Background
Bladder cancer is a common malignancy; it is the fourth most commonly diagnosed cancer globally and the eighth leading cause of cancer-related deaths in males worldwide [
1,
2]. Approximately 75–80% of bladder cancer patients are initially diagnosed as having nonmuscle-invasive bladder cancer (NMIBC), and a half of such patients experience recurrence within five years; in fact, up to 30% of NMIBC patients will progress to muscle-invasive bladder cancer (MIBC), and the latter initially occurs in approximately 25% of bladder cancer patients and has a poor prognosis [
3]. Treatment of MIBC patients usually involves surgical resection of the tumor followed by cisplatin-based chemotherapy [
3,
4]. The cisplatin-based chemotherapy causes severe toxicity and has a relatively low anticancer efficiency, with only 40–65% of metastatic bladder cancer patients showing a clinical response [
3,
4]. Thus, a better understanding of the molecular mechanisms underlying bladder cancer progression and recurrence will lead to the identification of novel anticancer targets for bladder cancer therapy.
Cullin-RING ligases (CRLs) are enzymes that target proteins for ubiquitin-mediated degradation, and altered CRL activity contributes to the development and progression of human cancers, including bladder cancer [
5]. CRL, also known as Skp1, cullin, or the F-box protein, belongs to the largest family of E3 ubiquitin ligases that mediate the proteasome-targeted degradation of 20% of ubiquitinated protein substrates, like the cell cycle-related, DNA replication, and signal transduction proteins as well as transcription factors [
5]. Experimentally, small molecule inhibitors, such as MLN4924, have been shown to suppress CRL activation by inhibition of cullin activity, and, in turn, to effectively reduce the growth of various human cancer cells in vitro [
6].
The regulator of cullins-1 (ROC1), also named as RING box protein-1 (RBX1), is a key CRL subunit that heterodimerizes with other cullins to form the CRL catalytic core [
5]. ROC1 contains a small zinc-binding domain (the RING finger), which is evolutionarily conserved and is essential for embryonic development, while aberrant ROC1 expression leads to CRL dysfunction and embryonic lethality [
7,
8]. ROC1 is also essential for maintenance of the genome integrity and cancer development [
8‐
12]. Our previous studies have demonstrated that ROC1 protein is overexpressed in bladder cancer tissues and that knockdown of ROC1 expression reduces the CRL activity, thus triggering the accumulation of its specific substrates (such as p21, p27, and DEPTOR) and leading to tumor growth suppression [
13,
14]. However, the mechanisms of ROC1 in the malignant proliferation of bladder cancer have not been fully elucidated. As accumulating evidence suggests an essential role of hedgehog signaling in tumor cell proliferation [
15], in this study, we explored the underlying molecular mechanisms by which ROC1 regulates the sonic hedgehog (SHH) pathway in bladder cancer using in vitro and in vivo experiments. The results of this study will form the molecular basis for the future development of a novel ROC1-based targeted therapy against bladder cancer.
Materials and methods
Cell culture and reagents
Human bladder cancer 5637 and T24 cell lines were obtained from the Chinese Academy of Science (Shanghai, China) and maintained in Roswell Park Memorial Institute medium-1640 (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin in a humidified 5% CO2 environment at 37 °C. Cycloheximide, the SHH signaling activator smoothened (SMO) agonist (SAG), the SMO antagonist GDC0449, and the Nedd8-activating enzyme inhibitor MLN4924 were purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (Sigma-Aldrich), and stored at − 20 °C as stock solutions. G418 was also from Sigma-Aldrich. The recombinant plasmid carrying hemagglutinin (HA)-tagged ubiquitin cDNA was purchased from Invitrogen (Shanghai, China).
Establishment of stable ROC1-overexpressed or -silenced bladder cancer cell lines
To establish a stable ROC1-overexpressed bladder cancer cell subline, we subcloned the full-length wild-type human ROC1 cDNA into the pcDNA3.1 vector (Invitrogen, Shanghai, China), named as pcDNA3.1-ROC1. After DNA sequence confirmation, this recombinant plasmid or pcDNA3.1 vector-only plasmid was transfected into bladder cancer cells using Lipofectamine 2000 (Invitrogen) for 48 h, and the cells were then cultured in G418-selecting cell culture medium at 100 μg/mL for 14 days. After that, individual G418-resistant monoclonal cells were selected and expanded in the 100 μg/mL G418-selective medium. The stable cell sublines were named as p-ROC1 or p-CONT. Furthermore, to knock down ROC1 or suppressor of fused homolog (SUFU) expression, we purchased siRNA oligonucleotides targeting ROC1 or SUFU from Invitrogen (Shanghai, China) and transfected them into bladder cancer cells, according to the manufacturer’s instructions. The ROC1 siRNA sequence was 5ʹ-GACTTTCCCTGCTGTTACCTAA-3ʹ; the SUFU siRNA sequence was 5ʹ-GCCATGACAATCGGAAATCTA-3ʹ; and the scrambled control siRNA sequence was 5ʹ-ACGTGACACGTTCGGAGAA-3ʹ.
Changed cell viability was assessed by using the Cell Counting Kit-8 kit (Beyotime, China), as previously described [
13]. For the colony formation assay, tumor cells were seeded in triplicate into 35-mm culture dishes at a density of 400 cells (for 5637 tumor cells) or 1000 cells (for T24 cells) per well and cultured for 9 days. The cells were fixed and stained with crystal violet in 50% methanol, and the number of cell colonies with more than 50 cells was counted.
Flow cytometry cell cycle distribution assay
Both ROC1-overexpressed and siRNA-transfected bladder cancer cells were detached from the cell culture dishes and fixed in ice-cold 70% ethanol overnight. One day later, the cells were washed twice with ice-cold phosphate-buffered saline (PBS), then stained with propidium iodide (Sigma-Aldrich) solution (20 mg/mL) for 5 min, and finally analyzed by using a BD FACScan flow cytometer (BD Biosciences, San Diego, CA, USA).
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was isolated with the Trizol reagent (Invitrogen) and reversely transcribed into cDNA with a PrimeScript Reverse Transcription kit (Takara, China), according to the manufacturers’ protocols. The resultant cDNA samples were then amplified using a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the SYBR Green master mix kit (Takara, Dalian, China) for detection of different genes using gene-specific primers (the detailed DNA sequences of each primer used in this study are available upon request). All measurements were performed in triplicate and quantified using the 2−∆∆Ct method.
Western blot and co-immunoprecipitation-Western blot
After the cells were subjected gene transfection or drug treatments, cell lysates were prepared and quantified according to a previous study [
14]. The western blot was carried out as described previously [
13], while the co-immunoprecipitation kit (Cat. #26419) from Thermo Scientific (Waltham, MA, USA) was used according to the manufacturer’s instructions with the following antibodies: anti-ROC1 (Abcam, Cambridge, MA, USA); anti-cyclin D1, anti-Cdc25c, anti-SUFU, anti-Gli1, anti-GAPDH, and anti-HA (Abcam, Hangzhou, China); and anti-Gli2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Immunofluorescence staining
Immunofluorescence staining was performed to assess Gli2 expression in cells, as described previously [
13]. Briefly, cells were grown on coverslips, fixed, and permeabilized, and then they were incubated with a primary antibody against Gli2 (Santa Cruz Biotechnology) followed by incubation with the Alexa 548-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA). Subsequently, the cells were counterstained by using 4,6-diamidino-2-phenylindole (Sigma) and analyzed under a Zeiss LSM500 confocal microscope (Zeiss International, Oberkochen, Germany).
In vivo tumor cell xenograft assay
An orthotopic tumor model of bladder cancer was used. In particular, tumor cells were cultured to reach 70–80% confluency, harvested, resuspended in PBS, and then mixed with Matrigel (Invitrogen) at a 1:1 vol/vol ratio. Next, mice (6-week-old, male, athymic, BALB/C nu/nu; n = 10 per group) were anesthetized by using 40 mg/kg sodium pentobarbital, and a small lower abdominal incision was made to expose the bladder for tumor cell injection. Tumor cells were then injected into the bladder wall using a 28-gauge needle; thereafter, the injection site was pressed with a cotton swab for 30 s, and the skin incision was then closed with the absorbable line. Tumor cell xenograft formation and growth were assessed by using the whole-body fluorescence imaging system weekly, with a Spectrum in vivo imaging system (Promega, Madison, WI, USA) with 470-nm excitation from an MT-20 light source. The emitted fluorescence signal was collected by using appropriate filters on a DP70 CCD camera and processed for contrast and brightness with Paint Shop Pro 8 (Corel, Ottawa, ON, Canada). Twelve weeks after the nude mice were inoculated with the pROC1 or pCONT tumor cells, the mice were sacrificed, and the xenograft tissues were resected. This study protocol was approved by the Animal Care and Use Committee of Yancheng First People’s Hospital (Jiangsu, China) and carried out following the Guidelines for the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research.
Human bladder tissue samples and immunohistochemistry
Bladder cancer tissue specimens were retrospectively collected from 93 bladder cancer patients who were cared for at Yancheng First People’s Hospital (Jiangsu, China) between January 2010 and May 2015. The patients included 79 males and 14 females with a median age of 67 years old (range: 45–87 years old); 43 of the patients underwent a transurethral resection, 12 underwent a partial cystectomy, and 38 underwent a radical cystectomy. Their tumor grade and stage were classified according to the World Health Organization 1973 criteria and the American Joint Committee on Cancer 2002 Tumor, Node, Metastasis system. This study of human subjects was approved by the Medical Ethics Committee of Yancheng First People’s Hospital (Permit Number: 2013KY004), and informed consent was obtained from each patient before enrolling into this study.
Paraffin-embedded tissue blocks were retrieved from the Pathology Department and used for the preparation of the tissue microarray and then immunostained with a primary antibody against ROC1 (Abcam), SUFU (Abcam), Ki67 (Boster, Wuhan, China), or Gli2 (Boster), according to our previous study [
13].
Statistical analysis
The western blot band intensities were quantified by using Image J software (National Institutes of Health, Bethesda, MD, USA). The data were expressed as means ± standard error of the mean (SEM) and statistically analyzed by using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). For multi-group comparisons, the Bonferroni t-test was used after one-way analysis of variance. Meanwhile, for two-group comparisons, the Student’s t-test was used. The correlation between ROC1 or SUFU and Gli2 expression was assessed by using Pearson’s χ2 test. A p value < 0.05 was considered statistically significant.
Discussion
Our present study demonstrated that ROC1 overexpression promoted the growth of bladder cancer 5637 and T24 cell lines in vitro and enhanced the growth of mouse orthotopic xenografts in nude mice. In contrast, knockdown of ROC1 expression had the opposite effects. Mechanistically, ROC1 targeted SUFU for ubiquitin-dependent degradation to release Gli2 from the SUFU complex, in turn activating the hedgehog pathway. These findings suggest that ROC1 plays an important role in the malignant proliferation of bladder cancer and that targeting ROC1 expression may help to control bladder cancer progression or recurrence in the future.
Dysregulation of cell proliferation is a landmark during tumorigenesis and progression, while induction of cell cycle arrest is an important strategy for the development of anticancer drugs that effectively control human cancers [
20]. Previous studies by others and us have demonstrated that knockdown of ROC1 expression is able to induce cancer cell G2/M arrest, autophagy, and senescence [
10,
13,
21]. In the current study, we revealed that exogenous overexpression of ROC1 promoted bladder cancer cell proliferation, whereas knockdown of ROC1 expression inhibited tumor growth through induction of G2/M arrest. Consistent with our current findings and further supporting the importance of CRL in the regulation of tumor cell proliferation, the suppression of CRL activity using FBXO22 silencing inhibited cancer progression through targeting of HDM2 for degradation in breast cancer [
12]. Mechanistically, DNA damage is the most common cause of inducing cell G2/M arrest [
20]. Indeed, ROC1 silencing was able to trigger the DNA damage response as a result of DNA re-replication [
8]. Furthermore, a recent study has shown that ROC1 silencing leads to deficiency in DNA double-strand break repair by exonuclease 1 excessive degradation [
22]. Taken together, we postulate that knockdown of ROC1 expression inhibited cancer cell proliferation by induction of cell cycle G2 arrest due to DNA damage.
The hedgehog pathway is important in cell differentiation and embryonic development [
15]. A variety of cancer types also have been linked to the aberrant activity of the hedgehog signaling pathway [
15,
16]. Gli1 and Gli2 are two important transcriptional factors in the hedgehog pathway, e.g., Gli1 acts as a transcriptional activator and is regulated by Gli2, while Gli2 acts as a strong activator of hedgehog signaling [
15]. Upon stabilization, Gli2 translocates into the cell nucleus to promote transcription of both Gli1 and Gli2 and other target genes [
23]. In general, Gli2 could play a more important role in bladder cancer [
24]. In the current study, we demonstrated that knockdown of ROC1 expression inhibited Gli2 expression but not Gli1 expression, whereas ROC1 overexpression promoted Gli2 expression in bladder cancer cells. These findings suggest that ROC1 regulates the hedgehog pathway in a Gli2-dependent manner. Notably, another study has revealed that the SPOP–CUL3–RBX1 E3 ubiquitin ligase complex can activate the hedgehog pathway through ZBTB3 degradation [
25]. Thus, CRL inactivation suppresses hedgehog activity via different pathways, but the exact mechanism remains unknown. We suppose that the mechanism may depend on the cell line or the cell treatment.
SUFU, a key negative regulator of the hedgehog pathway, can bind to Gli to inhibit the hedgehog pathway activity [
17], while SUFU protein can be degraded by the CRL
Fbxl17 E3 ubiquitin ligase [
18]. In the current study, we demonstrated that SUFU accumulation inactivated the hedgehog signaling upon ROC1 knockdown, while blockage of SUFU expression restored the hedgehog signaling suppression triggered by the ROC1 knockdown, indicating that the ROC1-regulated hedgehog signaling in bladder cancer was dependent on SUFU degradation. One conceivable explanation may be that with the cooperation of ROC1, the F-box protein Fbxl17 binds to SUFU and promotes degradation in the CRL complex. Moreover, we also observed that silencing of both ROC1 and SUFU expression only partially restored the hedgehog pathway activity, indicating that the SUFU–Gli2 axis is necessary but not sufficient for regulation of hedgehog activity and that other regulatory pathways may also be involved.
In addition, our current ex vivo data further revealed an association between ROC1 expression and the hedgehog pathway proteins, i.e., ROC1 expression was inversely associated with SUFU (
P < 0.001) but positively associated with Gli2 expression (
P < 0.001) in bladder cancer tissues. We speculate that ROC1 could be a promising prognostic biomarker for predicting bladder cancer progression. The small molecule MLN4924 (Pevonedistat), which abrogates neddylation of the cullin subunit of CRLs, has been identified as a promising anticancer drug [
6]. A recent study has shown that MLN4924 is able to synergistically enhance cisplatin cytotoxicity to urothelial carcinoma [
26]. Our current study revealed that knockdown of ROC1 expression induced the same effect as MLN4924; therefore, we hypothesized that the combination of cisplatin with hedgehog inhibition by ROC1 silencing would provide a novel strategy to control bladder cancer in the future.
The development of bladder cancer, like most other human cancers, is a multifactorial and multistage cell transformation and carcinogenic process. Nevertheless, the current study does have some limitations. For example, besides the orthotopic tumour xenograft, we also observed lung and liver metastases in the in vivo experiment. This finding may be attributed to long of an observation period of three months, and distant metastasis could not be completely ruled out. Secondly, our sample size in the current study was small, and the association between ROC1 expression and patient prognosis was not investigated. Therefore, a future study with a larger sample size and follow-up data is needed to verify our current data. The present study is just a proof-of-principle study, and more research is needed to better understand the molecular mechanisms.
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