ASB13 inhibits breast cancer metastasis through promoting SNAI2 degradation and relieving its transcriptional repression of YAP

Abstract

Transcription factor SNAI2 plays key roles during development and has also been known to promote metastasis by inducing invasive phenotype and tumor-initiating activity of cancer cells. However, the post-translational regulation of SNAI2 is less well studied. We performed a dual-luciferase-based, genome-wide E3 ligase siRNA library screen and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells promoted cell migration and decreased F-actin polymerization, while overexpression of ASB13 suppressed lung metastasis. Furthermore, ASB13 knockout decreased YAP expression, and such regulation is dependent on an increased protein level of SNAI2, which in turn represses YAP transcription. YAP suppresses tumor progression in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, cell migration in vitro, and lung metastasis in vivo. Clinical data analysis reveals that ASB13 expression is positively correlated with improved overall survival in breast cancer patients. These findings establish ASB13 as a suppressor of breast cancer metastasis by promoting degradation of SNAI2 and relieving its transcriptional repression of YAP.

Keywords: ASB13; Hippo–YAP pathway; SNAI2; breast cancer; metastasis; migration; ubiquitin proteasome system.

Figure 1.

A dual-luciferase system for the identification of SNAI2 targeting E3 ligase. (A) SUM159 cells were treated with 10 µM CHX for indicated time or MG132 for 6 h. The endogenous SNAI2 protein was detected by immunoblotting. β-ACTIN was used as internal loading control. (+) Treated with MG132, (−) no treatment. (B) LM2 cells were treated with 10 µM CHX for indicated time or MG132 for 6 h. The endogenous SNAI2 protein was detected by immunoblotting. β-ACTIN was used as internal loading control. (C) Illustration of the dual-luciferase reporter screening system for SNAI2 targeting E3 ligase. (CMV) Cytomegalovirus promoter, (LTR) long terminal repeat viral promoter. (D) Immunofluorescence staining of SNAI2-Luc in SUM159-SNAI2-Luc cell line with SNAI2 antibody. Scale bar, 50 µm. (E) In HEK293T cells stably expressing SNAI2-Luc, the turnover rate for SNAI2-Luc was determined by CHX pulse-chase assay and luciferase assay. Data are presented as mean ± standard error. (F) In SUM159 cells stably expressing SNAI2-Luc, the turnover rate for SNAI2-Luc was determined by CHX pulse-chase assays.

Figure 2.

A genome-wide siRNA library screen for E3 ubiquitin ligase(s) targeting the SNAI2 protein. (A) Experimental procedure flow chart for identification of E3 ubiquitin ligase(s) targeting SNAI2 protein. (B) Dual-luciferase-based siRNA library screen in HEK293T cells against human E3 ligases identified multiple E3 candidates. E3 ligase was considered to be a positive hit if its knockdown led to more than twofold increase in ff-luc/r-luc raito. (C) Lysates from HEK293T cells treated with 10 µM MG132 for 4 h were immunoprecipitated with FLAG beads, then immunoblotted with either HA antibody for E3 ligases or FLAG antibody for SNAI2 protein. The 50-kDa IgG bands in immunoprecipitated samples are labeled with an asterisk. Dashed line in input image indicates that the band for TRIM69 is above the 50-kDa IgG band. (D) HEK293 cells were transiently transfected with plasmids expressing HA-tagged ASB13 or TRIM3, and FLAG-tagged SNAI2 and stained with antibodies against HA or FLAG tag to visualize the cellular localizations of these proteins by immunofluorescence. Scale bar, 20 µm. (E) Two E3 ligase candidates or a control pLEX-vector were cotransfected with SNAI2-Luc plasmid into 293T cells, and a CHX pulse-chase assay was performed 48 h later. (F) Quantification of SNAI2 protein levels presented in E using ImageJ software. Vector versus TRIM3, P = 0.009; vector versus ASB13, P = 2.8 × 10⁻⁵.

Figure 3.

ASB13 targets SNAI2 protein for ubiquitination and degradation. (A) HEK293T cells were cotransfected with plasmids expressing HA-Ub, SNAI2-FLAG together with either a vector control, ASB13, or the ASB13-ΔSOCS plasmid. Cells were treated with MG132 for 6 h before IP using a denature IP protocol to pull down the SNAI2 protein, and the polyubiquitinated SNAI2 protein was detected by an anti-HA antibody. (B) Box plot showing normalized ASB13 mRNA levels in ER⁺ and ER⁻ breast cancer patients. Data are from the Wang et al. (2005) data set (GSE2034), and patients were free of lymph node invasion at the time of diagnosis. P = 0.0001 with unpaired two-tailed t-test. The middle line represents medium, the box represents 25%–75% values, while the error bar represents minimum and maximum without outlier. (C) Kaplan–Meier plot of overall survival of breast cancer patients stratified by the expression of ASB13 gene. Data were obtained from KMplot.com. (D,E) Endogenous ASB13 expression level was repressed during EMT inducer treatments like TGF-β, Wnt, and EGF signaling activation in MCF10A and EpRas cells. Cells were treated with TGF-β, LiCl (inhibitor of GSK3β kinase, activator of Wnt signaling pathway), and recombinant EGF protein, respectively. Data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01 by Student's t-test. (F) ASB13 (HA-tagged) was stably expressed in SUM159 and LM2 cells by lentivirus transduction, cell lysates were collected, and the SNAI2 protein level was detected by immunoblotting. (G) Quantification of SNAI2 protein levels presented in F using ImageJ software. Data are presented as mean ± SEM. (**) P < 0.01 by Student's t-test. (H) Pulse-chase experiments in ASB13 overexpressing SUM159 cells and LM2 cells. (I) Quantification of SNAI2 degradation dynamics presented in H using ImageJ software. For LM2 cells, vector versus ASB13, P = 0.006; for SUM159 cells, vector versus ASB13, P = 2.3 × 10⁻⁵.

Figure 4.

ASB13 inhibits breast cancer migration and metastasis through promoting SNAI2 degradation. (A) ASB13 was KO by CRISPR–Cas9 system in SUM159 and LM2 cells. Genomic DNA was purified and the targeted locus was amplified by PCR. Representative sanger sequencing and TIDE analysis was used to confirm the ASB13 KO efficiency. (Top panel) gRNA1 in SUM159 cells. (Bottom panel) gRNA2 in LM2 cells. (NG) Nontargeting gRNA. (B) The expression level of the SNAI2 protein was determined by immunoblotting after ASB13 KO by CRISPR-Cas9 in SUM159 and LM2 cells. β-ACTIN was used as internal loading control. (C) The SNAI2 mRNA level was determined by real time qPCR in ASB13 KO cells compared with that of NG cells. (B,C, top panels) SUM159 cells (gRNA1). (Bottom panels) LM2 cells (gRNA2). Data are presented as mean ± SEM. n = 3, not significant (n.s.) by Student's t-test. (D) Representative images of Boyden chamber migration assay for NG cells and ASB13 KO cells using SUM159 and LM2 cell line. Scale bar, 400 µm. (E) Quantification of migrated cells from experiment in D. Data are presented as mean ± SEM. SUM159 on the left and LM2 at the right. n = 3. (**) P < 0.01 by Student's t-test. (F) We intravenously injected 10⁵ vector control or ASB13-overexpressing LM2 cells into 6- to 8-wk-old female athymic nude mice to generate lung metastasis. Lung metastasis burden in mice were quantified weekly by bioluminescence imaging (BLI). (Left panel) Representative BLI images of each group at experimental endpoint. (Right panel) Normalized BLI signals of lung metastasis. Data are presented as mean ± SEM. n = 10. (*) P < 0.05 by Student's t-test. (G) Representative lung metastasis nodule images are presented from experiment in F. (H) Numbers of lung metastasis lesions of mice injected with the indicated LM2 cell lines. n = 10, (**) P < 0.01 by Mann–Whitney U-test.

Figure 5.

ASB13 inhibits cell migration through SNAI2 degradation and inhibiting Hippo–YAP pathway. (A) F-actin was stained with phalloidin–rhodamine dye in WT, ASB13 KO, and SNAI2-Luc overexpression SUM159 cell lines. Scale bar, 30 µm. (B) F-actin and G-actin from the indicated SUM159 cells expressing different constructs were segmented by ultra-speed centrifugation and analyzed by immunoblotting using corresponding antibodies. (C) Quantification of endogenous F-actin levels presented in B using ImageJ software. Data are presented as mean ± SEM. n = 3. (**) P < 0.01 by Student's t-test. (D) The YAP protein level was detected by immunoblotting in ASB13 KO cells and SNAI2-Luc-overexpressing cells, and compared with the vector control of SUM159 cells. (E) Two independent SNAI2 stable cell lines were generated by transducing SNAI2-expressing lentivirus into SUM159 cells. YAP and SNAI2 expression in these cells was determined by immunoblotting. β-ACTIN was used as internal loading control. (F) The mRNA level of YAP and SNAI2 was determined by real-time qPCR in SNAI2-Luc overexpressing SUM159 cells compared with that of vector control cells. Data are presented as mean ± SEM. n = 3, (**) P < 0.01 by Student's t-test. (G) Indicated plasmids were cotransfected into 293T cells. Firefly luciferase reporter activity was measured and normalized to renilla luciferase internal control. Data are presented as mean ± SEM. n = 3; P = 0.001; (**) P < 0.01 by Student's t-test. (H) Lentivirus encoding gRNA1-resistant ASB13 was transduced into ASB13 KO SUM159 cell line to re-express ASB13. SNAI2 and YAP protein levels were determined by immunoblotting. β-ACTIN was used as loading control.

Figure 6.

YAP functions as a tumor suppressor gene in breast cancer. (A) YAP KO cell lines generated in Supplemental Figure S6A were used to perform Boyden chamber migration assay, tumorsphere assay, and soft agar colony formation assay. Representative images are displayed. Scale bars, 500 µm. (B) Quantification of the number of migrated cells from Boyden chamber migration assay in A. Data are presented as mean ± SEM. n = 3, (**) P < 0.01 by Student's t-test. (C) Quantification of tumorsphere formation assay in A. Data are presented as mean ± SEM. n = 3, (**) P < 0.01 by Student's t-test. (D) Quantification of soft agar colony formation assay in A. Data are presented as mean ± SEM. n = 3, (**) P < 0.01 by Student's t-test. (E) A total of 2 × 10⁶ SUM159 or SUM159 YAP KO cells was mixed 1:1 by volume with Matrigel (BD Biosciences) per injection. Mice were injected orthotopically in both flanks. Tumor volume was measured and calculated as volume = (length × width²)/2. Data are presented as mean ± SEM. n = 10, (**) P < 0.01 by Student's t-test. (F) Representative lung metastasis nodule images are presented from experimental end point in E. (G) Number of lung metastasis lesions from experiment in E were quantified. n = 5, (*) P < 0.05 by Student's t-test. (H) Schematic of ASB13-SNAI2-YAP regulation mechanism in breast cancer progression. SNAI2 inhibits F-actin polymerization and YAP mRNA expression to promote cancer cell migration and metastasis. SNAI2 protein is recognized and ubiquitinated by ASB13 for UPS-mediated protein degradation.