Background
Pancreatic cancer is a highly malignant tumor of digestive system and its incidence is increasing rapidly in recent years. The early diagnosis is still very difficult for pancreatic cancer patients. Most pancreatic cancer patients are diagnosed with advanced stage and the prognosis is poor [
1]. For the patients suffering from pancreatic cancer, the 5-year relative survival rate is less than 8% [
2]. Therapy failure in most pancreatic cancer patients is mainly due to distant metastasis before surgical operation and limited efficiency of chemotherapy or radiation therapy [
3]. It is urgently necessary to elucidate the underlying mechanisms of pancreatic cancer progression and develop effective therapies.
Clinical studies have proven that pancreatic cancer has close relation with hyperglycemia. Diabetes mellitus have been proved to increase the incidence of pancreatic cancer compared with non-diabetes population [
4]. The mortality of pancreatic cancer patients associated with diabetes mellitus is significantly higher than those without diabetes [
5]. Moreover, pancreatic cancer patients with diabetes mellitus frequently showed larger tumors and reduced median survival [
6]. Unfortunately, the definite role and molecular mechanisms of hyperglycemia in the progression of pancreatic cancer have not been clearly elucidated until now.
The immune system plays an important role in the development of pancreatic ductal adenocarcinoma. Unfortunately, the immune system seems imbalanced in pancreatic cancer patients, facilitating spontaneous cancer development [
7]. Despite the presence of many immune cells in pancreatic cancer tissue, immune dysfunction is observed where the tumor microenvironment is immunosuppressive, leading to inhibited activation of immune effectors. Natural killer (NK) cells are vital components of innate immune system. NK cells can kill cancerous cells through recognizing the ligands expressed on the surface of tumor cells [
8]. NK cells are recognized as the first line of defense against cancer [
9], and have gained much attention in adoptive cancer immunotherapy. The killing effect of NK cells mainly relies on its activating receptors NKG2D, which can bind to NKG2D ligands (NKG2DLs) on target cells and mediate the cytotoxicity [
10]. MHC class I chain related molecules A/B (MICA/B) is a highly glycosylated membrane protein, belonging to NKG2DL family [
10]. As the ligand of NKG2D, MICA/B can activate NK cells specifically to induce immune killing. However, tumor cells can escape from immune surveillance mediated by NKG2D through shedding or weakening MHC class I chain related molecules (MIC) from the membranes of cancer cells [
11].
In this study, we demonstrated that high glucose inhibited the cell surface expression of MICA/B on pancreatic cancer cells and weaken the cytotoxicity of NK cells on pancreatic cancer. Moreover, high glucose promoted the expression of polycomb protein Bmi1, which increased GATA2 and inhibited cell surface MICA/B expression. Bmi1 is a major component of Polycomb Repressor Complex 1 (PRC1) family, and was originally identified as an oncogene associated with the development of murine lymphoma [
12]. In this study, we identified a novel role of Bmi1 in pancreatic cancer immune escape. Our results demonstrated a new pathway of AMPK-Bmi1-GATA2-MICA/B axis, which was activated under high glucose and shown to be essential for the immune escape of pancreatic cancer cells.
Methods
Cell culture
The pancreatic cancer cell lines, PANC-1 and SW1990, were obtained from ATCC, and were cultured in DMEM medium containing 10% fetal bovine serum and 100 U/ml penicillin/streptomycin mixture (Beyotime Biotechnology, Shanghai, China). NK cells were originally obtained from China Center for Type Culture Collection (CCTCC), and cultured in α-MEM containing 12.5% horse serum, 12.5% fetal bovine serum and 200 U/ml of recombinant human interleukin − 2 (rhIL-2). The cells were cultured in 37 °C with 5% CO2. The concentration of glucose was 5 mM for general cell culture and in order to simulate the high glucose environment, two levels of diabetogenic glucose concentration (15 mM and 25 mM) were chosen.
Western blot analysis
After washing three times with PBS, total cell lysates were extracted with RIPA lysis buffer. Quantitation of proteins was performed using the BCA protein concentration kit (Beyotime Biotechnology, Shanghai, China) and 30 μg of each sample was used to SDS-PAGE electrophoresis and transferred to the PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk for 1 h and incubated with primary antibody at 4 °C over night. After washing 3 times with TBST (10 min/ times), the membranes were incubated with second antibody at room temperature for 1 h. After washing another 3 times with TBST, they were visualized with enhanced chemiluminescence (Pierce, Thermo Fisher, Waltham, MA, USA). The primary antibodies against GAPDH (1:1000), Bmi1 (1:1000), p-AMPK (1:1000), and AMPK (1:1000) were purchased from CST (Cell Signaling Technology, Danvers, MA, USA). MICA/B (1:200) antibody was purchased from Santa Cruz (Santa Cruz Biotechnology, Texas, U.S.A.). GATA2 (1:1000) antibody was purchased from Abcam (Abcam, Cambridge, UK). GAPDH was used as the internal control. AMPK activator (A-769662) and AICAR were obtained from Selleck chemicals (Selleck.cn, Shanghai, China).
Quantitative real-time PCR
After washed with PBS, the total RNA of the treated cells was extracted using TRIzol. cDNA was obtained by reverse transcription through the reaction of 1 μg RNA and PrimeScript™ RT Master Mix (Takara Bio, Shiga, Japan). qRT-PCR is obtained according to the SYBR Green PCR Kit (Takara Bio, Shiga, Japan). The results were analyzed according to 2
-ΔΔCT, and GAPDH was used as control. Primer sequences for GAPDH, Bmi1, GATA2, MICA, and MICB were shown in Additional file
3: Table S1.
ChIP
Chromatin immunoprecipitation (ChIP) was performed using anti-GATA2 antibody and EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA), following the manufacturer’s protocol. The IgG was used as the internal control. After high glucose treatment, ChIP was performed by immunoprecipitation with IgG or GATA2 antibody. The bound DNA fragments were amplified with MICA/B promoter-specific primers. The PCR products were resolved by electrophoresis. Primer sequences for ChIP-qPCR were shown in Additional file
3: Table S1.
Cell transfection
Bmi1 over-expression cDNA (pcDNA3.1-Bmi1) and empty vector cDNA (pcDNA3.1-NC) were designed and synthesized by GenePharma (Shanghai, China). The GATA2 siRNA/ Bmi1 siRNA and NC siRNA were designed and synthesized by Ribobio (Guangzhou, China). Lipofectamine™2000 (Invitrogen, California, U.S.A.) was used in the cell transfection according to manufacturer’s protocol. After transfection for 6 h, the medium was replaced to normal medium. The siRNA sequences and negative control siRNA sequences were shown in Additional file
4: Table S2.
Flow cytometry analysis
After transfection for 48 h of Bmi1 plasmid or GATA2 siRNA/Bmi1 siRNA, the culture medium was discarded. The cells were digested with trypsin and centrifugated. After washing three times with PBS, they were made into monocellular suspension. Each test tube was given 10 μL MICA/B -PE antibody (R&D systems, Minnesota, U.S.A.) under the condition of dark light, and incubated for 30 min at 4 °C. After washing three times, the tubes were added with 200 μl PBS before analyzing. The results were obtained by flow cytometer analysis.
Lactate dehydrogenase (LDH) release assay
The killing ability of NK cells was analyzed by LDH release assay according to manufacturer’s protocol (Beyotime Biotechnology, Shanghai, China). Briefly, the target cell is 10 thousand, and the effective target ratio is 2.5:1, 5:1, 10:1 and 20:1 in 96-well plates. LDH release assay was performed after incubation for 4 h in 37°C and 5% CO2. The killing activity of NK cells was calculated as following: the killing activity (%) = (OD experimental group - OD natural release)/(OD maximum release - OD natural release)*100%.
Immunofluorescence
Pancreatic cancer cells were plated in 12-well plates with a density of 1 × 104 cells/well. After washing with cold PBS, the cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Then, they were permeabilized with 0.5% Triton-X, blocked with goat serum, incubated with Bmi1 primary antibody at 4 °C over night. After washing with PBS, the cells were incubated with fluorescent secondary antibody for 2 h at room temperature. Then the samples were stained with DAPI for 5 min and photographed with fluorescence microscopy.
Immunohistochemistry (IHC) analysis
We investigated the association between Bmi1 and MICA/B expression in cancer tissue using tissue microarrays. Two arrays (same set) contained 30 cases of pancreatic cancer tissues (Outdo Biotech, Shanghai, China) were obtained. The first array was stained with anti-Bmi1 antibody (Cell Signaling Technology, Danvers, MA, USA) and the second array was stained with anti-MICA/B (Santa Cruz Biotechnology, Texas, U.S.A.) antibody using standard IHC protocol.
Animal experiments
For the diabetic pancreatic cancer mice model, the 5-week male Balb/c athymic nude mice (Beijing Vital River Laboratory, Beijing, China) were randomly divided into Control, Hyperglycemia, Hyperglycemia + insulin, Control + NK, Hyperglycemia + NK, and Hyperglycemia + insulin + NK groups. For the diabetic groups, the mice were injected streptozocin (STZ) (Sigma, St. Louis, MO, USA) at a concentration of 175 mg/kg on Day 0 and Day 7, respectively and STZ was dissolved in cold sodium citrate buffer (pH = 4.5). Blood samples were taken from the tail vein and measured with SANNUO (Changsha, China). The mice with blood glucose > 300 mg/dL were included in our experiments (n = 5 in each group). After establishing the diabetes model, two groups were injected insulin (0.8 units/kg/day) to normalize blood glucose level. Then the six groups were all subcutaneously injected PANC-1 cells (3× 106 /100 μL/mouse) in the right flank. One week after subcutaneous implantation, NK cells (105/mouse) were injected into mice once a week for three weeks. The animals were sacrificed one week after the last NK cell injection. The tumor size was measured periodically and calculated by the formula 0.5 × length × width2. The expression levels of Bmi1, MICA/B and GATA2 were measured with IHC.
Statistical analysis
The results were shown as mean ± SD. The Western blot results were analyzed by Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA). Comparisons between the two treatments were evaluated using Student’s t test. Comparisons between multiple groups were performed with Two-way ANOVA analysis. The SPSS 21.0 software was used for statistical analysis and P < 0.05 was considered as statistically significant.
Discussion
Pancreatic cancer is one of the most malignant tumors featured with high mortality. Gene mutation, including K-RAS, TP53, SMAD4, and others, was involved in the molecular pathogenesis of pancreatic cancer [
19]. However, these discovered abnormalities to date limitedly contributed to the improvement in therapeutic efficacy or survival among pancreatic cancers patients. The pancreatic cancer has been considered to harbor unique microenvironments. Moreover, pancreatic tumor microenvironments confer highly malignant properties on pancreatic cancer cells and promote pancreatic cancer progression [
20]. In this study, we develop our hypothesis that high glucose affects the expression of Bmi1, AMPK, GATA2, and MICA/B and promotes pancreatic cancer cells to escape from immune surveillance. These findings constitute a new signal pathway in response to hyperglycemia, a condition frequently observed in pancreatic cancer patients and are associated with increased mortality and poor survival.
Recent studies suggest that hyperglycemia may play a previously underexplored role in promoting pancreatic cancer progression. Diabetes mellitus has been considered as a potential risk factor for pancreatic cancer and is closely related to the poor prognosis [
21,
22]. Accumulating evidences show positive correlation between diabetes mellitus and the increased incidence of cancers [
23,
24]. Among the cancers affected by diabetes mellitus, pancreatic cancer exhibits the most obvious correlation with high glucose [
5]. Excessive glucose may help cancer cells to maintain their high metabolism and non-controlled proliferation [
25]. Moreover, evidence shows that hyperglycemia promotes proliferation and metastasis of pancreatic cancer cells [
26]. Multiple mechanisms were involved in the biological association between hyperglycemia and cancer, such as uncontrolled proliferation, hyperinsulinaemia, inflammatory response, et al. [
27]. However, there existed sparse literatures regarding the immunological mechanism between hyperglycemia and pancreatic cancer.
In our study, we found that high glucose can inhibit the antitumor immunity by reducing the killing effect of NK cells on pancreatic cancer. This inhibition was related to the reduced MICA/B expression on pancreatic cancer cells. As an important component of NKG2DLs, MICA/B expression is restricted to tumor tissues and plays key roles in mediating the cytotoxicity of NK cells. Decreased MICA/B expression may facilitate cancer immune escape from natural killer (NK) cell-mediated cytotoxicity. Multiple mechanisms have been found to participate in regulating MICA/B expression. It has been reported that DNA damage response pathways, heat shock stress, BCR/ABL oncogene, and bacterial/viral infections can all participate in regulating MICA/B expression [
28‐
31]. In this experiment, we elucidate a new phenomenon that MICA/B can be down-regulated by tumor microenvironment such as high glucose. The mechanism may be one of the tactics that pancreatic cancer escape immune killing. One interesting finding in our study is that high glucose inhibits MICA/B by promoting Bmi1 expression. Abnormal expression of Bmi1 was seen in a variety of cancers, and was related to malignant behaviors of cancer [
32,
33]. We previously reported that overexpression of Bmi1 promotes proliferation, malignant transformation, and is related to a poor survival of pancreatic cancer [
34]. It has been reported that Bmi1 can enhance the immunomodulatory properties of human mesenchymal stem cells [
35]. However, few studies correlated Bmi1 with cancer immune escape, rendering the exploration of Bmi1 in cancer immunity a necessity. In current study, we confirmed that Bmi1 can inhibit anticancer immunity of pancreatic cancer via reducing NK cell killing through suppressing MICA/B expression. We further proved that high glucose can promote Bmi1 expression through inhibiting AMPK signaling pathway. These findings provide new insights of Bmi1 as a central node connecting high glucose and pancreatic cancer development and progression.
In this study, we demonstrated that Bmi1 suppresses MICA/B expression, and this inhibition can be achieved by enhancing GATA2 expression. GATA2 is a member of GATA family transcription factors and contains zinc fingers in its DNA binding domain. GATA2 is involved in the development and differentiation of different types of cells, for example hematopoietic stem cells [
36]. Previous study showed that GATA2 was involved in the escape of HBV
+ HCC cells from NK cell immune surveillance [
37]. In this study, we verified that GATA2 can bind to the MICA and MICB promoter and inhibit the transcription of MICA/B genes. Moreover, Bmi1 inhibits MICA/B expression through up-regulating of GATA2 in pancreatic cancer cells, contributing to the immune escape eventually. Our research may open a new avenue to GATA2 research in pancreatic cancer.
Being an abnormal physiological condition in microenvironment, high glucose may affect the biological behavior of cancer cells through changing multiple signaling pathways [
38]. We speculate that signaling pathways changed by high glucose may be involved in promoting Bmi1 expression and inhibiting MICA/B expression. AMPK is an important energy sensor which can regulate metabolic or energy homeostasis and participate in almost all aspects of cell function [
39,
40]. Moreover, AMPK signaling can be affected by energy metabolism in cells, and its activity was negatively correlated with the invasion ability of tumor cells [
41]. In our study, AMPK signaling pathway was inhibited in pancreatic cancer cells treated with high glucose. AMPK inhibition coincides with Bmi1 promotion, GATA2 promotion and MICA/B inhibition. After AMPK was activated, the expression of Bmi1 and GATA2 was inhibited, whereas MICA/B expression was recovered. Importantly, when AMPK signaling was activated, the NK cells could restore its killing effect on pancreatic cancer cells in hyperglycemic environment. Our results suggest that inhibition of AMPK signaling in high glucose can inhibit antitumor immune function by promoting Bmi1 expression and suppressing MICA/B expression. Since AMPK signaling plays a key role in mediating immune escape of pancreatic cancer, it is an ideal target for activating antitumor immunity.