Introduction
Aberrant expression of membrane receptor is a common feature observed in a variety of cancers. For example, the overexpression of epidermal growth factor receptor-1 (EGFR) in non-small cell lung cancer (Salomon et al., receptor-1995) and epidermal growth factor receptor-2 (HER2/neu) in breast cancer (Slamon et al., 1987) are well-characterized, respectively. Increased activation of membrane receptors promotes proliferation, survival and invasion ability of cancer cells via interacting with specific ligands or auto-activation. Importantly, the constitutive activation of signaling pathways also offers potential opportunities for pharmacological intervention. In breast cancer, trastuzumab (also known as (a.k.a.) Herceptin) targeting therapy has been used to treat HER2/neu+ tumor and significantly improves clinical outcome (Piccart-Gebhart et al., 2005). However, critical issues including developing drug resistance, limited response rate and cancer recurrence remain to be resolved. Thus, identification of novel cell surface receptors involved in breast tumorigenesis is urgently needed to offer new potential therapeutic targets.
During the last decade, accumulating evidence has suggested a strong association between chronic inflammation and cancer development among different types of cancer (Coussens and Werb, 2002). Cancer cells may take advantage of cytokine or cytokine receptor overexpression to benefit their own growth or invasive ability via autocrine or paracrine loop. In breast cancer, several pro-inflammatory cytokines, such as IL-1 (Singer et al., 2003), IL-6 (Sansone et al., 2007) and TGF-β (Taylor et al., 2013), have been reported to promote proliferation or invasion. IL-17A (a.k.a. IL-17) mainly secreted from TH17 cells activates downstream signaling of STAT3 to up-regulate several pro-survival genes, also promotes invasion of breast cancer (Wang et al., 2009; Zhu et al., 2008). The activating of IL-17A/IL-17RA axis is also required for the initiation and
progression of inflammation associated pancreatic intraepithelial neoplasia (PanIN) (McAllister et al., 2014). Compared to the role of IL-17A/IL-17RA in tumorigenesis, overexpression of IL-17RB in murine leukemia cells also implicates an oncogenic role of this receptor (Tian et al., 2000). However, the precise contribution of IL-17RB signaling in tumorigenesis remains to be substantiated.
The interaction among IL-17 ligands and receptors are intertwined. Previously, we found that IL-25 (a.k.a. IL-17E) secreted from non-malignant mammary epithelial cells induces breast cancer apoptosis (Furuta et al., 2011). The apoptotic activity of IL-25 is mediated by differential expression of its receptor, 25R, which is composed of IL-17RB and IL-17RA heterodimer (Rickel et al., 2008). IL-17RA and IL-IL-17RB are also the receptors for IL-17A and IL-17B, respectively (Shi et al., 2000; Yao et al., 1995). Thus, the ligands-receptors interaction may exert differential roles in a temporal and spatial manner. It is worth noting that high expression of IL17RB was found to correlate with poor prognosis in breast cancer patients (Furuta et al., 2011). However, the precise role of IL-17RB/IL-17B signal contributes to breast carcinogenesis remains unclear.
In this study, we affirmed that the amplified IL-17RB/IL-17B signal was critical for breast tumorigenesis by correlating its expression with poor prognosis based on two well-characterized independent cohorts of breast cancer patients. Gain or loss of function study of IL-17RB/IL-17B signal in non-malignant mammary epithelial cells and cancer cells further supported this notion. Amplified IL-17RB/IL-17B signal activated Bcl-2 expression to exert anti-apoptotic effect through NF-κB pathway. Importantly, treatment with IL-17RB/IL-17B specific antibodies significantly reduced tumorigenicity of breast cancer cells. These data indicate that the amplified IL-17RB/IL-17B signaling contributes to breast tumorigenesis and offers a potential therapeutic target for breast cancer.
Materials and Methods Cell lines
Human breast cancer cell lines MCF7, MB-157, MB-231, MDA-MB-361, MDA-MB-468, SKBR3 and SKBR3-hr were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics/antimycotics. Non-malignant mammary epithelial cell lines H184B5F5/M10 (M10) and MCF 10A cells were cultured in Minimal Essential Medium supplemented with 10% fetal bovine serum and Dulbecco’s modified Eagle’s medium/F12 supplemented with 5% horse serum, 20 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 μg/ml insulin and antibiotics/antimycotics in a humidified 37℃ incubator supplemented with 5% CO2. H184B5F5/M10 cell line was purchased from Bioresource Collection and Research Center (BCRC) in Taiwan, and others were purchased from ATCC.
Clinical specimens
All human samples were obtained from National Taiwan University Hospital (NTUH). The samples were encoded to protect patient confidentiality and used under protocols approved by the Institutional Review Board of Human Subjects Research Ethics Committee of Academia Sinica (AS-IRB02-98042) and NTUH, Taipei, Taiwan (#200902001R). Clinical information was obtained from pathology reports. Patients with at least 5 years follow-up were included in this study.
Soft agar colony formation assay
In one well of a 12-well plate, 2500 cells were seeded in culture medium containing 0.35% agar on top of a layer of culture medium containing 0.5% agar (M10
cells also used MCF 10A culture medium in soft colony formation assay). Cells were maintained in a humidified 37℃ incubator for 16 days and colonies were fixed with ethanol containing 0.05% crystal violet for quantification. For addition of rIL-17B protein or IL-17B/IL-17RB neutralization assays, human IL-17B (R&D Systems), anti-human IL-17RB antibodies or rIL-17B was added to the soft agar culture every 2 days.
Xenograft assay in NOD/SCID/γnull mice
Animal care and experiments were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica (IACUC#080085). 2 X 106 MDA-MB-361 breast cancer cells mixed with equal volume of Matrigel (BD bioscience) were injected into NOD/SCID/γnull fat pads (Shultz et al., 2005). Tumor volumes were evaluated every 4 days after initial detection. Student’s t-test was used to test the significant differences between shLacZ, shIL-17RB, and shIL-17B cells derived tumor growth. In vivo administration of IL-17RB antibody was initiated when tumors reached 50-100 mm3, and the mice were divided into a same group with comparable tumor size.
For each tumor, 10 μg of IL-17RB antibody in 20 μl sterile PBS was administrated by intratumoral injection. Non-linear regression (curve fit) was used to evaluate the statistical significance of tumor growth between control and treated mice in each group.
IL-17RB antibody
Recombinant IL-17RB extracellular domain that carried only a single N-linked GlcNAc at each glycosylation sites was generated by ectopic overexpression in a suspension cell culture of N-acetylglucosaminyltransferase I-deficient (GnTI-) strain HEK293 cells (Reeves et al., 2002). The resulting N-glycans, GlcNAc2Man5, was then treated with endoglycosidase Endo H to remove residual glycans. Polyclonal antibody
generated through this immunogen was used throughout the entire work.
Immunoblotting
Immunoblot analysis was performed after 8% or 12% SDS-PAGE, with overnight incubation of 1:2000 dilution of mouse polyclonal anti-IL17RB, anti-Bcl2 (OP60T, Merck), anti-Caspase3 (IMG144A, IMGENEX), or 1:1000 dilution of anti-IL17B (MAB1248, R&D Systems), anti-TRAF6 (Sc-8409, Santa Cruz), anti-HER2 (GTX61656, Genetex) antibodies followed by a 1:10000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (GeneTex). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Protein concentration was determined by the Bradford assay (Bio-Rad) before loading and verified by α-tubulin level using a 1:10000 dilution of anti-α-tubulin antibody (GTX72360, GeneTex). The intensity of western blot bands was quantified using Image J software (NIH).
Immunohistochemistry
Formalin-fixed paraffin embedded primary tumor tissue sections were used.
Antigen retrieval was performed using EDTA buffer (Trilogy) heated for 10 min in a microwave. Endogenous peroxidase activity was eliminated by 3％ H2O2. The slides were blocked in PBS containing 10％ FBS and then incubated with purified mouse anti-IL17RB polyclonal antibody (1:100) or anti-HER2 rabbit antibody (1:100) overnight at 4
℃. HRP conjugated rabbit/mouse polymer (Dako REAL EnVision) and liquid diaminobenzidine tetrahydrochloride plus substrate (DAB chromogen) were used for visualization. All slides were counterstained with hematoxylin, and the images were taken using an Aperio Digital Pathology System. Samples were identified as IL-17RB positive if more than 5% of the tumor cells were positive for membrane staining.
Three-dimensional morphogenesis Assay
In a well of an eight-well chamber slides (Labtek, Nunc), approximately 5000 M10 or MCF 10A cells were seeded in growth medium supplemented with 2% Matrigel on top of a layer of Growth Factor Reduced Matrigel (BD Biosciences) as described in (Debnath et al., 2003). The 3D morphogenesis was monitored by fluorescence microscopy confocal sectioning at day 16 after seeding.
Co-immunoprecipitation Assay
The whole cell protein extract was prepared using lysis buffer (10mM Tris-HCl, 150mM NaCl, 2mM MgCl2 and 1％ Triton X-100) at 4℃ and pre-cleaned with protein A/G beads (Santa Crus) for 60 min at 4℃. IL-17RB and TRAF6 were immunoprecipitated with 1 μg antibodies against IL-17RB and TRAF6 (8028, Cell Signaling), respectively, at 4℃ overnight. Normal mouse/rabbit IgG was used as a control. The immunoprecipitated protein complex were separated by SDS-PAGE, and followed by western blot analysis. In rIL-17B treatment experiment, the cells were serum starved for 6 h before treated with rIL-17B for 5 minutes.
NF-κB reporter assay
Cells of 80% confluence were transfected using Lipofectamine 2000 (Invitrogen).
For NF-κB reporter assay, 0.5 μg NF-κB luciferase reporter plasmid and 50 ng of the pGL4-74 Renilla luciferase plasmid (as a transfection efficiency control) were co-transfected into cells per well (24-well plate). Cell extracts were prepared at 24 h after transfection, and the luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) following the manufacturer’s instruction.
RNA isolation and reverse transcription
Total RNA from cell culture or clinical specimens were isolated using Trizol reagent (Invitrogen) and reverse-transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) for gene expression analysis according to instructions from the manufacturers.
Real-time PCR assay
Quantitative real-time RT–PCR was performed using Fast SYBR-Green master mix for gene expression according to the manufacturer’s instruction and analyzed on a StepOnePlus Real-Time PCR system (Applied Biosystems). The primer sequences were shown in Table S6, and β-actin mRNA was used as an internal control for mRNA expression. Relative expression levels were calculated according to the relative ΔCt method, and the specificity of each primer pairs were determined by dissociation curve analysis.
Recombinant human IL-17B protein expression and purification
The plasmid that includes the full-length IL-17B with a C-terminal six-histidine tag was transfected into human embryonic kidney 293 EBNA cell by using polyethyleneimine (Durocher et al., 2002). Transiently transfected cell was cultured in Freestyle 293 expression medium (Invitrogen) at 37℃ for 96 h. The supernatant containing secreted IL-17B was purified by nickel-affinity chromatography following the manufacture’s procedure. IL-17B was further purified with Superdex 200 size-exclusion chromatography (GE Healthcare) equilibrated in 50 mM HEPES, pH 7.5, and 150 mM NaCl.
Stable cell lines for IL-17RB
The cDNA clones of human IL-17RB-FL in pCMV6-XL5 and IL-17RB1 in pCMV-SPORT6 vector were obtained from OriGene and mammalian gene collection then cloned into pQCXIH retroviral vector (Clontech). The expression construct of IL-17RB2 was generated from IL-17RB-FL and cloned into pQCXIP retroviral vector (Clontech). GP2-293 retrovirus packaging cells were cotransfected with IL-17RB variants containing retroviral plasmids and pVSVG plasmids using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instruction. The retrovirus supernatant was harvested in the conditioned medium after 24 h post transfection. The M10 and MCF 10A cells were infected with retrovirus with addition of 8 μg/ml polybrene and then selected with 200 μg/ml Hygromycin B (Sigma) or 1 μg/ml puromycin (Sigma) to establish stable cell lines.
IL-17RB/IL-17B knockdown experiment
For the knockdown of endogenous IL-17RB/IL-17B, the lentiviral vector carrying IL-17RB/IL-17B specific shRNA was obtained from National RNAi Core Facility (Academia Sinica, Taiwan). The target sequence were
5’-CCATTAAGGTTCTTGTGGTTT-3’ for human IL-17RB and
5’-TCTTACCATTTCCATCTTCCT-3’ for human IL-17B. shRNA against β-galactosidase was used as a negative control (shLacZ). For lentivirus production, HEK-293T cells were cotransfected with shRNA containing lentiviral vector, envelope plasmid pMD.G and packaging plasmid pCMVΔR8.91. Virus containing supernatant was harvested at 24 and 48 h post transfection. MDA-MB-361and MCF7 cells were infected with lentivirus and then selected with 1 and 2 μg/ml puromycin, respectively.
Statistical methods
The association between IL-17RB gene expression and survival in breast cancer patients was evaluated using univariate Cox proportional-hazards regression analysis.
Multivariate Cox regression analysis was used to adjust the association between survival and IL-17RB gene expression level for varying clinical parameters including age, tumor size, lymph-node status, tumor grade and estrogen receptor expression. Hazard ratio was evaluated using the method of Grambsch and Therneau. No violation of the proportional assumption was detected. The optimal cut-off value of IL-17RB gene expression level for 5-year survival was determined using ROC (receiver operating characteristic) analysis. Kaplan-Meier method was plotted and log-rank test was used to evaluate the statistical significance between patients with high and low IL-17RB expression in survival.
Results
High expression of IL-17RB promotes breast tumorigenesis
We first examined the expression of IL-17RB in a panel of non-malignant mammary epithelial cells (H184B5F5/M10 and MCF 10A) and breast cancer cell lines (MCF7, MDA-MB-157, MDA-MB-231, MDA-MB-361 and MDA-MB-468) by Western blot and RT-PCR. Elevated expression of IL-17RB protein and mRNA were predominantly observed in many breast cancer cell lines (Fig. II-1A and 1B). Depletion of IL-17RB by its corresponding shRNA in two cell lines, MDA-MB-361 and MCF7, highly expressing IL-17RB (Fig. II-1C), resulted in a significant decrease in soft-agar colony formation (Fig. II-1D). IL-17RB depletion also significantly retarded tumor growth in a xenograft model using NOD/SCID/γnull mice (Fig. II-1E). Palpable tumors derived from the control (shLacZ) and IL-17RB depleted cells (sh17RB) were both observed in the first week. However, from Day 20 to 36, tumors from control cells grew faster and larger than those from IL-17RB depleted cells (Fig. II-1E). The wet weights of the tumors derived from IL-17RB depleted cells were only 40% of those from the control cells (Fig. II-1F and 1G), indicating that high expression of IL-17RB promotes tumor growth.
Membrane bound IL-17RB is critical for promoting breast tumorigenesis
The gene of human IL-17RB encodes two alternative spliced isoforms. Isoform 1 contains a transmembrane domain (refers to IL-17RB1 hereafter), and isoform 2 (IL-17RB2) is a secreted form without the transmembrane domain (Tian et al., 2000). The IL-17RB full-length (IL-17RB-FL) cDNA mainly transcribed IL-17RB1 and a very small amount of IL-17RB2 due to harboring an intron inside (Tian et al., 2000). To pinpoint which isoform is critical for breast tumorigenesis, the non-malignant mammary
epithelial cell line, M10, was transduced with retrovirus carrying IL-17RB1, IL-17RB2 or IL-17RB-FL, respectively (Fig. II-2A). These cells were seeded in the three-dimensional Matrigel culture for testing their acinar forming activity. The control and IL-17RB2 overexpressing M10 cells formed acinar structure with normal hollow lumens, but M10 cells expressing IL-17RB1 or IL-17RB-FL failed to develop a proper lumen-like structure (Fig. II-2B and 2C). In addition, only cells expressing the membrane bound IL-17RB promoted colony formation (Fig. II-2D). These results indicated that overexpression of the membrane bound IL-17RB1 contributes to the transformation of normal cells to cancerous phenotypes.
IL-17RB/IL-17B signaling activates NF-κB pathway and exerts anti-apoptosis via up-regulation of Bcl-2
To elucidate how IL-17RB promotes tumorigenesis in breast cancer, we performed differential expression profiling using IL-17RB overexpressing M10 cells and IL-17RB depleted MDA-MB-361 cells. We found 72 up-regulated and 70 down-regulated genes with differential expression ratio greater than 1.5 fold (Table II-1 and 2).
Use of the bioinformatics database, DAVID (http://david.abcc.ncifcrf.gov/) and KEGG, we found that apoptosis and focal adhesion pathway were most likely regulated by IL-17RB (Fig. II-3). The pro-apoptotic genes TNFSF10 (Wiley et al., 1995) and TRADD (Baker and Reddy, 1998) were up regulated in IL-17RB depleted cells. Conversely, anti-apoptotic gene Bcl2 (Catz and Johnson, 2001) was up regulated in IL-17RB overexpressing cells (Fig. II-4A). These results were further confirmed by real-time quantitative PCR (Q-PCR) and western blot analyses (Fig. II-4B and 4C). Since IL-17RB signaling activates NF-κB in human renal cell line (Lee et al., 2001) and NF-κB up-regulates Bcl-2 (Catz and Johnson, 2001), in breast cancer cells, it is likely that
overexpression of IL-17RB may block apoptosis via NF-κB-mediated Bcl-2 up-regulation in breast cancer cells. To test this possibility, we performed NF-κB reporter assay and found that the NF-κB promoter activity was up-regulated in 17RB1 and IL-17RB-FL overexpressing cells, but downregulated in IL-17RB depleted MDA-MB-361 cells (Fig. II-4D). Furthermore, Bcl-2, but not other NF-κB downstream pro-survival genes including XIAP (Tang et al., 2001) and Survivin (Ambrosini et al., 1997), were up-regulated in IL-17RB1 and IL-17RB-FL overexpressing cells (Fig. II-4B, 4C and 5).
When treating with the cytotoxic agent, etoposide (VP-16, topoisomerase II inhibitor), activation of apoptotic marker cleavage caspase-3 was reduced in IL-17RB overexpressing cells compared with the control (Fig. II-6A). In contrast, caspase-3 activation was enhanced in IL-17RB depleted cells (Fig. II-6B). These results suggested that overexpression of IL-17RB inhibited apoptosis via NF-κB-mediated Bcl-2 up-regulation.
IL-17B enhances tumorigenic activity through IL-17RB
IL-17B, the ligand of IL-17RB, was expressed in both normal and tumor cells by RT-PCR (Fig. II-7A); however, the level of the secreted ligand was barely detectable by ELISA. To test whether ectopic addition of IL-17B enhances tumorigenic activity of breast cancer cells, we generated recombinant IL-17B (rIL-17B) protein from mammalian cell expressing system (Fig. II-7B). Supplement with rIL-17B increased the colony formation of MDA-MB-361 cells, which express high endogenous IL-17RB, in a dose-dependent manner (Fig. II-7C). Similar results were also observed in M10 cells expressing IL-17RB-FL, but not the control (Fig. II-7D). In contrast, depletion of the endogenous IL-17B in MDA-MB-361 cells (Fig. II-8A) not only inhibited the colony formation (Fig. II-8B) but also decreased the NF-κB reporter activity (Fig. II-8C) and
Bcl2 expression (Fig. II-8D). Consistently, the tumor size and weight were both reduced in IL-17B knockdown cells compared to shLacZ control in the xenograft model (Fig. II-8E and 8F). These findings suggested that IL-17B contributes to breast tumorigenesis specifically via IL-17RB.
IL-17B signaling activates NF-κB by enhancing TRAF6 recruitment to IL-17RB Based on bioinformatics and amino acid sequences analysis in 17RA and IL-17RB, we determined two types of putative functional domains which may involve in IL-17RB signaling: (1) the extracellular ligand binding domains (LBD) (Thr28-Leu36, Thr89-Ser96, and Ser259-His264, Fig. II-9), and (2) the intracellular TRAF6 binding domain (from Pro339-Glu341), which is critical for IL-17RB signaling transduction (Maezawa et al., 2006). To affirm that IL-17B signal transduces through IL-17RB, two mutants, ΔLBD, deleted with ligand-binding domain of Thr89-Ser96 and the other, ΔTRAF6, deleted TRAF6 binding domain, were generated (Fig. II-10A). The expressions of these two mutants were comparable in M10 cells (Fig. II-10B). Compared with the wild-type receptor, expression of these two mutants abolished IL-17RB signaling leading to the reduction of colony formation and NF-κB promoter activity (Fig.
II-10C and 10D). Similarly, unlike the wild-type receptor, acinus formation of M10 cells expressing these mutants appeared to be unaffected (Fig. II-10E and 10F). Consistently, addition of rIL-17B to those cells failed to enhance their colony formation (Fig. II-10G).
To trace the downstream effectors of this signaling, we tested whether IL-17B promotes the recruitment of TRAF6 to IL-17RB. TRAF6 is the factor directly binding to the TRAF6 binding domain in IL-17RB receptor upon ligand addition. This recruitment is also critical for the NF-κB signaling transduction (Maezawa et al., 2006; Ye et al., 2002). Upon rIL-17B treatment, the association between IL-17RB and TRAF6 was
increased in a dose-dependent manner (Fig. II-11A). In contrast, both ΔTRAF6 and ΔLBD mutants failed to recruit TRAF6 (Fig. II-11B). These results suggested that IL-17B bound to the extracellular domain of IL-17RB and transduced the signal through its intracellular domain by recruiting TRAF6 to activate NF-κB activity.
Antibodies targeting to IL-17RB/IL-17B inhibit tumorigenicity of breast cancer cells expressing IL-17RB
To further assess the importance of the IL-17RB/IL-17B signaling, we used antibodies specific to IL-17RB and IL-17B to examine their biological consequences.
Addition of IL-17B antibodies to the M10 cells expressing IL-17RB or MDA-MB-361 cells inhibited their colony forming activity (Fig. II-12A and 12B). Similarly, addition of IL-17RB antibody inhibited colony formation of MDA-MB-361 cells (Fig. II-12C).
Furthermore, treatment with IL-17RB antibodies retarded tumor growth of MDA-MB-361 cells in the xenograft model (Fig. II-12D). These results suggested that disruption of IL-17RB/IL-17B signaling inhibits breast tumorigenicity and use of the specific antibodies may provide a potential therapeutic strategy to treat IL-17RB positive breast
Furthermore, treatment with IL-17RB antibodies retarded tumor growth of MDA-MB-361 cells in the xenograft model (Fig. II-12D). These results suggested that disruption of IL-17RB/IL-17B signaling inhibits breast tumorigenicity and use of the specific antibodies may provide a potential therapeutic strategy to treat IL-17RB positive breast