Inhibition of TPL2 by interferon-α suppresses bladder cancer through activation of PDE4D
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Drugs that inhibit the MEK/ERK pathway have therapeutic benefit in bladder cancer treatment but responses vary with patients, for reasons that are still not very clear. Interferon-α (IFN-α) is also used as a therapeutic agent for bladder cancer treatment but the response rate is low. It was found that IFN-α could enhance the cytotoxic effect of MEK inhibition. However, the potential mechanisms of that are still unclear. Understanding of the cross-talk between the IFN-α and MEK/ERK pathway will help enhance the efficacy of IFN-α or MEK inhibitors on bladder cancer.
Immunoprecipitation and pull-down assay were used to reveal the formation of signaling complex. The protein expressions were detected by western blot and immunohistochemistry. The cAMP level, Phosphodiesterase 4D (PDE4D) activity and Prostaglandin E2 (PGE2) concentration in cells, serum and tissues were detected by enzyme-linked immunosorbent assay. The role of PDE4D in bladder tumorigenesis in vivo was examined by the xenograft model. Tissue microarray chips were used to investigate the prognostic roles of PDE4D and tumor progression locus 2 (TPL2) in bladder cancer patients.
IFN-α down-regulated the cyclooxygenase-2 (COX-2) expression in bladder cancer cells through the inhibition of TPL2/NF-κB pathway; IFN-α also inhibited COX-2 expression by suppressing cAMP signaling through TPL2-ERK mediated PDE4D activity. Reduction of the intracellular cAMP level by PDE4D potentiated the antitumor effect of IFN-α against bladder cancer in vitro and in vivo. Further analysis of clinical samples indicated that low PDE4D expression and high level of TPL2 phosphorylation were correlated to the development and poor prognosis in bladder cancer patients.
Our data reveal that IFN-α can exert its antitumor effect through a non-canonical JAK-STAT pathway in the bladder cancer cells with low activity of IFN pathway, and the TPL2 inhibition is another function of IFN-α in the context of bladder cancer therapy. The antitumor effects of IFN-α and MEK inhibition also depend on the PDE4D-mediated cAMP level in bladder cancer cells. Suppression of the TPL2 phosphorylation and intracellular cAMP level may be possible therapeutic strategies for enhancing the effectiveness of IFN-α and MEK inhibitors in bladder cancer treatment.
KeywordsInterferon TPL2 PDE4D cAMP COX-2
Cyclic adenosine monophosphate
cAMP-response element binding protein
Extracellular signal-regulated kinases
Fibroblast growth factor receptor 3
Mitogen-activated protein kinase
- MEK, also known as MAPKK
Mitogen-activated protein kinase kinase
Muscle invasive bladder cancer
Nuclear factor kappa-light-chain-enhancer of activated B cells
Non-muscle invasive bladder cancer
Protein kinase B
Receptor for activated C kinase 1
Receptor tyrosine kinase
Tumor progression locus 2
Type I interferon receptor 2
Type I interferon receptor 1
Bladder cancer is the ninth most common cancer worldwide, particularly in highly developed countries . Compared with non-muscle invasive bladder cancer (NMIBC), the muscle invasive bladder cancer (MIBC) represents a more aggressive cancer type with a mere five-year survival period in < 50% of the cases . Multiple novel druggable target molecules in bladder cancer were identified, and among these, 45% belong to the receptor tyrosine kinase (RTK)-MAPK pathway [3, 4, 5, 6]. As a member of the MAPK cascade, TPL2 (also known as COT or MAP3K8) was reported to be a novel therapeutic target in certain inflammatory and cancerous disorders . The TPL2 phosphorylation primarily activates ERK through a MEK-dependent mechanism  and is involved in the NF-κB pathway regulation through IκB kinase (IKK) complex . Notably, both these pathways were identified to be associated with the grade, stage, and survival outcome of bladder cancer patients [4, 10]. Therefore, the inhibition of TPL2 activation might improve MIBC treatment; however, further studies are required to understand the underlying mechanisms.
Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin E2 (PGE2) production and COX-2 overexpression is associated with bladder neoplasia development . PGE2 level is frequently elevated at the tumor sites  and chemotherapy-induced apoptotic cells release PGE2, which in turn promotes tumorigenesis and resistance against therapeutic agents in bladder cancer treatment . Inhibition of COX-2-PGE2 pathway was reported to reduce drug resistance in xenograft models of urothelial cell carcinoma [10, 14]. In the NMIBC treatment, IFN-α is used clinically in combination with Bacillus Calmette-Guerin (BCG) and currently it is a preventive agent against distant metastases and local recurrence although the response rate of patients is merely 15% . However, the mechanism involved in the poor response of patients towards IFN treatment remains unclear. IFN-α was also found to enhance the efficacy of chemotherapeutic drugs by suppressing the NF-κB activity in advanced renal cell carcinoma . Therefore, IFN-α might suppress COX-2-PGE2 pathway through the inhibition of NF-κB activation and this mechanism needs to be further investigated in bladder cancer.
The cAMP is a key second messenger through which PGE2 exerts its physiological functions . Studies also showed that cAMP could stimulate the proliferation and cyst formation of renal epithelial cells [17, 18]. In a recent study, IFN-α suppressed cAMP level through MEK/ERK-mediated PDE4 activation and deactivated the suppressive function of human regulatory T cells , which was reported previously to reduce the risk of renal cancer progression . Moreover, the down-regulation of PDE4D7 was reported recently to promote the prostate cancer progression through compartmentalization of cAMP [21, 22]. IFN-α/β treatment was also found to strongly enhance the cytotoxic effect of MEK inhibition solely in melanoma cell lines with low activity of IFN pathway . Thus, we aimed to investigate whether the cross-talk between TPL2/MEK/ERK pathway and PDE4D/cAMP signaling mediates the antitumor effect of IFN-α and the potential of these molecules as biomarkers for targeted molecular therapy of bladder cancer.
Cell lines and reagents
T24 and HEK293A cells were obtained from Huaxi Hospital (Chengdu, China) and were authenticated using Short Tandem Repeat (STR) analysis. 5637 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). T24 and 5637 cells were cultured in Roswell Park Memorial Institute Medium (RPMI) 1640 (HyClone) containing 10% (V/V) fetal bovine serum (FBS, HyClone). HEK293A cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (V/V) FBS. All the cell lines were maintained in an incubator with 5% CO2 at 37 °C. Forskolin (S2449), PD98059 (S1177), and roflumilast (S2131) were purchased from Selleck Chemicals (Shanghai, China). TPL2 kinase inhibitor (#19710) was purchased from Cayman Chemical (Shanghai, China). Human IFN-α-2a (Z03003) and human EGF (Z02691) were purchased from Genscript., Ltd. (Nanjing, China). Antibodies used in this study as follow: STAT1 (#41462); STAT3 (#41465); p-STAT3 (Tyr705), (#11045); JAK1 (#35530); Tyk2 (#38374); COX-2 (#21679); p- IκBα (Ser32/36), (#11152); IκBα (#29054); CREB (#21052); ERK1/2 (#40901); p-ERK1/2 (Thr202/Tyr204), (#12082); TPL2 (#33235); PDE4D (#23049); IKKα/β (#1057); IFNAR2 (#32426); IFNAR1 (#32400) were purchased from Signalway Antibody, LLC. (Nanjing, China). p-STAT1 (Tyr701), (#9167); p-JAK1 (Tyr1034/1035), (#3331); p-TYK2 (Tyr1054/1055), (#68790); p-TPL2 (Ser400), (#4491); p-CREB (Ser133), (#9198); RACK1 (#5432); P-IKKα/β (Ser176/180), (#2697) were purchased from Cell Signaling Technology, lnc. (Shanghai, China). β-tubulin (#341002) was purchased from Zen Bio Science, Ltd. (Chengdu, China).
Western blot analysis
Whole cell lysates were extracted using RIPA buffer (Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail (Sigma, Shanghai, China). The protein concentration was measured using a BCA protein assay kit (Bestbio, Shanghai, China). Cell lysates were performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein bands were electrophoretically transferred onto nitrocellulose membranes. After incubation with the primary and secondary antibodies, protein bands were visualized by enhanced-chemiluminescence reaction (Amersham Biosciences, Piscataway, NJ, USA).
PDE4D overexpression and knockdown
PDE4D overexpression was performed in T24 and 5637 bladder cancer cells using the PDE4D-pReceiver-M11 (or the control) vectors according to the manufacturer’s instructions (Genecopoeia, Rockville, MD, USA). PDE4D knockdown in T24 and 5637 cells was performed using the siRNA sequences targeting PDE4D (stQ0007397) or the non-targeting control siRNA (stQ0007397–1) according to the manufacturer’s instructions (Ribobio, Guangdong, China).
Cell viability assay
T24 cells were seeded (5 × 103 cells/well) in 96-well plates using 100 μL medium and incubated overnight. The specific drugs used in the particular experiments were diluted in culture medium and added to cells and the plates were incubated for an additional 72 h. The cells of the control group were treated using 0.1% DMSO. Cell proliferation was measured as the absorbance at 450 nm using a cell counting kit-8 (CCK-8) according to the manufacturer’s instructions (Solarbio, China). Experiments were performed in triplicates.
Trans-well cell migration assay
The migration of T24 and 5637 bladder cancer cells were measured by trans-well assay according to the manufacturer’s instructions (Thermo Fisher, USA). Briefly, T24 and 5637 cells (1 × 105 cells/ml) were added into the trans-wells (100 μL/well) and allowed to migrate under different treatments for 6 h at 37 °C. Cotton swabs were used to remove cells from the upper surface of the trans-wells, and migratory cells attached to the undersurface were stained with crystal violet (0.5%). The numbers of migrated cells (5 different fields per well) were counted using an inverted microscope.
Enzyme-linked immunosorbent assay (ELISA)
The PGE2 levels in cell culture supernatants and serum of mice were estimated according to the manufacturer’s instructions using human PGE2 ELISA kit (Invitrogen, USA) and mouse PGE2 ELISA kit (Cusabio Technology, USA), respectively.
cAMP level and PDE4D activity analysis
The cAMP levels in cells and the xenograft tumor tissues were quantified according to the manufacturer’s instructions using a cAMP-Glo™ assay kit (Promega, USA). The enzyme activities of PDE4D isoforms that were immunoprecipitated from cells and xenograft tumor tissues were quantified according to the manufacturer’s instructions using a PDE-Glo™ phosphodiesterase assay kit (Promega).
Immunoprecipitation and pull-down assay
The extracts of T24 cells and xenograft tumor tissues were precleared with protein A/G agarose beads (Santa Cruz Biotechnology) and incubated with primary antibodies overnight at 4 °C. Later, these samples further incubated with protein A/G agarose beads for 2 h at 4 °C. The immunoprecipitate were suspended in sample buffer and detected by performing western blotting or activity analysis.
Mouse xenograft model
Female BALB/c (nu/nu) nude mice (aged 5-weeks) were purchased from Dashuo Laboratory Animal Technology, Ltd. (Chengdu, China) and were kept on a 12-h day/night cycle with free access to food and water. All the experiments and procedures were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). T24 or 5637 cells (5 × 106 cells/mouse) were subcutaneously injected into the flanks of mice in serum-free RPMI 1640 (100 μL). The tumor volume was calculated using the formula: volume = 1/2 (length × width2). When the tumor volume was approximately 100 to 150 mm3, mice were randomly segregated into six groups (seven mice per group) and treated using specific drugs or inhibitors. The tumor size was measured every 3rd day with a caliper. After 28 days (T24 cells) or 24 days (5637 cells), mice were sacrificed to surgically remove tumors and measure the tumor volume and weight. The serum of each mouse was collected to perform PGE2 analysis. To determine cAMP level and for PDE4D activity analyses, the lysates of xenograft tumor tissues were extracted using SDS lysis buffer (Beyotime Biotechnology, China). The expression levels of specific proteins in the xenograft tumor tissues were analyzed using tissue microarrays by Outdo Biotech, Ltd. (Shanghai, China).
Tissue microarray (TMA) and immunohistochemical (IHC) analysis
TMA chips that consisted of the bladder tumor tissue specimens (n = 126) and adjacent normal bladder tissue specimens (n = 40) were purchased from Outdo Biotech, Ltd. (Shanghai, China). Hematoxylin-eosin (H&E) staining was performed by following the routine method and IHC of TMA chips was performed using primary antibodies against PDE4D (1:150) and pTPL2 (1:150). The assignment of positive-staining score was based on the percentage of positive-staining (0% positive: 0, 1–25% positive: 1, 26–50% positive: 2, 51–75% positive: 3, and 76–100% positive: 4) and staining intensity score was based on the staining intensity (no intensity: 0, weak intensity: 1+, moderate intensity: 2+, and strong intensity: 3+). The final staining index was calculated using the formula: positive-staining score × staining intensity score. These scores were independently determined by two pathologists who were blinded to the clinical and pathological information. IHC staining of TMA using each antibody was performed in a single experiment by including the negative staining control.
The statistical significance of variations among the experimental groups was evaluated using Student t-test and one-way and two-way analyses of variance (ANOVA) tests in the analyses of cAMP levels, PGE2 production, cell viability, and PDE4D activity. Data are represented as mean ± standard deviation (SD). Survival analysis was performed using Kaplan-Meier method and compared with the log-rank test. Wilcoxon signed rank test (unpaired comparisons) was used to determine the significant variations among the expressions of particular proteins in the bladder tumor tissues and adjacent bladder tissues. Spearman’s rank correlation coefficient was used to analyze the correlation among the expressions of specific proteins and various clinicopathological features in patients. Data are represented as mean ± standard error of mean (SEM). P < 0.05 was considered as statistically significant value. SPSS 13.0 software (SPSS, Chicago, IL, USA) was used to perform survival and correlation analyses, while Prism version 6.07 (GraphPad Software) to perform other analyses.
IFN-α suppresses COX-2 expression by inhibition of the TPL2-mediated NF-κB activation and cAMP/CREB pathway
The cAMP/CREB pathway is another main modulator of COX-2 expression [26, 27]. The cAMP level is also regulated by IFN-α-induced MEK/ERK-mediated PDE4 activity . Therefore, we further investigated whether IFN-α decreases the COX-2 expression through TPL2-mediated cAMP/CREB pathway. In T24 cells, IFN-α down-regulated the intracellular cAMP level which was further decreased by the treatment with TPL2i or PD98059 (Fig. 1d). Consistent with the aforementioned result, the CREB phosphorylation was inhibited by IFN-α in the presence or absence of TPL2i or PD98059 accompanied by the down-regulation of COX-2 expression. Conversely, forskolin (a cAMP elevator) counteracted the down-regulation of COX-2 expression induced by IFN-α and TLP2i or PD98059 (Fig. 1e). Furthermore, the reduction of COX-2 expression by IFN-α was abrogated after the treatment with epidermal growth factor (EGF) that is known to activate ERK phosphorylation  (Fig. 1f). To determine whether the decrease of intracellular cAMP level inhibits the bladder cancer cell growth, we used TPL2i or PD98059, and forskolin to treat the respective bladder cancer cell groups. TPL2i or PD98059 treatment reduced the bladder cancer cell viability and this reduction was attenuated by forskolin. Furthermore, the cell growth was promoted after the individual treatment of forskolin (Fig. 1g). These data suggested that IFN-α inhibited COX-2 expression through TPL2-mediated inhibition of the NF-κB activation and cAMP/CREB pathway.
TPL2 regulates the cAMP-hydrolyzing activity of PDE4D at IFNAR2
Induction of PDE4D expression by roflumilast synergizes with IFN-α activity to reduce intracellular cAMP level
Roflumilast potentiates the anti-tumor effect of IFN-α in vivo
PDE4D expression and TPL2 phosphorylation levels are correlated with the human bladder cancer development
Unlike PDE4D expression, the level of TPL2 phosphorylation was found to be significantly higher in the bladder tumor tissues than that in the adjacent bladder normal tissues (P = 0.003) (Fig. 5e-g, Additional file 11: Figure S10, Additional file 10: Table S1), and the high level of pTPL2 was positively correlated to poor prognosis (Fig. 5h). The survival curves that correspond to each of the clinicopathologic features were also analyzed as the basic information about the tissue microarray chips (Additional file 12: Figure S11). Additionally, the low PDE4D expression and high pTPL2 levels were found to positively correlate with the age and TNM stages of bladder cancer patients (Additional file 10: Table S2). To further validate our TMA-IHC results, we analyzed the data of bladder cancer patients derived from The Cancer Genome Atlas (TCGA) database. In TCGA data, a significantly low (cancer vs. normal) PDE4D expression was observed in bladder cancer patients (Fig. 5i) and correlated with their poor prognosis (Fig. 5j). Moreover, the data also revealed that the PDE4B expression was significantly lower in the bladder cancer tissues than that in the bladder normal tissues (Additional file 13: Figure S12B); however, variations were not observed in the cases of PDE4A and PDE4C expressions (Additional file 13: Figure S12A and C). The data derived from Oncomine database indicated similar results of PDE4D expression (Additional file 14: Figure S13A-D); however, regarding the un-phosphorylated TPL2 expression, variations were not observed between the bladder mucosal and bladder tumors (Additional file 14: Figure S13E). Together, these results suggest that the low PDE4D expression and high pTPL2 levels are correlated to the MIBC development and poor prognosis in MIBC patients.
The NF-κB/COX-2 pathway is activated through IκB kinase (IKK) complex  and the overexpression of COX-2 plays a significant role in the bladder tumorigenesis . IKK activity is required to activate the TPL2-ERK axis , however, the serine400 phosphorylation of TPL2 also activates the IKK complex through NF-κB-inducing kinase (NIK) [9, 24]. Our results showed that IFN-α did not affect the constitutive TPL2 expression but inhibited the serine400 phosphorylation of TPL2 and subsequent IKKα/β phosphorylation, suggesting that IFN-α might inhibit the IKKα/β activation through TPL2. Notably, the serine400 residue of TPL2 is phosphorylated in a protein kinase B (AKT) dependent manner  and the mutational activation of phosphatidylinositol 3-kinase (PI3K)/AKT pathway is common in bladder cancer . This is another probable reason for the TPL2 activation and COX-2 overexpression in bladder cancer.
The intracellular cAMP was reported to promote the proliferation in renal epithelial cells and stimulates the cyst formation in diseased kidney cells [17, 18]. This is supported by our observation that an increase of cAMP level promotes the bladder cancer cell proliferation. The modulation of NF-κB pathway by cAMP/CREB is highly dependent on cell-type and -condition . The cAMP-activated IKK causes NF-κB activation [34, 35] and PKA (a main effector of cAMP) also activates NF-κB by the destabilization of protein phosphatase 2C beta (PP2Cβ; a negative regulator of NF-κB) . CREB is another major transcriptional factor involved in the regulation of COX-2 expression and CREB activation is regulated in a TPL2-dependent manner . We found that IFN-α also suppressed COX-2 expression by reducing the intracellular cAMP level through TPL2/ERK-mediated PDE4D activity in bladder cancer cells. IFN-α is clinically used in bladder cancer but the underlying mechanism of resistance against IFN-α therapy remains unclear . Consistent with a previous report , we also found that IFN-α barely affect JAK-STAT pathway in bladder cancer cells, which suggest that IFN-α might exert antitumor effect by inhibiting COX-2 expression independent of canonical JAK/STAT pathway. Thus, our findings are helpful to understand the antitumor effect of type I IFNs in cells with low activity of IFN pathway and could provide novel insight into the oncogenic role of TPL2 in bladder cancer.
The majority of bladder cancers are highly dependent on ERK that is activated by the alterations of FGFR, MAPK/MEK or Notch pathways [3, 5, 6]. Although the inhibitors of FGFR or MEK indicate promising improvement in bladder cancer treatment, responses vary with patients and the reasons are still not very clear [3, 4]. Furthermore, ERK activation was found to phosphorylate PDE4D at the catalytic region and thus causes the inhibition of cAMP-hydrolyzing activity . Here we showed that the PDE4D activity was repressed by the constitutive activation of TPL2/ERK in bladder cancer cells and the antitumor effect of IFN-α-induced TPL2/ERK inhibition partially depended on the PDE4D-mediated cAMP level. This is further supported by the observation that IFN-α/β enhances the cytotoxic efficiency of MEK inhibitors in melanoma cell lines with low IFN activity . Our finding provides a probable explanation for the response heterogeneity of MEK inhibition in cancer treatment  because the regulatory effect of MEK inhibition on PDE4D activity largely depends on the cell-type/environment [8, 39] and finally leads to the different change of cAMP level.
The recruitment of PDE4D to specific intracellular sites is important for the cAMP compartmentalization  and ERK was found to interact with IFNAR2 . In this study, we demonstrated that a signaling complex formed by TPL2, RACK1, and PDE4D at IFNAR2 facilitated IFN-α to inhibit TPL2 phosphorylation and enhance PDE4D activity, which in turn suppressed the NF-κB activation and intracellular cAMP level. RACK1 is a signaling scaffold protein and it binds with IFNAR2 to mediate the recruitment and activation of STAT1 protein by IFN . RACK1 also specifically recruits PDE4D through a helical domain but does not affect the PDE4D activity . Our results indicated that RACK1 bound to IFNAR2 and recruited PDE4D after the IFN-α stimulation, which facilitated IFN-α to enhance the PDE4D activity through TPL2/ERK. It suggested that the formation of a signaling complex at IFNAR2 might generate a local compartment of low cAMP concentration and assist IFN-α to exert its function. This provides new insight into the observations that the cAMP counteracts apoptosis and growth inhibition induced by IFN-α . Furthermore, RACK-1 was found to modulate NF-κB activation , indicating that RACK-1 might also involve in IFN-α induced IKK inhibition.
The expression of PDE4 isoforms could be induced by cAMP elevator including PDE4 inhibitors [30, 31]. Roflumilast is an FDA-approved PDE4 inhibitor that is orally administered. Recently, roflumilast was reported to induce PDE4B and PDE4D expression in human epithelial cells . In this study, we found that the induction of PDE4D expression by roflumilast synergized with IFN-α to reduce the cAMP level and potentiated the antiproliferation effect of IFN-α on bladder cancer in both the cell lines and mice xenograft model. The roflumilast-induced PDE4D did not alter the intracellular cAMP level after 12 h in bladder cancer cells, reinforcing the notion that the PDE4D activity is stringently regulated by compartmentalization in cells [38, 39, 45]. Moreover, both the IFN-α and roflumilast were found to inhibit the PGE2 production in mice serum. This observation is consistent with the findings that IFN-α or roflumilast inhibit the NF-κB activity and other inflammatory factors [16, 46].
The downregulation of PDE4D expression was found recently to increase the proliferation of prostate cancer cells and associated with the progression of prostate cancer [21, 22]. In this study, lower expression of PDE4D and higher TPL2 phosphorylation were found in the bladder tumor tissues than that in the adjacent normal tissues and correlated with poor prognosis. However, the total TPL2 expression did not vary between the bladder tumor and normal tissues. This suggested that the low PDE4D expression and high level of TPL2 phosphorylation might synergistically induce the cAMP level and promote MIBC development. Because the high level of TPL2 phosphorylation probably induces the COX-2 expression and activates the MEK/ERK pathway to increase cAMP level through the inhibition of PDE4D activity. Considering the important role of PDE4D in the downstream of TPL2-MEK/ERK pathway [19, 39], PDE4D expression might be a prognostic marker in bladder cancer patients with an aberrant MAPK activation.
We thank Yan Liang for her helpful comments on the immunohistochemical analysis. The staff at the Institute of Laboratory Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital are also greatly acknowledged.
This work was supported by National Natural Science Foundation of China (Nos. 21561142003, 21861142007, 21672207), Chinese Academy of Sciences President’s International Fellowship Initiative (Nos. 2015 PB049, 2015 PB061), Science & Technology Department of Sichuan Province (No. 2016JZ0022), and the National New Drug Innovation Major Project of China (2017ZX09101003–001-006).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
GLZ and FW initiated and designed the in vitro and in vivo studies. QZ performed the experiments and prepared the manuscript. ZYZ, TP and NS analyzed the data of immunohistochemistry from animal experiments. EMN and BA provided technical and material support. PZJ, WHC and WLL analyzed the clinical data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Tissue microarray chips were purchased from Outdo Biotech, Ltd. (Shanghai, China). All the experiments and procedures of animals were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
Consent for publication
The authors declare they have no competing interests.
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