Background
The global cancer statistics showed that bladder cancer (BC) caused an estimated 573,000 new cases and 213,000 deaths in the year 2020 [
1]. Traditionally, BC is categorized into non–muscle-invasive bladder cancer (NMIBC), which is characterized by a high recurrence rate, and muscle-invasive bladder cancer (MIBC), which is prone to metastasis and has a poor prognosis [
2,
3]. The 5-year survival rate of BC has not improved over the past three decades [
4]. Therefore, it is essential to understand the molecular mechanisms that underlie BC tumorigenesis and progression to develop more effective therapeutic strategies.
The SWI/SNF complex was first discovered and isolated from yeast. It is highly conserved across the eukaryotic kingdoms [
5]. Several previous studies show that researches have shown that in normal tissues, the SWI/SNF complex can functionally regulate genes associated with DNA repair, cell cycle, and cell division [
6]. The subunits of the SWI/SNF complex are frequently dysregulated in 25% of all carcinomas [
7], which underscores the significance of the SWI/SNF complex in carcinogenesis.
SNF5 (also known as SMARCB1) encodes a ~ 50 kDa protein, which is the core subunit of the SWI/SNF complex [
8]. Approximately 95% of the malignant rhabdoid tumors (MRT) harbor aberrations in the SNF5 subunit [
9,
10]. In vivo experiments using mouse models demonstrate that SNF5 loss results in rapid formation of tumors in all subjects at 11 weeks, which is half the time taken for TP53-induced tumorigenesis [
11,
12]. Several studies focused on understanding the SNF5-mediated suppression of tumorigenesis. Recently, a mechanistic study demonstrates that SNF5 antagonizes MYC, an oncoprotein transcription factor, by impairing its DNA-binding ability in MRT; SNF5 inactivation can synergistically accelerate tumor formation in combination with p53 loss [
13]. Similarly, in myeloid leukemia, SNF5 downregulation induces the activation of Rac GTPase, thereby promoting cell migration and survival [
14]. Given the abovementioned findings, SNF5 has long been considered a tumor suppressor. Nevertheless, SNF5 is upregulated in liver cancer and seems to play an oncogenic role [
15], suggestive of its dichotomy in tumorigenesis. To date, however, the effects of SNF5 on BC and the underlying mechanisms remain unknown.
Thus, this study aimed to investigate the functions of SNF5 and the mechanisms underlying SNF5 mediated pathological features in BC. Furthermore, the clinical potential of SNF5 in BC was evaluated by using public database and drug susceptibility tests. The findings may facilitate individualized clinical management of BC patients.
Methods
Cell culture and transfection
The T24, 5637, and UM-UC-3 human BC cell lines and the normal urothelial cell line SV-HUC-1 were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured in RPMI-1640 (HyClone, USA) or MEM medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C in 5% CO2. Lentiviruses for SNF5 knockdown and SNF5 overexpression were synthesized by GenePharma Co. Ltd. (Shanghai, China), and lentiviral transduction was performed according to the manufacturer’s instructions.
CCK-8 assay
CCK-8 assay was used for cell proliferation and drug susceptibility tests. Drugs used were AKT inhibitor VIII (HY-10355), gefitinib (HY-50895) and AZD-0530 (HY-10234), were purchased from MedChem Express (Monmouth Junction, NJ, USA). The cells were seeded in 96-well plates (800 or 5000 cells/well) and cultured for 4 days or 3 days. Cell viability was evaluated using the Cell Counting Kit 8 (CCK8; Dojindo, Japan) according to the manufacturer’s instructions. Briefly, 10 µL CCK-8 solution was added to each well at pre-determined time points, and the cells were incubated for 2 h. Absorbance at 450 nm was detected using a microplate reader (Bio-Rad, USA).
As described previously [
16], the cells were seeded in 6-well plates (800 cells/well) and cultured for approximately 14 days. The colonies thus obtained were washed and fixed with 4% paraformaldehyde, stained with crystal violet for 20 min, and air-dried after rinsing off the excess dye. The number of colonies that were visible to the naked eye was counted.
Wound-healing assay
The wound-healing assay was performed using the Culture-Inserts 2 Well system (Ibidi GmbH, Germany). Serum-starved cells were seeded into the inserts. The images were acquired at predetermined time points after the removal of the culture inserts. The cell migratory ability was calculated as follows:
Wound-healing percentage = (Area of the original scratch−Area of the scratch at pre-determined timepoint)/Area of the original scratch × 100. The Image J software (version 1.52, NIH, USA) was used to analyze the area.
Transwell chamber assay
Cell migration was also investigated using the Transwell chamber assay (Corning, USA), according to the manufacturer’s instructions. Briefly, serum-starved cells were resuspended in 0.2 mL serum-free medium and transferred into the upper chamber. In the lower chamber, 0.6 mL RPMI-1640 medium supplemented with 10% FBS was added as a chemoattractant. The cells on the upper surface of the chamber were scrubbed and those on the lower surfaces were fixed with 4% paraformaldehyde and stained with crystal violet. These cells were photographed, and counted in five random fields.
Tumorigenicity assays
An in vivo tumor xenograft model was established using five-week-old nude mice purchased from the Third Military Medical University (Chongqing, China). An equal number of T24 or 5637 live cells were injected subcutaneously into the root of the right thigh for each mouse after counting and excluding the trypan blue-stained cells. The tumor size was measured using vernier calipers and the tumor volume was calculated as follows:
Volume (mm3) = (length × width2)/2. The mice were euthanized intraperitoneally by injecting an overdose of pentobarbital sodium (150 mg/kg). Finally, the end-point tumors were excised.
Western blotting
Total protein was extracted from the cells using the RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and measured using a BCA kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein per sample were diluted in 5X SDS loading buffer, separated in 10% SDS-PAGE gel, and transferred to a PVDF membrane. The blots were incubated sequentially with the primary and corresponding secondary antibodies. The primary antibodies included SNF5 (CST, 91735S; 1:1000), Snail (CST, 3879S; 1:1000), E-cadherin (CST, 14472; 1:1000), Vimentin (Bioworld, 1491; 1:1000), GAPDH (Affinity, AF7021; 1:3000), signal transducer and activator of transcription (STAT3; CST, 9139S; 1:1000), and pSTAT3-Y705 (CST, 9145S; 1:1000). The protein bands were visualized using the ECL reagent (Bio-Rad, CA, United States), quantified by Image J (National Institutes of Health, USA) and the relative protein levels were normalized to that of GAPDH.
Immunohistochemistry (IHC)
IHC was performed as previously described [
17]. Briefly, tissues were fixed, paraffin-embedded, and sliced into 5 μm thick sections. The protein expression of SNF5 (CST, 91735, 1:1000), pSTAT3-Y705 (CST, 9145, 1:400), Ki67 (CST, 9027,1:500), and cleaved caspase 3 (CST, 9661,1:400) was detected and scored according to the above-mentioned reference.
The transcriptomic and clinicopathological data of BC patients were obtained from TCGA using UCSC Xena. GSE13507 was downloaded from the GEO database and normalized by the RMA package [
18]. The patient characteristics are summarized in Additional file
1: Tables S1 and Additional file
2: Tables S2. The gene set enrichment analysis (GSEA) was performed with GSEA-4.0 software (
http://software.broad institute.org/gsea/). The survival probability was evaluated with the Kaplan–Meier method, and the differences were determined by the log-rank test (
https://CRAN.R-project.org/package=survminer). The pROC package was used for receiver operating characteristic (ROC) analysis [
19]. Using the GDSC, we estimated the chemotherapeutic sensitivity of patients in the TCGA data set. The prediction process was conducted by the pRRophetic package [
20]. The half-maximal inhibitory concentration (IC
50) for each sample was calculated by ridge regression via tenfold cross-validation using the GDSC training set.
Statistical analyses
Statistical analyses were performed using Prism 8.0 and R software (version 4.0). All data were presented as mean ± SD, and the intergroup differences were analyzed using the two-tailed Student’s t-test. The differences among multiple groups were assessed by analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
Discussion
In this study, to the best of our knowledge, for the first time, we identified the function of SNF5 in BC. SNF5 expression was significantly downregulated in BC patients with lymphatic metastasis, and it conferred poor clinical outcomes in BC patients. Functional assays demonstrated that attenuation of SNF5 expression facilitated cell proliferation, both in vitro and in vivo, and enhanced cell migration. Our findings suggested that diminished SNF5 levels may be implicated in STAT3 activation and their cross-talk could promote cell proliferation and migration. Importantly, computational identification and drug-sensitivity experiments provided valuable clues for therapeutic approaches tailored for BC patients based on their SNF5 expression.
Interest in SNF5, the core member of the SWI/SNF complex, initially arose from its frequent inactivation by biallelic mutations in 95% of MRT cases, a highly aggressive and lethal cancer type [
9,
28]. Subsequent studies identified that SNF5 deletion occurring due to heterozygous deletions is quite common in other cancers, such as chronic myeloid leukemia and melanoma [
29,
30]. Previous studies indicated that SNF5 aberrations are restricted to MRT and there are rarely any mutations in solid tumors [
31]. Indeed, the mutation rate of SNF5 was only 4% in BC in TCGA dataset (data not shown), which decreased the likelihood of mutation-induced low SNF5 expression in BC. Moreover, Stachowiak et al. found a marked decrease in SNF5 protein levels in BC specimens [
32], but a higher SNF5 mRNA level in BC, which suggested that post-transcriptional mechanisms may be involved in the attenuation of SNF5 expression in BC.
Earlier studies have demonstrated that SNF5 is very closely related to the prognosis of several cancer tumors. We for the first-time report that low SNF5 expression is significantly associated with poor prognosis in BC. This result is in line with previous findings in melanoma, hepatocellular carcinoma and skull base chordoma [
30,
33] and further confirmed the tumor-suppressor role of SNF5 in BC. Dysregulated cell cycle is a key driver of uncontrolled cell proliferation in cancer. In this study, SNF5-induced accelerated cell proliferation was in line with previous findings, which have indicated that SNF5 loss results in cell cycle progression and enhanced cell proliferation [
34]. Moreover, in vivo data suggested that cyclin D1 is a critical down-stream regulator of carcinogenesis in the absence of SNF5 [
35]. Interestingly, cyclin D1 is also downstream of STAT3. Apoptosis is another determinant for tumor growth. In gastric carcinoma, overexpressing SNF5 in cells induce apoptosis by inhibiting Bcl-2 and upregulating Bax [
36]. Choudhari et.al show that deactivation of STAT3 can promote apoptosis in hepatocellular carcinoma [
37]. However, IHC staining for cleaved caspase 3 appeared to be inconsistent with the above findings. This contradiction suggested the involvement of other signaling pathways in apoptosis upon SNF5 downregulation in BC.
The correlation between the epithelial-mesenchymal transition (EMT) pathway and tumor metastasis has been well established. Compelling evidence demonstrates that STAT3 impacts the invasion and migration of cancer cells via EMT. Furthermore, we found that enhanced migration via EMT regulated by SNF5 depletion aligned with previous findings that suggset high migratory abilities in MRT cells having deletion mutation in SNF5 [
38]. Further studies, informed by this work, are needed to elucidate whether SNF5 has an impact on cell invasion in BC. However, we found no significant changes in SNF5-overexpressing T24 cells. This suggested that SNF5, a highly conserved gene, may be present sufficiently enough to exert its suppressive functions on oncogenic signaling pathways in BC.
In our study, the evidence that the aggressiveness of SNF5-depleted BC cells was compromised by STAT blockade, for the first time, provided a critical link between SNF5 and JAK/STAT signaling pathway in malignant phenotypes of BC. There are two possible modes for interaction between SNF5 and STAT3. A recent study shows that SNF5 directly binds to the oncogene, MYC, and impedes target gene recognition by MYC [
13]. This finding endows the possibility that the STAT3 signaling pathway is inhibited upon SNF5 binding in BC. Another possible explanation revolves around the indirect mechanism by which SNF5 may regulate the inhibitory upstream pathway of STAT3.
Notably, AKT inhibitor VIII, gefitinib, AZD-0530, and GSK126, could also be preferentially considered for patients with low SNF5 expression. Findings on the activation of AKT in various cancers, especially in SNF5-deficent MRT, are in line with the significant response sensitivity to AKT inhibitor VIII observed in the SNF5-knockdown T24 cells. A phospho-proteomic analysis identified phosphorylation of EGFR in SNF5 deficient cells and found that EGFR inhibition could exert better therapeutic effects in SNF5 deficient cells [
39]. Gefitinib, a selective EGFR inhibitor approved by the FDA for the treatment of lung cancer, consistently showed greater sensitivity in SNF5 knockdown BC cells. Currently, a randomized phase III trial is being conducted to investigate whether gefitinib exerts synergistic effects with Bacille Calmette-Guérin (BCG) immunotherapy in high-risk BC patients (Clinical Trial Identifier: NCT00352079). Src, together with EGFR, is involved in tumor development. AZD0530 (saracatinib), a potent inhibitor of Src kinase, can inhibit metastasis in an in vivo model of BC [
40]. However, earlier investigations have shown that Src activity had no significant effect on tumor cell growth [
41,
42], which helps in explaining the lack of significant difference in drug response for AZD-0530.
The antagonism between SNF5 and EZH2 has been well documented [
27], however, the EZH2 inhibitors are not included in pRRophetic R package. Therefore, we investigated whether GSK126, an inhibitor of EZH2, could provide additional benefit over the use of cisplatin alone. Interestingly, GSK126 could sensitize SNF5-depleted cells to cisplatin. Overall, the abovementioned findings provide a basis for the potential clinical application of EGFR-targeted chemotherapy or cisplatin plus EZH2 inhibitor regimens in BC based on SNF5 expression.
In summary, for the first time, we identified the functions of SNF5 and found an association between SNF5 and STAT3 in BC biological features, thereby, enhancing the understanding of mechanisms underlying BC progression. Importantly, the findings may provide valuable clues for the development of therapeutic approaches individually customized for BC patients based on their SNF5 expression in the future.
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