Background
Breast cancer is the most prevalent malignancy for women worldwide, with 1.67 million new cases were diagnosed in only 2012 [
1]. Breast cancer is characterized as a heterogeneous type of disease with various histopathological features and genetic variability, leading to complex prognostic outcomes. This diversity points the importance of understanding the biology of breast cancer and provides more precise and efficient therapy strategy. Now, breast cancers are classified into four major types as luminal A (ER
+and/or PR
+, HER2
−, Ki-67
+ < 20%), luminal B (ER
+ and/or PR
+, HER2
−, Ki-67
+ ≥ 20%), luminal B-HER2
+ (ER
+ and/or PR
+, HER2
+), HER2
+ (ER
−PR
−HER2
+), and triple-negative (TN; ER
−PR
−HER2
−), based on the molecular profile [
2].
HER2, also known as human epidermal growth factor receptor 2, is encoded by the oncogene
Erbb2 [
3]. It belongs to the epidermal growth factor receptor (EGFR) family, which contains four members: EGFR (HER1, ErbB1), HER2 (ErbB2, HER2/neu), HER3 (ErbB3), and HER4 (ErbB4). HER2 usually acts as an orphan receptor to be a heterodimer partner to other EGFR members upon growth factor binding, which triggers receptor tyrosine phosphorylation and the downstream kinases activation for intracellular signaling transduction [
4]. This signaling renders multiple critical cellular functions, including cell survival, proliferation, polarity change and migration, while the aberrant HER2 upregulation often occurs in about 20–30% of breast cancers as well as ovarian cancers with poor prognosis [
5‐
9].
HER2 upregulation is associated with aggressiveness and worse prognosis of breast cancer. Although the HER2 protein-targeted therapy with the specific antibody Herceptin (trastuzumab) has led to efficient therapy improvement in HER2-possitive patients along with the specific HER2 signal inhibition as well as the antibody-dependent cellular cytotoxicity [
10]. But the observed portion of intrinsic resistance or the acquired drug tolerance were easily developed for the later relapse. Thus, it is necessary to elucidate the underlying mechanisms of HER2 overexpression and its hyperactivation in breast cancers, in order to find an effective alternative or combined therapy.
SH3BGRL is a member of SH3BGR family which comprises of SH3BGR, SH3BGRL2, and SH3BGRL3 [
11]. SH3BGRL broadly expresses in many human tissues and organs, including bone marrow, heart, lung, liver and kidney [
12]. Our recent study thoroughly characterized the general expression patterns of SH3BGR family members during zebrafish embryo development [
13]. SH3BGRL encodes a protein of 114 amino acids with a conserved proline-rich PLPPQIF region, which includes both Homer EVH1-binding and SH3-binding motifs [
14]. As a scaffold protein, SH3BGRL should play important roles in the protein-protein interaction involved in signal transduction, membrane trafficking, cytoskeletal rearrangements and other key cellular processes [
15].
Our previous results unmasked a novel role of mouse SH3BGRL (mSH3BGRL) in driving colorectal cancer metastasis through c-Src activation, but the inverse role of human SH3BGRL as a tumor suppressor [
16]. The later study further verified the suppression role of human SH3BGRL in leukemogenesis [
17]. Clinically, SH3BGRL is highly upregulated in breast tumors and squamous oral carcinoma, implying its possible tumor-promoting role in these contexts [
15,
18,
19]. However, the specific mechanism of SH3BGRL in breast cancer is yet to know. A previous report indicated that SH3BGRL may bind to HER2 [
20], but the downstream events regarding the breast cancer occurrence was not addressed. In this study, we intend to thoroughly investigate the interaction between SH3BGRL and HER2 and unveil the exact novel role of SH3BGRL in HER2-positve breast tumors.
Methods
Cell lines, reagents and antibodies
HEK293T, MCF-7 and MDA-MB-453 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). HEK293T and MCF-7 cells were maintained in DMEM HIGH GLUCOSE (Hyclone, Los Angeles, USA) supplemented with 10% fetal bovine serum (Hyclone, Los Angeles, USA) and 1% Penicillin-Streptomycin solution (Hyclone, Los Angeles, USA). MDA-MB-453 cells were maintained in RPMI 1640 (Hyclone, Los Angeles, USA) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution. All cells were incubated at 37 °C in a humid incubator containing 5% CO2. The medium was changed at alternate days, and cells were split at ration 1:3 when reached 90% confluence. SH3BGRL specific monoclonal antibody was purchased from Santa Cruz. Antibodies, including HER2, p-HER2 (Y1196), p-HER2 (Y877), p-HER2 (Y1221/1222), Akt, p-Akt, ERK, p-ERK, Ki-67, GAPDH, γ-Tubulin and Na+/K + -ATPase were purchased from Cell Signaling Technology (Danvers, USA).
Clinical samples
Twenty three pairs of breast cancer tissues and the corresponding adjacent counterparts were collected with the informed consents, according to Sun Yat-Sen University health regulation, and the study was further approved by the Research Ethics Committee. Tissue microarrays including 76 HER2-positive breast cancer tissues were purchased from Alenabio Company (Xi’an, China) under the ethics regulation.
Plasmid constructs and transfection
The EGFP-SH3BGRL, sh-SH3BGRL and HER2-YFP plasmids were constructed as previously described [
16]. To construct HA-tagged SH3BGRL mutants, the mutagenesis PCR was performed with the listed pairs of primers (Supplemental Table S
1). Cell transfection was performed with Lipofectamine 2000 (Invitrogen, Waltham, USA) in accordance with the manufacturer’s instructions. SH3BGRL overexpression in MCF-7 cells was achieved with EGFP-SH3BGRL plasmids, and the empty vector was used as control. SH3BGRL knockdown in MDA-MB-453 cells was accomplished with two SureSlencing TM shRNA plasmids mixture for human SH3BGRL (TG309466; Origene, Beijing, China) with GFP label, and the scramble shRNA as control [
16]. After transfection, cells were selected with 400 μg/ml G418 (Sigma, St. Louis, USA) (for SH3BGRL overexpression) or 1 μg/ml Puromycin (Sigma, St. Louis, USA) (for SH3BGRL knock-down) for 3–4 weeks, and the single stable cell clones were picked under fluorescence Microscope (Nikon, Tokyo, Japan) to make the stable cell pools.
Western blotting
After the indicated treatments, cells were lysed in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). Equal amount of protein was loaded and simultaneously subjected to electrophoresis in SDS-polyacrylamide gel and transferred to 0.22 μm pore-sized PVDF membranes (Roche, Basel, Switzerland). Membranes were briefly blocked with 5% skim milk and incubated with the primary antibodies overnight at 4 °C, followed by incubation with the species-matched secondary antibody conjugated with HRP (Cell Signaling Technology, Danvers, USA) for 1 h at room temperature prior to chemiluminescence detection.
Cell proliferation, cell cycle and anti-cancer drug-induced apoptosis assays
Cells were seeded in 96-well plates at a density of 1000 cells per well. The growth rate of cells was evaluated by using the CCK-8 cell proliferation kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturers’ instructions. Cell-cycle analysis was carried out by flow cytometry (Beckman CytoFLEX, California, USA) after propidium iodide staining.
For analysis the role of SH3BGRL in anticancer drug-induced apoptosis, parental MCF-7 and SH3BGRL-overexpressing MCF-7 cells were treated with 10 μM Cisplatin (TOCRIS, Abingdon, OX, UK) for 12 h or with 150 ng Herceptin (Roche, Basel, Switzerland) for 96 h, respectively. Likely, MDA-MB-453 and MDA-MB-453 SH3BGRL knockdown cells were treated with 10 μM Cisplatin for 12 h, with 150 ng Herceptin for 96 h, PI3K/AKT inhibitor LY294002 (CST, Danvers, MA,USA) for 24 h, or Herceptin and ATK inhibitor combination for 48 h, respectively. Cell apoptosis was analyzed using the Annexin V/7-AAD Apoptosis Detection kit (Keygen Biotech, Nanjing, China), and the percentage of apoptotic cells was analyzed by flow cytometer (Beckman CytoFLEX, California, USA). The detailed procedures of all above assays were described as previously [
16].
Cell immunofluorescence staining
Cells were grown at low density on coverslips overnight, washed with PBS three times and fixed with 4% paraformaldehyde for 15 min. After permeabilization with 0.5% Triton X-100 for 20 min, cells were blocked with 5% BSA for 30 min at room temperature. Then the cells were incubated with the specific primary antibody overnight at 4 °C, followed by incubation with secondary antibody: goat anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 (Invitrogen, Waltham, USA) for 1 h at room temperature in the dark. Finally, the samples were mounted with Anti-fade reagent with DAPI (Invitrogen, Waltham, USA). Imaging was processed with confocal fluorescence microscope (Nikon, Tokyo, Japan).
Immunoprecipitation assay
For co-immunoprecipitation, HEK293T cells were co-transfected with each SH3BGRL mutant along with YFP-HER2 plasmids for 48 h. The transfected cells were harvested and lysed with lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 20 mM EDTA). For endogenous interaction of SH3BGRL with HER2, MDA-MB-453 cell lysates were directly used for co-immunoprecipitation. All mentioned cell lysates were incubated with SH3BGRL, HER2 specific antibody or normal mouse IgG overnight at 4 °C respectively, followed by mixing with protein A or protein G Magnetic beads (ThermoFisher, Waltham, USA) for another 3 h on a rotator. All beads were washed with lysis buffer for 6 × 5 min, and the precipitated protein complexes were eluted with loading buffer for western blotting analyses with proper antibodies.
Cell membrane and cytosol protein extraction
After washing with 1 ml of ice-cold PBS, cells were harvested and lysed with the membrane protein extraction reagent A (Plasma Membrane Protein Extraction Kit, Beyotime, China). After homogenization, the mixture was centrifuged at 700 g for 10 min at 4 °C and the supernatant was further centrifuged at 10,000 g for 30 min at 4 °C. The sediment contains the total membrane protein while the supernatant contains cytosol protein. Further, the pellet was mixed with membrane protein extraction reagent B for 20 min and centrifuged at 10,000 g for 30 min at 4 °C to collect the supernatant.
Molecular docking analysis of SH3BGRL with HER2
Protein tertiary structures of SH3BGRL(PDB:1U6T) and ERBB2 (PDB:1MFG) were downloaded from the protein structure database, and ZDOCK 3.0.2 was used to perform online molecular docking (
http://zdock.umassmed.edu/). Many possible docking modes of SH3BGRL and HER2 were obtained. Based on the ZDOCKScore ranking, the docking mode with the highest score was chosen as the interaction simulation between SH3BGRL and HER2, while the energy stability ranking was also considered. The Pymol software was used to analyze the possible binding sites of amino acids in the two proteins.
Immunodeficient xenograft mouse tumor model
BALB/c-nude mice (female, 4–5 weeks of age, 18-20 g in body weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Briefly, cells were trypisinized, washed and resuspended in phosphate buffered saline at a density of 107 cells/ml. Seven mice were inoculated subcutaneously with 5 × 106 cells of MD-MBA-453 Vector cells into the right dorsal flanks, and MD-MBA-453 SH3BGRL knockdown cells into the left dorsal flanks, respectively. Similarly, another three mice were inoculated subcutaneously with 5 × 106 MCF-7 parental (Vector) and MCF-7 SH3BGRL cells, respectively. For the growth of the MCF-7 derived tumor, 50 μg estrodiol cypionate (MedChemexpress, NJ, USA) was injected in each mouse once 7 days to sustain the stable estrogen level, till the end of experiment. Mice were sacrificed 30 days after tumor implantation, and the tumors were removed and weighed. For drug treatment, 5 × 106 cells of MD-MBA-453 parental cells (Vector) into the right dorsal flanks, and MD-MBA-453 SH3BGRL knockdown (KD) cells into the left dorsal flanks, respectively. Lapatinib, Herceptin or LY294002 were dosed as 40 mg/kg, 20 mg/kg or 50 mg/kg via i.p. injection twice a week. PBS was used as a negative control. After injection for one week, volumes of the formed tumors were measured once a week. Mice were sacrificed in the fourth week after tumor cell implantation, and the tumors were removed and weighed. GraphPad Prism 5 and paired t test were used for statistical analysis.
Immunohistochemical assay
All xenografted and patient tissues were fixed with 4% paraformaldehyde overnight at 4 °C, and then embedded in paraffin. The samples were subsequently sectioned into thin slices and mounted on slides, followed by deparaffinization in xylene and rehydration through a series of ethanol-water solutions. Antigen retrieval was carried out by immersing the sections in citrate acid buffer with heating with Microwave oven. Slides were then blocked with 3% hydrogen peroxide to block nonspecific activity. After rinsing, slides were blocked with 5% BSA, and then incubated with SH3BGRL antibody or p-HER2 (Y1196) antibody overnight at 4 °C. Immunohistochemical staining kit (
BOSTER Biological Technology, Wuhan, China) was used for color development. Images were captured and confirmed by professional pathologist under microscope (Nikon, Tokyo, Japan). The color intensity of slides was divided into four grades (points) to score SH3BGRL and p-HER2 (Y1196) expression level for statistical analysis.
Statistical analysis
The SPSS 20.0 were performed for the statistical analysis and the data are presented as mean ± SEM. Comparisons between groups were analyzed using Student’s t-test, chi-square test and Kaplan-Meier for survival analysis (data acquired from THE HUMAN PROTEINS ATLAS). The correlationship between SH3BGRL and p-HER2 was assessed using Spearman correlation analysis. Differences were considered to be statistically significant with p < 0.05.
Discussions
Intrinsic resistance of HER2-targeted therapy limits the survival of a portion of breast cancer patients, but the behind mechanism remains elusive. Protein interactomic landscape of HER2-positive breast cancers predicts that SH3BGRL may relate to HER2, but no any information on the physiological function of their interplaying was reported [
20]. Here we evidently characterized that SH3BGRL directly binds to HER2 via its motifs α1, α2 helixes and β3 sheet (core proline-rich PLPPQIF of SH3 domain) on cell membrane of breast tumor cells. This association stabilizes HER2 on cell membrane through delaying the endocytosis of HER2-containing dimers of EGFR family as well as the dimerization without ligand stimulation. This phenomenon subsequently contributes to the prolonged HER2 activation and function duration of the downstream signaling in breast tumor cells. Consequently, it results in the enhanced tumor cell proliferation and survival ability for breast tumor progression. Besides, this association between HER2 and SH3BGRL also provokes the intrinsic HER2-targeted therapy resistance.
Interestingly, we disclose the function of SH3BGRL in maintaining HER2 on cell membrane, which expands our knowledge in the delicate HER2 regulation in nature, but the detailed mechanism is yet to be resolved. Based on our results, we hypothesize that binding of SH3BGRL to HER2 may block some crucial interacting motifs of HER2 to clathrin or other factors on cell membrane, such as vacuole. Their interaction then leads to the insufficient endocytic vesicle formation for the consequent dimer internalization, as clathrin is necessary to HER2 endocytosis and intracellular transport [
22]. In this regard, our result may propose a novel mechanism underlying over-activated HER2 signaling in SH3BGRL-proficient breast cancer.
HER2 is well known as a dimerization partner with other EGFR family members, leading to specific tyrosine phosphorylation in its intracellular kinase region [
4]. Different partnership or growth factor stimulation may contribute to different tyrosine site phosphorylation, but no a confirmed example was reported. Here we demonstrated that binding of SH3BGRL to HER2 specifically enhanced the phosphorylation at Y877 in response to EGF stimulation, and Y1196 even without EGF stimulation in HER-2 high breast cancer cells. In parallel, SH3BGRL imposed no obvious effect on Y1221/1222 phosphorylations which were also important for downstream signaling. Therefore, we predict that SH3BGRL may function as a fine switcher by binding the specific HER2 domain(s) to discriminate HER2 signals from other stimulator through the specific tyrosine phosphorylation, which might provide a new way for blocking the specific tyrosine phosphorylation and the particular downstream signaling. Putatively, we speculate that binding of SH3BGRL to HER2 may cause HER2 conformation change to lead to the unique tyrosine phosphorylation maintenance.
Ras/Raf/MAPK and PI3K/AKT pathways are believed to be the downstream signaling triggered by HER2 phosphorylation [
23‐
27]. Consistently, we showed that SH3BGRL stimulated the AKT and ERK phosphorylation through the prolonged HER2 phosphorylation at Y1196, while silencing SH3BGRL abolished these effects, indicating the important effect of SH3GRL in HER2 Y1196 phosphorylation-dependent signaling in breast tumor cells. Targeting the precise HER2 phosphorylation site, such as p-Y1196, would be an effective and straightforward strategy, while can avoid the side effect from the non-selective inhibition of the necessary function of HER2. Therefore, SH3BGRL would be such a candidate. It is known that the appropriate HER2 phosphorylation at the particular tyrosine site is crucial to the normal cell metabolism and function, including breast, ovary tissues.
Given that SH3BGRL only structurally contains two adaptor domains, SH3 binding and homer EVH1 binding motifs, it should be an instinctive adaptor protein to link various proteins with the suitable binding domains, leading to the cross-talking of series of cell signaling pathways [
28,
29]. The aberrant expression of such scaffolding proteins thus are inevitably crucial to tumorigenesis and metastasis [
30], for instance, Grb2 [
31], 14–3-3 [
32], mda-9/Syntenin [
33] and p130Cas [
34], which all work in such manners in various tumors. Therefore, SH3BGRL expression level could be considered as a diagnostic marker for breast tumor based on our results.
We previously showed that mouse SH3BGRL promotes cancer metastasis as a novel c-Src activator, while human SH3BGRL, as an ortholog, suppresses tumor formation and metastasis [
16]. However, SH3BGRL is observed to be broadly upregulated in breast tumors, including squamous oral carcinoma, among which only rare patients contain somatic mutation, such as R76C which can mimic the mouse SH3BGRL to enhance tumorigenesis and metastasis. In contrast, low SH3BGRL expression is related to AML progression [
17], indicating the dual functions of SH3BGRL in cancer progression. Considering the adaptor character of SH3BGRL, this dual-sided effect might be attributed to the specific cell contexts in different tissues, including HER2 expression state in breast tumors.
HER2-positive breast tumors account for about 22% of all breast cancers [
35], which are usually metastatic with poor prognosis prior to occurrence of HER2-targeted therapy [
36]. With emergence of trastuzumab (Herceptin), HER2-positive breast cancers were successfully treated [
37]. However, there are patients instinctively resistant to HER2-targeted therapy, including the various HER2 mutations, hinting the incomplete understanding on HER2-positive breast tumors. Large TCGA data analyses further confirm that patients with higher SH3BGRL expression would present poor prognosis, which is in line with our results.
Functionally, Herceptin itself functions as a blocker to compete the ligand-binding site of HER2 on extracellular membrane region to inhibit HER2 activation [
38], and Lapatinib binds to HER2 ATP-binding domain to block its phosphorylation and kinase activity. Our results indicated that the binding of SH3BGRL to HER2 can activate HER2 phosphorylation at Y1196 and the downstream signaling to promote tumor cell proliferation and survival, regardless of EGF stimulation, uncovering that prior to ligand stimulation, SH3BGRL may promote HER2 to form dimers with other RGFR members. Therefore, the subsequent inhibition with Herceptin or Lapatinib should be ineffective in HER2-positive breast cancers with concomitant SH3BGRL overexpression. Therefore, Targeting SH3BGRL or its downstream signaling would be a promising and effective strategy, which is also validated by our xenograft tumor therapies.
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