Introduction
Breast cancer incidence has increased steadily during the past decades and is currently the most common malignancies in females worldwide [
1]. There are four major subtypes of breast cancer: Luminal A, Luminal B, HER2 and Triple-negative breast cancer (TNBC). TNBC, defined by lack of clinicopathological expression of hormonal receptors and Her2, is the most aggressive subtype that accounts for only 15% of incidence but approximate 30% of breast cancer-related deaths [
2,
3]. TNBC could not be treated with endocrine or targeted therapy [
4]. Despite recent advances in chemotherapy, TNBC treatments are either transient or effective in a limited set of patients, and can be associated with severe toxicity [
5]. Moreover, TNBC cells are characterized by exceeding mitotic, invasive, migratory features for distant dissemination, leading to frequent tumor recurrence [
6]. Thus, the field is in need of new biomarkers and therapeutic targets.
β-Catenin, central effector of Wnt signaling pathway, mainly serves as a transcriptional co-activator of T-cell factors/lymphoid enhancer factor (TCFs/LEF) to dictate downstream genes transcription, and plays crucial roles in development and progression of human diseases [
7]. Unsurprisingly, β-Catenin activity is enriched in BLBC and predicts poor prognosis of TNBC patients [
8,
9]. Studies have demonstrated aberrant activation of β-Catenin promotes cell migration, invasion and stem cell-like properties of TNBC which encourage metastasis [
10]. However, mutations of key components of Wnt/β-Catenin signaling pathway such as APC and β-Catenin that have been proved to contribute to colorectal cancer, and other malignancies are not involved in the enrichment of β-Catenin signal in TNBC [
11].
Crosstalk between growth-factor signal transduction and canonical Wnt pathway allows β-Catenin activation in Wnt-independent ways. As a common joint of multiple signaling pathways, β-Catenin can be phosphorylated on tyrosine residues upon the effective stimulation of growth factors, resulting in excessive β-Catenin nuclear translocation and transcriptional activation of targeted genes in the context of malignant disease [
12‐
14]. Nevertheless, whether the aggressive traits of TNBC cells could be attributed to the tyrosine phosphorylation of β-Catenin remain largely elusive.
HOMER family scaffolding proteins comprise three members (HOMER1-3) in mammals [
15,
16]. Whereas the carboxyl-terminal Coiled-coil self-assembly domain allows HOMER proteins to build multimeric platform for scaffolding function, the enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain located in amino-terminus is responsible for the recognition of different functional molecules such as phosphoinositide 3-kinase enhancer long isoform (PIKE-L), group 1 metabotropic glutamate receptors (mGluRs), IP3 receptors (IP3R) and Shanks to participate in intracellular calcium release and glutamatergic signaling transduction [
16‐
21]. These HOMER proteins predominately expressed in the nervous system, peripheral tissues, and particularly in human cancers to regulate cell growth, migration and apoptosis [
20,
22‐
25]. These pieces of evidence suggest that HOMER proteins may play important roles in carcinogenesis by affecting oncogenic pathways. However, the roles of HOMER family in human breast cancer, especially in TNBC, are still unclear.
In the present study, we find that, among the HOMER family members, HOMER3 is selectively overexpressed in TNBC and correlates with poor prognosis. Notably, HOMER3 plays a scaffolding function to simultaneously interact with c-Src and β-Catenin and promotes their efficient interactions, thus facilitating the growth factor-induced β-Catenin tyrosine phosphorylation and activation. HOMER3 is essential for EGF-mediated aggressiveness and metastasis both in vitro and in vivo. These findings uncover the role of HOMER3 in growth-factor-induced β-Catenin activation and TNBC metastasis, and open new avenues to prevent or overcome TNBC by targeting HOMER3.
Materials and methods
Cells
Breast cancer cell lines were obtained from the ATCC cultured following the provider’s recommendations. MCF-7, T47D, MDA-MB-231 were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS). ZR-75–1, BT-549 and 4T1 cells were maintained in RPMI-1640 medium with 10% FBS. SUM159PT cells were maintained in Ham's F-12 with 10% FBS. All cells were supplemented with penicillin/streptomycin, hydrocortisone, insulin, HEPES and L-glutamine, and maintained at 37 °C in 5% CO2. Cell lines were authenticated by short tandem repeat (STR) fingerprinting.
This study was conducted on a total of 347 paraffin-embedded breast cancer samples including 256 non-TNBCs and 91 TNBCs, which had been histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center from 2005 to 2013. Clinicopathological characteristics are summarized in Additional file
7: Table 1. Ethics approval and prior patient consent had been obtained from the Institutional Research Ethics Committee for the use of the clinical specimens for research purposes.
Immunohistochemistry (IHC)
IHC staining was performed on the 347 paraffin-embedded breast cancer tissue sections using HOMER3 and β-Catenin antibodies as previously reported [
26]. The results of the staining were evaluated and scored by two independent pathologists, who were blinded to the clinical outcome. The HOMER3 staining was graded with four scores, strong + 3, moderate + 2, weak + 1, and negative 0. Specimens with scores + 3, + 2 were defined as high expression; while the others scored as + 1 or 0 were low expression. On the other hand, specimens with > 10% nuclear β-Catenin expression were defined as nuclear β-Catenin-positive, and specimens with ≤ 10% nuclear β-Catenin expression were nuclear β-Catenin-negative. Details of the IHC method were provided in Additional file
7.
Xenograft tumor models
Breast cancer spontaneous metastasis model and lung colonization model were performed as previously described [
26]. Female BALB/c or BALB/c-nude mice (5–6 weeks old) were purchased and housed in barrier facilities on a 12-h light/dark cycle. The Institutional Animal Care and Use Committee of Sun Yat-sen University approved all experimental procedures. For spontaneous metastasis assays, 4T1-luciferase cells (2 × 10
5) with or without HOMER3 knockdown were orthotopically injected into the mammary fat pads of BALB/c mice and inoculated for 5 weeks. Metastasis was detected using the IVIS imagining system (Caliper) by blocking the orthotopic tumor signals.
In the lung colonization model, BALB/c-nude mice were intravenously injected with control or HOMER3 silencing MDA-MB-231-luc cells (1 × 106, n = 8/group). Lung metastasis burden of animals was monitored weekly using bioluminescent imaging (BLI). Mice were euthanized 9 weeks after inoculation, and lung metastases were evaluated.
In the in vivo experiments, mice were subcutaneously administered with EGF (1 mg/kg, mEGF for 4T1 and hEGF for MDA-MB-231) every other day as previously reported [
27]. For validation, lungs were fixed in formalin and embedded in paraffin using a routine method and subjected for H&E staining. Lung surface metastatic lesions were counted under a dissecting microscope and presented as the mean ± s.e.m..
Immunoprecipitation (IP) assays
Cell lysates were prepared from the indicated cells using lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 1% NP-40). Lysates were then incubated with anti-HOMER3 (Sigma-Aldrich, rabbit mAb), or anti-c-Src, or anti-β-Catenin (Cell Signaling Technology, mouse or rabbit mAb) antibody, and protein G-conjugated agarose, or Flag, Myc, HA affinity agarose (Sigma-Aldrich), at 4 °C overnight. Beads containing affinity-bound proteins were washed 6 times by IP wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 0.1% NP-40), followed by eluting using 1 M glycine (pH 3.0). The eluates were then mixed with sample buffer and denatured and used for the western blot analysis. Target proteins were blot with primary antibodies derived from biological sources different from those used in IP to avoid high background. For the detection of β-Catenin Tyr phosphorylation levels, cell lysates were first pulldown with anti-β-Catenin antibody (Cell Signaling Technology, mouse mAb) and subjected for western blot, followed by staining with p-Tyr specific antibody (Cell Signaling Technology, rabbit mAb).
Protein purification was performed as previously reported [
28]. Briefly, 5 × 10
7 293 T cells transfected with 200 μg Flag-HOMER3, or Flag-c-Src, or Flag-β-Catenin expressing plasmid was lysed using RIPA buffer (0.25% SDS). Lysates were then incubated with 200-μl Flag affinity agarose (Sigma-Aldrich) overnight at 4 °C. Beads containing affinity-bound proteins were washed six times by 5-ml RIPA buffer, followed by elution with 500 μl of Flag competing peptides (Sigma-Aldrich, 0.1 μg/μl) twice. The elutes were pooled and washed with 5 ml PBS using 3-kDa MW cutoff filter units (Millipore) to remove the competing peptides. The purities of HOMER3, c-Src and β-Catenin were examined by SDS-PAGE and Coomassie blue staining and then subjected for in vitro binding assays.
Cytokines and cell treatment
Recombinant proteins including Wnt3A, EGF and TGFα were purchased from R&D system (Minneapolis, MN, USA) and SinoBiological (Beijing, China). Before treatment, cells were changed to 1% FBS medium culturing for 1 h; cytokines were then added to a final concentration of 20 ng/ml for Wnt3A, 20 ng/ml for EGF, and 10 ng/ml for TGFα, respectively. For the matrigel 3-D spheroid formation assay, cells were replenished with 50-μl serum-free or EGF-supplemented medium every other day.
TOP/FOP flash activity assays
The wild-type (TOP) and mutant (FOP) LEF/TCF reporters were cloned into pGL3 luciferase constructs (Promega). Twenty thousand cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of TOP or FOP flash, plus 1 ng of pRL-TK Renilla plasmid (Promega), was transfected into cells using the Lipofectamine 3000 reagent according to the manufacturer's recommendation. Luciferase and Renilla signals were measured 24 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. The results were calculated as the ratio of specific TOP-Flash over non-specific FOP-Flash relative renilla luciferase units (RLU).
Statistical analysis
Statistical analyses were performed using the SPSS version 19.0 statistical software package. Statistical tests for data analysis included the log-rank test, χ2 test, Spearman-rank correlation test and Student’s t test (two-tailed). Multivariate statistical analysis was performed using a Cox regression model. P < 0.05 was considered statistically significant.
Additional information is provided in Supplementary Materials and Methods.
Discussion
Scaffolding proteins are required for the assembly of signal transduction complexes in response to extrinsic stimuli, such as growth factors, hormones and extracellular matrix components [
31,
32]. Given their ability to integrate and coordinate multiple signaling events, HOMER scaffolding proteins have emerged as crucial players in the control of cell proliferation, survival and differentiation [
20,
22,
33]. More importantly, it has been found that alterations of their expression levels result in aberrant signaling cascades, which promotes the development and progression of human diseases [
23‐
25,
34]. In this study, we find that HOMER3 uses its EVH1 domain to recognize c-Src and β-Catenin, thus providing a scaffolding platform to increase the efficiency of c-Src-mediated β-Catenin tyrosine phosphorylation and activation under growth factors. These findings uncover a novel role of HOMER3 in the crosstalk between growth factor receptor signal transduction and canonical Wnt pathway, and suggest an oncogenic role of HOMER3 in cancer metastasis.
Constitutive activation of β-Catenin has been proved to contribute to malignant progression of TNBC [
8,
9]. However, comprehensive genomic analysis revealed that activating mutations of certain Wnt/β-Catenin pathway components such as APC and β-Catenin were rarely observed in breast cancer, indicating that alternative mechanisms, probably Wnt-independent ways, exist to activate β-Catenin. Notably, β-Catenin could be phosphorylated on tyrosine residues by growth factors for excessive nuclear translocation and transcriptional activation of targeted genes in malignant cancers [
12‐
14]. Nevertheless, whether and how the aggressive traits of TNBC cells are regulated by tyrosine phosphorylation-induced activation of β-Catenin remain unclear. Herein, we showed that the levels of β-Catenin tyrosine phosphorylation were substantially increased in TNBC compared with non-TNBC cells. We later found that growth factor stimulated β-Catenin tyrosine phosphorylation was accelerated by HOMER3 overexpression but impaired by silencing of HOMER3. HOMER3 promotes tumor aggressiveness and metastasis of TNBC cells via facilitating β-Catenin tyrosine phosphorylation. Thus, these findings provide a novel Wnt-independent mechanism for β-Catenin activation in TNBC, and suggest HOMER3 as a targeting vulnerability of β-Catenin signaling.
Undeniably, molecular-targeted therapy is integral for modern breast cancer treatment. Effective targeting therapy could transform deadly metastatic breast cancer into a controllable chronic disease to some extent. However, considering that TNBC tumors lack expression of hormone and Her2 receptors, and these patients cannot benefit from endocrine therapy or targeted medicine such as trastuzumab [
35]. Although, conventional radiotherapy and chemotherapy have demonstrated efficacy in the treatment of TNBC, these treatments are either transient or effective in a limited set of patients due to frequent and early occurrence of metastasis [
5]. In this study, our results show that HOMER3 is selectively overexpressed in TNBC and correlate earlier tumor metastasis and shorter patient survival. Importantly, silencing of HOMER3 robustly inhibits the invasion and metastatic outgrowth of TNBC cells, suggesting that HOMER3 might be a potential therapeutic target against TNBC.
Notably, a recent report indicates that genomic amplification contributes to HOMER3 overexpression in esophageal squamous cell carcinoma [
24,
36]. However, no significant copy number alteration of the HOMER3 genomic locus could be observed from the TCGA breast cancer dataset [
11]. Interestingly, a research dedicated onto the spine and dendrite degeneration in spinocerebellar ataxia presented by Ruegsegger et al. identified HOMER3 as a downstream target of mTORC1 since ablation of mTORC1 signaling leads to reduced HOMER3 levels in cerebellar Purkinje cells and vice versa [
37]. However, we here did not observe any significant effects of EGF/TGF-α on the expression of HOMER3, suggesting that HOMER3 itself is not a direct downstream gene of EGFR signaling. Future investigation of the mechanism for the specific upregulation of HOMER3 in TNBC might provide new clues for the targeting of HOMER3.
In summary, our study reveals that HOMER3 is a critical regulator in growth factor-induced β-Catenin activation and promotes metastasis in breast cancer. Understanding the precise role of HOMER3 in breast cancer pathogenesis and in the assembly of c-Src/β-Catenin complex promises to increase our knowledge of the biological basis of TNBC malignant progression and may also facilitate the development of new therapeutic strategies against TNBC.
Conclusions
Triple-negative breast cancer (TNBC) is the most challenging subtype of human breast cancer, demanding new biomarkers and therapeutic targets. HOMER family proteins (HOMER1-3) are scaffolding proteins that regulate the assembly of signal transduction complexes in response to extrinsic stimuli. However, the role of HOMER protein in breast cancer, especially the potential in TNBC metastasis remains unclear. In this study, we identified that HOMER3 was selectively overexpressed in TNBC and associated with poor prognosis. Intriguingly, HOMER3 was essential for growth factor-mediated but not canonical Wnt-induced β-Catenin activation. HOMER3 used its EVH1 domain to recognize and interact with both c-Src and β-Catenin, and utilized the coiled-coil domain to provide a multimeric scaffolding platform to facilitate c-Src-induced β-Catenin tyrosine phosphorylation under growth factor stimulation.
Importantly, silencing of HOMER3 robustly inhibited TNBC metastasis in vivo. These findings uncover a novel role of HOMER3 in growth factor-mediated constitutive activation β-Catenin in TNBC and suggest that HOMER3 might be a targetable vulnerability of TNBC.
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