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Cancer Research 66, 11399, December 1, 2006. doi: 10.1158/0008-5472.CAN-06-2386
© 2006 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Protein Kinase C ß Enhances Growth and Expression of Cyclin D1 in Human Breast Cancer Cells

Haiyang Li1,3 and I. Bernard Weinstein1,2,3

1 Herbert Irving Comprehensive Cancer Center, 2 Department of Medicine, and 3 Institute of Human Nutrition, Columbia University, New York, New York

Requests for reprints: I. Bernard Weinstein, Herbert Irving Comprehensive Cancer Center, Columbia University, 701 West 168th Street, New York, NY 10032. Phone: 212-305-6921; Fax: 212-305-6889; E-mail: ibw1{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although alterations in the expressions of protein kinase C (PKC) have been implicated in breast carcinogenesis, the roles of specific isoforms in this process remain elusive. In the present study, we examined the specific roles of PKCß1 and ß2 in growth control in human breast cancer cell lines. The PKCß-specific inhibitor LY379196 significantly inhibited growth of the breast cancer cell lines MCF-7, MDA-MB-231, and BT474, but not the normal mammary epithelial cell line MCF-10F. Treatment of MCF-7 cells with LY379196 caused an increase in the fraction of cells in the G1 phase of the cell cycle. To explore the roles of PKCß1 and ß2, we used cDNA expression vectors that encode wild-type and constitutively activated or dominant negative mutants of these two proteins. When compared with vector controls, derivatives of MCF-7 cells that stably overexpress wild-type PKCß1 or PKCß2 displayed a slight increase in growth rate; derivatives that stably express the constitutively active mutants of PKCß1 or PKCß2 displayed a marked increase in growth rate; and derivatives that stably express a dominant negative mutant of PKCß1 or ß2 displayed inhibition of growth. The derivatives of MCF-7 cells that stably express the constitutively activated mutants of PKCß1 or ß2 were more resistant to growth inhibition by LY379196 than the vector control MCF-7 cells. Immunoblot analysis indicated that MCF-7 cells that stably overexpress wild-type or constitutively activated mutants of PKCß1 or ß2 had higher cellular levels of cyclin D1 than vector control cells, whereas cells that express a dominant negative mutant had decreased levels of cyclin D1. The derivatives that stably express the constitutively activated mutants of PKCß1 or ß2 also displayed increased cyclin D1 promoter activity in transient transfection luciferase reporter assays, and this induction of activity requires activator protein 1. Constitutively activated PKCß1 and ß2 also enhanced the transcription of c-fos in transient transfection luciferase reporter assays. Thus, PKCß1 and ß2 may play important positive roles in the growth of at least a subset of human breast cancers. Therefore, inhibitors of these isoforms may be useful in breast cancer chemoprevention or therapy. (Cancer Res 2006; 66(23): 11399-408)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein kinase Cs (PKC) comprise a family of at least 10 mammalian isozymes that are lipid-dependent serine/threonine protein kinases with a broad range of tissue distribution and differential cellular localizations (1). The PKC family can be divided into three subgroups: conventional PKCs ({alpha}, ß1, ß2, {gamma}), novel PKCs ({delta}, {varepsilon}, {eta}, {theta}), and atypical PKCs ({lambda}/{iota} and {zeta}). PKC transduces signals from a plethora of extracellular receptors (2, 3). The role of PKC has repeatedly been implicated in breast cancer (4, 5). It has been shown that the total levels of PKC enzymatic activity are elevated in malignant breast tumors when compared with normal breast tissues (6, 7). Furthermore, estrogen receptor–negative (ER–), hormone-independent breast tumors tend to have higher levels of total PKC activity than estrogen receptor–positive (ER+) tumors (8). This inverse relationship between the levels of ER and PKC was observed both in primary breast tumors and in immortalized breast cancer cell lines (9, 10). Treatment of MCF-7 and other breast cancer cell lines with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which is a potent activator of PKC, under conditions that induced down-regulation of PKC activity, resulted in growth inhibition and enhanced expression of variables associated with a more differentiated phenotype (11). These findings suggest that alterations in PKC-mediated signal transduction pathways may be involved in the pathogenesis of breast cancer. However, the precise roles of specific isoforms of PKC in human breast cancer cells have not been elucidated, mainly because these cells contain multiple PKC isoforms, and it has been difficult to distinguish the roles of individual isoforms.

The ß isoforms of PKC have been implicated in several types of cancer especially in colon cancer. Increased expression of PKCß2 is an early event in colon carcinogenesis in mice (12). Increased expression of PKCß2 in the colon of transgenic mice causes hyperproliferation and increased susceptibility to colon carcinogenesis due, at least in part, to repression of expression of the transforming growth factor-ß (TGF-ß) type II receptor (13). Overexpression of PKCß2 also renders transgenic mice more susceptible to carcinogen-induced colonic hyperproliferation, aberrant crypt foci formation, and colon carcinogenesis (13). PKCß2 also induces the expression of cyclooxygenase type 2 in rat intestinal epithelial cells in vitro and in transgenic PKCß2 mice in vivo (14). Overexpression of either PKCß1 or ß2 in human colon carcinoma cell lines causes a 2- to 5-fold increase in the levels of transforming growth factor-{alpha} (TGF{alpha}) in the growth medium (15). However, previous studies have not specifically addressed the roles of PKCß1 or PKCß2 in the proliferation of human breast cancer cells.

Specific isoforms of PKC, including PKCß1 and ß2, regulate the transcriptional control of cyclin D1 (CCND1) expression, which plays a critical role in the progression of mammalian cells through the G1 phase of the cell cycle (16). The G1 to S transition is a critical step because abnormalities in this step can enhance cell proliferation, genome instability, and tumor progression. The importance of cyclin D1 in breast cancer is evidenced by the fact that overexpression of this cyclin is seen in ~60% of human breast cancers (17, 18). In ~15% of the cases, it is due to gene amplification; in the remaining 45%, it is due to increased transcription (19). Furthermore, overexpression of cyclin D1 has been linked with poor prognosis in breast cancer (20, 21). The cyclin D1 promoter is one of the major targets for several growth stimulatory signaling pathways (22, 23). For example, signaling through the Wnt as well as mitogen-activated protein kinase (MAPK) pathways potently induce cyclin D1 gene transcription (22–24). Therefore, in the present study we examined the effects of PKCß1 and ß2 on both cell proliferation and cyclin D1 expression in human breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and antibodies. The compound LY379196 was provided by Eli Lilly, Inc. (Indianapolis, IN) and its properties have previously been described (25). PKCß1 and ß2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); the PKC{delta} antibody from PanVera (Madison, WI); the cyclin D1 antibody (06-137) from Upstate Biotechnology, Inc. (Lake Placid, NY); the c-fos and actin antibodies from Sigma (St. Louis, MO); and the hemagglutinin antibody from Covance (Richmond, CA).

Cell lines and growth curves. The MCF-10F normal mammary epithelial cell line and the MCF-7, MDA-MB-231, and BT474 human breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). Unless indicated otherwise, the cells were grown in MEM with 10% fetal bovine serum (FBS) purchased from Invitrogen Corp. (Carlsbad, CA) in a 100% humidified incubator at 37°C with 5% CO2.

For growth curve analysis, cells were plated in triplicate at a density of 5 x 104 per well in six-well (35-mm) plates with 2 mL of MEM containing 10% FBS. Geneticin (800 µg/mL; Invitrogen) was added to the growth media for derivatives of MCF-7 cells overexpressing various PKC constructs. The cells were refed with fresh medium every 2 days. Cell numbers were counted using a Coulter counter (Beckman Coulter, Inc., Fullerton, CA) everyday for the subsequent 5 days after suspension with trypsin.

Growth inhibition was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. In brief, cells were plated in triplicate into 96-well plates (5 x 103 per well) with 100 µL MEM containing 10% FBS and cultured overnight to allow for cell attachment. Cells were then treated with 10 nmol/L LY379196 dissolved in DMSO for 48 hours in MEM containing 10% FBS and assayed using a MTT assay kit (Roche Diagnostic Corp., Indianapolis, IN). The number of viable cells was quantified spectrophotometrically using an ELISA reader. All assays were done in triplicate and repeated experiments yielded similar results.

Flow cytometry and cell cycle analysis. Cells were treated with 0.1% v/v DMSO as the solvent control or with LY379196 (10 nmol/L, dissolved in DMSO) for 24 or 48 hours. Both adherent and floating cells were collected, washed with PBS, resuspended in 1 mL of PBS, and fixed with 5 mL of 70% ethanol, centrifuged, resuspended in 1 mL of PBS containing 2 mg/mL RNase (Sigma), and stained with 0.2 mg/mL of propidium iodide (Sigma) in the dark for 30 minutes. The cell suspension was then filtered through a 60-µm Spectra mesh filter (Spectrum Medical Industries, Houston, TX). Samples of 10,000 to 20,000 cells were then analyzed for DNA histograms and cell cycle phase distributions using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and the data were analyzed using CellQuest software (Becton Dickinson) according to the manual provided by the manufacturer.

Immunoblot analysis. Exponentially growing cells were harvested and sonicated for 10 seconds in modified radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L DTT, 25% glycerol, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L NaF, 0.1 mmol/L Na3VO4, 10 mmol/L ß-glycerophosphate, and 1 mmol/L phenylmethylsulfonyl fluoride]. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Protein samples, 20 µg per lane, were subjected to SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The membrane was blocked with 5% skim milk and incubated with the indicated primary antibodies for 1 hour at room temperature or overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase–conjugated antimouse immunoglobulin G (IgG) or antirabbit IgG secondary antibodies (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) as indicated. Protein bands were visualized with the enhanced chemiluminescence immunoblotting system (Amersham Pharmacia Biotech). Immunoblotting for actin was done to verify equivalent amounts of loaded protein. Repeat studies gave similar results.

Plasmids. The pcDNA3 plasmid DNA (Invitrogen) was used as a control vector and used as needed to achieve the same total amount of plasmid DNA per transfection. The expression plasmids for wild-type and constitutively activated and dominant negative mutant forms of PKC ß1, ß2, and {delta} were previously described (16). The cyclin D1 promoter-luciferase reporters (22), the c-fos promoter-luciferase reporter (26), and the c-fos serum response element luciferase reporter plasmid pSRE-luc (27) were previously described.

Colony formation assay. MCF-7 and MDA-MB-231 cells were transfected with the control vector pcDNA3 or expression plasmids for various PKC isoforms using the Lipofectin reagent (Invitrogen). After 24 hours, the cells were replated into 100-mm plates at 1 x 105 per plate, and the cells were cultured in the presence of 1 mg/mL Geneticin (Invitrogen) for 3 weeks to select for cells that stably expressed the transfected plasmid. The colonies were fixed with formalin, stained with Giemsa (Sigma), and counted. The results are presented as mean values of triplicate assays.

Generation of cloned derivatives of MCF-7 cells that stably overexpress various PKCß constructs. MCF-7 cells were transfected with either the control vector pcDNA3 or wild-type or mutant PKCß constructs and selected as described above. The cells were either pooled and serially passaged, or individual clones were selected. For colony selection, for each transfection, 20 clones were individually selected and serially passaged in MEM containing 10% FBS and 800 µg/mL Geneticin. Two weeks later, immunoblot analysis was done on each clone to detect expression of the hemagglutinin-tagged protein using an anti-hemagglutinin-tag primary antibody. Clones that expressed high levels of the indicated PKCß constructs were then used in further studies.

Transient transfection reporter assays. Triplicate samples of 1 x 105 MCF-7 cells in 35-mm plates containing MEM plus 10% FBS were cultured overnight to allow for cell attachment. The cells were then transfected using Lipofectin in Opti-MEM (Invitrogen) with 1 µg of the indicated reporter plasmid, 0.5 to 2 µg of various expression vectors, and 0.5 µg of the control reporter plasmid pCMV-ß-galactosidase. The pcDNA3 plasmid DNA was added to the transfections to achieve the same total amount of plasmid DNA per transfection. Eighteen hours after transfection, cells were refed with phenol red–free MEM (Invitrogen) with 0.5% charcoal-stripped FBS. Cell extracts were prepared after a 24-hour incubation in the phenol red–free MEM with 0.5% charcoal-stripped FBS. Cell extracts were prepared and luciferase assays were done using the Luciferase Assay System (Promega Corp., Madison, WI). Luciferase activities were normalized with respect to parallel ß-galactosidase activities to correct for differences in transfection efficiency. ß-Galactosidase assays were done using the ß-Galactosidase Enzyme Assay System (Promega). The mean values and SDs were calculated from triplicate samples. Similar experiments were done thrice and representative results from one of these experiments are presented.

Because the cyclin D1-luc reporters, the c-fos-luc reporter, and the pSRE-luc can be activated by growth factors that are present in serum, we used a low concentration of 0.5% charcoal-stripped FBS to minimize the stimulatory effects caused by serum. In addition, because phenol red in tissue culture media is a weak estrogen (28), to reduce nonspecific effects on the estrogen-responsive MCF-7 cells, phenol red–free MEM was used in the above transient transfection luciferase reporter assays.

Reverse transcription-PCR analysis. MCF-7 cells grown in 60-mm plates were transfected with Transfectin (Bio-Rad) in phenol red–free MEM with 0.5% charcoal-stripped FBS, with 5 µg of the indicated plasmids encoding wild-type and mutant forms of PKC. Cells were refed with fresh phenol red–free MEM with 0.5% charcoal-stripped FBS and incubated for an additional 48 hours. Total RNA was purified using Trizol reagent (Invitrogen) following the protocol of the manufacturer. cDNA was synthesized from 1 µg total RNA using the iScript kit (Bio-Rad) and amplified by PCR using Choice Taq DNA Polymerase (Denville, Metuchen, NJ). The primers used for amplification are, for cyclin D1, 5'-CCCTCGGTGTCCTACTTCAA-3' and 5'-GGGGATGGTCTCCTTCATCT-3', and for c-fos, 5'-CCAACCTGCTGAAGGAGAAG-3' and 5'-ATGATGCTGGGAACAGGAAG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–specific PCR products (using primers 5'-CGAGATCCCTCCAAAATCAA-3' and 5'-TGTGGTCATGAGTCCTTCCA-3') from the same RNA samples were amplified and served as internal controls. Each amplification cycle consisted of 30 seconds at 94°C for denaturation, 30 seconds at 60°C for primer annealing, and 1 minute at 72°C for extension. After PCR amplification, the fragments were analyzed by agarose gel electrophoresis and stained with ethidium bromide.

Statistical analysis. Data are analyzed using Student's t test. A difference between groups with P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKCß is required for the growth of human breast cancer cells. To study the role of PKCß1 and ß2 in human breast cancer, we first determined their levels of protein expression in exponentially growing human breast cancer cells and normal mammary epithelial cells. We chose the MCF-10F normal mammary epithelial cell line and three human breast cancer cell lines in this assay. The MCF-7 cells are ER+ and do not overexpress Her2/neu or epidermal growth factor receptor (EGFR); the MDA-MB-231 cells are ER– and overexpress EGFR; and the BT474 are ER– and overexpress Her2/neu. Therefore, each of them represents a clinically relevant subtype of human breast cancer. These cells were harvested and protein extracts were examined by immunoblot analysis. We found that PKCß1 and ß2 were expressed at high levels in all three human breast cancer cells, but not in the normal mammary epithelial cells (Fig. 1A ). The expression levels of PKCß1 in all three breast cancer cell lines tested were >2-fold higher than that of the MCF-10F normal mammary epithelial cell line (Fig. 1B). The expression levels of PKCß2 in these breast cancer cell lines were 2- to 7-fold higher than that of the MCF-10F normal mammary epithelial cell line (Fig. 1C). In contrast, the levels of expression of PKC{delta} varied among the three human breast cancer cell lines and were not increased in the breast cancer cells when compared with the normal MCF-10F cells (Fig. 1A).


Figure 1
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  		Figure 1. A, immunoblot analysis of the expression of endogenous PKCs ß1, ß2, and {delta} in exponential cultures of MCF-10F, MCF-7, MDA-MB-231, and BT474 cells. B, densitometry analysis of the levels of PKCß1 protein in exponential cultures of MCF-10F, MCF-7, MDA-MB-231, and BT474 cells. The expression ratios were normalized with respect to levels of actin. Three independent analyses were done and SDs were calculated. *, P < 0.05, comparing the normalized ratios to those in MCF-10F cells. C, similar densitometry analysis of the protein expression levels of PKCß2 in exponential cultures of MCF-10F, MCF-7, MDA-MB-231, and BT474 cells. **, P < 0.01, compared with normalized ratios of MCF-10F cells.

 
To examine whether the breast cancer cell lines require PKCß for growth, we treated the three breast cancer cell lines, as well as the MCF-10F cells, with the PKCß-specific inhibitor LY379196, which has >60-fold selectivity for PKCß versus other PKC isoforms (25). This inhibitor is not, however, selective for PKCß1 or ß2 (25). Although the growth rates of these breast cancer cells were different, treatment with LY379196 for 24 hours significantly inhibited growth of all three breast cancer cell lines (P < 0.05), and at 48 and 96 hours caused ~80% and 90% decreases, respectively, in cell viability in the three human breast cancer cells when compared with the control DMSO solvent–treated cells (P < 0.005 and P < 0.002, respectively). However, this concentration of LY379196 did not cause significant growth inhibition in MCF-10F cells (Fig. 2A ). Using cell cycle analysis, we found that in MCF-7 cells, treatment with 10 nmol/L LY379196 caused an increase of cells in the G1 and a decrease of cells in the S phase of the cell cycle (Fig. 2B and C; P < 0.05), suggesting that PKCß plays an important role in the G1 to S transition.


Figure 2
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  		Figure 2. A, growth inhibition caused by 10 nmol/L LY379196 in MCF-10F, MCF-7, MDA-MB-231, and BT474 cells. Percent growth inhibition was calculated by comparing LY379196-treated cells with control DMSO solvent–treated cells. B, cell cycle analysis of MCF-7 cells after treatment with 10 nmol/L LY379196. The analyses were done at least thrice and yielded similar results. C, statistical analysis of the cell cycle distribution of MCF-7 cells after treatment with LY379196. *, P < 0.05; **, P < 0.005; ***, P < 0.001, compared with the control DMSO solvent–treated cells.

 
To further examine the specific the roles of PKCß1 and ß2 in the growth of human breast cancer cells, we employed expression plasmids that encode wild-type and mutant forms of PKCß1 or ß2. Because the NH2-terminal domain of PKC contains a pseudosubstrate region that binds to the COOH-terminal catalytic domain and inhibits its activity, truncation mutants that contain only the COOH-terminal catalytic domain are constitutively activated (CAT; ref. 16). The dominant negative (KR) mutants encode a full-length PKC with a point mutation (K371R) that abolishes the ATP-binding ability (16). We transfected MCF-7 and MDA-MB-231 cells with the control vector pcDNA3 or various expression plasmids that encode the wild-type (WT) or constitutively activated (CAT) or dominant negative (KR) mutants of PKCß1 or ß2, and then did colony formation assays. We found that both MCF-7 and MDA-MB-231 cells transfected with PKCß1-WT or PKCß2-WT formed more colonies than cells transfected with the control vector pcDNA3 (P < 0.05). With both cell lines, cells transfected with PKCß1-CAT or PKCß2-CAT formed even a larger number of colonies than cells transfected with the control vector (P < 0.02 or P < 0.01). Cells transfected with PKCß1-KR or PKCß2-KR formed fewer colonies than the control cells, but the difference was not statistically significant, possibly because the levels of the expression of these dominant negative mutants were not high enough to inhibit the endogenous proteins (Fig. 3A ). In contrast to the results obtained with the PKCß isoforms, MCF-7 cells transfected with the constitutively activated PKC{delta} plasmid formed fewer colonies when compared with cells transfected with the control vector (P < 0.01). Similar results were also seen in BT474 breast cancer cells (data not shown). These findings are consistent with evidence that PKC{delta} often exerts growth inhibitory and/or apoptotic effects (29) and confirm the isoform specificity of our PKC constructs.


Figure 3
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  		Figure 3. A, colony formation efficiency of MCF-7 or MDA-MB-231 cells after transfection with the control vector pcDNA3 or the indicated expression plasmids of PKCs. All assays were done in triplicate and the results were expressed as percent of control (i.e., the number of colonies obtained compared with the vector control plasmid). B, immunoblot analysis of the expression of PKCß wild-type and mutant proteins in clones of MCF-7 derivatives. Actin was used as a loading control. C, growth curves of two clones of MCF-7 derivatives that stably expressed a constitutively activated (CAT) mutant of PKCß1. D, growth curves of clones of MCF-7 derivatives that stably expressed the indicated PKCß2 constructs. E, growth curves of MCF-7 derivatives treated with 10 nmol/L LY379196. Cell viability was determined with MTT assays and expressed as percent of the control DMSO solvent–treated cells. F, growth curves of MCF-7 derivatives done in the absence of serum. *, P < 0.05; **, P < 0.02; ***, P < 0.01, compared with vector control cells.

 
In view of the above results, it was of interest to isolate individual clones of MCF-7 cells that stably overexpress wild-type or mutant forms of PKCß1 and ß2 and further examine their properties. After transfection with the respective plasmids and selection with Geneticin, 20 individual clones were isolated and serially passaged. Extracts of these clones were then examined by immunoblot analysis for stable expression of the related proteins using an antibody to the hemagglutinin tag (Fig. 3B). Two clones (designated MCF-7-PKCß1-CAT-1 and MCF-7-PKCß1-CAT-2) were identified that stably express high levels of PKCß1-CAT. One clone (designated MCF-7-PKCß2-WT-7) was identified that stably expresses a high level of PKCß2-WT. Three clones (designated MCF-7-PKCß2-CAT-2, MCF-7-PKCß2-CAT-3, and MCF-7-PKCß2-CAT-4) were identified that stably express high levels of PKCß2-CAT. One clone (designated MCF-7-PKCß2-KR-9) was identified that stably expresses a high level of PKCß2-KR.

To investigate the effects of overexpression of these forms of PKCß1 or ß2 on the growth of MCF-7 cells, we did growth curve analysis. When compared with vector controls, the two clones of MCF-7 cells that stably express a constitutively active mutant of PKCß1 (CAT) displayed a marked increase in growth rate (Fig. 3C; P < 0.01). The clone that stably overexpresses wild-type PKCß2 displayed a slight increase in growth rate (Fig. 3D; P < 0.02). The three clones that express a constitutively active mutant of PKCß2 (CAT) also displayed a marked increase in growth rate (Fig. 3D; P < 0.01). It is of interest that the clone that expresses a dominant negative mutant (KR) of PKCß2 displayed inhibition of growth (Fig. 3D; P < 0.01).

We also attempted to overexpress wild-type and constitutively activated mutants of PKC{delta} in MCF-7 cells to compare the effects of overexpression of different PKC isoforms. However, after Geneticin selection, none of the clones that were picked and examined by immunoblot analysis expressed the corresponding PKC{delta} protein. Presumably this reflects the cytotoxicity of PKC{delta} to MCF-7 cells, which is consistent with previous studies on PKC{delta} (29).

Because the control MCF-7 cells were very sensitive to growth inhibition by the PKCß-specific inhibitor LY379196, we examined the effects of this compound on the growth of derivatives of MCF-7 cells that stably overexpress various constructs of PKCß1 or ß2. The derivatives that stably express the constitutively activated mutants of PKCß1 or ß2 were ~3-fold more resistant to growth inhibition by LY379196 than the vector control cells or cells that expressed the wild-type or dominant negative (KR) forms of PKCß2 (Fig. 3E; P < 0.05).

Pooled populations, rather than individual clones of MCF-7 derivatives that stably overexpress wild-type PKCß1 or ß2, or express the constitutively activated mutant of PKCß1 or ß2, also displayed greater tolerance to serum starvation because there was reduced growth inhibition when these cells were serum starved when compared with the vector control cells (Fig. 3F; P < 0.01).

PKCß1 and ß2 activate the expression of cyclin D1. Because in MCF-7 cells the PKCß-specific inhibitor LY379196 caused cell cycle arrest at the G1 phase, we investigated whether PKCß1 or ß2 plays a role in regulating expression of cyclin D1 because it plays a critical role in the G1-S transition (20). Using immunoblot analysis, we found that MCF-7 derivatives that overexpress constitutively activated mutants of PKCß1 or ß2 had higher cellular levels of cyclin D1 than vector control cells, whereas cells that expressed a dominant negative mutant of PKCß2 had decreased levels of cyclin D1 (Fig. 4A ). Using transient transfection cyclin D1 promoter-luciferase reporter assays, we found that the derivatives that stably express the constitutively activated mutants of PKCß1 or ß2 also displayed increased cyclin D1 promoter activity, and in these derivatives the cyclin D1 promoter could be activated in the absence of serum (Fig. 4B; P < 0.05 or P < 0.01).


Figure 4
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  		Figure 4. A, cell extracts were prepared from exponentially dividing cultures of the clones of MCF-7 cells that stably express constitutively activated PKCß1 or ß2 and examined by immunoblot analysis with a cyclin D1 antibody. Actin was used as a loading control. B, transient transfection reporter assays. Each of the indicated clones was transiently transfected with a cyclin D1 promoter-luciferase reporter plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum. Extracts were prepared and luciferase activity was determined and normalized with respect to ß-gal activity. C, each of the indicated clones was transiently transfected with a cyclin D1 promoter-luciferase reporter plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum, with or without 30 nmol/L LY379196. Luciferase activity was determined and normalized with respect to ß-gal activity. Results are expressed as percent of promoter activity obtained in the drug-treated cells when compared with the same cell line treated with DMSO solvent (control), which was assigned a value of 100%. D, MCF-7 cells were treated with 10 nmol/L LY379196 and cell lysates were prepared after the indicated time and examined by immunoblot analysis with a cyclin D1 antibody. Actin was used as a loading control. E, MCF-7 cells were transiently cotransfected with a cyclin D1 promoter-luciferase reporter plasmid and the indicated PKC expression plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum. Luciferase activity was determined and normalized with respect to ß-gal activity. F, analysis of cyclin D1 mRNA levels by reverse transcription-PCR (RT-PCR) after MCF-7 cells were transiently transfected with the indicated PKC constructs. GAPDH was used as an internal control. *, P < 0.05; **, P < 0.02; ***, P < 0.01.

 
In view of the above findings indicating that PKCß1 and ß2 stimulate the transcriptional activity of the cyclin D1 promoter (Fig. 4B) and the fact that LY379196 specifically inhibits the activity of PKCß1 and ß2, we examined the effects of LY379196 on cyclin D1 promoter activity. The various derivatives of MCF-7 cells described above were transiently transfected with a cyclin D1 promoter-luciferase reporter plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum, with or without 30 nmol/L of LY379196. Treatment with LY379196 caused inhibition of cyclin D1 promoter activity in the vector control cells and in the derivatives. It is of interest that the PKCß2-WT-7 cells were significantly more resistant to the inhibitory effects of the drug, but this was not seen with the CAT mutant–expressing cells (Fig. 4C; P < 0.05). This may reflect the different properties of the intact versus the mutant proteins. We further analyzed the cyclin D1 protein level after treating cells with LY379196 and found that LY379196 caused a decrease in cyclin D1 protein level after 6 hours of treatment (Fig. 4D).

To extend the evidence that PKCß1 and ß2 stimulate cyclin D1 promoter activity in MCF-7 cells, and to further elucidate which element in the cyclin D1 promoter PKCß1 and ß2 might activate, we carried out transient transfection reporter assays in which we cotransfected pcDNA3, PKCß2-WT, PKCß2-CAT, PKC{delta}-WT, or PKC{delta}-CAT plasmids together with a series of cyclin D1 promoter-luciferase reporter plasmids. These cyclin D1 promoter constructs included the full-length promoter –1745CD1, a truncated –964CD1 promoter that includes an activator protein 1 (AP-1) site, and a truncated –964(AP1m)CD1 promoter with a mutated AP1 site (22). We found that transient transfection of the wild-type PKCß1 or ß2 stimulated the activity of both the full-length cyclin D1 promoter and the truncated –964CD1, but not the activity of the AP-1 site mutated promoter (Fig. 4E). Transient transfection of the constitutively activated mutants of PKCß1 or ß2 also stimulated the activity of both the full-length cyclin D1 promoter and the truncated –964CD1, but did not stimulate the activity of the AP1 site mutated promoter (Fig. 4E). These findings provide evidence that the AP1 site in the cyclin D1 promoter is required for the stimulation of transcription of cyclin D1 by PKCß1 or ß2. In contrast, transient transfection of the wild-type or the constitutively activated mutant of PKC{delta} inhibited cyclin D1 promoter activity (Fig. 4E), again indicating the isoform specificity of our constructs. In addition, we also examined the mRNA levels of cyclin D1 after transfection with the above-described PKC constructs. We confirmed that the mRNA levels were increased after transient transfection with the wild-type or constitutively activated forms of PKCß1 or ß2 (Fig. 4F). In contrast, transfection with the constitutively activated forms of PKC{delta} decreased the mRNA levels in MCF-7 cells (Fig. 4F).

PKCß1 and ß2 activate the serum responsive element in the c-fos gene. Because the above findings suggest that the AP-1 site in the cyclin D1 promoter is required for the induction of transcription mediated by PKCß1 and ß2, and because induction of the early response gene c-fos has been shown to be enhanced by PKCß (30, 31), it was of interest to determine whether overexpression of PKCß1 or ß2 activates c-fos transcription in our MCF-7 derivatives. Therefore, MCF-7 derivatives that express wild-type or mutant forms of PKCß were transfected with the c-fos promoter-luciferase reporter plasmid. Eighteen hours after growing the transfected cells in serum-free medium, cell extracts were prepared and assayed for luciferase activity. We found that the derivatives that express the constitutively activated PKCß1 or ß2 displayed increased transcriptional activity of the c-fos promoter, and this effect was seen even in the absence of serum (Fig. 5A ). The serum responsive element (SRE) transcriptional control element in the c-fos promoter region plays an important role in the transcription of c-fos. Using a pSRE-luc reporter plasmid (32), we found that in the MCF-7 derivatives that express constitutively activated PKCß1 or ß2, there was an increase in the activity of the SRE transcriptional control element (Fig. 5B). The derivative that overexpressed wild-type PKCß2 also displayed increased SRE activity (Fig. 5B), and this activity was more resistant to inhibition by LY379196 than the vector control MCF-7 cells (Fig. 5C). Consistent with the activation of the c-fos promoter in the MCF-7 derivatives expressing the constitutively activated PKCß1 or ß2, the c-Fos protein levels were also increased in these cells (Fig. 5D). We also carried out transient transfection reporter assays in which we cotransfected pcDNA3, PKCß2-WT, PKCß2-CAT, PKC{delta}-WT, or PKC{delta}-CAT plasmids together with the pSRE-luc plasmid. We found that transient transfection of the wild-type PKCß1 or ß2 stimulated the activity of SRE, and transient transfection of the constitutively activated PKCß1 or ß2 further stimulated SRE activity (Fig. 5E). When we examined the mRNA levels of c-fos after transfection with the above-described PKC constructs, we found that the mRNA levels were increased after transient transfection with the wild-type or constitutively activated forms of PKCß1 or ß2 (Fig. 5F). In contrast, transfection with the constitutively activated forms of PKC{delta} decreased the mRNA levels of c-fos in MCF-7 cells (Fig. 5F). These findings, with respect to increased cyclin D1, c-fos, and SRE promoter activity, suggest that in MCF-7 cells, PKCß1 and ß2 enhance and possibly integrate multiple pathways of signal transduction and gene expression that stimulate cell proliferation.


Figure 5
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  		Figure 5. A, each of the indicated clones was transiently transfected with a c-fos promoter-luciferase reporter plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum. Luciferase activity was determined and normalized with respect to ß-gal activity. B, each of the indicated clones was transiently transfected with a SRE promoter-luciferase reporter plasmid for 18 hours, together with a ß-gal reporter plasmid. Then cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum. Luciferase activity was determined and normalized with respect to ß-gal activity. C, each of the indicated clones was transiently transfected with a SRE promoter-luciferase reporter plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum, with or without 30 nmol/L LY379196. Luciferase activity was determined and normalized with respect to ß-gal activity. Results are expressed as percent of promoter activity obtained in the drug-treated cells when compared with the same cell line treated with DMSO solvent (control), which was assigned a value of 100%. D, c-Fos protein levels in PKCß clones. E, MCF-7 cells were transiently cotransfected with a SRE promoter-luciferase reporter plasmid and the indicated PKC expression plasmid for 18 hours, together with a ß-gal reporter plasmid. Then the cells were grown for 24 hours in phenol red–free MEM with 0.5% charcoal-stripped serum. Luciferase activity was determined and normalized with respect to ß-gal activity. F, analysis of c-fos mRNA levels by RT-PCR after MCF-7 cells were transiently transfected with the indicated PKC constructs. GAPDH was used as an internal control. *, P < 0.05; **, P < 0.02; ***, P < 0.01, compared with the vector control cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The precise roles of specific isoforms of PKC in growth control and cell cycle regulation in human breast cancer cells have not previously been elucidated, in part because these cells contain multiple isoforms of PKC and it has been difficult to distinguish the roles of individual isoforms. We focused on the ß isoforms of PKC because of their ubiquitous expression in various cell types and the previous evidence that they might play important roles in other types of cancer (12, 33–36). We used the drug LY379196 that specifically inhibits the activities of PKCß1 and ß2 to address this question. In addition, as a separate approach, we constructed a series of mammalian expression vectors that encode wild-type or mutant forms of PKCß1 and ß2. These constructs were then used to generate and characterize derivatives of the MCF7 human breast cancer cell line that stably overexpress the wild-type or constitutively activated or dominant negative mutants of these two isoforms of PKC. PKCß1 and ß2 are splice variants of the same gene, which differ only by amino acid sequences in the last 50 residues in the COOH end of catalytic domain. Therefore, it is likely that our constitutively activated (CAT) mutants retain specificity between PKCß1 and ß2.

We found that endogenous PKCß1 and ß2 are expressed at relatively high levels in the human breast cancer cell lines MCF-7, MDA-MB-231, and BT474 when compared with normal MCF-10F mammary epithelial cells (Fig. 1). The PKCß-specific inhibitor LY379196 markedly inhibited the growth of MCF-7 (ER+, HER2–), MDA-MB-231 (ER–, HER2+, EGFR overexpressing), and BT474 (ER+, HER2 overexpressing) cells (Fig. 2A), and caused slight arrest of the cells at the G1 phase of the cell cycle (Fig. 2B and C). This suggests that the ß isoforms of PKC may be indispensable for the growth of both estrogen-dependent and estrogen-independent breast cancer cells. Stable overexpression of wild-type PKCß1 or ß2 or expression of the constitutively activated mutants of PKCß1 or ß2 stimulated the growth of MCF-7 cells (Fig. 3C and D) and increased the protein levels of cyclin D1 and c-Fos (Figs. 4A and 5D), whereas stable expression of a dominant negative mutant of PKCß2 inhibited growth (Fig. 3D) and decreased the protein levels of cyclin D1 and c-Fos (Figs. 4A and 5D). In addition, transient transfection of parental MCF-7 cells with wild-type or constitutively activated mutants of PKCß1 or ß2 stimulated cyclin D1 and c-fos promoter activity (Figs. 4E and 5E) and increased the mRNA levels of these two genes (Figs. 4F and 5F), whereas transient transfection with wild-type or constitutively activated mutants of PKC{delta} exerted the opposite effect (Figs. 4E-F and 5E-F). Furthermore, in parental MCF-7 cells, the PKCß-specific inhibitor LY379196 inhibited transcriptional activation of the cyclin D1 promoter and the SRE element in the c-fos promoter (Figs. 4C and 5C).

Cyclin D1 plays a critical role in regulating the progression of cells through the G1 phase of the cell cycle by binding to and activating cyclin-dependent kinase (CDK)-4 or CDK6. This results in phosphorylation of the pRb protein, leading to activation of the E2F family of transcription factors, which, in turn, enhances the progression of cells into the S phase. Thus, increased expression of cyclin D1 can enhance cell cycle progression and cell proliferation (37). Therefore, it is of interest that increased expression of cyclin D1 occurs in ~60% of human breast cancers (17, 18). And in some studies, this is associated with a poor prognosis (20, 21). We found that the AP-1 enhancer element in the cyclin D1 promoter is required for activation of the cyclin D1 promoter by PKCß1 or ß2. Furthermore, constitutively activated mutants of PKCß1 or ß2 activated the SRE element, which is present in the promoter region of the c-fos gene (Figs. 4E and 5B). AP-1 activity is regulated by Fos, Jun, and the activating transcription factor subfamily of transcription factors, which in turn are activated by upstream MAPKs. These findings suggest that PKCß1 and ß2 might exert their stimulatory effects at the level of MAPKs, which is consistent with previous findings from our laboratory (32) and those of other investigators (38, 39). However, the exact site(s) at which PKCß1 and ß2 exert their primary effects in breast cancer cells remains to be determined. We should also emphasize that although some of our studies focused on cyclin D1 (Fig. 4), it is likely that the growth-stimulating effects of PKCß1 and ß2 on breast cancer cells are also exerted via other pathways. Furthermore, our results do not exclude the possibility that other isoforms of PKC might also play a role in enhancing the proliferation of breast cancer cells, although this does not seem to be the case for PKC{delta} (Figs. 1A, 3A, 4E-F, and 5E-F).

Although PKCß1 and ß2 are derived from a single gene by alternative splicing, there is evidence that in some cell systems they may play different roles in cell growth, apoptosis, and cell transformation (40, 41). Thus, in a human gastric cancer cell line, transfection with a PKCß1 antisense oligonucleotide resulted in aberrant cell morphology, suppression of cell growth, and decreased colony-forming ability in vitro (42). In addition, this antisense oligonucleotide inhibited the growth of gastric tumor xenografts in nude mice (42). However, in cells transfected PKCß2 antisense oligonucleotide, there were no significant changes in cell morphology, cell growth, or tumorigenecity (42). Another example is that increased expression of PKCß2 in the colon of transgenic mice leads to hyperproliferation and increased susceptibility to colon carcinogenesis (12), and in gastric cancer cells PKCß1 acts as a survival factor in response to chemotherapeutic agents that induce apoptosis (43, 44). However, in our studies, we did not observe major differences between PKCß1 and ß2 with respect to their in vitro effects on human breast cancer cells. On the other hand, our findings do not exclude the possibility that these two isoforms might exert other differential effects on breast cancers in vivo.

In view of our results, we searched the Oncomine DNA microarray database for relative levels of expression of PKCß mRNA in human breast cancers (45). In one study (46), the levels of this RNA were significantly higher in breast carcinomas than in breast fibroadenomas and normal breast tissues. In another study (47), PKCß expression was significantly higher in lobular carcinoma than in fibroadenoma or human mammary epithelial cells growing in culture. In both studies (46, 47), the data suggest that lobular carcinoma had higher expression of PKCß than did ductal carcinoma or other histologic subtypes of breast cancer. There was, however, no difference in terms of PKCß expression between ER+ and ER– subtypes of breast cancer. However, BRCA1 mutation–positive breast cancers had significantly higher PKCß expression levels than BRCA2 mutation–positive or sporadic breast cancers (48). Thus, BRCA1 mutations may lead to overexpression of PKCß, and this may warrant further studies.

In summary, our findings provide evidence that increased expression of PKCß1 or ß2 in human breast cancer cells may play an important role in enhancing cell cycle progression and cell proliferation. The fact that PKCß1 and PKCß2 stimulated the growth of both ER+ MCF-7 cells and ER– MDA-MB-231 cells suggests that the stimulation of breast cancer cell growth by these isoforms of PKC is independent of ER status. Therefore, inhibitors that target PKCß1 and/or PKCß2 might be useful in the treatment of both ER+ and ER– breast cancers.


   Acknowledgments
 
Grant support: National Cancer Institute grant CA26056-24 and awards from the T. J. Martell Foundation and the National Foundation for Cancer Research (I.B. Weinstein).

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.

Received 6/29/06. Revised 8/28/06. Accepted 9/25/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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