Cancer Letters 361 (2015) 271–281
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Kindlin-2 interacts with and stabilizes EGFR and is required for EGF-induced breast cancer cell migration Baohui Guo a,b, Jianchao Gao a,b, Jun Zhan a,b, Hongquan Zhang a,b,* a Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China b Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Beijing 100191, China
A R T I C L E
I N F O
Article history: Received 3 February 2015 Received in revised form 10 March 2015 Accepted 10 March 2015 Keywords: Kindlin-2 Epidermal growth factor receptor Breast cancer Protein degradation Cell migration
A B S T R A C T
Epidermal growth factor receptor (EGFR) mediates multiple signaling pathways that regulate cell proliferation, migration and tumor invasion. Kindlin-2 has been known as a focal adhesion molecule that binds to integrin to control cell migration and invasion. However, molecular mechanisms underlying the role of Kindlin-2 in breast cancer progression remain elusive. Here we report that Kindlin-2 interacts with EGFR and mediates EGF-induced breast cancer cell migration. We found that EGF treatment dramatically increases Kindlin-2 expression at both mRNA and protein levels in a variety of cancer cells. Inhibitors specific for EGFR or PI3K blocked Kindlin-2 induction by EGF. Importantly, Kindlin-2 interacted with EGFR kinase domain, which was independent of Kindlin-2 binding to integrin cytoplasmic domain. Intriguingly, Kindlin-2 stabilized EGFR protein by blocking its ubiquitination and degradation. Depletion of Kindlin-2 impaired EGF-induced cell migration. Our results demonstrated that Kindlin-2 participates in EGFR signaling and regulates breast cancer progression. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Breast cancer is one of the most commonly identified cancers and also the third cause of cancer death in women [1]. Genomic studies have identified five breast cancer intrinsic subtypes: Luminal A, Luminal B, HER2, basal-like and normal-like breast cancers [2,3]. Basal-like tumors are often resistant to chemotherapy and are able to develop distant metastases to the lung and brain [4]. Basal-like breast cancers exhibit high histologic grades, aggressive clinical features and poor prognosis [5,6] and are often identified by enhanced expression of the epidermal growth factor receptor (EGFR) [7]. EGFR, a transmembrane glycoprotein, exists on the surface of cells and contains extracellular, transmembrane and tyrosine kinase domains [8]. EGFR is not only important for the normal growth of breast tissues but also involved in the initiation and progression of breast cancers [9,10]. Previous studies have shown a strong correlation between high expression of EGFR and the aggressive potential of breast tumors [11–15]. EGFR overexpression is also correlated with poor overall survival in breast cancer patients [16,17]. EGFR is activated by binding to its ligands such as EGF, which leads to the receptor dimerization, autophosphorylation and activation of several downstream signaling pathways, enabling cell proliferation, spreading,
differentiation and migration [18]. The activated receptor recruits adapter proteins including GRB2, PLC and Cbl which in turn activate downstream signaling cascades including the RAS-RAF-MEKERK, PI3K-AKT, PLC-PKC and STATs before degradation [19,20]. However, proteins associated with EGFR may differ from the signaling pathways they mediated, suggesting that EGFR signaling is complicated and new interactors need to be characterized. Here we identified EGFR interacts with Kindlin-2, a newly identified integrininteracting focal adhesion molecule both in vivo and in vitro. Kindlin-2, also known as mig-2 (mitogen inducible gene-2), has been reported as a scaffold protein which enhances Talin mediated integrin activation [21,22]. Kindlin-2 also binds to membranes enriched in phosphoinositides and enhances integrin mediated cell adhesion and spreading [23]. Several studies have linked Kindlin-2 to cancer progression [24–26]. How Kindlin-2 participates in breast cancer progression remains to be further elucidated. Our previous findings implicated that high expression of Kindlin-2 promotes genome instability [25], and inhibits microRNA 200 family members in breast cancer cells [26]. In this study we provided evidence that Kindlin-2 and EGFR cooperate in invasive breast cancers. Materials and methods Cell lines and cell culture
* Corresponding author. Tel.: +008610 82802424; fax: +008610-82802424. E-mail address: [email protected] (H. Zhang). http://dx.doi.org/10.1016/j.canlet.2015.03.011 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
Human cell lines (HeLa, HEK-293T, SUM159, MDA-MB-231) were obtained from American Type Culture Collection (ATCC, USA). Cells were cultured in DMEM or RPMI 1640 medium (Invitrogen, USA) supplemented with 10% FBS (Hyclone, USA). Cells
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were grown at 37 °C in humidified conditions with 5% CO2. For transfection, cells were incubated in Opti-MEM medium (Invitrogen, USA) containing plasmid DNA, Lipofectamine (Invitrogen, USA) or PEI (Polysciences, USA) for 6 h. Subsequently, the medium was replaced by DMEM or RPMI 1640 medium and incubated for 24 h before further experiments. Cells were starved in serum-free medium for 24 h, then treated with EGF (100 ng/ml) for various time periods as indicated before harvest. Plasmids and siRNAs Flag–EGFR and pEGFP–EGFR were gifts from Professor Zhijie Chang (Tsinghua University, China). Flag–Kindlin-2 and pEGFP-Kindlin-2 were generated as previously reported [24]. Point mutations of Kindlin-2 were generated using a Stratagene Quick Change site-directed mutagenesis kit. All sequences were verified by DNA sequencing. Kindlin-2 specific siRNAs target sequences 1: 5′AAGCUGGUGGAGAAACUCG-3′; 2: 5′-CAGCGAGAAUCUUGGAGGC-3′. Negative control siRNA: 5′-UUCUCCGAACGUGUCACG-3′. Reagents and antibodies P38 inhibitor SB203580, MEK inhibitor U0126 and PI3K inhibitor Wortmannin were obtained from EMD (USA). EGF and AG1478 were purchased from Sigma (USA). Anti-Flag, HA, GFP antibodies, cyclohexamide (CHX) and proteasome inhibitor MG132 were purchased from Sigma (USA). Secondary antibodies were purchased from Santa Cruz Biotechnology (USA). EGFR Rabbit mAb was from Cell Signaling Technology (USA). Kindlin-2 mouse antibody (Millipore, USA), Alexa-568 goat anti-rabbit and Alexa488 goat anti-mouse (Life Technology, USA) for confocal microscopy; phosphoEGFR and phospho-ERK rabbit antibodies were obtained from Cell Signaling Technology (USA). Immunoprecipitation and immunoblotting Cells were grown in 10-cm dishes to 80% confluence and lysed in RIPA buffer containing protease inhibitor cocktails. Cell lysates were clarified by centrifugation at 12,000 × rpm for 15 min at 4 °C, and total protein concentration was determined using the BCA method. For each immunoprecipitation assay, 2 mg protein and 2 μg antibody were added for each reaction. Normal mouse or rabbit IgG was used as an irrelevant immunoprecipitation control. Immobilized antibodies were mixed with equal amounts of protein lysate and incubated with rotation at 4 °C overnight followed by addition of 50 μl 50% protein A or G Sepharose slurry and rotating for 4 h. Beads were washed with PBS buffer containing 1% NP-40 four times. Immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblotting. The membranes were detected by the Super Signal chemiluminescence kit (Pierce, USA). Software Image J was used for quantification analysis of the band density of target proteins in Western blot assays. In vitro pull-down assay Glutathione S-transferase-tagged EGFR fusion proteins were expressed in Escherichia coli. MBP–Kindlin-2 was generated and expressed as reported previously [27]. Each purified GST-fused EGFR protein or MBP-fused protein was immobilized on 40 μl of Glutathione-Sepharose 4B beads (GE Healthcare, USA) and equilibrated in the pull-down binding buffer consisting of 50 mM HEPES, 50 mM NaCl, 0.1% NP40, and Protease Inhibitors. The purified complex was added to each affinity beads and the binding reactions were incubated at 4 °C for 3 h. The beads were washed three times using the binding buffer. The bound proteins were analyzed by SDSPAGE followed by Coomassie staining. Immunofluorescence and confocal microscopy Cells were plated on collagen type I coated coverslips. After serum starvation overnight, cells were treated with 100 ng/ml EGF for the time indicated, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in BSA, and stained with the indicated antibodies overnight at 4 °C. Antigen-antibody complexes were detected by staining with Alexa-coupled secondary antibodies for 1 h. Stained cells were visualized with a 63 × 1.4 oil objective using a Zeiss LSM780 laser scanning confocal microscope. Images were representatives of three independent experiments. Cell migration assay The Transwell cell migration system consisted of cell culture champers with an 8.0 μM pore size in 24-well plate (BD, USA). Cells were seeded on 6 well plates, serum starved overnight and treated with either vehicle or 100 ng/ml EGF for 6 h, After incubation, cells were resuspended with serum-free DMEM containing vehicle or 100 ng/ml EGF, and 1 × 104 cells were separately seeded in the champers. PBS containing 3 μg/ml collagen type I was loaded into the lower chamber and incubated for 6 h. Migrated cells were fixed with 4% formaldehyde for 15 min and stained with 0.5% crystal violet for 15 min. Migrated cells were counted in 15 different microscopic fields by 10 × objective using a Nikon microscope. All assays were performed at least three times.
Statistical analysis Correlation analysis between Kindlin-2 and EGFR expression in human breast cancer samples was analyzed for significance using Prism 5.0 software (GraphPad Software, USA) with Pearson r test, where p < 0.05 was considered statistically significant. For other experiments, results are expressed as mean ± SD. Statistical analyses were performed using Student’s t test. P < 0.05 was considered statistically significant. All experiments were repeated at least three times.
Results Increased expression of Kindlin-2 is correlated with breast cancer progression To determine the role of Kindlin-2 in breast cancer progression, Oncomine datasets were analyzed (Fig. 1A). Results showed that Kindlin-2 mRNA expression was increased in invasive ductal breast carcinoma compared with noninvasive ductal breast carcinoma (p = 8.17E-04, fold change: 2.537). Moreover, the poor prognostic effect of Kindlin-2 expression was supported by Kaplan– Meier Plotter analysis (www.kmplot.com), which showed that high Kindlin-2 abundance correlated with a poor relapse-free survival (RFS, p = 1.8E-05, HR=1.29) using microarray data from breast cancer patients (n = 3455 ) (Fig. 1B). These results indicate that Kindlin-2 plays a role in invasive breast carcinomas. Kindlin-2 expression is correlated with EGFR expression in breast cancer tissues Furthermore, EGFR overexpression is frequently observed in most basal-like breast cancer cell lines and an important marker in the immunohistochemical identification of basal-like breast cancers [14]. Similarly, we found high Kindlin-2 expression in basal-like breast cancer cell lines including MDA-MB-231, SUM159 and HS578T [25]. Then we set out to assess the association between EGFR and Kindlin-2 expression. We analyzed the correlation of Kindlin-2 and EGFR mRNA expression in two independent datasets, 43 (GSE22035) and 60 breast cancer patient samples (GSE1378) (Fig. 1C and D). Positive correlation was identified between EGFR and Kindlin-2 mRNA expression in breast cancer tissues (GSE22035, n = 43, R = 0.5689, p < 0.0001; GSE1378, n = 60, R = 0.4055, p < 0.0013). To determine the clinical significance of Kindlin-2 in breast cancer patients versus normal breast tissues, we conducted an Oncomine analysis to characterize gene expression using massive microarray data from breast cancers. Our search for Kindlin-2 gene expression was based on 53 datasets and finally revealed 24 datasets with significant p values. In all of these cases, Kindlin-2 was consistently decreased in cancers compared with normal breast tissues. Moreover, a similar pattern of EGFR expression that was decreased in carcinomas was also observed. Search for EGFR gene expression was based on 51 datasets and revealed 24 datasets with significant p values, further supporting the correlation between Kindlin-2 and EGFR in breast cancer patients (Supplementary Fig. S1). A recent study reported that Kindlin-2 expression is reduced in breast cancer samples [28], consistent with our results. These data indicated that Kindlin-2 functions in breast cancer progression. Activation of EGFR signaling promotes Kindlin-2 expression through the EGFR–PI3K signaling pathway Kindlin-2 was identified as a mitogen-inducible gene firstly [29], we therefore validated EGF effect on Kindlin-2 expression by Western blot and quantitative PCR. EGFR-positive HeLa cells were treated with EGF for various times. We found that EGF-induced Kindlin-2 expression in HeLa cells is in a time-dependent manner (Fig. 2A). Quantitative PCR results showed that EGF also increased Kindlin-2 mRNA levels in HeLa cells (Supplementary Fig. S2A). Similar results
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Fig. 1. Kindlin-2 expression is correlated with EGFR expression in breast cancer tissues. (A) Analysis of expression of Kindlin-2 mRNA in 14 breast cancer patients’ samples of ductal breast carcinomas in situ versus invasive ductal breast carcinomas. Data were obtained from Oncomine in Schuetz breast, Pearson R test. (B) Kaplan–Meier survival plots demonstrating the poor prognostic effect of Kindlin-2 gene high expression correlated with a short RFS in breast cancer patients (n = 3455). (C) Analysis of correlation of Kindlin-2 and EGFR mRNA expression in two independent datasets, 43 breast cancer patient samples from GEO (GSE22035) and (D) 60 breast cancer patient samples from GEO (GSE1378). All data are log transformed and median centered.
were found in human basal-like breast cancer cell lines MDA-MB231 and SUM159, which showed high EGFR expression. EGF treatment triggered EGFR and ERK activation as well as Kindlin-2 upregulation (Fig. 2B, Supplementary Fig. S2B). To determine whether the EGFR signaling induced Kindlin-2 expression was directly regulated at the transcriptional level, MDAMB-231 cells were treated with 1 μg/ml transcription inhibitor actinomycin D (AD) or 100 nM protein synthesis inhibitor cycloheximide (CHX) for 1 h followed by EGF treatment. Results showed that AD prevented EGF-induced Kindlin-2 expression in MDA-MB231 cells while CHX did not (Fig. 2C). These results suggested that EGFR signaling upregulates Kindlin-2 expression at the transcriptional level and is independent of protein synthesis. In response to EGF stimulation, EGFR triggers several downstream signaling cascades, including MAPK/ERK, phosphatidylinositol 3-kinase (PI3K)/AKT, and Stat pathways [19]. Therefore, we determined the downstream signaling pathway of EGFR that leads to Kindlin-2 upregulation by a series of specific chemical inhibitors that were known to target EGFR signaling. MDA-MB-231 cells were pretreated with an EGFR inhibitor AG1478 (AG), ERK1/2 inhibitor U0126 (U), p38 inhibitor SB203580 (SB), or PI3K inhibitor Wortmannin (W) for 1 h followed by EGF treatment for 6 h. We observed that only the EGFR or PI3K inhibitor abrogated EGF-induced Kindlin-2 expression (Fig. 2D), suggesting that Kindlin-2 induction employs an EGFR–PI3K signaling cascade. Kindlin-2 interacts with EGFR in vitro and in vivo Given the strong mRNA expression correlation between EGFR and Kindlin-2, we hypothesized that Kindlin-2 might interact with EGFR. The interaction between Kindlin-2 and EGFR was
confirmed by different approaches, using overexpressed Kindlin-2 and EGFR as well as endogenous proteins. Firstly, HEK-293T cells were co-transfected with Flag–EGFR and GFP–Kindlin-2 (Fig. 3A) or co-transfected with Flag–Kindlin-2 and GFP–EGFR (Fig. 3B). Immunoprecipitation with anti-Flag antibody showed that EGFR was co-immunopecipitated with Kindlin-2. These data indicated that Kindlin-2 interacts with EGFR. To examine the endogenous interaction between EGFR and Kindlin-2, we performed co-immunoprecipitation experiments. Results showed that EGFR is able to be co-immunoprecipitated by Kindlin-2 antibody and Kindlin-2 is able to be co-immunoprecipitated by EGFR antibody but not the control IgG (Fig. 3C and D). To determine whether EGFR binds to Kindlin-2 in vitro, purified MBP–Kindlin-2 protein was incubated with MDA-MB-231 cell lysates. MBP and MBP–Kindlin-2 were stained by Coomassie blue as loading control (Fig. 3E, below). Western blot analysis revealed that Kindlin-2 binds to EGFR in vitro (Fig. 3E). Kindlin-2 is known to bind to integrin to mediate integrin outsidein signaling [21,22], we thus wonder if Kindlin-2 binding to integrin is required for its interaction with EGFR. A Flag–Kindlin-2 QW mutant deficient in binding to integrin was generated. Both the wild-type (WT) and mutant Flag–Kindlin-2 were transfected into HEK-293T cells. Results indicated that the interaction between Kindlin-2 and EGFR is independent of Kindlin-2 binding to integrin (Supplementary Fig. S3A). Given that EGF promotes the interaction of EGFR with several proteins, we thus wanted to examine the effect of EGF on the interaction of EGFR with Kindlin-2. Interestingly, we found that EGFR binds to Kindlin-2 under conditions with or without EGF (Supplementary Fig. S3B, left). However, EGF treatment weakened the interaction between the two (Supplementary Fig. S3B, right).
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Fig. 2. Activation of EGFR signaling promotes Kindlin-2 expression through the EGFR–PI3K pathway. (A) HeLa cells were serum starved overnight and then were stimulated with 100 ng/ml EGF for the indicated time period. The expression of Kindlin-2 was analyzed by Western blot (left). Results were demonstrated by histograms to quantify the expression levels at the indicated time period from a representative experiment (right). (B) Western blot analysis of Kindlin-2 induction by EGF in basal-like breast cancer cell line SUM159. Cells were serum starved and treated with 100 ng/ml EGF for the indicated time period. Expressions of phosphorylated EGFR and phosphorylated ERK were also analyzed to authenticate the effectiveness of EGF stimulation. (C) After 12 h of serum starvation, MDA-MB-231 cells were pretreated with actinomycin D (AD) or CHX for 1 h, followed by 100 ng/ml EGF treatment for 1 h. The expression of Kindlin-2 was analyzed by Western blot. Results were demonstrated by histograms (bottom) to quantify the expression levels. (D) MDA-MB-231 cells were serum starved overnight and pretreated with DMSO (−), MEK inhibitor U0126 (U), EGFR inhibitor AG1478 (AG), PI3K inhibitor Wortmannin (W), p38 inhibitor SB203580 (SB), or a transcription inhibitor actinomycin D (AD) for 1 h followed by EGF treatment for 1 h, Kindlin-2 expression was analyzed by Western blot. Results were demonstrated by histograms to quantify the expression levels (bottom). Data were presented as mean ± SD (vertical bars) from three independent experiments. *** p < 0.001. NS: not significant.
Mapping of the interacting region between Kindlin-2 and EGFR EGFR is composed of extracellular, transmembrane (TM), juxtamembrane (JM), kinase (KD) and the C-terminal flexible domains. To determine which domain of EGFR was required for the interaction with Kindlin-2, HEK-293T cells were transfected with Flag–EGFR-A or Flag–EGFR-B and co-immunoprecipitation was performed (Supplementary Fig. S4A). Results showed that TM, JM and KD are the major binding domains of EGFR with Kindlin-2 (Supplementary Fig. S4B). To further confirm these results, different GST-tagged EGFR fragments were expressed (Fig. 4A) and purified using Glutathione Sepharose 4B beads, then the beads were incubated with MDA-MB-231 cell lysates. No interaction between the C-terminal flexible domain of EGFR with Kindlin-2 was detected (Fig. 4B), indicating that JM and KD of EGFR were required for its interaction with Kindlin-2. Similarly, Kindlin-2 was divided into three fragments and FERM and F0 domains were found to bind to EGFR
(Fig. 4C and D). Collectively, the N-terminus of Kindlin-2 is able to bind to EGFR juxtamembrane and kinase domains. Co-localization of Kindlin-2 with EGFR in breast cancer cells To further characterize the interaction between Kindlin-2 and EGFR, we examined the subcellular localization of endogenous Kindlin-2 and EGFR proteins in cells by immunofluorescence assay. Kindlin-2 has been reported to locate at focal adhesions to mediate integrin activation and cell adhesion [30]. Interestingly, in serum starved MDA-MB-231 cells, we found that in addition to focal adhesions, Kindlin-2 also located at the lamellipodia, the dynamic membrane structures of the leading edge of motile cells. Kindlin-2 distribution at the lamellipodia partially overlaps with EGFR, suggesting that the two proteins co-localize with each other at the lamellipodia (Fig. 5A). Given that EGF is able to induce rapid EGFR endocytosis, we therefore examined the changes of EGFR localization.
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Fig. 3. Kindlin-2 interacts with EGFR in vivo and in vitro. (A) HEK-293T cells were transiently transfected with Flag–EGFR and GFP–Kindlin-2 or (B) transfected with Flag– Kindlin-2 and GFP–EGFR. After cell lysis, equal amounts of protein were immunoprecipitated with anti-Flag antibody or IgG. Both unprocessed lysates (Input) and immunoprecipitates (IP) were resolved by SDS-PAGE and transferred onto PVDF membranes. Identical blots were probed with anti-EGFR, anti-Flag or anti-Kindin-2 antibodies. (C) MDA-MB-231 cells were lysed and equal amounts of protein lysates were immunoprecipitated with anti-EGFR antibody or IgG and probed with anti-Kindlin-2 antibody. (D) MDA-MB-231 cells were lysed and equal amounts of protein lysates were immunoprecipitated with anti-Kindlin-2 antibody or IgG and probed with anti-EGFR antibody. (E) Purified MBP– Kindlin-2 or MBP protein alone was incubated with MDA-MB-231 cell lysates at 4 °C overnight. Beads were washed and the remaining proteins were resolved by SDS-PAGE and further analyzed by Western blot using anti-EGFR antibody (upper). The MBP and MBP–Kindlin-2 were stained by Coomassie blue (below).
To follow the possible translocation of Kindlin-2 and EGFR in living cells, MDA-MB-231 cells were treated with EGF for 10 or 60 min. We found that EGF induced translocation of EGFR from the periphery of cells to the cytoplasm and nucleus at a time point of 60 min, while Kindlin-2 showed only partial translocation into the nucleus. These results suggest that EGF treatment impaired the co-localization of EGFR and Kindlin-2, consistent with the previous immunoprecipitation data that EGF can weaken the interaction between EGFR with Kindlin-2 (Supplementary Fig. S3B). EGFR and Kindlin-2 co-localized at the lamellipodia in MDAMB-231 cells, which showed few cell–cell junctions. For adherent SUM159 cells, Kindlin-2 was diffusely distributed throughout the cytoplasm with prominent accumulation at cell–cell junctions. EGFR was also found primarily at cell–cell junctions. Merged images showed that Kindlin-2 and EGFR co-localized at cell–cell junctions under conditions of serum starvation (Fig. 5B, upper), and co-localized at lamellipodia upon EGF treatment (Fig. 5B, middle). EGF induced translocation of EGFR into the cytoplasm, and the co-localization was also decreased after 60 min EGF treatment (Fig. 5B, bottom).
degradation is mediated by ubiquitination of the receptor, endocytosis, and finally EGFR is degradated by both proteasomal and lysosomal hydrolases [31]. Treatment of cells with the proteasome inhibitor MG132 led to raised the protein level of EGFR, an effect that can be realized by exogenous expression of Kindlin-2 (Fig. 6B). However, the presence of both MG132 and Kindlin-2 expression could not further raise the level of EGFR (Fig. 6B), suggesting that MG132 and Kindlin-2 may target the same mechanism, indicating that EGFR upregulation in Kindlin-2 expressing cells is due to the reduced degradation of EGFR. In addition, overexpression of Kindlin-2 prolonged the half-life of EGFR (Fig. 6C), while knockdown of Kindlin-2 reduced the half-life of EGFR (Fig. 6D), clearly indicating that Kindlin-2 regulates EGFR protein turnover. Given that ubiquitination is an important step in mediating EGFR degradation, we therefore carried out ubiquitination assays to examine whether Kindlin-2 is responsible for the regulation of EGFR ubiquitination. To this end, the level of ubiquitylated EGFR was examined and was found to be decreased in Kindlin-2 overexpressing HEK-293T cells (Fig. 6E). These results support our hypothesis that Kindlin-2 upregulated EGFR is mediated by decreased ubiquitination of EGFR.
Kindlin-2 stabilizes EGFR protein by blocking EGFR ubiquitination and degradation Kindlin-2 is required for EGF-induced breast cancer cell migration Overexpression of Kindlin-2 in HEK-293T cells led to upregulation of EGFR (Fig. 6A). Given that Kindlin-2 altered only the protein but not the mRNA levels of EGFR (data not shown), we hypothesized that Kinlin-2 is involved in the stability of EGFR protein. EGFR
SUM159 and MDA-MB-231 are aggressive breast cancer cell lines and exhibit high migratory capacity compared with other breast cancer cell lines [32]. Stimulation of cancer cells with various growth
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Fig. 4. Mapping of the interaction between Kindlin-2 and EGFR. (A) The schematic diagram of different GST-tagged EGFR fragments. Human EGFR including extracellular domain which binds with the ligands, transmembrane domain (TM), juxtamembrane domain (JM), kinase domain (KD) and the C-terminal flexible region. (B) GST–EGFR fragments were purified using Glutathione Sepharose 4B beads, then the beads was incubated with MDA-MB-231 cell lysate at 4 °C overnight. Beads were washed and the remaining proteins were resolved by SDS-PAGE and analyzed by Western blot using anti-Kindlin-2 antibody. (C) The schematic diagram of different Flag tagged Kindlin-2 fragments used in the mapping. (D) The truncated segments were transfected into HEK-293T cells. Twenty-four hours later cells were harvested. Then the segments were pulled down using purified GST–EGFR-A and analyzed by Western blot using anti-Flag antibody.
factors including EGF increases cell migration [33,34]. Inhibitors of EGFR are able to reduce breast cancer metastasis to the bone and brain in mouse models [35,36], suggesting that the EGFR signaling is involved in the regulation of breast cancer metastasis. To uncover the functional implication of the EGFR–Kindlin-2 interaction, we examined the role of Kindlin-2 in EGF-induced cell motility in breast cancer cells. Control or Kindlin-2 siRNAs were used to inhibit endogenous and EGF-induced Kindlin-2 expression in basal-like breast cancer cell SUM 159. Immunoblotting confirmed the knockdown of Kindlin-2 in SUM 159 cells (Fig. 7A). We found that EGF stimulation further enhanced SUM159 cell migration. In addition, depletion of Kindlin-2 by siRNA significantly inhibited cell migration caused not only by Kindlin-2 but also by EGF (Fig. 7B), indicating that Kindlin-2 is required for EGF-stimulated breast cancer cell migration. Furthermore, we observed a similar requirement of Kindlin-2 in EGF-induced cell migration using MDA-MB-231 cells stably expressing control or short hairpin RNA against Kindlin-2 (Fig. 7C and D). Collectively, these results strongly indicated that Kindlin-2 is required for EGF-induced breast cancer cell migration. Discussion Metastatic breast cancers are highly devastating diseases with few effective treatments. However, mechanisms underlying breast cancer metastasis are still unclear. Previous studies have linked all
Kindlin family members, including Kindlin-1, -2 and -3, with breast cancer progression. Kindlin-1 was found to regulate TGF β signaling and played a role in breast cancer lung metastasis [37]. Kindlin-3 was also found to be involved in breast cancer progression and metastasis [38]. Our previous studies showed that Kindlin-2 binds directly to the TβRI receptor and Smad to activate TGF β signaling [39]. Kindlin-2 can also stabilize β-catenin and plays a role in Wnt signaling [27]. Kindlin-2 was found to regulate malignant mesothelioma cell adhesion and migration [40]. However, the detailed mechanism of Kindlin-2 in the regulation of breast cancer progression is still unknown. Kindlin-2 regulates cancer cell migration via multiple mechanisms including EGFR signaling pathway as reported here. Kindlin-2 also cooperates with integrin β3 and migfilin to regulate cell migration [21], and is involved in TGF β and Wnt pathways to regulate cell migration [27,41]. In addition, Kindlin-2 promotes carcinogenesis by inducing genome instability [25]. Importantly, Kindlin-2 functions as a scaffold molecule of cellular focal adhesive structures [21–23]. Furthermore, Kindlin-2 also functions as a regulatory molecule controlling various signaling pathways [24–27]. Interestingly, we identified that Kindlin-2 is highly expressed in mesodermderived organs [42]. An exception was seen that Kindlin-2 is strongly expressed in tissues that are highly mobile during embryonic development. Therefore, normal breast tissues still express higher levels of Kindlin-2 accordingly to maintain the structure of the epithelium.
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Fig. 5. Co-localization of Kindlin-2 with EGFR in breast cancer cells. (A) MDA-MB-231 cells or (B) SUM-159 cells were plated on collagen type I coated coverslips, and were subjected to serum starvation overnight and then stimulated with 100 ng/ml EGF or vehicle for the indicated time period. Cells were fixed and double-stained with rabbit anti-EGFR/Alexa Fluor 568-conjugated secondary antibody and mouse anti-Kindlin-2/Alexa Fluor 488-conjugated secondary antibody. The nuclei were stained with Hoechst 33342. Slides were analyzed by Zeiss LSM confocal laser microscopy. Merged pictures represent the composite of both channels, with yellow regions indicative of colocalization. The images are representative of three independent experiments, with at least six fields examined in each assay.
In non-metastatic breast cancer Kindlin-2 expression is lower than the normal breast tissue, which may be due to the insufficiency of stimulating factors. However, the role of Kindlin-2 in breast cancer progression is elusive. Gozgit et al. found that Kindlin-2 expression
is higher in an aggressive subset than a nonaggressive subset of MCF-7 cells, and knockdown of Kindlin-2 inhibited breast cancer cell invasion [43]. However, Kindlin-2 showed heterogeneity in a panel of 34 formalin-fixed breast tumors, and only half of the
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Fig. 6. Kindlin-2 stabilizes EGFR protein by attenuating EGFR ubiquitination and degradation. (A) HEK-293T cells were transfected with GFP empty vector, Flag–EGFR or GFP–Kindlin-2, Flag–EGFR separately. EGFR expression was analyzed by Western blot. (B) SUM159 cells were transfected with Flag empty vector or Flag–Kindlin-2 and then treated with Vehicle or 10 μM MG132 for 6 h before harvesting. EGFR expression was analyzed by Western blot. (C) SUM159 cells were transfected with GFP or GFP– Kindlin-2 or (D) transfected with control siRNA or Kindlin-2 siRNA, and treated with 100 nM CHX and 100 ng/ml EGF for the indicated time. EGFR expression was analyzed by Western blot. (E) HEK-293T cells were co-transfected with HA-Ub, Flag–EGFR, GFP empty vector or GFP–Kindlin-2 plasmids and treated with 10 μM MG132 for 6 h before harvesting. EGFR ubiquitination was detected by immunoprecipitation with Flag antibody and immunoblotting with HA antibody.
patients showed Kindlin-2 overexpression and the other half showed no detectable Kindlin-2 [43]. In Oncomine data mining Kindlin-2 expression was found to be downregulated in nonmetastatic breast cancer patients but was upregulated in patients with invasiveness. It is possible that Kindlin-2 functions differentially in benign and malignant breast tumors. In normal breast tissues or benign breast diseases Kindlin-2 expression is highly expressed and is localized at membrane-anchored structures, i.e., focal adhesions. In nonmetastatic breast cancer patients Kindlin-2 expression is lower than the normal breast tissues and is localized at the cytoplasm. Kindlin-2 expression was raised in metastatic breast cancer patients, in which epithelial to mesenchymal transition (EMT) has taken
place. Taken together, Kindlin-2 is a versatile molecule that plays a differential role in breast cancer progression. Our data suggested that Kindlin-2 regulates breast cancer progression by maintaining EGFR protein level via controlling EGFR ubiquitination and its degradation. A previous study has shown that Kindlin-2 can be induced by serum [29]. Here, we demonstrated that EGF induces Kindlin-2 expression through the EGFR–PI3K signaling pathway in breast cancer cells. Importantly, our Oncomine data mining showed that Kindlin-2 co-expresses with EGFR in breast carcinomas. Although EGFR has been known as a marker for basal-like breast cancer, it is still not clear how EGFR modulates the aggressive behaviors of breast cancer. When EGF binds to the receptor, it
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Fig. 7. Kindlin-2 is required for EGF-induced breast cancer cell migration. (A) SUM159 cells were transfected with control or Kindlin-2 siRNAs and (B) MDA-MB-231 stable cell pooled clones expressing control or short hairpin RNA against Kindlin-2 cells were seeded in the Transwell. After 12 h serum starvation, the cells were incubated in the presence or absence of EGF for 6 h. Cells that migrated were stained and counted. Images are representative of three independent experiments, with at least 15 fields examined in each assay. *p < 0.05; **p < 0.01; ***p < 0.001. (C) A working model depicting that Kindlin-2 is upregulated by EGF through the EGFR–PI3K pathway, and thereby raised Kindlin-2 in turn stabilizing EGFR protein. Thus, Kindlin-2 is required for EGF-induced breast cancer cell migration.
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triggers a variety of signaling pathways, leading to cell proliferation and migration [33]. Several adaptor molecules are recruited to the receptor to transduce the outside-in signaling. We found that Kindlin-2 could bind to EGFR to the kinase domain and mediates EGF-induced cell migration by stabilizing EGFR protein. The effect of Kindlin-2 on maintaining the stability of EGFR values Kindlin-2 as a potential novel target for basal-like breast cancer therapy. Therefore, targeting Kindlin-2–EGFR interaction may represent a hopeful direction for anti-basal-like breast cancer drug screening. Overexpression of EGFR has been shown to promote breast cancer cell migration [44]. However, the mechanism for the initiation and maintenance of cell motility remains unclear. EGFR upregulation occurs via multiple mechanisms including gene amplification, C-terminal truncation, transcriptional activation and posttranscriptional modifications [45]. A previous study showed that Kindlin-2 could bind to integrin and depletion of Kindlin-2 triggers integrin degradation [46]. In this report, we identified a novel function of Kindlin-2 in the control of EGFR degradation. A mechanistic explanation for this function of Kindlin-2 is that Kindlin-2 can decrease EGFR ubiquitination and degradation by binding to the EGFR kinase domain. However, for now we do not know exactly how their interaction leads to the decreased EGFR ubiquitination. It is possible that Kindlin-2 may compete out the binding sites of some E3 ligases, for example, c-Cbl binding to EGFR and reduces c-Cbl action on EGFR. Apparently these mysteries remain for future investigations. In summary, we identified a novel role of Kindlin-2 by demonstrating that Kindlin-2 interacts with the kinase domain of EGFR and prevents EGFR from degradation via decrease of EGFR ubiquitination in human breast cancer cells. Kindlin-2 is required for EGF-induced breast cancer cell migration. Our findings point out that targeting Kindlin-2 and EGFR interaction may shed light on basal-like breast cancer therapeutics. Acknowledgments This study was supported by grants from the Beijing Natural Science Foundation 7120002, National Natural Science Foundation of China 81230051, 30830048, 31170711 and 81321003, and the Ministry of Science and Technology of China 2015CB553906, 2013CB910501, and 2013ZX09401-004-006, the 111 Project of the Ministry of Education, Peking University grants BMU20120314 and BMU20130364, and a Leading Academic Discipline Project of Beijing Education Bureau to H.Z. Conflict of interest The authors declare that there is no competing financial interest in relation to the work described. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.03.011. References [1] R. Siegel, et al., Cancer statistics, 2014, CA Cancer J. Clin. 64 (1) (2014) 9–29. [2] Cancer Genome Atlas Network, Comprehensive molecular portraits of human breast tumours, Nature 490 (7418) (2012) 61–70. [3] C.M. Perou, et al., Molecular portraits of human breast tumours, Nature 406 (6797) (2000) 747–752. [4] E.F. Solomayer, et al., Metastatic breast cancer: clinical course, prognosis and therapy related to the first site of metastasis, Breast Cancer Res. Treat. 59 (3) (2000) 271–278. [5] L.J. van ‘t Veer, et al., Gene expression profiling predicts clinical outcome of breast cancer, Nature 415 (6871) (2002) 530–536. [6] M. Smid, et al., Subtypes of breast cancer show preferential site of relapse, Cancer Res. 68 (9) (2008) 3108–3114.
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Rimawi, et al., Epidermal growth factor receptor expression in breast cancer association with biologic phenotype and clinical outcomes, Cancer 116 (5) (2010) 1234–1242. [14] G. Viale, et al., Invasive ductal carcinoma of the breast with the “triple-negative” phenotype: prognostic implications of EGFR immunoreactivity, Breast Cancer Res. Treat. 116 (2) (2009) 317–328. [15] T. Badovinac-Crnjevic, et al., Significance of epidermal growth factor receptor expression in breast cancer, Med. Oncol. 28 (Suppl. 1) (2011) S121–S128. [16] H.W. Lo, et al., Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer, Cancer Res. 65 (1) (2005) 338–348. [17] R.I. Nicholson, J.M. Gee, M.E. Harper, EGFR and cancer prognosis, Eur. J. Cancer 37 (Suppl. 4) (2001) S9–S15. [18] C.M. Warren, R. Landgraf, Signaling through ERBB receptors: multiple layers of diversity and control, Cell. Signal. 18 (7) (2006) 923–933. [19] A. Citri, Y. Yarden, EGF-ERBB signalling: towards the systems level, Nat. Rev. Mol. Cell Biol. 7 (7) (2006) 505–516. [20] K.M. Ferguson, Structure-based view of epidermal growth factor receptor regulation, Annu. Rev. Biophys. 37 (2008) 353–373. [21] Y.Q. Ma, et al., Kindlin-2 (Mig-2): a co-activator of 3 integrins, J. Cell Biol. 181 (3) (2008) 439–446. [22] E. Montanez, et al., Kindlin-2 controls bidirectional signaling of integrins, Genes Dev. 22 (10) (2008) 1325–1330. [23] H. Qu, et al., Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins, J. Cell Sci. 124 (Pt 6) (2011) 879–891. [24] J. Gao, et al., A feedback regulation between Kindlin-2 and GLI1 in prostate cancer cells, FEBS Lett. 587 (6) (2013) 631–638. [25] T. Zhao, et al., Kindlin-2 promotes genome instability in breast cancer cells, Cancer Lett. 330 (2) (2013) 208–216. [26] Y. Yu, et al., Kindlin 2 promotes breast cancer invasion via epigenetic silencing of the microRNA200 gene family, Int. J. Cancer 133 (6) (2013) 1368–1379. [27] Y. Yu, et al., Kindlin 2 forms a transcriptional complex with beta-catenin and TCF4 to enhance Wnt signalling, EMBO Rep. 13 (8) (2012) 750–758. [28] V. Gkretsi, et al., Mitogen-inducible Gene-2 (MIG2) and migfilin expression is reduced in samples of human breast cancer, Anticancer Res. 33 (5) (2013) 1977–1981. [29] M. Wick, et al., Identification of serum-inducible genes: different patterns of gene regulation during G0→S and G1→S progression, J. Cell Sci. 107 (Pt 1) (1994) 227–239. [30] J.E. Lai-Cheong, et al., Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome, J. Invest. Dermatol. 128 (9) (2008) 2156–2165. [31] G. Levkowitz, et al., Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1, Mol. Cell 4 (6) (1999) 1029–1040. [32] K.C. Nannuru, R.K. Singh, Tumor-stromal interactions in bone metastasis, Curr. Osteoporos. Rep. 8 (2) (2010) 105–113. [33] X. Cui, et al., Epidermal growth factor induces insulin receptor substrate-2 in breast cancer cells via c-Jun NH(2)-terminal kinase/activator protein-1 signaling to regulate cell migration, Cancer Res. 66 (10) (2006) 5304–5313. [34] M.L. Burness, T.A. Grushko, O.I. Olopade, Epidermal growth factor receptor in triple-negative and basal-like breast cancer: promising clinical target or only a marker?, Cancer J. 16 (1) (2010) 23–32. [35] W.W. Du, et al., Versican G3 promotes mouse mammary tumor cell growth, migration, and metastasis by influencing EGF receptor signaling, PLoS ONE 5 (11) (2010) e13828. [36] F. Nie, et al., Involvement of epidermal growth factor receptor overexpression in the promotion of breast cancer brain metastasis, Cancer 118 (21) (2012) 5198–5209. [37] S. Sin, et al., Role of the focal adhesion protein kindlin-1 in breast cancer growth and lung metastasis, J. Natl. Cancer Inst. 103 (17) (2011) 1323–1337. [38] K. Sossey-Alaoui, et al., Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis, FASEB J. 28 (5) (2014) 2260–2271. [39] X. Wei, et al., Kindlin-2 mediates activation of TGF-beta/Smad signaling and renal fibrosis, J. Am. Soc. Nephrol. 24 (9) (2013) 1387–1398. [40] Z. An, et al., Kindlin-2 is expressed in malignant mesothelioma and is required for tumor cell adhesion and migration, Int. J. Cancer 127 (9) (2010) 1999–2008. [41] J. 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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Kindlin-2 interacts with and stabilizes EGFR and is required for EGF-induced breast cancer cell migration Baohui Guo a,b, Jianchao Gao a,b, Jun Zhan a,b, Hongquan Zhang a,b,* a Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, and State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China b Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Beijing 100191, China
A R T I C L E
I N F O
Article history: Received 3 February 2015 Received in revised form 10 March 2015 Accepted 10 March 2015 Keywords: Kindlin-2 Epidermal growth factor receptor Breast cancer Protein degradation Cell migration
A B S T R A C T
Epidermal growth factor receptor (EGFR) mediates multiple signaling pathways that regulate cell proliferation, migration and tumor invasion. Kindlin-2 has been known as a focal adhesion molecule that binds to integrin to control cell migration and invasion. However, molecular mechanisms underlying the role of Kindlin-2 in breast cancer progression remain elusive. Here we report that Kindlin-2 interacts with EGFR and mediates EGF-induced breast cancer cell migration. We found that EGF treatment dramatically increases Kindlin-2 expression at both mRNA and protein levels in a variety of cancer cells. Inhibitors specific for EGFR or PI3K blocked Kindlin-2 induction by EGF. Importantly, Kindlin-2 interacted with EGFR kinase domain, which was independent of Kindlin-2 binding to integrin cytoplasmic domain. Intriguingly, Kindlin-2 stabilized EGFR protein by blocking its ubiquitination and degradation. Depletion of Kindlin-2 impaired EGF-induced cell migration. Our results demonstrated that Kindlin-2 participates in EGFR signaling and regulates breast cancer progression. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Breast cancer is one of the most commonly identified cancers and also the third cause of cancer death in women [1]. Genomic studies have identified five breast cancer intrinsic subtypes: Luminal A, Luminal B, HER2, basal-like and normal-like breast cancers [2,3]. Basal-like tumors are often resistant to chemotherapy and are able to develop distant metastases to the lung and brain [4]. Basal-like breast cancers exhibit high histologic grades, aggressive clinical features and poor prognosis [5,6] and are often identified by enhanced expression of the epidermal growth factor receptor (EGFR) [7]. EGFR, a transmembrane glycoprotein, exists on the surface of cells and contains extracellular, transmembrane and tyrosine kinase domains [8]. EGFR is not only important for the normal growth of breast tissues but also involved in the initiation and progression of breast cancers [9,10]. Previous studies have shown a strong correlation between high expression of EGFR and the aggressive potential of breast tumors [11–15]. EGFR overexpression is also correlated with poor overall survival in breast cancer patients [16,17]. EGFR is activated by binding to its ligands such as EGF, which leads to the receptor dimerization, autophosphorylation and activation of several downstream signaling pathways, enabling cell proliferation, spreading,
differentiation and migration [18]. The activated receptor recruits adapter proteins including GRB2, PLC and Cbl which in turn activate downstream signaling cascades including the RAS-RAF-MEKERK, PI3K-AKT, PLC-PKC and STATs before degradation [19,20]. However, proteins associated with EGFR may differ from the signaling pathways they mediated, suggesting that EGFR signaling is complicated and new interactors need to be characterized. Here we identified EGFR interacts with Kindlin-2, a newly identified integrininteracting focal adhesion molecule both in vivo and in vitro. Kindlin-2, also known as mig-2 (mitogen inducible gene-2), has been reported as a scaffold protein which enhances Talin mediated integrin activation [21,22]. Kindlin-2 also binds to membranes enriched in phosphoinositides and enhances integrin mediated cell adhesion and spreading [23]. Several studies have linked Kindlin-2 to cancer progression [24–26]. How Kindlin-2 participates in breast cancer progression remains to be further elucidated. Our previous findings implicated that high expression of Kindlin-2 promotes genome instability [25], and inhibits microRNA 200 family members in breast cancer cells [26]. In this study we provided evidence that Kindlin-2 and EGFR cooperate in invasive breast cancers. Materials and methods Cell lines and cell culture
* Corresponding author. Tel.: +008610 82802424; fax: +008610-82802424. E-mail address: [email protected] (H. Zhang). http://dx.doi.org/10.1016/j.canlet.2015.03.011 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
Human cell lines (HeLa, HEK-293T, SUM159, MDA-MB-231) were obtained from American Type Culture Collection (ATCC, USA). Cells were cultured in DMEM or RPMI 1640 medium (Invitrogen, USA) supplemented with 10% FBS (Hyclone, USA). Cells
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were grown at 37 °C in humidified conditions with 5% CO2. For transfection, cells were incubated in Opti-MEM medium (Invitrogen, USA) containing plasmid DNA, Lipofectamine (Invitrogen, USA) or PEI (Polysciences, USA) for 6 h. Subsequently, the medium was replaced by DMEM or RPMI 1640 medium and incubated for 24 h before further experiments. Cells were starved in serum-free medium for 24 h, then treated with EGF (100 ng/ml) for various time periods as indicated before harvest. Plasmids and siRNAs Flag–EGFR and pEGFP–EGFR were gifts from Professor Zhijie Chang (Tsinghua University, China). Flag–Kindlin-2 and pEGFP-Kindlin-2 were generated as previously reported [24]. Point mutations of Kindlin-2 were generated using a Stratagene Quick Change site-directed mutagenesis kit. All sequences were verified by DNA sequencing. Kindlin-2 specific siRNAs target sequences 1: 5′AAGCUGGUGGAGAAACUCG-3′; 2: 5′-CAGCGAGAAUCUUGGAGGC-3′. Negative control siRNA: 5′-UUCUCCGAACGUGUCACG-3′. Reagents and antibodies P38 inhibitor SB203580, MEK inhibitor U0126 and PI3K inhibitor Wortmannin were obtained from EMD (USA). EGF and AG1478 were purchased from Sigma (USA). Anti-Flag, HA, GFP antibodies, cyclohexamide (CHX) and proteasome inhibitor MG132 were purchased from Sigma (USA). Secondary antibodies were purchased from Santa Cruz Biotechnology (USA). EGFR Rabbit mAb was from Cell Signaling Technology (USA). Kindlin-2 mouse antibody (Millipore, USA), Alexa-568 goat anti-rabbit and Alexa488 goat anti-mouse (Life Technology, USA) for confocal microscopy; phosphoEGFR and phospho-ERK rabbit antibodies were obtained from Cell Signaling Technology (USA). Immunoprecipitation and immunoblotting Cells were grown in 10-cm dishes to 80% confluence and lysed in RIPA buffer containing protease inhibitor cocktails. Cell lysates were clarified by centrifugation at 12,000 × rpm for 15 min at 4 °C, and total protein concentration was determined using the BCA method. For each immunoprecipitation assay, 2 mg protein and 2 μg antibody were added for each reaction. Normal mouse or rabbit IgG was used as an irrelevant immunoprecipitation control. Immobilized antibodies were mixed with equal amounts of protein lysate and incubated with rotation at 4 °C overnight followed by addition of 50 μl 50% protein A or G Sepharose slurry and rotating for 4 h. Beads were washed with PBS buffer containing 1% NP-40 four times. Immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblotting. The membranes were detected by the Super Signal chemiluminescence kit (Pierce, USA). Software Image J was used for quantification analysis of the band density of target proteins in Western blot assays. In vitro pull-down assay Glutathione S-transferase-tagged EGFR fusion proteins were expressed in Escherichia coli. MBP–Kindlin-2 was generated and expressed as reported previously [27]. Each purified GST-fused EGFR protein or MBP-fused protein was immobilized on 40 μl of Glutathione-Sepharose 4B beads (GE Healthcare, USA) and equilibrated in the pull-down binding buffer consisting of 50 mM HEPES, 50 mM NaCl, 0.1% NP40, and Protease Inhibitors. The purified complex was added to each affinity beads and the binding reactions were incubated at 4 °C for 3 h. The beads were washed three times using the binding buffer. The bound proteins were analyzed by SDSPAGE followed by Coomassie staining. Immunofluorescence and confocal microscopy Cells were plated on collagen type I coated coverslips. After serum starvation overnight, cells were treated with 100 ng/ml EGF for the time indicated, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked in BSA, and stained with the indicated antibodies overnight at 4 °C. Antigen-antibody complexes were detected by staining with Alexa-coupled secondary antibodies for 1 h. Stained cells were visualized with a 63 × 1.4 oil objective using a Zeiss LSM780 laser scanning confocal microscope. Images were representatives of three independent experiments. Cell migration assay The Transwell cell migration system consisted of cell culture champers with an 8.0 μM pore size in 24-well plate (BD, USA). Cells were seeded on 6 well plates, serum starved overnight and treated with either vehicle or 100 ng/ml EGF for 6 h, After incubation, cells were resuspended with serum-free DMEM containing vehicle or 100 ng/ml EGF, and 1 × 104 cells were separately seeded in the champers. PBS containing 3 μg/ml collagen type I was loaded into the lower chamber and incubated for 6 h. Migrated cells were fixed with 4% formaldehyde for 15 min and stained with 0.5% crystal violet for 15 min. Migrated cells were counted in 15 different microscopic fields by 10 × objective using a Nikon microscope. All assays were performed at least three times.
Statistical analysis Correlation analysis between Kindlin-2 and EGFR expression in human breast cancer samples was analyzed for significance using Prism 5.0 software (GraphPad Software, USA) with Pearson r test, where p < 0.05 was considered statistically significant. For other experiments, results are expressed as mean ± SD. Statistical analyses were performed using Student’s t test. P < 0.05 was considered statistically significant. All experiments were repeated at least three times.
Results Increased expression of Kindlin-2 is correlated with breast cancer progression To determine the role of Kindlin-2 in breast cancer progression, Oncomine datasets were analyzed (Fig. 1A). Results showed that Kindlin-2 mRNA expression was increased in invasive ductal breast carcinoma compared with noninvasive ductal breast carcinoma (p = 8.17E-04, fold change: 2.537). Moreover, the poor prognostic effect of Kindlin-2 expression was supported by Kaplan– Meier Plotter analysis (www.kmplot.com), which showed that high Kindlin-2 abundance correlated with a poor relapse-free survival (RFS, p = 1.8E-05, HR=1.29) using microarray data from breast cancer patients (n = 3455 ) (Fig. 1B). These results indicate that Kindlin-2 plays a role in invasive breast carcinomas. Kindlin-2 expression is correlated with EGFR expression in breast cancer tissues Furthermore, EGFR overexpression is frequently observed in most basal-like breast cancer cell lines and an important marker in the immunohistochemical identification of basal-like breast cancers [14]. Similarly, we found high Kindlin-2 expression in basal-like breast cancer cell lines including MDA-MB-231, SUM159 and HS578T [25]. Then we set out to assess the association between EGFR and Kindlin-2 expression. We analyzed the correlation of Kindlin-2 and EGFR mRNA expression in two independent datasets, 43 (GSE22035) and 60 breast cancer patient samples (GSE1378) (Fig. 1C and D). Positive correlation was identified between EGFR and Kindlin-2 mRNA expression in breast cancer tissues (GSE22035, n = 43, R = 0.5689, p < 0.0001; GSE1378, n = 60, R = 0.4055, p < 0.0013). To determine the clinical significance of Kindlin-2 in breast cancer patients versus normal breast tissues, we conducted an Oncomine analysis to characterize gene expression using massive microarray data from breast cancers. Our search for Kindlin-2 gene expression was based on 53 datasets and finally revealed 24 datasets with significant p values. In all of these cases, Kindlin-2 was consistently decreased in cancers compared with normal breast tissues. Moreover, a similar pattern of EGFR expression that was decreased in carcinomas was also observed. Search for EGFR gene expression was based on 51 datasets and revealed 24 datasets with significant p values, further supporting the correlation between Kindlin-2 and EGFR in breast cancer patients (Supplementary Fig. S1). A recent study reported that Kindlin-2 expression is reduced in breast cancer samples [28], consistent with our results. These data indicated that Kindlin-2 functions in breast cancer progression. Activation of EGFR signaling promotes Kindlin-2 expression through the EGFR–PI3K signaling pathway Kindlin-2 was identified as a mitogen-inducible gene firstly [29], we therefore validated EGF effect on Kindlin-2 expression by Western blot and quantitative PCR. EGFR-positive HeLa cells were treated with EGF for various times. We found that EGF-induced Kindlin-2 expression in HeLa cells is in a time-dependent manner (Fig. 2A). Quantitative PCR results showed that EGF also increased Kindlin-2 mRNA levels in HeLa cells (Supplementary Fig. S2A). Similar results
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Fig. 1. Kindlin-2 expression is correlated with EGFR expression in breast cancer tissues. (A) Analysis of expression of Kindlin-2 mRNA in 14 breast cancer patients’ samples of ductal breast carcinomas in situ versus invasive ductal breast carcinomas. Data were obtained from Oncomine in Schuetz breast, Pearson R test. (B) Kaplan–Meier survival plots demonstrating the poor prognostic effect of Kindlin-2 gene high expression correlated with a short RFS in breast cancer patients (n = 3455). (C) Analysis of correlation of Kindlin-2 and EGFR mRNA expression in two independent datasets, 43 breast cancer patient samples from GEO (GSE22035) and (D) 60 breast cancer patient samples from GEO (GSE1378). All data are log transformed and median centered.
were found in human basal-like breast cancer cell lines MDA-MB231 and SUM159, which showed high EGFR expression. EGF treatment triggered EGFR and ERK activation as well as Kindlin-2 upregulation (Fig. 2B, Supplementary Fig. S2B). To determine whether the EGFR signaling induced Kindlin-2 expression was directly regulated at the transcriptional level, MDAMB-231 cells were treated with 1 μg/ml transcription inhibitor actinomycin D (AD) or 100 nM protein synthesis inhibitor cycloheximide (CHX) for 1 h followed by EGF treatment. Results showed that AD prevented EGF-induced Kindlin-2 expression in MDA-MB231 cells while CHX did not (Fig. 2C). These results suggested that EGFR signaling upregulates Kindlin-2 expression at the transcriptional level and is independent of protein synthesis. In response to EGF stimulation, EGFR triggers several downstream signaling cascades, including MAPK/ERK, phosphatidylinositol 3-kinase (PI3K)/AKT, and Stat pathways [19]. Therefore, we determined the downstream signaling pathway of EGFR that leads to Kindlin-2 upregulation by a series of specific chemical inhibitors that were known to target EGFR signaling. MDA-MB-231 cells were pretreated with an EGFR inhibitor AG1478 (AG), ERK1/2 inhibitor U0126 (U), p38 inhibitor SB203580 (SB), or PI3K inhibitor Wortmannin (W) for 1 h followed by EGF treatment for 6 h. We observed that only the EGFR or PI3K inhibitor abrogated EGF-induced Kindlin-2 expression (Fig. 2D), suggesting that Kindlin-2 induction employs an EGFR–PI3K signaling cascade. Kindlin-2 interacts with EGFR in vitro and in vivo Given the strong mRNA expression correlation between EGFR and Kindlin-2, we hypothesized that Kindlin-2 might interact with EGFR. The interaction between Kindlin-2 and EGFR was
confirmed by different approaches, using overexpressed Kindlin-2 and EGFR as well as endogenous proteins. Firstly, HEK-293T cells were co-transfected with Flag–EGFR and GFP–Kindlin-2 (Fig. 3A) or co-transfected with Flag–Kindlin-2 and GFP–EGFR (Fig. 3B). Immunoprecipitation with anti-Flag antibody showed that EGFR was co-immunopecipitated with Kindlin-2. These data indicated that Kindlin-2 interacts with EGFR. To examine the endogenous interaction between EGFR and Kindlin-2, we performed co-immunoprecipitation experiments. Results showed that EGFR is able to be co-immunoprecipitated by Kindlin-2 antibody and Kindlin-2 is able to be co-immunoprecipitated by EGFR antibody but not the control IgG (Fig. 3C and D). To determine whether EGFR binds to Kindlin-2 in vitro, purified MBP–Kindlin-2 protein was incubated with MDA-MB-231 cell lysates. MBP and MBP–Kindlin-2 were stained by Coomassie blue as loading control (Fig. 3E, below). Western blot analysis revealed that Kindlin-2 binds to EGFR in vitro (Fig. 3E). Kindlin-2 is known to bind to integrin to mediate integrin outsidein signaling [21,22], we thus wonder if Kindlin-2 binding to integrin is required for its interaction with EGFR. A Flag–Kindlin-2 QW mutant deficient in binding to integrin was generated. Both the wild-type (WT) and mutant Flag–Kindlin-2 were transfected into HEK-293T cells. Results indicated that the interaction between Kindlin-2 and EGFR is independent of Kindlin-2 binding to integrin (Supplementary Fig. S3A). Given that EGF promotes the interaction of EGFR with several proteins, we thus wanted to examine the effect of EGF on the interaction of EGFR with Kindlin-2. Interestingly, we found that EGFR binds to Kindlin-2 under conditions with or without EGF (Supplementary Fig. S3B, left). However, EGF treatment weakened the interaction between the two (Supplementary Fig. S3B, right).
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Fig. 2. Activation of EGFR signaling promotes Kindlin-2 expression through the EGFR–PI3K pathway. (A) HeLa cells were serum starved overnight and then were stimulated with 100 ng/ml EGF for the indicated time period. The expression of Kindlin-2 was analyzed by Western blot (left). Results were demonstrated by histograms to quantify the expression levels at the indicated time period from a representative experiment (right). (B) Western blot analysis of Kindlin-2 induction by EGF in basal-like breast cancer cell line SUM159. Cells were serum starved and treated with 100 ng/ml EGF for the indicated time period. Expressions of phosphorylated EGFR and phosphorylated ERK were also analyzed to authenticate the effectiveness of EGF stimulation. (C) After 12 h of serum starvation, MDA-MB-231 cells were pretreated with actinomycin D (AD) or CHX for 1 h, followed by 100 ng/ml EGF treatment for 1 h. The expression of Kindlin-2 was analyzed by Western blot. Results were demonstrated by histograms (bottom) to quantify the expression levels. (D) MDA-MB-231 cells were serum starved overnight and pretreated with DMSO (−), MEK inhibitor U0126 (U), EGFR inhibitor AG1478 (AG), PI3K inhibitor Wortmannin (W), p38 inhibitor SB203580 (SB), or a transcription inhibitor actinomycin D (AD) for 1 h followed by EGF treatment for 1 h, Kindlin-2 expression was analyzed by Western blot. Results were demonstrated by histograms to quantify the expression levels (bottom). Data were presented as mean ± SD (vertical bars) from three independent experiments. *** p < 0.001. NS: not significant.
Mapping of the interacting region between Kindlin-2 and EGFR EGFR is composed of extracellular, transmembrane (TM), juxtamembrane (JM), kinase (KD) and the C-terminal flexible domains. To determine which domain of EGFR was required for the interaction with Kindlin-2, HEK-293T cells were transfected with Flag–EGFR-A or Flag–EGFR-B and co-immunoprecipitation was performed (Supplementary Fig. S4A). Results showed that TM, JM and KD are the major binding domains of EGFR with Kindlin-2 (Supplementary Fig. S4B). To further confirm these results, different GST-tagged EGFR fragments were expressed (Fig. 4A) and purified using Glutathione Sepharose 4B beads, then the beads were incubated with MDA-MB-231 cell lysates. No interaction between the C-terminal flexible domain of EGFR with Kindlin-2 was detected (Fig. 4B), indicating that JM and KD of EGFR were required for its interaction with Kindlin-2. Similarly, Kindlin-2 was divided into three fragments and FERM and F0 domains were found to bind to EGFR
(Fig. 4C and D). Collectively, the N-terminus of Kindlin-2 is able to bind to EGFR juxtamembrane and kinase domains. Co-localization of Kindlin-2 with EGFR in breast cancer cells To further characterize the interaction between Kindlin-2 and EGFR, we examined the subcellular localization of endogenous Kindlin-2 and EGFR proteins in cells by immunofluorescence assay. Kindlin-2 has been reported to locate at focal adhesions to mediate integrin activation and cell adhesion [30]. Interestingly, in serum starved MDA-MB-231 cells, we found that in addition to focal adhesions, Kindlin-2 also located at the lamellipodia, the dynamic membrane structures of the leading edge of motile cells. Kindlin-2 distribution at the lamellipodia partially overlaps with EGFR, suggesting that the two proteins co-localize with each other at the lamellipodia (Fig. 5A). Given that EGF is able to induce rapid EGFR endocytosis, we therefore examined the changes of EGFR localization.
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Fig. 3. Kindlin-2 interacts with EGFR in vivo and in vitro. (A) HEK-293T cells were transiently transfected with Flag–EGFR and GFP–Kindlin-2 or (B) transfected with Flag– Kindlin-2 and GFP–EGFR. After cell lysis, equal amounts of protein were immunoprecipitated with anti-Flag antibody or IgG. Both unprocessed lysates (Input) and immunoprecipitates (IP) were resolved by SDS-PAGE and transferred onto PVDF membranes. Identical blots were probed with anti-EGFR, anti-Flag or anti-Kindin-2 antibodies. (C) MDA-MB-231 cells were lysed and equal amounts of protein lysates were immunoprecipitated with anti-EGFR antibody or IgG and probed with anti-Kindlin-2 antibody. (D) MDA-MB-231 cells were lysed and equal amounts of protein lysates were immunoprecipitated with anti-Kindlin-2 antibody or IgG and probed with anti-EGFR antibody. (E) Purified MBP– Kindlin-2 or MBP protein alone was incubated with MDA-MB-231 cell lysates at 4 °C overnight. Beads were washed and the remaining proteins were resolved by SDS-PAGE and further analyzed by Western blot using anti-EGFR antibody (upper). The MBP and MBP–Kindlin-2 were stained by Coomassie blue (below).
To follow the possible translocation of Kindlin-2 and EGFR in living cells, MDA-MB-231 cells were treated with EGF for 10 or 60 min. We found that EGF induced translocation of EGFR from the periphery of cells to the cytoplasm and nucleus at a time point of 60 min, while Kindlin-2 showed only partial translocation into the nucleus. These results suggest that EGF treatment impaired the co-localization of EGFR and Kindlin-2, consistent with the previous immunoprecipitation data that EGF can weaken the interaction between EGFR with Kindlin-2 (Supplementary Fig. S3B). EGFR and Kindlin-2 co-localized at the lamellipodia in MDAMB-231 cells, which showed few cell–cell junctions. For adherent SUM159 cells, Kindlin-2 was diffusely distributed throughout the cytoplasm with prominent accumulation at cell–cell junctions. EGFR was also found primarily at cell–cell junctions. Merged images showed that Kindlin-2 and EGFR co-localized at cell–cell junctions under conditions of serum starvation (Fig. 5B, upper), and co-localized at lamellipodia upon EGF treatment (Fig. 5B, middle). EGF induced translocation of EGFR into the cytoplasm, and the co-localization was also decreased after 60 min EGF treatment (Fig. 5B, bottom).
degradation is mediated by ubiquitination of the receptor, endocytosis, and finally EGFR is degradated by both proteasomal and lysosomal hydrolases [31]. Treatment of cells with the proteasome inhibitor MG132 led to raised the protein level of EGFR, an effect that can be realized by exogenous expression of Kindlin-2 (Fig. 6B). However, the presence of both MG132 and Kindlin-2 expression could not further raise the level of EGFR (Fig. 6B), suggesting that MG132 and Kindlin-2 may target the same mechanism, indicating that EGFR upregulation in Kindlin-2 expressing cells is due to the reduced degradation of EGFR. In addition, overexpression of Kindlin-2 prolonged the half-life of EGFR (Fig. 6C), while knockdown of Kindlin-2 reduced the half-life of EGFR (Fig. 6D), clearly indicating that Kindlin-2 regulates EGFR protein turnover. Given that ubiquitination is an important step in mediating EGFR degradation, we therefore carried out ubiquitination assays to examine whether Kindlin-2 is responsible for the regulation of EGFR ubiquitination. To this end, the level of ubiquitylated EGFR was examined and was found to be decreased in Kindlin-2 overexpressing HEK-293T cells (Fig. 6E). These results support our hypothesis that Kindlin-2 upregulated EGFR is mediated by decreased ubiquitination of EGFR.
Kindlin-2 stabilizes EGFR protein by blocking EGFR ubiquitination and degradation Kindlin-2 is required for EGF-induced breast cancer cell migration Overexpression of Kindlin-2 in HEK-293T cells led to upregulation of EGFR (Fig. 6A). Given that Kindlin-2 altered only the protein but not the mRNA levels of EGFR (data not shown), we hypothesized that Kinlin-2 is involved in the stability of EGFR protein. EGFR
SUM159 and MDA-MB-231 are aggressive breast cancer cell lines and exhibit high migratory capacity compared with other breast cancer cell lines [32]. Stimulation of cancer cells with various growth
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Fig. 4. Mapping of the interaction between Kindlin-2 and EGFR. (A) The schematic diagram of different GST-tagged EGFR fragments. Human EGFR including extracellular domain which binds with the ligands, transmembrane domain (TM), juxtamembrane domain (JM), kinase domain (KD) and the C-terminal flexible region. (B) GST–EGFR fragments were purified using Glutathione Sepharose 4B beads, then the beads was incubated with MDA-MB-231 cell lysate at 4 °C overnight. Beads were washed and the remaining proteins were resolved by SDS-PAGE and analyzed by Western blot using anti-Kindlin-2 antibody. (C) The schematic diagram of different Flag tagged Kindlin-2 fragments used in the mapping. (D) The truncated segments were transfected into HEK-293T cells. Twenty-four hours later cells were harvested. Then the segments were pulled down using purified GST–EGFR-A and analyzed by Western blot using anti-Flag antibody.
factors including EGF increases cell migration [33,34]. Inhibitors of EGFR are able to reduce breast cancer metastasis to the bone and brain in mouse models [35,36], suggesting that the EGFR signaling is involved in the regulation of breast cancer metastasis. To uncover the functional implication of the EGFR–Kindlin-2 interaction, we examined the role of Kindlin-2 in EGF-induced cell motility in breast cancer cells. Control or Kindlin-2 siRNAs were used to inhibit endogenous and EGF-induced Kindlin-2 expression in basal-like breast cancer cell SUM 159. Immunoblotting confirmed the knockdown of Kindlin-2 in SUM 159 cells (Fig. 7A). We found that EGF stimulation further enhanced SUM159 cell migration. In addition, depletion of Kindlin-2 by siRNA significantly inhibited cell migration caused not only by Kindlin-2 but also by EGF (Fig. 7B), indicating that Kindlin-2 is required for EGF-stimulated breast cancer cell migration. Furthermore, we observed a similar requirement of Kindlin-2 in EGF-induced cell migration using MDA-MB-231 cells stably expressing control or short hairpin RNA against Kindlin-2 (Fig. 7C and D). Collectively, these results strongly indicated that Kindlin-2 is required for EGF-induced breast cancer cell migration. Discussion Metastatic breast cancers are highly devastating diseases with few effective treatments. However, mechanisms underlying breast cancer metastasis are still unclear. Previous studies have linked all
Kindlin family members, including Kindlin-1, -2 and -3, with breast cancer progression. Kindlin-1 was found to regulate TGF β signaling and played a role in breast cancer lung metastasis [37]. Kindlin-3 was also found to be involved in breast cancer progression and metastasis [38]. Our previous studies showed that Kindlin-2 binds directly to the TβRI receptor and Smad to activate TGF β signaling [39]. Kindlin-2 can also stabilize β-catenin and plays a role in Wnt signaling [27]. Kindlin-2 was found to regulate malignant mesothelioma cell adhesion and migration [40]. However, the detailed mechanism of Kindlin-2 in the regulation of breast cancer progression is still unknown. Kindlin-2 regulates cancer cell migration via multiple mechanisms including EGFR signaling pathway as reported here. Kindlin-2 also cooperates with integrin β3 and migfilin to regulate cell migration [21], and is involved in TGF β and Wnt pathways to regulate cell migration [27,41]. In addition, Kindlin-2 promotes carcinogenesis by inducing genome instability [25]. Importantly, Kindlin-2 functions as a scaffold molecule of cellular focal adhesive structures [21–23]. Furthermore, Kindlin-2 also functions as a regulatory molecule controlling various signaling pathways [24–27]. Interestingly, we identified that Kindlin-2 is highly expressed in mesodermderived organs [42]. An exception was seen that Kindlin-2 is strongly expressed in tissues that are highly mobile during embryonic development. Therefore, normal breast tissues still express higher levels of Kindlin-2 accordingly to maintain the structure of the epithelium.
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Fig. 5. Co-localization of Kindlin-2 with EGFR in breast cancer cells. (A) MDA-MB-231 cells or (B) SUM-159 cells were plated on collagen type I coated coverslips, and were subjected to serum starvation overnight and then stimulated with 100 ng/ml EGF or vehicle for the indicated time period. Cells were fixed and double-stained with rabbit anti-EGFR/Alexa Fluor 568-conjugated secondary antibody and mouse anti-Kindlin-2/Alexa Fluor 488-conjugated secondary antibody. The nuclei were stained with Hoechst 33342. Slides were analyzed by Zeiss LSM confocal laser microscopy. Merged pictures represent the composite of both channels, with yellow regions indicative of colocalization. The images are representative of three independent experiments, with at least six fields examined in each assay.
In non-metastatic breast cancer Kindlin-2 expression is lower than the normal breast tissue, which may be due to the insufficiency of stimulating factors. However, the role of Kindlin-2 in breast cancer progression is elusive. Gozgit et al. found that Kindlin-2 expression
is higher in an aggressive subset than a nonaggressive subset of MCF-7 cells, and knockdown of Kindlin-2 inhibited breast cancer cell invasion [43]. However, Kindlin-2 showed heterogeneity in a panel of 34 formalin-fixed breast tumors, and only half of the
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Fig. 6. Kindlin-2 stabilizes EGFR protein by attenuating EGFR ubiquitination and degradation. (A) HEK-293T cells were transfected with GFP empty vector, Flag–EGFR or GFP–Kindlin-2, Flag–EGFR separately. EGFR expression was analyzed by Western blot. (B) SUM159 cells were transfected with Flag empty vector or Flag–Kindlin-2 and then treated with Vehicle or 10 μM MG132 for 6 h before harvesting. EGFR expression was analyzed by Western blot. (C) SUM159 cells were transfected with GFP or GFP– Kindlin-2 or (D) transfected with control siRNA or Kindlin-2 siRNA, and treated with 100 nM CHX and 100 ng/ml EGF for the indicated time. EGFR expression was analyzed by Western blot. (E) HEK-293T cells were co-transfected with HA-Ub, Flag–EGFR, GFP empty vector or GFP–Kindlin-2 plasmids and treated with 10 μM MG132 for 6 h before harvesting. EGFR ubiquitination was detected by immunoprecipitation with Flag antibody and immunoblotting with HA antibody.
patients showed Kindlin-2 overexpression and the other half showed no detectable Kindlin-2 [43]. In Oncomine data mining Kindlin-2 expression was found to be downregulated in nonmetastatic breast cancer patients but was upregulated in patients with invasiveness. It is possible that Kindlin-2 functions differentially in benign and malignant breast tumors. In normal breast tissues or benign breast diseases Kindlin-2 expression is highly expressed and is localized at membrane-anchored structures, i.e., focal adhesions. In nonmetastatic breast cancer patients Kindlin-2 expression is lower than the normal breast tissues and is localized at the cytoplasm. Kindlin-2 expression was raised in metastatic breast cancer patients, in which epithelial to mesenchymal transition (EMT) has taken
place. Taken together, Kindlin-2 is a versatile molecule that plays a differential role in breast cancer progression. Our data suggested that Kindlin-2 regulates breast cancer progression by maintaining EGFR protein level via controlling EGFR ubiquitination and its degradation. A previous study has shown that Kindlin-2 can be induced by serum [29]. Here, we demonstrated that EGF induces Kindlin-2 expression through the EGFR–PI3K signaling pathway in breast cancer cells. Importantly, our Oncomine data mining showed that Kindlin-2 co-expresses with EGFR in breast carcinomas. Although EGFR has been known as a marker for basal-like breast cancer, it is still not clear how EGFR modulates the aggressive behaviors of breast cancer. When EGF binds to the receptor, it
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Fig. 7. Kindlin-2 is required for EGF-induced breast cancer cell migration. (A) SUM159 cells were transfected with control or Kindlin-2 siRNAs and (B) MDA-MB-231 stable cell pooled clones expressing control or short hairpin RNA against Kindlin-2 cells were seeded in the Transwell. After 12 h serum starvation, the cells were incubated in the presence or absence of EGF for 6 h. Cells that migrated were stained and counted. Images are representative of three independent experiments, with at least 15 fields examined in each assay. *p < 0.05; **p < 0.01; ***p < 0.001. (C) A working model depicting that Kindlin-2 is upregulated by EGF through the EGFR–PI3K pathway, and thereby raised Kindlin-2 in turn stabilizing EGFR protein. Thus, Kindlin-2 is required for EGF-induced breast cancer cell migration.
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triggers a variety of signaling pathways, leading to cell proliferation and migration [33]. Several adaptor molecules are recruited to the receptor to transduce the outside-in signaling. We found that Kindlin-2 could bind to EGFR to the kinase domain and mediates EGF-induced cell migration by stabilizing EGFR protein. The effect of Kindlin-2 on maintaining the stability of EGFR values Kindlin-2 as a potential novel target for basal-like breast cancer therapy. Therefore, targeting Kindlin-2–EGFR interaction may represent a hopeful direction for anti-basal-like breast cancer drug screening. Overexpression of EGFR has been shown to promote breast cancer cell migration [44]. However, the mechanism for the initiation and maintenance of cell motility remains unclear. EGFR upregulation occurs via multiple mechanisms including gene amplification, C-terminal truncation, transcriptional activation and posttranscriptional modifications [45]. A previous study showed that Kindlin-2 could bind to integrin and depletion of Kindlin-2 triggers integrin degradation [46]. In this report, we identified a novel function of Kindlin-2 in the control of EGFR degradation. A mechanistic explanation for this function of Kindlin-2 is that Kindlin-2 can decrease EGFR ubiquitination and degradation by binding to the EGFR kinase domain. However, for now we do not know exactly how their interaction leads to the decreased EGFR ubiquitination. It is possible that Kindlin-2 may compete out the binding sites of some E3 ligases, for example, c-Cbl binding to EGFR and reduces c-Cbl action on EGFR. Apparently these mysteries remain for future investigations. In summary, we identified a novel role of Kindlin-2 by demonstrating that Kindlin-2 interacts with the kinase domain of EGFR and prevents EGFR from degradation via decrease of EGFR ubiquitination in human breast cancer cells. Kindlin-2 is required for EGF-induced breast cancer cell migration. Our findings point out that targeting Kindlin-2 and EGFR interaction may shed light on basal-like breast cancer therapeutics. Acknowledgments This study was supported by grants from the Beijing Natural Science Foundation 7120002, National Natural Science Foundation of China 81230051, 30830048, 31170711 and 81321003, and the Ministry of Science and Technology of China 2015CB553906, 2013CB910501, and 2013ZX09401-004-006, the 111 Project of the Ministry of Education, Peking University grants BMU20120314 and BMU20130364, and a Leading Academic Discipline Project of Beijing Education Bureau to H.Z. Conflict of interest The authors declare that there is no competing financial interest in relation to the work described. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.03.011. References [1] R. Siegel, et al., Cancer statistics, 2014, CA Cancer J. Clin. 64 (1) (2014) 9–29. [2] Cancer Genome Atlas Network, Comprehensive molecular portraits of human breast tumours, Nature 490 (7418) (2012) 61–70. [3] C.M. Perou, et al., Molecular portraits of human breast tumours, Nature 406 (6797) (2000) 747–752. [4] E.F. Solomayer, et al., Metastatic breast cancer: clinical course, prognosis and therapy related to the first site of metastasis, Breast Cancer Res. Treat. 59 (3) (2000) 271–278. [5] L.J. van ‘t Veer, et al., Gene expression profiling predicts clinical outcome of breast cancer, Nature 415 (6871) (2002) 530–536. [6] M. Smid, et al., Subtypes of breast cancer show preferential site of relapse, Cancer Res. 68 (9) (2008) 3108–3114.
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