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[Cancer Research 66, 6296-6303, June 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Protein Kinase C{zeta} Mediates Epidermal Growth Factor–Induced Growth of Head and Neck Tumor Cells by Regulating Mitogen-Activated Protein Kinase

Ezra Eddy Wyssam Cohen1, Mark W. Lingen1,3, Bangmin Zhu2, Hongyan Zhu2, Michael Wayne Straza1, Carolyn Pierce1, Leslie E. Martin3 and Marsha Rich Rosner2

1 Section of Hematology/Oncology, Department of Medicine; 2 Ben May Institute for Cancer Research, Center for Integrative Sciences; and 3 Department of Pathology and Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois

Requests for reprints: Marsha Rich Rosner, Ben May Institute for Cancer Research, Center for Integrative Sciences, University of Chicago, 5841 South Greenwood Avenue, MC6027, Chicago, IL 60637. Phone: 312-702-0380; Fax: 312-702-4634; E-mail: mrosner{at}ben-may.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein kinase C (PKC) {zeta} has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKC{zeta} is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKC{zeta} expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKC{zeta} expression from normal to malignant tissue. PKC{zeta} activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKC{zeta} using either kinase-dead PKC{zeta} mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKC{zeta} inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKC{zeta} is associated with SCCHN progression, (b) PKC{zeta} mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKC{zeta} mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKC{zeta} inhibitors function additively with other inhibitors that target similar or complementary signaling pathways. (Cancer Res 2006; 66(12): 6296-303)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The epidermal growth factor (EGF) receptor (EGFR), which is nearly universally expressed in squamous cell carcinomas of the head and neck (SCCHN), has been associated with cancer cell growth, survival, and metastasis. In nonmalignant tissues, the EGFR signaling pathway has been shown to require specific protein kinase C (PKC) isoforms for effective signaling (1–3). The PKC family of serine-threonine kinases consists of 10 members that are classified by their requirements for activation (4). Classic PKCs ({alpha}, ß, and {gamma}) require both Ca2+ and diacylglycerol (DAG); novel PKCs ({delta}, {varepsilon}, {eta}, and {theta}) are Ca2+ independent but still require DAG; and atypical PKCs ({zeta} and {iota}/{lambda}) are Ca2+, DAG, and phorbol ester independent.

Relatively, little is known about PKC expression, function, and effects of inhibition in SCCHN. Previously, we have shown, in a neuronal model, that PKC{zeta} is necessary for EGF-induced mitogen-activated protein kinase (MAPK) activation, whereas PKC{delta} is required for basic fibroblast growth factor stimulation (2). However, the role of PKC{zeta} or other isoforms in SCCHN signaling has not been determined.

Therefore, we undertook the current study to characterize PKC{zeta} expression, activation, and function in normal and malignant head and neck tissues. We show that PKC{zeta} is highly expressed in head and neck tumors, and inhibition of PKC{zeta} reduces MAPK activation in normal human adult epidermal keratinocytes (NHEK) and five of seven head and neck tumor cell lines. Furthermore, SCCHN cell proliferation and viability is reduced by inhibition of PKC{zeta}. Finally, PKC{zeta} inhibition potentiates the action of other growth inhibitors in SCCHN. The findings of this study thus implicate PKC{zeta} as a relevant target in SCCHN and suggest that PKC{zeta} inhibition is a viable therapeutic strategy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. SQ20B, SCC61, SCC25, and JSQ3 cell lines were provided by Dr. Ralph Weichselbaum (University of Chicago, Chicago, IL). HN5 cells were provided by the Ludwig Institute for Cancer Research (London, United Kingdom). CCL 138 cells were purchased from the American Type Culture Collection (Manassas, VA). MSK 921 cells were provided by Dr. David Raben (University of Colorado Health Sciences Center, Aurora, CO). NHEK were purchased from Cambrex Corp. (East Rutherford, NJ). All cell lines were maintained as described previously (5–7). Myristoylated PKC pseudosubstrate (Myr-PS), bisindolylmaleimide 1 (BIM), chelerythrine chloride, and U0126 were purchased from EMD Biosciences (San Diego, CA). TAT-conjugated pseudosubstrate (TAT-PS; GRKKRRQRRRPPSIYRRGARRWRKL) and TAT-conjugated scrambled (TAT-Scr; GRKKRRQRRRPPRLYRKRIWRSAGR) peptides (8) were purchased from Tufts University Core Facility (Boston, MA). ZD1839 was provided by AstraZeneca Pharmaceuticals (Alderley Park, Cheshire, United Kingdom). Phosphorylated specific extracellular signal-regulated kinase (ERK) antibody (Thr202/Tyr204 ERK 1/2), all phosphorylated specific PKC antibodies, and total ERK antibody were purchased from Cell Signaling (Beverly, MA). Total PKC isoform, {alpha}-tubulin, actin, and anti-mouse horseradish peroxidase (HRP)–conjugated antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human epidermal growth factor was obtained from Biomedical Technologies (Stoughton, MA). Recombinant PKC proteins were purchased from Invitrogen Corp. (Long Island, New York). Recombinant {lambda} phosphatase and phosphatase reaction solution were obtained from New England Biolabs (Beverly, MA). The expression vector for kinase-dead PKC{zeta} (KD-PKC{zeta}) was generated as described previously (2).

Immunohistochemistry. Immunohistochemistry for PKC{zeta} and phosphorylated PKC{zeta}/{lambda} were done on custom human tissue microarrays. For PKC{zeta} detection, deparaffinized sections were microwaved in citrate buffer, rabbit primary antibody (Santa Cruz Biotechnology) was applied at a dilution of 1:100 for 1 hour at room temperature, and the rabbit EnVision+ kit (DAKO, Carpinteria, CA) was used for detection. Phosphorylated PKC{zeta}/{lambda} was detected using a rabbit primary antibody (Cell Signaling) and HRP-conjugated secondary antibody to rabbit (Envision+, DAKO). All sections were counterstained with hematoxylin. All specimens were scored on a 1+ to 4+ scale.

Western blotting. Cells were treated and analyzed as described previously (2). All PKC isoform antibodies were used at 1:1,000 dilutions except the anti-PKC{theta} antibody (1:500). All phosphorylated specific PKC antibodies were used at dilutions of 1:500 except the phosphorylated PKC{zeta} antibody (1:1,000). Recombinant PKC proteins were activated for use as positive controls as described previously (9).

Receptor tyrosine kinase immunoblotting. Proteome profiler human phosphorylated receptor tyrosine kinase (RTK) array that assays multiple phosphotyrosine receptors was purchased from the manufacturer (R&D Systems, Minneapolis, MN) and developed as instructed.

DNA plasmid electroporation. After trypsinization, SCC61 and SQ20B cells were counted and resuspended in cell line transfection buffer (Amaxa, Inc., Gaithersburg, MD) at 20 million/mL, and program U030 was utilized.

Construction of recombinant lentiviral vector containing the KD-PKC{zeta} gene. DNA encoding COOH-terminal hemagglutinin (HA)–tagged PKC{zeta}-K281R was obtained from Jae-Won Soh (Columbia University, New York, NY). pCDH expression lentivectors were purchased from System Biosciences (Mountain View, CA). pCDH-PKC{zeta}-K281R was generated from the HA-tagged PKC{zeta}-K281R as template by PCR using the 5' forward primer (5'-AGCTCTAGAGCCACCATGCCCAGCAGGACCGGC-3') and 3' reverse primer (5'-GCGGAATTCCGCTCAGGCGTAGTCAGGCACGTC-3'). The resulting PCR product was digested with XbaI and EcoRI and cloned into pCDH-MCS1-EF1-copGFP.

Transduction with lentiviral vectors. Lentivirus was produced from 293T cells by transient cotransfection of either 8.5 µg pCDH-MCS1-EF1-copGFP empty vector or 8.5 µg pCDH-MCS1-EF1-copGFP-PKC{zeta}-K281R with 4 µg pVSV-G and 6.4 µg pCMV{Delta}R8.2 (from Naldini and Trono; Salk Institute, La Jolla, CA) to make pseudoviral particle. TransIT-LT1 reagent (Mirus Bio, Madison, WI) was used to transfect 293T cells. SCC61 cells were transduced according to the manufacturer's directions.

Bromodeoxyuridine proliferation assay. A bromodeoxyuridine (BrdUrd) proliferation assay kit (EMD Biosciences) was used for all experiments. Cells plated in quadruplicate in 96-well plates at 1 x 103 to 2 x 103 per well were starved and incubated with respective reagents for 24 hours. During the last 6 hours of treatment, 20 µL BrdUrd label was added to each well at a 1:2,000 dilution. After cell fixation and incubation with antibodies, HRP substrate plates were read using the Synergy HT Multidetection microplate reader (Bio-Tek, Winooski, VT) at dual wavelengths of 450 to 540 nm.

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. A (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (Roche Diagnostics Corp., Indianapolis, IN) was used according to the manufacturer's instructions. Cells plated in quadruplicate in 96-well plates at 1 to 2 x 103 per well were starved and incubated with reagents. Plates were read using the Synergy HT Multidetection microplate reader (Bio-Tek) at dual wavelengths at 600 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PKC{zeta} in normal, dysplastic, and malignant oral epithelium. Initially, we examined the expression of total PKC{zeta} in normal human oral mucosa, dysplastic oral mucosa, and head and neck tumor biopsies by immunohistochemistry. The same samples were also stained with a phosphorylated specific antibody for the PKC{zeta} activation loop at Thr410 (10, 11). Analysis of staining intensity reveals that the expression of both total and activated PKC{zeta} increased significantly from normal to malignant tissue (P < 0.0001, Cuzick's trend test; Fig. 1A ). In addition, only samples from malignant tumors showed membranous staining of phosphorylated PKC{zeta}, implicating its involvement in receptor signaling (Fig. 1B). These results indicate that activated PKC{zeta} is associated with malignant progression of SCCHN.


Figure 1
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  		Figure 1. PKC expression in normal, dysplastic, and malignant SCCHN tissues and cell lines. A, tissue samples immunostained with either anti-PKC{zeta} or anti-phosphorylated PKC{zeta} antibodies. Staining intensity is expressed on a four-point scale from 1+ to 4+ (see Materials and Methods). Representative staining intensities (1+, 2+, and 4+) from the different tissues. B, relative distribution for PKC{zeta} and phosphorylated PKC{zeta} expression from a low of 1+ to a high of 4+ in normal, dysplastic, and malignant SCCHN tissues. P < 0.0001, Cuzick's trend test. C, expression of classic and novel PKC isoforms. Normal human epidermal keratinocytes (K), SQ20B (SQ), SCC61 (61), SCC25 (25), and JSQ3 (JS) tumor cells were analyzed for PKC expression. +, recombinant PKC proteins used as positive controls. Immunoblotting with PKC isoform-specific and anti-{alpha}-tubulin antibodies was done as described in Materials and Methods. D, expression of phosphorylated novel and classic PKC isoforms. Phosphorylated PKC{delta} was assessed using two phosphorylated specific antibodies directed against Thr505 or Ser643. E, PKC{zeta} expression in NHEK and SCCHN cell lines. Left, lysates from SCCHN cells in either serum-free conditions (–) or stimulated with 100 ng/mL EGF (+) were analyzed by immunoblotting with anti-PKC{zeta} or anti-phosphorylated PKC{zeta} antibodies; right, SQ20B cell lysates were incubated with or without phosphatase (PTPase) before immunoblotting with anti-phosphorylated PKC{zeta} antibody. Representative of at least three independent experiments.

 
Expression of PKC isoforms in human keratinocytes and SCCHN cell lines. The high PKC{zeta} expression in malignant tissue specimens prompted the characterization of PKC{zeta} relative to other PKC isoforms in normal and malignant cells. A panel of PKC-specific antibodies was used to detect total and phosphorylated PKC isoform expression in NHEK. Expression of {alpha}, {delta}, {varepsilon}, {theta}, and {zeta} (Fig. 1C and D) as well as phosphorylation of most of these isoforms ({alpha}, {delta}, {theta}, and {zeta}; Fig. 1D and E, lane K) was observed. NHEK cells expressed the phosphorylated forms of PKC{theta} and PKC{delta} but not PKC{varepsilon}. These results reveal that most of the expressed PKC isoforms, with the exception of PKC{varepsilon}, are phosphorylated and potentially activated in growing NHEK cells.

To determine the relative importance of PKC isoforms as mediators of head and neck tumor growth, we analyzed their expression in four radioresistant, EGFR-overexpressing SCCHN cell lines (SQ20B, SCC61, SCC25, and JSQ3; ref. 5) by immunoblotting with anti-PKC antibodies. Similar to NHEK cells, the PKC isoforms {alpha}, {delta}, {varepsilon}, {theta}, and {zeta} are expressed at varying levels, whereas isoforms ß1, ß2, {gamma}, and {eta} do not seem to be expressed in these cell lines (Fig. 1C and E, lanes SQ, 61, 25, and JS). In addition, isoforms {alpha}, {varepsilon}, and {zeta} are phosphorylated at activation sites to some degree in these lines (Fig. 1D and E). Interestingly, it seems that PKC{zeta} is constitutively phosphorylated at Thr410 in the head and neck cell lines (Fig. 1E, left), as incubation with EGF did not alter phosphorylation. Treatment with phosphatase confirmed the phosphorylated specificity of the antibody, resulting in a loss of anti-phosphorylated PKC{zeta} immunoreactivity (Fig. 1E, right). These results indicate that only a subset of PKC isoforms, including PKC{zeta}, are expressed and potentially activated in both keratinocytes and head and neck tumor cells.

Inhibition of PKC{zeta} blocks ERK activation by EGF in NHEK and SCCHN cells. To determine whether PKC{zeta} regulates EGF-stimulated MAPK (ERK1/2) activation in keratinocytes, NHEK cells were starved and exposed to EGF in the presence or absence of specific PKC{zeta} inhibitors Myr-PS or TAT-PS. Incubation with either PKC{zeta} inhibitor abrogated EGF-stimulated ERK1/2 phosphorylation (Fig. 2A ). These results indicate that activation of ERK1/2 by EGF in normal human keratinocytes is dependent on PKC{zeta} kinase activity.


Figure 2
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  		Figure 2. Inhibition of PKC{zeta} decreases ERK activation in keratinocytes and five of seven squamous cell carcinoma cell lines. A, suppression of ERK activation by PKC{zeta} inhibitors in NHEK cells. NHEK cells were preincubated with 15 µmol/L Myr-PS or TAT-PS before preincubation in either serum-free (control) medium or 100 ng/mL EGF as described in Materials and Methods. Lysates were analyzed by immunoblotting with anti-phosphorylated ERK and anti-ERK antibodies. Columns, mean of three samples; bars, SE. ERK activity represents the ratio of pERK/ERK intensity on immunoblots. Representative of three independent experiments. B, suppression of ERK activation by PKC{zeta} inhibitors in SCC61 and SQ20B cells. Cells were pretreated with either Myr-PS (15 µmol/L) or BIM (200 nmol/L) and then incubated in serum-free (control) or 100 ng/mL EGF. Columns, mean of three samples; bars, SE. ERK activity represents the ratio of pERK/ERK intensity on immunoblots. Representative of three independent experiments. C, suppression of ERK activation by PKC{zeta} inhibitors in HN5, CCL 138, and MSK 921 cells. Cells were pretreated with Myr-PS (15 µmol/L) and then incubated in serum-free (control) or 100 ng/mL EGF. Representative of two independent experiments. D, lack of suppression of ERK activation by PKC{zeta} inhibitors in SCC25 and JSQ3 cells. Cells were pretreated with either Myr-PS (15 µmol/L) or BIM (200 nmol/L) and then incubated in serum-free (control) or 100 ng/mL EGF. Representative of three independent experiments. Columns, mean of three samples; bars, SE. ERK activity represents the ratio of pERK/ERK intensity on immunoblots.

 
Because the majority of SCCHN cells overexpress the EGF receptor, we determined whether inhibition of PKC{zeta} influences MAPK signaling downstream of the EGF receptor in SCCHN cell lines. Specific PKC{zeta} inhibitors, Myr-PS or TAT-PS, were incubated with starved or EGF-stimulated SCCHN cells. In five of the cell lines (SCC61, SQ20B, HN5, CCL-138, and MSK-921), the inhibition of PKC{zeta} effectively reduced ERK activation as in NHEK cells (Fig. 2B and C). In contrast, two cell lines (SCC25 and JSQ3) were resistant to inhibition of MAPK by Myr-PS (Fig. 2D), suggesting that they have developed alternative pathways for MAPK activation by EGF. These results indicate that the majority of head and neck tumor cells retain their dependence on PKC{zeta} for MAPK activation by EGF.

Previous studies have shown that regulation of the Raf/MAPK cascade by specific PKC isoforms is dependent on the cell type and growth factor stimulus (2, 12). To determine whether other PKC isoforms mediate EGF-induced ERK1/2 phosphorylation, four of the cancer cell lines (SCC61, SQ20B, JSQ3, and SCC25) and NHEK cells were stimulated with EGF and incubated with an inhibitor of both classic and novel PKC isoforms, BIM (Fig. 3B and D ; data not shown). Inhibition of either classic or novel PKC isoforms failed to block ERK activation in any of the cell lines following EGF stimulation, although BIM was effective at inhibiting phosphorylation of the classic and novel PKC substrate, myristolated alanine-rich C kinase substrate (data not shown). Taken together, these results show that inhibition of PKC{zeta}, but not other PKC isoforms, blocks EGF-stimulated MAPK activation in normal keratinocytes, and the majority of head and neck cell lines were evaluated.


Figure 3
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  		Figure 3. Transfection of SCCHN cells with KD-PKC{zeta} inhibits ERK activation and DNA synthesis and potentiates suppression of DNA synthesis by MEK and PKC inhibitors. A, suppression of EGF-induced ERK activation by KD-PKC{zeta} in SQ20B and SCC61 cells. Cells were transfected with KD-PKC{zeta} and then incubated in serum-free (control) or 100 ng/mL EGF. Samples were resolved by SDS-PAGE and immunoblotting with anti-phosphorylated ERK and anti-ERK antibodies. B, transfection efficiency of KD-PKC{zeta} expression in SQ20B (62%) and SCC61 (54%) cells. Cells were cotransfected with expression vectors for GFP and KD-PKC{zeta} and examined 4 days later for GFP expression by fluorescence microscopy and cell number by phase microscopy. C, suppression of DNA synthesis by KD-PKC{zeta} in SQ20B and SCC61 cells. Cells were transfected with KD-PKC{zeta} or control vector and then incubated in serum-free medium (No EGF) or 100 ng/mL EGF. BrdUrd incorporation assays were done as described in Materials and Methods. Columns, mean of three independent experiments; bars, SE. D, inhibition of DNA synthesis by U0126 in SQ20B or SCC61 cells transfected with either KD-PKC{zeta} (dashed line) or control (solid line) vector. Cells were transfected with KD-PKC{zeta} or control vector and then incubated in 100 ng/mL EGF. BrdUrd incorporation assays were done as described in Materials and Methods. Representative of three independent experiments. Columns, mean of quadruplicate samples; bars, SE. E, inhibition of DNA synthesis by chelerythrine chloride (CC) in SQ20B or SCC61 cells transfected with either KD-PKC{zeta} (dashed line) or control (solid line) vector. Cells were transfected with KD-PKC{zeta} or control vector and then incubated in 100 ng/mL EGF. BrdUrd incorporation assays were done as described in Materials and Methods. Representative of three independent experiments. Columns, mean of quadruplicate samples; bars, SE. *, P < 0.05.

 
EGFR and MAPK are required for DNA synthesis in SCCHN cell lines. Because PKC{zeta} regulates EGF signaling and EGF receptors are highly expressed in SCCHN, we investigated the role of activated EGF receptors in tumor cell DNA synthesis. Analysis of 80 RTKs by immunoblotting for tyrosine phosphorylation revealed that the EGFR (ErbB1) is constitutively activated in the absence of serum in all cells tested (Supplementary Fig. S1A; data not shown). We also observed that other members of the EGFR family were activated, such as ErbB3, in some of the tumor cell lines. To determine whether inhibition of the EGF-RTK activity is sufficient to block DNA synthesis of the cells in serum, the SQ20B and SCC61 cell lines were exposed to ZD1839, an EGFR tyrosine kinase inhibitor (13). Phosphorylation of the EGFR and the downstream ERKs in these cells was inhibited in a dose-dependent manner by ZD1839 (Supplementary Fig. S1B). ZD1839 also reduces both autocrine- and serum-stimulated DNA synthesis in these cells as measured by BrdUrd incorporation (Supplementary Fig. S1C). These results indicate that EGFR is selectively activated and regulates DNA synthesis in SCCHN cells.

MAPK activation has been found to correlate with EGF receptor expression in SCCHN and to regulate cell proliferation in EGFR-overexpressing tumor cell lines (14). To determine whether activation of the Ras-Raf-MAPK pathway is critical to DNA synthesis in SCCHN, the SQ20B and SCC61 cell lines were exposed to an inhibitor of MAPK/ERK kinase (MEK; U0126). The MEK inhibitor blocked both EGF-stimulated ERK (Supplementary Fig. S1D) and DNA synthesis (Supplementary Fig. S1E). These results indicate that MAPK, a downstream effector of the EGFR, is required for DNA synthesis by the radiation-resistant, EGFR-dependent SCCHN cells.

Inhibition of PKC{zeta} reduces DNA synthesis. Because PKC{zeta} is required for MAPK activation by EGF, we examined the role of PKC{zeta} in autocrine SCCHN cell DNA replication. The Myr-PS peptide was nonspecifically toxic during prolonged incubation, and RNA interference approaches were ineffective at PKC{zeta} depletion in these cell lines (data not shown). Therefore, a KD-PKC{zeta} construct was transfected into SQ20B and SCC61 cells and was able to inhibit ERK activation (Fig. 3A). Cotransfection with green fluorescent protein (GFP) revealed that the transfection efficiency of the electroporated KD-PKC{zeta} expression vector is at least 50% in SQ20B and SCC61 cells (Fig. 3B). When the KD-PKC{zeta}-transfected cells were assayed for BrdUrd incorporation, DNA synthesis in both SCC61 and SQ20 cells was significantly reduced (Fig. 3C). The rate of DNA synthesis was similar in the presence or absence of exogenous EGF; however, in SQ20B cells, KD-PKC{zeta} was less effective in the presence of exogenous EGF. Consistent with the suppressive effects of EGFR and MEK inhibitors, these results indicate that KD-PKC{zeta} reduces both autocrine and exogenous EGF-stimulated EGFR signaling in SCCHN cell lines.

KD-PKC{zeta} potentiates the inhibitory effects of U0126 and chelerythrine chloride. We determined whether suppression of PKC{zeta} can potentiate the action of other cell proliferation inhibitors by investigating the effect of KD-PKC{zeta} on the potency of U0126, a more general inhibitor of the MAPK cascade. As shown in Fig. 3D, electroporation of either SQ20B or SCC61 cells with KD-PKC{zeta} reduces the concentration of the MEK inhibitor required to achieve a specific reduction in DNA synthesis. Because other PKC isoforms ({alpha}, {delta}, and {varepsilon}) are also expressed and phosphorylated in these SCCHN cells and a broad PKC inhibitor, chelerythrine chloride, can inhibit tumor growth in mouse xenograft models (15), we tested the effect of electroporating KD-PKC{zeta} on the inhibition of EGF-induced DNA synthesis by chelerythrine chloride. As shown in Fig. 3E, an additive reduction in DNA synthesis was observed compared with control vector. These results indicate that PKC{zeta} suppression enhances the action of other inhibitors that target similar or complementary signaling pathways.

PKC{zeta} inactivation reduces SCCHN proliferation. Lentivirus expressing KD-PKC{zeta} or TAT-PS peptide also inhibited PKC{zeta} activity in SQ20B or SCC61 cells. Immunoblotting for PKC{zeta} confirmed expression of the lentiviral KD-PKC{zeta} protein in the SCCHN cells (Fig. 4A ). As shown using SCC61 cells, introduction of KD-PKC{zeta} almost completely abrogated serum-stimulated DNA synthesis (Fig. 4B). Similarly, exposure of SQ20B and SCC61 cells to low but repeated doses of the TAT-PS peptide also significantly inhibited DNA synthesis compared with a scrambled TAT-fusion control peptide (Fig. 4C; data not shown). Taken together, these results indicate that PKC{zeta} inhibition by multiple approaches is sufficient to completely inhibit DNA synthesis in aggressive head and neck tumor cell lines.


Figure 4
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  		Figure 4. PKC{zeta} inactivation reduces SCCHN proliferation. A, expression of the KD-PKC{zeta} protein in lentivirus-infected cells. SCC61 cells were mock infected (mock) or infected with pCDH-EV expression lentivirus (vector) or with pCDH-KD-PKC{zeta}-expressing lentivirus (KD-PKC{zeta}) and harvested at 48 hours after infection. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using anti-PKC{zeta} antibody as described in Materials and Methods. B, suppression of DNA synthesis by KD-PKC{zeta} in SCC61 cells infected with pCDH-EV-expressing lentivirus (control vector) or pCDH-KD-PKC{zeta}-expressing lentivirus (KD-PKC{zeta}). Cells were infected for 48 hours with KD-PKC{zeta} or control vector and then incubated in serum-free medium (control) or 20% serum-containing medium for 24 hours. BrdUrd incorporation assays were done as described in Materials and Methods. Representative of two independent experiments. Columns, mean of quadruplicate samples; bars, SE. C, suppression of DNA synthesis by TAT-PS inhibitory peptide in SQ20B cells. Cells were grown in serum-free or 100 ng/mL EGF-containing medium (EGF) for 24 hours. TAT-PS or Tat-Scr (2 µmol/L; control TAT) was then added every 8 hours for 24 hours. BrdUrd incorporation assays were done as described in Materials and Methods. Representative of two independent experiments. Columns, mean of quadruplicate samples; bars, SE. D, reduction of cell proliferation by MEK inhibitor. SCC61 cells were grown in 20% serum-containing medium for 72 hours and incubated with increasing concentrations of U0126. MTT assays were done as described in Materials and Methods. Representative of at least three independent experiments. Columns, mean of quadruplicate samples; bars, SE. E, reduction of cell proliferation by KD-PKC{zeta} in SCC61 cells infected with pCDH-EV-expressing lentivirus (control vector) or pCDH-KD-PKC{zeta}-expressing lentivirus (KD-PKC{zeta}). Cells were grown in 20% serum-containing medium for 72 hours. MTT assays were done as described in Materials and Methods. Representative of two independent experiments. Columns, mean of quadruplicate samples; bars, SE.

 
We also examined whether inhibition of PKC{zeta} was able to reduce the number of metabolically active SCCHN cells using the MTT assay. Paralleling its effects on DNA synthesis, but to a lesser degree, the MEK inhibitor, U0126, reduced proliferating SCC61 cells in a dose-dependent manner (Fig. 4D). Lentiviral transfection of SCC61 cells with KD-PKC{zeta} similarly reduced cell metabolism (Fig. 4E) consistent with its role in MAPK activation. Overall, these results show that PKC{zeta} is a critical mediator of SCCHN proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we explored the role of one specific isoform, PKC{zeta}, in normal oral mucosa and keratinocytes as well as SCCHN tumors and cell lines. The expression of activated PKC{zeta} increases dramatically with malignant progression in normal and SCCHN tissue. PKC{zeta} inhibition can reduce EGFR-mediated MAPK signaling, DNA synthesis, and cell viability and can act in an additive fashion with other more general inhibitors in radioresistant SCCHN cell lines. These results identify a key role for PKC{zeta} in the development of aggressive SCCHN tumors and highlight an alternative strategy for selective suppression of EGF-mediated SCCHN growth.

Our work extends previous studies of PKC expression in NHEK and SCCHN cells and tissues. Although similar expression of PKCs ({alpha}, {delta}, {varepsilon}, {eta}, and {zeta}) in normal human keratinocytes was observed (16), different expression patterns have also been reported (17). There is limited data available about PKC expression in SCCHN, and the results are conflicting (18, 19). Our study would suggest that the novel isoforms ({delta}, {varepsilon}, and {theta}) are highly expressed in SCCHN cell lines. Furthermore, we report a successive increase in total and phosphorylated PKC{zeta} expression in normal, dysplastic, and malignant SCCHN tissue. PKC isoform phosphorylation, necessary but not sufficient for activity, is currently the best measure of activation available in archival tissue specimens. The increase in total PKC{zeta} along with phosphorylation in the malignant specimens underscores a significant function for this isoform in SCCHN.

In addition to mediating EGFR-dependent cell proliferation, it is likely that PKC{zeta} plays other critical roles in SCCHN tumor progression. In breast cancer cell lines, inhibition of PKC{zeta} reduced EGF-induced chemotaxis, whereas inhibition of other PKC isoforms had minimal effects (20). In a renal cancer model, PKC{zeta} activated hypoxia-inducible factor (HIF)-1{alpha} and HIF-2{alpha} and promoted their association with p300 (21). Therefore, PKC{zeta} mediates several key steps in tumor progression, including cell proliferation, survival, cell migration, and angiogenesis.

Our studies indicate that the requirement for PKC{zeta} to activate MAPK can be overridden, as two of the SCCHN cell lines were not sensitive to inhibition of PKC{zeta}. The dysregulation of PKC{zeta}-dependent MAPK activation has not been described previously and raises the possibility that loss of regulation by this enzyme through alternative mechanisms of MAPK activation plays an important role in the maintenance of the malignant state in specific SCCHN cell lines.

Inhibition of PKC{zeta} in SCCHN cells potentiates the action of MEK and PKC inhibitors. Because U0126 blocks MEK activation of MAPK, whereas PKC{zeta} inhibits Raf kinase activation (2), the two inhibitors act at different steps along the pathway but seem to be additive. Similar results were obtained when combining KD-PKC{zeta} with chelerythrine chloride, a broad spectrum PKC inhibitor that is most potent against classic and novel isoforms. Previous studies by Chmura et al. reported that chelerythrine chloride was effective at suppressing the growth of SQ20B xenografts in mice (15). The use of chelerythrine chloride does not allow identification of the specific isoform responsible for the additivity, although it is noteworthy that only PKC{alpha}, among the classic and novel isoforms, is consistently expressed and phosphorylated in NHEK and SCCHN cells. Thus, it seems that targeting the same pathway or complementary pathways by different mechanisms lowers the amount of active enzyme and the threshold necessary to reduce DNA synthesis and cell proliferation.

The current study would suggest that PKC{zeta} is a valid target in SCCHN based on its expression pattern in malignant disease, its critical role in EGF-induced MAPK activation, and its ability to inhibit SCCHN proliferation. Suppression of the proliferative stimulus from the EGFR signaling cascade by PKC{zeta} should significantly lower the threshold for effective repression by other less selective and perhaps dose-limited inhibitors.


   Acknowledgments
 
Grant support: National Cancer Institute, NIH grants RO1 CA 109278 (M.R. Rosner), 3 P50 DE11921-0551 (E.E.W. Cohen), DE12322 (M.W. Lingen), and DE00470 (M.W. Lingen); American Society of Clinical Oncology Young Investigator Award (E.E.W. Cohen); Francis L. Lederer Foundation (E.E.W. Cohen); Cornelius Crane Trust for Eczema Research (M.R. Rosner); and AstraZeneca Pharmaceuticals (E.E.W. Cohen).

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.

We thank Drs. Anning Lin, Jia Hong, and Masha Koshiginsky for their assistance.


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

Received 9/ 8/05. Revised 3/15/06. Accepted 3/24/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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