Development Of Gemcitabine Resistance Research Articles

Epigenetic Activation of the CMTM6-IGF2BP1-EP300 Positive Feedback Loop Drives Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a dismal prognosis. Gemcitabine-based chemotherapy has emerged as a first-line treatment for PDAC. However, the development of gemcitabine resistance often results in therapeutic failure. In order to uncover the underlying mechanisms of gemcitabine resistance, gemcitabine-resistant PDAC cell lines and patient-derived xenograft (PDX) models are established and subjected to RNA sequencing. It is found that CMTM6 is closely related to gemcitabine resistance in PDAC. Multi-omics analysis revealed that EP300-mediated H3K27ac modification is involved in the transcriptional activation of CMTM6, which maintains IGF2BP1 expression by preventing its ubiquitination. The m6A reader IGF2BP1 stabilizes the EP300 and MYC mRNAs by recognizing m6A modifications, forming a positive feedback loop that enhances tumor stemness and ultimately contributes to PDAC resistance. The combined application of the EP300 inhibitor inobrodib and gemcitabine exerts a synergistic effect on PDAC. Overall, these findings reveal that the EP300-CMTM6-IGF2BP1 positive feedback loop facilitates gemcitabine resistance via epigenetic reprogramming and the combined use of inobrodib and gemcitabine represents a promising strategy for overcoming chemoresistance in PDAC, warranting further investigation in clinical trials.

Exploring epigenetic dynamics unveils a super-enhancer-mediated NDRG1-β-catenin axis in modulating gemcitabine resistance in pancreatic cancer

Chemoresistance remains a formidable challenge in pancreatic ductal adenocarcinoma (PDAC) treatment, necessitating a comprehensive exploration of underlying molecular mechanisms. This work aims to investigate the dynamic epigenetic landscape during the development of gemcitabine resistance in PDAC, with a specific focus on super-enhancers and their regulatory effects. We employed well-established gemcitabine-resistant (Gem-R) PDAC cell lines to perform high-throughput analyses of the epigenome, enhancer connectome, and transcriptome. Our findings revealed notable alterations in the epigenetic landscape and genome architecture during the transition from gemcitabine-sensitive to -resistant PDAC cells. Remarkably, we observed substantial plasticity in the activation status of super-enhancers, with a considerable proportion of these cis-elements becoming deactivated in chemo-resistant cells. Furthermore, we pinpointed the NDRG1 super-enhancer (NDRG1-SE) as a crucial regulator in gemcitabine resistance among the loss-of-function super-enhancers. NDRG1-SE deactivation induced activation of WNT/β-catenin signaling, thereby conferring gemcitabine resistance. This work underscores a NDRG1 super-enhancer deactivation-driven β-catenin pathway activation as a crucial regulator in the acquisition of gemcitabine-resistance. These findings advance our understanding of PDAC biology and provide valuable insights for the development of effective therapeutic approaches against chemoresistance in this malignant disease.

Niban apoptosis regulator 1 promotes gemcitabine resistance by activating the focal adhesion kinase signaling pathway in bladder cancer.

Although intravesical gemcitabine (GEM) chemotherapy (IGC) can effectively reduce the recurrence risk of non-muscle invasive bladder cancer (NMIBC), the development of GEM resistance may occur and result in cancer recurrence and disease progression. Herein, a label-free proteomics approach was used to characterize the proteomic profiles of primary/post-IGC recurrent NMIBC. A total of 218 proteins were found to be differentially expressed in paired primary and post-IGC recurrent NMIBC. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that multiple signaling pathways including “focal adhesion” were highly enriched in recurrent NMIBC. Niban apoptosis regulator 1 (NIBAN1) was identified as the top upregulated protein in recurrent NMIBC. Highly increased NIBAN1 expression was observed in a number of GEM-resistant cancer cell lines and in post-IGC recurrent NMIBC specimens. Manipulation of NIBAN1 expression affected the chemosensitivity to GEM in bladder cancer cell models. Moreover, NIBAN1 also regulated focal adhesion/focal adhesion kinase (FAK) signaling activation in bladder cancer cell lines. Highly elevated FAK (pY397) expression was observed in post-IGC recurrent NMIBC specimens, which was positively correlated with NIBAN1 expression. Knockdown of FAK markedly attenuated GEM resistance in GEM-resistant bladder cancer cells. In vivo studies demonstrated that knockdown of NIBAN1 disrupted FAK signaling and sensitized GEM-resistant bladder cancer cells to GEM treatment. Our findings suggest that NIBAN1 might regulate FAK signaling activation to promote GEM resistance in bladder cancer. Targeting NIBAN1/FAK signaling may help sensitize bladder cancer cells to GEM treatment.

Synergistic Interactions between GW8510 and Gemcitabine in an In Vitro Model of Pancreatic Cancer.

One of the main reasons for the poor survival rates of pancreatic cancer patients is the development of gemcitabine resistance, indicating that novel treatment strategies that have the ability to improve gemcitabine sensitivity are in need to combat this devastating disease. TCGA PAAD data was used to determine the clinicopathological significance of high RRM2 (Ribonucleotide reductase subunit M2) expression for Pancreatic Ductal Adenocarcinoma (PDAC). The effects of GW8510 and gemcitabine on PANC-1 cell viability were determined using WST-8 assay. The potential synergistic interaction between GW8510 and gemcitabine was evaluated by the Combination Index (CI) analysis. The effects of GW8510 treatment on apoptosis, cell cycle, and cell migration, either in combination with gemcitabine or alone, were investigated. The effect of GW8510 on RRM2 protein levels was evaluated using ELISA assay. RRM2 is significantly over-expressed in PDAC compared to healthy pancreatic tissues (p <0.0001). RRM2 mRNA expression was found to be significantly correlated with the overall survival rate of patients (HR=2.17 [1.44-3.27], p=0.00016) and the pathological stages of the disease (p=0.0054). GW8510 significantly decreased the RRM2 protein levels compared to the control. Cell viability analysis showed that GW8510 has a similar effect to gemcitabine in inhibiting PANC-1 cell viability. GW8510 was found to synergize with gemcitabine to inhibit PANC-1 cell viability and migration. However, the effects of GW8510 on PANC-1 cells could not be explained by induction of apoptosis or cell cycle arrest. Targeting RRM2 using GW8510 may have the potential to increase gemcitabine sensitivity in pancreatic cancer.

Secretion of fibronectin by human pancreatic stellate cells promotes chemoresistance to gemcitabine in pancreatic cancer cells

BackgroundGemcitabine remains a cornerstone in chemotherapy of pancreatic ductal adenocarcinoma (PDAC) despite suboptimal clinical effects that are partly due to the development of chemoresistance. Pancreatic stellate cells (PSCs) of the tumor stroma are known to interact with pancreatic cancer cells (PCCs) and influence the progression of PDAC through a complex network of signaling molecules that involve extracellular matrix (ECM) proteins. To understand tumor-stroma interactions regulating chemosensitivity, the role of PSC-secreted fibronectin (FN) in the development of gemcitabine resistance in PDAC was examined.MethodsPSC cultures obtained from ten different human PDAC tumors were co-cultured with PCC lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1 and SW-1990) either directly, or indirectly via incubation with PSC-conditioned medium (PSC-CM). Gemcitabine dose response cytotoxicity was determined using MTT based cell viability assays. Protein expression was assessed by western blotting and immunofluorescence. PSC-CM secretome analysis was performed by proteomics-based LC-MS/MS, and FN content in PSC-CM was determined with ELISA. Radiolabeled gemcitabine was used to determine the capacity of PCCs to uptake the drug.ResultsIn both direct and indirect co-culture, PSCs induced varying degrees of resistance to the cytotoxic effects of gemcitabine among all cancer cell lines examined. A variable degree of increased phosphorylation of ERK1/2 was observed across all PCC lines upon incubation with PSC-CM, while activation of AKT was not detected. Secretome analysis of PSC-CM identified 796 different proteins, including several ECM-related proteins such as FN and collagens. Soluble FN content in PSC-CM was detected in the range 175–350 ng/ml. Neither FN nor PSC-CM showed any effect on PCC uptake capacity of gemcitabine. PCCs grown on FN-coated surface displayed higher resistance to gemcitabine compared to cells grown on non-coated surface. Furthermore, a FN inhibitor, synthetic Arg-Gly-Asp-Ser (RGDS) peptide significantly inhibited PSC-CM-induced chemoresistance in PCCs via downregulation of ERK1/2 phosphorylation.ConclusionsThe findings of this study suggest that FN secreted by PSCs in the ECM plays a key role in the development of resistance to gemcitabine via activation of ERK1/2. FN-blocking agents added to gemcitabine-based chemotherapy might counteract chemoresistance in PDAC and provide better clinical outcomes.

The distinct role of CD73 in the progression of pancreatic cancer

Recent studies have shown that the non-enzymatic function of CD73 plays a key role in tumor progression, but this function of CD73 in pancreatic cancer cells has not been studied. Furthermore, little is known about the mechanism involved in CD73 regulation in tumors. Here, we found that CD73 expression was upregulated in pancreatic ductal adenocarcinoma (PDAC) and that its expression correlated with poor prognosis. CD73 knockdown inhibited cell growth and induced G1 phase arrest via the AKT/ERK/cyclin D signaling pathway. We also found that tumor necrosis factor receptor (TNFR) 2 was involved in CD73-induced AKT and ERK signaling pathway activation in PDAC. Further, miR-30a-5p overexpression significantly increased the cytotoxic effect of gemcitabine in pancreatic cancer by directly targeting CD73 messenger RNA (mRNA), suggesting that regulation of the miR-30a-5p/CD73 axis may play an important role in the development of gemcitabine resistance in pancreatic cancer. In summary, this regulatory network of CD73 appears to represent a new molecular mechanism underlying PDAC progression, and the mechanistic interaction between miR-30a-5p, CD73, and TNFR2 may provide new insights into therapeutic strategies for pancreatic cancer.Key messagesCD73 was upregulated in PDAC and correlated with poor prognosis.CD73 knockdown inhibited cell growth and induced G1 phase arrest.TNFR2 was involved in CD73-induced AKT and ERK signaling pathway.miR-30a-5p targeted CD73 and increased the sensitivity to gemcitabine.

Down-regulation of miR-30a-5p is Associated with Poor Prognosis and Promotes Chemoresistance of Gemcitabine in Pancreatic Ductal Adenocarcinoma.

MicroRNA-30a-5p (miR-30a-5p) plays an important role in many biological and pathological processes, and therefore has been studied extensively. However, its expression and function in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Furthermore, whether miR-30a-5p affects sensitivity of PDAC cells to gemcitabine (GEM) is worthy of further exploration. The results showed that miR-30a-5p expression in pancreatic cancer was decreased and the down-regulated expression correlated with poor prognosis, while up-regulating miR-30a-5p suppressed tumor cell proliferation, cell cycle and increased apoptosis. MiRNA expression profiles between gemcitabine-resistant pancreatic cancer cells and parental pancreatic cancer cells showed significant change of miR-30a-5p expression. Besides, up-regulating miR-30a-5p in PDAC significantly increased the chemosensitivity of gemcitabine. Furthermore, FOXD1 is a direct target of miR-30a-5p and the miR-30a-5p/FOXD1/ERK axis may play an important role in the development of gemcitabine resistance in pancreatic cancer. In summary, our study showed that miR-30a-5p increases the sensitivity of pancreatic cancer to gemcitabine, and it may be a potential therapeutic target to overcome gemcitabine resistance.

MiR-129-5p inhibits gemcitabine resistance and promotes cell apoptosis of bladder cancer cells by targeting Wnt5a.

Gemcitabine resistance is a major obstacle for effective treatment of bladder cancer. This study was aimed to investigate the potential role of miR-129-5p in the development of gemcitabine resistance in bladder cancer cells and its underlying mechanism. The IC50 for gemcitabine in 20 bladder cancer cells was first profiled from Genomics of Drug Sensitivity in Cancer. miR-129-5p level and gene mRNA expression were detected using quantitative real-time PCR (qRT-PCR). Cell viability, apoptosis, and gene protein level were assessed by MTT, flow cytometry, and Western blot, respectively. Regulatory relationship between Wnt5a and miR-129-5p was determined using luciferase reporter assay. We found that down-regulated miR-129-5p level contributed to gemcitabine resistance in bladder cancer cells and tissues. We also observed restoration of miR-129-5p could significantly increase cell sensitivity to gemcitabine and promote cell apoptosis. Mechanism analysis revealed that Wnt5a is a direct target gene of miR-129-5p and knock-down of Wnt5a reversed gemcitabine resistance. Taken together, our findings indicate that miR-129-5p and Wnt5a may be novel therapeutic targets for overcoming gemcitabine resistance in bladder cancer treatment.

RNA interference targeting ETS-1 downregulates RRM2 and enhances gemcitabine sensitivity in pancreatic cancer cells

Aberrant expression of the Ets-1 transcription factor is increasingly associated with the progression of several human cancers. Previously we reported Ets-1 over-expression in gemcitabine resistance pancreatic cancer cell lines. In the present study, we identified Ribonucleotide reductase–M2 subunit, a key enzyme involved in the DNA synthesis is elevated in gemcitabine-resistant PANC1 cells. Silencing of Ets-1 expression in resistant cell line diminishes the Ribonucleotide reductase–M2 expression. The RAS/ERK pathway is commonly activated in carcinomas and promotes oncogenesis by altering transcriptional programs. Ets-1 silencing abrogates the expression of Ribonucleotide reductase–M2 levels in parental cells treated with epidermal growth factor to further activate the RAS/ERK pathway. Our findings clearly demonstrate the involvement of RAS/ERK-mediated Ets-1 pathway in the development of gemcitabine resistance by over-expressing Ribonucleotide reductase–M2.

MiRNA-21 induces epithelial to mesenchymal transition and gemcitabine resistance via the PTEN/AKT pathway in breast cancer.

Acquisition of gemcitabine resistance in breast cancer has not been fully clarified. Prior studies suggest that miRNAs are important to chemoresistance in solid tumors and we confirmed that miR-21 is involved in the development of gemcitabine resistance. Epithelial-to-mesenchymal transition (EMT) and AKT pathway activation were noted to be important to this resistance as well. PTEN, a direct target gene of miR-21, was significantly downregulated in gemcitabine-resistant breast cancer cells and restoration of PTEN expression blocked miR-21-induced EMT and gemcitabine resistance. Our data offer novel insight into gemcitabine resistance in breast cancer and suggest that miR-21 may be used to predict optimal breast cancer therapy and may be a potential therapeutic target for reversing gemcitabine resistance.

Identifying microRNA-mRNA regulatory network in gemcitabine-resistant cells derived from human pancreatic cancer cells.

Pancreatic cancer is unresectable in over 80 % of patients owing to difficulty in early diagnosis. Chemotherapy is the most frequently adopted therapy for advanced pancreatic cancer. The development of drug resistance to gemcitabine (GEM), which is always used in standard chemotherapy, often results in therapeutic failure. However, the molecular mechanisms underlying the gemcitabine resistance remain unclear. Therefore, we sought to explore the microRNA-mRNA network that is associated with the development of gemcitabine resistance and to identify molecular targets for overcoming the gemcitabine resistance. By exposing SW1990 pancreatic cancer cells to long-term gemcitabine with increasing concentrations, we established a gemcitabine-resistant cell line (SW1990/GEM) with a high IC50 (the concentration needed for 50 % growth inhibition, 847.23 μM). The mRNA and microRNA expression profiles of SW1990 cells and SW1990/GEM cells were determined using RNA-seq analysis. By comparing the results in control SW1990 cells, 507 upregulated genes and 550 downregulated genes in SW1990/GEM cells were identified as differentially expressed genes correlated with gemcitabine sensitivity. Gene ontology (GO) analysis showed that the differentially expressed genes were related to diverse biological processes. The upregulated genes were mainly associated with drug response and apoptosis, and the downregulated genes were correlated with cell cycle progression and RNA splicing. Concurrently, the differentially expressed microRNAs, which are the important player in drug resistance development, were also examined in SW1990/GEM cells, and 56 differential microRNAs were identified. Additionally, the expression profiles of selected genes and microRNAs were confirmed by using Q-PCR assays. Furthermore, combining the differentially expressed microRNAs and mRNAs as well as the predicted targets for these microRNAs, a core microRNA-mRNA regulatory network was constructed, which included hub microRNAs, such as hsa-miR-643, hsa-miR-4644, hsa-miR-4650-5p, hsa-miR-4455, hsa-miR-1261, and hsa-miR-3676. The predicted targets of these hub microRNAs in the microRNA-mRNA network were also observed in the identified differential genes. As a result, a differential gene and microRNA expression pattern was constructed in gemcitabine-resistant pancreatic cancer cells. Therefore, these data may be useful for the detection and treatment of drug resistance in pancreatic cancer patients, and the microRNA-mRNA network-based analysis is expected to be more effective and provides deep insights into the molecular mechanism of drug resistance.

Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells

Gemcitabine, a novel pyrimidine nucleoside analog, has become the standard chemotherapeutic agent for pancreatic cancer patients. The clinical impact of gemcitabine remains modest owing to the high degree of inherent and acquired resistance. There are various lines of evidence that confirm the role of Ets-1, a proto-oncoprotein, in tumor invasion, progression, and chemoresistance. This study examines a hypothesis that implicates Ets-1 in the development of gemcitabine-resistance in pancreatic cancer cells. Ets-1 protein expression was assessed in parental pancreatic cancer cells and their gemcitabine-resistant clones. Western blot analysis revealed elevated levels of Ets-1 protein expression in gemcitabine-resistant PANC1GemRes (4.8-fold increase; P < 0.05), MIA PaCa2GemRes (3.2-fold increase; P < 0.05), and Capan2GemRes (2.1-fold increase; P < 0.05) cells as compared to their parental counterparts. A time course analysis was conducted to determine the change in Ets-1 expression in the parental cells after incubation with gemcitabine. Reverse transcriptase quantitative real-time PCR (RT-qPCR) and Western blot analysis revealed a significant increase in Ets-1 expression. All the three parental cells incubated with gemcitabine showed elevated Ets-1 protein expression at 6 h. By 24 h, the expression level had decreased. Using small interfering RNA (siRNA) against Ets-1 in gemcitabine-resistant cells, we demonstrated a reversal in gemcitabine chemosensitivity and also detected a marked reduction in the expression of the Ets-1 target genes MMP1 and uPA. Our novel finding demonstrates the significance of Ets-1 in the development of gemcitabine chemoresistance in pancreatic cancer cells. Based on these results, a new siRNA-based therapeutic strategy targeting the Ets-1 genes can be designed to overcome chemoresistance.

Gemcitabine chemoresistance and molecular markers associated with gemcitabine transport and metabolism in human pancreatic cancer cells

To identify predictive molecular markers for gemcitabine resistance, we investigated changes in the expression of four genes associated with gemcitabine transport and metabolism during the development of acquired gemcitabine resistance of pancreatic cancer cell lines. The expression levels of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), RRM1, and RRM2 mRNA were analysed by real-time light cycler-PCR in various subclones during the development of acquired resistance to gemcitabine. Real-time light cycler-PCR demonstrated that the expression levels of either RRM1 or RRM2 progressively increased during the development of gemcitabine resistance. Expression of dCK was slightly increased in cells resistant to lower concentrations of gemcitabine, but was decreased below the undetectable level in higher concentration-resistant subclones. Expression of hENT1 was increased in the development of gemcitabine resistance. As acquired resistance to gemcitabine seems to correlate with the balance of these four factors, we calculated the ratio of hENT1 × dCK/RRM1 × RRM2 gene expression in gemcitabine-resistant subclones. The ratio of gene expression decreased progressively with development of acquired resistance in gemcitabine-resistant subclones. Furthermore, the expression ratio significantly correlated with gemcitabine sensitivity in eight pancreatic cancer cell lines, whereas no single gene expression level correlated with the sensitivity. These results suggest that the sensitivity of pancreatic cancer cells to gemcitabine is determined by the ratio of four factors involved in gemcitabine transport and metabolism. The ratio of the four gene expression levels correlates with acquired gemcitabine-resistance in pancreatic cancer cells, and may be useful as a predictive marker for the efficacy of gemcitabine therapy in pancreatic cancer patients.