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. Cancer Res; 70(5); 2105–14

The canonical function of cyclin D1 is to promote progression through the G1-S phase of the cell cycle by binding to cyclin-dependent kinase 4 (cdk4) to phosphorylate and inactivate the retinoblastoma protein and release E2F transcription factors. Several human cancers, including breast, colon, and prostate, as well as hematopoietic malignancies, overexpress the cyclin D1 gene (13). However, there is no correlation between cyclin D1 overexpression and regulation of E2F target genes by microarray analysis nor between cdk-dependent cyclin D1 activity and tumor development, suggesting that the role of cyclin D1 in oncogenesis is at least partially independent of its function as a cell cycle regulator (4, 5). Cyclin D1 has recently been associated with cell adhesion and motility in primary bone macrophages (6). Studies in cyclin D1−/− mouse embryo fibroblasts revealed that cyclin D1 inhibits Rho-activated kinase II and thrombospondin 1 to promote cell migration (7). The cdk inhibitor p16INK4a has also been shown to inhibit the migration of erythroleukemia and endothelial cells (8, 9). Indeed, p16INK4a colocalized in the ruffles and lamellipodia of migrating endothelial cells together with cyclin D1, cdk4/6, and the αvβ3-integrin machinery.

Filamin A (FLNa), a member of the nonmuscle actin-binding protein family, is a widely expressed molecular scaffold protein that regulates signaling events involved in cell motility and invasion by interacting with integrins, transmembrane receptor complexes, adaptor molecules, and second messengers (10, 11). FLNa has recently been shown to bind cyclin B1/cdk1 in a yeast two-hybrid system using recombinant glutathione S-transferase (GST)-cyclin B1 protein as bait and a 10.5-day-old embryonic mouse library as prey (12). Using truncated recombinant FLNa and cyclin B1 protein fragments, the regions of interaction between FLNa and cyclin B1 were shown to be located within amino acids 1–40 of cyclin B1 and the FLNa NH2-terminal region in repeat 9. In addition to cyclin B1, filamins have been reported to bind with more than 30 proteins, and because many filamin-interacting proteins are membrane receptors for cell signaling molecules, filamins may be involved in coordinating a variety of signal transduction pathways (13). For example, FLNa has been shown to be a substrate for calcium-calmodulin–dependent kinase II, interacting with filamentous actin to promote migration of human neck squamous cell carcinoma cells (14). FLNa has also been shown to interact with prostate-specific antigen and regulate androgen receptor (15, 16). In addition, FLNa has been shown to be a key element in transforming growth factor-β signaling through its association with SMADs (17) and in A549 lung carcinoma cells undergoing epithelial-mesenchymal transition (18). FLNa has also been associated with a variety of cancers including prostate cancer, melanoma, human bladder cancer, and neuroblastoma (10, 19, 20).

We hypothesized that elevated cyclin D1 facilitates motility in the highly invasive and metastatic MDA-MB-231 breast cancer cell line. Although there are many proteins that have been shown to affect migration and invasion in this cell line, our focus on these molecules is due to the fact that many of the known proteins affect mitogenic signals that affect the levels of cyclin D1, and there are several known kinases affecting FLNa and the increasing evidence that FLNa plays a key role in many processes such as epithelial-mesenchymal transition. In our studies, we found that the cell motility of MDA-MB-231 cells can be affected by altering cyclin D1 levels or cyclin D1-cdk4/6 kinase activity. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we found that cyclin D1 coimmunoprecipitates with the actin cytoskeleton protein FLNa and that the phosphorylation state of FLNa was concomitantly affected when either cyclin D1 levels or cyclin D1-cdk4/6 kinase activity was altered. We also found that lower levels of cyclin D1 are associated with decreased phosphorylation of FLNa at Ser2152 and Ser1459. We also analyzed the effects of decreasing cyclin D1 and cyclin D1-cdk4/6 activity on the global phosphoproteome. Our analyses revealed changes in protein expression in many proteins related to cytoskeletal function, biomolecular synthesis, organelle biogenesis, and calcium regulation concomitant with decreased cell motility induced by decreased cyclin D1 and cyclin D1-cdk4/6 activity.

Cell culture

Human breast carcinoma MDA-MB-231 cells (American Type Culture Collection) were grown in DMEM containing penicillin and streptomycin (each 100 mg/L) and supplemented with 10% fetal bovine serum (FBS) at 37°C in 5.0% CO2. The Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC) Flex DMEM (Invitrogen) was prepared as per manufacturer's recommendation using heavy amino acids 13C615N4 Arg and 13C6 Lys, and the cells were used in SILAC-based mass spectrometry experiments (21).

Cell invasion/migration assay

The cell invasion assay was conducted using BD Biocoat Matrigel 24-well invasion chambers with filters coated with extracellular matrix on the upper surface (BD Biosciences). The experiments were done according to the manufacturer's protocol. Experiments were done in triplicate (mean ± SE). Cells (2.5 × 104) were added to the upper chamber and allowed to invade for 24 h. The experiments were done according to the manufacturer's protocol.

Wound healing assay

MDA-MB-231 cells were grown to 70% confluence in six-well plates. Linear scratches were made with a micropipette tip across the diameter of the well, and dislodged cells were rinsed with PBS. Cell culture medium was replaced with fresh DMEM containing 10% FBS. The cells were allowed to grow and the width of the wound was monitored at the specified times for the degree of wound healing.

Fluorescent immunocytochemistry

Cells were prepared as in the wound healing assay and probed with mouse anti–cyclin D1 (DCS-6, 1:50; Cell Signaling Technology) and anti-FLNa (1:100; Santa Cruz Biotechnology) primary antibodies in 1% bovine serum albumin (BSA) at 4°C overnight. Then cells were incubated with goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 647 (1:2,000; Invitrogen Corporation) in 1% BSA for 1 h. Cells were stained with 100 ng/mL 4′,6′-diamidino-2-phenylindole hydrochloride in PBS for 2 min to visualize the nuclei. Images were captured digitally using a Zeiss LSM 510 META confocal microscope.

Lysis buffers, immunoprecipitation, and Western blot

Whole-cell lysates for Western blots were prepared in a modified radioimmunoprecipitation assay buffer [25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L sodium chloride, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS] supplemented with 1 mmol/L sodium orthovanadate and protease inhibitor cocktail (Complete EDTA-free protease inhibitor cocktail from Roche Applied Science). For the immunoprecipitation studies, whole-cell lysates were prepared in COPRE lysis buffer [20 mmol/L HEPES (pH 7.9), 50 mmol/L sodium chloride, 0.1% NP40, 10% glycerol, 1 mmol/L DTT]. COPRE lysates (500 μg) were incubated with 1 μg of antibodies for 1 h at 4°C before adding 40 μL of protein G magnetic beads (Invitrogen) for an overnight incubation at 4°C. The following antibodies were used for Western blotting: anti–cyclin D1 (Ab-3) polyclonal antibody (Lab Vision/Neomarker); anti–cyclin D1 (DCS-6) monoclonal antibody, anti-cdk4 (C-22) and anti-actin (C-11) polyclonal antibodies, anti-FLNa and anti–phospho-FLNa (Ser2152) polyclonal antibodies (Cell Signaling Technology); and ImmunoPure horseradish peroxidase–conjugated goat anti-rabbit antibodies (Pierce Biotechnology). Primary antibodies were used at 1:1,000 dilution and secondary horseradish peroxidase–conjugated antibodies were used at 0.05 μg/mL.

Perturbation of the cyclin D1-cdk complex by p16INK4 peptides

Peptides corresponding to amino acids 84–103 of human p16INK4a protein with a COOH-terminal sequence of 16 amino acids encoding the Antennapedia homeodomain (Penetratin) were synthesized. Peptide 20 (DAAREGFLATLVVLHRAGARRQIKIWFQNRRMKWKK) with the substitution of Asp92 with alanine has a lower IC50 to inhibit cyclin D1-cdk4/6 phosphorylation of a GST-pRb protein in vitro and to arrest cell cycle progression in G1 than the corresponding peptide containing the wild-type sequence, and peptide 21 (DAAREGFLDTLAALHRAGARRQIKIWFQNRRMKWKK) carrying the substitution of Val95 with alanine and Val96 with alanine has an increased IC50in vitro and has lost ∼60% of the cell cycle inhibitory capacity (22, 23). These peptides were added to the cell culture medium at a concentration of 20 μmol/L.

Perturbation of the cyclin D1-cdk complex by cyclin D1 siRNA

Cyclin D1 expression was inhibited using validated Stealth siRNAs (Invitrogen). Two different inhibiting RNAs were used in our studies and were found to have different efficiencies of cyclin D1 knockdown. They were CCND1(51) (AUGGUUUCCACUUCGCAGCACAGGA) and CCND1(52) (UUAGAGGCCACGAACAUGCAAGUGG). The Invitrogen predesigned negative control siRNAs were also used. Transfections were done with Lipofectamine 2000 as per manufacturer's recommendation, 200 pmol of siRNA and 5 μL of Lipofectamine 2000 for each well of a six-well plate (Invitrogen).

Proteomics

The identification of phosphoproteins was accomplished using two complementary methods. For both methods, cells were first grown in SILAC medium as described. In the first method, after treatment with the inhibitory peptides, phosphoproteins were isolated using the Qiagen PhosphoProtein Purification kit as per manufacturer's instructions. In the second method, phosphopeptides were isolated using PhosSelect (Sigma-Aldrich) as per manufacturer's instructions. Proteins separated on gels were identified using LC-MALDI on a 4800 Proteomics Analyzer (Applied Biosystems, Inc.) and the phosphopeptides were analyzed using electrospray ionization-mass spectrometry (ESI-MS) on a Proteome X workstation (Thermo-Fisher). Peptide identification was done on an in-house Mascot Server for the LC-MALDI spectra and on Sequest for the ESI-MS spectra. Additional information can be found in Supplementary Data.

Bioinformatics analysis

Function information of identified phosphoproteins was obtained from the Swiss-Prot database. To determine if any types of proteins are overrepresented, enrichment analysis of their gene ontology (GO) terms was done. To find statistically overrepresented GO categories among phosphoproteins identified in this study, we used the BiNGO plugin for Cytoscape (24). The required data set files were created as described in the BiNGO User Guide. The enrichment analysis was done using the “HyperGeometric test” with correction for multiple hypothesis testing using the following parameters: GO_Biological_Process ontology, annotation for H. sapiens. GO terms that were significant with P value of <0.05 were determined to be overrepresented.

Decreased Cyclin D1 and Cyclin D1-cdk4/6 Kinase Activity Reduces the Invasion and Migration Potential of MDA-MB-231 Breast Cancer Cells

Previous studies have shown that cyclin D1−/− mouse embryo fibroblasts display increased cellular adherence, defective motility, and impaired wound response compared with those with restored cyclin D1 levels (7). To determine if cyclin D1 also helps control motility in the highly migratory MDA-MB-231 breast cancer cells, cyclin D1 mRNA and protein expression was inhibited using two available siRNAs, CCND1(51) and CCND1(52). A control siRNA with random sequence was used as negative control. Western blot data show that both cyclin D1–specific siRNAs inhibit cyclin D1 protein expression, albeit at different levels. CCND1(52) inhibits cyclin D1 by ∼50% and CCND1(51) inhibits cyclin D1 by >90% compared with cells transfected with control siRNA (Fig. 1A).

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 cyclin D1–specific siRNA compared with scrambled siRNA. The amount of cdk4 and FLNa was not affected, whereas the amounts of pFLNa(Ser2152) (A) and pFLNa(Ser1459) (B) decreased in cells transfected with cyclin D1–specific siRNA, with greater effect with CCND1(51). FLNa is a binding partner of cyclin D1. C, anti–cyclin D1 immunoprecipitations were done using MDA-MB-231 protein lysate. Phosphorylated FLNa was detected by Western blot of the immunoprecipitated fraction. D, anti-FLNa immunoprecipitations were done using MDA-MB-231 protein lysate. Cyclin D1 was detected by Western blot of the immunoprecipitated fraction.

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 cyclin D1–specific siRNA compared with scrambled siRNA. The amount of cdk4 and FLNa was not affected, whereas the amounts of pFLNa(Ser2152) (A) and pFLNa(Ser1459) (B) decreased in cells transfected with cyclin D1–specific siRNA, with greater effect with CCND1(51). FLNa is a binding partner of cyclin D1. C, anti–cyclin D1 immunoprecipitations were done using MDA-MB-231 protein lysate. Phosphorylated FLNa was detected by Western blot of the immunoprecipitated fraction. D, anti-FLNa immunoprecipitations were done using MDA-MB-231 protein lysate. Cyclin D1 was detected by Western blot of the immunoprecipitated fraction.

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Using these siRNAs, the role of cyclin D1 in MDA-MB-231 cell migration was assessed with a wound healing assay. In Table 1, we report the average width of the wound for the cells treated with the two cyclin D1–specific siRNAs and that for the cells treated with control siRNA relative to the initial wound width 12 hours after scratching. Cells treated with the cyclin D1–specific siRNAs had significantly wider wounds compared with cells treated with control siRNA (P < 0.001).

Table 1.

Summary of wound healing and invasion assays using MDA-MB-231 cells transfected with cyclin D1–specific siRNA or p16INK4a peptides

TreatmentWidth of wounds after 12 h (% of initial ± SD)No. of invading cells after 24 h (avg ± SD)
Scr siRNA (M) 0.12 ± 0.08 38.4 ± 11.6 
CCND1(51) 0.72 ± 0.22* 0.15 ± 0.48* 
CCDN1(52) 0.42 ± 0.16* 0.2 ± 0.52* 
 
Treatment Width of wounds after 24 h (% of initial ± SD) 
wt 0.04 ± 0.02 
p20 0.53 ± 0.07* 
p21 0.38 ± 0.04* 
TreatmentWidth of wounds after 12 h (% of initial ± SD)No. of invading cells after 24 h (avg ± SD)
Scr siRNA (M) 0.12 ± 0.08 38.4 ± 11.6 
CCND1(51) 0.72 ± 0.22* 0.15 ± 0.48* 
CCDN1(52) 0.42 ± 0.16* 0.2 ± 0.52* 
 
Treatment Width of wounds after 24 h (% of initial ± SD) 
wt 0.04 ± 0.02 
p20 0.53 ± 0.07* 
p21 0.38 ± 0.04* 

*P < 0.05.

We next examined the role of cyclin D1 in the ability of MDA-MB-231 cells to invade in Matrigel-coated modified Boyden invasion chambers. Transfection with either CCND1(51) or CCND1(52) siRNA almost completely abolished the ability of MDA-MB-231 cells to cross the membrane. Very few cells transfected with either cyclin D1–specific siRNA crossed the membrane (<1 cell per field of view) when assayed 12 hours after transfection (Table 1). In contrast, in cells transfected with control scrambled siRNA, an average of 38.4 cells were seen per field of view.

To determine if cell migration is also dependent on cdk4/6 activity, we inhibited kinase activity by introducing two peptides derived from p16INK4a into the culture media of MDA-MB-231 cells (22, 23). The p16 INK4 family of proteins inhibit cdk4 and cdk6 kinase activities through direct interaction with the kinase subunit only. The p16INK4a p21 peptide contains two alanine substitutions at Val95 and Val96 of the p16INK4a protein, and the p16INK4a p20 peptide contains a substitution of Asp92 to alanine. These peptides have been shown to be taken up from the tissue culture medium (22). In these studies, cyclin D1-cdk4/6 kinase activity was first blocked by incubating cells with 20 μmol/L of p20 or p21 p16INK4a peptide for 24 hours. The monolayer was then scratched to create a wound, and the monolayer washed and incubated with peptides for an additional 24 hours. The wound was completely healed in the untreated cells after 24 hours, whereas in the cells treated with either p20 or p21 peptide, there was incomplete wound healing and the difference for p20- and p21-treated cells and wild-type was statistically significant, with P < 4.0 × 10-5 (Table 1). The p20 peptide has been shown to be a stronger kinase inhibitor than p21 (22). Consistent with this, p20 was more effective than p21 at inhibiting the wound healing activity of these cells.

FLNa Binds to Cyclin D1 In vitro

Although the mechanism by which cyclin D1 influences cellular migration is not well understood, several studies using cells from cyclin D1–deficient mice have been reported (6, 7, 22). To identify proteins that interact directly with cyclin D1, immunoprecipitation experiments were conducted using MCF-7 cells transfected with FLAG-tagged cyclin D1. The immunoprecipitated proteins were first separated on an SDS-PAGE gel and stained with Coomassie R250. Bands were excised and digested with trypsin, and the proteins identified by MALDI-MS/MS. A novel binding partner, the actin cytoskeleton protein FLNa, was identified in a band that migrated above the 220-kDa molecular weight marker, consistent with the molecular weight of 280 kDa of FLNa. FLNa has been shown to be critical for cellular motility, as it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins and various transmembrane proteins.

Because of the role of FLNa in cell motility and also because FLNa is a known phosphoprotein, we hypothesized that our observation of the cyclin D1/cdk4 effect on cell migration is mediated through phosphorylation of FLNa. Whereas there are many potential sites of phosphorylation on FLNa, only two (Ser2152 and Ser2523) of the 28 have been associated with cytoskeletal reorganization.1

Phosphorylation at Ser2152 has been shown to be required for Pak1-mediated membrane ruffling and for regulation of cellular migration by ribosomal S6 kinase, a key kinase in the Ras-mitogen-activated protein kinase pathway (25, 26). We therefore checked to see if FLNa phosphorylated at Ser2152 immunoprecipitates with endogenous cyclin D1 in MDA-MB-231 cells. We found that FLNa phosphorylated at Ser2152 (pFLNa) coprecipitated with cyclin D1 using an antibody against phosphorylated FLNa in a Western blot of the cyclin D1 precipitate (Fig. 1C), establishing the interaction of phosphorylated FLNa and endogenous cyclin D1 in MDA-MB-231 cells. As further proof, we immunoprecipitated endogenous FLNa and then used an antibody against cyclin D1 in a Western blot of the FLNa precipitate and found that cyclin D1 coprecipitated with FLNa, verifying the interaction between FLNa and cyclin D1 (Fig. 1D). These results show that cyclin D1 and pFLNa (Ser2152) are binding partners in MDA-MB-231 protein lysates.

Cyclin D1 and FLNa colocalize in MDA-MB-231 cell ruffles

Although cyclin D1 is primarily located in the nucleus, there have been some reports that there are significant levels in the cytoplasm, particularly near the cell membrane. To further investigate if cyclin D1 and FLNa are interacting at the cell membrane and that this colocalization is likely a functional interaction related to migration, we performed immunofluorescence double labeling of cyclin D1 and FLNa. In Fig. 2, we show using confocal microscopy that the signal of cyclin D1 was mainly shown in the nucleus, with lower but significant signal in the cytoplasm and cell membrane. FLNa was observed to be localized primarily in the cytoplasm and cell membrane. We observed that cyclin D1 and FLNa proteins colocalize strongly in cell ruffles in those cells that migrated into the gaps created by wounding, indicating that the colocalization of cyclin D1 with FLNa is in migrating cells, but not in nonmigrating cells.

Figure 2.

Cyclin D1 and FLNa colocalize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (green), FLNa (red), and DNA (blue). Colocalization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. Bar, 10 μm.

Figure 2.

Cyclin D1 and FLNa colocalize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (green), FLNa (red), and DNA (blue). Colocalization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. Bar, 10 μm.

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Mechanism of Cyclin D1–Dependent Migration and Invasion

We have shown that decreased cyclin D1 protein expression and cyclin D1-cdk4/6 activity decrease the invasion and migration potential of MDA-MB-231 cells. We have also shown that cyclin D1 and FLNa precipitate together in an immunoprecipitation assay. Because cyclin D1 is known as a regulatory subunit of a dimeric holoenzyme that includes cdk4, we next measured the effects of cyclin D1 knockdown on the levels of protein expression of cdk4, FLNa, and pFLNa. MDA-MB-231 cells were transfected with either control siRNA or cyclin D1–specific siRNA [CCND1(51) and CCND1(52)] for 48 hours and protein expression was then measured by Western blot (Fig. 1A). As expected, cyclin D1 protein expression was lower in cells transfected with cyclin D1–specific siRNA (when compared with the actin loading control, it is reduced by 80% and 40%, respectively), but neither FLNa nor cdk4 protein expression was affected. However, the level of phosphorylated FLNa was lower in cells transfected with both cyclin D1–specific siRNAs. These data support the hypothesis that FLNa phosphorylation is cyclin D1 dependent.

Identification of Other Cyclin D1/cdk4–Dependent Phosphoproteins

To identify other proteins whose phosphorylation status is dependent on cyclin D1 and cdk4/6, we used the SILAC in vivo labeling strategy combined with phosphoprotein enrichment to identify proteins that are differentially expressed 48 hours after introduction of the p16INK4a p20 peptide to inhibit cyclin D1-cdk4/6. We identified approximately 150 phosphoproteins. By comparing the peak intensities of the isotopically labeled peptides, we were able to determine that 44 were upregulated and 19 were downregulated, responding with at least a 1.5-fold change in expression level in response to cyclin D1-cdk4/6 inhibition (Table 2). The majority of these phosphoproteins have been previously associated with some type of cancer either in vitro or in vivo. We used Biological Network Gene Ontology tool (BiNGO), a Java-based tool, to determine which Gene Ontology (GO) categories are statistically overrepresented in the set of identified phosphoproteins (24). Although it is currently not possible to analyze the entire phosphoproteome in a single experiment and the enrichment process may result in an unbalanced enrichment of certain types of proteins, we assume that particular functional groups containing a larger-than-expected number of affected proteins are isolated based on the underlying physiologic process and not due to the enrichment process. Figure 3 shows the graphical representation of the results. The colored nodes are those determined to be overrepresented with statistical significance. We found that by looking at the closest branch points of the overrepresented GO_biological processes, four major categories are represented: (a) cellular organization and biogenesis/localization, which contains cytoskeleton-related processes; (b) metabolic processes; (c) cell motility; and (d) metabolic processes. Table 3 lists the Gene ID residing under each of these categories. Many of these proteins are classified under multiple processes. Specifically, many of the proteins classified under cellular organization and biogenesis/localization are involved with cell motility as well as cytoskeletal-related processes such as actin- and microtubule-based movement and actin binding, suggesting that increased protein synthesis and processes related to organelle and cytoskeletal components are important in the decreased cell migration phenotype resulting from cyclin D1-cdk4/6 repression. Many of the proteins categorized under metabolism are involved in protein synthesis.

Table 2.

Phosphoproteins whose level of expression was altered by at least 1.5-fold, listed by inhibition of cyclin D1-cdk4/6 activity using p16INK4a peptides in MDA-MB-231 cells

Gene symbolNameGenBank accession no.Fold change (p16INK4 peptide vs no peptide)Association with cancer
c11orf30 EMSY (chromosome 11 ORF 30) Q7Z589 2.3 
CALU Calumenin precursor (crocalbin) O43852 2.9 
EEF1B2 Eukaryotic translation elongation factor 1β2 P24534 2.4 
EIF5 Eukaryotic translation initiation factor 5 P55010 2.8 
ENO1 Enolase 1α P06733 2.4 
ERBB2IP Erbb2 interacting protein Q96RT1 2.6 
FAM120A Family with sequence similarity 120A Q9NZB2 2.4  
GATA4 GATA binding protein 4 P43694 2.8 
HSPB1 Heat shock 27-kDa protein 1 P04792 2.2 
ITIH2 Inter-α (globulin) inhibitor H2 P19823 2.5 
ITSN2 Intersectin2 (ITSN2, SH3P18) Q9NZM3 2.3  
KRT1 Keratin 1 (epidermolytic hyperkeratosis, CK1) P04264 2.1 
KRT10 Keratin 10 (epidermolytic hyperkeratosis) P13645 2.2 
LAMB2 Laminin β2 (laminin S) P55268 7.9 
LASP1 LIM and SH3 protein 1 Q14847 2.9 
LRIT1 Leucine-rich repeat–containing protein 21 precusor (LRC21) Q9P2V4 2.2  
MARCKS Myristoylated alanine-rich protein kinase C substrate P29966 2.2 
MLL Myeloid/lymphoid or mixed lineage leukemia Q03164 2.7 
MYO7A Myosin VIIA Q13402 2.1  
NOL1 Nucleolar protein 1, 120 kDa P46087 2.5 
PCYT1A Phosphate cytidylyltransferase 1, choline, α P49585 2.3  
PCYT1B Phosphate cytidylyltransferase 1, choline, β Q9Y5K3 2.1  
PDLIM4 PDZ and LIM domain 4 P50479 
PKP4 Plakophilin-4 Q99569 2.5 
PPFIA2 Liprin-α2 (LIPA2, PTPRF-interacting protein α2) Q75334 2.4 
PRSS1 Protease, serine, 1 (trypsin 1) Q07477 3.7 
RCN1 Reticulocalbin-1 precusor Q15293 2.5 
RHPN2 Rhophilin-like protein Q8IUC4 10.1  
RPL10 60S ribosomal protein L10 P27635 2.4 
RPL13 Ribosomal protein L13 P26373 3.6 
RPL27A Ribosomal protein L27a P46776 2.2 
RPS6 40S ribosomal protein S6 P62753 
SEC22A Vesicle-trafficking protein SEC22a Q96IW7  
SEC61B Protein transport protein Sec61 β-subunit P60468 2.1  
TALDO1 Transaldolase 1 P37837 6.3 
TPI1 Triosephosphate isomerase 1 P60174 1.7  
TUBA1B Tubulin, α-ubiquitous chain P68363 2.1  
TUBA1C Tubulin α6 Q9BQE3 3.1  
TUBB1 Tubulin β1 P07436 2.3 
TUBB5 Tubulin β5 Q7JJU6 2.4 
TUFM Tu translation elongation factor, mitochondrial (EFTU) P49411 2.4  
VPS13D Vacuolar protein sorting-associated protein 13D Q5THJ4 3.3  
YLPM1 YLP motif–containing protein 1 (YLPM1, ZAP113) P49750 2.6  
ZNF148 Zinc finger protein 148 Q9UQR1 3.2 
AFAP1 Actin filament–associated protein Q8N556 −3.3  
AHI1 Abelson helper integration site 1 protein homolog (AHI-1, Jouberin) Q8N157 −2.2 
AHNAK AHNAK nucleoprotein isoform 1 Q09666 −2.5  
ARHGEF2 Rho/rac guanine nucleotide exchange factor 2 Q9H023 −2.7 
BCLAF1 BCL2-associated transcription factor 1 Q9NYF8 untreated only  
C11orf58 Small acidic protein O00193 −7.1  
CLIC6 Chloride intracellular channel 6 Q96NY7 untreated only  
CST4 Cystatin-S precusor P01036 −2.1  
DYNC1LI1 Dynein light chain-A Q9Y6G9 −2.2  
EHA2 Ephrin receptor EphA2 P29317 untreated only 
ERRFI1 Mitogen-inducible gene 6 protein Q9UJM3 −2.6 
FAM129B Hypothetical protein LOC64855 Q5VVW7 −5.3  
FAM82C Family with sequence similarity 82, member C Q96TC7 untreated only  
FLNA Filamin 1 (actin binding protein-280) P21333 −1.5 
G3BP1 Ras-GTPase–activating protein SH3-domain–binding protein Q13283 −3.7 
HN1 Hematologic and neurologic expressed 1 isoform 1 Q9UK76 −6.7 
HRNR Hornerin Q86YZ3 −2.9 
MACF1 Microtubule-actin cross-linking factor 1 Q96PK2 −2.1 
NCOA3 Nuclear receptor coactivator 3 Q9Y6Q9 −2.1 
NOP5/NOP58 Nucleolar protein NOP5/NOP58 Q9Y2X3 −2.8 
Gene symbolNameGenBank accession no.Fold change (p16INK4 peptide vs no peptide)Association with cancer
c11orf30 EMSY (chromosome 11 ORF 30) Q7Z589 2.3 
CALU Calumenin precursor (crocalbin) O43852 2.9 
EEF1B2 Eukaryotic translation elongation factor 1β2 P24534 2.4 
EIF5 Eukaryotic translation initiation factor 5 P55010 2.8 
ENO1 Enolase 1α P06733 2.4 
ERBB2IP Erbb2 interacting protein Q96RT1 2.6 
FAM120A Family with sequence similarity 120A Q9NZB2 2.4  
GATA4 GATA binding protein 4 P43694 2.8 
HSPB1 Heat shock 27-kDa protein 1 P04792 2.2 
ITIH2 Inter-α (globulin) inhibitor H2 P19823 2.5 
ITSN2 Intersectin2 (ITSN2, SH3P18) Q9NZM3 2.3  
KRT1 Keratin 1 (epidermolytic hyperkeratosis, CK1) P04264 2.1 
KRT10 Keratin 10 (epidermolytic hyperkeratosis) P13645 2.2 
LAMB2 Laminin β2 (laminin S) P55268 7.9 
LASP1 LIM and SH3 protein 1 Q14847 2.9 
LRIT1 Leucine-rich repeat–containing protein 21 precusor (LRC21) Q9P2V4 2.2  
MARCKS Myristoylated alanine-rich protein kinase C substrate P29966 2.2 
MLL Myeloid/lymphoid or mixed lineage leukemia Q03164 2.7 
MYO7A Myosin VIIA Q13402 2.1  
NOL1 Nucleolar protein 1, 120 kDa P46087 2.5 
PCYT1A Phosphate cytidylyltransferase 1, choline, α P49585 2.3  
PCYT1B Phosphate cytidylyltransferase 1, choline, β Q9Y5K3 2.1  
PDLIM4 PDZ and LIM domain 4 P50479 
PKP4 Plakophilin-4 Q99569 2.5 
PPFIA2 Liprin-α2 (LIPA2, PTPRF-interacting protein α2) Q75334 2.4 
PRSS1 Protease, serine, 1 (trypsin 1) Q07477 3.7 
RCN1 Reticulocalbin-1 precusor Q15293 2.5 
RHPN2 Rhophilin-like protein Q8IUC4 10.1  
RPL10 60S ribosomal protein L10 P27635 2.4 
RPL13 Ribosomal protein L13 P26373 3.6 
RPL27A Ribosomal protein L27a P46776 2.2 
RPS6 40S ribosomal protein S6 P62753 
SEC22A Vesicle-trafficking protein SEC22a Q96IW7  
SEC61B Protein transport protein Sec61 β-subunit P60468 2.1  
TALDO1 Transaldolase 1 P37837 6.3 
TPI1 Triosephosphate isomerase 1 P60174 1.7  
TUBA1B Tubulin, α-ubiquitous chain P68363 2.1  
TUBA1C Tubulin α6 Q9BQE3 3.1  
TUBB1 Tubulin β1 P07436 2.3 
TUBB5 Tubulin β5 Q7JJU6 2.4 
TUFM Tu translation elongation factor, mitochondrial (EFTU) P49411 2.4  
VPS13D Vacuolar protein sorting-associated protein 13D Q5THJ4 3.3  
YLPM1 YLP motif–containing protein 1 (YLPM1, ZAP113) P49750 2.6  
ZNF148 Zinc finger protein 148 Q9UQR1 3.2 
AFAP1 Actin filament–associated protein Q8N556 −3.3  
AHI1 Abelson helper integration site 1 protein homolog (AHI-1, Jouberin) Q8N157 −2.2 
AHNAK AHNAK nucleoprotein isoform 1 Q09666 −2.5  
ARHGEF2 Rho/rac guanine nucleotide exchange factor 2 Q9H023 −2.7 
BCLAF1 BCL2-associated transcription factor 1 Q9NYF8 untreated only  
C11orf58 Small acidic protein O00193 −7.1  
CLIC6 Chloride intracellular channel 6 Q96NY7 untreated only  
CST4 Cystatin-S precusor P01036 −2.1  
DYNC1LI1 Dynein light chain-A Q9Y6G9 −2.2  
EHA2 Ephrin receptor EphA2 P29317 untreated only 
ERRFI1 Mitogen-inducible gene 6 protein Q9UJM3 −2.6 
FAM129B Hypothetical protein LOC64855 Q5VVW7 −5.3  
FAM82C Family with sequence similarity 82, member C Q96TC7 untreated only  
FLNA Filamin 1 (actin binding protein-280) P21333 −1.5 
G3BP1 Ras-GTPase–activating protein SH3-domain–binding protein Q13283 −3.7 
HN1 Hematologic and neurologic expressed 1 isoform 1 Q9UK76 −6.7 
HRNR Hornerin Q86YZ3 −2.9 
MACF1 Microtubule-actin cross-linking factor 1 Q96PK2 −2.1 
NCOA3 Nuclear receptor coactivator 3 Q9Y6Q9 −2.1 
NOP5/NOP58 Nucleolar protein NOP5/NOP58 Q9Y2X3 −2.8 
Figure 3.

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

Figure 3.

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

Close modal
Table 3.

Categorization of overrepresented phosphoproteins under the major GO_processes identified by BiNGO analysis

Cell motilityCellular component organization and biogenesisLocalizationMetabolic processes
FLNa ARHGEF2 LASP1 SH3KBP1 ARHGEF2 MYO7A TUBA1C ARHGEF2 RPL27A 
HSPB1 C11ORF30 MAACF1 STMN1 ERBB2IP NOP5/NOP58 TUBB1 EEF1B2 RPS6 
LAMB2 EIF5 MLL SYTL4 FLNa NUP153 TUBB5 EIF5 STMN1 
MACF1 EIF5B MYO7A TLN1 G3BP1 SEC22A TXNDC1 EIF5B TALDO1 
MARCKS ENO1 NOP5/NOP58 TUBA1B HSPB1 SEC61B VPS13C ENO1 TPI1 
TLN1 ERBB2IP NUUP153 TUBA1C ITSN2 SH3KBP1 VPS13D HSPB1 TUBA1B 
TUBB1 FLNa PLEC1 TUBB1 LAMB2 STMN1  PCYT1A TUBA1C 
TUBB5 HSPB1 SEC22A TUBB5 LASP1 SYTL4  PCYT1B TUBB1 
 ITSN2 SEC61B TXNDC1 MACF1 TLN1  PGM2L1 TUBB5 
 LAMB2 9-Sep  MARCKS TUBA1B  RPL10 TUFM 
       RPL13  
Cell motilityCellular component organization and biogenesisLocalizationMetabolic processes
FLNa ARHGEF2 LASP1 SH3KBP1 ARHGEF2 MYO7A TUBA1C ARHGEF2 RPL27A 
HSPB1 C11ORF30 MAACF1 STMN1 ERBB2IP NOP5/NOP58 TUBB1 EEF1B2 RPS6 
LAMB2 EIF5 MLL SYTL4 FLNa NUP153 TUBB5 EIF5 STMN1 
MACF1 EIF5B MYO7A TLN1 G3BP1 SEC22A TXNDC1 EIF5B TALDO1 
MARCKS ENO1 NOP5/NOP58 TUBA1B HSPB1 SEC61B VPS13C ENO1 TPI1 
TLN1 ERBB2IP NUUP153 TUBA1C ITSN2 SH3KBP1 VPS13D HSPB1 TUBA1B 
TUBB1 FLNa PLEC1 TUBB1 LAMB2 STMN1  PCYT1A TUBA1C 
TUBB5 HSPB1 SEC22A TUBB5 LASP1 SYTL4  PCYT1B TUBB1 
 ITSN2 SEC61B TXNDC1 MACF1 TLN1  PGM2L1 TUBB5 
 LAMB2 9-Sep  MARCKS TUBA1B  RPL10 TUFM 
       RPL13  

A majority of these proteins have a known role either directly in affecting cell motility or in a related role such as cell adhesion and cytoskeletal relationship. Many of these proteins also play roles in transcription, translation, and Ca2+ binding, suggesting that these mechanisms are important in the increased cell motility phenotype induced by p16INK4a.

In addition to looking at the global changes in phosphorylated proteins, we looked for specific changes in FLNa phosphorylation. We identified decreased levels of phosphorylated FLNa at Ser1459 on cyclin D1-cdk4/6 repression [Supplementary Fig. S1 shows the spectrum of the singly and doubly charged daughter ions of phosphorylated FLNa peptide (m/z 650.2, in the doubly charged state)]. Ser1459 has been previously identified as a site of FLNa phosphorylation but has never been linked to a specific kinase (27). Incubation with p16INK4a or cyclin D1 knockdown using siRNA caused a decrease of ∼50% of pFLNa(Ser2152) as determined via Western blot and of pFLNa(Ser1459) in the mass spectrometry experiments. To validate this result, we obtained an antibody specific to pFLNa(Ser1459), and as shown in Fig. 1B, treatment of the cells with siRNA against cyclin D1 caused an ∼50% decrease in the amount of pFLNa(Ser1459).

We showed that cyclin D1 siRNA and inhibition of the cyclin D1-cdk4/6 kinase complex through peptide treatment resulted in decreased motility and an impaired wound healing response in the invasive MDA-MB-231 breast cancer cell line. We also showed that cyclin D1 bound to the actin binding protein FLNa and that the amount of phosphorylated FLNa(Ser2152) and FLNa(Ser1459) decreased concomitant with the decreased migration resulting from cyclin D1 siRNA transfection. We found that cyclin D1 and FLNa coimmunoprecipitated as well as colocalized in migrating cells, which suggested that the interaction was likely functional as the interaction occurred at the cell membrane where phosphorylated FLNa could recruit other molecules that were required for cytoskeleton reorganization (11). These findings identify a migration-related function of cyclin D1 in breast cancer cells and also provide new information about the mechanism for affecting motility in these cells.

Recent studies suggest a role for cyclin D1 in cellular migration (6, 28). The bone marrow macrophages isolated from cyclin D1–deficient mice displayed decreased motility (6). It was also shown that cyclin D1−/− mouse embryo fibroblasts exhibited increased adherence and decreased cellular motility compared with wild-type cells (7). Further evidence showed that these cyclin D1–related phenotypic changes were regulated through a member of the Rho family of small GTPases that is known to play an important role in the regulation of cell motility. In these cells, the activity of Rho-activated kinase (ROCKII) was increased. Thrombospondin 1, a matrix glycoprotein that inhibits cellular metastasis, was also shown to be regulated by cyclin D1 in these cells. The cdk inhibitor p27KIP has also been determined to be required for cyclin D1 regulation of cellular migration mouse embryo fibroblasts (28). In T47D breast cancer cells, cyclin D1 was shown to act similarly. A fewer number of T47D cells transfected with cyclin D1 siRNA were able to across the membrane in the Boyden chamber migration assay compared with control cells (29).

In our studies, we find that decreased cyclin D1 levels in MDA-MB-231 cells also lead to decreased wound healing capacity as well as decreased invasion potential. We also provide evidence that cyclin D1 and pFLNa coimmunoprecipitate and cyclin D1 and FLNa colocalize in MDA-MB-231. The amounts of two phosphorylated forms of FLNa, pFLNa(Ser1459) and pFLNa(Ser2152), are dependent on the level of cyclin D1 protein. To our knowledge, this is the first report of a mediator of phosphorylation at Ser1459. FLNa has been shown to interact with the COOH-terminal pleckstrin homology domain of ROCK and that this complex localizes at protrusive cell membranes (30). The known association of FLNa with other small GTPases regulates actin remodeling, formation of filopodia, and membrane ruffles, which, taken together with binding of FLNa to cyclin D1, suggests the mechanism for increased activity of ROCKII.

At least 28 sites of phosphorylation, including Ser2152 and Ser1459, have been identified in human FLNa.2

With regard to Ser2152, FLNa has been reported to be a substrate for the serine/threonine kinase p21-activated kinase (Pak1) identified by yeast two-hybrid screening using the NH2-terminal 1–270 amino acids of Pak1 as bait (31). In fibroblasts, FLNa has been identified as a substrate for one member of the Ras/mitogen-activated kinase pathway, p90 ribosomal S6 protein kinase 2 (25, 26). Ser1459 as a site of phosphorylation of FLNa has been identified in HeLa cell lysate that was enriched for phosphoproteins; however, the associated kinase was not identified (27, 32). Herein we have found that inhibition of cyclin D1-cdk4/6 activity resulted in a reduction of pFLNa(Ser1459) via mass spectrometry experiments and validated by Western blotting. A recent report by Lee et al. showed that cyclin B1/cdk1 can interact with and partially regulate the function of FLNa by phosphorylating Ser1436 in vitro and decrease its ability to cross-link actin in vitro. However, more experiments must be conducted to determine whether cyclin D1/cdk4 kinase directly phosphorylates Ser1459 or if it is part of a larger signaling pathway. An alternative hypothesis is that cyclin D1/cdk4 and FLNa are part of a complex and that phosphorylation of FLNa is accomplished by another molecule. FLNa could also be a potential scaffold for some unknown transmembrane receptors or signaling molecules.

We have identified approximately 65 phosphorylated proteins whose level of expression was significantly different in cells transfected with the p16INK4a peptide inhibitor of cyclin D1-cdk4/6 activity (Table 2). The majority of these phosphoproteins have been reported to play a role in some type of cancer either in vitro or in vivo. BiNGO was used to determine that localization, metabolism, cell motility, and cellular component organization/biogenesis Gene Ontology (GO) categories are statistically overrepresented in our set of identified phosphoproteins. Many of the proteins categorized under metabolism are involved in protein synthesis. Regulation of translation is critical for proper protein expression. Deregulation of protein translation is associated with abnormal gene expression leading to altered physiology, including cell growth and possibly cancer (33). Eukaryotic initiation factor 4E (eIF-4E) is one of the several initiation factors responsible for the regulation of translation in eukaryotes, and elevated level of eIF-4E is associated with many solid tumors including breast, prostate, and cervix cancers (33). In this study, we identify several other proteins related to protein translation that may play a role in increased cell motility. Signaling pathways identified as deregulated in some cancers are also represented in our data set.

In addition to FLNa, the other proteins associated specifically with cell motility are tubulin β1 and tubulin β5 (TUBB1 and TUBB5), microtubule-acting cross-linking factor 1 (MACF1), heat shock 27-kDa protein 1 (HSPB1), myristolyated alanine-rich protein kinase C substrate (MARCKS), talin 1 (TLN1), and laminin β2 (LAMB2). Except for TUBB1 and TUBB5, which, with actin and intermediate filaments, make up the cytoskeleton, and laminin β2, a component of the extracellular matrix, all of the proteins in this category are actin binding proteins. These data suggest that FLNa is only one of several other actin binding proteins involved in the cyclin D1–driven cell motility in MDA-MB-231 cells.

Taken together, our results show for the first time a direct interaction (and involvement) of cyclin D1 with the cytoskeletal protein FLNa, which is necessary for cell remodeling—a feature critical to cancer cell motility and hence migration and extravasation to other sites. Consistent with our results, there are reports that FLNa, in particular its phosphorylated form, plays an important role in breast cancer cell migration. FLNa phosphorylated at Ser2152 has been identified as a target of caveloin-1 in insulin-like growth factor-I–stimulated migration of MCF-7 breast cancer cells (34), and FLNa has been identified in the plasma of patients with metastatic breast cancer (35).

A.A. Quong: employment, Food and Drug Administration. The other authors disclosed no potential conflicts of interest.

Grant Support: Pennsylvania Department of Health Breast and Cervical Cancer Section grant #05-07-09 (J.N. Quong and A.A. Quong), the Breast Cancer Research Foundation (A.A. Quong and J.N. Quong), and NIH grantsR01CA70896, R01CA75503, R01CA86072, and R01CA107382 (R.G. Pestell). The Kimmel Cancer Center was supported by NIH Cancer Center Core grant P30CA56036 (R.G. Pestell). This project is funded in part by the Dr. Ralph and Marian C. Falk Medical Research Trust and a grant from the Pennsylvania Department of Health (R.G. Pestell). The Department specifically disclaims responsibility for any analysis, interpretations, or conclusions.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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