MIA PaCa-2 Research Articles

Inhibition of phospholipase D1 reduces pancreatic carcinogenesis in mice partly through a FAK-dependent mechanism.

Phospholipase D (PLD) plays a critical role in cancer progression. However, its role in pancreatic cancer remains unclear. Thus, we evaluated the role of PLD1, one of two classical isoforms of PLD, in pancreatic carcinogenesis in vivo. The role of PLD1 in tumor growth was evaluated by subcutaneously transplanting human MIA PaCa-2 cells expressing endogenous PLD1 levels (Ctr KD cells) or cells in which PLD1 was knocked down (Pld1 KD cells) into immunodeficient mice. Twenty days post-implantation, tumors that arose from Pld1-KD cells were significantly smaller, compared to controls (Ctr KD). Then, we assessed the role of PLD1 in the tumor microenvironment, by subcutaneously implanting mouse LSL-KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) cells into wild-type (WT) or PLD1 knockout (Pld1-/-) mice. Compared to WT, tumor growth was attenuated in Pld1-/- mice by 39%, whereas treatment of Pld1-/- mice with gemcitabine reduced tumor growth by 79%. When PLD1 was ablated in LSL-KrasG12D;Ptf1Cre/+ (KC) mice, no reduction in acinar cell loss was observed, compared to KC mice. Finally, treatment of KC mice with a small molecule inhibitor of PLD1 and PLD2 (FIPI) significantly reduced acinar cell loss and cell proliferation, compared to vehicle-treated mice. Mechanistically, the effect of PLD on tumor growth is mediated, partly, by the FAK pathway. In conclusion, while PLD1 is a critical regulator of pancreatic xenograft and allograft growth, playing an important role at the tumor and at the microenvironment levels, inhibition of PLD1 and PLD2 are necessary to reduce pancreatic carcinogenesis in KC mice, and might represent a novel therapeutic target.

Activation of autophagy, paraptosis, and ferroptosis by micheliolide through modulation of the MAPK signaling pathway in pancreatic and colon tumor cells

Micheliolide (MCL), a naturally occurring sesquiterpene lactone, has demonstrated significant anticancer properties through the induction of various programmed cell death mechanisms. This study aimed to explore MCL's effects on autophagy, paraptosis, and ferroptosis in pancreatic and colon cancer cells, along with its modulation of the MAPK signaling pathway. MCL was found to substantially suppress cell viability in these cancer cells, particularly in MIA PaCa-2 and HT-29 cell lines. The study identified that MCL induced autophagy by enhancing the levels of autophagy markers such as Atg7, p-Beclin-1, and Beclin-1, which was attenuated by the autophagy inhibitor 3-MA. Furthermore, MCL was found to facilitate paraptosis, indicated by decreased Alix and in-creased ATF4 and CHOP levels. It also promoted ferroptosis, as demonstrated by the reduced expression of SLC7A11, elevated TFRC levels, and increased intracellular iron. Additionally, MCL activated the MAPK signaling pathway, marked by the phosphorylation of JNK, p38, and ERK, linked with an increase in ROS production that is vital in regulating these cell death mechanisms. These findings propose that MCL is a versatile anticancer agent, capable of activating various cell death pathways by modulating MAPK signaling and ROS levels. These results emphasize the therapeutic promise of MCL in treating cancer, pointing to the necessity of further in vivo investigations to confirm these effects and determine its potential clinical uses.

Ferrocenyl β-Diketonate Compounds: Extended Ring Systems for Improved Anticancer Activity.

A library of ferrocenyl β-diketonate compounds with varying degrees of aromatic functionality have been synthesized and fully characterized. This includes cyclic voltammetry and the analysis of four new structures by single crystal X-ray diffraction. The compounds cytotoxic potential has been determined by MTT screening against pancreatic carcinoma (MIA PaCa-2), ovarian adenocarcinoma (A2780), breast adenocarcinomas (MDA-MB-231 and MCF-7) and normal epithelial retinal (ARPE-19). The compounds show a general trend, where increasing the number of aromatic rings in the molecule yields an increase in cytotoxicity and follows the trend anthracenyl > naphthyl > phenyl > methyl. The compounds are particularly sensitive to the triple negative cancer cell line MDA-MB-231, and the potential modes of action have been studied by production of reactive oxygen species using fluorescence microscopy and cell morphology using Scanning Electron Microscopy. All assays highlight the ferrocenyl β-diketonate with an anthracenyl substituent to be the lead compound in this library. The decomposition was also observed within cells, to give a cytotoxic fluorescent molecule, which has been visualized by confocal microscopy.

The function of p97/valosin-containing protein (VCP) and small VCP-interacting protein (SVIP) in invasion and migration of pancreatic cancer cells.

Misfolded proteins are eliminated by a process known as endoplasmic reticulum-associated protein degradation (ERAD). ERAD has an impact on a variety of illnesses, such as diabetes, cystic fibrosis, cancer, and neurological conditions. As one of the many proteins involved in ERAD, this study is focused on p97/valosin-containing protein (VCP) and small VCP-interacting protein (SVIP). The existence and function of SVIP and p97/VCP in various types of pancreatic cancer have not yet been investigated. The study's objectives are to examine the expressions of SVIP and p97/VCP in two pancreatic cancer types and to show whether these proteins aid in the invasion and migration of pancreatic cancer cells. In this work, MIA PaCa-2 and PANC-1 human cell lines were examined. Immunocytochemistry and immunofluorescence were performed to detect the cellular localization and presence of p97/VCP and SVIP in pancreatic cancer cells. Following p97/VCP siRNA and SVIP siRNA transfection of the cells, protein expressions were assessed using Western blot analysis. The effects of this suppression on cell invasion and migration were determined using the xCELLigence real-time analysis system (RTAC). In the nucleus and cytoplasm of MIA PaCa-2 and PANC-1 cells, p97/VCP and SVIP immunoexpressions were seen. The decrease in protein expressions of p97/VCPsi and SVIPsi was significant in pancreatic cells compared to the controlsi. The p97/VCP siRNA transfection reduced the invasion and migration of MIA PaCa-2 and PANC-1 cells. In addition, the SVIP siRNA suppression resulted in increasing the invasion and migration ability of both cells. This study also demonstrated, for the first time, SVIP expression in MIA PaCa-2 and PANC-1 cells. Overall, the findings show the differential expression and function of p97/VCP and SVIP in pancreas ductal adenocarcinoma cells. The potential of the pancreatic cancer cells to migrate and invade altered when the two cell lines were transfected with p97/VCPsi and SVIPsi.

Unraveling the hepatoprotective and anti-pancreatic cancer potential of Caralluma europaea: a comprehensive in vivo, in vitro and in silico evidence

Caralluma europaea Guss. (C. europaea) is a medicinal plant used for cancer treatment. However, these treatments may be associated with complications that need to be investigated. This work aims to evaluate not only the chemical composition but also the hepatoprotective and anticancer properties of C. europaea extracts. The chemical constitution of the hydroethanolic extract was explored using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The hydroethanolic extract, flavonoids, and polyphenols-rich extract at 100, 15, and 50 mg/kg, respectively, were administered to acetaminophen-treated rats for seven days. We used Western blotting and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) to determine the protein and the mRNA levels of cancer stemness markers in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 exposed to increasing doses of C. europaea extracts. In silico analysis was used to evaluate the effects of phenolic compounds revealed in C. europaea on caspase-3 and HSP90, and on liver damage on CYP2E1. The primary phenolics detected by GC-MS and HPLC were ferulic acid and benzofurazan. The positive control group showed an increase in AST, ALT, ALP, triglycerides, and VLDL levels. C. europaea extracts demonstrated hepatoprotective effects by ameliorating acetaminophen-induced alterations of biochemical and hispathological parameters. Immunoblotting and RT-qPCR profiling of cancer stemness markers indicated a reduction in the expression levels of Oct-4 and Nanog proteins, as well as a reduction in the mRNA levels of CD133 by 50–60% and Sox2 by 80–90% in pancreatic cancer cells. Molecular docking showed that naringenin presented the highest docking Gscore on CYP2E1 (−8.199) and HSP90 (−7.742). In conclusion, C. europaea extracts could be considered as a safe and promising therapeutic strategy to sensitize pancreatic cancer cells to chemotherapy.

Branched oncolytic peptides target HSPGs, inhibit metastasis, and trigger the release of molecular determinants of immunogenic cell death in pancreatic cancer.

Immunogenic cell death (ICD) can be exploited to treat non-immunoreactive tumors that do not respond to current standard and innovative therapies. Not all chemotherapeutics trigger ICD, among those that do exert this effect, there are anthracyclines, irinotecan, some platinum derivatives and oncolytic peptides. We studied two new branched oncolytic peptides, BOP7 and BOP9 that proved to elicit the release of damage-associated molecular patterns DAMPS, mediators of ICD, in pancreatic cancer cells. The two BOPs selectively bound and killed tumor cells, particularly PANC-1 and Mia PaCa-2, but not cells of non-tumor origin such as RAW 264.7, CHO-K1 and pgsA-745. The cancer selectivity of the two BOPs may be attributed to their repeated cationic sequences, which enable multivalent binding to heparan sulfate glycosaminoglycans (HSPGs), bearing multiple anionic sulfation patterns on cancer cells. This interaction of BOPs with HSPGs not only fosters an anti-metastatic effect in vitro, as demonstrated by reduced adhesion and migration of PANC-1 cancer cells, but also shows promising tumor-specific cytotoxicity and low hemolytic activity. Remarkably, the cytotoxicity induced by BOPs triggers the release of DAMPs, particularly HMGB1, IFN-β and ATP, by dying cells, persisting longer than the cytotoxicity of conventional chemotherapeutic agents such as irinotecan and daunorubicin. An in vivo assay in nude mice showed an encouraging 20% inhibition of tumor grafting and growth in a pancreatic cancer model by BOP9.

Design, synthesis and pharmacological evaluation of 1,2,3,4-tetrahydrobenzofuro[2,3-c]pyridine derivatives as p21-activated kinase 4 inhibitors for treatment of pancreatic cancer

The p21-activated kinase 4 (PAK4), a key regulator of malignancy, is negatively correlated with immune infiltration and has become an emergent drug target of cancer therapy. Given the lack of high efficacy PAK4 inhibitors, we herein reported the identification of a novel inhibitor 13 bearing a tetrahydrobenzofuro[2,3-c]pyridine tricyclic core and possessing high potency against MIA PaCa-2 and Pan02 cell lines with IC50 values of 0.38 and 0.50 μmol/L, respectively. This compound directly binds to PAK4 in a non-ATP competitive manner. In the mouse Pan02 model, compound 13 exhibited significant tumor growth inhibition at a dose of 100 mg/kg, accompanied by reduced levels of PAK4 and its phosphorylation together with immune infiltration in mice tumor tissue. Overall, compound 13 is a novel allosteric PAK4 inhibitor with a unique tricyclic structural feature and high potency both in vitro and in vivo, thus making it worthy of further exploration.

Development of Novel ROCK Inhibitors via 3D-QSAR and Molecular Docking Studies: A Framework for Multi-Target Drug Design.

Background/Objectives: Alterations in the actin cytoskeleton correlates to tumor progression and affect critical cellular processes such as adhesion, migration and invasion. Rho-associated coiled-coil-containing protein kinases (ROCK1 and ROCK2), important regulators of the actin cytoskeleton, are frequently overexpressed in various malignancies. The aim of this study was therefore to identify the key structural features of ROCK1/ROCK2 inhibitors using computer-aided drug design (CADD) approaches. In addition, new developed ROCK inhibitors provided a significant framework for the development of multitarget therapeutics-ROCK/HDAC (histone deacetylases) multitarget inhibitors. Methods: 3D-QSAR (Quantitative structure-activity relationship study) and molecular docking study were employed in order to identify key structural features that positively correlate with ROCK inhibition. MDA-MB-231, HCC1937, Panc-1 and Mia PaCa-2 cells were used for evaluation of anticancer properties of synthesized compounds. Results: C-19 showed potent anti-cancer properties, especially enhancement of apoptosis and cell cycle modulation in pancreatic cancer cell lines. In addition, C-19 and C-22 showed potent anti-migratory and anti-invasive effects comparable to the well-known ROCK inhibitor fasudil. Conclusions: In light of the results of this study, we propose a novel multi-target approach focusing on developing dual HDAC/ROCK inhibitors based on the structure of both C-19 and C-22, exploiting the synergistic potential of these two signaling pathways to improve therapeutic efficacy in metastatic tumors. Our results emphasize the potential of multi-target ROCK inhibitors as a basis for future cancer therapies.

Abstract C027: Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models

Abstract Background/Objective: Prognosis remains grim for localized or metastasized pancreatic ductal adenocarcinoma (PDAC) with 5-year survival rates of 3-16%. More than 90% of PDAC cases harbor KRAS mutations. KRASG12C inhibitors recently developed have limited use for PDAC because only 3% of cases express this mutation. We developed a novel pan-RAS inhibitor, ADT-1004 and evaluated antitumor activity in tumor models established from murine or human PDAC cell lines or patient-derived xenografts (PDX). ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Murine PDAC cells harboring the KRASG12D mutation (KPC-Luc or 2838C3-Luc) were orthotopically implanted into the pancreas of C57BL/6J mice. Four PDX PDAC tumors harboring KRAS mutations were implanted orthotopically in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental KRASG12C MIA PaCa-2 PDAC cells and a resistant derivative of MIA PaCa-2 cells were implanted subcutaneously. To assess selectivity, mice were implanted with RASWT BxPC-3 cells SC. Mice were treated orally with ADT-1004 (40mg/kg) 5x/week for 4 weeks. Sotorasib and adagrasib KRASG12C inhibitors were administered at the same dose, route, and schedule as ADT-1004. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines in vitro. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCa-2 PDAC cells was 2 nM compared to 2500 nM for RASWT BxPC-3 PDAC cells. Growth inhibition by ADT-007 was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at doses up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 exceeding growth IC50 values. ADT-1004 displayed robust antitumor activity in mouse tumor models using human- or mouse-derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing selective inhibitory effects on ERK phosphorylation in tumors but not in normal tissues. ADT-1004 also inhibited growth of KRASG12C mutant MIA PaCa-2 tumors to a comparable extent as sotorasib and adagrasib. Sotorasib and adagrasib were ineffective against xenografted tumors of resistant cells derived from MIA PaCa-2 cells, while ADT-1004 strongly inhibited tumor growth. Target selectivity of ADT-1004 was confirmed in vivo, as ADT-1004 did not affect the growth of RASWT BxPC-3 tumors. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDAC tumors by blocking activated RAS to disrupt downstream MAPK/AKT signaling. Side-by-side comparisons reveal antitumor efficacy advantages of ADT-1004 over mutant-specific KRAS inhibitors. These results highlight the promise of ADT-1004 for treatment of PDAC and other RAS-driven cancers by addressing the complex genetic landscape of many cancers and limitations from resistance to mutant specific KRAS inhibitions. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C027.

Abstract A011: Enhanced Radiosensitivity of Pancreatic Cancer Achieved through Inhibition of Cyclin-Dependent Kinase 1

Abstract Introduction: One of the major challenges in treating Pancreatic Ductal Adenocarcinoma (PDAC) is overcoming radioresistance. This study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through both genetic and pharmaceutical inhibition as a strategy to increase the radiosensitivity of PDAC cells. Methods: We conducted mass spectrometry and phosphoproteomics analyses to compare engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) with their parental controls. Additionally, we examined the TCGA PDAC database to correlate clinical outcomes, such as overall survival (OS), with CDK1 mRNA expression levels in PDAC patients. Furthermore, we developed a microRNA-based TET-on inducible shRNA to specifically inhibit CDK1 expression in PDAC cell lines. In an orthotopic PDAC model, we evaluated the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We also employed a selective CDK1 inhibitor, RO-3306, to target CDK1 activation, followed by in vitro experiments using immunoblotting, immunocytochemistry, and clonogenic assays. Results: Our phosphoproteomics analysis revealed a significant increase in phospho-CDK1 (Tyr15) levels in the radiation-resistant cell lines. High CDK1 expression was associated with poorer OS in PDAC patients. Additionally, we observed heightened CDK1 phosphorylation at the threonine tyrosine 14 (Tyr14) residue following radiation exposure. Through our in vivo and in vitro experiments, we demonstrated that CDK1 inhibition, in conjunction with radiation therapy, delayed tumor growth and enhanced radiosensitivity in PDAC cells. Treatment with RO-3306 caused a substantial shift of cells into the G2/M phase and disrupted DNA repair post-radiation exposure, further enhancing radiosensitivity. Conclusion: Our findings underscore the critical role of CDK1 in PDAC radioresistance. Targeting CDK1 in combination with radiotherapy shows promise for future exploration as a potential treatment strategy in PDAC. Citation Format: Stetson Van Matre, Saaimatul Huq, Lokesh Akana, Oscar Zuniga, Henrique Rodrigues, Adam Wolfe. Enhanced Radiosensitivity of Pancreatic Cancer Achieved through Inhibition of Cyclin-Dependent Kinase 1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A011.

Abstract C035: MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion

Abstract Introduction: Melanoma-associated antigens (MAGEs) are linked to aggressive disease progression and treatment resistance in many cancers, but their role in pancreatic cancer is not well understood. Our preliminary TCGA data analysis suggested that 10% of pancreatic tumors express MAGEA3/6 and potentially help pancreatic cancer cells survive and grow under nutrient stress. This study aims to uncover the molecular mechanisms of MAGEA3/6 function in pancreatic cancer cell metabolism and disease progression under nutrient-poor conditions. Methods: To understand the role of highly homologous genes MAGEA3 and MAGEA6 in pancreatic cancer, we expressed them in highly glycolytic MIA PaCa-2 pancreatic cancer cells. Cells were then exposed to cancer-related nutritional stressors, including glycolysis inhibition by 2-deoxy-D-glucose (2DG) treatment and glutamine depletion. We analyzed the effect of MAGEA6 expression on cell viability and cell metabolism by Alamar Blue viability assay (BioRad), Seahorse (Agilent), Phenotype MicroArray Mammalian analysis (Biolog), transmission electron microscopy (TEM), and RNA sequencing. To confirm our results, we performed loss-of- function studies in MAGEA3/6-positive cells, including PANC1. Results: We found that, compared to control GFP-expressing cells, MAGEA6-expressing MIA PaCa-2 cells were resistant to glycolysis inhibition with 2-DG. In contrast, they were more susceptible to glutamine depletion, suggesting the important role of MAGEA3/6 in metabolic phenotype and adaptations of pancreatic cancer cells. Further, we showed that MAGEA6-expressing cells exhibited increased ATP production and mitochondrial oxidative phosphorylation, especially under 2DG stress. After 48 hours of 2DG treatment, TEM images revealed significant differences in nuclear, mitochondrial, and ribosomal structures in MAGEA6-cells, further suggesting MAGEA6's role in metabolic adaptation. The Phenotype MicroArray assay demonstrated that α-D-Glucose is the primary nutrient source consumed by both GFP and MAGEA6-expressing cells among the 93 nutrients tested. Additionally, A6 cells utilize Pyruvic Acid as an alternative nutrient source more than GFP cells. To uncover molecular underpinning, we performed RNA sequencing and found that MAGEA6 expression affects transcriptome under glutamine depletion and 2DG treatment. Intriguingly, one of the major differentially regulated biological processes was associated with the upregulation of several signaling pathways, including MAPK and AMPK. Conclusion: Our findings suggest that in pancreatic cancer, MAGEA6 recruits stress and growth factor- dependent MAPK and AMPK pathways to overcome glycolysis inhibition, even in glutamine- depleted conditions, providing a growth advantage. This implies that patients with MAGEA6- positive tumors may benefit from combination therapy targeting metabolism and signaling pathways. Citation Format: Sima Tozandehjani, Barbara Breznik, Klementina Fon Tacer, Miloš Vittori, Juan Sebastian Solano, Sara Uhan. MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C035.

Abstract C055: Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element

Abstract Genome-wide association studies (GWAS) have identified independent signals at 5p15.33 across numerous cancers, with protective alleles for one cancer often conferring risk for another. Many of these associations are thought to act via allele-specific alterations in the cis- regulation of target genes. Transcriptomic analyses in multiple tissue types implicate two plausible target genes: TERT and CLPTM1L. However, the mechanisms linking cancer susceptibility alleles to their presumed target genes, along with the observed antagonistic pleiotropy between different cancers remain elusive. With this in mind, we employed a comprehensive approach, integrating statistical fine-mapping of GWAS summary statistics with massively parallel reporter assays (MPRA) and a densely tiled CRISPRi screen performed across eight cell lines, representative of four distinct cancers associated with 5p15.33 alleles – pancreatic, melanoma, lung, and bladder cancer. Integrative screening identified up to 30 variants across multiple signals in the locus with putative allele-specific transcriptional activity. Furthermore, a variable number tandem repeat (VNTR) in intron 9 of CLPTM1L emerged as a potent transcriptional enhancer in CRISPRi screening. Leveraging PacBio sequencing, VNTR alleles of European ancestry samples from the 1000 Genomes Project were genotyped and found to be highly polymorphic, with longer VNTRs linked to the haplotype tagged by rs31490-A (risk increasing for pancreatic cancer). Subsequent imputation into a pancreatic cancer GWAS of 9,040 cases and 12,496 controls revealed an association between longer VNTRs and risk of pancreatic cancer (OR for VNTR in the 75th percentile = 1.17, 95% CI = 1.13-1.22, P = 2.21x10-12). The significance of the association for the lead SNP rs31490 after conditioning on the VNTR genotype dropped to P= 2.20x10-5, suggesting that VNTR alleles merit consideration as part of the credible variant set for pancreatic cancer. Targeted CRISPRi assays suggested VNTR-mediated cis-regulation of both TERT and CLPTM1L in PANC-1 and MIA PaCa-2 pancreatic cancer cell lines, while dual luciferase reporter assays in the same cell lines revealed that VNTR enhancer activity is mediated by Hippo pathway transcription factors. In ongoing experiments, we aim to expand on luciferase reporter assays and DNA-protein binding assays in relevant cell lines with the aim of not only deciphering how longer VNTRs differentially exert their transcriptional effects compared to shorter alleles but also characterizing the divergent directions of effect between different cancer types. These findings underscore the proposition that pancreatic cancer susceptibility at the 5p15.33 locus may be mediated by both SNPs and an uncharted class of variants, yet to be characterized in the post-GWAS landscape. Citation Format: Aidan O'Brien, Hyunkyung Kong, Minal Patel, Katelyn E Connelly, Mai Xu, Irene Collins, Jun Zhong, Jason Hoskins, Michelle Ho, Brenen Papenburg, Mark M Iles, Matthew H Law, Maria-Teresa Landi, Rachael Stolzenber- Solomon, Brian M Wolpin, Alison P Klein, Nick Orr, Stephen J Chanock, Sara Lindström, Marc-Henri Stern, Ludmila Prokunina-Olsson, Jieyon Choi, Kevin M Brown, Laufey T Amundadottir. Unraveling pancreatic cancer susceptibility at 5p15.33: Functional characterization of a novel VNTR element [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C055.

Combination of S-1 and the oral ATR inhibitor ceralasertib is effective against pancreatic cancer cells.

In our previous study, we found that the Chk1 inhibitor prexasertib enhances the antitumour effect of the oral anticancer drug S-1 against pancreatic cancer cells. In this study, we investigated the effect of combining S-1 and ceralasertib, an oral inhibitor of ATR, which is located upstream of Chk1. Ceralasertib is currently being investigated in multiple clinical trials for various cancers. The cell-proliferation inhibitory effect was measured by MTT assay, using the pancreatic cancer cell lines BxPC-3, SUIT-2, PANC-1, and MIA PaCa-2, while apoptosis was measured by flow cytometry using PI/Annexin staining. The mechanism underlying the combined effect was analysed using western blotting, and the antitumor effect was analysed using a mouse xenograft model. MTT assay revealed that the combination of S-1 and ceralasertib had a synergistic effect, leading to the suppression of cell proliferation. Measurement with PI/Annexin staining revealed that the combination of S-1 and ceralasertib induced apoptosis more efficiently than either drug alone. Western blotting results showed that ceralasertib inhibited S-1-induced activation of ATR and Chk1. The average estimated tumour volume after 3weeks of administration was 601 mm3 in the S-1 group, 580 mm3 in the ceralasertib group, and 298 mm3 in the combination group. The combination of S-1 and ceralasertib demonstrated a high antiproliferative effect in inhibiting tumour growth in vitro.

Characterizing Extracellular Vesicles Generated from the Integra CELLine Culture System and Their Endocytic Pathways for Intracellular Drug Delivery.

Extracellular vesicles (EVs) have attracted great attention as promising intracellular drug delivery carriers. While the endocytic pathways of small EVs (sEVs, <200 nm) have been reported, there is limited understanding of large EVs (lEVs, >200 nm), despite their potential applications for drug delivery. Additionally, the low yield of EVs during isolation remains a major challenge in their application. Herein, we aimed to compare the endocytic pathways of sEVs and lEVs using MIA PaCa-2 pancreatic cancer cell-derived EVs as models and to explore the efficiency of their production. The cellular uptake of EVs by MIA PaCa-2 cells was assessed and the pathways were investigated with the aid of endocytic inhibitors. The yield and protein content of sEVs and lEVs from the Integra CELLine culture system and the conventional flasks were compared. Our findings revealed that both sEVs and lEVs produced by the Integra CELLine system entered their parental cells via multiple routes, including caveolin-mediated endocytosis, clathrin-mediated endocytosis, and actin-dependent phagocytosis or macropinocytosis. Notably, caveolin- and clathrin-mediated endocytosis were more prominent in the uptake of sEVs, while actin-dependent phagocytosis and macropinocytosis were significant for both sEVs and lEVs. Compared with conventional flasks, the Integra CELLine system demonstrated a 9-fold increase in sEVs yield and a 6.5-fold increase in lEVs yield, along with 3- to 4-fold higher protein content per 1010 EVs. Given that different endocytic pathways led to distinct intracellular trafficking routes, this study highlights the unique potentials of sEVs and lEVs for intracellular cargo delivery. The Integra CELLine proves to be a highly productive and cost-effective system for generating EVs with favourable properties for drug delivery.

Design, Synthesis, and Biological Evaluation of 5-Amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide Derivatives as Novel and Potential MEK/RAF Complex Inhibitors Based on the "Clamp" Strategy.

Herein, we described the rational drug design and synthesis of a series of 5-amino-4-fluoro-1H-benzo[d]imidazole-6-carboxamide derivatives that inhibit MEK and RAF kinases. The detailed screening cascades revealed that 16b was a preferred compound, which might act like a "clamp" to stabilize the MEK/RAF complex, thereby effectively inhibiting MEK1, BRAF, and BRAFV600E with IC50 values of 28, 3, and 3 nM, respectively. 16b possessed an excellent selectivity over other 312 human-related kinases at 1 μM. In vitro, 16b showed potent antiproliferative activities against MIA PaCa-2 (G12C KRAS), HCT116 (G13D KRAS), and C26 (G12D KRAS) cells with IC50 values of 0.011, 0.079, and 0.096 μM, respectively. CoIP experiments demonstrated that 16b could induce MEK/RAF complex formation. Most importantly, in the C26 syngeneic colorectal and HCT116 mice xenograft tumor models, 16b demonstrated tumor growth inhibition of 70 and 93%, respectively, suggesting that 16b may be a promising MEK/RAF complex inhibitor and worthy of further development.

5-nitro-thiophene-thiosemicarbazone derivative induces cell death, cell cycle arrest, and phospho-kinase shutdown in pancreatic ductal adenocarcinoma cells

IntroductionPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatment options. This study explores the potential of novel 5-nitro-thiophene-thiosemicarbazone derivatives as therapeutic agents for PDAC. MethodsWe evaluated the cytotoxicity of seven derivatives in peripheral blood mononuclear cells (PBMCs) and PDAC cell lines. Promising candidates (PR12 and PR17) were further analyzed for their effects on colony formation, cell cycle progression, and reactive oxygen species (ROS) production. PR17, the most promising derivative, was subjected to additional investigation, including analysis of autophagy-related genes and protein kinase inhibition. ResultsThree derivatives (PR16, PR19, and PR20) displayed cytotoxicity towards PBMCs. PR12 reduced colony formation and G0/G1 cell cycle arrest in PDAC cells. Notably, PR17 exhibited potent activity in MIA PaCa-2 cells, inducing S-phase cell cycle arrest, downregulating autophagy genes, and inhibiting key protein kinases. ConclusionPR17, a 5-nitro-thiophene-thiosemicarbazone derivative, demonstrates promising antineoplastic activity against PDAC cells by potentially modulating cell cycle progression, autophagy, and protein kinase signaling. Further studies are warranted to elucidate the detailed mechanism of action and explore its efficacy in vivo.

Enhanced radiosensitivity of pancreatic cancer achieved through inhibition of Cyclin-dependent kinase 1

Background and purposeOvercoming radioresistance is a critical challenge in pancreatic ductal adenocarcinoma (PDAC). Our study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through genetic and pharmaceutical inhibition to radiosensitize PDAC cells. Materials and MethodsMass spectrometry and phosphoproteomics were used to analyze engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) compared to parental controls. The TCGA PDAC database was queried for clinical outcomes and patients were dichotomized based on the median CDK1 mRNA expression. We generated a microRNA-based TET-on inducible shRNA to inhibit CDK1 expression in two PDAC cell lines. We used an orthotopic model of PDAC to test the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We targeted CDK1 activation with a selective CDK1 inhibitor, RO-3306, followed by in vitro experiments employing immunoblotting, immunocytochemistry, and clonogenic assays. ResultsPhosphoproteomics analysis revealed that phospho-CDK1 (Tyr15) was significantly elevated in the resistant clones. We found that high CDK1 expression was associated with worse OS in PDAC patients. Radiation exposure increased CDK1 phosphorylation. In MIA PaCa-2 and PANC-1 cells, CDK1 inhibition synergized with radiation therapy to delay tumor growth in vivo. CDK1 inhibition via. RO-3306 resulted in a significant shift of cells into the G2/M phase and disrupted DNA repair after radiation exposure. In vitro, pre-treatment with RO-3306 led to enhanced radiosensitivity of PDAC cells. ConclusionCDK1 plays a crucial role in PDAC radioresistance. Targeting CDK1 with radiotherapy holds promise for further investigation in PDAC treatment.

A Cyclometalated Iridium(III) Complex Exerts High Anticancer Efficacy via Fatty Acid Beta-Oxidation Inhibition and Sphingolipid Metabolism Reprogramming.

Given the extensive role of lipids in cancer development, there is substantial clinical interest in developing therapies that target lipid metabolism. In this study, we identified one cyclometalated iridium complex (Ir2) that exhibits potent antiproliferation activity in MIA PaCa-2 cells by regulating fatty acid metabolism and sphingolipid metabolism simultaneously. Ir2 also efficiently overcomes cisplatin resistance in vitro. Satisfyingly, the generated Ir2@F127 carriers, as a temperature-sensitive in situ gelling system of Ir2, showed effective cancer treatment with minimal side effects in an in vivo xenograft study. To the best of our knowledge, Ir2 is the first reported cyclometalated iridium complex that exerts anticancer activity in MIA PaCa-2 cells by intervening in lipid metabolism, which provides an alternative pathway for the anticancer mechanism of cyclometalated iridium complexes.

LINC01857 promotes cell proliferation and migration while dampening cell apoptosis in pancreatic cancer by upregulating CDC42EP3 via miR-450b-5p

BackgroundPancreatic cancer (PC) is a devastating human malignancy with a poor survival outcome (5-year survival less than 10%). In recent years, the regulatory roles of long non-coding RNAs (lncRNAs) in various types of cancers have been widely reported. Based on bioinformatics analysis, LINC01857 is shown to be highly expressed in PC tissue. Nevertheless, the role of LINC01857 in PC is limitedly reported. Hence, this study aimed to explore the effects of lncRNA LINC01857 on PC cell process and the related mechanism. MethodsRT-qPCR and fluorescence in situ hybridization (FISH) assay were conducted to measure LINC01857 expression and distribution in PANC-1 and MIA PaCa-2 cells. Colony formation and wound healing assays as well as flow cytometry analyses were employed to estimate the proliferation, migration, and apoptosis of PC cells transfected with pcDNA3.1-LINC01857 or si-LINC01857 compared with the behavior of PC cells transfected with empty pcDNA3.1 vector (control) or si-negative control (NC). Furthermore, RNA pulldown and luciferase reporter assays were utilized to demonstrate the interaction of LINC01857 and miR-450b-5p or to validate the binding of miR-450b-5p and cell division cycle 42 effector protein 3 (CDC42EP3). ResultsLINC01857 was highly expressed in PANC-1 and MIA PaCa-2 cells in contrast to its expression in pancreatic ductal epithelial cells (8.9 folds and 7.1 folds, p<0.001). Silencing LINC01857 significantly reduced cell proliferation and migration while enhancing apoptosis (p<0.0005). In contrast, overexpression of LINC01857 markedly (p<0.05) accelerated these malignant behavior of PC cells. MiR-450b-5p was targeted and inversely regulated by LINC01857. Moreover, CDC42EP3 was verified to be targeted by miR-450b-5p, and CDC42EP3 was correlated to LINC01857 in a positive manner (p<0.001). Rescue experiments manifested that silencing CDC42EP3 effectively (p<0.05) reversed the promoting effect of LINC01857 on malignant behavior of PC cells. ConclusionLINC01857 promotes PC cell proliferation and migration while obstructing cell apoptosis by binding to miR-450b-5p and thus regulating CDC42EP3 expression. The study presents a novel and promising regulatory axis, which holds potential for the identification of biomarkers and development of therapeutic strategies for PC treatment.

Prenylated xanthones from mangosteen (Garcinia mangostana) target oxidative mitochondrial respiration in cancer cells

Mangosteen (Garcinia mangostana) is well-known for its nutritional value and health benefits. Breast cancer is the most common cancer and the leading cause of cancer-related mortality among females worldwide. Here we show that the prenylated xanthones, α-mangostin, γ-mangostin, 9-hydroxycalabaxanthone (9-HCX), and garcinone E from the mangosteen pericarp exhibit cytotoxicity against a panel of human cancer cell lines including lung adenocarcinoma (A549), cervical carcinoma (HeLa), prostatic carcinoma (DU 145), pancreatic carcinoma (MIA PaCa-2), hepatocellular carcinoma (Hep G2), bladder urothelial cancer (5637), as well as the triple-negative breast cancer cells MDA-MB-231. In line with its higher predicted bioactivity score compared to other prenylated xanthones, 9-HCX induced the strongest antiproliferative and proapoptotic effects in MDA-MB-231 breast cancer xenografts in vivo. In different in vitro models, we demonstrate that prenylated xanthones from G. mangostana target mitochondria in cancer cells by inhibition of the mitochondrial respiratory chain complex II (α-mangostin, γ-mangostin, and garcinone E) and complex III (9-HCX) as shown in isolated mitochondria. Accordingly, oxidative mitochondrial respiration (OXPHOS) was inhibited, mitochondrial proton leak increased, and adenosine triphosphate (ATP) synthesis decreased as analyzed by Seahorse assay in MDA-MB-231 cells. Hence, the prenylated xanthones increased mitochondrial superoxide levels, induced mitochondrial membrane permeabilization, and initiated caspase 3/7-mediated apoptosis in MDA-MB-231 triple-negative breast cancer cells. Thus, prenylated xanthones from Garcinia mangostana exhibit anticancer activity based on interference with the mitochondrial respiration.