Resistance Of Pancreatic Cancer Cells Research Articles

Hypoxia activated HGF expression in pancreatic stellate cells confers resistance of pancreatic cancer cells to EGFR inhibition.

Epidermal growth factor receptor (EGFR) is an essential target for cancer treatment. However, EGFR inhibitor erlotinib showed limited clinical benefit in pancreatic cancer therapy. Here, we showed the underlying mechanism of tumor microenvironment suppressing the sensitivity of EGFR inhibitor through the pancreatic stellate cell (PSC). The expression of alpha-smooth muscle actin (α-SMA) and hypoxia marker in human pancreatic cancer tissues were detected by immunohistochemistry, and their correlation with overall survival was evaluated. Human immortalized PSC was constructed and used to investigate the potential effect on pancreatic cancer cell lines in hypoxia and normoxia. Luciferase reporter assay and Chromatin immunoprecipitation were performed to explore the potential mechanisms invitro. The combined inhibition of EGFR and Met was evaluated in an orthotopic xenograft mouse model of pancreatic cancer. We found that high expression levels of α-SMA and hypoxia markers are associated with poor prognosis of pancreatic cancer patients. Mechanistically, we demonstrated that hypoxia induced the expression and secretion of HGF in PSC via transcription factor HIF-1α. PSC-derived HGF activates Met, the HGF receptor, suppressing the sensitivity of pancreatic cancer cells to EGFR inhibitor in a KRAS-independent manner by activating the PI3K-AKT pathway. Furthermore, we found that the combination of EGFR inhibitor and Met inhibitor significantly suppressed tumor growth in an orthotopic xenograft mouse model. Our study revealed a previously uncharacterized HIF1α-HGF-Met-PI3K-AKT signaling axis between PSC and cancer cells and indicated that EGFR inhibition plus Met inhibition might be a promising strategy for pancreatic cancer treatment. This study was supported by The National Natural Science Foundation of China.

ARID1A promotes chemosensitivity to gemcitabine in pancreatic cancer through epigenetic silencing of RRM2.

Pancreatic cancer is one of the most common malignancies with very poor prognosis due to its broad resistance to chemotherapy. ARID1A, a subunit of SWI/SNF complex, is involved in pancreatic carcinogenesis through epigenetic silencing of oncogenes. In this study, we aimed to explore whether ARID1A was implicated in the gemcitabine resistance in pancreatic cancer patients via regulating RRM2. We examined the effect of ARID1A depletion on the gemcitabine sensitivity in pancreatic cancer cells and explored the role of RRM2 in ARID1A-mediated pancreatic cancer cells chemosensitivity to gemcitabine. We found that Knockout of ARID1A led to gemcitabine resistance in pancreatic cancer cells, effect of which could be reversed by RRM2, a gemcitabine resistance related gene. ARID1A decreased the transcription of RRM2, and directly bound to the promoter of RRM2. Moreover, expression of RRM2 was negatively correlated with ARID1A in pancreatic cancer tissues. Thus, ARID1A-mediated RRM2 epigenetic suppression is crucial for enhancement of pancreatic cancer chemosensitivity to gemcitabine, and ARID1A could be used as a biomarker to guide the gemcitabine chemotherapy of pancreatic cancer.

Gemcitabine resistance of pancreatic cancer cells is mediated by IGF1R dependent upregulation of CD44 expression and isoform switching

Chemoresistance in pancreatic cancer cells may be caused by the expansion of inherently resistant cancer cells or by the adaptive plasticity of initially sensitive cancer cells. We investigated how CD44 isoforms switching contributed to gemcitabine resistance. Treating CD44 null/low single-cell clones with increasing amounts of gemcitabine caused an increase in expression of CD44 and development of gemcitabine resistant (GR) cells. Drug sensitivity, invasiveness, and EMT process was evaluated by MTT, Matrigel invasion assays, and western blots. Genetic knockdown and pharmacological inhibitors were used to examine the roles of CD44 and IGF1R in mediating gemcitabine resistance. CD44 promoter activity and its interactive EMT-related transcription factors were evaluated by luciferase reporter assay and chromatin immunoprecipitation assay. Kaplan–Meier curve was created by log-rank test to reveal the clinical relevance of CD44 and IGF1R expression in patients. We found silence of CD44 in GR cells partially restored E-cadherin expression, reduced ZEB1 expression, and increased drug sensitivity. The gemcitabine-induced CD44 expressing and isoform switching were associated with an increase in nuclear accumulation of phosphor-cJun, Ets1, and Egr1 and binding of these transcription factors to the CD44 promoter. Gemcitabine treatment induced phosphorylation of IGF1R and increased the expression of phosphor-cJun, Ets1, and Egr1 within 72 h. Stimulation or suppression of IGF1R signaling or its downstream target promoted or blocked CD44 promoter activity. Clinically, patients whose tumors expressed high levels of CD44/IGF1R showed a poor prognosis. This study suggests that IGF1R-dependent CD44 isoform switching confers pancreatic cancer cells to undergo an adaptive change in response to gemcitabine and provides the basis for improved targeted therapy of pancreatic cancer.

GABRP promotes CD44s-mediated gemcitabine resistance in pancreatic cancer.

BackgroundPancreatic ductal adenocarcinoma (PDAC) has the worst five-year overall survival rate among all cancer types. Acquired chemoresistance is considered one of the main reasons for this dismal prognosis, and the mechanism of chemoresistance is unknown.MethodsWe previously identified a subpopulation of chemoresistant CD44high-expressing PDAC cells. Subsequently, we selected the candidate gene, gamma-aminobutyric acid receptor subunit Pi (GABRP), from three Gene Expression Omnibus datasets as the potential CD44 downstream target mediating the gemcitabine resistance. Loss and gain of function such as stable knockdown of CD44 by small hairpin (sh) RNA-mediated silencing technique and overexpression (O/E) of CD44s had been studied for comparing the gemcitabine resistance among CD44high-expressing cells, shCD44 cells, CD44low-expressing cells and O/E CD44s expressing cells. Functional assays including cell viability, colony formation, invasion, quantitative PCR and western blotting techniques were performed to validate the roles of CD44 and GABRP playing in mediating the gemcitabine resistance in pancreatic cancer cells.ResultsCD44s depletion significantly reduced gemcitabine resistance in shCD44 single clone cells compared to CD44high-expressing cells. Knockdown of CD44 cells formed less colonies, became less invasive and remarkably decreased the mRNA level of GABRP. While overexpression of CD44s had the opposite effect on gemcitabine resistance, colony formation and invasive property. Of note, long term gemcitabine resistant pancreatic cancer cells detected increased expression of CD44 and GABRP. Clinically, GABRP expression was significantly upregulated in the tissues of patients with pancreatic cancer compared to the normal samples, and the overall survival rate of patients with low GABRP expression was longer. CD44 and GABRP co-expression was positively correlated in 178 pancreatic cancer patients.ConclusionOur findings suggest that GABRP may serve as a CD44s downstream target to diminish gemcitabine resistance in pancreatic cancer, and both CD44s and GABRP molecules have the potential to become prognostic biomarkers for PDAC patients with gemcitabine resistance.

E2F2 enhances the chemoresistance of pancreatic cancer to gemcitabine by regulating the cell cycle and upregulating the expression of RRM2.

Both pro-oncogenic and anti-oncogenic effects of E2F2 have been revealed in different malignancies. However, the precise role of E2F2 in pancreatic cancer, in particular in relation to therapeutic intervention with gemcitabine, remains unclear. In this study, the effect of E2F2 on the proliferation and cell cycle modulation of pancreatic cancer cells, and whether E2F2 plays a role in the treatment of pancreatic cancer cells by gemcitabine, were investigated. The expression of E2F2 in pancreatic cancer was assessed by various methods including bioinformatics prediction, Western blotting, and real-time PCR. The effect of E2F2 on the proliferation and cell cycling of pancreatic cancer cells was analyzed by tissue culture and flow cytometry. In addition, the effect of E2F2 on the intervention of pancreatic cancer by gemcitabine was investigated using both in vitro and in vivo approaches. The expression of E2F2 was found to be significantly increased in pancreatic cancer tissues and cell lines. The pathogenic capacity of E2F2 lied in the fact that this transcription factor promoted the transformation of pancreatic cancer cell cycle from G1-phase to S-phase, thus enhancing the proliferation of pancreatic cancer cells. Furthermore, the expression of E2F2 was increased in pancreatic cancer cells in the presence of gemcitabine, and the augmented expression of E2F2 upregulated the gemcitabine resistance-related gene RRM2 and its downstream signaling molecule deoxycytidine kinase (DCK). The resistance of pancreatic cancer cells to gemcitabine was confirmed using both in vitro and in vivo models. In this study, E2F2 has been demonstrated for the first time to play a pro-oncogenic role in pancreatic cancer by promoting the transition of the cell cycle from G1-phase to S-phase and, therefore, enhancing the proliferation of pancreatic cancer cells. E2F2 has also been demonstrated to enhance the chemotherapy resistance of pancreatic cancer cells to gemcitabine by upregulating the expression of RRM2 and DCK that is downstream of RRM2.

Abstract 6349A: Mechanical stress activates autophagy to induce drug resistance in pancreatic cancer cells

Abstract INTRODUCTION: One of the most fundamental problems that oncologists are currently facing in pancreatic cancer patient care is the high resistance to all types of chemotherapies or targeted therapies. Thus, there is an urgent need for increasing patient response to treatment. A common characteristic among most pancreatic cancer patients that could serve as potential target for treatment, is the biomechanically abnormal tumor microenvironment (TME). This altered TME is characterized by an increased stiffness which among others, is responsible for the development of mechanical stresses in the tumor interior. Even though several studies have investigated the effect of mechanical stress on cancer cells and tumor progression, it has not yet defined whether it can affect drug sensitivity and what are the mechanisms involved. HYPOTHESIS: Based on our recent findings, obtained by a Reverse Phase Protein Assay, we observed that mechanical stress activates a resistance response, employing autophagy activation, in pancreatic cancer cells. To this end, we hypothesize that mechanical stress developed during tumor growth, can induce autophagy activation to eventually promote drug resistance in pancreatic tumors. Therefore, inhibition of this mechanism can improve therapy. METHODOLOGY: We use two pancreatic cancer cells lines, MIA PaCa-2 and PANC-1, to apply mechanical stress, either in a 2D experimental setup or by employing tumor spheroids in a 3D agarose matrix. In both cases, cells are exposed to the most common chemotherapeutic agents used for pancreatic cancer treatment, gemcitabine and 5-Fluorouracil (5FU). The cytotoxic effect of each drug, as well as autophagy- and signaling pathway- activation are then analyzed. Finally, in vitro assays and treatments, along with in vivo animal models of pancreatic tumors, are performed to test the efficacy of an autophagy inhibitor combined with chemotherapy and a mechanotherapeutic that modulates intratumoral mechanical stresses. RESULTS: Proteomic analysis, together with pharmacological inhibition and siRNA treatments, revealed that mechanical stress activates autophagy to impair cell apoptosis and promote cancer cell survival. This stress-adaptation mechanism in turn impairs the cytotoxic effect of both gemcitabine and 5FU. A combined treatment employing chemotherapy with an autophagy inhibitor, and a mechanotherapeutic agent, showed promising results in increasing the overall-survival of pancreatic tumor-bearing mice. CONCLUSION: Our results provide solid evidence that alleviation of intratumoral mechanical stresses with mechanotherapeutics combined with autophagy inhibitors can improve the efficacy of chemotherapeutic agents in pancreatic cancer. Citation Format: Maria Kalli, Ruxuan Li, Ioannis Zervantonakis, Triantafyllos Stylianopoulos. Mechanical stress activates autophagy to induce drug resistance in pancreatic cancer cells [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 6349A.

Abstract 6367: Activation of vitamin D/VDR signaling reverses gemcitabine resistance of pancreatic cancer cells through inhibition of MUC1 expression

Abstract Objective: Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis, largely due to its resistance to currently available therapies. Identifying the determinants of and signaling pathways regulating this resistance is essential for the development of effective therapeutic modalities to improve the outcome of PDA. The purpose of this study is to define the role of vitamin D/VDR signaling and underlying mechanisms in PDA pathogenesis and therapeutic resistance. Materials & Methods: We used an unbiased reverse phase protein array (RPPA) to identify Vitamin D3 downstream molecules; we also used a variety of cellular and molecular biological approaches to determine the impact of vitamin D/VDR signaling on the expression of MUC1 mucin and the cellular response to gemcitabine treatment in PDA cells. We used in vivo experimental systems to determine the effect of combination use of gemcitabine with Vitamin D analogue on pancreatic tumorigenicity. Additionally, we used ultra-high resolution mass spectrometry (HRMS) to analyze the relative abundance of intracellular dFdCTP in tumor tissue samples. Results: We identified that MUC1 protein expression was significantly reduced in AsPC-1 human pancreatic cancer cells after treatment with vitamin D3 or its analogue calcipotriol (Cal) by the RPPA analysis, which was further confirmed in MiaPaca-2 and Colo357 pancreatic cancer cells. VDR negatively regulated MUC1 expression in pancreatic cancer cells in both gain- and loss-of-function assays. We established gemcitabine (Gem) resistant AsPC-1-GemR and Colo357-GemR cells and found that AsPC-1-GemR and Colo357-GemR cells had much lower levels of VDR but higher levels of MUC1 protein expression compared to their parental cells, Vitamin D3 or its analogue significantly induced VDR and inhibited MUC1 expression in AsPC-1-GemR and Colo357-GemR cells, and sensitized the resistant cells to gemcitabine treatment. The results of siRNA knockdown experiments indicated that inhibition of MUC1 expression was essential for Cal’s sensitization of PDA cells to Gem treatment in vitro. In keeping with these findings, administration of the Vitamin D3 analogue, paricalcitol (PA) significantly enhanced the therapeutic efficacy of Gem compared to chemotherapy alone in xenograft and syngeneic mouse models of pancreatic cancer without any significant systemic toxicity. The improved combination treatment correlated with increased intratumoral concentration of dFdCTP, the biological active metabolite of gemcitabine. Conclusions: This study reveals a previously unidentified vitamin D/VDR-MUC1 signaling pathway involved in the regulation of gemcitabine resistance of PDA. Our results suggest that combinational therapies which include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA. Citation Format: Daoyan Wei, Margarete Hafley, Liang Wang, Yi Liu, Peiying Yang, Xiangsheng Zuo, Robert S. Bresalier. Activation of vitamin D/VDR signaling reverses gemcitabine resistance of pancreatic cancer cells through inhibition of MUC1 expression [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 6367.

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.

Abstract 4027: Dual MEK and AKT inhibition suppresses pancreatic cancer growth and migration

Abstract Pancreatic cancer is one of the deadliest cancers, due to late diagnosis and very few available therapeutic treatments. The Kras gene is frequently mutated in pancreatic cancer, but previous clinical trials using RAS inhibitors have proven ineffective. Additionally, many drug treatments targeting the MAPK pathway have shown little success due to the inherent drug resistance of pancreatic cancer cells and to acquired resistance through activation of alternative proliferative pathways such as JAK-STAT and PI3K-AKT. Our pathway analysis after MEK inhibition in pancreatic cancer cells displayed sustained and/or upregulation of AKT activity in multiple cell lines. Therefore, we hypothesize that a combination treatment of Trametinib and Omipalisib would block two prominent mitogenic pathways (MEK and AKT, respectively) to suppress cancer cell proliferation and migration. Multiple analyses including Western blot, clonogenic, 5-Ethynyl-2’-Deoxyuridine (EDU), and scratch migration assays were employed to determine the augmentation of cellular function in murine and anthropic pancreatic cancer cell lines after single or combination treatment with Trametinib and/or Omipalisib. Results demonstrated that application of Omipalisib alone was successful at blocking pAKT but failed to suppress pERK. Conversely, Trametinib alone selectively inhibited pERK but did not affect pAKT levels. The combination treatment successfully suppressed both pathways in relatively low quantities, indicating their efficacy as a dual therapeutic. Dual therapy was further effective at inhibiting cell growth as evidenced by clonogenic and EdU assays. Recovery and closure from an in vitro scratch wound was also significantly inhibited with combination of Omipalisib and Trametinib. In vivo studies demonstrated that both the pancreatic xenograft mean tumor growth and final tumor size were significantly reduced in response to the combination treatment compared to vehicle. In conclusion, dual therapy with Omipalisib and Trametinib showed a greater anti-tumor efficacy than treatment with either drug alone. Currently, we are determining the prolonged effectiveness of this combination treatment program in genetically-engineered mouse models. If successful, a pharmacological application of Omipalisib and Trametinib has the potential to provide a beneficial therapeutic option to pancreatic cancer patients. Citation Format: Jarrid Jack, Alexandra Pierce, Bailey Bye, McKinnon Walsh, Prabhakar Chalise, Michael N. VanSaun. Dual MEK and AKT inhibition suppresses pancreatic cancer growth and migration [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 4027.

NNK from tobacco smoking enhances pancreatic cancer cell stemness and chemoresistance by creating a β2AR-Akt feedback loop that activates autophagy.

Low responsiveness to chemotherapy is an important cause of poor prognosis in pancreatic cancer. Smoking is a high‐risk factor for pancreatic cancer and cancer resistance to gemcitabine; however, the underlying mechanisms remain unclear. 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is the main metabolite of tobacco burning and has been shown to be associated with cancer development and chemoresistance. However, in pancreatic cancer, its mechanism remains poorly understood. In this study, we found that NNK promoted stemness and gemcitabine resistance in pancreatic cancer cell lines. Moreover, NNK increased autophagy and elevated the expression levels of the autophagy‐related markers autophagy‐related gene 5 (ATG5), autophagy‐related gene 7 (ATG7), and Beclin1. Furthermore, the results showed that NNK‐promoted stemness and gemcitabine resistance was partially dependent on the role of NNK in cell autophagy, which is mediated by the β2‐adrenergic receptor (β2AR)‐Akt axis. Finally, we proved that NNK intervention could not only activate β2AR, but also increase its expression, making β2AR and Akt form a feedback loop. Overall, these findings show that the NNK‐induced β2AR‐Akt feedback loop promotes stemness and gemcitabine resistance in pancreatic cancer cells.

Solute Carrier Family 35 Member F2 Regulates Cisplatin Resistance and Promotes Malignant Progression of Pancreatic Cancer by Regulating RNA Binding Motif Protein 14.

We aimed to explore the role of Solute Carrier Family 35 Member F2 (SLC35F2) in pancreatic cancer (PCa) and to further study whether SLC35F2 regulates cisplatin resistance of PCa cells through the modulation of RNA binding motif protein 14 (RBM14) expression. SLC35F2 expression in 60 pairs of PCa tissues and adjacent ones was studied by RT-PCR analysis. Meanwhile, SLC35F2 expression levels in PCa cell lines were also evaluated by qPCR assay. In addition, SLC35F2 knockdown models were constructed in PCa cisplatin-resistant cells. Furthermore, we determined the interaction between SLC35F2 and RBM14 via luciferase assay. The findings of the present study demonstrated that SLC35F2 was significantly upregulated in PCa tissues. High level of SLC35F2 indicated higher incidence of metastasis and shorter survival rates. In vitro cell experiments revealed that knockdown of SLC35F2 suppressed cell invasion and metastasis capacity of cisplatin-resistant PCa cell lines PANC-1/DDP and CFPAC-1/DDP. It was also suggested that the key protein RBM14 in the SLC35F2 knockdown group was remarkably reduced. SLC35F2 can bind to RBM14 specifically. Overexpression of RBM14 partially reversed the effects of knockdown of SLC35F2 on the development of PCa. SLC35F2 expression in PCa tissues and cell lines is remarkably increased. In addition, it was also suggested that SLC35F2 may regulate cisplatin resistance of PCa cells through modulating RBM14 expression. In conclusion, it is conceivable from the study that SLC35F2 was remarkably upregulated in PCa and promoted the malignancy of PCa via regulating RBM14.

Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters to reverse gemcitabine resistance in pancreatic cancer

BackgroundAlthough gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro.MethodsImmunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178.ResultsWe found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan–Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters.ConclusionsOur results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.

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.

HNRNPC regulates RhoA to induce DNA damage repair and cancer-associated fibroblast activation causing radiation resistance in pancreatic cancer.

Pancreatic cancer (PC) is one of the most lethal types of cancer due to its asymptomatic nature in the early stages and consequent late diagnosis. Its mortality rate remains high despite advances in treatment strategies, which include a combination of surgical resection and adjuvant therapy. Although these approaches may have a positive effect on prognosis, the development of chemo‐ and radioresistance still poses a significant challenge for successful PC treatment. Heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) and RhoA have been implicated in the regulation of tumour cell proliferation and chemo‐ and radioresistance. Our study aims to investigate the mechanism for HNRNPC regulation of PC radiation resistance via the RhoA pathway. We found that HNRNPC and RhoA mRNA and protein expression levels were significantly higher in PC tissues compared to adjacent non‐tumour tissue. Furthermore, high HNRNPC expression was associated with poor patient prognosis. Using HNRNPC overexpression and siRNA interference, we demonstrated that HNRNPC overexpression promoted radiation resistance in PC cells, while HNRNPC knockdown increased radiosensitivity. However, silencing of RhoA expression was shown to attenuate radiation resistance caused by HNRNPC overexpression. Next, we identified RhoA as a downstream target of HNRNPC and showed that inhibition of the RhoA/ROCK2‐YAP/TAZ pathway led to a reduction in DNA damage repair and radiation resistance. Finally, using both in vitro assays and an in vivo subcutaneous tumour xenograft model, we demonstrated that RhoA inhibition can hinder the activity of cancer‐related fibroblasts and weaken PC radiation resistance. Our study describes a role for HNRNPC and the RhoA/ROCK2‐YAP/TAZ signalling pathways in mediating radiation resistance and provides a potential therapeutic target for improving the treatment of PC.

Macrophage C/EBPδ Drives Gemcitabine, but Not 5-FU or Paclitaxel, Resistance of Pancreatic Cancer Cells in a Deoxycytidine-Dependent Manner.

Treatment of pancreatic ductal adenocarcinoma (PDAC), a dismal disease with poor survival rates, is hampered by the high prevalence of chemotherapy resistance. Resistance is accompanied by macrophage infiltration into the tumor microenvironment, and infiltrated macrophages are key players in chemotherapy resistance. In the current manuscript, we identify CCAAT/enhancer-binding protein delta (C/EBPδ) as an important transcription factor driving macrophage-dependent gemcitabine resistance. We show that conditioned medium obtained from wild type macrophages largely diminishes gemcitabine-induced cytotoxicity of PDAC cells, whereas conditioned medium obtained from C/EBPδ-deficient macrophages only minimally affects gemcitabine-induced PDAC cell death. Subsequent analysis of RNA-Seq data identified the pyrimidine metabolism pathway amongst the most significant pathways down-regulated in C/EBPδ-deficient macrophages and size filtration experiments indeed showed that the low molecular weight and free metabolite fraction most effectively induced gemcitabine resistance. In line with a role for pyrimidines, we next show that supplementing macrophage conditioned medium with deoxycytidine overruled the effect of macrophage conditioned media on gemcitabine resistance. Consistently, macrophage C/EBPδ-dependent resistance is specific for gemcitabine and does not affect paclitaxel or 5-FU-induced cytotoxicity. Overall, we thus show that C/EBPδ-dependent deoxycytidine biosynthesis in macrophages induces gemcitabine resistance of pancreatic cancer cells.

Automated biophysical classification of apoptotic pancreatic cancer cell subpopulations by using machine learning approaches with impedance cytometry.

Unrestricted cell death can lead to an immunosuppressive tumor microenvironment, with dysregulated apoptotic signaling that causes resistance of pancreatic cancer cells to cytotoxic therapies. Hence, modulating cell death by distinguishing the progression of subpopulations under drug treatment from viable towards early apoptotic, late apoptotic, and necrotic states is of interest. While flow cytometry after fluorescent staining can monitor apoptosis with single-cell sensitivity, the background of non-viable cells within non-immortalized pancreatic tumors from xenografts can confound distinction of the intensity of each apoptotic state. Based on single-cell impedance cytometry of drug-treated pancreatic cancer cells that are obtained from tumor xenografts with differing levels of gemcitabine sensitivity, we identify the biophysical metrics that can distinguish and quantify cellular subpopulations at the early apoptotic versus late apoptotic and necrotic states, by using machine learning methods to train for the recognition of each phenotype. While supervised learning has previously been used for classification of datasets with known classes, our advancement is the utilization of optimal positive controls for each class, so that clustering by unsupervised learning and classification by supervised learning can occur on unknown datasets, without human interference or manual gating. In this manner, automated biophysical classification can be used to follow the progression of apoptotic states in each heterogeneous drug-treated sample, for developing drug treatments to modulate cancer cell death and advance longitudinal analysis to discern the emergence of drug resistant phenotypes.

Hsa_circ_0074298 promotes pancreatic cancer progression and resistance to gemcitabine by sponging miR-519 to target SMOC.

Objective: To investigate the expression of hsa_circ_0074298 (circular RNA) and the molecular mechanism that promotes tumor growth and enhances the chemoresistance of pancreatic cancer.Methods: Real-time reverse transcription-PCR was used to detect hsa_circ_0074298 expression in pancreatic cancer. The intracellular localization of hsa_circ_0074298 was determined by RNA in situ hybridization. The CCK8 method, colony formation assay, Transwell assay, and flow cytometry were used to evaluate the effects of hsa_circ_0074298 on the proliferation, migration, invasion, cell cycle, apoptosis of pancreatic cancer cells. Bioinformatics analysis and dual luciferase assays were employed to detect the association of hsa_circ_0074298 and miR-519d and the binding of miR-519d to the target gene SMOC2. A subcutaneous xenograft model was established to observe the effect of hsa_circ_0074298 in vivo.Results: The hsa_circ_0074298 was mainly localized in the cytoplasm. Hsa_circ_0074298 was highly expressed in pancreatic cancer tissues and cell lines. The expression of hsa_circ_0074298 was significantly correlated with pancreatic cancer tumor size, lymph node metastasis, and pathological grade. hsa_circ_0074298 could sponge miR-519, and miR-519d bound to SMOC2. Downregulation of hsa_circ_0074298 expression significantly inhibited cell proliferation, migration, invasion, colony forming ability and promoted cell cycle arrest, apoptosis and chemo-resistance of pancreatic cancer in vitro and vivo. However, the effects could be reversed by a miR-519d inhibitor or SMOC2 overexpression.Conclusion: By sponging miR-519 and targeting SMOC2, hsa_circ_0074298 promotes the growth and metastasis of pancreatic cancer and increases the resistance of pancreatic cancer cells to gemcitabine.

Acquired chemoresistance can lead to increased resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus

Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV) against different malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies have demonstrated that VSV-based OVs are effective against the majority of tested human PDAC cell lines. However, some PDAC cell lines are resistant to VSV. PDAC is one of the deadliest types of human malignancies in part due to intrinsic or acquired chemoresistance. Here, we investigated how acquired chemoresistance impacts the efficacy of VSV-based OV therapy. Using an experimental evolution approach, we generated PDAC cell lines with increased resistance to gemcitabine and examined their responsiveness to oncolytic virotherapy. We found that gemcitabine-resistant PDAC cells become more resistant to VSV. The cross-resistance correlated with upregulated levels of a subset of interferon-stimulated genes, resembling the interferon-related DNA damage resistance signature (IRDS), often associated with resistance of cancer cells to chemotherapy and/or radiation therapy. Analysis of ten different PDAC cell lines showed that four PDAC cell lines most resistant to VSV were also highly resistant to gemcitabine, and they all displayed IRDS-like expression in our previous reports. Our study highlights a possible interaction between two different therapies that should be considered in the future for the development of rational treatment regimens.

MiR-499a-5p promotes 5-FU resistance and the cell proliferation and migration through activating PI3K/Akt signaling by targeting PTEN in pancreatic cancer.

BackgroundPancreatic cancer (PC) can be considered a representative cancer type of the human body. As demonstrated by some studies, microRNA (miR)-499 is dysregulated in various cancer types including PC, for which chemotherapy involving 5-fluorouracil (5-FU) has long been considered the first-line therapy. However, there are complex and comprehensive mechanisms related to 5-FU, which have not been fully elucidated. This study thus aimed to examine the molecular mechanisms of 5-FU resistance through miR-499a-5p in PC.MethodsThe expression of miR-499a-5p in PC was measured using quantitative polymerase chain reaction (PCR). MiR-499a-5p was examined in-vivo for its effects on the malignant phenotypes of PC cells.ResultsThe results of the present study demonstrated miR-499a-5p to be upregulated in PC and 5-FU resistant PC tissues. According to in vitro assays in PC cells (PANC1/FR), miR-499a-5p was found to affect adenosine triphosphate (ATP) binding cassette subfamily B member 1 (P-gp), ATP binding cassette subfamily C member 1 (MRP1), and ATP binding cassette subfamily G member 2 (BCRP), thereby facilitating 5-FU resistance in PC cells. Functions assays indicated that suppressed miR-499a-5p expression inhibited the proliferation and migration of cells but facilitated apoptosis in the PC cell line; by contrast, miR-499a-5p overexpression triggered the inverse phenotypic changes of cells. Concerning the mechanisms involved, miR-499a-5p increased PI3K/Akt signaling by targeting phosphatase and tensin homolog (PTEN).ConclusionsTaken together, these findings demonstrate that miR-499a-5p can be potentially applied to PC therapy.

Exosomal lncRNA UCA1 Derived From Pancreatic Stellate Cells Promotes Gemcitabine Resistance in Pancreatic Cancer via the SOCS3/EZH2 Axis.

ObjectivePancreatic cancer is associated with poor prognosis and dismal survival rates. This study aims to investigate roles of lncRNA UCA1-loaded exosomes secreted by pancreatic stellate cells (PSCs) in Gemcitabine (Gem) resistance of pancreatic cancer under hypoxia, which involves the methylation of SOCS3 and EZH2 recruitment.MethodsThe exosomes were isolated from PSCs and hypoxic PSCs (HPSCs), and co-cultured with pancreatic cancer cells transduced with manipulated lncRNA UCA1, EZH2, and SOCS3. The interaction among lncRNA UCA1, EZH2, and SOCS3 was characterized by RIP and ChIP assays. Next, MTT assay, flow cytometry and TUNEL staining and Transwell assay were used to detect cell viability, apoptosis, invasion, and migration. Gem-resistant pancreatic cancer cell line (GemMIA-R3) was established, which was applied in a mouse xenograft model of pancreatic cancer, with MTT assay to determine Gem sensitivity.ResultsLncRNA UCA1 was highly expressed, while SOCS3 was poorly expressed in pancreatic cancer tissues. Hypoxia induced activation of PSCs and promoted release of exosomes. LncRNA UCA1 delivered by hypoxic PSC-derived exosomes (HPSC-EXO) regulated histone methylation level in SOCS3 gene region through recruitment of EZH2. In vitro and in vivo experimental results confirmed that lncRNA UCA1-loaded HPSC-EXO promoted malignant phenotypes, inhibited apoptosis, and promoted Gem resistance of pancreatic cancer cells as well as tumorigenesis in mice.ConclusionUnder hypoxic conditions, exosomes secreted by hypoxia-induced PSCs deliver lncRNA UCA1 into pancreatic cancer cells, where lncRNA UCA1 recruits EZH2 and regulates histone methylation level in SOCS3 gene region, thereby augmenting pancreatic cancer resistance to Gem.