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Cancer Research 67, 5308, June 1, 2007. doi: 10.1158/0008-5472.CAN-06-3936
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Protein Kinase C{alpha} Determines HER2 Fate in Breast Carcinoma Cells with HER2 Protein Overexpression without Gene Amplification

Alessandra Magnifico1, Luisa Albano1, Stefano Campaner3, Manuela Campiglio1, Silvana Pilotti2, Sylvie Ménard1 and Elda Tagliabue1

1 Molecular Targeting Unit, Department of Experimental Oncology and 2 Department of Pathology, National Cancer Institute, Foundation IRCCS; 3 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

Requests for reprints: Sylvie Ménard, Molecular Targeting Unit, Department of Experimental Oncology, National Cancer Institute, Via Venezian 1, Milan 20133, Italy. Phone: 39-02-23902572; Fax: 39-02-23903073; E-mail: sylvie.menard{at}istitutotumori.mi.it.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In some HER2-positive breast tumors, cell surface overexpression of HER2 is not associated with gene amplification but may instead rest in altered gene transcription, half-life, or recycling of the oncoprotein. Here, we show that HER2 overexpression in HER2 2+ carcinomas is associated with neither an increase in gene transcription nor a deregulation in the ubiquitin-dependent pathways, but instead seems to be regulated by protein kinase C{alpha} (PKC{alpha}) activity. The stimulation of PKC{alpha} up-regulated HER2 expression, whereas PKC{alpha} inhibition by pharmacologic treatments and PKC{alpha}-specific small interfering RNA led to a dramatic down-regulation of HER2 levels only in breast cancer cells HER2 2+. Consistent with the in vitro data, our biochemical analysis of HER2 2+ human primary breast specimens revealed significantly higher levels of phosphorylated PKC{alpha} compared with HER2-negative tumors. Inhibition of HER2 activation by the tyrosine kinase inhibitor lapatinib led to decreased levels of PKC{alpha} phosphorylation, clearly indicating a cross-talk between PKC{alpha} and HER2 molecules. These data suggest that HER2 overexpression in HER2 2+ carcinomas is due to an accumulation of the recycled oncoprotein to the cell surface induced by activated PKC{alpha}. [Cancer Res 2007;67(11):5308–17]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer remains the most frequent malignancy among North American and European women. One of the genetic abnormalities underlying the progression from normal breast epithelia to invasive cancer cells is amplification of the HER2 oncogene (1, 2). Recent data, including microarray results, have shown that HER2-positive breast carcinomas represent a particular subset with distinct clinical and biological characteristics. HER2-positive tumors can be classified as HER2 1+, HER2 2+, and HER2 3+ depending on the level of HER2 expression based on immunohistochemical analysis (3). Whereas 90% of HER2 3+ breast tumors show oncogene amplification, 80% of HER2 2+ breast tumors do not show this amplification (4). HER2-positive tumors are particularly aggressive, with increased proliferation and metastatic potential (5–8), and a better understanding of the biology of these tumors is crucial in improving and optimizing their treatment. In fact, not all patients with HER2-positive tumors are responsive to new therapies targeting the oncoprotein (9). Specifically, HER2 2+ tumors do not respond to therapeutic treatment with Herceptin, the humanized monoclonal antibody (mAb) specific for HER2.

Here, we examined the possible biological mechanisms underlying HER2 expression in HER2-overexpressing tumor cells without gene amplification (2+) compared with HER2-overexpressing breast carcinoma cell lines with gene amplification (3+) and HER2 low-expressing breast carcinoma cell lines (1+). The serine/threonine protein kinase C (PKC) enzymes have been linked to tumorigenesis based on the observation that PKC activators, such as phorbol 12-myristate 13-acetate (PMA), can act as tumor promoters. Potentiation of the malignant phenotype may be mediated by activation of selective PKC isoenzymes or through altered isoenzyme expression profiles (10). The PKC{alpha} and PKCß isoenzymes have often been linked to malignant phenotype (11), whereas PKC{delta} is thought to mediate anticancer effects (12). PKC{alpha} has been implicated in the regulation of a variety of cellular functions, such as proliferation, differentiation, and apoptosis, in response to a diverse range of stimuli. The regulatory effects of PKC{alpha} activity on these functions are modulated by the functional interaction of the enzyme with several proto-oncogenes (13, 14). In addition, PKC{alpha} has been proposed to divert internalized HER1 molecules from a degradative fate to a recycling pathway (15). Considering PKC{alpha} as the major player of HER2 fate in HER2 2+ tumors, we show here that (a) PKC{alpha} activity regulates HER2 expression levels of HER2 2+ cells; (b) the two proteins are constitutively associated; (c) only the active form of PKC{alpha} determines the dynamic recycling of HER2 on the tumor cell surface; and (d) HER2 constitutively activates PKC{alpha} enzyme activity. Our findings point to the potential therapeutic usefulness of tyrosine kinase inhibitors (TKI) in HER2 2+ breast tumors to block the positive feedback between HER2 and PKC{alpha}.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human breast cancer cell lines MDA-MB175, BT20 (16), MDA-MB453, SKBr3, MCF7, MDA-MB231, and human embryonic kidney cell line HEK-293 (all from American Type Culture Collection) were grown in RPMI 1640 (Invitrogen) supplemented with 4 mmol/L glutamine and 10% FCS (Invitrogen) at 37°C in a 5% CO2. For culture of human breast cancer cell line ZR75-1 (16), medium was supplemented with 10 mmol/L HEPES and 1 mmol/L sodium pyruvate. In all experiments, cells were used when 70% confluent.

Antibodies and reagents. Anti-HER2 mouse mAb Ab3 (Calbiochem) was used for Western blotting at 1 µg/mL. mAb MGR2, raised against the extracellular domain of HER2, was developed in our laboratory (17) and used in immunofluorescence assay at 10 µg/mL and in immunoprecipitation assay at 5 µg/mg whole-cell lysates. Anti-human leucocyte antigen (HLA) mAb (Gene Tex) was used in immunofluorescence at 10 µg/mL. Anti-PKC{alpha} mAb (H-7) was used for Western blotting at 1 µg/mL; anti-PKC{delta} polyclonal antibody (C17) was used for Western blotting at 1 µg/mL; and anti-c-Cbl (C15) was used for Western blotting at 1 µg/mL (all from Santa Cruz Biotechnology). Anti-PKC{alpha} mAb (BD Bioscences Pharmingen) was used for immunoprecipitation at 5 µg/mg whole-cell lysates. Polyclonal anti–phosphorylated PKC{alpha} (P-PKC{alpha}; Ser657) antibody was used for Western blotting at 2 µg/mL and anti–phosphorylated tyrosine (clone 4G10) mAb was used for Western blotting at 1 µg/mL (both from Upstate Biotechnology). Anti-Src mAb (clone GD11) at 1 µg/mL, anti–phosphorylated Src (P-Src; Y416) polyclonal antibody at 1 µg/mL, anti–phosphorylated p44/42 mitogen-activated protein kinase (P-MAPK; Thr202/Tyr204; E10) at 0.1 µg/mL, and anti-p44/42 MAPK polyclonal antibody at 0.1 µg/mL were used in Western blotting (all from Cell Signaling). Anti-{alpha}-tubulin clone B-5-1-2 and anti-vinculin clone hVIN-1 mAbs were used for Western blotting at 0.4 µg/mL (both from Sigma).

Epidermal growth factor (EGF; 20 ng/mL), PMA (500 nmol/L), rottlerin (5 µmol/L), and GÖ6976 (1 µmol/L) were from Sigma. Lapatinib (100 nmol/L) was from Glaxo Smith-Kline Research. Herceptin (20 nmol/L) was from Roche. 4',6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma.

Indirect immunofluorescence. Cells were grown on six-well plates, starved overnight, and treated with PMA, rottlerin, GÖ6976, or EGF. After washing twice with PBS containing protease and phosphatase inhibitors, cells were detached by EDTA/trypsin and incubated with primary antibodies [in PBS + 0.05% bovine serum albumin (BSA)] for 1 h on ice. After three further washings, samples were incubated with secondary antibody in PBS + 0.05% BSA for 45 min on ice, washed twice, and analyzed by flow cytometry with FACSCalibur (Becton Dickinson).

Chromosome preparation and HER2 fluorescence in situ hybridization. HER2 fluorescence in situ hybridization (FISH) was done on metaphases prepared from suspensions of breast carcinoma cell lines according to standard protocol (18). Each slide was observed using a Zeiss Axioscope equipped with a 100 W mercury lamp and DAPI, Spectrum Green, and Spectrum Orange filters (Vysis). HER2 amplification was assessed based on Vysis criteria as described (19). Chromosome 17 was stained in green and HER2 gene (located on chromosome 17) was stained in red. The ratio between the number of red spots and the number of green spots per cell (>2) is indicative of the level of gene amplification. HER2 FISH on breast primary specimens was done as already described (20).

RNA extraction and real-time PCR. RNA was extracted using the Total Quick RNA Cells and Tissue kit (Talent) according to the manufacturer's protocol. First-strand cDNA was synthesized using the SuperScript II RT kit (Invitrogen) according to the manufacturer's instructions. Real-time PCRs (RT-PCR) were run on an ABI Prism 7900 RT-PCR machine (Applied Biosystems) using the following cycling conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s each followed by termination at 60°C for 1 min. Each sample contained 50 ng template cDNA, 10 µL of 2x Taqman Universal Master Mix (Applied Biosystems), 100 nmol/L of each primer, and 200 nmol/L probe in a 20 µL volume. Amplification primers and probe used for wild-type HER2 have been described (21). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene used as an endogenous control was amplified using the Taqman gene expression assay (Applied Biosystems). Retrotranscribed Universal Reference total RNA (Stratagene) was amplified as an internal control in all runs. Data from triplicate samples were analyzed with SDS 2.1 software (Applied Biosystems) and relative HER2 mRNA expression levels were calculated using the {Delta}{Delta}CT method (22).

Immunoprecipitation and Western blotting analysis. Cells were grown in 10-cm culture dishes, washed briefly with ice-cold PBS, and solubilized for 20 min at 4°C with lysis buffer [50 mmol/L Tris-HCl (pH 6.8), 300 mmol/L NaCl, 0.5% Triton X-100, 0.5% ß-octoglucoside, 10% glycerol, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, 10 mmol/L NaF, 2 mmol/L sodium orthovanadate]. Cellular lysates were cleared and incubated with MGR2 followed by incubation with Sepharose-protein A+G beads (Sigma). Immunoprecipitates were washed mixed with gel sample buffer, heated, and subjected to electrophoresis on precast 7% polyacrylamide gels (Invitrogen). Western blots were developed using the enhanced chemiluminescence method (Amersham Pharmacia Biotech). Autoradiographic signals were measured using a Bio-Rad scanning densitometer (Bio-Rad; ChemiDoc/XRS, Bio-Rad). Data were acquired and analyzed using Quantity One version 4.6.1 software.

Primary breast cancer solubilization. Lysis buffer was added to ~150 mg tissue from each of 15 frozen primary breast tumor samples and the mixtures were disrupted using a Tissue-Lyser machine (Qiagen) in three cycles for 2 min each. The soluble part of each sample was analyzed for protein content by Coomassie staining and analyzed for PKC{alpha} and P-PKC{alpha} protein levels by immunoprecipitation and Western blotting.

Statistical analysis. The Student's t test was used to compare data from the study of active PKC{alpha} in primary breast specimens. Values were expressed as a mean ± SD; differences were considered significant at P < 0.05.

Silencing of PKC{alpha} by small interfering RNA transfection. Cells seeded in 12-well plates (60–80% confluence) were transfected with either a small interfering RNA (siRNA) specific for human PKC{alpha} (pKD-PKC{alpha}-v4, final concentration of 50 nmol/L; Upstate Cell Signaling) or a pool of control RNA duplexes using Fugene 6 transfection reagent (Roche). Cells were harvested at 72 h after transfection.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of HER2 expression. To identify additional cellular models for investigating the biological mechanism responsible of HER2 expression in HER2 2+ tumors, we analyzed three representative cell lines, MDA-MB453, MCF7, and MDA-MB175, for HER2 protein expression and HER2 gene amplification. Flow cytometry analysis of HER2 protein expression revealed the highest HER2 expression in MDA-MB453 cells (Fig. 1A, line 1 ), an intermediate level in MDA-MB175 cells (Fig. 1A, line 2), and almost no expression in MCF7 cells (Fig. 1A, line 3). The standardized HercepTest (data not shown) scored MDA-MB453 cells as HER2 3+, MDA-MB175 cells as HER2 2+, and MCF7 cells as HER2 1+. The three cell lines were analyzed by FISH to exclude the possibility that HER2 protein overexpression in MDA-MB175 cells was due to gene amplification (Fig. 1B). Only MDA-MB453 cells displayed HER2 gene amplification (Fig. 1B). Chromosome 17 was tetraploid in all three cell lines.


Figure 1
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  		Figure 1. HER2 status assessment. A, immunofluorescence analysis of HER2 cell surface expression on 3+ MDA-MB453 (1), 2+ MDA MDA-MB175 (2), and 1+ MCF7 (3) cells. B, FISH analysis of HER2 gene amplification in 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells. Green stain, chromosome 17; red stain, HER2 gene located in chromosome 17. C, RT-PCR analysis of HER2 and housekeeping GAPDH mRNA levels in the three breast cancer cell lines using 50 ng of total RNA. Results are given as ratio between HER2 and GAPDH genes relative to the internal control. D, immunoprecipitation analysis of HER2, P-HER2, P-Src, and Src levels in 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells. The bands below the Src signal represent the heavy chains of anti-Src antibodies; whole-cell lysates (WCL) of 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells were analyzed by Western blotting for MAPK and P-MAPK.

 
Analysis of HER2 mRNA levels by RT-PCR indicated 2-fold higher levels in MCF7 cells and 10-fold higher levels in MDA-MB453 cells compared with levels in MDA-MB175 cells (Fig. 1C). Together, these results indicate that the overexpression of HER2 protein in MDA-MB175 cells is not associated with either gene amplification or increased HER2 mRNA expression.

Immunoprecipitation followed by Western blotting analysis of HER2 (Fig. 1D, top) indicated an intermediate HER2 expression in MDA-MB175, high in MDA-MB453, and low in MCF7 cells. However, in MDA-MB175 cell, phosphorylated HER2 levels (i.e., activated, P-HER2) were 3- and 8-fold higher than those expressed in MDA-MB453 and MCF7 cells, respectively (Fig. 1D, top). To investigate HER2 signaling, the three cell lines were also analyzed for the expression of Src and its active form, phosphorylated on the Tyr416 (23). MDA-MB175 cells displayed 2-fold higher levels of Src levels compared with MCF7 cells, whereas MDA-MB453 cells had undetectable levels of Src protein (Fig. 1D, middle) as shown previously (24). The high levels of activated HER2 in MDA-MB175 cells generated a continuous activation of MAPK proteins (Fig. 1D, bottom). Thus, MDA-MB175 cells express high levels of active HER2, high levels of active Src, and high levels of P-MAPK, the HER2-dependent mitogenic signal.

c-Cbl expression in breast cancer cell lines. Based on the reported ability of c-Cbl proteins to target HER family members to the lysosomes (25, 26), we tested whether down-modulation of c-Cbl might account for the overexpression of HER2 in 2+ cell line. Western blotting analysis indicated similar levels of c-Cbl protein in the three cell lines, whereas immunoprecipitation analysis showed that the ratio of c-Cbl associated to HER2 was similar in the 2+ and 3+ cell lines, but 1+ cells presented almost undetectable levels of c-Cbl associated to HER2 (Fig. 2A ). Biochemical analysis did not reveal the 110-kDa phosphorylated c-Cbl (P-c-Cbl) species in any of the cell lines (Fig. 2A). These data indicate that the levels of c-Cbl protein associated to HER2 in all cell lines tested do not account for the different pattern of HER2 expression.


Figure 2
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  		Figure 2. Analysis of PKC{alpha} status. A, Western blotting analysis of whole-cell lysates from 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells for c-Cbl and {alpha}-tubulin levels and immunoprecipitation analysis for c-Cbl, P-c-Cbl, and HER2 levels. B, top, immunoprecipitation analysis of soluble extracts from 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells for PKC{alpha} and P-PKC{alpha} levels and Western blotting analysis for PKC{alpha} and {alpha}-tubulin levels. Middle, immunoprecipitation analysis of homogenized total breast tumor tissues (7 classified as HER2 1+ and 8 as HER2 2+ without gene amplification) for PKC{alpha} and P-PKC{alpha} levels. Results are given as densitometry values of PKC{alpha} and P-PKC{alpha} bands for HER2 2+ and HER2 1+ primary breast tumors. Columns, mean; bars, SD. The P-PKC{alpha} protein levels differed significantly (P = 0.01, unpaired Student's t test) in the two tumor subgroups. Bottom, immunofluorescence analysis of HER2 expression in untreated cells (light gray area) and rottlerin-treated cells (4 h; dark gray area, 2+ MDA-MB175 cells; black area, 1+ MCF7 cells; white, 3+ MDA-MB453 cells). Boxed area, immunoprecipitation analysis of soluble extracts from 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells for PKC{delta} levels. C, immunoprecipitation analysis of soluble extract from 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells for HER2, coassociated PKC{alpha}, and P-PKC{alpha} or for PKC{alpha}, P-PKC{alpha}, and coassociated HER2.

 
PKC{alpha} expression in breast cancer cell lines and tumor specimens. PKC{alpha} has been proposed to divert internalized HER1 molecules from a degradative fate to a recycling pathway (15). Analysis of PKC{alpha} expression levels in the three representative breast tumor cell lines showed that 2+ MDA-MB175 cells expressed the highest levels (Fig. 2B, top) and displayed 3-fold higher levels of activated PKC{alpha} (P-PKC{alpha}) compared with 1+ MCF7 cells. Immunoprecipitation and Western blotting analysis of surgical specimens from 15 human primary breast tumors (7 classified as HER2 1+ and 8 as HER2 2+ without gene amplification as shown by FISH analysis; data not shown) revealed significantly higher levels of both PKC{alpha} and P-PKC{alpha} (P = 0.01) in the HER2 2+ compared with HER2 1+ specimens (Fig. 2B, middle). PKC{delta}, which is involved in the HER2-dependent mitogenic signal but not in receptor recycling (27), showed similar protein levels in all three cell lines (Fig. 2B, bottom, boxed area). Moreover, inhibition of PKC{delta} activity by rottlerin did not affect HER2 levels in the three cell lines (Fig. 2B, bottom). These data suggest that 2+ breast carcinoma cells express the highest levels of active PKC{alpha} in association with high levels of HER2 expression.

Determination of physical association between PKC{alpha} and HER2. Immunoprecipitation followed by Western blotting analysis of cell pellets solubilized with different detergents revealed a physical association between PKC{alpha} and HER2 only when ß-octoglucoside detergent was used. Indeed, PKC{alpha} was present in HER2 precipitates of 2+ MDA-MB175 and 1+ MCF7 cells, whereas no band at 85 kDa was found in HER2 precipitates from 3+ MDA-MB453 cells (Fig. 2C). Western blotting analysis showed a higher phosphorylation levels of PKC{alpha} associated to HER2 in 2+ MDA-MB175 cells than in 1+ MCF7 cells (Fig. 2C), and 3-fold more HER2 was found coassociated to PKC{alpha} in 2+ MD-MB175 compared with 1+ MCF7 cells (Fig. 2C). The association between HER2 and PKC{alpha} was confirmed in two additional 2+ cell lines (BT20 and ZR75-1 cells), whereas no association was detected in 3+ SKBr3 or 1+ MDA-MB231 cells (Supplementary Fig. S1).

Role of PKC{alpha} activity in HER2 cell surface expression. To investigate the role of PKC{alpha} in HER2 expression, cell lines were treated with PMA, an activator of serine/threonine kinases (28). In 2+ MDA-MB175 cells, HER2 expression doubled on 5-min PMA stimulation (Fig. 3A , PMA, black line; untreated, gray area), decreasing over time as reported previously (15). By contrast, 3+ MDA-MB453 and 1+ MCF7 cells showed no changes in HER2 levels after PMA stimulation, regardless of the duration of the treatment (Fig. 3A, PMA, black line). Thus, serine/threonine kinase activation modulated HER2 surface expression in 2+ but not in 3+ and 1+ cell lines.


Figure 3
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  		Figure 3. Modulation of PKC{alpha} activity. A, immunofluorescence analysis of HER2 cell surface expression in 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells treated with PMA (black line) or with GÖ6976 (4 h, black dotted line; 12 h, dotted gray line) or left untreated (gray areas). B, immunoprecipitation analysis of P-PKC{alpha} and PKC{alpha} levels in soluble extracts from 2+ MDA-MB175, 3+ MDA-MB453, and 1+ MCF7 cells treated with GÖ6976 or PMA or left untreated (NT). C, immunofluorescence analysis of HER2 expression in 2+ BT20, ZR75-1, 3+ SKBr3, and 1+ MDA-MB231 cells treated with PMA (black line) or with GÖ6976 (4 h, black dotted line; 12 h, dotted gray line) or left untreated (gray areas). D, Western blotting analysis of HER2, PKC{alpha}, and {alpha}-tubulin levels in soluble extracts from 2+ MDA-MB175, BT20, ZR75-1, and 3+ MDA-MB453 cells treated with PKC{alpha}-siRNA or left untreated.

 
In other experiments, HER2 expression was assessed in cells treated with a specific inhibitor of PKC{alpha} activity, GÖ6976. 2+ MDA-MB175 cells showed a 78% decrease in HER2 expression after 4-h GÖ6976 treatment (Fig. 3A, GÖ6976, black dotted line), whereas a 12-h treatment completely abolished HER2 cell surface expression (Fig. 3A, GÖ6976, dotted gray line). By contrast, GÖ6976 treatment had no effect on HER2 expression in 3+ MDA-MB453 or 1+ MCF7 cells (Fig. 3A). Western blotting analysis of HER2 levels in 2+ MDA-MB175 cells treated with GÖ6976 revealed a 50% reduction compared with untreated cells, whereas HER2 levels remained stable in 3+ MDA-MB453 cells after GÖ6976 treatment (Supplementary Fig. S2A). Although the very low levels of HER2 in 1+ MCF7 cells made it difficult to assess the effect of PKC{alpha} activity modulators on HER2 expression by flow cytometric or by biochemical analysis, longer exposure of Western blots did reveal a GÖ6976-dependent down-regulation of total HER2 (Supplementary Fig. S2, boxed area). Analysis of activated PKC{alpha} to confirm the efficacy of our treatments showed that after GÖ6976 treatment P-PKC{alpha} levels in 2+ MDA-MB175 and 1+ MCF7 cells were reduced by 60% and 30%, respectively, whereas PMA increased P-PKC{alpha} levels by 50% in both cell lines. Levels of PKC{alpha} did not change significantly in all treatments (Fig. 3B). Analysis of a larger panel of cell lines (Fig. 3C) showed that HER2 levels increased by 50% and 30% in 2+ BT20 and ZR75-1 cells, respectively, on PMA stimulation (Fig. 3C, black line), whereas HER2 levels on 4-h GÖ6976 treatment decreased by 70% and 60% (Fig. 3C, black dotted line); after 12-h GÖ6976 treatment, HER2 levels were undetectable in all 2+ cell lines (Fig. 3C, gray dotted line). Biochemical analysis showed that GÖ6976 treatment reduced the levels of P-PKC{alpha} by 40%, whereas PMA treatment increased P-PKC{alpha} levels by 30% in the 2+ cell lines (Supplementary Fig. S2B); however, in 3+ SKBr3 and 1+ MDA-MB231 cells, HER2 levels remained unchanged despite the reduced activity of PKC{alpha} after GÖ6976 treatment and the increased activity after PMA treatment (Fig. 3C). Overall, these data indicate a direct correlation between PKC{alpha} activity and HER2 cell surface expression only in HER2 2+ cell lines. Further evidence for this conclusion came from experiments in which PKC{alpha} was silenced using siRNA (Fig. 3D); siRNA-transfected 2+ MDA-MB175 cells showed a 50% decrease in PKC{alpha} expression and a 40% decrease in HER2 expression levels compared with control cells. 2+ ZR75-1 and BT20 cells transfected with the same PKC{alpha}-specific siRNA showed a 50% reduction in PKC{alpha} levels and a 30% decrease in HER2 expression, whereas MDA-MB453 cells, which are negative for PKC{alpha}, showed similar levels of HER2 in PKC{alpha} siRNA-treated or control cells (Fig. 3D). No changes were detected on treatment with a pool of control RNA duplexes. These data, together with the results obtained with pharmacologic modulators of PKC{alpha} activity, strongly indicate that the inhibition of PKC{alpha} expression/activity down-regulates HER2 expression specifically in HER2 2+ cells.

PKC{alpha} activity on EGF stimulation. The effect of PKC{alpha} on EGF-activated HER2 is currently unknown. We followed the fates of HER1 and HER2 on EGF stimulation in the presence of the PKC{alpha} inhibitor GÖ6976. Flow cytometry analysis of 2+ MDA-MB175 cells indicated a 70% reduction in HER1 expression on GÖ6976 treatment (Fig. 4, lines b and e) or on EGF stimulation (Fig. 4, lines c and f) compared with the untreated sample (Fig. 4, line a). The combination of EGF and GÖ6976 treatment for 4 or 12 h (Fig. 4, lines d and g) reduced HER1 expression by 84% compared with untreated cells.


Figure 4
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  		Figure 4. Differential effect of PKC{alpha} inhibition on EGF-stimulated HER1 versus HER2 expression in 2+ cells. Immunofluorescence was used to detect HER1 and HER2 cell surface levels in 2+ MDA-MB175 cells either untreated (line a) or treated with GÖ6976 for 4 h (line b) or 12 h (line e); stimulated with EGF for 4 h (line c) or 12 h (line f); or treated simultaneously with EGF and GÖ6976 for 4 h (line d) or 12 h (line g).

 
Similar analysis showed that HER2 cell surface expression was reduced by 77% and 80% on 4- and 12-h GÖ6976 treatment (Fig. 4, lines b and e), whereas EGF stimulation did not down-regulate HER2 levels at either time (Fig. 4, lines c and f). Treatments with EGF in combination with GÖ6976 reduced HER2 levels by 80% (Fig. 4, lines d and g). These data might indicate that PKC{alpha} determines HER2 fate in EGF-stimulated cells by recycling HER2 back to the cell surface. Conversely, activated HER1 seems to be preferentially down-regulated through lysosomal degradation.

PKC{alpha} activation by HER2. To further analyze the relationship between HER2 and PKC{alpha} activities, 2+ MDA-MB175, BT20, ZR75-1, 3+ MDA-MB453, SKBr3, and 1+ MCF7 cells were treated with two HER2 inhibitors: lapatinib, a TKI that reduces P-HER2 levels, or Herceptin, a humanized anti-HER2 mAb (9). Treatment of 2+ MDA-MB175 cells with lapatinib for 48 h led to a 54% reduction in activated HER2 levels and a 50% reduction in P-PKC{alpha} levels (Fig. 5 ). After 72 h of lapatinib treatment, no activated HER2 was detectable, whereas P-PKC{alpha} was decreased by 50% compared with untreated cells. When 2+ BT20 and ZR75-1 cells were treated with lapatinib for 48 and 72 h, no activated HER2 was detected (Fig. 5, top). The levels of HER2 were similar in both treated and untreated samples. After 72 h of lapatinib treatment, P-PKC{alpha} levels were reduced by 60%, whereas the total levels of PKC{alpha} remained unchanged (Fig. 5). The effect of TKI on HER2 activity was confirmed by a significant reduction in the levels of P-MAPK in all treatments (Fig. 5). To definitively show that the effect of lapatinib on PKC{alpha} activity was confined to 2+ breast carcinoma cell lines, we extended the study to 3+ SKBr3 cells. A 48-h lapatinib treatment reduced P-HER2 levels by 60%, whereas after 72 h, P-HER2 levels decreased by 50% (Fig. 5, bottom). The levels of HER2 remained unchanged. The expression of total and active PKC{alpha} did not change on lapatinib treatments. Similar results were obtained with 3+ MDA-MB453 cells. MCF7 cells did not respond to lapatinib treatment (Fig. 5, bottom). Treatment of HEK-293 cells, which display undetectable levels of HER2, with lapatinib did not affect P-PKC{alpha} levels (Supplementary Fig. S3), further showing that HER2 activity is responsible for PKC{alpha} activation.


Figure 5
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  		Figure 5. Effect of HER2 inhibition by lapatinib on PKC{alpha} expression. 2+ MDA-MB175, BT20, ZR75-1, 3+ MDA-MB453, SKBr3, and 1+ MCF7 cells were treated with lapatinib for 48 and 72 h and analyzed by immunoprecipitation for HER2 and P-HER2 levels and for PKC{alpha} and P-PKC{alpha} levels. Whole-cell lysates were analyzed by Western blotting for MAPK, P-MAPK, and {alpha}-tubulin levels.

 
In experiments using Herceptin, which induces HER2 down-modulation, treatment of 3+ SKBr3 cells for 48 or 72 h led to a 70% reduction in HER2 levels and a 40% decrease in P-HER2 levels (Fig. 6, bottom ); levels of activated and total PKC{alpha} remained unchanged following Herceptin treatment. Similar results were obtained with 3+ MDA-MB453 cells (Fig. 6, bottom). In Herceptin-treated 2+ MDA-MB175 cells, levels of HER2 were reduced by 20%, whereas P-HER2 was unchanged (Fig. 6, top); P-PKC{alpha} and PKC{alpha} levels were unaffected by Herceptin treatment. Similar results were obtained with 2+ BT20 and ZR75-1 cells (Fig. 6, top). 1+ MCF7 cells showed a 50% down-regulation of HER2, but P-PKC{alpha} and PKC{alpha} levels were unaffected by the drug treatment (Fig. 6, bottom).


Figure 6
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  		Figure 6. Effect of HER2 inhibition by Herceptin on PKC{alpha} expression. 2+ MDA-MB175, BT20, ZR75-1, 3+ MDA-MB453, SKBr3, and 1+ MCF7 cells were treated with Herceptin for 48 and 72 h and analyzed by immunoprecipitation for HER2 and P-HER2 levels and for PKC{alpha} and P-PKC{alpha} levels. Whole-cell lysates were analyzed by Western blotting for MAPK, P-MAPK, and {alpha}-tubulin levels.

 
These data, indicating that lapatinib completely down-regulates P-HER2 levels only in HER2 2+ cells, thus inducing a partial decrease in P-PKC{alpha} levels, whereas Herceptin affects neither P-HER2 nor P-PKC{alpha} levels, show that only in HER2 2+ breast carcinoma cell lines is PKC{alpha} activity regulated by the HER2 tyrosine kinase activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we show that activated PKC{alpha} accounts for the constitutive recycling of HER2 in breast carcinoma cell lines overexpressing HER2 without gene amplification (2+). The increased levels of activated PKC{alpha} detected in HER2 2+ human primary breast tumor tissue samples without gene amplification are consistent with this finding, although the results obtained with primary tumors were less dramatic than those in tumor cell lines because the specimens were not microdissected before solubilization and thus contained cells other than tumor cells in the extracts. The use of a PKC{alpha}-specific siRNA and a pharmacologic inhibitor showed the link between HER2 expression and PKC{alpha} activity because HER2 levels were significantly reduced only in HER2 2+ tumor cells silenced for PKC{alpha} activity. Moreover, treatment of cells with PMA, which can activate canonical ({alpha}, ß, and {gamma}) and noncanonical ({delta}, {varepsilon}, {eta}, and {theta}) PKC proteins (29), led to a 2-fold increase in HER2 cell surface expression only in the HER2 2+ cells. The effect of inhibited PKC{alpha} activity seems to be confined to HER2 expression because HLA antigen expression was unchanged by the drug treatments.

HER2, which is resistant to ubiquitin-dependent degradation pathways (30, 31), was shunted back to the cell surface by activated PKC{alpha} in cells stimulated with EGF, suggesting that the HER1-HER2 heterodimer is a substrate for PKC{alpha} recycling activity (32). Indeed, we showed a specific physical association of PKC{alpha} with HER2 using solubilized lipid rafts. Accordingly, recent data indicate that HER2 resides in protrusions rich in lipid rafts in which HER2 is maintained in a signaling-competent form (33) as shown for the T-cell receptor (34). These lipid rafts are sphingolipid-enriched membrane domains where different signaling proteins reside, including growth factor receptors, PKC, Ras, Grb10, and Nedd4 (35–38), and where mitogenic signals can initiate and can be modulated (39, 40). Activated growth factor receptors can be internalized in canonical endocytic vesicles enriched in clathrin and in non-clathrin–containing vesicles (39, 41). In addition, it has been proposed that HER family members are present on the cell surface in a dynamic state (42).

The association between HER2 and PKC{alpha} can be mediated by Src proteins or by adaptor proteins, such as Grb, because these proteins reside in lipid rafts (43). In fact, PKC{alpha} can coprecipitate with Src proteins and its activity can be modulated by general Src inhibitors (44). HER2 2+ cell lines show a constitutive activation of Src proteins that likely mediates the indirect activation of PKC{alpha} by active HER2. Thus we cannot exclude the involvement of Src in our cellular model. Src is also implicated in the activation of Cbl-dependent pathways (45–47). HER2 1+ cells showed no detectable levels of c-Cbl coassociated to HER2, and HER2 3+ and 2+ cells showed similar levels of c-Cbl coassociated to HER2, but c-Cbl was not active. Overall, these data argue against a deregulation of ubiquitin-dependent degradative pathways in HER2 2+ cell lines.

Thus, HER2 is the primary determinant of its fate in HER2 2+ cells. The dramatic decrease in P-PKC{alpha} levels following inhibition of HER2 phosphorylation by TKI (lapatinib) shows the presence of cross-talk between HER2 and PKC{alpha} activities. Downstream proteins, such as P-MAPK, were down-regulated, and mitogenic signaling was consequently switched off. By contrast, Herceptin did not affect the levels of P-HER2 and P-PKC{alpha} in the 2+ breast carcinoma cell lines.

Consistent with the in vitro data, HER2 2+ primary breast tumor tissue displayed increased PKC{alpha} activity due to increased PKC{alpha} expression levels. Thus, it seems that an activation loop is created in these tumors, in which activation of HER2 induces activation of PKC{alpha} that recycles HER2 on the membrane. It remains unclear why such a loop does not occur in HER2 1+ tumors especially because they express PKC{alpha}. One possibility is that in these tumors, HER2 is not present in an active state, so that low activation of PKC{alpha} and no HER2 accumulation occur. In HER2 3+ tumors, the inability of PKC{alpha} to affect HER2 fate might be due to the preferentially formation of HER2-HER2 homodimers, which do not associate with PKC{alpha} for recycling fate.

Patients with HER2 2+ breast tumors have a poor prognosis and most of these tumors are unresponsive to therapy with Herceptin. An improved understanding of the mechanism underlying HER2 accumulation on the tumor cell membrane is needed in devising strategies to disrupt this accumulation. Our data showing that HER2 is a constitutive PKC{alpha} substrate and that PKC{alpha} is a HER2 substrate in a regulated loop raise the possibility that TKIs instead of Herceptin might be used therapeutically in HER2 2+ breast tumors to block the positive feedback between HER2 and PKC{alpha} and at least transform these tumors into less aggressive HER2 1+ tumors.


   Acknowledgments
 
Grant support: Associazione Italiana per la Ricerca sul Cancro.

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 10/25/06. Revised 3/22/07. Accepted 3/27/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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