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Up‐regulation of glycolysis promotes the stemness and EMT phenotypes in gemcitabine‐resistant pancreatic cancer cells

Abstract Cancer stem cells (CSCs) and epithelial–mesenchymal transition (EMT)‐type cells are considered as underlying causes of chemoresistance, tumour recurrence and metastasis in pancreatic cancer. We aimed to describe the mechanisms – particularly glycolysis – involved in the regulation of the CSC and EMT phenotypes. We used a gemcitabine‐resistant (GR) Patu8988 cell line, which exhibited clear CSC and EMT phenotypes and showed reliance on glycolysis. Inhibition of glycolysis using 2‐deoxy‐D‐glucose (2‐DG) significantly enhanced the cytotoxicity of gemcitabine and inhibited the CSC and EMT phenotypes in GR cells both in vitro and in vivo. Intriguingly, the use of the reactive oxygen species (ROS) scavenger N‐acetylcysteine (NAC) restored the CSC and EMT phenotypes. H2O2 produced changes similar to those of 2‐DG, indicating that ROS were involved in the acquired cancer stemness and EMT phenotypes of GR cells. Moreover, doublecortin‐like kinase 1 (DCLK1), a pancreatic CSC marker, was highly expressed and regulated the stemness and EMT phenotypes in GR cell. Both 2‐DG and H2O2 treatment suppressed DCLK1 expression, which was also rescued by NAC. Together, these findings revealed that glycolysis promotes the expression of DCLK1 and maintains the CSC and EMT phenotypes via maintenance of low ROS levels in chemoresistant GR cells. The glycolysis‐ROS‐DCLK1 pathway may be potential targets for reversing the malignant behaviour of pancreatic cancer.


Introduction
Pancreatic cancer is a highly aggressive malignant disease with a 5year survival rate of less than 8% [1]. As the most important part of chemotherapy, gemcitabine alone or in combination with other therapeutics has been adopted as the standard chemotherapy for advanced pancreatic cancer, despite a very limited progression-free survival interval [2,3]. One of the important causes of the disappointing outcome of patients with pancreatic cancer is the acquired drug resistance to gemcitabine [2,4].
The existence of CSCs is one of the fundamental drivers of chemoresistance in pancreatic cancer [4][5][6][7]. CSCs are a subpopulation of tumour cells that are endowed with the capacity for selfrenewal. They are also considered to be an underlying cause of tumorigenesis, tumour recurrence and metastasis [7][8][9]. Previous presence of oxygen [13]. Although aerobic glycolysis is a well-recognized hallmark of cancer cells, the metabolic signatures particular to chemoresistant cells and their parental cells remain elusive. Recently, it has been reported that cancer cell stemness can be epigenetically regulated by metabolic reprogramming [14]. In addition, a recent study demonstrated that radioresistant nasopharyngeal carcinoma CSCs exhibited a greater reliance on glycolysis than did their parental cells and that their stem cell-like properties could be curbed via inhibition of glycolysis [15]. Therefore, it is important to elucidate the role of metabolism change in chemoresistance associated with CSC and EMT characteristics in pancreatic cancer cells.
Unlike normal cells, cancer cells maintain high ROS levels and suffer from oxidative stress [16]. However, CSCs have lower levels of ROS than do cancer cells in general. In fact, the maintenance of low ROS levels has been found to be essential for maintaining stemness and EMT properties in CSCs [17][18][19][20]. Studies have shown that glycolysis accounts for the maintenance of low ROS levels in CSCs [19,21]. ROS have also been reported to link glucose metabolism to CSC and the EMT phenotypes in breast cancer [19]. In the light of these observations, we attempt to characterize chemoresistant pancreatic cancer cells from a ROS-mediated metabolism perspective.
Emerging evidence suggests that DCLK1, a well-established putative pancreatic CSC marker, regulates the EMT phenotype [22] and facilitates tumour invasion and metastasis [23]. However, to the best of our knowledge, studies on the relationship between glycolysis and DCLK1 were not reported. We also explored the roles of glycolysis and ROS involved in the regulation of DCLK1.
In this study, we demonstrated that GR Patu8988 cells were more glycolytic than parental gemcitabine-sensitive (GS) cells. In addition, glycolysis maintained gemcitabine-induced CSC and EMT phenotypes via maintaining ROS at low levels. Additionally, ROS negatively regulated the expression of DCLK1 which in turn regulated the stemness and EMT properties of GR cells. We conclude that inhibition of glycolysis, up-regulation of ROS and knockdown of DCLK1 may eradicate CSCs, reverse the EMT phenotype and therefore enhance the chemosensitivity. These findings may open the door for new and innovative therapies for patients with pancreatic cancer.

Cell lines and culture conditions
The human pancreatic cancer line Patu8988 was originated from KeyGEN (China) [Correction added on 14th June 2017, after first online publication: the origin of the cell PATU78988 was incorrect and updated on this version]. GR Patu8988 cells were derived as described previously [10,12]. In short, Patu8988 cells were cultured with increasing concentrations of gemcitabine (Selleck.cn, Shanghai, China) from 20 nM to a final 1000 nM for up to 12 weeks and were finally cultured in 1 lM gemcitabine during multiple passaging. The duration of cultivation in 1 lM gemcitabine was 9 months when the cells completely adapted to the treatment. The resultant cells were termed as GR cells. Both cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Beijing, China) supplemented with 10% foetal bovine serum (Gibco Invitrogen, Grand Island, NY, USA).

Cell viability assay
This was conducted as described previously [24]. Cells (6000/well) were seeded in 96-well plates overnight. The cells were then treated with different agents for the indicated time. As for the proliferation of the transfected GR cells, cells (2.5 9 10 3 ) were seeded and transfected in 96-well plate. Cell growth was observed for 5 days. MTT (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated for another 4 hrs. The absorbance was read at 490 nm using a microplate photometer after adding DMSO (Sigma-Aldrich). Details are shown in supplementary materials and methods of Data S1.

Western blot analysis
Cells were washed twice with cold PBS and lysed with a radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Shanghai, China) at 4°C for 30 min. The total protein was extracted, and the concentration of each sample was determined using a BCA protein assay kit (Beyotime) according to the manufacturer's instructions. Equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) which were then blocked with 5% non-fat milk powder dissolved in Tris-buffered saline with Tween-20 (TBST) for 1 hr and incubated with primary antibodies over night at 4°C. The membranes were washed with TBST three times (10 min. each), incubated with secondary horseradish peroxidasecoupled antibodies (Aspen, Wuhan, China) and visualized using ECL substrate (ThermoFisher, Waltham, MA, USA). The antibodies were provided in the supplementary materials and methods Data S1.

Quantitative real-time PCR assay
Cellular RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). cDNA was obtained by reverse transcription with 0.5 lg of RNA with Pri-meScript RT Master Mix (Takara Bio, Kusatsu, Shiga, Japan). Quantitative real-time PCR (qRT-PCR) was performed using a quantitative SYBR Green PCR Kit (Takara Bio). Each sample was set up in triplicate wells. The mRNA levels of the targeted genes were expressed with the 2 ÀDDCT method and normalized to GAPDH. Primer sequences are listed in Table S1.

Migration and invasion assays
Cells (5 9 10 4 cells) in 200 lL DMEM plus 0.1% foetal bovine serum (FBS) were plated into the upper compartment of a Transwell chamber (Corning, Costar, NY, USA) coated with ECM gel (Sigma-Aldrich) or left uncoated. The lower chamber was filled with 700 lL DMEM plus 20% FBS. After the cells were cultured for 24 hrs, the cells in the upper chamber were removed with a cotton swab. The invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for visualization. Cells were counted in five respective fields at 2009 magnification using a microscope (Olympus, Tokyo, Japan). stained with fluorescein isothiocyanate-conjugated CD24 antibody (BD Pharmingen, San Diego, CA, USA) or phycoerythrin-conjugated CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) or isotype control IgG (Biolegend, San Diego, CA, USA) and were analysed using a flow cytometer (BD, Biosciences, Franklin Lakes, NJ, USA). Levels of intracellular ROS were determined using an ROS assay kit (Beyotime) according to the manufacturer's instructions. Details are shown in supplementary materials and methods of Data S1.

Sphere-formation assay
Cells (5 9 10 3 ) with different treatments were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM)/Ham's nutrient mixture (F12) (1:1) medium (Invitrogen) supplemented with 20 ng/ml of epithelial growth factor (PeproTech, Rocky Hill, NJ, USA) and 10 ng/ml of basic fibroblast growth factor (PeproTech) for 14 days. Twice a week, half of the culture medium was replaced and the medium was supplemented. Spheres larger than 50 lm were counted using a microscope (Olympus) [6].

Small interfering RNA-mediated knockdown of DCLK1
Small interfering RNA (siRNA) (RiboBio Co. Guangzhou, China) sequence targeting the coding region of DCLK1 (siDCLK1#1: CAGAGGTGCGAGAGAATAA and siDCLK1#2: CTGGAAAGATAAAGAAGCA) and negative control siRNA (siNC) not matching any of the human genes were obtained. Knockdown of DCLK1 was performed by transfecting cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The efficiency of the transfection was confirmed by Western blot and qRT-PCR analyses.
Glucose uptake and lactate production assays Glucose uptake and lactate production were performed according to previously published methods [26]. The complete medium was replaced with a glucose-free medium and incubated for 2 hrs. Cells were then incubated with the fluorescence-labelled glucose analogue (2-(N-(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) 2-NBDG (Cayman Chemical, Ann Arbor, MI, USA) with a final concentration of 10 lM for 30 min. at 37°C. The uptake of 2-NBDG was analysed with flow cytometry and fluorescence microscope. For assessment of lactate production, cells (2.5 9 10 5 ) were cultured in complete medium. After 36 hrs, the supernant was collected and centrifugated. Lactate production was assessed using a lactic acid assay kit (Nanjing Jiancheng Bio. Nanjing, China) according to the manufacturer's protocol and was corrected for the total protein in each sample.

Animal experiment
All animal experiments were handed in compliance with the Institutional Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology. For subcutaneous tumour formation, GS or GR cells with different treatments were subcutaneously injected into the right flank of nude mice. In addition, an orthotopic transplantation pancreatic cancer model was performed to assess liver metastasis as described previously [27]. The detailed treatment protocols are shown in supplementary materials and methods of Data S1.

Statistical analysis
All data were analysed using GraphPad Prism 5.0 statistical software (San Diego, CA, USA). Mann-Whitney U-test was used to calculate the significant difference between two groups. For data set containing more than two groups, one-way ANOVA with Bonferroni post-tests was carried out. Two-way ANOVA was used to determine differences over different drug treatments. Results were expressed as means AE S.E.M. P values <0.05 were considered statistically significant.

GR cells display enhanced aerobic glycolysis and glycolytic dependency
Cell viability assays showed that the half-maximal inhibitory concentrations (IC50) of gemcitabine for GR and GS cells were 134.3 lM and 2.8 lM, respectively (Fig. 1A). GR cells, therefore, showed 48fold greater resistance to gemcitabine than did GS cells.
To further examine the dependence of pancreatic cancer cells on glycolysis for survival, we evaluated cell viability upon addition of 2-DG, an inhibitor of glycolysis. The IC 50 of gemcitabine decreased from 134.3 lM to 3.9 lM in GR cells and from 2.8 lM to 1.4 lM in GS cells after the introduction of 2-DG ( Fig. 1A and E). Furthermore, cell viability decreased significantly more in GR cells than in GS cells after treatment with 2-DG alone (Fig. 1F), further indicating that GR cells were more dependent on glycolysis for survival.
In accordance with our finding that GR cells have high glycolytic flux, assays of both protein and mRNA expressions showed that the levels of the glycolytic proteins and enzymes glucose transporter 1 (GLUT1), hexokinase-II (HK-II), lactate dehydrogenase A (LDHA) and pyruvate kinase M2 (PKM2) were significantly higher in GR cells ( Fig. 1G and H).

GR cells demonstrate the CSC and EMT phenotypes compared with GS cells
We found that GR cells displayed a CSC-like phenotype ( Fig. 2A-D). Flow cytometric analysis using antibodies against pancreatic CSC  surface markers showed that GR cells had an obviously higher expression of the stemness markers CD24 and CD133 on their cell surfaces than did GS cells ( Fig. 2A). Intriguingly, both Western blot and qRT-PCR analyses indicated that the pluripotency markers Nanog and Sox2 were expressed at higher levels in GR cells than in GS cells ( Fig. 2B and C). In addition, the sphere-formation assay showed that GR cells formed more and larger spheres than did GS cells (Fig. 2D). Taken together, these findings suggest that chemoresistant GR cells possess enhanced stem-like cell characteristics than their GS parental cells.
GR cells, which exhibit a loss of cell-cell adhesion, increased formation of pseudopodia, and spindle-shaped morphology, were morphologically distinct from GS cells (Fig. 2E). The MTT assay showed that there was no obvious change in the proliferation between GS and GR cells (Fig. 2F). We next investigated markers of EMT. Assays of both protein and mRNA levels showed that GR cells had lower 2059 expression of the epithelial marker E-cadherin and higher expression of the mesenchymal marker Vimentin and the transcriptional factor zinc finger protein SNAI1 (Snail 1) than did GS cells (Fig. 2G and H). One of the important characteristics of mesenchymal-like cells is their enhanced capacity for migration and invasion [10]. Consistent with our morphological observations, we found that GR cells demonstrated significantly greater capacity for migration and invasion than did GS cells (Fig. 2I and J). These observations suggest that GR cells possess the EMT property, becoming more invasive in vitro. To further observe whether the biological characteristics of GR cells were reversible, we cultured GR cells in gemcitabine-free medium up to 7 days. Interestingly, these GR cells still showed enhanced chemoresistance and invasive and self-renewal capacities compared with their parental GS cells (data not shown), indicating that GR cells underwent stable biological transformation during the long-term induction.

Glycolysis regulates the gemcitabine-induced CSC and EMT phenotype via maintaining low ROS levels
To further elucidate the role of ROS in the acquired stemness and EMT phenotypes of GR cells, we used flow cytometry to assess ROS levels. We found that ROS levels were obviously lower in GR cells than in GS cells (Fig. 3A), indicating that the gemcitabine-induced stemness and EMT phenotypes may be associated with intrinsically low ROS levels. Because it has been reported that 2-DG treatment can inhibit glycolysis and induce cytotoxicity via oxidative stress and that 2-DG's cytotoxicity can be suppressed by the antioxidant NAC [28][29][30][31], we first investigated these effects of 2-DG treatment. As we expected, 2-DG treatment induced ROS production, which could be inhibited by treatment with NAC (Fig. 3B). In addition, cell viability assay showed that the viability was not significantly affected within 24 hrs in GR cells treated with or without 2-DG in the presence or absence of NAC. However, NAC inhibited the cytotoxicity induced by treatment with 2-DG at 48 hrs (Fig. 3C). In the light of these observations, we further investigated the effect of oxidative stress induced by glycolytic inhibition on the stemness and EMT phenotypes in GR cells.
GR cells were treated with or without the glycolytic inhibitor 2-DG in the presence or absence of NAC for the indicated time. We found that 2-DG effectively decreased the expression of the pancreatic cancer stemness markers CD24 and CD133 (Fig. 3D) and reduced the levels of pluripotency markers Nanog and Sox2 in both protein and mRNA levels (Fig. 3E). 2-DG treatment also suppressed the sphere-forming ability of GR cells (Fig. 3F). However, compared with cells treated with 2-DG alone, cells treated with a combination of 2-DG and NAC exhibited increased levels of CD24 and CD133 (Fig. 3D), higher Nanog and Sox2 protein levels (Fig. 3E, upper), increased mRNA expression of Nanog and Sox2 (Fig. 3E, lower) and enhanced sphereformation ability (Fig. 3F).
In addition, both Western blot and qRT-PCR analyses indicated that E-cadherin expression was up-regulated and that Vimentin and Snail1 were down-regulated after 2-DG treatment (Fig. 4A and B). Additionally, GR cells treated with 2-DG showed a significantly inhibited capacity for migration and invasion ( Fig. 4C and D). However, NAC combined with 2-DG treatment also restored the EMT phenotype as indicated by the significant down-regulation of E-cadherin expression and up-regulation of Vimentin and Snail1 expression compared with the levels in GR cells treated with 2-DG alone ( Fig. 4A and B). Finally, the increased capacity for migration and invasion that had been inhibited in GR cells by 2-DG treatment was also restored after the introduction of NAC ( Fig. 4C and D).
To further investigate the role of ROS in the maintenance of the CSC and EMT phenotypes, we introduced exogenous H 2 O 2 to induce an increase in ROS levels comparable to that induced by 2-DG in GR cells (Fig. 5A). We also assessed the oxidative stress induced by H 2 O 2 treatment on cell viability. Similar to 2-DG treatment, the cell viability was not significantly affected within 24 hrs in GR cells. NAC also inhibited the cytotoxicity induced by treatment with H 2 O 2 at 48 hrs (Fig. 5B). Like 2-DG treatment, H 2 O 2 treatment decreased the protein expression of the pluripotency markers Nanog and Sox2 and the EMT markers Vimentin and Snail1. However, the addition of NAC blocked the down-regulation of these proteins (Fig. 5C). We also found that H 2 O 2 significantly inhibited sphere formation and reduced the migratory and invasive capacity of GR cells. NAC prevented the H 2 O 2 -induced loss of the CSC and EMT phenotypes (Fig. 5D-F). All these results suggest that glycolysis maintains the gemcitabineinduced CSC and EMT phenotypes via down-regulating ROS production in GR cells.

The glycolysis-ROS-DCLK1 pathway accounts for the maintenance of gemcitabine-induced CSC and EMT phenotypes
We found that GR cells had a higher expression level of DCLK1 than did GS cells in protein and mRNA levels (Fig. 6A). To confirm the involvement of DCLK1 in the maintenance of stemness and the EMT  phenotypes in GR cells, we transfected siRNA-targeting DCLK1 (siDCLK1#1 and siDCLK1#2) into GR cells. The successful transfection of siDCLK1 was verified by a reduction in the expression of both the DCLK1 protein (Fig. 6B, left) and DCLK1 mRNA (Fig. 6B, right) expressions compared with negative siRNA controls (siNC). Both protein and mRNA levels of the pluripotency markers Nanog and Sox2 were decreased after siDCLK1 transfection (Fig. 6C and D). In addition, the sphere-forming ability of GR cells was significantly suppressed by siDCLK1 transfection (Fig. 6E). The MTT assay showed that silencing DCLK1 inhibited the proliferation of GR cells (Fig. 6F). DCLK1 knockdown also inhibited EMT phenotype. The migratory capability and invasive capability of GR cells were reduced after siDCLK1 transfection (Fig. 6G and H). In addition, the protein expression and mRNA levels of the epithelial marker E-cadherin were upregulated, while the mesenchymal marker Vimentin and the transcription factor Snail1 were down-regulated after siDCLK1 transfection ( Fig. 6I and J).
We next examined the roles of glycolysis and ROS in DCLK1 expression. H 2 O 2 administration decreased the expression of DCLK1 in GR cells; the introduction of NAC restored DCLK1 expression (Fig. 6K), indicating the involvement ROS in DCLK1 expression. Interestingly, the glycolysis inhibitor 2-DG decreased the expression of DCLK1 in GR cell, and this expression was also recovered by the introduction of NAC (Fig. 6K). Together, these data suggest that glycolysis regulates DCLK1 expression via downregulating ROS production in GR cells exhibiting the CSC and EMT phenotypes.

Glycolysis promotes tumorigenesis and metastasis in vivo
We further investigate the role of glycolysis in tumour formation and metastasis in vivo. We did not observe significant differences in tumour latency, tumour weight (19.0 AE 1.8 mg versus 20.0 AE 2.3 mg, P > 0.05) and tumour incidence in mice inoculated with GS or GR cells ( Fig. 7A and B). However, tumours originated from GR cells had enhanced staining for DCLK1 (Fig. 7C). In addition, in our orthotopic transplantation pancreatic cancer model, GR cells gave rise to a 13-fold increase in formation of metastatic liver nodules compared with GS cells (Fig. 7D-F). These findings suggest that GR cells are more invasive and acquired increased metastatic ability in vivo.
To further illuminate the role of glycolysis in tumorigenesis and metastasis, we treated GR cells with or without 2-DG. GR cells formed tumour nodules in 5 of 5 injected mice, whereas 2-DGtreated GR cells formed tumour nodules in 4 of 5 injected mice ( Fig. 7G and H). In addition, 2-DG-treated cells formed reduced tumour weight (6.2 AE 1.8 mg versus 19.58 AE 4.1 mg, P < 0.05) compared with GR cells (Fig. 7G). Furthermore, tumours formed by 2-DG-treated cells showed reduced staining of DCLK1 (Fig. 7I). Moreover, 2-DG-treated cells resulted in obviously decreased liver metastases (Fig. 7J-L). These observations suggest that glycolysis is involved in the tumorigenesis and metastasis of GR cells in vivo.

Discussion
Acquired resistance to chemotherapeutic agents is an obstacle in treating pancreatic cancer [2]. Accumulating evidence has shown that a small population of pancreatic cancer cells with stem cell-like features are responsible for chemoresistance, tumorigenesis, progression and metastasis [7,32]. In addition, EMT-type cells are also believed to play critical roles in drug resistance in pancreatic cancer [33]. In this study, we for the first time demonstrated the functional role of glycolysis in the maintenance of chemoresistance, the CSC and EMT phenotypes of GR pancreatic cancer cells via down-regulation of ROS levels.
It has been reported that energy metabolism plasticity determines stemness potential [34]. Cancer cells undergo a metabolic shift in which they become more dependent on glycolysis for survival than are normal cells [13]. Therefore, it is reasonable to speculate that glycolysis plays a role in the acquired stemness phenotype of pancreatic cancer cells. Shen et al. demonstrated that radioresistant nasopharyngeal carcinoma cells exhibited a greater reliance on glycolysis than their parental cells and the stem cell-like properties could be curbed via inhibition of glycolysis [15]. We found that GR cells showed a higher glycolysis flux than their parental cells. We also found that the glycolytic inhibitor 2-DG significantly reinforced the cytotoxicity of gemcitabine towards GR cells. Importantly, 2-DG treatment led to the inhibition of CSC and the EMT phenotypes in GR cells both in vitro and in vivo. Therefore, a combination of a glycolytic inhibitor with conventional chemotherapeutic drugs might effectively enhance chemosensitivity in chemoresistant pancreatic cancer cells and help prevent recurrence and metastasis after chemotherapy [21].
ROS, as an intracellular messenger, play various roles in regulating important cell functions. The connection between ROS homeostasis and energy metabolism is complex [16]. ROS have been reported to link glucose metabolism to the CSC and EMT phenotypes in breast cancer [19]. Moreover, evidence suggests that 2-DG treatment can cause oxidative stress in cancer cells [30,31]. In the light of these findings, we have elucidated the effects of oxidative stress in the inhibition of stemness and EMT phenotypes in chemoresistant pancreatic cancer cells. As expected, the introduction of the ROS scavenger NAC restored the 2-DG-induced loss of CSC and EMT phenotypes in GR cells. Furthermore, the application of NAC also recovered the H 2 O 2 -induced loss of the CSC and EMT phenotypes in GR cells, further demonstrating that glycolysis is responsible for the maintenance of the gemcitabine-induced CSC and EMT phenotypes via maintaining ROS at relatively low levels.
Previous studies have shown that CSCs possess relatively low intracellular ROS levels [17,35,36]. In line with these studies, we found that GR cells also displayed lower ROS levels than did their parental GS cells. The elevation of ROS may lead to the differentiation of CSCs and the loss of stem cell markers [37]. Therefore, disrupting ROS homeostasis and promoting ROS production may be an effective approach to eradicating CSCs and reversing chemoresistance of pancreatic cancer.
Our study has further elucidated the underlying mechanisms by which ROS affect the stemness in GR cells. We focused on DCLK1   because it is a newly identified CSC marker which is functionally involved in driving cancer carcinogenesis and cancer progression [38]. One novel findings of this study is that DCLK1 is highly expressed in GR cells; this finding is consistent with the finding that GR cells have stem cell-like features. We also found that tumours formed by GR cells had obviously increased staining of DCLK1 compared with their GS parental cell. In addition, we found that DCLK1 was responsible for the maintenance of stemness and the EMT phenotypes in pancreatic cancer cells, which was in line with a previous study [39]. Additionally, our results showed that H 2 O 2 or 2-DG treatment decreased DCLK1 expression, which was restored by NAC, indicating that ROS negatively regulate the expression of DCLK1 in GR cells. Furthermore, tumours formed by GR cells treated with 2-DG showed a decreased staining for DCLK1. Collectively, these findings reveal that glycolysis-related ROS regulate the CSC and EMT phenotypes by targeting DCLK1 in GR cells, suggesting that DCLK1 is a promising target in treating pancreatic cancer.
In our in vivo study, we did not observe the differences of tumour growth between GS and GR cells although GR cells showed increased expression of stem cell-like markers and sphere-formation ability, indicating that other mechanisms other than glycolysis may be involved in the regulation of tumorigenesis in vivo. The present observation needs further investigation for the complex mechanisms involved in chemoresistance in cancer cells.
In conclusion, our data provide the first evidence that glycolytic signalling plays a functional role in the maintenance of CSC and the EMT phenotypes via down-regulation of ROS production and up-regulation of DCLK1 expression in GR pancreatic cancer cells. Therefore, inhibition of glycolysis signalling, up-regulation of ROS generation and/or silencing of DCLK1 may be effective approaches to eliminating CSCs, reversing the EMT phenotype and thus eradicating chemoresistant cancer cells to improve the prognosis of patients with pancreatic cancer.