Hypoxia potentiates gemcitabine-induced stemness in pancreatic cancer cells through AKT/Notch1 signaling
Profound chemoresistance remains an intractable obstacle in pancreatic cancer treatment. Pancreatic cancer stem cells (CSCs) and the ubiquitous hypoxic niche have been proposed to account for drug resistance. However, the mechanism involved requires further exploration. This study investigated whether the hypoxic niche enhances gemcitabine-induced stemness and acquired resistance in pancreatic cancer cells by activating the AKT/Notch1 signaling cascade. The therapeutic effects of blockading this signaling cascade on gemcitabine-enriched CSCs were also investigated.
The expression levels of CSC-associated markers Bmi1 and Sox2 as well as those of proteins involved in AKT/Notch1 signaling were measured by Western blot analysis. The expression level of the pancreatic CSC marker CD24 was measured by flow cytometry. Change in gemcitabine sensitivity was evaluated by the MTT assay. The ability of sphere formation was tested by the sphere-forming assay in stem cell medium. The ability of migration and invasion was detected by the transwell migration/invasion assay. A mouse xenograft model of pancreatic cancer was established to determine the effect of Notch1 inhibition on the killing effect of gemcitabine in vivo. The ability of metastasis was investigated by an in vivo lung metastasis assay.
Gemcitabine promoted pancreatic cancer cell stemness and associated malignant phenotypes such as enhanced migration, invasion, metastasis, and chemoresistance. The AKT/Notch1 signaling cascade was activated after gemcitabine treatment and mediated this process. Blockading this pathway enhanced the killing effect of gemcitabine in vivo. However, supplementation with hypoxia treatment synergistically enhanced the AKT/Notch1 signaling pathway and collaboratively promoted gemcitabine-induced stemness.
These findings demonstrate a novel mechanism of acquired gemcitabine resistance in pancreatic cancer cells through induction of stemness, which was mediated by the activation of AKT/Notch1 signaling and synergistically aggravated by the ubiquitous hypoxic niche. Our results might provide new insights for identifying potential targets for reversing chemoresistance in patients with pancreatic cancer.
KeywordsGemcitabine Hypoxia Cancer stem cell AKT Notch1
Cancer stem cells
Fetal bovine serum
Glyceraldehyde 3-phosphate dehydrogenase
NOTCH1 intracellular domain
Stem cell medium
Pancreatic cancer is one of the most lethal cancers worldwide, with a 5-year survival rate that has remained at less than 10% for the past few decades [1, 2]. For many patients, there is little choice other than chemotherapy, especially in the advanced stage . However, gemcitabine, a first-line anticancer drug for pancreatic cancer, provides a limited survival advantage in treated patients . Numerous strategies have been proposed to improve the therapeutic effect of gemcitabine, but the prognosis for patients with pancreatic cancer remains disappointing . Therefore, identifying new chemotherapeutic agents or adjuvant therapies is necessary to enhance the effectiveness of chemotherapy and reduce tumor recurrence.
Accumulating evidence indicates that tumors harbor a subpopulation of cells, termed cancer stem cells (CSCs), that are responsible for initiating tumor growth and driving relapse after chemotherapy [6, 7]. CSC-associated markers Bmi1 and Sox2 sufficiently enhance self-renewal and dedifferentiation and endow pancreatic cancer cells with stemness [8, 9]. Further, the pancreatic CSC marker CD24 increases the ability of cells to migrate and invade and has a close correlation with a poor prognosis [10, 11, 12]. Our previous results suggested that gemcitabine can enhance the stemness of pancreatic cancer cells ; however, the exact mechanism remains to be determined. Clarifying the mechanism involved in this process will help identify adjuvant agents for enhancing the killing effect of gemcitabine chemotherapy.
A hypoxic microenvironment has been verified in many malignances, including pancreatic cancer; it plays a critical role in the resistance of cancer cells to anticancer drugs [14, 15]. Recent studies have shown that, under hypoxic conditions, pancreatic cancer cells exhibit substantial apoptosis resistance induced by gemcitabine ; however, the mechanism remains elusive. Hypoxia signaling has a close correlation with the induction and maintenance of stemness phenotypes, such as self-renewal, undifferentiated condition, sphere-forming ability [16, 17, 18]. In a preliminary study, we also found that hypoxia promotes the expression of CSC-associated markers Bmi1 and Sox2 in pancreatic cancer cells. We, therefore, speculate that the hypoxic niche might synergistically enhance the acquired gemcitabine chemoresistance through stemness induction.
Notch signaling is evolutionarily conserved and critically implicated in cell fate, including proliferation, differentiation, and apoptosis [19, 20]. Mounting evidence indicates that the release of the Notch intracellular domain (NICD) from its ligands leads to aberrant activation of Notch signaling in a variety of malignancies [21, 22, 23]. Reports concerning the correlation between Notch1 signaling and CSC phenotype have increased in recent times. It has been suggested that aberrant activation of Notch1 helps cells acquire epithelial–mesenchymal transition and CSC self-renewal properties and is associated with pancreatic cancer treatment failure [24, 25]. However, the role and mechanism of Notch1 signaling in acquired gemcitabine resistance remain elusive. PI3K/AKT signaling is extensively activated in many tumors, including pancreatic cancers [26, 27]. AKT inhibition induces apoptosis of pancreatic cancer cells and enhances the killing effect of gemcitabine . Moreover, there exists a reciprocal regulation between the Notch1 and AKT signaling pathways , by which both of them interactively regulate chemoresistance and maintenance of stemness.
In this study, we verified that the hypoxic niche synergistically enhances gemcitabine-induced stemness and acquired resistance in pancreatic cancer cells by activating the AKT/Notch1 signaling cascade. Furthermore, a chemotherapeutic combination involving the blockade of such a signaling pathway weakens the gemcitabine-enriched CSC population, providing a new therapeutic strategy against acquired chemoresistance.
Cell culture and treatments
The human pancreatic cancer cell line PANC-1 was obtained from the American Type Culture Collection (Manassas, VA, USA). The Patu8988 cell line was purchased from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). Both cell lines were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator with 5% CO2 at 37 °C. After reaching a 60–80% confluence level, the cells were treated with different concentrations of gemcitabine (Selleck, Houston, TX, USA) for 24 h. To examine the role of the Notch1 or AKT signaling pathway in enhancing stemness, the pancreatic cancer cells were pretreated with 10 μM DAPT (γ-secretase inhibitor; Selleck) for 24 h or 20 μM LY294002 (AKT inhibitor; Beyotime Biotechnology, Shanghai, China) for 2 h before gemcitabine treatment. To clarify the effect of hypoxia on pancreatic cancer cell stemness, the cells were treated with 1% O2 for different time intervals or with various doses of CoCl2 (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. To test the synergistic effect of hypoxia and gemcitabine, the cells were co-treated with optimal doses of gemcitabine and CoCl2 (as indicated in the pertinent figure legends) for 24 h.
Western blot analysis
Western blot analysis was performed as previously described . In brief, total cell lysates were electrophoresed in a sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skim milk and incubated overnight with primary antibodies. After washing, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase, and the proteins were visualized by adding an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies against Bmi1, Notch1, NICD1, AKT, p-AKT (phosphorylated AKT), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) were purchased from Cell Signaling Technology (Danvers, MA, USA), and those against Sox2 and HIF-1α (hypoxia-inducible factor-1α) were purchased from Abcam (Boston, MA, USA).
Transwell migration/invasion assay
Migration and invasion assays were performed in 24-well Transwell chambers (Corning, Fisher Scientific). For the transwell invasion assay, the upper compartment of the chamber was precoated with Matrigel (Sigma-Aldrich). Equal amounts of approximately 10 × 104 cells were seeded into each upper chamber. The upper and lower chambers were filled with culture medium containing 0.1 and 30% FBS, respectively. After about 24 h, the migratory and invasive cells on the lower surface of the membrane were fixed, stained with 0.1% crystal violet, and then counted in five random fields under a light microscope.
The MTT assay was performed as previously described . After different treatments, the pancreatic cancer cells were seeded into 96-well plates and further incubated with various concentrations of gemcitabine (Selleck) for 48 h. Then, 20 μL of MTT solution (5 mg/mL; Sigma-Aldrich) was added to each well. The plates were incubated for 4 h, after which the medium was replaced with 150 μL of dimethyl sulfoxide (Sigma-Aldrich). The optical density was detected at 490 nm. Each concentration of gemcitabine was set up in five replicate wells.
Flow cytometry analysis
Flow cytometry analysis was performed as previously described . Anti-CD24–FITC antibody was purchased from BD Pharmingen (San Diego, CA, USA).
Sphere-forming ability assay
The sphere-forming ability assay was performed in stem cell medium (SCM) as previously described . Briefly, after different treatments, the pancreatic cancer cells were washed three times and suspended in SCM, which consisted of Dulbecco’s modified Eagle’s medium/F12 medium supplemented with bovine serum albumin (0.4%; Sigma-Aldrich), Insulin-Transferrin-Selenium (ITS; 1×; Sigma-Aldrich), basic fibroblast growth factor (10 ng/mL; PeproTech, Rocky Hill, NJ, USA), and epidermal growth factor (20 ng/mL; PeproTech). Approximately 1 × 104 cells per well were seeded into ultralow-attachment 6-well plates (Corning), and the medium was changed every 3 days. After 15 to 20 days, the formed spheres (diameter ≥ 50 μm) were counted under a light microscope. The efficiency of sphere formation was calculated on the basis of the ratio of number of spheres to total number of cells.
Xenografts were formed by subcutaneously injecting PANC-1 cancer cells into the right flank of 3- to 4-week-old athymic mice (2 × 106 cells per 100 μL per mouse) (HFK Bioscience Co., Beijing, China). Approximately 6 days after subcutaneous implantation, the mice were randomly separated into the control, GEM (gemcitabine), GEM+DAPT, and DAPT groups (n = 5 per group). Gemcitabine (20 mg/kg) and DAPT (10 mg/kg) were intraperitoneally injected every 3 days and every day, respectively. Tumor volume was measured periodically by using the following formula: Volume = 0.5 × length × width2. The experimental protocol complied with the “Guide for the Care and Use of Animals in Wuhan University”.
In vivo lung metastasis assay
PANC-1 cells were separated into four groups (control, GEM, GEM+DAPT, and GEM+LY294002) and treated as indicated above. After treatment, approximately 4 × 106 cells suspended in 0.2 mL phosphate-buffered saline were injected into the lateral tail vein of 7- to 8-week-old nude mice (HFK Bioscience Co.; n = 5 per group). After about 4 weeks, the mice were euthanized, and the lungs were completely resected and photographed. For hematoxylin and eosin (H&E) staining, the lungs were fixed with 4% paraformaldehyde and cut into 5-μm sections. The specimens were then stained with H&E, and the number of metastases was detected microscopically. All mice were handled in accordance with the protocols approved by the “Guide for the Care and Use of Animals in Wuhan University”.
The data in our study were expressed as mean ± standard deviation. Student’s t-test was used to compare differences between two groups. Values were considered statistically significant at P < 0.05.
Gemcitabine promotes Notch1 activation and pancreatic cancer cell stemness
Notch1 signaling mediates gemcitabine-induced stemness
Notch1 inhibition enhances the killing effect of gemcitabine and suppresses metastasis in vivo
AKT promotes pancreatic cancer cell stemness partly by mediating Notch1 activation
Hypoxia synergistically enhances gemcitabine-induced stemness
AKT/Notch1 signaling mediates the synergistic enhancement of the stemness induced by gemcitabine and hypoxia co-treatment
Hypoxia has been reported to activate AKT expression . Our results also demonstrated a similar effect in pancreatic cancer cells (Fig. 6a and b). In addition, synergistically augmented p-AKT expression was more evident after gemcitabine and CoCl2 co-treatment (Fig. 6c), whereas AKT suppression significantly suppressed the collaboratively inductive effect of the co-treatment on Bmi1 and Sox2 expression (Fig. 6d). In line with these changes, the enhanced sphere-forming ability of the cells was also dramatically reduced after AKT inhibition (Fig. 6e-g). Moreover, the induction of NICD1 expression by gemcitabine and CoCl2 co-treatment was significantly abolished by pretreatment with an AKT inhibitor in both evaluated cells (Fig. 6d), which suggests the role of AKT regulation in Notch1 activation in cells treated with gemcitabine and CoCl2. Collectively, these results suggest that AKT/Notch1 signaling plays a role in promoting the synergistic induction of stemness by gemcitabine and hypoxia.
The Notch1 signaling pathway plays an essential role in the maintenance and self-renewal of CSCs in a variety of malignancies [38, 39]. It has been suggested that a fraction of pancreatic CSCs are enriched as a consequence of the killing effect of gemcitabine treatment, with a small residual cells exhibiting an activated Notch1 signaling pathway . In our study, gemcitabine (a relatively low dose) might not have had a cytotoxic effect because of the high rate of cell survival after gemcitabine treatment. We found that gemcitabine treatment increased Notch1 and NICD1 expression in a dose-dependent manner. Therefore, we believe that the activation of Notch1 resulted mainly from the “inductive effect” of gemcitabine and not just from the “enrichment” of CSCs. In addition, pretreatment with the γ-secretase inhibitor DAPT significantly inhibited the molecules and behaviors (such as migration, invasion, and chemoresistance) associated with gemcitabine-induced stemness. Further, combination treatment with DAPT enhanced the killing effect of gemcitabine and inhibited gemcitabine-induced metastasis in vivo. Our results suggest that induction of Notch1 signaling plays an important role in gemcitabine-enhanced stemness.
AKT activation plays an important role in the migration, invasion, metastasis, chemoresistance, and CSC-like phenotype of pancreatic cancer cells, and it is closely correlated with the prognosis [27, 40, 41, 42]. AKT activation overlaps in function and entails mutual cooperation with Notch1 [29, 43]. In our study, we showed that low-dose gemcitabine can facilitate AKT activation, which is in line with our findings on the enhanced stemness of pancreatic cancer cells. Further, AKT inhibition significantly attenuated gemcitabine-activated Notch1 expression as well as the consequent CSC-like behaviors of the cells. Our results suggest a potentially adverse collaboration between Notch1 and AKT, in which the two signaling pathways are intertwined in increasing the stemness of pancreatic cancer cells upon gemcitabine chemotherapy. Therefore, our results are consistent with previous findings and suggest the role of AKT signaling in promoting gemcitabine-induced Notch1 activation and pancreatic cancer cell stemness.
Hypoxia is a frequent condition in pancreatic cancer. It contributes greatly to the apoptosis resistance of cells to chemotherapy . However, the exact mechanism remains obscure. Increasing evidence has demonstrated that the hypoxic niche plays an important role in maintaining the self-renewal property and undifferentiated condition of various CSCs [18, 44]. In our study, we also showed that hypoxia synergistically promotes the gemcitabine-induced CSC-like phenotype, with an increased expression of stemness-associated molecules and enhancement of sphere-forming ability, which might explain the persistence of gemcitabine resistance in pancreatic cancer. Hypoxia activates Notch-responsive promoters and promotes the expression of downstream genes . Our study also demonstrated an increase in both NICD1 expression and AKT activation after hypoxia treatment. In addition, inhibition of AKT abolished Notch1 activation and pancreatic cell stemness induced by co-treatment with gemcitabine and hypoxia. Therefore, our results demonstrate that hypoxia collaboratively promotes gemcitabine-induced stemness through the AKT/Notch1 signaling pathway.
In conclusion, our data show a novel mechanism of acquired gemcitabine resistance in pancreatic cancer through stemness induction, which is aggravated by the ubiquitous hypoxic niche in cancer cells. Thus, strategies aimed at eliminating pancreatic CSCs might present a promising approach for overcoming gemcitabine resistance and developing effective treatments for pancreatic cancer. Moreover, our results highlight the important role of the AKT/Notch1 signaling pathway in mediating this process. We provide evidence that combination treatment with adjuvant drugs targeting such signaling pathways offers a better therapeutic benefit against pancreatic cancer in vitro and in vivo, suggesting AKT/Notch1 as attractive targets for eliminating pancreatic CSCs. Altogether, our study provides new insights into strategies for reversing chemoresistance in patients with pancreatic cancer.
We would like to thank Prof. Shun-chang Zhou, Laboratory Animal Unit, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, for offering help with establishing the tumor xenograft.
This study was supported by grants from the Scientific Research Foundation of Wuhan University (Grant number: 2042018kf0118) and the Natural Science Foundation of China (Grant number: 81803030).
Availability of data and materials
The datasets used and analysed during the study are available from the corresponding author on reasonable request.
JT was responsible for the experimental design and supervision. ZLZ and HH performed the experiments, analyzed the data, and wrote the paper. YPR and KFZ performed some of the experiments and analyzed the data. ZCZ, ZGT, and CLX participated in revising and polishing the manuscript. All authors have read and approved the final version of manuscript.
Ethics approval and consent to participate
This study was approved by the ethical review board of Renmin Hospital, Wuhan University (Wuhan, China).
Consent for publication
The authors declare that they have no competing interests.
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