Acquisition Of Gemcitabine Resistance Research Articles

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.

Abstract C052: Repurposing Statins to Target Gemcitabine Resistance in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDA therapy, but resistance remains a major hurdle in clinical care. Metabolic reprogramming is an important driver of chemoresistance. Accordingly, targeting these altered metabolic pathways can improve response to treatment. To investigate metabolic vulnerabilities, we generated models of acquired PDA gemcitabine resistance. Screening common metabolic inhibitors we observed that gemcitabine-resistant cells were exquisitely susceptible to statins, inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This is due to a downregulation of the mevalonate biosynthesis pathway in gemcitabine-resistant cells. Importantly, the statin sensitivity correlates with the extent of the gemcitabine resistance, suggesting this metabolic vulnerability is gradually gained during acquisition of resistance. Rescue experiments showed that statins induce apoptosis by reduced protein geranylation of small GTPases. Finally, we find that pitavastatin inhibits growth of gemcitabine resistant tumors in immune competent models. 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 treatment combination of gemcitabine and pitavastatin, two widely used compounds in patients, has potential to translate into an immediate clinical benefit and improve survival in this deadly disease. Citation Format: Sabrina Calderon, Alica K. Beutel, Ethan Nghiem, Rima Singh, Natalie Yousefian, Cecily Anaraki, Kevin Gulay, Dennis Juarez, Alexander Kleger, Thomas Seufferlein, Alexander Muir, Cholsoon Jang, Herve Tiriac, David Fruman, Christopher Halbrook. Repurposing Statins to Target Gemcitabine Resistance in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C052.

The Natural Product Parthenolide Inhibits Both Angiogenesis and Invasiveness and Improves Gemcitabine Resistance by Suppressing Nuclear Factor κB Activation in Pancreatic Cancer Cell Lines.

We previously established pancreatic cancer (PaCa) cell lines resistant to gemcitabine and found that the activity of nuclear factor κB (NF-κB) was enhanced upon the acquisition of gemcitabine resistance. Parthenolide, the main active ingredient in feverfew, has been reported to exhibit antitumor activity by suppressing the NF-κB signaling pathway in several types of cancers. However, the antitumor effect of parthenolide on gemcitabine-resistant PaCa has not been elucidated. Here, we confirmed that parthenolide significantly inhibits the proliferation of both gemcitabine-resistant and normal PaCa cells at concentrations of 10 µM and higher, and that the NF-κB activity is significantly inhibited, even by 1 µM parthenolide. In Matrigel invasion assays and angiogenesis assays, the invasive and angiogenic potentials were higher in gemcitabine-resistant than normal PaCa cells and were inhibited by a low concentration of parthenolide. Furthermore, Western blotting showed suppressed MRP1 expression in gemcitabine-resistant PaCa treated with a low parthenolide concentration. In a colony formation assay, the addition of 1 µM parthenolide improved the sensitivity of gemcitabine-resistant PaCa cell lines to gemcitabine. These results suggest that parthenolide may be used as a novel therapeutic agent for the treatment of gemcitabine-resistant PaCa.

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.

Abstract 6386: Phosphomimetic Dicer rewires glutamine metabolism and gemcitabine resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy that shows high cancer-mortality and poor prognosis, in the absence of obvious symptoms and biomarkers for early diagnosis. Gemcitabine is a type of chemotherapy drug which commonly used to treat PDAC, however, the rapid acquisition of resistance to gemcitabine treatment has been observed in many of PDAC patients. The miRNA-producing enzymes Dicer is crucial for the maturation of miRNAs, and we found previously that ERK/Sp1 signaling transactivate both gene and protein expressions of Dicer, leading an acquisition of gemcitabine resistance in pancreatic cancer cells both in vitro and in vivo. In this study, we further identified that phosphomimetic S1016E of Dicer may regulate miRNAs maturation, resulting an imbalance of glutaminase (GLS) and glutamine synthetase (GS/GLUL). Metabolism analysis revealed that gemcitabine-resistant PANC-1 (PANC-1/GEM) cells have high glutamine dependence and GLS/GLUL ratio. Our study suggests that phosphomimetic Dicer S1016E plays a critical role in glutamine metabolism reprogramming and gemcitabine resistance of pancreatic cancer. Citation Format: Ching-Feng Chiu, Ji Min Park, Yu-Shiuan Shen, Chia-Ying Lin, Tung-Wei Hsu, Yen-Hao Su, Hsin-An Chen. Phosphomimetic Dicer rewires glutamine metabolism and gemcitabine resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6386.

GAS41 mediates proliferation and GEM chemoresistance via H2A.Z.2 and Notch1 in pancreatic cancer.

GAS41 is a YEATS domain protein that binds to acetylated histone H3 to promote the chromatin deposition of H2A.Z in non-small cell lung cancer. The role of GAS41 in pancreatic cancer is still unknown. Here, we aimed to reveal this role. GAS41 expression in pancreatic cancer tissues and cell lines was examined using qRT-PCR, Western blotting and immunohistochemistry. MTT, colony formation, spheroid formation and in vivo tumorigenesis assays were performed to assess the proliferation, tumorigenesis, stemness and gemcitabine (GEM) resistance of pancreatic cancer cells. Mechanistically, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) assays were used to evaluate the roles of GAS41, H2A.Z.2 and Notch1 in pancreatic cancer. We found that GAS41 is overexpressed in human pancreatic cancer tissues and cell lines, and that its expression increases following the acquisition of GEM resistance. We also found that GAS41 up-regulates Notch, as well as pancreatic cancer cell stemness and GEM resistance in vitro and in vivo. We show that GAS41 binds to H2A.Z.2 and activates Notch and its downstream mediators, thereby regulating stemness and drug resistance. Depletion of GAS41 or H2A.Z.2 was found to down-regulate Notch and to sensitize pancreatic cancer cells to GEM. Our data indicate that GAS41 mediates proliferation and GEM resistance in pancreatic cancer cells via H2A.Z.2 and Notch1.

Glycogen synthase kinase-3β participates in acquired resistance to gemcitabine in pancreatic cancer.

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)‐3β, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine‐sensitive BxPC‐3 cell‐derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3β‐specific inhibitor alone attenuated the viability and proliferation of the gemcitabine‐resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine‐resensitizing effect of GSK3β inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro‐oncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.

Oblongifolin C reverses GEM resistance via suppressing autophagy flux in bladder cancer cells.

A number of previous studies have demonstrated that inhibiting autophagy can increase the cellular cytotoxicity of chemotherapeutic agents in urothelial cancer cells. However, the mechanistic roles of autophagy in gemcitabine (GEM) resistant bladder cancer cells have not been thoroughly investigated. In the present study, immunohistochemistry staining of autophagy marker LC3 was performed in bladder cancer and healthy control tissues and demonstrated an essential role of autophagy in cancer development. A GEM-resistant cell line was established to assess the effects of autophagy on the acquisition of GEM resistance. Western blotting of autophagy markers in GEM-resistant bladder cancer cells suggested that GEM resistance was caused, at least partially, by GEM-induced autophagy. GEM resistance was demonstrated to be reversed by the inhibition of autophagy by 3-methyladenine. In addition, oblongifolin C (OC), a novel autophagic flux inhibitor purified from traditional Chinese medicine, was found to enhance the efficiency of GEM in GEM-resistant bladder cancer cells by inhibiting autophagic flux. In conclusion, data from the present study suggest that autophagy serves an important role in bladder cancer development and GEM resistance. OC treatment has the ability to reverse GEM-resistance in bladder cancer cells by suppressing autophagic flux, thereby providing a potential adjunctive therapeutic option for bladder cancer GEM treatment.

Integrin β1 promotes gemcitabine resistance in pancreatic cancer through Cdc42 activation of PI3K p110β signaling

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignancies with very poor prognosis due to its broad resistance to chemotherapy. Our previous study showed that integrin β1 expression is upregulated in PDAC and confers gemcitabine resistance in PDAC cells via the signaling pathway including Cdc42 and AKT activation. But the accurate signal transductions are not clear. Here, we aimed to illuminate the signal transductions of integrin β1 in the acquisition of gemcitabine resistance in PDAC. Drug-resistance (DR) cells from AsPC-1 parent cell line (PCL) were selected. Integrin β1 expression was determined using western blot assay. Changes in drug response and the activity of phosphatidylinositol 3-kinase (PI3K) signaling after knockdown of integrin β1, Cdc42 or p110β were evaluated using MTT, cleaved caspase-3 immunofluorescence and western blot assay. Western blot assays also detected the variations in Cdc42 activity and p110β expression after integrin β1 knockdown. The interaction between Cdc42 and p110β was determined by Glutathione S-transferase (GST) pull-down assay. The results showed that integrin β1 expression was upregulated in DR-AsPC-1 cells, and integrin β1 knockdown significantly decreased the activity of Cdc42, a target molecule of integrin β1, and p110β expression. Knockdown of anyone of integrin β1, Cdc42 and p110β inhibited the activity of PI3K signaling, and sensitized DR-AsPC-1 cells to gemcitabine. GST pull-down assay showed that GTP-Cdc42 interacted with p110β. Collectively, these data indicated that integrin β1 promoted gemcitabine resistance in PDAC through Cdc42 activation of PI3K p110β signaling. In vivo experiments also confirmed this conclusion. These findings contribute to a better understanding the molecular mechanism of chemoresistance and facilitate the development of more targeted and effective treatment strategy for PDAC.

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.

Abstract 5473: Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene

Abstract Gemcitabine (GEM) has shown clinical activity in several solid tumors such as pancreatic and biliary tract cancers, urinary cancer, and non-small cell lung cancers, but a substantial number of patients acquire GEM chemoresistance. GEM functions after phosphorylation by deoxycytidine kinase (DCK) and its inactivation is one of the important mechanisms for acquisition of gemcitabine resistance. We established eight GEM resistant cell lines including four pancreatic, two biliary tract, and two gastrointestinal cancers and investigated mechanisms for acquisition of GEM resistances; six GEM resistant cell lines harbored complete inactivating mutations of DCK by protein-truncating mutation or complete or partial loss of the gene, and one gastric cancer cell line harbored a missense mutation that inactivate DCK activity. One pancreatic cancer cell line lost GEM sensitivity by unknown mechanism. Our present results strongly suggest that genetic alteration of DCK can be the major cause of acquisition of GEM resistance. Further studies analyzing primary tumors will give us (1) the fully understanding of acquisition of GEM resistance, and (2) desirable clues to overcome the acquisition of GEM resistance. Citation Format: Akira Horii, Yuriko Saiki, Tomohiro Nakano, Chiharu Kudo, Makoto Sunamura. Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5473. doi:10.1158/1538-7445.AM2015-5473

Gemcitabine Treatment Causes Deregulation of Epithelial to Mesenchymal Transition Transcription Factors Transcription and Translation in Pancreatic Ductal Adenocarcinoma Cells

Context Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease, characterised by limited response to chemotherapeutic treatment and early metastasis, leading to very poor prognosis. Epithelial to mesenchymal transition (EMT), a process finely regulated both at transcription and splicing level, contributes to PDAC invasion and affects the response to chemotherapeutic drugs. The expression of the EMT transcription factor ZEB1 is inversely related to sensitivity cells to gemcitabine treatment. Notably, ZEB1 encodes multiple splice variants that mainly differ in the 5’ untranslated region (UTR). However, the biological role of these splice variants in EMT and drug resistance is currently unknown. Objective Characterization of the molecular events involved in the acquisition of gemcitabine resistance in PDAC cells. Methods PCR analysis of EMT genes; Western blot analysis of proteins of the mTOR pathway; 7-mGTP cap assay of cap-dependent translation; polysomal-RNPs fractioning for analysis of mRNA translation. Results PDAC cells exposed to gemcitabine for 72 hours up-regulated mesenchymal genes, including ZEB1, which is known to confer chemoresistance. This response is accompanied by inhibition of mTOR pathway and cap-dependent translation, as confirmed by reduced assembly of the translation initiation complex eIF4F. Conversely, cap-independent translation is not impaired by the drug. In this context, ZEB1 splice variants containing different 5’ UTRs are differentially loaded on polysomes, suggesting that expression of specific variants allows ZEB1 translation during drug treatment. Conclusion Our results show that treatment with gemcitabine alters the expression of EMT genes and that these events are concomitant to important alteration in the translational program. Together, these processes can drive to different translational patterns in presence of gemcitabine, as shown for the ZEB1 variants, which may take part to the mechanisms leading to chemoresistance of PDAC cells.

CD44‐positive cells are responsible for gemcitabine resistance in pancreatic cancer cells

Accumulating evidence suggests that tumors are composed of a heterogeneous cell population with a small subset of cancer stem cells (CSCs) that sustain tumor formation and growth. Recently, there have been efforts to explain drug resistance of cancer cells based on the concept of CSCs having an intrinsic detoxifying mechanism. In the present study, to investigate the role of CSCs in acquiring chemoresistance in pancreatic cancer, gemcitabine-resistant cells were established by exposure to serially escalated doses of gemcitabine in HPAC and CFPAC-1 cells. Gemcitabine-resistant cells were more tumorigenic in vitro and in vivo, and had greater sphere-forming activity than parental cells. After high-dose gemcitabine treatment to eliminate most of the cells, CD44(+) cells proliferated and reconstituted the population of resistant cells. CD44(+)CD24(+)ESA(+) cells remained as a small subset in the resistant cell population. Among ATP-binding cassette (ABC) transporters, which are known as the mechanism of drug resistance in CSCs, ABCB1 (MDR1) was significantly augmented during the acquisition of drug resistance. ABC transporter inhibitor verapamil resensitized the resistant cells to gemcitabine in a dose-dependent manner and RNA interference of CD44 inhibited the clonogenic activity of resistant cells. In human pancreatic cancer samples, CD44 expression was correlated with histologic grade and the patients with CD44-positive tumors showed poor prognosis. These data indicate that cancer stem-like cells were expanded during the acquisition of gemcitabine resistance and in therapeutic application, targeted therapy against the CD44 or ABC transporter inhibitors could be applied to overcome drug resistance in the treatment of pancreatic cancer.