Viability Of Pancreatic Cancer Cells Research Articles

Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

Statins, the drugs used for the treatment of hypercholesterolemia, have come into the spotlight not only as chemoadjuvants, but also as potential stem cell modulators in the context of regenerative therapy. In our study, we compared the in vitro effects of all clinically used statins on the viability of human pancreatic cancer (MiaPaCa-2) cells, non-cancerous human embryonic kidney (HEK 293) cells and adipose-derived mesenchymal stem cells (ADMSC). Additionally, the effect of statins on viability of MiaPaCa-2 and ADMSC cells spheroids was tested. Furthermore, we performed a microarray analysis on ADMSCs treated with individual statins (12 μM) and compared the importance of the effects of statins on gene expression between stem cells and pancreatic cancer cells. Concentrations of statins that significantly affected cancer cells viability (< 40 μM) did not affect stem cells viability after 24 h. Moreover, statins that didn´t affect viability of cancer cells grown in a monolayer, induce the disintegration of cancer cell spheroids. The effect of statins on gene expression was significantly less pronounced in stem cells compared to pancreatic cancer cells. In conclusion, the low efficacy of statins on non-tumor and stem cells at concentrations sufficient for cancer cells growth inhibition, support their applicability in chemoadjuvant tumor therapy.

TAGLN2 targeted control of ARPC5-mediated activation of the MEK/ERK signaling pathway influences the proliferation, invasion, and metastasis of pancreatic cancer cells

PurposePancreatic cancer (PC) is a common tumor of the digestive tract with an insidious onset and high malignancy potential. Currently, surgery is the only effective treatment modality. Therefore, it is crucial to discover new targeted therapeutic modalities. We studied whether transgelin 2 (TAGLN2) targeted control of actin-related protein 2/3 complex subunit 5 (ARPC5)-mediated activation of the MEK/ERK signaling pathway to Influences the proliferation, invasion, and metastasis of pancreatic cancer cells. MethodsThe effects of TAGLN2 overexpression and knockdown on the proliferative viability and invasive metastatic ability of pancreatic cancer cells were verified through in vitro and in vivo assays via constructing a stable lentiviral transfection of human pancreatic cancer cell lines PANC-1 and SW1990. Bioinformatics analysis was used to predict the relationship between TAGLN2 and ARPC5. These findings were subsequently verified through protein profiling, immunofluorescence (IF), and coimmunoprecipitation (CO-IP) assays. In vitro experiments were also conducted to confirm the effect of TAGLN2 modulation on ARPC5 expression, which subsequently affects the proliferation and invasive metastatic ability of pancreatic cancer cells. The study analyzed the relationship between TAGLN2 and the MEK/ERK signaling pathway through bioinformatics and in vitro experiments with the MEK signaling pathway inhibitor U0126. ResultsTAGLN2 is expressed at high levels in pancreatic cancer cell lines, and its expression is positively correlated with poor prognosis of pancreatic cancer. ARPC5 is a direct target of TAGLN2 and is associated with the MEK/ERK signaling pathway. In vivo and ex vivo experiments confirmed that overexpression of TAGLN2 promoted the proliferation, invasion, and metastasis of pancreatic cancer cells, and silencing ARPC5 reversed these effect. ConclusionOur research revealed that TAGLN2 protein binds to ARPC5 protein and contributes to increased ARPC5 expression and activation of the MEK/ERK signaling pathway. This activation promotes pancreatic cancer cell growth, infiltration, and spread. Hence, TAGLN2 is a potential viable therapeutic target in pancreatic cancer and represents a novel therapeutic approach.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Abstract LB274: Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression

Abstract HIKESHI, under heat stress conditions, has been reported to transport molecular chaperone HSP70 into the nucleus to prevent cancer cell death from heat stress. The tumor-supporting roles of molecular chaperones in various solid tumors are explored extensively. Yet, the sole targeting of the molecular chaperones could not significantly impact anticancer treatment. Thus, we hypothesized that the specific transporter protein HIKESHI might improve this outcome. However, understanding of the clinical relevance of HIKESHI in pancreatic cancer remained unclear, and few studies addressed the HIKESHI role in cancer and whether targeting can inhibit pancreatic cancer cell viability and the other HSPs. Here, we first investigated the expression pattern of the HIKESHI protein in surgically resected pancreatic cancer samples (n=142) and analyzed the clinical relevance of the HIKESHI. We found that the HIKESHI protein was overexpressed in cancer compared to the adjacent normal tissue, and positive expression of cancer HIKESHI was related to poor overall survival. In addition, HIKESHI-positive expression in cancer was identified as an independent prognostic marker for overall survival. Next, using shRNA, we established the stable HIKESHI-suppressed pancreatic cancer cell line (PANC-1). HIKESHI suppression inhibited cell growth and colony formation ability in vitro. The expression and nuclear translocation of HSP70 and HSP110/105 were suppressed in HIKESHI-suppressed cells compared to the control shRNA cells despite no heat stress conditions. Furthermore, we found that the tumor growth, HIKESHI, and Ki67 expression in HIKESHI-suppressed tumors were inhibited compared to the control shRNA tumors. Next, we performed Cap Analysis of Gene Expression and IP-MS analysis to discover the potential downstream and novel interactive partner of the HIKESHI protein. The extensive mechanism of these analyses is under investigation. Our findings suggest that targeting HIKESHI may be a promising therapeutic strategy against refractory pancreatic cancer. Citation Format: Bilguun Erkhem-Ochir, Takehiko Yokobori, Gendensuren Dorjkhorloo, Haruka Okami, Takaomi Seki, Norifumi Harimoto, Ryo Muranushi, Kouki Hoshino, Kei Hagiwara, Norihiro Ishii, Mariko Tsukagoshi, Kenichiro Araki, Hiroshi Saeki, Ken Shirabe. Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB274.

Abstract 4495: Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells

Abstract Background: The identification of novel compounds that specifically target cancer cells while sparing normal tissues is crucial. The orphan tyrosine kinase-like receptor 1 (ROR1) is one such target expressed exclusively in various human malignancies, including pancreatic cancer, but not in normal adult tissues. Inhibiting ROR1 signaling shows promise in halting pancreatic cancer cell proliferation and invasion. In this study, lead compounds CSF1R-IN-1 and MCULE-8715589337 were found as potential inhibitors of the ROR1 and were further evaluated for its potential effect on pancreatic cancer cell viability. Method: We curated a compound library of 6 million from PubChem, 2 million lead-like compounds from ZINC15, and 2 million compounds from diverse vendors. Employing Glide’s HTVS followed by XP, we refined the selection based on docking scores lower than the reference molecule, Ponatinib. Compounds were shortlisted by pre-simulation MMGBSA calculations with Schrödinger Prime MM-GBSA module, focusing on compounds with energies below -100 kcal/mol. Then we performed MD simulation for 100ns. Post-simulation MMGBSA on the last 1 ns of simulation assessed change in ligands’ binding energies. The selected leads underwent further validation utilizing two pancreatic cancer cell lines, MiaPaCa-2 and PANC-1. Half-maximal inhibitory concentration (IC50) was assessed using MTS cell viability assay. Result: Comparing pre- and post-simulation MMGBSA energies, the reference compound showed increased, while CSF1R-IN-1 and MCULE-8715589337 exhibited decreased energies. The reference ligand's RMSD showed stability with fluctuations of 3 to 3.5 Å, whereas CSF1R-IN-1 and MCULE-8715589337 had stable and lower RMSD values (approximately 3 Å and 2.4 Å). Test compounds, CSF1R-IN-1 and MCULE-8715589337 showed better molecular interactions with ROR1 compared to reference. The reference compound formed hydrogen bonds with ILE555 and ASP633, along with pi-pi stacking with PHE552. CSF1R-IN-1 forms a hydrogen bond with ILE555 and ASP633, pi-pi stacking with PHE552, and interaction with SER632 through a water bridge. Additionally, compound MCULE-8715589337 forms hydrogen bonds with ASP633, SER632, LYS506, and GLU523. CSF1R-IN-1 notably inhibited proliferation in MiaPaCa-2 cells (IC50: 1.29 μM) while no efficacy in PANC-1 cells (IC50: 78.41 μM). MCULE-8715589337 showed relatively similar IC50 values of 39.06 μM and 37.73 μM in MiaPaCa-2 and PANC-1 cells, respectively. Conclusion: The reduction in MMGBSA energies, low RMSD values, and a higher number of interactions highlight the better characteristics of both compounds compared to the reference. Our findings show CSF1R-IN-1 and MCULE-8715589337 have efficacy in pancreatic cancer cells, but further studies are required to validate and to understand the mechanism of action. Citation Format: Indrakant Kumar Singh, Shradheya RR Gupta, Pooja Mittal, Shivani Soni, Jae Ho Lo, Francesca Battaglin, Yan Yang, Lesly Torres-Gonzalez, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Joshua Millstein, Heinz Josef Lenz. Structure-based screening for potential ROR1 inhibitors and their potential as antitumor agents in pancreatic cancer cells [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 4495.

Abstract 6001: Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells

Abstract Background: Myeloid cell leukemia 1 (MCL-1) is an anti-apoptotic member of the BCL-2 protein family which is involved in intrinsic (mitochondrial) pathway of apoptosis and is deregulated in several cancers. Current MCL-1 specific inhibitors have limitations in terms of efficiency, tolerability, and off-target effects and challenges associated with shallow binding groove of MCL-1. In this study, dihydroergotamine mesylate (DHE), an approved anti-migraine drug, was found as a potential ligand of the MCL-1 BH3-binding pocket and was further evaluated for its potential MCL-1 inhibition and effect on pancreatic cancer cells. Methods: PubChem Database was used for library preparation and virtual screening (OpenBabel and AutoDock Vina). The 55 crystal structures available for MCL-1 in Protein Data Bank (PDB) were clustered using principal component analysis with Bio3D R-package to select representative PDBs for further screening. Consensus voting and k-means clustering was used to shortlist compounds for Molecular dynamics (MD) simulations. MD simulations (100 nanoseconds) by GROMACS were employed to measure stability of ligands in MCL-1 binding pocket. DHE efficacy on cell proliferation was evaluated with MTS and clonogenic assays and on apoptosis using the FITC/Annexin V flow cytometry assay in MiaPaCa-2 and PANC-1 pancreatic cancer cell lines. Effect of DHE treatment on MCL-1 gene and protein expression was analyzed using qRT-PCR and western blotting, respectively. Results: Computational screening identified DHE (PubChem ID: 12308974) as a top hit exhibiting consistent interactions within the MCL-1 BH3-binding pocket. Notably, DHE displayed stable RMSD and formed four hydrogen bonds throughout MD simulations. In vitro results showed that DHE significantly reduced viability of MiaPaCa-2 and PANC-1 pancreatic cancer cells with IC50 values of 31 μM and 47 μM, respectively. DHE significantly (P&amp;lt;0.001) suppressed colony formation by pancreatic cancer cells in a dose-dependent (10μM, 20μM, and 40μM) manner. The percentage of Annexin V-positive and Propidium iodide-negative fraction in MiaPaCa-2 cells treated with DHE also increased in a dose-dependent manner. DHE did not have significant effect on MCL-1 mRNA expression. However, western blotting demonstrated MCL-1 protein levels were significantly reduced in both pancreatic cancer cell lines upon DHE treatment. Conclusion: Our findings showed DHE suppresses the pancreatic cancer cells viability and proliferation, and significantly increased early apoptotic cells. DHE treatment had no effect on MCL-1 at the transcription level but significantly reduced the protein, suggesting posttranslational modifications, and thus future mechanism of action studies is required. The present study reports that the anti-migraine drug DHE might be a novel therapeutic agent for pancreatic cancer and needs to be explored further. Citation Format: Pooja Mittal, Jae Ho Lo, Shivani Soni, Francesca Battaglin, Shradheya RR Gupta, Lesly Torres-Gonzalez, Yan Yang, Sandra Algaze, Priya Jayachandran, Karam Ashouri, Alexandra Wong, Wu Zhang, Jian Yu, Lin Zhang, Joshua Millstein, Indrakant Kumar Singh, Heinz Josef Lenz. Repurposing dihydroergotamine mesylate as a potential MCL-1 inhibitor and its antitumor effect in pancreatic cancer cells [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 6001.

Abstract 3322: Sigma-2-Erastin inhibits PC tumor growth through lipid ROS-DUSP6-MAPK signaling

Abstract Introduction: Pancreatic cancer (PC) is a malignancy with poor prognosis and high mortality with a very low 5-year survival rate. Currently, apoptosis and autophagy are the central mechanisms of programmed cell death (PCD) that maintain cellular homeostasis and regulate cell fate. With the recent emergence of ‘ferroptosis’ as a new type of PCD and its close association with the pathophysiological processes of many diseases including tumors, it is viewed as a potential mechanism to improve systemic treatment of PC. Considering the opportunity to regulate both apoptosis and ferroptosis by a single agent, our group has developed a cancer-targeted small molecule consisting of a sigma-2 ligand (a known apoptotic inducer) delivery moiety linked to an inhibitor of the cystine importer xCT (dm-Erastin, a known ferroptotic inducer) and named it as Sigma-2-Erastin (S2E) or ACXT-3102. The study presented here is aimed at determining the efficacy of ACXT-3102 in PC and defining its underlying mechanism of action. Methods: Using in vitro and in vivo tumor xenograft models of human PC, we determined the effects of ACXT-3102 treatments in regulating PCD of PC cells and defined the signaling pathway involved in mediating the effects. Results: As assessed by TitreGlo assays, we observed dose-dependent inhibition of PC cell (BxPC-3, AsPC-1 &amp; PANC-1) viability. Following the WES-analysis of signaling proteins, we observed that ACXT-3102 induces PC cell death by combinatorial regulation of apoptotic and ferroptotic PCD via lipid ROS-DUSP6-MAPK/ERK-mediated signaling pathway. Interestingly, the results of this study demonstrated a hyperactivation of MAPK/ERK signaling by increased phosphorylation instead of inhibition involved in inducting apoptotic cell death as represented by significant increases in expression of cleaved Capase-3/7 and PARP. In addition, ACXT-3102 treatments deceased GPX4 expression along with an increase in lipid ROS production as key markers of inducing ferroptotic cell death. Results from the murine model of human tumor xenografts using AsPC-1 cells further demonstrated significant decreases in tumor volume and increases in survival proportions in ACXT-3102-treated mice. Conclusions: ACXT-3102, an orally administered conjugate of sigma-2 ligand and Erastin induces PCD of human PC by uniquely regulating apoptosis and ferroptosis together. Therefore, ACXT-3102 offers a novel and improved therapeutic strategy for treating patients of fatal human carcinomas, especially those, which are difficult to diagnose at an early stage. Citation Format: Kumar S. Bishnupuri, Kenneth F. Newcomer, Qingqing Gong, Li Ye, Suwanna Vangveravong, Rony Takchi, Bradley T. Keller, Dirk M. Spitzer, William G. Hawkins. Sigma-2-Erastin inhibits PC tumor growth through lipid ROS-DUSP6-MAPK signaling [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 3322.

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.

MicroRNA-485-5p targets keratin17 to regulate pancreatic cancer cell proliferation and invasion via the FAK / SRC / ERK pathway.

Background: It is crucial to probe into the biological effect and mechanism of miRNA-485-5p regulating keratin 17 (KRT17) in pancreatic cancer (PC) to understand its pathogenesis and identify potential biological targets. Methods: The bioinformatics means were used to evaluate the clinical significance of KRT17 expression in the Cancer Genome Atlas (TCGA) database. TargetScan database analysis in conjunction with dual luciferase and RNA Immunoprecipitation (RIP) experiments was used to probe the interaction relationship of miRNA-485-5p with KRT17. The expression of miRNA-485-5p and KRT17 in PC tissue and cancer cell lines was detected by Q-PCR paired with western blot assay. The biological function of miRNA-485-5p in regulating KRT17 was investigated in the PC cell line via gene silencing/overexpression technique. A western blot experiment was utilized to investigate the regulatory effect of KRT17 on cell cycle-related proteins and the FAK/Src/ERK signal pathway. Results: The level of KRT17 was increased in PC tissues and this significantly decreased the survival rate of PC patients. TargetScan in combination with dual luciferase and RIP experiments verified the miRNA-485-5p target KRT17. The expression of KRT17 was high in the PC cell line, although the expression of miRNA-485-5p was low. Silencing KRT17 or overexpression of miRNA-485-5p significantly inhibited PC cell viability, proliferation, invasion, and colony formation, while promoting apoptosis. Overexpression of KRT17 drastically reversed the function of miRNA-485-5p. The silenced KRT17 remarkably downregulated the expression of cyclinD1, Cyclin Dependent Kinase 1 (CDK1), CDK2, Phospho-Focal Adhesion Kinase (p-FAK), p-Src, and p-ERK proteins in the PC cells. Conclusion: Generally, an essential signaling cascade of miRNA-485-5p/KRT17/FAK/Src/ERK influences the biological functions of PC cells.

COL10A1 promotes tumorigenesis by modulating CD276 in pancreatic adenocarcinoma

BackgroundPancreatic adenocarcinoma (PAAD) is a lethal malignant tumour. Further study is needed to determine the molecular mechanism and identify novel biomarkers of PAAD.MethodsGene expression data from the GSE62165 microarray were analysed with the online software Morpheus to identify differentially expressed genes (DEGs). The STRING database was used to generate a protein‒protein interaction (PPI) network for these DEGs. Hub genes were identified with Cytoscape. COL10A1 expression in PAAD was analysed via the GEPIA database. COL10A1 expression in pancreatic cancer cell lines was measured by using qRT‒PCR. The LinkedOmics database was utilized to perform survival analysis of pancreatic adenocarcinoma patients grouped based on COL10A1 expression level. CCK-8, wound healing, and Transwell assays were used to study the role of COL10A1 in pancreatic cancer cell viability, migration, and invasion. Differentially expressed genes that were related to COL10A1 in PAAD were analysed via the LinkedOmics portal. After COL10A1 was knocked down, CD276 expression was assessed by western blotting.ResultsCOL10A1 was identified as one of the hub genes in PAAD by bioinformatics analysis of the GSE62165 microarray with Morpheus, the STRING database and Cytoscape. GEPIA revealed elevated expression of COL10A1 in PAAD samples vs. normal samples. COL10A1 expression was also increased in pancreatic cancer cells vs. control cells. Survival analysis of PAAD patients via LinkedOmics revealed that high expression of COL10A1 was associated with a poorer prognosis. Knockdown of COL10A1 inhibited the proliferation, migration, and invasion of cells in functional assays. Furthermore, mechanistic studies indicated that CD276 was a target of COL10A1 and that knockdown of COL10A1 decreased CD276 expression. Overexpression of CD276 in cells reversed COL10A1 knockdown-induced repression of proliferation and migration.ConclusionsOur research suggests that COL10A1 promotes pancreatic adenocarcinoma tumorigenesis by regulating CD276. This study provides new insight into biomarkers and possible targets for pancreatic cancer treatment.

Inhibitory effect of liriopesides B in combination with gemcitabine on human pancreatic cancer cells

Gemcitabine (GEM) is a standard chemotherapeutic agent for patients with pancreatic cancer; however, GEM-based chemotherapy has a high rate of toxicity. A combination of GEM and active constituents from natural products may enhance its therapeutic efficacy and reduce its toxicity. This study investigated the synergistic effects of the combination of liriopesides B (LirB) from Liriope spicata var. prolifera and GEM on human pancreatic cancer cells. The results of our study showed that the combination of LirB and GEM synergistically decreased the viability of pancreatic cancer cells. The combination also caused a strong increase in apoptosis and a strong decrease in cell migration and invasion. Furthermore, LirB combined with GEM had potent inhibitory effects on pancreatic cancer stem cells (CSCs). Studies on the mechanisms of action showed that the combination more potently inhibited protein kinase B (Akt) and nuclear factor kappa B (NF-κB), as well as the downstream antiapoptotic molecules B-cell lymphoma 2 (Bcl-2) and survivin than either agent used alone. The results of this study suggest that the combination of LirB with GEM may improve the efficacy of GEM for the treatment of pancreatic cancer.

Intercellular Mechanisms of Therapeutic Resistance at the Tumor-Stromal Interface Using Ultra High-Plex Single-Cell Spatial Transcriptomics and Genetically-Engineered Tumoroids

There is a major gap in knowledge regarding how intercellular interactions in the tumor microenvironment (TME) mediate therapeutic resistance. Achievement of this goal has been limited by a lack of (1) spatial context in dissociated single-cell methods; (2) single-cell resolution in spatial profiling approaches; (3) high quality data and yield with FFPE patient specimens; and (4) computational methods for ligand-receptor analyses that consider both gene expression and spatial coordinates. We developed an innovative spatial biology paradigm that combines cutting-edge experimental and computational methods to enable high-resolution, spatially-guided discovery of critical mediators of therapeutic resistance. We applied this approach to dissect the single-cell spatial transcriptomic landscape of untreated vs. chemoradiotherapy-treated primary human pancreatic ductal adenocarcinoma (PDAC; n = 21) using ultra-high plex spatial molecular imaging (SMI) optimized for high-sensitivity, subcellular detection of up to 6000 gene transcripts in FFPE sections-an order of magnitude greater than contemporary methods. We recovered over 1,000,000 high-quality single cells in situ representing more than 20 distinct cell types, including epithelial, immune, endothelial, endocrine, and diverse stromal cells. We developed an optimal transport-based computational method to infer cell-cell communication at the cancer-stromal interface. Treatment with chemoradiotherapy was associated with the largest increase in fibroblast-malignant interactions. Comparing the SMI data with orthogonal single-nucleus RNA-sequencing and digital spatial profiling data, we identified CLCF1-CNTFR as the fibroblast-malignant interaction most associated with resistance to chemoradiotherapy in PDAC. CLCF1 is a gp130-family cytokine that activates Jak-STAT signaling and acts as a potent neurotrophic factor. Notably, the CLCF1-CNTRF (fibroblast-malignant) interaction has prominent pro-oncogenic effects in lung adenocarcinoma and an engineered CNTFR decoy receptor with therapeutic potential has been developed. To functionally validate the role of the CLCF1-CNTFR (fibroblast-malignant) interaction in mediating resistance to cytotoxic therapy, we created CRISPR-engineered cancer-fibroblast tumoroids and modulated expression of this ligand-receptor pair. Pancreatic cancer cell viability in the presence of 5-fluorouracil was better maintained with increased CLCF1-CNTFR signaling. In this study, we integrated ultra high-plex single-cell spatial transcriptomics, optimal transport ligand-receptor predictions, and genetically-engineered stromal tumoroids to identify and validate CLCF1-CNTFR as an important intercellular mechanism of resistance to chemoradiotherapy in PDAC-pioneering a paradigm for translating single-cell spatial biology to clinical oncology.

Lycorine inhibits pancreatic cancer cell growth and neovascularization by inducing Notch1 degradation and downregulating key vasculogenic genes

Pancreatic cancer is highly metastatic and lethal with an increasing incidence globally and a 5-year survival rate of only 8%. One of the factors contributing to the high mortality is the lack of effective drugs in the clinical setting. We speculated that effective compounds against pancreatic cancer exist in natural herbs and explored active small molecules among traditional Chinese medicinal herbs. The small molecule lycorine (MW: 323.77) derived from the herb Lycoris radiata inhibited pancreatic cancer cell growth with an IC50 value of 1 μM in a concentration-dependent manner. Lycorine markedly reduced pancreatic cancer cell viability, migration, invasion, neovascularization, and gemcitabine resistance. Additionally, lycorine effectively suppressed tumor growth in mouse xenograft models without obvious toxicity. Pharmacological studies revealed that the levels and half-life of Notch1 oncoprotein in the pancreatic cancer cells Panc-1 and Patu8988 were notably reduced. Moreover, the expression of the key vasculogenic genes Semaphorin 4D (Sema4D) and angiopoietin-2 (Ang-2) were also significantly inhibited by lycorine. Mechanistically, lycorine strongly triggered the degradation of Notch1 oncoprotein through the ubiquitin–proteasome system. In conclusion, lycorine effectively inhibits pancreatic cancer cell growth, migration, invasion, neovascularization, and gemcitabine resistance by inducing degradation of Notch1 oncoprotein and downregulating the key vasculogenic genes Sema4D and Ang-2. Our findings provide a new therapeutic candidate and treatment strategy against pancreatic cancer.

Circ_103809 Aggravates the Malignant Phenotype of Pancreatic Cancer Through Modulating miR-197-3p/TSPAN3 Axis.

Pancreatic cancer (PC) is a malignant tumor with insidious clinical manifestations and dismal prognosis. Emerging reports have demonstrated that circRNAs exert pivotal biological function in PC. Here, we investigated the crucial biological role and underlying regulatory mechanisms of differentially expressed circ_103809 in PC. In this study, hsa_circ_103809 (hsa_circ_0072088) was identified as the research object via analyzing and screening the aberrantly expressed circRNAs in PC by GSE69362 dataset. The levels of circ_103809 in PC tissues and cells were assessed via qRT-PCR. Functional assays were conducted to monitor the impacts of circ_103809 on PC cells. Additionally, the downstream molecular targets and regulatory networks of circ_103809 were predicted by bioinformatics and validated using luciferase assays and rescue experiments. We found that circ_103809 was substantially upregulated in PC tissues and cells. Silencing circ_103809 restrained the growth viability, clonogenic rate, migration, and invasion capabilities of PC cells. Further mechanistic exploration disclosed that miR-197-3p was the downstream gene of circ_103809, while Tetraspanin-3 (TSPAN3) was a direct target of miR-197-3p. The suppressive effect of circ_103809 knockdown on malignant processes of PC cells was eliminated by miR-197-3p downregulation or TSPAN3 upregulation. Our study demonstrated that circ_103809 served as an innovative positive regulator in the growth and metastasis of PC cells. Furthermore, circ_103809 mediated the miR-197-3p/TSPAN3 axis to modulate the malignant progression of PC cells, which was prospected to be a probable biomarker and an efficient therapeutic target for PC.

Xanthoxyletin blocks the RANK/RANKL signaling pathway to suppress the growth of human pancreatic cancer cells.

Xanthoxyletin is a vital plant-derived bioactive coumarin. It has been shown to exhibit anticancer effects against different human cancers. Nonetheless, the anticancer effects of xanthoxyletin against human pancreatic cancer cells have not been evaluated. Against this backdrop, the present study was designed to evaluate the anticancer effects of xanthoxyletin in human pancreatic cancer cells and to decipher the underlying molecular mechanisms. The results revealed a significant (p < 0.05) upregulation of receptor activator of NF-kappaB (RANK), receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human pancreatic tissues and cell lines at both transcriptional and translational levels. The administration of pancreatic cancer cells with xanthoxyletin diminished the viability of Capan-2 cells in a concentration-dependent manner and led to a significant decline in RANK, RANKL, and OPG expression. Silencing of RANK and xanthoxyletin treatment declined the viability of Capan-2 pancreatic cancer cells via induction of apoptosis. However, pancreatic cancer cells overexpressing RANK could rescue the growth inhibitory effects. Collectively, xanthoxyletin targets the RANK/RANKL signaling pathway in pancreatic cancer cells to induce cell apoptosis and may prove to be an important lead molecule.

PNU-74654 Induces Cell Cycle Arrest and Inhibits EMT Progression in Pancreatic Cancer.

Background and Objectives: PNU-74654, a Wnt/β-catenin pathway inhibitor, has an antiproliferative effect on many cancer types; however, its therapeutic role in pancreatic cancer (PC) has not yet been demonstrated. Here, the effects of PNU-74654 on proliferation and cell cycle phase distribution were studied in PC cell lines. Materials and Methods: The cancer-related molecular pathways regulated by PNU-74654 were determined by a proteome profiling oncology array and confirmed by western blotting. Results: The cell viability and proliferative ability of PC cells were decreased by PNU-74654 treatment. G1 arrest was observed, as indicated by the downregulation of cyclin E and cyclin-dependent kinase 2 (CDK2) and the upregulation of p27. PNU-74654 inhibited the epithelial-mesenchymal transition (EMT), as determined by an increase in E-cadherin and decreases in N-cadherin, ZEB1, and hypoxia-inducible factor-1 alpha (HIF-1α). PNU-74654 also suppressed cytoplasmic and nuclear β-catenin and impaired the NF-κB pathway. Conclusions: These results demonstrate that PNU-74654 modulates G1/S regulatory proteins and inhibits the EMT, thereby suppressing PC cell proliferation, migration, and invasion. The synergistic effect of PNU-74654 and chemotherapy or the exclusive use of PNU-74654 may be therapeutic options for PC and require further investigation.

Development of novel 1,2,4-triazole containing compounds with anticancer and potent anti-CB1 activity

There is still an unmet need for novel and improved anti-cancer compounds. Nitrogen atoms have heterocyclic ring moieties, which have been shown to have powerful anticancer properties in both natural and synthetic derivatives. Due to their dipole character, hydrogen bonding capacity, rigidity and solubility, 1,2,4-triazoles are particularly effective pharmacophores, interacting with biological receptors with high affinity. Thus, novel 1,2,4-triazole-containing molecular derivatives were synthesized using green chemistry methods, microwave irradiation and ultrasonication, and these methods’ operational simplicity and maximum greener synthetic efficiency with green chemistry metrics calculations will be attractive for academic and industrial research and tested against three distinct human cancer cell lines including PANC1 (pancreatic cancer), DU145 (prostate cancer), MCF7 (breast cancer) and one fibroblast cell line (HDF). Here, we showed that compounds 5e and 5f were similar to CB1 antagonists in structure, binding affinity and poses. In addition, compounds 5e–g decreased the viability of pancreatic and prostate cancer cells, albeit with cytotoxicity to HDF cells. The IC50 values for PANC1 cells were between 5.9 and 7.3 µM for compounds 5e–g. Cell cycle analysis showed that the effect of compounds 5e–g in cancer cell growth was largely due to cell cycle arrest at S-phase. In sum, novel 1,2,4-triazole-containing compounds with anticancer and potent anti-CB1 activity have been developed. Communicated by Ramaswamy H. Sarma

Silencing of IGHG1 reverses the resistance of pancreatic cancer to multidrug chemotherapy by modulating autophagy.

Pancreatic cancer is one of the most aggressive tumors with a high-mortality rate. First-line drugs include 5-fluorouracil (5-FU), gemcitabine (GEM), and oxaliplatin (OXA). Resistance to 5-FU, GEM, and OXA is a major challenge. Immunoglobulin heavy chain F1 (IGHG1) participates in the regulation of cancer progression. It is still unclear how IGHG1 affects 5-FU, GEM, and OXA in pancreatic cancer. The expression status of IGHG1 in pancreatic cancer was analyzed through bioinformatics tools. IGHG1 expression in pancreatic cancer tissues and cells was determined via RT-qPCR. Cell counting kit 8 assays, and flow cytometry analysis were utilized to detect the impact of IGHG1,5-FU, GEM, and OXA on cell proliferation and apoptosis. Western blotting was utilized to detect changes in the levels of the autophagy-associated proteins LC3, Beclin-1, p62, and ATG5. Immunofluorescence assays were employed to determine LC3 expression in cells. Xenograft experiments were conducted on nude mice to study tumor growth. IGHG1 was overexpressed in pancreatic cancer cells and tissues. IGHG1 expression was downregulated by 5-FU, GEM, or OXA treatment in cells. Treatment with 5-FU, GEM, or OXA repressed viability and promoted apoptosis and autophagy in pancreatic cancer cells. IGHG1 silencing exhibited the same results. Furthermore, IGHG1 depletion notably strengthened the effects of 5-FU, GEM, and OXA on pancreatic cancer cell viability, apoptosis, and autophagy. The combination of IGHG1 depletion with 5-FU, GEM, or OXA significantly reduced tumor growth in vivo. Silencing of IGHG1 could enhance 5-FU, GEM, or OXA function in pancreatic cancer and reverse resistance by regulating apoptosis and autophagy.

MiR-4299 inhibits tumor progression in pancreatic cancer through targeting ADAM17.

Pancreatic cancer (PC) is one of the most aggressive malignant tumors in human beings. Tumor capacity of evading immune-mediated lysis is a critical step in PC malignant progression. We aimed to evaluate the underlying regulatory mechanism of miR-4299 in the proliferation, metastasis, apoptosis, and immune escape in PC. miR-4299 and ADAM17 expressions in PC tissues and cell lines were detected using qRT-PCR. MTT assay and flow cytometry were used to detect cell viability and apoptosis, respectively. A luciferase reporter gene assay was conducted to confirm the targeted relationship between miR-4299 and ADAM17. Xenograft tumors in nude mice were used to detect tumorigenesis in vivo. PC cells were co-cultured with NK cells for determining the immune escape ability. NKG2D-positive rate of NK cells was detected using flow cytometry; NK cell-killing ability was detected using MTT assay. miR-4299 was downregulated in PC tissues and cell lines. miR-4299 inhibited PC cell proliferation and invasion, promoted cell apoptosis, and reduced PC tumor growth in vivo. ADAM17 3'UTR directly bound to miR-4299. ADAM17 overexpression could reverse miR-4299 effects on PC cell viability, invasion, apoptosis, and immune escape. miR-4299 exerted suppressive effects on PC cell proliferation, invasion, and immune escape via targeting ADAM17 expression. This study revealed a novel miR-4299/ADAM17 axis-modulating PC progression and proposed to concern the immune regulatory mechanism of miRNAs in PC development.

The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress

The ubiquitin-specific peptidase 10 (USP10) plays a context-specific, pro or anti-tumorigenic role in different malignancies. However, the role of USP10 in pancreatic cancer remains unclear. Our protein and RNA level analysis from archived specimens and public databases show that USP10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and expression correlates with poor overall patient survival. Phenotypically, silencing USP10 decreased viability, clonal growth and invasive properties of pancreatic cancer cells. Mechanistically, silencing USP10 upregulated BiP and induced endoplasmic reticulum (ER) stress that led to an unfolded protein response (UPR) and upregulation of PERK, IRE1α. Decreased cell viability of USP10 silenced cells could be rescued by a chemical chaperone that promotes protein folding. Our studies suggest that USP10 by protecting pancreatic cancer cells from ER stress may support tumor progression.