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Carcinogenesis Advance Access originally published online on March 2, 2006
Carcinogenesis 2006 27(8):1538-1546; doi:10.1093/carcin/bgl002
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Overexpression of PKC{alpha} is required to impart estradiol inhibition and tamoxifen-resistance in a T47D human breast cancer tumor model

Xin Lin, Yanni Yu, Huiping Zhao, Yiyun Zhang, Jessica Manela1 and Debra A. Tonetti*

Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago Chicago, IL, USA
1 Robert H. Lurie Comprehensive Cancer Center, Northwestern University Chicago, IL, USA

*To whom correspondence should be addressed at: Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 833 S Wood Street (M/C 865), Chicago, IL 60612, USA. Tel: +1 312 413 1169; Fax: +1 312 996 1698; Email: dtonetti{at}uic.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
We previously reported that stable overexpression of protein kinase C alpha (PKC{alpha}) in hormone responsive T47D:A18 breast cancer cells produces a hormone-independent/tamoxifen (TAM)-resistant and 17ß-estradiol (E2)-inhibitory phenotype in vivo. Furthermore, overexpression of PKC{alpha} in T47D:A18 cells also results in cross-upregulation of PKCs ß and {delta}. In this study, we further characterized the contribution of PKC isozymes {alpha}, ß and {delta} to this complex phenotype. To determine whether downregulation of PKC{alpha} is sufficient to restore the hormone-dependent phenotype in T47D:A18/PKC{alpha} cells, PKC{alpha} was selectively knocked down using short hairpin RNA (shRNA). To determine the contribution of PKCß or {delta} to the hormone-independent/TAM-resistant and E2-inhibitory phenotype, stable T47D:A18/PKCß and T47D:A18/PKC{delta} clones were established. Downregulation of PKC{alpha} by shRNA in T47D:A18/PKC{alpha}20 cells also resulted in reduced PKCß protein expression in vitro. Tumors established from a T47D:A18/PKC{alpha}/shRNA stable clone exhibit 50% reduction of PKC{alpha} protein without concomitant reduction in PKCß, and exhibit partial reversal of the TAM-resistant and E2-inhibitory phenotype in vivo. Furthermore, stable overexpression of neither PKCß nor PKC{delta} in T47D:A18 cells are sufficient to produce hormone-independent growth in vitro or in vivo, nor TAM-resistant and E2-inhibited growth in vivo. Taken together, these results suggest that PKC{alpha} is required to impart the TAM-resistant and E2-inhibitory phenotype in vivo.

Abbreviations: ER{alpha}, estrogen receptor alpha; NS-shRNA, non-silencing shRNA; PKC, protein kinase C; TAM, tamoxifen; shRNA, short hairpin RNA


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tamoxifen (TAM) is an effective endocrine therapy in ~60% of all estrogen receptor alpha (ER{alpha}) positive/progesterone receptor positive breast cancers. The inhibitory effect of TAM is observed almost exclusively in breast tumors that are ER{alpha} positive because estrogen is the major growth stimulator for these types of tumors. However after prolonged antiestrogen hormonal therapy, breast cancer often progresses from an estrogen-sensitive state to an estrogen-insensitive state (1). Although the development of TAM resistance may be a consequence of ER{alpha} loss, TAM resistance most often occurs despite expression of ER{alpha} (2–4). In these cases, constitutive overexpression of autocrine growth factor or growth factor receptor by tumor cells has been proposed as one possible mechanism of TAM resistance (5–14). Increased autocrine or paracrine growth factor signaling networks bypass the classical ER-mediated signaling pathway in human breast cancer cells and result in failure of antiestrogen therapy. For example, preclinical and clinical studies have reported a decreased efficacy of TAM in tumors overexpressing c-ErbB2 (6,14–16) and PC cell-derived growth factor (PCDGF/GP88) (17). Activation of the ras/mitogen-activated protein kinase (MAPK/ERK1/2) pathway known to lie downstream of growth factor receptor signaling can cause ligand-independent activation of ER{alpha} (18–21). Protein kinase C (PKC) isozymes are known to modulate MAPK/ERK1/2 signaling pathways (22,23); PKC-mediated activation of MAPK/ERK1/2 can directly phosphorylate ER Ser-118 (18,21), a site normally phosphorylated in response to estrogen itself by a MAPK-independent mechanism (24). These studies provide additional mechanistic explanations for how signaling by growth factor receptors can result in hormone independence/TAM resistance.

PKC is a family of serine/threonine protein kinases, which mediate a multitude of effects regulating cellular proliferation and differentiation (25). The family consists of 12 isozymes, {alpha}, ßI, ßII, {gamma}, {delta}, {varepsilon}, {xi}, {theta}, {eta}, {iota}, µ and {nu}, which can be classified into three subgroups—conventional, novel and atypical (26). Conventional isozymes are activated by diacylglycerol (DAG), phorbol esters and calcium, and include isozymes {alpha}, ßI, ßII and {gamma}, novel isozymes {delta}, {varepsilon}, {eta}, and {theta} are activated by DAG/phorbol esters but are calcium-independent and atypical isozymes including {zeta} and {iota}/{lambda} are unresponsive to DAG/phorbol esters and calcium. In addition to differences in their cofactor requirements, these isozymes also differ in tissue expression, sub-cellular localization,, and substrate specificity. It is well documented that ER and PKC activity are inversely related in breast cancer cell lines (27) and PKC is elevated in malignant versus normal breast tissue (27–29).

We have shown previously that the ER{alpha}-negative/hormone-independent human breast cancer cell line T47D:C42 has elevated PKC{alpha} protein expression compared with the ER{alpha}-positive/hormone-dependent T47D:A18 clone (30). Stable overexpression of PKC{alpha} in T47D:A18 cells produces clones that are able to grow in the absence of hormonal stimulation and, thus, exhibit autonomous growth in vitro (30). Tumors derived from T47D:A18/PKC{alpha} clones also display autonomous growth, are TAM-resistant and growth inhibited by 17ß-estradiol (E2) in vivo (31). Interestingly, stable overexpression of PKC{alpha} also results in increased endogenous expression of PKC isozymes ß and {delta} (30). It was reported previously that stable transfection of PKC{alpha} results in upregulation of PKCß in MCF-7 cells (32,33) and PKC{delta} in murine lymphoma and promylelocytic cells (34). To study the contribution of PKCs {alpha}, ß and {delta} to the growth phenotype of T47D:A18/PKC{alpha} cells and tumors, two approaches were employed: (i) PKC{alpha} expression in the T47D:A18/PKC{alpha}20 clone was selectively knocked down by using short hairpin RNA (shRNA) technology and (ii) Stable PKCs ß and {delta} clones were established in T47D:A18 and the hormone-responsive phenotypes of these clones were examined.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines and culture conditions
T47D:A18 is a hormone-dependent human breast cancer cell clone that was described previously (35). T47D:A18/neo and T47D:A18/PKC{alpha}20 clones have been described previously (30). T47D:A18, T47D:A18/neo and T47D:A18/PKC{alpha}20 clones were maintained in RPMI-1640 phenol-red medium supplemented with 10% fetal bovine serum (FBS). T47D:A18/neo and T47D:A18/PKC{alpha}20 clones were also supplemented with G418 (500 µg/ml). Prior to cell proliferation assays, all cell lines were maintained in phenol red-free RPMI-1640 supplemented with 10% 3x dextran-coated charcoal (DCC)-treated FBS (estrogen-depleted media) for 3 days. Prior to transient transfection experiments for northern blots, all cell lines were grown in complete medium containing 10% FBS.

RNA isolation and northern blot
PKCs {alpha}, ß and {delta} cDNAs were cloned in order to synthesize the antisense riboprobes. Total RNA was isolated from the T47D:A18 clones using the Triazol reagent (Invitrogen, Carlsbad, CA). PKCs ß and {delta} cDNA fragments were obtained from total RNA by RT–PCR by using the following primers sets: PKCß-forward (5'-CAGAAGGCCAGTGTTGATGGCTGG-3') and PKCß-reverse (5'-TCTGTTGCCATTGTTGTCAAATTT-3'); PKC{delta}-forward (5'-GCCTTCAACTCCTATGAGCTGGGC-3') and PKC{delta}-reverse (5'-GTCCTCCAGGAAATACTGAACAGA-3'). The 1.3 kb EcoRI fragment of the PKC{alpha} gene was obtained by digestion of the PKC{alpha} clone (obtained form ATCC, Rockville, MD) with EcoRI. The resulting DNA fragments were cloned into the pGEM-T vector (Promega, Madison, WI) to obtain the PKCs {alpha}, ß and {delta} recombinant plasmids. After sequencing and determination of the insert orientation, the recombinant plasmids were used to synthesize DIG-labeled antisense riboprobes by the RNA labeling kit according the manufacturer's instructions (Roche Diagnostics Corporation, Indianapolis, IN). For gene expression studies, total RNA (10–20 µg) was isolated from T47D cell lines and electrophoresed on 1% agarose gels containing 2.2 M formaldehyde, transblotted onto nylon membranes using the TransBlot system (BioRad Laboratories, Hercules, CA) and fixed to the membrane by UV cross-linking. Hybridization was carried out at 68°C for 16 h in 50% formamide hybridization solution containing 20 ng/ml RNA probe. Following hybridization, the membrane was washed twice at room temperature with 2x SSC buffer (30 mM sodium citrate (pH 7.0) and 300 mM NaCl) containing 0.1% SDS and twice at 68°C with 0.5x SSC buffer containing 0.1% SDS. For detection, anti-DIG-AP antibody was used in conjunction with CDP-star chemiluminescent reagent. Bands were quantified by Scion image software (Scioncorp.com).

Construction of shRNA plasmids
The pSUPER vector, which directs the synthesis of shRNAs, was a gift from Dr Yin Mo (Southern Illinois University) (36). The shRNA sequences of human PKC{alpha} corresponded to the coding regions 492–510 relative to the first nucleotide of the start codon (37). The sense oligonucleotide sequence containing the human PKC{alpha} shRNA pair (underlined sequence) is as follows: 5'-GATCCCC(AAAGGCTGAGGTTGCTGAT)TTCAAGAGA(ATCAGCAACCTCAGCCTTT)TTTTTGGAAA-3', and the antisense oligonucleotide sequences containing the human PKC{alpha}shRNA sequence is AGCTTTTCCAAAAA(AAAGGCTGAGGTTGCTGAT)TCTCTTGAA(ATCAGCAACCTCAGCCTTT)GGG-3'. The non-silencing shRNA control sequences were designed according to the non-silencing shRNA (NS-shRNA) sequence available at www.qiagen.com. The sense oligonucleotide sequence containing NS-shRNA pair (underlined sequence) is GATCCCC(TTCTCCGAACGTGTCACGT)TTCAAGAGA(ACGTGACACGTTCGGAGAA) TTTTTGGAAA, and antisense oligonucleotide containing NS-shRNA pair (underlined sequence) is 5'-AGCTTTTCCAAAAA(TTCTCCGAACGTGTCACGT)TCTCTTGAA(ACGTGACACGTTCGGAGAA)GGG-3'.

To generate shRNA duplexes, 0.2 µM sense and antisense oligonucleotides were annealed by incubating the mixed oligonucleotides in the PCR thermocycler using the following profile: 95°C for 30 min, 80°C for 30 min, 70°C for 30 min and room temperature for another 30 min. Double stranded oligonucleotides were subsequently cloned into the pSUPER plasmid in the frame of the BglII and HindIII sites. The inserts were screened by restriction enzyme digestion with EcoRI and HindIII and confirmed by sequencing with M13 forward and reverse primers (DNA Sequencing Facility, University of Chicago).

Partial PKC purification and western blot
A PKC-enriched fraction was purified from at least 4 x 108 cells by DEAE-cellulose anion exchange column chromatography as previously described (38). Protein concentration was measured using the BCA assay (Pierce, Rockford, IL). An 8% SDS–polyacrylamide gel was loaded with equal amounts of protein (50–100 µg/lane) and biotin-labeled molecular weight standards to approximate protein size. Following electrophoresis, protein was transferred to Hybond-ECL nitrocellulose membranes (Amersham Life Sciences, Buchinghamshire, England) by semi-dry electroblot transfer. PKC isozyme polyclonal antibodies {alpha}, ß and {delta} (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted 1:500 in TBS-T [20 mM Tris (pH 7.6), 137 mM NaCl and 0.1% Tween-20] containing 5% dry milk. The Supersignal western blotting detection system (Pierce, Rockford, IL) was used to visualize immunoreactive bands. Equal loading of total protein was assessed by stripping and reprobing the membrane with a ß-actin monoclonal antibody (Sigma Chemical. St Louis. MO).

Generation of T47D:A18/PKCß, T47D:A18/PKC{delta} and T47D:A18/PKC{alpha}/shRNA stable transfectants
The pSPKCs ßII and {delta} expression plasmids were generously provided by Eli Lilly (39). pSUPER-PKC{alpha}shRNA and pSUPER-NS-shRNA plasmids were constructed as described in the previous section. To generate the stable PKC{alpha}-shRNA clones in T47D:A18/PKC{alpha}20 cells, the pSUPER-PKC{alpha}-shRNA construct was co-transfected with pcDNA3.1/LacZ plasmid (Invitrogen, Carlsbad, CA) into T47D:A18/PKC{alpha}20 cells by electroporation (250 V, 950 µF). pSUPER or pSUPER-NS-shRNA was also transfected by electroporation into T47D:A18/PKC{alpha}20 cells as vector or non-silencing shRNA control. The transfection mixture was added to 10 ml RPMI-1640 media (phenol red +) containing 10% FBS and seeded into 100 mm tissue culture dishes. Following incubation for 2 days, the medium was replaced with medium containing both G418 (500 µg/ml) and hygromycin B (100 µg/ml). Individual colonies were picked following 3 weeks of selection and screened for PKCs {alpha}, ß and {delta} expression by western blot analysis. To generate PKCß or {delta} stable clones in T47D:A18 cells, pSPKCs ßII, {delta} or pSVHNX-neo (control) (39) were transfected into T47D:A18 cells by electroporation. Clones designated T47D:A18/neo, T47D:A18/ß13, T47D:A18/ß16, T47D:A18/{delta}14, T47D:A18/PKC{alpha}20/shRNA#19 and /PKC{alpha}shRNA#20 were chosen for further characterization.

Proliferation assay
The cell clones T47D:A18/neo, T47D:A18/PKC{alpha}20, T47D:A18/PKCß13 or 16, T47D:A18/{delta}14 and T47D:A18/PKC{alpha}20/shRNA#19 or /shRNA#20 were seeded at 3 x 104 cells/ml estrogen-depleted media into T25 tissue culture flasks. The following day (day 1) medium containing either ethanol (control), E2 (10–9 M) or 4-hydroxytamoxifen (4-OHT) (10–7 M) was added. All compounds were dissolved in 100% ethanol and added to the medium at a 1:1000 dilution. Cells were counted on Day 2–10.

Establishment of T47D:A18/PKCß, PKC{delta}, T47D:A18/PKC{alpha}/shRNA#19 and/shRNA#20 tumors in athymic mice
T47D:A18/neo, T47D:A18/PKCß13, T47D:A18/PKC{delta}14, T47D:A18/PKC{alpha}20 and T47D:A18/PKC{alpha}20/PKC{alpha}shRNA#19 or /shRNA#20 cells were injected s.c. (1 x 107 cells/site) into the axillary mammary fat pads of ovariectomized 5–6-week-old BALB/c athymic mice (Harlan Sprague Dawley, Madison, WI). Mice were divided into three treatment groups consisting of 10 mice/group: control (no treatment); E2 (E2 capsule); or TAM. E2 was administered via silastic capsules (1.0 cm) implanted s.c. between the scapules and the capsules were replaced every 8 weeks. TAM was administered p.o. at a dose of 1.5 mg/animal daily for 5 days as described previously (31). Tumor cross-sectional area was determined weekly by Vernier calipers and calculated using the formula: (length/2) x (width/2) x {pi}. Mean tumor area was plotted against time in weeks to monitor tumor growth. The mice were killed by CO2 inhalation and cervical dislocation. Tumors were excised and snap frozen in liquid nitrogen and stored at –80°C.

Statistical analysis
When comparing with one group, data were analyzed using unpaired t-test. When comparing groups, data were analyzed using One-way Analysis of Variance (ANOVA) followed by the Dunnett multiple comparison test. All statistics were performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software, San Diego California USA). Significant differences were indicated when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
shRNA-mediated degradation of the PKC{alpha} mRNA in T47D:A18/PKC{alpha}20 cells
To determine whether the pSUPER-PKC{alpha}-shRNA construct could produce shRNA specific to PKC{alpha} mRNA degradation, T47D:A18/PKC{alpha}20 cells were transiently transfected with pSUPER, pSUPER-NS-shRNA and pSUPER-PKC{alpha}-shRNA constructs. The cells were harvested 48 or 60 h post-transfection, and PKCs {alpha}, ß and {delta} mRNA levels were measured by northern blot analysis. More than 80% of the PKC{alpha} mRNA was degraded when cells were transfected with the pSUPER-PKC{alpha}-shRNA construct (10 µg) for 48 h (Figure 1A). PKC{alpha} mRNA was not affected by either pSUPER vector or non-silencing RNA construct (pSUPER-NS-shRNA) at the same dose for the same period of time. Interestingly, PKCß mRNA expression was reduced to 61% when the cells were transfected with 10 µg of the pSUPER-PKC{alpha}-shRNA construct compared with the pSUPER-NS-shRNA control vector at 48 h post-transfection and this reduction was statistically significant (Figure 1C). However, no effect on PKCß mRNA expression was observed at 60 h post-transfection suggesting the knockdown was modest and transient. Expression of PKC{delta} mRNA was not affected. (Figure 1A). These results suggest that the PKC{alpha}-shRNA also targeted PKCß mRNA destruction, even though the target sequence was in a region not homologous to PKCß. Alternatively, PKCß mRNA may be transcriptionally downregulated secondary to the decrease in PKC{alpha} expression. There is evidence in the literature for cross-regulation between PKC isozymes (32–34).


Figure 1
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  		Fig. 1 shRNA-mediated degradation of PKC{alpha} mRNA in T47D:A18/PKC{alpha}20 cells. T47D:A18/PKC{alpha}20 cells were transiently transfected with pSUPER, pSUPER-NS-shRNA and pSUPER-PKC{alpha}-shRNA constructs. PKCs {alpha}, ß and {delta} mRNA levels were measured 48 and 60 h post-transfection by northern blot analysis. (A), northern blot analysis representative of three independent experiments; (B), ethidium bromide (EtBr) stained rRNA. (C), fold PKCß mRNA expression relative to pSUPER vector and pSUPER-NS-shRNA control vectors from three independent northern blot analyses. *P < 0.05 using ANOVA. **P < 0.05 using the Dunnett multiple comparisons test post-test.

 
Generation T47D:A18/PKC{alpha}20/shRNA stable transfectants
To verify that reduction of the PKC{alpha} mRNA corresponded to a decrease in PKC{alpha} protein, pSUPER-PKC{alpha}-shRNA and pcDNA3.1/LacZ were stably co-transfected into T47D:A18/PKC{alpha}20 cells, and individual clones were selected in the medium containing G418 (500 µg/ml) and hygromycin (100 µg/ml) for 2 months. Several stable clones were screened by western blot analysis for PKC isozyme expression. T47D:A18/PKC{alpha}20/shRNA stable clone nos 19 and 20 exhibited a 50% reduction of PKC{alpha} protein expression compared with parental T47D:A18/PKC{alpha}20 cells (Figure 2). Consistent with the observed effect of PKC{alpha}-shRNA on PKCß mRNA expression, endogenous PKCß protein was also decreased in these stable clones when using a PKCß1 antibody from Santa Cruz Biotechnology (Figure 2). However, a PKCß monoclonal antibody from BD Biosciences produced variable expression of PKCß protein in the stable cell lines and in some instances there was no difference in PKCß expression (results not shown). Therefore, in light of the northern blot result that shows PKC{alpha}-shRNA downregulates PKCß mRNA expression, and consistent protein PKCß downregulation with at least one PKCß-specific antibody, we feel investigation of the role of PKCß is important.


Figure 2
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  		Fig. 2 Western blot analysis of PKC isozyme expression in T47D:A18/PKC{alpha}20/shRNA stable cell clones. (A), PKC protein was partially purified from T47D:A18 (lane 1), T47D:A18/neo (lane 2), T47D:A18/PKC{alpha}20 (lane 3), T47D:A18/PKC{alpha}20/shRNA#19 (lane 4) and T47D:A18/PKC{alpha}20/shRNA#20 (lane 5) stable clones by DEAE-cellulose chromatography as described in Materials and methods. Western blot analysis was performed using specific antibodies to PKCs {alpha}, ßI and {delta} isozymes. Equal protein loading was assessed by re-probing with a ß-actin monoclonal antibody. (B), Quantitation of the bands in the panel A by Scion image software. The results are expressed as mean ± SE. Asterisk indicates statistical difference compared with T47D:A18/PKC{alpha}20 cells.

 
T47D:A18/PKC{alpha}20/shRNA stable clones do not reverse autonomous growth in vitro
We have reported that overexpression of PKC{alpha} in T47D:A18 produces hormone-independent growth in cell culture or autonomous growth (30). PKC{alpha}-shRNA clones exhibited downregulation of both PKCs {alpha} and ß, therefore reversal of the autonomous growth phenotype of the T47D:A18/PKC{alpha}20/shRNA stable clones were assessed by proliferation assays. As expected, all clones grew in the presence of E2 (Figure 3A). However, in the absence of E2, although both shRNA clones grow at a slower rate, neither of the clones exhibited reversal of autonomous growth (Figure 3B). These results suggest that 50% reduction of PKCs {alpha} and ß protein expression is not sufficient to reverse the autonomous growth of T47D:A18/PKC{alpha} cells in vitro.


Figure 3
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  		Fig. 3 Proliferation rate of T47D:A18/PKC{alpha}/shRNA clones grown in (A) E2-containing medium and (B) E2-depleted medium. Proliferation rate was assessed by cell counting as described in Materials and methods. The results are expressed as total cell number ± SE for each time point. These results are representative of three independent experiments.

 
T47D:A18/PKC{alpha}20/shRNA stable clones exhibit partial reversal of the TAM-resistant and E2 inhibitory phenotypes in vivo
We have reported that tumors derived from the T47D:A18/PKC{alpha}20 clone grow in the absence of E2 (autonomous growth), in the presence of TAM, and these tumors are growth inhibited by E2 (31). To determine whether downregulation of PKCs {alpha} and ß can reverse this phenotype in vivo, we injected T47D:A18/PKC{alpha}20/shRNA stable clones into the mammary fat pads of ovariectomized athymic mice. The mice were divided into three treatment groups; no treatment (controls), E2 (1.0-cm capsule) or TAM (1.5 mg/day, p.o. five times per week). The T47D:A18/PKC{alpha}/shRNA#19 clone exhibited partial reversal of the TAM-resistant and E2 inhibitory phenotype in vivo (Figure 4A). However, the T47D:A18/PKC{alpha}/shRNA#20 clone remained TAM-resistant, hormone-independent and growth inhibited by E2 (Figure 4B). The partial phenotypic change exhibited by the T47D:A18/PKC{alpha}/shRNA#19 tumor correlated with 50% reduction of PKC{alpha} expression, whereas no reduction of PKC{alpha} expression is evident in the T47D:A18/PKC{alpha}/shRNA# 20 tumors (Figure 4C and D). Therefore, reduction in PKC{alpha} protein correlates with the partial reversal of phenotype observed with the T47D:A18/PKC{alpha}/shRNA#19 tumor. Interestingly, downregulation of PKCß was not observed in either of the PKC{alpha}-shRNA tumors compared with parental T47D:A18/PKC{alpha}20 tumors (Figure 4C and D). These results suggest that 50% reduction of PKC{alpha} alone is sufficient to partially reverse the TAM-resistant and E2-inhibitory phenotype of T47D:A18/PKC{alpha} tumors. However, the partial knockdown achieved in the T47D:A18/PKC{alpha}/shRNA#19 clone may not be sufficient to reverse the autonomous growth phenotype either in vivo (Figure 4A) or in vitro (Figure 3B).


Figure 4
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  		Fig. 4 Initiation and growth of tumors in athymic mice. (A), T47D:A18/PKC{alpha}20/shRNA#19, or (B), T47D:A18/PKC{alpha}20/shRNA#20. A total of 30 mice received injections of each cell line and were treated (10 mice/group) for 7 weeks with a 1 cm E2 capsule (E2 group; open circles), 1.5 mg p.o. TAM (TAM group; closed triangles), or left untreated (control group; closed circles). (C), western blot analysis of PKC isozymes partially purified from tumors derived from T47D:A18/PKC{alpha}20 (lane 1); T47D:A18/PKC{alpha}20/shRNA#19 (lane 2); T47D:A18/PKC{alpha}20/shRNA#20 (lane 3). (D), Bands were quanitified using Scion image software. Results represent three independent experiments, and are expressed as mean ± SE. Asterisk indicates statistical difference compared with T47D:A18/PKC{alpha}20 cells.

 
Establishment of PKCs ß and {delta} stable transfectants in T47D:A18
We have reported previously that stable transfection of PKC{alpha} in T47D:A18 cells results in concomitant upregulation of PKCs ß and {delta} (30). Other laboratories have also documented cross-regulation of other PKC isozymes by the introduction of PKC{alpha} (32–34). To ascertain the contribution of PKCs ß and {delta} in the manifestation of the autonomous, TAM-resistant and E2-inhibitory phenotype, we have established T47D:A18/PKCß and T47D:A18/PKC{delta} stable clones and examined PKC isozyme expression by western blot analysis. T47D:A18/PKCß13 and T47D:A18/PKCß16 clones exhibit upregulation of both PKCs ßI and ßII, whereas the T47D:A18/PKC{delta}14 clone shows upregulation of PKC{delta} as well as cross-upregulation of endogenous PKCßI (Figure 5). The stable PKCs ß and {delta} clones express minimal or undetectable levels of PKC{alpha} protein compared with the T47D:A18/PKC{alpha}20 clone (Figure 5).


Figure 5
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  		Fig. 5 Western blot analysis of PKC isozymes expression in T47D:A18/PKCß and /PKC{delta} stable cell clones. PKC protein was partially purified from T47D:A18/neo, T47D:A18/PKC{alpha}, T47D:A18/PKCß and T47D:A18/PKC{delta} stable clones by DEAE-cellulose chromatography as described in Materials and methods. Western blot analysis was performed using specific antibodies to PKCs {alpha}, ß and {delta} isozymes proteins. Equivalent protein loading was assessed by re-probing with ß-actin antibody.

 
To determine the contribution of PKCs ß and {delta} to the autonomous growth phenotype, hormone responsiveness was determined in T47D:A18/PKCß and /PKC{delta} stable clones by cell proliferation assay. T47D:A18/neo, T47D:A18/PKCß and /PKC{delta} clones were unable to grow in estrogen-depleted medium or in the presence of 4-hydroxytamoxifen (4-OHT) (Figure 6A, C and D). However, these clones grow in response to E2 treatment, thereby exhibiting a hormone-dependent growth phenotype. Only T47D:A18/PKC{alpha}20 clones were able to grow in estrogen-depleted medium and in medium containing 4-OHT and E2 (Figure 6B), exhibiting autonomous growth and partial TAM-resistance.


Figure 6
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  		Fig. 6 Proliferation rate of stable clones. (A), T47D:A18/neo; (B), T47D:A18/PKC{alpha}20; (C), T47D:A18/PKCß13 and (D), T47D:A18/PKC{delta}14 stable clones grown in E2-depleted medium with or without treatment with E2 or 4-OHT. The results are expressed as total cell number ± SE for each time point. These results are representative of three independent experiments. Asterisk indicates statistical difference among groups at P < 0.05.

 
PKCs ß or {delta} stable clones exhibit a hormone-dependent phenotype in vivo
We have shown that overexpression of PKCß and/or {delta} in T47D:A18 results in a hormone-dependent phenotype in cell culture. To determine whether this phenotype is maintained in vivo, we injected T47D:A18/PKCß13 or T47D:A18/PKC{delta}14 cells into the mammary fat pads of ovariectomized athymic mice. Seven weeks after the injection of T47D:A18/neo cells, E2 significantly stimulated tumor growth compared with the control and TAM groups (Figure 7A), which is consistent with previous findings (31). Eight weeks post-injection of T47D:A18/PKCß or /PKC{delta} cells, tumor growth was observed only in the E2 group (Figure 7B, C). No tumor was detected in any of the mice in the Control or TAM groups, exhibiting similar growth characteristics as the parental T47D:A18 cells. These results indicate that overexpression of PKCß and/or {delta} is not sufficient to impart the phenotype observed in the PKC{alpha} overexpressing tumors in vivo.


Figure 7
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  		Fig. 7 Initiation and growth of tumors derived from (A), T47D:A18/neo; (B), T47D:A18/PKCß13; or (C), T47D:A18/PKC{delta}14 grown in athymic mice (mean ± SE). A total of 30 mice received injections of each cell line and were treated (10 mice/group) for 7–8 weeks with a 1 cm E2 capsule (E2 group; open circles), 1.5 mg TAM, p.o. (TAM group; closed triangles), or left untreated (control group; closed circles). (A) and (C), E2 significantly stimulated tumor growth [(A), Weeks 4–7, P < 0.01; (C) Weeks 2–8, P < 0.05] compared with untreated control or TAM-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
TAM resistance is a considerable obstacle in the management of breast cancer. Therefore, identification of key molecules involved in TAM-resistance will be the critical first step in the development of a logical targeted therapy (31). We recently reported that elevated PKC{alpha} expression may be predictive of TAM treatment failure (40). Our in vivo T47D:A18/PKC{alpha}20 tumor model exhibits autonomous, TAM-resistant and E2-inhibitory growth characteristics (31). However, in addition to PKC{alpha}, the T47D:A18/PKC{alpha}20 clone also exhibits increased endogenous expression of PKCs ß and {delta} (30). To explore the contribution of PKCs {alpha}, ß and {delta} to the autonomous, TAM-resistant and E2-inhibitory growth phenotype, two approaches were taken; (i) PKC{alpha} was specifically downregulated in T47D:A18/PKC{alpha}20 cells by shRNA and (ii) T47D:A18 cells were stably transfected with either PKCß or {delta}.

RNA silencing or interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded (dsRNA) molecules (41). To determine whether downregulation of PKC{alpha} alone is sufficient to reverse the growth characteristics of T47D:A18/PKC{alpha}20 cells, shRNA technology was utilized. The pSUPER-PKC{alpha}-shRNA construct produced shRNA that knocked down PKC{alpha} mRNA and protein by 80 and 50%, respectively. In cell culture, we found that PKC{alpha}-shRNA also resulted in downregulation of PKC ß mRNA and protein, whereas PKC{delta} expression was unaffected. One possible explanation for this observation is the cross-regulation of PKC isozymes known to exist in cell systems as reported previously (34). Reduction in PKCs {alpha} and ß in cell culture is not sufficient to reverse autonomous growth in vitro (Figure 3). Interestingly, although tumors were derived from both T47D:A18/ PKC{alpha}/shRNA constructs, only the T47D:A18/PKC{alpha}/shRNA#19 clone produced tumors exhibiting 50% knockdown of PKC{alpha} protein. Furthermore, these tumors do not display concomitant PKCß downregulation (Figure 4C). It is only these T47D:A18/PKC{alpha}/shRNA#19 tumors that also exhibit reversal of TAM-resistance and E2-inhibition (Figure 4A). The growth characteristics of the T47D:A18/PKC{alpha}/shRNA#20 tumors are not altered and this correlates with no reduction in PKC{alpha} or ß protein (Figure 4B–D). Despite the reduction of PKC{alpha} protein observed in the T47D:A18/PKC{alpha}/shRNA#19 tumors, these tumors retain the autonomous growth characteristic and are able to grow in the absence of hormonal stimulation. This finding is consistent both in vitro and in vivo (Figures 3B and 4B) and suggests that perhaps 50–80% PKC{alpha} downregulation is not sufficient to reverse this phenotype. Since the T47D:A18 cells have virtually undetectable levels of PKC{alpha} protein as determined by western blot analysis [Figure 5; (30)], it is not surprising that a 50% reduction of PKC{alpha} would not restore the parental phenotype entirely. The apparent lack of PKC{alpha} knockdown in the T47D:A18/PKC{alpha}/shRNA#20 tumors may be due to loss of the PKC{alpha}-shRNA construct during development of the tumors. These results suggest that PKC{alpha} is required to mediate the TAM-resistant and E2-inhibitory phenotype in T47D:A18 cells. The partial reversal of the TAM-resistant and E2-inhibitory phenotype may be due to the incomplete knockdown of PKC{alpha} and/or potential synergistic effects with PKCß and/or {delta}. This possibility is supported by studies showing that both PKCs {alpha} and {delta} are highly expressed in three out of four antiestrogen resistant MCF-7 cell lines (42). However, in our cell and tumor models, PKC{alpha} appears to play a dominant role in the acquisition of TAM-resistance, autonomous growth and E2 inhibitory growth characteristics. Interestingly, the PKC{alpha} gene, PRKCA, maps to chromosome 17q22–q23.2 according to the genome-draft assignment, and was fine-mapped by flourescence in situ hybridization (FISH) analysis to be located at 17q24 (43). Several genes involved in the pathogenesis of malignant breast cancer are found on chromosome 17 including p53, Her-2/neu/ERBB2, BRCA1 and nm23 (44). It is interesting to speculate whether upregulation of PKC{alpha} occurs along with alterations of these well-known breast-cancer-associated genes in this highly mutation-susceptible locus in breast cancer. Using dominant negative mutants of PKC{alpha} in MCF-7 cells, PKC{alpha}-specific downstream signaling targets were identified (45). In these experiments, the dominant negative PKC{alpha} construct downregulated the anti-apoptotic bcl-2 protein and sensitized cells to apoptosis in response to TAM. In addition, the cyclin-dependent kinase inhibitor, p21cip1, was also downregulated. The authors conclude that PKC{alpha} overexpression leads to upregulation of both bcl-2 and p21cip1, thus, protecting cells from apoptosis. These effects were not observed using dominant negative PKC{varepsilon} or {zeta} constructs. Other PKC{alpha} downstream signaling molecules implicated in TAM-resistance include AP-1 (46–48), PI3K/Akt (49,50), MAPK (51,52) and NF{kappa}B (53–55). We demonstrated that the T47D:A18/PKC{alpha}20 cells exhibit elevated basal AP-1 activity compared with the T47D:18/neo vector control cells (30). In addition, elevated phospho-Akt protein levels were observed in TAM-treated T47D:A18/PKC{alpha}20 tumors compared with tumors treated with E2 (manuscript submitted). However no difference in expression of total and phosphorylated ERK1/2 (phospho-p44/42) nor NF{kappa}B signaling components (phospho-NF{kappa}Bp65, Ser32phospho-I{kappa}B) was observed in T47D:A18/PKC{alpha}20 and T47D:A18/neo cells (unpublished observations). We continue to explore and attempt to connect the molecular signaling pathways initiated by PKC{alpha} leading to TAM-resistant, E2-inhibitory, autonomous growth.

In summary, we find that PKCß and/or {delta} overexpression is not sufficient to impart autonomous, TAM-resistant and E2-inhibited growth to T47D:A18 cells and tumors. However, partial suppression of PKC{alpha} results in partial reversal of TAM-resistance and E2-growth inhibition. This finding may have important therapeutic implications. TAM has been used to treat all stages of ER{alpha}-positive breast cancer (56); however, high-dose estrogen also causes tumor regression in post-menopausal women with ER{alpha}-positive breast cancer (57,58). We reported previously that overexpression of PKC{alpha} may predict TAM treatment failure (40) and, therefore, may be a prognostic indicator for TAM-resistance. Furthermore, the E2-induced tumor regression observed in our pre-clinical xenograft model suggests that perhaps selection of patients with PKC{alpha}-overexpressing tumors may greatly improve the efficacy of high-dose estrogen treatment, a therapeutic approach likely to be superior to TAM in this situation. Perhaps treatment of breast cancer with the PKC{alpha} antisense oligonucleotide Affinitak (LY900003/ISIS 3521) (59) can be improved by identification of patients who exhibit PKC{alpha} overexpression. Finally, the recent favor of aromatase inhibitor therapy over TAM (60–62) is another potential concern for patients harboring tumors that overexpress PKC{alpha}. Eliminating all estrogen may in fact cause tumor growth. We are currently in the process of developing a pre-clinical model to address the utility of aromatase inhibitors in this setting and determining the signaling pathway mediated by PKC{alpha} leading to E2-induced tumor regression. Elucidation of the mechanism is likely to lead to additional therapeutic targets for the treatment of breast cancer.


   Acknowledgments
 
The generous gift of the pSPKC expression plasmids was provided by Lilly Research Laboratories (Indianapolis, IN). We thank Dr Yin Mo for providing the pSUPER vector and technical advice in the construction of pSUPER-shRNA plasmids. The authors would like to thank Luci Wan for assistance with isolation of total RNA, Su-Young Choi for the assistance with northern blot analyses, Margaret Sharp for assistance with the generation of PKCs {alpha}, ß and {delta} RNA probes, and Ahmed Mahafzah for technical assistance. This work was supported in part by NIH RO1 CA79847 and the University of Illinois Campus Research Board.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 7, 2005; revised February 21, 2006; accepted February 25, 2006.


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