the reprogramming of normal pancreatic ductal epithelial cell line (PDEC) and PDAC derived from patients through the Sendai virus, which can most robustly transfer genes regardless of cell types. While control cells (e.g., acinar cells and fibroblast) were fully reprogrammed into iPSC cells, both PDEC and PDAC were impeded from being fully reprogrammed into pluripotency. Intriguingly, a subset of normal PDEC cells as well as some PDAC cell acquired a “partial reprogramming marker” upon reprogramming, suggesting that PDEC cells are stalled in early developmental states. Therefore, we now conclude that normal and cancerous pancreatic ductal cell states have an intrinsic mechanism that blocks complete reprogramming into a pluripotent state, and the oncogenic pathway is poised for activation, despite the nature of the high plasticity of PDAC. Identifying the factors responsible for maintaining this impenetrable cancer chromatin landscape is ideal for nominating therapeutic targets for intercepting PDAC plasticity. This work is supported by OHSU/CEDAR project award 68182-939-000. Citation Format: Dmytro Grygoryev, Taelor Ekstrom, Jason M. Link, Rosalie C. Sears, Jungsun Kim. Restricted cellular plasticity of human pancreatic ductal cells into a pluripotent state [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 B046.

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.

Pharmacologic Ascorbate as Radioprotector of Normal Tissue in Pancreatic Cancer Treatment

INTRODUCTION: Radiation (IR) is an integral part of pancreatic ductal adenocarcinoma (PDAC) treatment but is limited by toxicity to nearby organs. Pharmacologic ascorbate (P-AscH–, high-dose, intravenous vitamin C) synergizes with IR to increase toxicity in PDAC cells but may protect normal cells. We hypothesize that inhibition of oxidative stress in normal cells is involved in the radioprotective mechanism of P-AscH–. METHODS: Duodenal samples from pancreaticoduodenectomy specimens of patients who underwent IR and gemcitabine were compared with patients who received P-AscH– with gemcitabine and IR. Trichrome staining and 4HNE as a marker of protein oxidation were evaluated. PDAC cells (MIA PaCa-2) and normal pancreatic ductal epithelial cells (H6c7) were treated with IR at 2, 4, 6 Gy with and without P-AscH–, probed with BODIPY C-11 to determine lipid peroxidation. RESULTS: Average villi length was significantly greater in the duodenum from patients who received P-AscH- (5.3 ± 0.3 vs 6.3 ± 0.4, means ± SEM, p = 0.05). 4HNE immunofluorescence was also lower in the duodenum of patients that received P-AscH– treatment (23.9% vs 10.1%, p = 0.04). In vitro MIA PaCa-2 cells had increased BODIPY oxidation with P-AscH- at 2, 4, and 6 Gy (ANOVA p < 0.05), but H6c7 cells showed no meaningful changes between all groups. CONCLUSION: Consistent with previous in vivo studies, P-AscH– ameliorates IR-induced damage to normal cells. This mechanism of radioprotection of normal cells by P-AscH– may be due to inhibition of oxidative stress.