DBC2/RhoBTB2 functions as a tumor suppressor protein via Musashi-2 ubiquitination in breast cancer

Abstract

The gene encoding 'deleted in breast cancer 2' (DBC2), also referred to as RHOBTB2 (Rho-related BTB domain-containing protein 2), is classified as a tumor suppressor gene. DBC2 is a substrate-specific adaptor protein for a novel class of Cullin-3 (CUL3)-based E3 ubiquitin ligases; however, it is unclear if the substrate adaptor function of DBC2 is required for its tumor suppressor activity. Furthermore, the key substrates of DBC2-mediated ubiquitination have yet to be identified. In the present study, we established a genome-wide human cDNA library-based in vitro ubiquitination target screening assay and identified Musashi-2 (MSI2) as a novel ubiquitination target protein of DBC2. MSI2 directly interacted with DBC2, and this interaction promoted MSI2 polyubiquitination and proteasomal degradation in breast cancer cells. Overexpression and knockdown experiments demonstrated that DBC2 suppressed MSI2-associated oncogenic functions and induced apoptosis. Immunohistochemistry analysis of a breast cancer tissue microarray revealed that DBC2 and MSI2 protein levels are inversely correlated in both normal breast tissues and breast cancer tissues. Taken together, these findings provide evidence that DBC2 suppresses tumorigenesis in breast cancer by ubiquitinating MSI2.

Figure 1

The interaction between DBC2 and MSI2. (a) DBC2 directly binds MSI2 in vitro. GST, GST-MSI2 and ³⁵S-methionine-labeled DBC2 were coincubated, and the samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresi (SDS–PAGE). (b) DBC2 complexes in MDA-MB-231 cells. The cells were transfected with FLAG-DBC2 and Myc-MSI2, and the protein complexes were immunoprecipitated (IP) using a control antibody and anti-FLAG. Western blot assays were conducted using the indicated antibodies. (c) DBC2 colocalizes with MSI2 in MDA-MB-231 cells. Cells cotransfected with dsRed-DBC2 (red fluorescence) and EGFP-MSI2 (green fluorescence) were treated with the proteasome inhibitor MG132 and analyzed using confocal microscopy. The nuclei were labeled with DAPI (4', 6-diamidino-2-phenylindole). The histogram presents the fluorescence intensity along the white arrow in the merged panel. (d) The interaction between endogenous DBC2 and MSI2. Protein complexes were immunoprecipitated from MDA-MB-231 cell lysates using control antibodies, anti-DBC2 and anti-MSI2. The protein interaction was confirmed using immunoblotting assays with the indicated antibodies. (e) Constructs of DBC2 functional domains and DBC2 mutant proteins. The Y284D DBC2 mutant protein (red line) is defective for CUL3 binding. The numbers indicate the amino-acid positions. (f) MDA-MB-231 cells were co-transfected with the indicated plasmids, and the interactions between DBC2 mutants and MSI2 were evaluated using immunoprecipitation and western blot assays with the indicated antibodies. The DBC2 C terminus and DBC2 Y284D mutant interact with MSI2. IVT, in vitro transcribed; WCL, whole-cell lysates.

Figure 2

DBC2 induces MSI2 ubiquitination and degradation in breast cancer cells. (a) DBC2 induces MSI2 ubiquitination. MDA-MB-231 cells were co-transfected with the indicated plasmids and treated with the proteasome inhibitor MG132. MSI2 pull-down experiments were conducted using Ni-NTA beads, and the samples were analyzed using western blot assays with the indicated antibodies. Ub, ubiquitin. (b) DBC2 inhibits MSI2 protein expression. MDA-MB-231 cells were transfected with Myc-tagged DBC2 and DBC2-MT (Y284D mutant). MSI2 protein levels in cell lysates were detected using western blot assays with the indicated antibody. (c) DBC2-mediated downregulation of MSI2 is enhanced by ROC1 and CUL3 overexpression. MDA-MB-231 cells were co-transfected with the indicated plasmids, and the cell lysates were analyzed using western blot assays. (d) DBC2-mediated downregulation of MSI2 is inhibited by MG132. Cells transfected with plasmids expressing DBC2 and DBC2-MT were treated with 10 μM MG132 for 4 h and analyzed using western blot assays. (e) DBC2-mediated ubiquitination of endogenous MSI2 is dependent on CUL3-binding. MDA-MB-231 cells co-transfected with plasmids expressing DBC2, DBC2-MT and ubiquitin were treated with MG132. Protein complexes were immunoprecipitated from cell lysates using anti-MSI2. MSI2 ubiquitination was evaluated using western blot assays with an antibody against ubiquitin. (f) DBC2 inhibits MSI2 protein stability. MDA-MB-231 cells transfected with control and Myc-DBC2-expressing plasmids were treated with 40 μg/ml cycloheximide (CHX) for 3, 6, 12 or 24 h. DBC2 and MSI2 protein levels were evaluated using western blot assays. The graph shows the relative normalized intensities of the MSI2 bands. The data are presented as the mean±s.d. of triplicate samples from at least two independent experiments. Student's t-test was used to determine statistical significance (*P<0.05).

Figure 3

Endogenous DBC2 regulates MSI2 ubiquitination and stability in breast cancer cells. (a and b) MSI2 protein levels increased in DBC2-knockdown cells in the absence of changes in MSI2 mRNA levels. MCF7 cells were stably infected with lentiviral vectors expressing two different shRNA constructs targeting DBC2 (nos. 1 and 2). DBC2 and MSI2 protein (a) and mRNA levels (b) were analyzed using western blot and quantitative reverse transcriptase–PCR (qRT–PCR), respectively. (c) DBC2 knockdown inhibits MSI2 ubiquitination. Control (shScramble) and shDBC2-knockdown cells transfected with a plasmid expressing HA-ubiquitin were treated with MG132. Endogenous MSI2 ubiquitination was evaluated using immunoprecipitation and western blot. The asterisk indicates a nonspecific band. (d) DBC2 knockdown enhances MSI2 protein stability. Control (shScramble) and shDBC2-knockdown cells were treated with cycloheximide (CHX) for the indicated period of time, and DBC2 and MSI2 protein levels were evaluated using western blot. The normalized intensity of each MSI2 band is expressed as the mean±s.d. of triplicate samples from at least two independent experiments. Student's t-test was used to determine statistical significance (*P<0.05).

Figure 4

DBC2 regulates the effect of MSI2 on breast cancer cell survival. (a) DBC2 inhibits MSI2 function. Cells were co-transfected with plasmids expressing DBC2, DBC2-MT and MSI2. DBC2, MSI2 and p21 protein levels were analyzed using western blot. (b and c) DBC2 regulates MSI2-mediated cell growth and proliferation. MDA-MB-231 cells were co-transfected with plasmids expressing DBC2, DBC2-MT (Y284D mutant) and MSI2. Cell viability and proliferation were evaluated using the WST-1 assay (b) and BrdU (5-bromo-2'-deoxyuridine) incorporation assay (c), respectively. The results are represented as the mean±s.d. of triplicate samples from at least two independent experiments. Student's t-test was used to determine statistical significance (*P<0.05). (d and e) DBC2 overexpression and shRNA-mediated MSI2 knockdown induced cell cycle arrest and apoptosis. Cell cycle progression in control, shMSI2 knockdown and DBC2-overexpressing cells stained with propidium iodide (PI) was evaluated using flow cytometry. Apoptosis was analyzed using Annexin V and PI labeling and flow cytometry. (f) DBC2 inhibits apoptosis in MSI2-depleted cells. MSI2-knockdown cells and DBC2-overexpressing cells were analyzed using western blot with the indicated antibodies.

Figure 5

DBC2 inhibits MSI2-dependent clonogenic growth and cell migration. (a) MSI2-induced clonogenic growth is inhibited by DBC2 in a CUL3 binding-dependent manner. MDA-MB-231 cells were transfected with plasmids expressing MSI2, DBC2 and DBC2-MT. Cells were treated with G418 (500 μg/ml) for 2 weeks, stained with crystal violet and visualized. The number of colonies is expressed as the mean±s.d. of triplicate experiments. (b) DBC2 inhibits cell migration via MSI2 degradation. MDA-MB-231 cells transfected with plasmids expressing DBC2, DBC2-MT and MSI2 were grown to 90% confluence. The cells were then wounded with a scratch, and wound closure was visualized and imaged. Wound closure (%) is expressed as the mean±s.d. of triplicate experiments. (c) DBC2 knockdown enhances the clonogenic growth of breast cancer cells in an MSI2 dose-dependent manner. MDA-MB-231 cells were transduced with plasmids expressing shScramble (control), shDBC2 and shMSI2, and clonogenic growth was subsequently evaluated. (d) DBC2 knockdown-associated increase in cell migration is MSI2-dependent. The cells were transduced with plasmids expressing shScramble (control), shDBC2 and shMSI2, and wound closure was visualized and imaged. Wound closure (%) is expressed as the mean±s.d. of triplicate experiments. *P<0.05, Student's t-test. NS, nonsignificant.

Figure 6

Inverse correlation between DBC2 and MSI2 protein levels in breast cancer cell lines, normal breast tissues and breast cancer tissues. (a) DBC2 and MSI2 protein levels in breast cancer cell lines were evaluated using western blot assays. (b) DBC2 and MSI2 protein levels in normal breast tissue and breast cancer tissue. Immunohistochemistry (IHC) analysis of a breast cancer tissue microarray was conducted using antibodies against and DBC2 and MSI2. H&E, hematoxylin and eosin staining. (c) Inverse correlation between the staining intensity of DBC2 and MSI2 in normal breast tissue and breast cancer tissue. The staining intensity was assigned a score from 0 to 3 (0=no staining, 1=weak staining, 2=moderate staining, 3=strong staining). The data are represented as the mean immunoreactive score±s.d. **P<0.005 compared with normal breast tissue, using two-tailed Student's t-test. (d) The IHC staining intensity of DBC2 and MSI2 according to cancer stage; 0 and 1 were defined as low score, and 2 and 3 were defined as high scores. *P<0.05 compared with high staining intensity, usingtwo-tailed Student's t-test. (e) DBC2 protein levels are inversely correlated with MSI2 protein levels in breast cancer tissues, IHC score 0, (+) IHC score 1, (++) IHC scores 2 and 3. Spearman's ρ correlation coefficient between MSI2 and DBC2 (P=0.018, r=−0.263).