Gemcitabine-resistant Pancreatic Cancer Research Articles

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

ENO1 contributes to the gemcitabine resistance of pancreatic cancer through the YAP1 signaling pathway.

Pancreatic cancer (PC), a leading cause of cancer-related deaths, has a 5-year survival rate of approximately 10%. α-Enolase (ENO1) is a junction channel protein involved in tumor cell apoptosis and chemoresistance. However, the role of ENO1 in PC remains unclear. The expression and prognosis of ENO1 levels were determined in PC using public databases based on The Cancer Genome Atlas (TCGA) data sets. Cell viability, half maximal inhibitory concentration (IC50), autophagy, apoptosis, and autophagy markers were examined using cell counting kit-8 (CCK-8), transmission electron microscope, flow cytometry assays, and immunoblot, respectively. Using the Gene Expression Omnibus (GEO) and TCGA data sets, we found that ENO1 was significantly enriched in PC tumor tissues, and high expression levels of ENO1 were associated with an unfavorable prognosis. Whereas ENO1 silencing suppressed proliferation, autophagy, and induced cell apoptosis in PC cells, and inhibited tumor growth in vivo. Mechanistically, knockdown of ENO1 enhanced cellular cytotoxicity of gemcitabine (GEM), as well as reducing the expression of yes-associated protein 1 (YAP1), a major downstream effector of the Hippo pathway in vitro. YAP1 promoted autophagy and protected PC cells from GEM-induced apoptotic cell death. Furthermore, YAP1 overexpression attenuated the inhibition effects of ENO1 silencing. Our results suggest that ENO1 overexpression promotes cell growth and tumor progression by increasing the expression of YAP1 in PC. Further studies are required to understand the detailed mechanisms between ENO1 and YAP1 in PC.

Abstract 4613: Tumor-derived interleukin 35 facilitates the gemcitabine resistance in pancreatic adenocarcinoma by mediating the dissemination of chemoresistance among PDAC cells

Abstract Rapid drug resistance after chemotherapy is a key cause of treatment failure in human pancreatic ductal adenocarcinoma (PDAC). Here, we found that tumor-derived interleukin 35 (IL-35) mediated the rapid resistance of PDAC to gemcitabine (GEM). The role of IL-35 in GEM resistance was determined by molecular and cell biology approaches in PDAC cells and in patient-derived tumor xenograft (PDX) models. We used whole genome RNA sequencing, molecular cloning and other molecular biological methods to study the molecular mechanism of IL-35 mediated GEM resistance. An anti-IL-35 neutralizing antibody was used in mouse models to investigate the feasibility of targeting IL-35 to overcome drug resistance in pancreatic cancer. We observed the phenomenon that GEM resistance could spread from GEM resistant PDAC cells to GEM sensitive ones. The results of sequencing and experiments confirmed that the IL-35 secretion is responsible for the dissemination of chemoresistance. IL-35 secreted from GEM resistant cells activated the IL35 expression in GEM sensitive cells and upregulated SOD2 expression via GP130-STAT1 signaling, which scavenges reactive oxygen species (ROS) and then leads to GEM resistance.Furthermore, we found that a neutralizing antibody against IL-35 significantly enhanced the tumor suppressive effect of GEM. These findings collectively reveal that IL-35 plays crucial roles in mediating GEM resistance in pancreatic cancer. Moreover, IL-35 might serve as a promising therapeutic target for combating PDAC chemoresistance. Citation Format: Chongbiao Huang, Huizhi Sun, Antao Chang, Jihui Hao. Tumor-derived interleukin 35 facilitates the gemcitabine resistance in pancreatic adenocarcinoma by mediating the dissemination of chemoresistance among PDAC 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 4613.

Abstract 7202: Strategy of targeting PKCβΙ protein to overcoming the acquired gemcitabine resistant pancreatic cancer cells

Abstract Pancreatic cancer (PC) is one of the most malignant cancers with limitation of treatment options such as resection surgery, radiation therapy and gemcitabine-based chemotherapy. To treating PC patients, gemcitabine-based chemotherapy is common and effective for PC, acquired gemcitabine resistance is one of the major reasons for treatment failure. Therefore, there is a need to suggest effective strategies and novel therapeutic approach for gemcitabine-resistant PC. Protein Kinase C β1 (PKC β1) is known as involved in multiple signal transduction related to oncogenic functions in cancer. We suggested that PKC β as one of the novel biomarker of drug resistance and further investigated the molecular mechanism. Among the natural products as small molecule inhibitor, we found that evodiamine has potent antitumor activity and various biological activities. Based on various biological activities of evodiamine, the scaffold was synthesized and investigated for antiproliferative and antitumor activities against gemcitabine resistant PANC-1 (PANC-GR) cells. Also, target validation experiment was employed to elucidate the molecular mechanism and verifying the target to overcoming gemcitabine resistance. Mechanistically, evodiamine derivatives have shown that inhibition of PKC β1 can modulate the proliferation of PANC-GR cells and exhibited tumor growth inhibitory activity in xenograft mouse model. Keywords: Pancreatic cancer cell, Gemcitabine resistance, Protein Kinase C β1 (PKC β1), Molecular mechanism Acknowledgements: This research was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (RS-2023-00273240). Citation Format: EunSeo Bae, Yijae Lim, Junhwa Hong, Woong Sub Byun, Simin Chun, Suckchang Hong, Sang Kook Lee. Strategy of targeting PKCβΙ protein to overcoming the acquired gemcitabine resistant 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 7202.

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.

Polyunsaturated fatty acids-induced ferroptosis suppresses pancreatic cancer growth

Despite recent advances in science and medical technology, pancreatic cancer remains associated with high mortality rates due to aggressive growth and no early clinical sign as well as the unique resistance to anti-cancer chemotherapy. Current numerous investigations have suggested that ferroptosis, which is a programed cell death driven by lipid oxidation, is an attractive therapeutic in different tumor types including pancreatic cancer. Here, we first demonstrated that linoleic acid (LA) and α-linolenic acid (αLA) induced cell death with necroptotic morphological change in MIA-Paca2 and Suit 2 cell lines. LA and αLA increased lipid peroxidation and phosphorylation of RIP3 and MLKL in pancreatic cancers, which were negated by ferroptosis inhibitor, ferrostatin-1, restoring back to BSA control levels. Similarly, intraperitoneal administration of LA and αLA suppresses the growth of subcutaneously transplanted Suit-2 cells and ameliorated the decreased survival rate of tumor bearing mice, while co-administration of ferrostatin-1 with LA and αLA negated the anti-cancer effect. We also demonstrated that LA and αLA partially showed ferroptotic effects on the gemcitabine-resistant-PK cells, although its effect was exerted late compared to treatment on normal-PK cells. In addition, the trial to validate the importance of double bonds in PUFAs in ferroptosis revealed that AA and EPA had a marked effect of ferroptosis on pancreatic cancer cells, but DHA showed mild suppression of cancer proliferation. Furthermore, treatment in other tumor cell lines revealed different sensitivity of PUFA-induced ferroptosis; e.g., EPA induced a ferroptotic effect on colorectal adenocarcinoma, but LA or αLA did not. Collectively, these data suggest that PUFAs can have a potential to exert an anti-cancer effect via ferroptosis in both normal and gemcitabine-resistant pancreatic cancer.

Tumor-derived interleukin 35 mediates the dissemination of gemcitabine resistance in pancreatic adenocarcinoma.

Rapid development of drug resistance after chemotherapy is a major cause of treatment failure in individuals with pancreatic ductal adenocarcinoma (PDAC). In this study, we illustrate that tumor-derived interleukin 35 (IL-35) mediates the accelerated resistance of PDAC to gemcitabine (GEM). We observe that GEM resistance can spread from GEM-resistant PDAC cells to GEM-sensitive cells, and that IL-35 is responsible for the propagation of chemoresistance, which is supported by sequencing and experimental data. Additionally, we discover that GEM-resistant cells have significantly higher levels of IL-35 expression. Mechanistically, aberrantly expressed IL-35 triggers transcriptional activation of SOD2 expression via GP130-STAT1 signaling, scavenging reactive oxygen species (ROS) and leading to GEM resistance. Furthermore, GEM treatment stimulates IL-35 expression through activation of the NF-κB pathway, resulting in acquired chemoresistance. In the mouse model, a neutralizing antibody against IL-35 enhances the tumor suppressive effect of GEM. Collectively, our data suggests that IL-35 is critical in mediating GEM resistance in pancreatic cancer, and therefore could be a valuable therapeutic target in overcoming PDAC chemoresistance.

Abstract A056: Targeting the mevalonate biosynthesis pathway in gemcitabine resistant pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a low survival rate. A lack of durable responses to chemotherapy renders its treatment particularly challenging and largely contributes to the devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDA therapy, but resistance remains a major hurdle in clinical care. Multiple mechanisms of chemoresistance have been attributed to rewired metabolic pathways in PDA cells. Accordingly, we hypothesized that altered metabolism in gemcitabine resistance represents a therapeutic vulnerability that can be targeted to improve response to treatment. To investigate metabolic vulnerabilities in gemcitabine resistance, we generated gemcitabine resistant murine PDA cell lines and assembled patient-derived gemcitabine high versus low responder organoids. Transcriptomic profiling revealed a downregulation of the mevalonate biosynthesis pathway and its rate-limiting enzyme, HMG-CoA reductase, upon the acquisition of gemcitabine resistance. Upregulation of this pathway has been proposed as a primary resistance mechanism to statins, HMG-CoA reductase inhibitors that are frequently prescribed to treat patients with hyperlipidemia. Accordingly, we postulated that statins would be more potent in gemcitabine resistant PDA cells than in their sensitive counterparts. Indeed, resistant cells were exquisitely susceptible to inhibition of the mevalonate pathway, an observation consistent across multiple clinically available statins. Downstream rescue experiments suggested that the main impact of blocking the mevalonate biosynthesis pathway is through lowered levels of geranylgeranyl pyrophosphate and reduced protein geranylation. To validate our findings in vivo, we implanted gemcitabine resistant murine PDA cells into syngeneic mice that were fed with a modified diet precluding geranylgeraniol. Here, the treatment combination gemcitabine and pitavastatin inhibited tumor growth of resistant allografts. Collectively, these data suggest that targeting the HMG-CoA reductase is a metabolic vulnerability that is gained in PDA cells upon the acquisition of gemcitabine resistance. The combination therapy gemcitabine and pitavastatin, two widely used compounds in patients, has potential to translate into a clinical benefit and improve survival in this deadly disease. Citation Format: Alica K. Beutel, Sabrina Calderon, Ethan Nghiem, Rima Singh, Cecily Anaraki, Kevin Gulay, Herve Tiriac, Alexander Kleger, Thomas Seufferlein, Alexander Muir, Cholsoon Jang, Dennis Juarez, David Fruman, Christopher Halbrook. Targeting the mevalonate biosynthesis pathway in gemcitabine resistant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A056.

Cancer-associated fibroblasts promote stemness maintenance and gemcitabine resistance via HIF-1α/miR-21 axis under hypoxic conditions in pancreatic cancer.

Gemcitabine (GEM) resistance affects chemotherapy efficacy of pancreatic cancer (PC). Cancer-associated fibroblasts (CAFs) possess the ability of regulating chemoresistance. This study probed the mechanism of hypoxia-treated CAFs regulating cell stemness and GEM resistance in PC. Miapaca-2/SW1990 were co-cultured with PC-derived CAFs under normoxic/hypoxic conditions. Cell viability/self-renewal ability was determined by MTT/sphere formation assays, respectively. Protein levels of CD44, CD133, Oct4, and Sox2 were determined by western blot. GEM tumoricidal assay was performed. PC cell GEM resistance was evaluated by MTT assay. CAFs were cultured at normoxia/hypoxia. HIF-1α and miR-21 expression levels were assessed by RT-qPCR and western blot, with their binding sites and binding relationship predicted and verified. CAF-extracellular vesicles (EVs) were incubated with Miapaca-2 cells. The RAS/AKT/ERK pathway activation was detected by western blot. PC xenograft models were established and treated with hypoxic CAF-EVs and GEM. CAFs and PC cell co-culture increased cell stemness maintenance, GEM resistance, cell viability, stem cell sphere number, and protein levels of CD44, CD133, Oct4, and Sox2, and weakened GEM tumoricidal ability to PC cells, with the effects further enhanced by hypoxia. Hypoxia induced HIF-1α and miR-21 overexpression in CAFs. Hypoxia promoted CAFs to secrete high-level miR-21 EVs via the HIF-1α/miR-21 axis, and activated the miR-21/RAS/AKT/ERK pathway. CAF-EVs promoted GEM resistance in PC via the miR-21/RAS/ATK/ERK pathway in vivo. Hypoxia promoted CAFs to secrete high-level miR-21 EVs through the HIF-1α/miR-21 axis, and activated the miR-21/RAS/AKT/ERK pathway via EVs to trigger stemness maintenance and GEM resistance in PC.

Antiproliferative Activity of Piceamycin by Regulating Alpha-Actinin-4 in Gemcitabine-Resistant Pancreatic Cancer Cells.

Although gemcitabine-based regimens are widely used as an effective treatment for pancreatic cancer, acquired resistance to gemcitabine has become an increasingly common problem. Therefore, a novel therapeutic strategy to treat gemcitabine-resistant pancreatic cancer is urgently required. Piceamycin has been reported to exhibit antiproliferative activity against various cancer cells; however, its underlying molecular mechanism for anticancer activity in pancreatic cancer cells remains unexplored. Therefore, the present study evaluated the antiproliferation activity of piceamycin in a gemcitabine-resistant pancreatic cancer cell line and patient-derived pancreatic cancer organoids. Piceamycin effectively inhibited the proliferation and suppressed the expression of alpha-actinin-4, a gene that plays a pivotal role in tumorigenesis and metastasis of various cancers, in gemcitabine-resistant cells. Long-term exposure to piceamycin induced cell cycle arrest at the G0/G1 phase and caused apoptosis. Piceamycin also inhibited the invasion and migration of gemcitabine-resistant cells by modulating focal adhesion and epithelial-mesenchymal transition biomarkers. Moreover, the combination of piceamycin and gemcitabine exhibited a synergistic antiproliferative activity in gemcitabine-resistant cells. Piceamycin also effectively inhibited patient-derived pancreatic cancer organoid growth and induced apoptosis in the organoids. Taken together, these findings demonstrate that piceamycin may be an effective agent for overcoming gemcitabine resistance in pancreatic cancer.

LncRNAs as nodes for the cross-talk between autophagy and Wnt signaling in pancreatic cancer drug resistance.

Pancreatic cancer is a malignancy with high mortality. In addition to the few symptoms until the disease reaches an advanced stage, the high fatality rate is attributed to its rapid development, drug resistance and lack of appropriate treatment. In the selection and research of therapeutic drugs, gemcitabine is the first-line drug for pancreatic cancer. Solving the problem of gemcitabine resistance in pancreatic cancer will contribute to the progress of pancreatic cancer treatment. Long non coding RNAs (lncRNAs), which are RNA transcripts longer than 200 nucleotides, play vital roles in cellular physiological metabolic activities. Currently, our group and others have found that some lncRNAs are aberrantly expressed in pancreatic cancer cells, which can regulate the process of cancer through autophagy and Wnt/β-catenin pathways simultaneously and affect the sensitivity of cancer cells to therapeutic drugs. This review presents an overview of the recent evidence concerning the node of lncRNA for the cross-talk between autophagy and Wnt/β-catenin signaling in pancreatic cancer, together with the practicability of lncRNAs and the core regulatory factors as targets in therapeutic resistance.

Synergistic effects of resveratrol with gemcitabine in pancreatic cancer chemotherapy by inhibiting the c-Met/PARP1 axis

Objective: Pancreatic cancer is a highly malignant tumor of the digestive tract with a dismal prognosis. A key challenge of pancreatic cancer is its resistance to chemotherapy. The c-Met/PARP1 axis plays an important role in the therapeutic resistance of breast cancer and hepatocellular carcinoma. Therefore, this study aims to explore potential therapeutic targets for improving chemotherapy for pancreatic cancer and the underlying mechanisms. Methods: Gemcitabine-resistant pancreatic cancer cell lines were constructed by our laboratory using a continuous low-concentration gemcitabine induction method. The proliferation and apoptosis of combination therapy were examined using flow cytometry and comet assay. Synergistic effects of two drugs were determined by Chou-Talalay's combination index (CI). The interactions between proteins were predicted using the AutoDock model and detected through Coimmunoprecipitation assays. Results: The combination of resveratrol and gemcitabine inhibited proliferation and promoted apoptosis of pancreatic cancer cells. We found that c-Met and PARP1 were highly expressed in gemcitabine-resistant pancreatic cancer cells. However, resveratrol inhibited their expression and improved the effectiveness of gemcitabine-induced DNA damage. In addition, our data demonstrated that resveratrol and gemcitabine had synergistic effects (CI < 1). Furthermore, the protein interactions between c-Met and PARP1 were attenuated after the early stage of resveratrol intervention. Using AutoDock models, we predicted the potential binding sites where resveratrol could impact the protein interaction between c-Met (tyrosine kinase domain) and PARP1 (CAT domain). Conclusion: Our results suggested that the synergistic effects of resveratrol with gemcitabine depend on the c-Met/PAPR1 axis. Using resveratrol as a combined chemotherapy agent may have clinical benefits for patients with refractory pancreatic cancer.

Prognostic value of RRM1 and its effect on chemoresistance in pancreatic cancer.

Pancreatic cancer (PC) remains a lethal disease, and gemcitabine resistance is prevalent. However, the biomarkers suggestive of gemcitabine resistance remain unclear. Bioinformatic tools identified ribonucleotide reductase catalytic subunit M1 (RRM1) in gemcitabine-related datasets. A cox regression model revealed the predictive value of RRM1 with clinical features. An external clinical cohort confirmed the prognostic value of RRM1. RRM1 expression was validated in gemcitabine-resistant cells in vitro and in orthotopic PC model. CCK8, flow cytometry, transwell migration, and invasion assays were used to explore the effect of RRM1 on gemcitabine-resistant cells. The CIBERSORT algorithm investigated the impact of RRM1 on immune infiltration. The constructed nomogram based on RRM1 effectively predicted prognosis and was further validated. Moreover, patients with higher RRM1 had shorter overall survival. RRM1 expression was significantly higher in PC tissue and gemcitabine-resistant cells in vitro and in vivo. RRM1 knockdown reversed gemcitabine resistance, inhibited migration and invasion. The infiltration levels of CD4 + T cells, CD8 + T cells, neutrophils, and plasma cells correlated markedly with RRM1 expression, and communication between tumor and immune cells probably depends on NF-κB/mTOR signaling. RRM1 may be a potential marker for prognosis and a target marker for gemcitabine resistance in PC.

Efficacy of HMJ-38, a new quinazolinone analogue, against the gemcitabine-resistant MIA-PaCa-2 pancreatic cancer cells.

Gemcitabine is frequently utilized to treat pancreatic cancer. The purpose of our study was to create a gemcitabine-resistant MIA-PaCa-2 pancreatic cancer cell line (MIA-GR100) and to evaluate the anti-pancreatic cancer efficacy of HMJ-38, a new quinazolinone analogue. Compared to their parental counterparts, MIA-PaCa-2, established MIA-GR100 cells were less sensitive to gemcitabine. MIA-GR100 cell viability was not affected by 10, 50 and 100 nM gemcitabine concentrations. HMJ-38 reduced MIA-GR100 cell growth and induced autophagy and apoptosis. When stained with monodansylcadaverine (MDC), acridine orange (AO), and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), MIA-GR100 cells shrunk, punctured their membranes, and produced autophagy vacuoles and apoptotic bodies. Combining chloroquine (CQ) and 3-methyladenine (3-MA) with HMJ-38 dramatically reduced cell viability, indicating that autophagy function as a cytoprotective mechanism. MIA-GR100 cells treated with both z-VAD-FMK and HMJ-38 were much more viable than those treated with HMJ-38 alone. HMJ-38 promotes apoptosis in MIA-GR100 cells by activating caspases. Epidermal growth factor receptor (EGFR) is one of HMJ-38's principal targets, as determined via in silico target screening with network prediction. HMJ-38 also inhibited EGFR kinase activity and EGFR-associated signaling in MIA-GR100 cells. HMJ-38 may be an effective chemotherapeutic adjuvant for gemcitabine-resistant pancreatic cancer cells, in which it induces an antitumor response.

Shikonin reverses cancer-associated fibroblast-induced gemcitabine resistance in pancreatic cancer cells by suppressing monocarboxylate transporter 4-mediated reverse Warburg effect

BackgroundGemcitabine is a first-line chemotherapeutic agent for pancreatic cancer (PC); however, most patients who receive adjuvant gemcitabine rapidly develop resistance and recurrence. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor stroma that contribute to gemcitabine-resistance. There is thus an urgent need to find a novel therapeutic strategy to improve the efficacy of gemcitabine in PC cells under CAF-stimulation. PurposeTo investigate if shikonin potentiates the therapeutic effects of gemcitabine in PC cells with CAF-induced drug resistance. MethodsPC cell-stimulated fibroblasts or primary CAFs derived from PC tissue were co-cultured with PC cells to evaluate the ability of shikonin to improve the chemotherapeutic effects of gemcitabine in vitro and in vivo. Glucose uptake assay, ATP content analysis, lactate measurement, real-time PCR, immunofluorescence staining, western blot, and plasmid transfection were used to investigate the underlying mechanism. ResultsCAFs were innately resistant to gemcitabine, but shikonin suppressed the PC cell-induced transactivation and proliferation of CAFs, reversed CAF-induced resistance, and restored the therapeutic efficacy of gemcitabine in the co-culture system. In addition, CAFs underwent a reverse Warburg effect when co-cultured with PC cells, represented by enhanced aerobic glycolytic metabolism, while shikonin reduced aerobic glycolysis in CAFs by reducing their glucose uptake, ATP concentration, lactate production and secretion, and glycolytic protein expression. Regarding the mechanism underlying these sensitizing effects, shikonin suppressed monocarboxylate transporter 4 (MCT4) expression and cellular membrane translocation to inhibit aerobic glycolysis in CAFs. Overexpression of MCT4 accordingly reversed the inhibitory effects of shikonin on PC cell-induced transactivation and aerobic glycolysis in CAFs, and reduced its sensitizing effects. Furthermore, shikonin promoted the effects of gemcitabine in reducing the growth of tumors derived from PC cells and CAF co-inoculation in BALB/C mice, with no significant systemic toxicity. ConclusionThese results indicate that shikonin reduced MCT4 expression and activation, resulting in inhibition of aerobic glycolysis in CAFs and overcoming CAF-induced gemcitabine resistance in PC. Shikonin is a promising chemosensitizing phytochemical agent when used in combination with gemcitabine for PC treatment. The results suggest that disrupting the metabolic coupling between cancer cells and stromal cells might provide an attractive strategy for improving gemcitabine efficacy.

Differential Sensitivity to Ionizing Radiation in Gemcitabine-Resistant and Paclitaxel-Resistant Pancreatic Cancer Cells

IntroductionChemoresistance remains a major challenge in treating pancreatic ductal adenocarcinoma (PDAC). While chemoradiation has proven effective in other tumor types, such as head-and-neck squamous cell carcinoma, its role in PDAC and its impact on acquired chemoresistance have yet to be fully explored. In this study, we investigated the sensitivity of gemcitabine- and paclitaxel-resistant PDAC cells to ionizing radiation (IR) and their underlying mechanisms. Materials and MethodsGemcitabine-resistant (GR) and paclitaxel-resistant (PR) clones were generated from PANC-1, PATU-T and SUIT2-007 pancreatic cancer cell lines. Cell survival after radiation was assessed using clonogenic assay, SRB assay, apoptosis and spheroid growth by bioluminescence while radiation-induced DNA damage was evaluated with Western blot, XL-PCR, ROS production and immunofluorescence. Autophagy and modulation of Hippo pathway were investigated using proteomics, Western blot, immunofluorescence and RT-qPCR. ResultsIn both 2D and 3D settings, PR cells were more sensitive to IR and showed decreased β-globin amplification indicating more DNA damage accumulation as compared to GR or wild-type (WT) cells after 24 hours. Proteomics analysis of PATU-T PR revealed that the protein MST4, a kinase involved in autophagy and Hippo signaling pathway, was highly downregulated. Differential association was found between autophagy and radiation treatment depending on the cell model. Interestingly, increased YAP nuclear localization and downstream Hippo pathway target gene expression were observed in in SUIT2-007 PR cells in response to IR, as compared to WT and GR. ConclusionThis is the first study investigating the potential of IR in targeting PDAC cells with acquired chemoresistance. Our results demonstrate that PR cells exhibit enhanced sensitivity to IR due to greater accumulation of DNA damage. Additionally, depending on the specific cellular context, radiation-induced modulation of autophagy and the Hippo pathway emerged as potential underlying mechanisms, and these findings have the potential to inform personalized treatment strategies for patients with acquired chemoresistance.

SLC38A5 Modulates Ferroptosis to Overcome Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic cancer is characterized by a poor prognosis, with its five-year survival rate lower than that of any other cancer type. Gemcitabine, a standard treatment for pancreatic cancer, often has poor outcomes for patients as a result of chemoresistance. Therefore, novel therapeutic targets must be identified to overcome gemcitabine resistance. Here, we found that SLC38A5, a glutamine transporter, is more highly overexpressed in gemcitabine-resistant patients than in gemcitabine-sensitive patients. Furthermore, the deletion of SLC38A5 decreased the proliferation and migration of gemcitabine-resistant PDAC cells. We also found that the inhibition of SLC38A5 triggered the ferroptosis signaling pathway via RNA sequencing. Also, silencing SLC38A5 induced mitochondrial dysfunction and reduced glutamine uptake and glutathione (GSH) levels, and downregulated the expressions of GSH-related genes NRF2 and GPX4. The blockade of glutamine uptake negatively modulated the mTOR-SREBP1-SCD1 signaling pathway. Therefore, suppression of SLC38A5 triggers ferroptosis via two pathways that regulate lipid ROS levels. Similarly, we observed that knockdown of SLC38A5 restored gemcitabine sensitivity by hindering tumor growth and metastasis in the orthotopic mouse model. Altogether, our results demonstrate that SLC38A5 could be a novel target to overcome gemcitabine resistance in PDAC therapy.

Dysregulation of the circ_0087502/miR-1179/TGFBR2 pathway supports gemcitabine resistance in pancreatic cancer

ABSTRACT Background Circular RNAs (circRNAs) are a cohort of non-coding RNAs generated by back-splicing events. Accumulating evidence supports the crucial role of circRNAs in human tumorigenesis, metastasis, and chemoresistance. However, the role and mechanism of circRNA circ_0087502 in pancreatic cancer are yet unknown. Methods The expression and function of circ_0087502 in pancreatic cancer were investigated using qRT-PCR and cell experiments. The predicted binding between circ_0087502 and microRNA-1179 (miR-1179), and between miR-1179 and TGFBR2, were examined using reporter assays. Results Pancreatic cancer tissues and cell lines were discovered to express circ_0087502 at higher levels. Patients with pancreatic cancer who express circ_0087502 at high levels have a worse prognosis. In addition, circ_0087502 knockdown reduced the proliferation, migration, and invasion of pancreatic cancer cells and made them more sensitive to gemcitabine treatment. We found that circ_0087502 worked as a sponge for miR-1179, allowing miR-1179 to bind to the critical oncogene TGFBR2 in its 3’-untranslated region (3’−UTR). Pancreatic cancer cells were highly resistant to gemcitabine and had increased proliferation, migration, and invasion when miR-1179 was inhibited or overexpressed. Conclusion These results confirm that circ_0087502 activates the miR-1179/TGFBR2 axis to promote gemcitabine resistance in pancreatic cancer. Thus, our data might lay the groundwork for developing novel therapeutic strategies targeting circ_0087502 in pancreatic cancer patients.

The role of LOXL2 induced by glucose metabolism-activated NF-κB in maintaining drug resistance through EMT and cancer stemness in gemcitabine-resistant PDAC

Gemcitabine is considered a standard treatment for pancreatic cancer, but developing drug resistance greatly limits the effectiveness of chemotherapy and increases the rate of recurrence. Lysyl oxide-like 2 (LOXL2) is highly expressed in pancreatic cancer and is involved in carcinogenesis and EMT regulation. However, studies on the role of LOXL2 in drug resistance are limited. Here, we investigated the mechanism of LOXL2 induction and the effect of LOXL2 on EMT and CSC in gemcitabine-resistant pancreatic cancer. Glucose metabolism was activated in gemcitabine-resistant pancreatic cancer cells, and NF-κB signaling was regulated accordingly. Activated NF-κB directly induces transcription by binding to the promoters of LOXL2 and ZEB1. The EMT process was significantly inhibited by the coregulation of ZEB1 and LOXL2. In addition, LOXL2 inhibition reduced the expression of cancer stemness markers and stemness by regulating MAPK signaling activity. LOXL2 inhibits tumor growth of gemcitabine-resistant pancreatic cancer cells and increases the sensitivity to gemcitabine in mouse models.Key messagesWe identified a specific mechanism for inducing LOXL2 overexpression in gemcitabine-resistant pancreatic cancer. Taken together, our results suggest LOXL2 has an important regulatory role in maintaining gemcitabine resistance and may be an effective therapeutic target to treat pancreatic cancer.

Photodynamic eradication of intratumoral microbiota with bacteria‐targeted micelles overcomes gemcitabine resistance of pancreatic cancer

AbstractIncreasing evidence suggests that intratumoral microbiota plays a pivotal role in tumor progression, immunosurveillance, metastasis, and chemosensitivity. Particularly, in pancreatic ductal adenocarcinoma, tumor‐resident Gammaproteobacteria could transform the chemotherapeutic drug gemcitabine (Gem) into its inactive form, thus rendering chemotherapy ineffective. Herein, a strategy for selectively eradicating intratumoral bacteria was described for overcoming Gem resistance in a pancreatic cancer animal model. An antimicrobial peptide was linked with photosensitizer through a poly (ethylene glycol) chain, which can self‐assemble into micelles with a diameter of ∼20 nm. The micelles could efficiently kill bacteria under light irradiation by inducing membrane depolarization, thereby inhibiting Gem metabolism. In a bacteria‐resident pancreatic cancer animal model, the selective photodynamic eradication of intratumoral bacteria was demonstrated to efficiently reverse Gem resistance. This research highlights antibacterial photodynamic therapy as a promising adjuvant strategy for cancer therapy by modulating intratumoral microbiota.