The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

Abstract

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

Keywords: Abi1; Actin cytoskeleton; Cbl; E3 ubiquitinligase; EGFR; Metastasis; Proteolysis; Src family tyrosine kinases; WRC.

Fig. 1

Abl tyrosine kinases dependent degradation of Abi Proteins in Bcr-Abl-positive leukemic cells. A. Expression of p185^Bcr-Abl in Ba/F3 cells induces down regulation of Abi2. Total lysates from 1 × 10⁶ Ba/F3 and Ba/F3 expressing p185^Bcr-Abl cells were analyzed by western blot using indicated antibodies. B. Abl tyrosine kinase inhibitor imatinib (IM) reverts Bcr-Abl-induced down-regulation of Abi and WAVE2 proteins. Ba/F3 p185^Bcr-Abl and K562 cells were treated with or without 5 μM Abl kinase inhibitor imatinib (IM) for 8 h. Total lysates of 1 × 10⁶ cells were analyzed by western blot using indicated antibodies. C. Imatinib (IM) treatment increases Abi1 protein level in p185^Bcr-Abl-positive leukemic cells. The p185^Bcr-Abl cells expressing HA-tagged Abi1 were treated with or without 5 μM Abl kinase inhibitor imatinib (IM), as indicated, at the presence of 50 μM cycloheximide (CHX) for indicated hours. Total lysates of 1 × 10⁶ cells were analyzed by western blot using indicated antibodies. D. Proteasome inhibitors revert Bcr-Abl-induced Abi2 down regulation. The p185^Bcr-Abl cells were treated with proteasome inhibitors MG132 (20 μM) and lactacystin (10 μM), lysosome inhibitors bafilomycin A1 (1 μM) and chloroquine (100> μM), and calpain inhibitor ALLN (25 μM), as indicated, for 5 >h. Total lysates from 1 × 10⁶ cells were subjected to western blot analysis.

Fig. 2

The phosphorylation of tyrosine 213 is required for Bcr-Abl-induced degradation of Abi1 and Abi2. A. Phosphorylation of Abi1 tyrosine 213 (Y213) in p185^Bcr-Abl cells. Ba/F3 cells and Ba/F3 cells expressing p185^Bcr-Abl or p185^Bcr-Abl plus green fluorescence protein (GFP)-tagged Abi1, as indicated, were treated with or without 5 μM imatinib (IM) for 8 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-Abi1 antibody and the immunoprecipitates were analyzed by western blotting using an antibody raised against a peptide flanking phosphorylated tyrosine 213 (pY213). B. A mutation of Y213 to phenylalanine (Y213F) increases Abi1 protein stability in Bcr-Abl-positive leukemic cells. The p185^Bcr-Abl cells expressing the HA-tagged wild type Abi1 (p185 HA-Abi1wt, upper panel) or Y213F mutant (p185 HA-Abi1Y213F, lower panel) were treated with 50 μM cycloheximide for indicated hours. Total lysates of 1 × 10⁶ cells were analyzed by western blot using indicated antibodies. C. A mutation of Y213 to phenylalanine (Y213F) abolishes Bcr-Abl-induced degradation of Abi2. The p185^Bcr-Abl cells stably expressing the HA-tagged wild type Abi2 (HA-Abi2 wt) or Y213F mutant (HA-Abi2Y213F) were treated with or without 5 μM imatinib (IM) for 8 h. Total lysates of 1 × 10⁶ cells were analyzed by western blot using indicated antibodies. D. The Y213F mutation reduces Abi2 ubiquitination in p185^Bcr-Abl cells. The p185^Bcr-Abl cells stably expressing the HA-tagged wild type Abi2 (HA-Abi2 wt) or Y213F mutant (HA-Abi2Y213F) were treated with or without 50 μM MG132 for 5 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-HA antibody and the immunoprecipitates were analyzed by western blotting using the indicated antibodies.

Fig. 3

Abi proteins contain a Cbl-TKB binding motif and Bcr-Abl induced downregulation of WRC requires Cbl E3 ubiquitin ligase. A. The alignment of amino acid sequences found in Abi1, Abi2, and various proteins that have been shown to bind to the Cbl-TKB domain. The consensus motif found in a cohort of protein tyrosine kinases (PTK) family is shown at the bottom. The highly conserved aspartic acid (D)/asparagine (N), tyrosine (Y), and proline (P) are in green, red, and purple, respectively, whereas the X represents any amino acid. B. Double knockout of c-Cbl and Cbl-B in p185^Bcr-Abl cells rescues Abi2 from Bcr-Abl-induced degradation. Total lysates from 1 × 10⁶ Ba/F3, p185^Bcr-Abl control (p185 ctrl), and two independent lines of p185^Bcr-Abl cells in which c-Cbl (Cbl KO1 and KO2), Cbl-B (Cbl-B KO1 and KO2), or both c-Cbl and Cbl-B (Cbl/Cbl-B KO1 and KO2) have been knocked out by CRISPR/Cas9-mediated gene editing were subjected to western blot analysis using the indicated antibodies. C. Double knockout of c-Cbl and Cbl-B in p185^Bcr-Abl cells increases protein levels of Abi1, Abi2, and WAVE2. Total lysates from 1 × 10⁶ p185^Bcr-Abl control (p185 ctrl) and two independent lines of p185^Bcr-Abl c-Cbl/Cbl-B double knockout cells (Cbl/Cbl-B KO1 and KO2) were subjected to western blot analysis using the indicated antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Abi1 depletion downregulates WAVE2 and inhibits Akt and Lyn pathways in Bcr-Abl positive leukemic cells. A. Downregulation of WAVE2 in Abi1-depleted K562 cells. Total lysates from 1 × 10⁶ control K562 (K562 ctrl) and three independent clones of Abi1-depleted K562 cells (K562 KO B4, E4, and E3) were analyzed by western blot using indicated antibodies. B. Depletion of Abi1 in p185^Bcr-Abl cells (left panel) and K562 cells (right panel) reduces Lyn activation. Total lysates from 1 × 10⁶ p185^Bcr-Abl control cells (p185 ctrl), K562 control cells (K562 ctrl), two independent clones of Abi1-depleted p185^Bcr-Abl cells (Abi1 KO2.3 and Abi1 KO6.2), and two independent clones of Abi1-depleted K562 (K562 KO B4 and KO E4) were analyzed by western blot using indicated antibodies. C. Abi1 knockout in K562 cells inhibits the activation of Akt pathway. Total lysates from 1 × 10⁶ K562 control (K562 ctrl) and three clones of Abi1-depleted K562 cells (K562 KO B4, KO E4 and KO E3) were analyzed by western blot using indicated antibodies.

Fig. 5

Abi1 is involved in the regulation of the EGF/EGFR signaling in metastatic breast cancer cells. A. Abi1 binds to EGFR in metastatic MDA-MB-231 human breast cancer cells upon EGF stimulation. A brain metastatic subline of MDA-MB-231 cells (231Br) was stimulated with or without 50 ng/ml EGF for 30 min. The total lysates were subjected to immunoprecipitation (IP) followed by western blot (WB) analysis using antibodies as indicated. B. Abi1 is tyrosine-phosphorylated and the Y213 is a major site phosphorylated by Abl tyrosine kinases in metastatic breast cancer cells. Left panel: lysates of human breast cancer cell lines MCF-7 (MCF-7), MDA-MB-231 (MB231), and a brain metastatic subline of MDA-MB-231 (231Br) cells were subjected to immunoprecipitation (IP) followed by western blot (WB) analysis using indicated antibodies. Right panel: A lung metastatic subline of MDA-MB-231 cells, LM2-4175 was transduced with retroviruses expressing GFP-tagged wild type Abi1 (WT) or Abi1 Y213F mutant (Y213F). The cells were treated with or without 5 μM imatinib (IM), as indicated, for 8 h and total cell lysates were subjected to immunoprecipitation (IP) followed by western blot (WB) analysis using indicated antibodies. C and D. CRISPR/Cas9 mediated knockout of Abi1 in LM2-4175 breast cancer cells leads to EGFR down regulation. The control LM2-4175 cells (475 ctrl) and Abi1-depleted LM2-4175 cells (4175 KO) were stimulated with 50 ng/ml EGF for 60 min (C) or indicated time (D). Total cell lysates were analyzed by western blotting with indicated antibodies.

Fig. 6

Abi1 depletion impairs LM2-4175 breast cancer cells invasion in vitro. A. A 3D tumor spheroid invasion assay of control LM2-4175 cells (4175 Control) and 2 individual clones of LM2-4175 Abi1 knockout cells (4175 Abi1 KO1 and 4175 Abi1 KO2). The data is a representative of three independent experiments. B. Comparison of invasive area of control LM2-4175 cells (4175 Ctrl) and LM2-4175 Abi1 knockout cells (4175 Abi1 KO1 and 4175 Abi1 KO2) in a triplicate experiment. The invasion area was calculated using Adobe Photoshop software. Unpaired student t test was used to compare the knockout cells with the 4175 controls. * P = 0.18; ** P < 0.05; *** P < 0.01

Fig. 7

Abi1 knockout impedes LM2-4175 breast cancer cells metastasis to lung in vivo. A. Lung weight of the mice injected intracardiacally with control LM2-4175 cells (4175 Ctrl, n = 5) and 2 individual clones of LM2-4175 Abi1 knockout cells (4175 KO1, n = 5; and 4175 KO2, n = 5; *P<0.05 compared to 4175 Ctrl). B. H&E staining of the representative lungs from the mouse injected with saline (control), LM2-4175 control cells (4175 Ctrl), and two clones of LM2-4175 Abi1 knockout cells (4175 KO1 and 4175 KO2). C. Bioluminescence imaging of mice at 2, 7, 10, 14, 17, and 20 days after intracardiac injection of LM2-4175 (LM2-4175 Ctrl) or LM2-4175 Abi1 knockout (LM2-4175 Abi1 KO) cells. Scale bar denotes radiance in photons per second per square centimeter per steradian (p/sec/cm²/sr). D. The quantitative bioluminescence imaging as the value of total flux (photons/sec) for mice injected with LM2-4175 control (red) and Abi1 knockout (4175 Abi1 KO, blue) cells. E. Survival curve of the mice injected with LM2-4175 cells (red, n = 5) and two individual clones of LM2-4175 Abi1 knockout cells, 4175 KO1 and 4175 KO2 (blue and purple, respectively, n = 5 for each group). Log-rank test P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)