BRCA1 proteins regulate growth of ovarian cancer cells by tethering Ubc9

Abstract

Mutation in the BRCA1 gene is associated with increased risk for hereditary breast and ovarian cancers. In sporadic ovarian tumors, BRCA1 dysfunction is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein. Our group has previously reported that BRCA1 proteins, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins, bind the sole SUMO E2-conjugating enzyme Ubc9. In this study, we examined the result of altered Ubc9 binding and knockdown on the sub-cellular localization and growth inhibitory function of BRCA1 proteins in ovarian cancer cells. Using live imaging of YFP, RFP-tagged BRCA1 and BRCA1a proteins, our results show enhanced cytoplasmic localization of K109R and C61G mutant BRCA1 proteins in ES-2, NIHOVCAR3 and UWB 1.289 ovarian cancer cells. Down-regulation of Ubc9 in ovarian cancer cells using Ubc9 siRNA resulted in cytoplasmic localization of BRCA1 and BRCA1a proteins. These mutant BRCA1a proteins were impaired in their capacity to inhibit growth of ES-2 ovarian cancer cells. Several ovarian cancer cells, including a BRCA1-null ovarian cancer cell line, showed higher levels of expression of Ubc9. This is the first study demonstrating the physiological link between loss of Ubc9 binding and loss of growth suppression of disease-associated mutant BRCA1a proteins in ovarian cancer cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of ovarian cancers.

Keywords: BRCA1; BRCA1a; Growth suppression; Ovarian cancer; RING domain mutants; Ubc9; nuclear import.

Figure 1

Ubc9 is required for nuclear localization of BRCA1 and BRCA1a proteins in ovarian cancer cells. The subcellular localization of BRCA1, BRCA1a and its mutants in various ovarian cancer cells by immunoflourescence and live imaging. A, ES-2 cells. B and D. NIHOVCAR3 cells and C. UWB1.289 cells. ES-2 and NIHOVCAR3 cells were seeded into 6-well plates and transfected with pRFP-BRCA1a or pRFP-BRCA1a Mut#1 or pRFP-BRCA1a Mut#4 or YFP-BRCA1 (Yellow fluorescent protein) NLSm with and without Ubc9 using Lipofectamine 2000 (Invitrogen) or X-tremeGENE 9 DNA transfection reagent (Roche). The nuclei were visualized with DNA staining dye Hoechst 24 hours after transfection. The live images of cells were taken by fluorescent microscope (20X) (Olympus). For immunoflourescence twenty four hours later, the cells were fixed in methanol and probed with BRCA1 antibody (Santa Cruz, Ab1 1/100) followed by Alexa Fluor 488 labeled secondary antibody (Invitrogen, 1/100) staining as described [31]. The nuclei were visualized by 4, 6-diamidino-2-phenylindole (DAPI) staining. The images were taken using fluorescent microscope (100X, oil)) (Olympus). Graphs show the proportion of transfected NIH: OVCAR3 cells showing predominantly nuclear (N), nuclear/cytoplasmic (NC), cytoplasmic (C) BRCA1 NLS (Nuclear Localization Signal) mutant. Bars represent the mean ± SE of at least two experiments.

Figure 2

Effect of Ubc9 knockdown on subcellular localization of endogenous and YFP and RFP tagged BRCA1 and BRCA1a proteins in ovarian cancer cells. Immunoflorescence (A) and (B) or live imaging using BRCA1 Ab1 antibody on ES-2 and NIHOVCAR3 cells transfected with control or Ubc9 siRNA and YFP-BRCA1 (C) RFP-BRCA1a (D). The nuclei were visualized with DAPI or blue fluorescent Hoechst dye staining using a fluorescent microscope (20X or 100X). Western blot analysis of Ubc9 expression in siRNA treated ES-2 and NIHOVCAR3 cells. The control and Ubc9 siRNA treated ES-2 and NIHOVCAR3 cells were lysed and probed with Ubc9 antibody. β-Actin was used as an internal control. The stoichiometry of Ubc9 protein levels is shown. The signal of Ubc9 protein band was quantified using software MultiGauge and the values were used to plot efficiency of Ubc9 knockdown in ES-2 and NIHOVCAR3 cells.

Figure 3

Altered Ubc9 binding results in loss of growth suppression by BRCA1a in ovarian cancer cells. ES-2 cells were transfected with pCDNA3 or pCDNA3-BRCA1a or pCDNA3-BRCA1a Mut#1 or pCDNA3-BRCA1a Mut#4 and selected with G418 and the colonies were stained with crystal violet and counted as described [14]. The number of colonies obtained by pCDNA3 was considered as 100%. Each experiment was repeated 3 times and the bars shown represent s.d.

Figure 4

Ubc9 is expressed at high levels in several ovarian cancer cells lines and UWB1.289 ovarian cancer cells and MCF10A normal mammary epithelial cells. Expression of Ubc9 in ES-2, NIHOVCAR3, and SKOV3 cells by western blot analysis. Protein concentrations were normalized using β-actin (A). Working hypothetical model showing how BRCA1/1a, by binding to Ubc9 inhibits the growth of ovarian cancer cells. Mutant BRCA1/1a is unable to interact with Ubc9 causing deregulated levels of UBC9 resulting in ovarian cancer (B).