Background
Globally, bladder cancer is the 10th most prevalent cancer type, with about 549,000 newly diagnosed patients in 2018 [
1]. Unfortunately, up to 70% of these patients will relapse upon transurethral resection of bladder cancer [
2], which greatly increases the suffering of patients.
Cancer recurrence is highly associated with cancer cell drug resistance and high tumorigenic capability [
3]. Moreover, these characteristics could be examined in a small cell subpopulation in bladder cancer tissue, which are called the cancer stem cells (CSC) or the tumor initiating cells (TICs) [
3‐
5]. TICs, with high self-renewal potential, are considered to contribute to tumor heterogeneity [
6,
7]. More and more researches support the viewpoint that TICs participate in tumorigenesis and regional recurrence in bladder cancer subgroups [
4,
8,
9]. Accordingly, targeting these TIC-like features may be an available therapy for decreasing tumor recurrence.
Ectopic stimulation of the Wnt/β-catenin pathway is required for drug-resistance and self renewal in colorectal TICs, which confers a poor prognosis [
10,
11]. In bladder cancer, the Wnt/β-catenin pathway takes part in epithelium stem cell maintenance [
12], indicating that changes in the vital pathway may lead to the tumorigenic potential of bladder cancer. Furthermore, genetic variants in this stem cell pathway are proven to modulate the etiology of bladder cancer [
13]. Therefore, investigation on the mechanisms underpinning the ectopic stimulation of Wnt/β-catenin pathway in bladder cancer may provide valuable therapy target for preventing recurrence.
Activating transcription factor 5 (ATF5) belongs to the basic leucine zipper family of transcription factors. ATF5 participates in transcriptional responses of multiple cellular stressors, such as cytosolic heat shock response [
14], endoplasmic reticulum unfolded protein response [
15]. Several studies have reported high expression of ATF5 in undifferentiated neural progenitor cells/stem cells [
16‐
19]. It plays a vital role in regulating cellular differentiation in various tissues, including the brain [
20], liver [
21], fat [
22], and bone [
23]. These functions that regulate the growth and differentiation in progenitor/stem cells indicate the potential role of ATF5 in the self-renewal and differentiation of TICs. Furthermore, the molecular mechanism underpinning the oncogenic role of ATF5 is largely unclear.
Herein, ATF5 was detected to be significantly upregulated in bladder urothelial carcinoma (BLCA) tissues, especially in recurrent BLCA. Ectopic ATF5 was significantly related to the clinicopathological characteristics and relapse-free survival rate of BLCA patients. Over-expressing ATF5 enhanced, whereas silencing ATF5 decreased, tumorigenic capability of BLCA in vitro and in vivo. Mechanistically, ATF5 transcriptionally upregulated DVL1 expression by binding its promoter region. This study suggests that ATF5 plays a key role in tumorigenic capability of BLCA, and might be a valuable prognosis marker and potential therapeutic target for BLCA patients.
Materials and methods
Cell culture
Bladder cancer cells SW780 and UM-UC-3 were bought from the American Type Culture Collection (ATCC, USA) and grown in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, California, USA) with fetal bovine serum (10%; HyClone, Logan, Utah, USA). Human lymphatic endothelial cells (HLECs) were acquired from ScienCell Research Laboratories (Carlsbad, California, USA) and maintained based on the manufacturer’s directions. Cell incubation was done at 37 °C in a 95% air and carbon dioxide (5%) environment.
Patients and collections of tissue samples
In this study, 140 primary BLCA tissues that had been embedded in paraffin were used. Diagnosis had been histopathologically performed at The Third Affiliated Hospital of Southern Medical University and Affiliated Cancer Hospital & Institute of Guangzhou Medical University from 2010 to 2015. Clinical as well as clinic-pathological classification and staging was evaluated based on the American Joint Committee on Cancer criteria. Clinically relevant information for the 140 study participants was listed in Table
1. Additionally, four fresh non-relapse BLCA specimens and four relapse BLCA specimens were acquired. The Clinical Trials Ethics Committee (The Third Affiliated Hospital of Southern Medical University, and Affiliated Cancer Hospital & Institute of Guangzhou Medical University) approved this research.
Table 1
Clinicopathological characteristics of clinical samples and expression of ATF5 in 140 bladder cancer
Age (years) |
≤ 60 | 62 | (44.3) |
> 60 | 78 | (55.7) |
Gender |
Male | 106 | (75.7) |
Female | 34 | (24.3) |
Grade |
Low grade | 103 | (73.6) |
High grade | 37 | (26.4) |
Stage |
Ta, T1 | 110 | (78.6) |
T2–T4 | 30 | (21.4) |
Tumor size |
≤ 3 cm | 105 | (75.0) |
> 3 cm | 35 | (25.0) |
Multiplicity |
1 | 97 | (69.3) |
> 1 | 43 | (30.7) |
Expression of ATF5 |
High expression | 65 | (46.4) |
Low expression | 75 | (53.6) |
Recurrence |
Yes | 38 | (27.1) |
No | 102 | (72.9) |
Progression |
Yes | 26 | (18.6) |
No | 114 | (81.4) |
RNA extraction and quantitative real-time PCR (qRT-PCR)
Isolation of total RNA from cells was done using the TRIzol reagent (Invitrogen, Cat No. 15596018) as instructed by the manufacturer and used for cDNA synthesis with random primers. Normalize the expression data to GAPDH gene. Expressions were calculated using the 2−[(Ct of gene)−(Ct of GAPDH)] method, whereby Ct denotes each transcripts’ threshold cycles.
Western blot (WB) assay
Western blot assay was conducted as reported previously [
24]. The antibodies used were: anti-ATF5 (Cat. No.HP001912, Sigma, St. Louis, MO, USA), anti-active-β-catenin (Cat. No.05-665-25UG, Pharmingen/BD Biosciences, Bedford, MA, USA), anti-DVL1 (Cat. No.ab106844, Abcam, Cambridge, MA, USA), anti-ABCG2 (Abcam, Cat. No. ab108312), anti-SOX2 (Abcam, Cat. No.ab92494) and anti-GAPDH antibody (Cat. No.T6199, Sigma-Aldrich).
Immunohistochemistry (IHC)
IHC was carried out in 140 BLCA tissues, which were detected with an anti-ATF5 antibody (Cat. No.HP001912, Sigma-Aldrich) as previously reported [
24]. Immunostaining degree, separately scored by two pathologists working independently and who had been blinded to histopathological features as well as patient information, was evaluated by staining indices (SI). The SI was determined as the product of staining intensity score and tumor grade cell proportions. Grading of tumor cell proportions was as: 0, absent positive tumor cells (PTC); 1, < 2% PTC; 2, 2–8% PTC; 3, 8–20% PTC; and 4, > 20% PTC. Assessment of staining intensities was as: 1, absent staining; 2, weakly (light yellow) stained; 3, moderately (yellow–brown) stained; and 4, strongly (brown) stained. Protein expression evaluated by SI had possible scores of 0, 1, 2, 3, 4, 6, 8, 9, 12 or 16. The sample with SI ≥ 8 was defined as highly expressed, while the sample with SI < 8 was defined as low expressed. Determination of the cut-off value was done according to the heterogeneity measure using the log rank test in accordance with overall survival.
Plasmids, retroviral infection, and transfection
Amplification of the human
ATF5 gene from cDNA was done by PCR and later cloned into a pcDNA4 lentiviral vector. For silencing ATF5, cloning of 2 human ATF5-targeting short hairpins RNA (shRNA) sequences into a PLKO.1 (OligoEngine, Washington, USA) was done to enable the generation of corresponding pSUPER. retro. ATF5-RNAi(s). SW780 or UM-UC-3 cells were seeded in culture plates (2 × 10
6 cells/p100 plate). Transfection was done using 10 ug of designated plasmids. Cells stably expressing ATF5 or ATF5 shRNA were obtained through retroviral infection of the 293FT cells. These cells were treated for 3 days using puromycin (3.33 µg/mL). The promoter region of the human
DVL1 gene, which had been generated via PCR-amplification from SW780 cells were cloned into NheI/BglII sites of the pGL3-basic luciferase reporter plasmids (Promega,Madison, WI, USA) in order to establish DVL1 luciferase reporters. Tables
2 and
3 shows the primers used in this study.
Table 2
Primers used for gene detection
ATF5 | F: TGGCTCGTAGACTATGGGAAA |
R: ATCAACTCGCTCAGTCATCCA |
GAPDH | F: TGTGGGCATCAATGGATTTGG |
R: ACACCATGTATTCCGGGTCAAT |
DVL1 | F: GAGGGTGCTCACTCGGATG |
R: GTGCCTGTCTCGTTGTCCA |
NANOG | F: TCCCGAGAAAAGATTAGTCAGCA |
R: AGTGGGGCACCTGTTTAACTT |
SOX2 | F: CTCGTGCAGTTCTACTCGTCG |
R: AGCTCTCGGTCAGGTCCTTT |
ABCG2 | F: CAGGTGGAGGCAAATCTTCGT |
R: ACCCTGTTAATCCGTTCGTTTT |
TCF1 | F: CAGAGGAGAGGAACCAAGCTA |
R: GCAACTCGGGACATAAAGCC |
CCND1 | F: GCTGCGAAGTGGAAACCATC |
R: CCTCCTTCTGCACACATTTGAA |
CD44 | F: CTGCCGCTTTGCAGGTGTA |
R: CATTGTGGGCAAGGTGCTATT |
JUN | F: CATTGTGGGCAAGGTGCTATT |
R: ACAGAGCGAGTGAAAATGTGTAT |
Table 3
The siRNA targets sequence of DVL1
siDVL1#1 | sense: CCAAGAUUAUCUACCACAUTT |
antisense: AUGUGGUAGAUAAUCUUGGTT |
siDVL1#2 | sense: CCAAGCUUCCCUGCUUCAATT |
antisense: UUGAAGCAGGGAAGCUUGGTT |
siDVL1#3 | sense: GCAUCUACAUUGGCUCCAUTT |
antisense: AUGGAGCCAAUGUAGAUGCTT |
Cells (5 × 102) were plated in 6-well ultralow cluster plates and incubated for 10 to 12 days. Incubation of tumor spheres was done in serum free DMEM/F12 (Invitrogen, Cat. No.88215) with epidermal growth factor (EGF, 20 ng/mL, Cat. No.37000015, PeproTech, Rocky Hill, USA), B27 (2%, Cat. No.12587010, Invitrogen), basic fibroblast growth factor (20 ng/mL, Cat. No.100-18B, bFGF, PeproTech), insulin (5ug/mL, Cat. No.100-11, PeproTech), and BSA (0.4%, Cat. No.A1933-1G, Sigma-Aldrich). After 10–12 days, tumor spheres (spherical, tight, non-adherent masses that were larger than 50 µm in diameter) were counted and imaged by inverse microscopy. The efficiency of sphere formation was calculated using the formulacolonies/input cells × 100%.
Luciferase reporter test
Luciferase reporter test was conducted as described previously. In short, in triplicates, bladder cancer cells (3 × 104 cells per well) were plated in 24-well plates and incubated for 24 h. Transfection of specified plasmids as well as a pRL-TK Renilla plasmid (1.5 ng) was done using the Lipofectamine 3000 Reagent (Cat. No.L3000008, Thermo Fisher Scientific). Then, 48 h after transfection, the luciferase as well as Renilla signals were assessed by the Dual Luciferase Reporter Assay Kit (Cat. No.E1980, Promega) as instructed by the manufacturer.
Chromatin immunoprecipitation-qPCR (ChIP-qPCR)
The ChIP assay was carried out as previously reported [
24]. In short, crosslinking was carried out using formalin (1%) followed by cell lysis in sodium dodecyl sulfate (SDS) buffer. Then DNA fragmentation was achieved by sonication. ChIP for ATF5 was carried out with a Flag antibody (Sigma, SAB4301135). Fragments of DNA that had been eluted were detected by qPCR. The primer used for ChIP assay was shown as follow:
DVL1primer 5:5′-TTGGAATGAGGCACAGGG-3′, 5'-GACAGAAAACTGCCCACC-3′.
Tumor xenograft
Male BALB/c nude mice (5–6-weekold, 16–18 g) were bought from Guangdong Experimental Animal Center (Guangdong, China) and housed in an animal holding facility on a 12 h dark/light cycle. Randomly, mice were allocated in to four groups (n = 6 per group). Inguinal folds of mice were inoculated with SW780 cells (1 × 106 cells), which had been stably transfected with RNAi-vector, ATF5-RNAi#1, ATF5 or vector, with Matrigel (to a final concentration of25%). An external caliper was used to assess tumor volumes, which were determined by the equation (L × W2)/2. Then, 36 d post-inoculation, mice were euthanized by quick intraperitoneal injection of 100 μg/g pentobarbital sodium, and sacrificed for tumor excision.
index.jsp) software program (version 2.2.3).
Statistical analysis
The SPSS v.21.0 software (SPSS Inc., New York, NY, USA) was used for all analyses. Comparison of means between groups was done by a two-tailed paired Student’s t-test. The association between ATF5 levels and clinic-pathological characteristics was calculated by χ2 test. Kaplan Meier was used for establishment of survival curves, which were compared by log-rank test. Then, survival data were calculated by univariate as well as multivariate Cox regression analyses. The bivariate correlation between variables was evaluated by Spearman's rank correlation coefficients. The P-value < 0.05 was the threshold for significance.
Discussion
This study revealed that ATF5 facilitates the formation of tumor sphere, promotes tumorigenicity and stimulates the Wnt/β-catenin pathway in bladder cancer. Furthermore, ATF5 was upregulated in human BLCA and elevated ATF5 was related to relapse-free survival outcomes, implying that ATF5 might be a potential prognosis marker for BLCA recurrence.
ATF5 has been demonstrated to be highly expressed in undifferentiated neural progenitor/stem cells [
16‐
19]. It seems to repress the differentiation of bone and brain tissues [
20,
23], while targeted abrogation of ATF5 leads to normal differentiation in neural progenitor cells [
18‐
20]. These studies support the potential regulation of ATF5 in the differentiation and self-renewal of TICs. In a variety of cancers, ATF5 has also been characterized to be upregulated, such as leukemia, breast cancer and gliomas [
25‐
27]. ATF5, as a transcription factor, functions as an oncogenic role in enhancing cell survival, migration and radioresistance of cancer cells [
28,
29]. Herein, ATF5 alteration significantly changed the expression of stemness-associated genes SOX2 and OCT4. SOX2 and OCT4 are transcription factors that control the transcriptional regulatory network in embryonic stem cells [
30]. They were commonly used as the markers of CSC in multiple cancers [
31,
32], including bladder cancer [
33,
34]. Our results indicated that ATF5 up-regulation could promote the stemness of bladder cancer cells. Furthermore, we found that overexpressions of ATF5 in bladder cancer cells promoted the formation of tumor sphere, which was correlated with the relapse-free survival of BLCA patients. On the contrary, the down-regulation of ATF5 inhibited the formation of spheres, which was correlated with the improvement of survival rate. These results indicate that ATF5 overexpression increases tumorigenicity and enhances the TIC-like phenotype in bladder cancer cells, thereby providing hope for developing novel therapeutic strategy to prevent BLCA recurrence.
The Wnt/β-catenin signaling, known as the canonical Wnt pathway, is essential for development of the embryo as well as tissue self-renewal of tissues [
35,
36]. Abnormal activations of this pathway can lead to unrestrained cells proliferation and malignant transformation [
35,
36]. As one of the most relevant pathways associated with TICs, this pathway is often abnormally stimulated in various cancers, including bladder cancer [
13,
37]. Stimulation of Wnt/β-catenin pathway by miR-543-3p could increase [
38], whereas inhibition of this signaling by miR-139-5p may inhibit [
39] TIC-like phenotype of BLCA cells, supporting the vital roles of Wnt/β-catenin pathway in regulating TIC-like phenotype of bladder cancer. Consistent with these studies, we detected that Wnt/β-catenin signaling was abnormally activated in BLCA. We showed that ATF5 could directly target and positively regulate
DVL1, leading to the stimulation of Wnt/β-catenin signaling.
DVL1, as a main component of the Wnt pathway, takes part in transduction of Wnt signals to β-catenin, and then stimulates downstream effector factors [
40]. In this study, we found that ATF5 could directly bind to
DVL1 promoter and stimulate its expression, and then activate the downstream genes of the Wnt/β-catenin pathway, including active β-catenin, MYC, CD44, JUN as well as CCND1, whereas down-regulating ATF5 reduced the expression of DVL1 and these factors. These findings demonstrate a novel mechanism underpinning hyperactivation of the Wnt/β-catenin pathway in BLCA. Herein, this study indicates that ATF5 could simulate the Wnt/β-catenin pathway and promote tumorigenic capability.
Conclusion
The present study reveals that overexpression of ATF5 in BLCA directly promotes DVL1 expression and stimulates the Wnt/β-catenin signaling, therefore increasing tumorigenicity, enhancing a TIC-like phenotype as well as predicting poor survival. Evaluation of the role of ATF5 in BLCA will broaden our understanding of the mechanism underpinning the high recurrence rate of BLCA, and establish whether ATF5 serves as a prognosis marker or potential treatment target for BLCA recurrence.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.