Introduction
Cancer stem cells (CSCs) are a small population of cells in tumors that possess characteristics associated with normal stem cells, such as self-renewal and differentiation. Therefore, CSCs drive tumor recurrence, metastasis, and drug resistance by generating new tumor cells [
1,
2]. In addition, CSC also possess a high degree of metabolic plasticity, which allows them to respond to metabolic changes and maintain proliferation, self-renewal and viability [
3]. Pancreatic cancer is one of the most aggressive solid malignancies, which has become the fourth leading cause of cancer related deaths in the world [
4]. The typical malignant characteristics of pancreatic cancer, such as rapid progression, easy recurrence, and poor drug response, are thought to be related to the strong stemness of pancreatic cancer cells [
5,
6]. Given the critical role of stemness in the progression of pancreatic cancer, identification of new targets that enhance the stemness of pancreatic cancer and development of specific therapies targeting stemness may significantly improve the prognosis of pancreatic cancer patients.
The Interleukin (IL)-20 subfamily plays a crucial role among the diverse mechanisms that facilitate stemness. For example, the expression of IL20RA and IL22RA1 promotes stemness in breast cancer [
7] and pancreatic cancer [
8], respectively. IL-22 has been reported to promote stemness in colorectal cancer [
9] and Kras-mutant lung cancer [
10]. Interleukin-20 receptor subunit beta (IL20RB) is a subunit of the IL-20 subfamily receptor. It forms a complete heterodimeric receptor with IL20RA or IL22RA1, respectively, and the ligands mainly include IL-19, IL-20 and IL-24 [
11]. We found that IL20RB is highly expressed in pancreatic cancer through a previous study by our group, not published online, but the role of IL20RB in pancreatic cancer and whether it promotes pancreatic cancer stemness are unknown.
Here, we examined the correlation between IL20RB expression and clinical characteristics of pancreatic cancer, and explored the underlying mechanisms of how IL20RB modulates pancreatic cancer stemness and chemotherapy resistance. We also evaluated the effect of IL20RB knockdown in mouse models, and the results suggested that IL20RB can be a promising therapeutic target for pancreatic cancer.
Materials and methods
Cell culture
The pancreatic cancer cell lines in this study (PANC-1 and MIA PaCa-2) were purchased from the American Type Culture Collection (ATCC). PANC-1 and MIA PaCa-2 cells were cultured using Gibco™ Dulbecco's modified eagle medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA). The complete culture medium contained 10% fetal bovine serum (FBS, NEWZERUM, New Zealand).
Western blot test
In short, the proteins in the cells were extracted, quantified, and denatured. Total protein was subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). PVDF membrane was sealed with 5% milk for 1 h at room temperature. In a cool store at 4 ℃, the PVDF membrane was incubated with the first antibody for a whole night. On the second day, the PVDF membrane was incubated with the second antibody at room temperature for 1 h and then exposed to a chemiluminescence instrument to obtain protein bands. Antibodies: rabbit anti-IL20RB antibody (WB: dil. 1:1000, Proteintech, 20,521-1-AP); rabbit anti-NANOG antibody (WB: dil. 1:1000, Proteintech, 14,295–1-AP); rabbit anti-SOX2 antibody (WB: dil. 1:1000, Proteintech, 11,064-1-AP); mouse anti-STAT3 antibody (WB: dil. 1:1000, Cell Signaling Technology, 9139); rabbit phospho-Stat3 (Tyr705) antibody (WB: dil. 1:2000, Cell Signaling Technology, 9145); mouse anti-β-ACTIN antibody (WB: dil. 1:20,000, Proteintech, 66,009-1-Ig) (dil., dilution).
Real-time quantitative PCR test
Total RNA was extracted from cell lines using Invitrogen
™ TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The first strand cDNA was synthesized with random primers by the first strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Relative RNA level was determined by real-time quantitative PCR on a Light Cycler 480 II (Roche Diagnostics, Mannheim, Germany) using the SYBR Green method. β-ACTIN was used as an internal control for mRNA levels of IL20RB and associated genes. There were three biological replicates per experiment. The relative expression level of RNA was calculated by the comparative CT method. The sequences of the genespecific primers are listed in Additional file
1: Table S1.
Plasmids and lentivirus production and transduction
IL20RB overexpression and ShIL20RB knockdown plasmids were successfully constructed in Guangzhou Ruibo Biotechnology Co., Ltd. as target plasmids, and commercially available psPAX2 and pMD2.G as packaging plasmids. The control vector and recombinant plasmids (psPAX2: PMD2.G: IL20RB ratio is 3:1:4) were transfected into 293 T cells to produce lentivirus and infect PANC-1 and MIA PaCa-2 cells, respectively. After 6 h, the supernatant was replaced with a complete medium, and the cells were screened with purinomycin.
The well-grown log-stage PANC-1 and MIA PaCa-2 pancreatic cancer cells were digested, centrifuged, and re-suspended. They were uniformly distributed in the six-well plate with a density of 2000–4000 cells per well. The cells were incubated at 37 ℃ for 7 to 10 days until the cell colonies were visible to the naked eye. They were stained with crystal violet solution and then photographed. Subsequently, the number of clones formed was calculated using the ImageJ software.
The sphere-forming medium was composed of DMEM/F12 + 1*B27 + 20 ng/ml bFGF + 20 ng/ml EGF. Ultra-low attachment six-well plates (3471, Corning) were selected, and cells were distributed at a density of 2000–5000 cells per well (depending on pelleting efficiency). Each well was supplemented with 4 ml medium. The culture lasted 7–10 days, during which the images were captured by an inverted microscope. In each well, three fields of view were photographed, and the number of cell spheres with a diameter greater than 75 µm was counted.
Flow cytometry analysis of side population (SP) cells
PANC-1 and MIA PaCa-2 pancreatic cancer cells were inoculated in six-well plates, centrifuged after digestion, suspended in 1 mL PBS, then added with 5 μg/mL Hoechst 33,342, and incubated in a warm box for 60 min. Flow cytometry analysis was conducted for these cells using a Beckman flow cytometer. The ultraviolet laser was used to excite Hoechst 33,342, and the emitted fluorescence signals were detected by the Hoechst Blue and Red fluorescence channels at 450/65 nm and 670/30 nm, respectively. The data were analyzed using FlowJo software.
Immunofluorescence staining
Pancreatic cancer tissues from patients were embedded into paraffin wax and sliced using a microtome. The slices were roasted, dewaxed with xylene and rehydrated with graded ethanol series. They were immersed in the citric acid solution for antigen retrieval and then treated with 3% hydrogen peroxide to deactivate endogenous peroxidases. The tissues were covered with serum and kept in a wet box at 37 ℃ for 1 h; after that, they were incubated at 4 ℃ overnight in a wet box with a suitable concentration of primary antibodies. Following the addition of second antibodies, the tissues were incubated at room temperature for 1 h away from light. Immediately after the treatment with a DAPI fluorescence-preserving mounting medium, the tissues were observed under a fluorescence microscope. Antibodies: rabbit anti-IL20RB antibody (dil. 1:50, Proteintech, 20,521-1-AP); rabbit anti-NANOG antibody (dil. 1:500, Proteintech, 14,295-1-AP); rabbit anti-SOX2 antibody (dil. 1:150, Proteintech, 11,064-1-AP); rabbit phospho-Stat3 (Tyr705) antibody (dil. 1:300, Cell Signaling Technology, 9145); mouse anti-pan-CK antibody (dil. 1:500, Abcam, ab215838); rabbit anti-IL-19 antibody (dil. 1:500, Bioss, bs-10087R) (dil., dilution).
Immunohistochemistry (IHC) analysis
Paraffin-embedded tissues of mouse tumors were sliced for this examination. The tissue culture plates were stained with NANOG, SOX2 and pan-CK antibodies. The expression level was evaluated according to staining intensity and percentage of cells stained positive. The staining intensity was negative, weak, medium and strong, respectively, corresponding to scores of 1, 2, 3 and 4. The percentage of cells stained positive was 0–25%, 26–50%, 51–75% and 76–100%, respectively, with scores of 1, 2, 3 and 4. The IHC score was obtained by multiplying the cell percentage score and the staining intensity score. Antibodies: rabbit anti-NANOG antibody (dil. 1:500, Proteintech, 14,295-1-AP); rabbit anti-SOX2 antibody (dil. 1:50, Proteintech, 11,064-1-AP); mouse anti-pan-CK antibody (dil. 1:200, Abcam, ab215838) (dil., dilution).
Animal experiments
Female BALB/c nude mice aged 4–6 weeks were used for this experiment. They were raised in the Huangpu Experimental Animal Center of Sun Yat-sen University Cancer Center with free access to food and water. The mice (five in each group) were injected subcutaneously with 0.1 ml of cell suspension containing 1 × 105, 1 × 106 or 1 × 107 cells. When the tumor was palpable, its length and width were measured periodically, and the volume was calculated according to the formula: volume = length × width2 × 0.5. The animal test of this study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center (Approval Number: L025504202301010).
Statistical analysis
Results are expressed as mean ± SD for experiments performed in triplicate or more. Student t-test was used for statistical analysis of the mean comparison between the two groups. Nonparametric test was used for statistical analysis of abnormal distribution data. Kaplan–Meier method was used for survival analysis. Spearman’s correlations was used to analyze the correlation between IL20RB levels and clinicopathological characteristics. All statistical analyses were performed using SPSS software package (version 25.0; IBM SPSS). P < 0.05 was considered significant for all statistical analyses.
Discussion
Cancer stemness refers to the characteristics of CSCs capable of proliferation, self-renewal and multidirectional differentiation [
1,
2]. Finding the signaling pathways regulating cancer stemness is crucial for the treatment of cancer. Previous studies have found that the oncogene MYC drives stemness in breast and pancreatic cancer [
20,
21]. Cytokines in the tumor microenvironment (TME), such as TGF-β1 and IL-6, have also been reported to promote the stemness of various cancer cells [
22,
23].
IL20RB is expressed in a variety of normal cells, including keratinocytes, fibroblasts, monocytes, T cells and endothelial cells [
11]. High expression of IL20RB was reported in lung cancer bone metastases [
24], clear cell renal cell carcinoma [
25] and papillary renal cell carcinoma [
26]. In this study, we found that IL20RB was highly expressed in pancreatic cancer samples and correlated with poor prognosis. Increasing studies purported the potential of IL-20 subfamily members in promoting cancer stemness. For example, it has been demonstrated that IL-22 acts on colorectal cancer cells to promote the activation of transcription factor STAT3 and expression of histone H3 lysine 79 methyltransferase, consequently increasing cancer stemness and tumorigenic potential [
9]. IL22RA1 has been reported to promote the stemness and tumorigenicity of pancreatic cancer cells by activating STAT3 [
8]. In the present paper, we found a positive correlation between the protein expression of stemness markers (NANOG and SOX2) and IL20RB in pancreatic cancer samples, suggesting that IL20RB may promote tumor stemness. We also found that IL20RB overexpression in pancreatic cancer cells increased the number of spheroids, the proportion of SP cells and the expression of stemness markers, indicating that IL20RB resulted in stronger stemness. The in vivo studies further confirmed that IL20RB enhanced the tumorigenic ability of pancreatic cancer cells. Tumor stemness is an important factor leading to chemoresistance in pancreatic cancer [
27]. Drug resistance is thought to be an intrinsic property of normal stem cells and CSCs and is acquired through multiple independent mechanisms, such as the upregulation of drug efflux pumps, superior DNA repair capacity, or enhanced protection against Reactive Oxygen Species [
28‐
30]. To further understand whether IL20RB regulates chemotherapy resistance, we performed clone formation assays on gemcitabine-treated pancreatic cancer cells and found that IL20RB overexpression enhanced drug resistance; in contrast, knockdown of IL20RB weakened drug resistance in pancreatic cancer cells. The in vivo study also confirmed that IL20RB knockdown combined with gemcitabine treatment further reduced tumor volume and weight.
IL20RB exerts its effects by binding to IL-19, IL-20 and IL-24 [
11,
31]. IL-19 and IL-20 were identified as IL-10 homologs in the expressed sequence tag database [
18,
32]. IL-24 was detected in terminally differentiated human melanoma cells induced by interferon β and the protein kinase C activator mezerein [
33]. It has been reported [
34,
35] that the major source of IL-19, IL-20 and IL-24 is myeloid cells, followed by epithelial cells. We found that IL-19 most significantly increased the proportion of SP cells and the expression of stemness markers in pancreatic cancer cells in vitro, indicating that IL-19 plays a major role in promoting stemness in pancreatic cancer. Immunofluorescence analysis confirmed the presence of IL-19 in the TME of clinical pancreatic cancer samples, which is consistent with findings in previous studies [
18,
19], suggesting the mechanism by which TME factors promote the stemness of pancreatic cancer cells. Moreover, IL20RB knockdown reversed the IL-19-mediated spheroid augmentation and upregulation of stemness markers, indicating that IL20RB is responsible for the effect of IL-19.
The STAT protein family include STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B) and STAT6 [
36]. STAT3 is involved in the proliferation of tumor cells, inhibition of apoptosis and promotion of stemness and chemotherapy resistance in cancer cells. STAT3 over-activation induces immunosuppression and tumor invasion [
37‐
39]. In this sense, STAT3 has emerged as a promising target in cancer treatment. In the present study, we found that the STAT3 signaling pathway played an important role in mediating the function of IL20RB in promoting cancer stemness and chemoresistance, and IL20RB-STAT3 signaling promoted the expression of NANOG, SOX2 and POU5F1. In addition, the co-expression of IL20RB and PSTAT3 was observed in clinical pancreatic cancer samples.
However, this work has some limitations. Firstly, the mechanism by which IL20RB exerted its effects was explored only in vitro. Secondly, the specific cells producing IL-19 in the TME need to be further investigated. Thirdly, this study lacks therapeutic experiments targeting IL20RB, and the effectiveness of pancreatic cancer therapies targeting IL20RB requires verification.
In conclusion, the present study is a pioneering work probing into the role of IL20RB in pancreatic cancer. IL20RB was found to be highly expressed in pancreatic cancer and enhanced the stemness properties of pancreatic cancer cells and confer resistance to chemotherapy. Mechanistically, this effect is mediated through the activation of the downstream STAT3 pathway by IL20RB. Notably, microenvironment-derived IL-19 serves as the primary ligand initiating the signaling cascade mediated by IL20RB in pancreatic cancer.
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