Cyclin D1/cyclin-dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells

Abstract

Cyclin D1 belongs to a family of proteins that regulate progression through the G1-S phase of the cell cycle by binding to cyclin-dependent kinase (cdk)-4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon, and prostate, overexpress the cyclin D1 gene. However, the correlation of cyclin D1 overexpression with E2F target gene regulation or of cdk-dependent cyclin D1 activity with tumor development has not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A (FLNa), a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. FLNa has also been associated with a variety of cancers including lung cancer, prostate cancer, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. Using mass spectrometry, we find that cyclin D1 and FLNa coimmunoprecipitate and that lower levels of cyclin D1 are associated with decreased phosphorylation of FLNa at Ser2152 and Ser1459. We also identify many proteins related to cytoskeletal function, biomolecular synthesis, organelle biogenesis, and calcium regulation whose levels of expression change concomitant with decreased cell motility induced by decreased cyclin D1 and cyclin D1-cdk4/6 activities.

Figure 1

MDA-MB-231 cells were transfected with scrambled, CCND1(51), and/or CCND1(52) siRNA. The amount of cyclin D1 protein was lower after transfection with the cyclin D1-specific siRNA compared to scrambled siRNA. The amount of CDK4 and FLNa was not affected while the amount of pFLNa (ser-2152) (A) and pFLNa(ser-1459) (B) decreased in cells transfected with the cyclin D1-specific siRNA with greater effect with CCND1(51). Filamin A (FLNa) is a binding partner of cyclin D1. (C) Anti-cyclin D1 immunoprecipitations were performed using MDA-MB-231 protein lysate. Phosphorylated FLNa was detected by Western blot of the immunoprecipitated fraction. (D) Anti-FLNa immunoprecipitations were performed using MDA-MB-231 protein lysate. Cyclin D1 was detected by Western blot of the immunoprecipitated fraction.

Figure 2

Cyclin D1 and FLNa co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.

Figure 3

Pathway output from BiNGO analysis. Colored circles indicate GO_processes determined to be over-represented with statistical significance as depicted in the legend.