This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Regala, R. P. Articles by Fields, A. P. Search for Related Content PubMed PubMed Citation Articles by Regala, R. P. Articles by Fields, A. P.

Atypical Protein Kinase C Is an Oncogene in Human Non–Small Cell Lung Cancer

¹ Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center; ² Department of Pathology, Mayo Clinic, Jacksonville, Florida; and ³ Department of Medicine and ⁴ Division of Biostatistics, Mayo Clinic, Rochester, Minnesota

Requests for reprints: Alan P. Fields, Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Griffin Cancer Research Building, Room 312, 4500 San Pablo Road, Jacksonville, FL 32224. Phone: 904-953-6160; Fax: 904-953-0277; E-mail: fields.alan{at}mayo.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein kinase C (PKC) isozymes have long been implicated in carcinogenesis. However, little is known about the functional significance of these enzymes in human cancer. We recently showed that the atypical PKC (aPKC) isozyme PKC is overexpressed in human non–small cell lung cancer (NSCLC) cells and that PKC plays a critical role in the transformed growth of the human lung adenocarcinoma A549 cell line in vitro and tumorigenicity in vivo. Here we provide compelling evidence that PKC is an oncogene in NSCLC based on the following criteria: (a) aPKC is overexpressed in the vast majority of primary NSCLC tumors; (b) tumor PKC expression levels predict poor survival in patients with NSCLC; (c) the PKC gene is frequently amplified in established NSCLC cell lines and primary NSCLC tumors; (d) gene amplification drives PKC expression in NSCLC cell lines and primary NSCLC tumors; and (e) disruption of PKC signaling with a dominant negative PKC allele blocks the transformed growth of human NSCLC cells harboring PKC gene amplification. Taken together, our data provide conclusive evidence that PKC is required for the transformed growth of NSCLC cells and that the PKC gene is a target for tumor-specific genetic alteration by amplification. Interestingly, PKC expression predicts poor survival in NSCLC patients independent of tumor stage. Therefore, PKC expression profiling may be useful in identifying early-stage NSCLC patients at elevated risk of relapse. Our functional data indicate that PKC is an attractive target for development of novel, mechanism-based therapeutics to treat NSCLC.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lung cancer is the leading cause of cancer death in the United States accounting for an estimated 160,440 deaths in 2004 (1). The 5-year survival rate is only 14%, underscoring the need for more effective modalities for prevention, diagnosis, prognosis, and treatment. Most lung cancer (80%) is classified as non–small cell lung cancer (NSCLC), with most remaining cases (18%) being SCLC (2). Early-stage NSCLC tumors are often treated with surgery and radiotherapy, whereas advanced-stage metastatic disease often receives combination chemotherapy (2). Unfortunately, despite surgical removal and adjuvant therapy, many early-stage NSCLCs relapse and become fatal.

Many genetic and epigenetic changes conspire to produce a malignant, invasive NSCLC (3). Common molecular changes include oncogenic mutation of K-ras (50%), up-regulation of c-myc (20-30%), mutation of p53 (50%), deletion of p16^Ink4A/Arf, and mutation of fragile histidine triad (4, 5). NSCLCs often overexpress the epidermal growth factor receptor (EGFR), ErbB2 (HER-2/neu), and/or the hepatocyte growth factor/scatter factor receptor (Met). Cytogenetic analysis reveals frequent chromosomal losses at 3p, 6q, 8p, 9p, 13q, 17p, and 19q and gains at 1q, 3q, 5p, and 8q (6).

The atypical protein kinase C (aPKC) isozymes PKC and PKC are a structurally and functionally distinct subclass of PKCs. aPKCs exhibit diacylglycerol-, calcium-, and phosphatidylserine-independent catalytic activity due to a unique NH₂-terminal regulatory domain (7). aPKC activity is regulated by phosphoinositide-dependent kinase (PDK1) phosphorylation (8) and by interactions with upstream effectors including Ras (9). aPKCs function in the establishment of cell polarity (10) and in cell survival (9, 11, 12) through direct interactions with downstream adaptor molecules involving a PB1 domain within the NH₂-terminal regulatory region of aPKC (13). aPKCs establish epithelial cell polarity through regulation of Rac1 and/or cdc42 (13) and promote cell survival through the canonical nuclear factor-B (NF-B) pathway (14) to activate transcription of the antiapoptotic survival genes Bcl-XL (15) and c-IAP1 and c-IAP2 (16).

We recently showed that PKC is overexpressed in many established human NSCLC cell lines (17). Furthermore, disruption of PKC signaling blocks the transformed growth of A549 lung adenocarcinoma cells in vitro and tumorigenicity in vivo (17). Molecular dissection of signaling downstream of PKC revealed that PKC is necessary for transformed growth by activating a Rac1/Pak/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) signaling axis, whereas PKC is not tightly coupled to NF-B activity in A549 cells (17). Herein, we provide compelling evidence that PKC is a major cancer gene in human NSCLC. We find that PKC is overexpressed in the vast majority of primary NSCLC tumors, that the PKC gene is a target for NSCLC tumor-specific amplification, that gene amplification is an important mechanism by which PKC expression is regulated in NSCLC, and that PKC expression is a prognostic indicator of poor survival in NSCLC patients independent of tumor stage. Mechanistically, we show that disruption of PKC signaling blocks the transformed growth of human NSCLC cells harboring PKC gene amplification. We conclude that PKC is a critical oncogene involved in human lung tumorigenesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents. Antibodies were from the following sources and were used at the indicated concentrations: anti-PKC (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100), PKC (BD PharMingen, San Diego, CA; 1:4,000), actin (Santa Cruz Biotechnology, 1:2,000), and the FLAG epitope (Sigma, St. Louis, MO; 1:2,000). Recombinant human PKC and PKC protein were from Upstate Biochemical (Charlottesville, VA).

Processing and analysis of human lung cancer tissues. H&E-stained sections of matched normal and lung tumor tissues were analyzed by a pathologist to confirm initial diagnosis, staging, and overall integrity of the tissue samples. Seventy-four cases of NSCLC [37 lung adenocarcinoma (LAC) and 37 squamous cell carcinoma (SCC)] and matched normal lung tissues were chosen for extraction of DNA, RNA, and protein. Ten 10-µm-thick slices were cut from each frozen tissue block. DNA was isolated in phenol/chloroform, total RNA isolated using RNAqueous 4PCR kit (Ambion, Austin, TX), and protein isolated by direct solubilization in SDS-PAGE sample buffer.

Analysis of PKC protein expression in human lung tissue. Protein from human tumor samples was quantified using nitric acid mediated nitration of tyrosine (18). Equal amounts of protein (30 µg) from each sample was resolved in 12% SDS-PAGE gels (Invitrogen, Carlsbad, CA), transferred to polyvinylidene difluoride (PVDF) membrane (Immobilin-P, Millipore, Billerica, MA), and subjected to immunoblot analysis using the appropriate antibodies and Enhanced Chemiluminescence (ECL) Plus detection (Amersham, Piscataway, NJ) as described previously (19, 20). Images were obtained on X-omat AR film and antigens quantified by fluorescence detection using a Typhoon 9410 Variable Mode Imager. The fluorescent signal was analyzed using ImageQuant 5.2 software (Amersham).

Immunohistochemistry was done on paraffin-embedded sections of primary tumor and normal lung tissues. The tissue was deparaffinized by placing slides into three changes of xylene and rehydrated in a graded ethanol series. The rehydrated tissue samples were rinsed in water and subjected to antigen retrieval in citrate buffer (pH 6.0) as described by the manufacturer (DAKO, Carpinteria, CA). Slides were treated with 3% H₂O₂ for 5 minutes to reduce endogenous peroxidase activity and washed with PBS containing 0.5% (w/v) Tween 20. PKC was detected using PKC antibody at a 1:100 dilution in PBS/Tween and visualized using the Envision Plus Dual Labeled Polymer Kit following the manufacturer's instructions (DAKO). Images were captured and analyzed using ImagePro software.

Statistical and survival analysis. Cancer-specific survival was estimated using the Kaplan-Meier method. The duration of follow-up was calculated from the sample date to the date of death or last follow-up. The associations of the clinical and pathologic features studied with death from NSCLC were assessed using Cox proportional hazards regression models and summarized with risk ratios (RR) and 95% confidence intervals (95% CI). Natural logarithmic transformations were explored if the distributions of continuously scaled variables were not approximately normal. In addition, the relationships between continuously scaled variables and death from NSCLC were investigated using martingale residuals from the Cox model (21). Statistical analyses were done using the SAS software package (SAS Institute, Cary, NC) and Ps 0.05 were considered statistically significant.

Real-time PCR analysis for PKC gene amplification. Genomic DNA from each sample or cell line was analyzed for amplification of PKC using Taqman technology on an Applied Biosystems (Foster City, CA) 7900HT sequence detection system. The human RNaseP1 gene was used as a DNA template control and for normalization of results to total DNA. The primer/probe set for the human PKC gene was forward primer, 5'-GGCTGCATTCTTGCTTTCAGA-3'; reverse primer, 5'-CCAAAAATATGAAGCCCAGTAATCA-3'; and probe, 5'-CAATCTTACCTGCTTTCT-3'. The primer/probe set for the RNaseP1 gene was designed and provided by ABI Assay on Demand. A tumor cell line or primary tumor was scored positive for PKC gene amplification when analysis revealed a minimum of one additional PKC allele when compared with the matched normal sample.

Cell culture, plasmids, transfections, and drug treatments. Human ChaGo, H520, H1299, and SK-MES-1 NSCLC cell lines as well as the nontransformed HBE4 lung epithelial cell line were obtained from American Type Culture Collection (Manassas, VA) and maintained as suggested by the supplier. The cells were maintained in a humidified tissue culture incubator at 37°C in 5% CO₂. H1299 and ChaGo cells were stably transfected with recombinant pBabe retroviruses containing Flag-tagged human full-length wild-type PKC (wtPKC), kinase dead PKC (kdPKC), or empty vector pBabe as described previously (19). Expression of FLAG-epitope-tagged PKC and total PKC was analyzed by immunoblot analysis as described previously (19).

Adherent growth kinetics of H1299 cells transfected with empty pBabe, pbabe/kdPKC, and pbabe/wtPKC were determined by plating cells (1 x 10⁴ cells per well) into multiwell culture dishes and monitoring cell growth daily over a 7-day period. Each day, cells from triplicate wells were trypsinized and counted using a hemocytometer.

Immunoblot analysis of non–small cell lung cancer cell lines. Cells were harvested by washing with PBS and scraping off the plate. The cell pellet was lysed in SDS sample buffer. Protein lysates were quantitated by using the nitration of tyrosine in nitric acid (18). Equal amounts of protein (20 µg) were loaded for each sample, resolved in 12% SDS-PAGE gels (Invitrogen), and transferred to PVDF membrane (Millipore Immobilin-P). A solution of 5% milk and PBS/Tween 20 was used for blocking. Western blot analysis was done with appropriate antibodies and detected using ECL Plus (Amersham).

Soft agar growth assays. Anchorage-independent growth was assayed by the ability of H1299 and ChaGo cell transfectants to form colonies in soft agar. The bottom agar consisted of growth medium containing 10% fetal bovine serum (FBS) and 0.75% agarose in 60-mm tissue culture dishes. Nine hundred cells were resuspended in growth medium containing 10% FBS and 0.75% agarose and plated on top of the bottom agar. The cells were incubated at 37°C in 5% CO₂. Cell colonies were visualized and quantified under a dissecting microscope (Olympus, Melville, NY) after 4 to 6 weeks in culture.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC is overexpressed in non–small cell lung cancer. We have implicated PKC in cellular transformation in animal models (19, 20) and more recently in human NSCLC cells (17). To determine whether PKC is relevant to human disease, we assessed PKC expression in primary NSCLC tumors. Seventy-four cases of NSCLC equally representing the two major subtypes of NSCLC (37 LAC and 37 SCC) and matched normal lung tissues were selected for analysis using the following inclusion criteria: (a) patients had received no cancer therapy before tissue collection; (b) staging and diagnosis was verified by pathologic examination; (c) tumor samples were highly enriched in tumor cells (>90%) with no necrosis; and (d) sufficient tumor and matched normal tissue was available for analysis. The clinical and pathologic features of the NSCLC cases analyzed are given in Table 1.

View larger version (39K): [in this window] [in a new window]   Figure 1. PKC is overexpressed in NSCLC. A, immunoblot analysis of 10 representative primary NSCLCs and matched normal lung tissue for PKC, PKC, and actin. Recombinant human PKC was included to assure antibody specificity. B, quantitative analysis of PKC expression in primary NSCLCs (n = 74). NSCLCs exhibit a statistically significant increase in PKC protein expression. *, P < 0.001, compared with matched normal lung epithelium. C, quantitative analysis of PKC mRNA abundance in primary NSCLCs by quantitative reverse transcriptase-PCR (n = 39). *, P < 0.008, compared with matched normal lung epithelium.

View larger version (55K): [in this window] [in a new window]   Figure 2. Overexpression of PKC is confined to lung cancer cells and correlates with poor survival in NSCLC patients. A, immunohistochemistry of normal lung, LAC, and SCC for PKC. Bar, 100 µm. B, intracellular PKC staining pattern in normal lung epithelial cells and lung adenocarcinoma cells. Bar, 10 µm. C, Kaplan-Meier survival curves. NSCLC patients were divided into two groups based on PKC expression as described in Materials and Methods. High PKC expression correlates with poor survival.

We next subjected the NSCLC cases to martingale residual analysis to determine a useful cut point to assess the relationship between protein expression and death from NSCLC. Based on this analysis, the NSCLC cases were divided into two groups based on the level of PKC expression (see Materials and Methods). Patients in the high PKC expression group (PKC >5, n = 12) were 2.6 times more likely to die from NSCLC than patients in the low PKC expression group (RR, 2.58; 95% CI, 0.99-6.74; P = 0.05; Fig. 2C). The prognostic value of PKC expression was comparable to tumor stage, which is currently one of the best prognostic indicators in NSCLC (22). Within our data set, patients with late-stage disease (stages III and IV) were 2.4 times more likely to die than those with early-stage disease (stages I and II; RR, 2.35; 95% CI, 1.03-5.36; P = 0.043). Interestingly, patients with early-stage disease had PKC levels comparable with patients with late-stage disease because the median PKC expression in low-stage tumors (stages I and II) is 3.1 (n = 27) compared with 3.2 for high-stage tumors (P = 0.688, n = 9).

We next assessed whether PKC expression in early-stage disease was a useful predictor of outcome. In this subset of patients, those with high PKC expression (>5) were almost 11 times more likely to die from their disease than patients with low PKC expression (RR, 10.87; 95% CI, 0.96-122.66; P = 0.05). Therefore, PKC expression in NSCLC patients predicts poor survival independent of tumor stage, indicating that PKC expression is useful in identifying patients with early-stage disease at elevated risk of relapse.

PKC gene amplification regulates PKC expression in non–small cell lung cancer. Given the prevalence of PKC overexpression in NSCLC tumors, we wished to investigate the mechanism by which tumor-specific overexpression is achieved. Of immediate interest to us was the fact that the PKC gene resides at chromosome 3q26. Amplification of chromosome 3q26 is among the most frequent cytogenetic changes observed in NSCLC, occurring in 20% of all NSCLC (6, 23) predominantly in SCC. Because the PKC gene resides at 3q26, it is possible that gene amplification is a mechanism by which PKC is overexpressed in NSCLC tumors. To assess this possibility, we conducted quantitative real-time PCR to assess PKC gene copy number in four established human lung squamous cell carcinoma cell lines Ski-Mes1, H520, H1299, and ChaGo. Our analysis revealed PKC amplification in three of the four established human SCC cell lines tested (H520, H1299, and ChaGo cells) but not in Sk-Mes1 or nontransformed HBE4 lung epithelial cells (Fig. 3A). The presence of PKC gene amplification correlated with the presence of chromosome 3q26 amplification in these cell lines (24), indicating that PKC is part of the 3q26 amplicon. Furthermore, there is a positive correlation between PKC gene copy number, PKC mRNA abundance, and PKC protein expression in these four cell lines, showing that gene amplification is an important mechanism driving PKC expression in SCC cells (Fig. 3A).

View larger version (27K): [in this window] [in a new window]   Figure 3. The PKC gene is frequently amplified in NSCLC cell lines and primary tumors. A, analysis of the human NSCLC Sk-Mes1, H520, H1299, and ChaGo cell lines and the nontransformed HBE4 lung epithelial cell line for PKC gene copy number, mRNA abundance, and protein expression. PKC gene amplification correlates with PKC mRNA abundance and PKC protein expression. B, primary lung adenocarcinomas rarely exhibit PKC gene amplification. PKC gene copy number was determined by quantitative real-time PCR as described in Materials and Methods. PKC gene copy values were normalized to the value of the single copy gene RNaseP in the same samples. Gene amplification was defined as a PKC gene copy number that is more than 1 SD above the PKC gene copy number determined in patient-matched normal lung tissues (n = 74). Horizontal line, cutoff for defining gene amplification based on this criterion. C, primary squamous cell carcinomas frequently harbor PKC gene amplification. Gene amplification was defined as stated in (B).

PKC

PKC

PKC

PKC

PKC

To determine whether PKC gene amplification drives PKC overexpression in SCC, we analyzed primary SCC tumors for PKC protein expression. Immunohistochemical analysis showed that SCCs harboring PKC gene amplification expressed higher levels of PKC protein than did SCC tumors without PKC amplification (Fig. 4A). Quantitative analysis of PKC expression of all SCC tumor samples (n = 37) showed that tumors harboring PKC gene amplification expressed significantly higher levels of PKC protein than SCC tumors without PKC gene amplification (Fig. 4B). In addition, there is a positive correlation between PKC gene copy number and PKC protein expression in SCC tumors (Fig. 4C). Taken together, these data show that gene amplification drives PKC expression in a significant subset of SCC tumors. Interestingly, there was no statistically significant difference in PKC protein expression in LAC and SCC tumors (mean PKC expression of 3.0 in LAC versus 2.9 in SCC; P = 0.53) despite the fact that PKC gene amplification is largely confined to SCC tumors. These data suggest that PKC expression may be regulated by distinct mechanisms in these tumor types.

View larger version (74K): [in this window] [in a new window]   Figure 4. PKC gene amplification drives PKC protein expression in SCCs. A, immunohistochemistry of representative primary SCC tumors that either do or do not harbor PKC gene amplification. B, SCC tumors that harbor PKC gene amplification express higher PKC protein levels than tumors without PKC gene amplification (n = 74). C, PKC gene copy number correlates with PKC protein expression in SCC (n = 37).

PKC is required for the transformed phenotype of squamous cell carcinoma cells harboring PKC gene amplification.

PKC

PKC

PKC

kdPKC

PKC

View larger version (29K): [in this window] [in a new window]   Figure 5. PKC is required for transformation of H1299 and ChaGo lung cancer cells that harbor PKC gene amplification. A, immunoblot analysis of H1299 cells stably transfected with pBabe, wtPKC, or kdPKC for Flag, PKC, and actin. B, growth kinetics of H1299 cell transfectants in adherent culture in the presence of 10% serum. C, anchorage-independent growth of H1299 cell transfectants in soft agar. D, immunoblot analysis of ChaGo cells stably transfected with empty pBabe or kdPKC for Flag, PKC, and actin. Analysis of anchorage-independent growth of ChaGo cell transfectants in soft agar.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Second, PKC expression predicts poor survival in NSCLC patients. PKC expression is a useful prognostic indicator of poor survival independent of tumor stage, currently the most reliable prognostic marker for NSCLC (22). Therefore, PKC expression holds considerable promise as a novel prognostic tool to identify early-stage NSCLC patients at elevated risk of progression or relapse. Elevated risk patients could be candidates for aggressive adjuvant therapy and diligent surveillance after surgical removal of their primary tumor.

Third, like many other oncogenes, PKC is a target for frequent, tumor-specific somatic genetic alteration by gene amplification. The PKC gene resides at chromosome 3q26, the most frequent site of chromosomal gain in NSCLC (6, 23). PKC gene amplification occurs frequently and correlates with PKC expression in both established human NSCLC cell lines and primary NSCLC tumors. Therefore, PKC gene amplification is an important mechanism driving PKC overexpression in NSCLC. Because PKC is required for NSCLC cell growth and tumorigenicity in vivo, our data provide compelling evidence that PKC is a critical target for gene amplification within the 3q26 amplicon that promotes NSCLC carcinogenesis. Our results take on even broader significance given the fact that chromosome 3q26 amplification also occurs frequently in SCC of the head and neck (28), esophagus (25, 29), cervix (30), and ovary (6, 31). It is very likely that the PKC gene is also amplified in these tumors.

Fourth, we show that PKC is required for the transformed domain of NSCLC cells harboring PKC gene amplification. Expression of a dominant-negative, kinase-deficient PKC allele blocks the transformed growth of both H1299 and ChaGo cells in vitro. These results are consistent with our recent finding that PKC is required for the transformed growth and tumorigenicity of human A549 lung adenocarcinoma cells (17), indicating that PKC is required for transformation of many human NSCLC cells. We recently identified Rac1 as a critical downstream effector of PKC-dependent transformation and elucidate a PKC Rac1 Pak MEK1,2 ERK1,2 signaling pathway that is necessary for A549 cell tumor growth in vivo (17). These results are interesting in light of those obtained in other cell systems. In chronic myelogenous leukemia cells, we showed that PKC is required for Bcr-Abl-mediated transformation and identified NF-B as a requisite downstream effector of PKC-dependent cell survival (11, 12, 32). In contrast, in rat intestinal epithelial cells, we identified the small molecular weight GTPase Rac1 as a critical downstream effector of oncogenic Ras-mediated, PKC-dependent transformation (19). Thus, it seems that PKC can contribute to transformation through activation of at least two different signaling pathways depending upon cellular context. It will be of interest to determine whether the same or different PKC signaling pathways are operative in other human tumor types.

In conclusion, this is the first report since the discovery of PKC almost 30 years ago to conclusively show that a PKC isozyme possesses the properties of a human oncogene. PKC is overexpressed in NSCLC tumors. PKC expression is predictive of poor clinical outcome. The PKC gene is functionally targeted for genetic alteration in a significant subset of NSCLC tumors. PKC activity is required for the transformed phenotype of NSCLC cells. Finally, PKC activates a critical signaling pathway that is required for NSCLC tumor growth. Our finding that inhibition of PKC signaling has a profound inhibitory effect on transformed growth, while having little or no effect on adherent cell growth, suggests that PKC may be a particularly attractive target for the development of novel, mechanism-based therapies for the treatment of NSCLC. In this regard, we predict that NSCLC tumors harboring PKC gene amplification may be particularly responsive to PKC-directed therapies.

	Acknowledgments

 
Grant support: NIH grant CA81436 (A.P. Fields) and Mayo Clinic Foundation.

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 Pam Kreinest for expert histology and immunohistochemical services, Aaron Bungum for coordinating human patient data and tissue access, and Dr. Wilma Lingle and the staff of the Tissue and Cell Molecular Analysis Shared Resource of the Mayo Clinic Comprehensive Cancer Center for processing of human patient tissues.

Received 7/ 7/05. Accepted 8/ 2/05.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. American Cancer Society. Surveillance research cancer statistics. Atlanta: American Cancer Society; 2004.
2. Schiller JH. Current standards of care in small-cell and non-small-cell lung cancer. Oncology 2001;61 Suppl 1:3–13.
3. Minna JD, Gazdar AF. Translational research comes of age. Nat Med 1996;2:974–5.[CrossRef][Medline]
4. Gazdar AF, Minna JD. Molecular detection of early lung cancer. J Natl Cancer Inst 1999;91:299–301.[Free Full Text]
5. Salgia R, Skarin AT. Molecular abnormalities in lung cancer. J Clin Oncol 1998;16:1207–17.[Abstract]
6. Balsara BR, Sonoda G, du Manoir S, Siegfried JM, Gabrielson E, Testa JR. Comparative genomic hybridization analysis detects frequent, often high-level, overrepresentation of DNA sequences at 3q, 5p, 7p, and 8q in human non-small cell lung carcinomas. Cancer Res 1997;57:2116–20.[Abstract/Free Full Text]
7. Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y. Protein kinase C subspecies from rat brain: its structure, expression, and properties. Proc Natl Acad Sci U S A 1989;86:3099–103.[Abstract/Free Full Text]
8. Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 1998;281:2042–5.[Abstract/Free Full Text]
9. Moscat J, Diaz-Meco MT. The atypical protein kinase Cs. Functional specificity mediated by specific protein adapters. EMBO Rep 2000;1:399–403.[CrossRef][Medline]
10. Suzuki A, Akimoto K, Ohno S. Protein kinase C / (PKC/): a PKC isotype essential for the development of multicellular organisms. J Biochem (Tokyo) 2003;133:9–16.[Abstract/Free Full Text]
11. Jamieson L, Carpenter L, Biden TJ, Fields AP. Protein kinase C activity is necessary for Bcr-Abl-mediated resistance to drug-induced apoptosis. J Biol Chem 1999;274:3927–30.[Abstract/Free Full Text]
12. Murray NR, Fields AP. Atypical protein kinase C protects human leukemia cells against drug-induced apoptosis. J Biol Chem 1997;272:27521–4.[Abstract/Free Full Text]
13. Etienne-Manneville S, Hall A. Cell polarity: Par6, aPKC and cytoskeletal crosstalk. Curr Opin Cell Biol 2003;15:67–72.[CrossRef][Medline]
14. Sanz L, Sanchez P, Lallena MJ, Diaz-Meco MT, Moscat J. The interaction of p62 with RIP links the atypical PKCs to NF-B activation. EMBO J 1999;18:3044–53.[CrossRef][Medline]
15. Wang CY, Guttridge DC, Mayo MW, Baldwin AS, Jr. NF-B induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol 1999;19:5923–9.[Abstract/Free Full Text]
16. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS, Jr. NF-B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 1998;281:1680–3.[Abstract/Free Full Text]
17. Regala RP, Weems C, Jamieson L, Copland JA, Thompson EA, Fields AP. Atypical protein kinase C plays a critical role in human lung cancer cell growth and tumorigenicity. J Biol Chem. Epub 2005 July 1.
18. Bible KC, Boerner SA, Kaufmann SH. A one-step method for protein estimation in biological samples: nitration of tyrosine in nitric acid. Anal Biochem 1999;267:217–21.[CrossRef][Medline]
19. Murray NR, Jamieson L, Yu W, et al. Protein kinase C is required for Ras transformation and colon carcinogenesis in vivo. J Cell Biol 2004;164:797–802.[Abstract/Free Full Text]
20. Zhang J, Anastasiadis PZ, Liu Y, Thompson EA, Fields AP. Protein kinase C ßII induces cell invasion through a Ras/MEK-, PKC/RAC 1-dependent signaling pathway. J Biol Chem 2004;279:22118–23.[Abstract/Free Full Text]
21. Therneau TM, Grambsch PM. Modeling survival data: extending the Cox model. 1st ed. Ann Arbor: Springer-Verlag; 2000.
22. Pastorino U, Andreola S, Tagliabue E, et al. Immunocytochemical markers in stage I lung cancer: relevance to prognosis. J Clin Oncol 1997;15:2858–65.[Abstract]
23. Brass N, Racz A, Heckel D, Remberger K, Sybrecht GW, Meese EU. Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung. Cancer Res 1997;57:2290–4.[Abstract/Free Full Text]
24. Yokoi S, Yasui K, Iizasa T, Imoto I, Fujisawa T, Inazawa J. TERC identified as a probable target within the 3q26 amplicon that is detected frequently in non-small cell lung cancers. Clin Cancer Res 2003;9:4705–13.[Abstract/Free Full Text]
25. Imoto I, Pimkhaokham A, Fukuda Y, et al. SNO is a probable target for gene amplification at 3q26 in squamous-cell carcinomas of the esophagus. Biochem Biophys Res Commun 2001;286:559–65.[CrossRef][Medline]
26. Singh B, Reddy PG, Goberdhan A, et al. p53 regulates cell survival by inhibiting PIK3CA in squamous cell carcinomas. Genes Dev 2002;16:984–93.[Abstract/Free Full Text]
27. Osada H, Takahashi T. Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 2002;21:7421–34.[CrossRef][Medline]
28. Snaddon J, Parkinson EK, Craft JA, Bartholomew C, Fulton R. Detection of functional PTEN lipid phosphatase protein and enzyme activity in squamous cell carcinomas of the head and neck, despite loss of heterozygosity at this locus. Br J Cancer 2001;84:1630–4.[CrossRef][Medline]
29. Pimkhaokham A, Shimada Y, Fukuda Y, et al. Nonrandom chromosomal imbalances in esophageal squamous cell carcinoma cell lines: possible involvement of the ATF3 and CENPF genes in the 1q32 amplicon. Jpn J Cancer Res 2000;91:1126–33.[Medline]
30. Sugita M, Tanaka N, Davidson S, et al. Molecular definition of a small amplification domain within 3q26 in tumors of cervix, ovary, and lung. Cancer Genet Cytogenet 2000;117:9–18.[CrossRef][Medline]
31. Sonoda G, Palazzo J, du Manoir S, et al. Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas. Genes Chromosomes Cancer 1997;20:320–8.[CrossRef][Medline]
32. Lu Y, Jamieson L, Brasier AR, Fields AP. NF-B/RelA transactivation is required for atypical protein kinase C-mediated cell survival. Oncogene 2001;20:4777–92.[CrossRef][Medline]

This article has been cited by other articles:

				C. Chabu and C. Q. Doe Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression Development, August 15, 2008; 135(16): 2739 - 2746. [Abstract] [Full Text] [PDF]

				R. P. Regala, E. A. Thompson, and A. P. Fields Atypical Protein Kinase C{iota} Expression and Aurothiomalate Sensitivity in Human Lung Cancer Cells Cancer Res., July 15, 2008; 68(14): 5888 - 5895. [Abstract] [Full Text] [PDF]

				M. Lee and V. Vasioukhin Cell polarity and cancer - cell and tissue polarity as a non-canonical tumor suppressor J. Cell Sci., April 15, 2008; 121(8): 1141 - 1150. [Abstract] [Full Text] [PDF]

				D. Chen, C. Gould, R. Garza, T. Gao, R. Y. Hampton, and A. C. Newton Amplitude Control of Protein Kinase C by RINCK, a Novel E3 Ubiquitin Ligase J. Biol. Chem., November 16, 2007; 282(46): 33776 - 33787. [Abstract] [Full Text] [PDF]

				M. Kim, A. Datta, P. Brakeman, W. Yu, and K. E. Mostov Polarity proteins PAR6 and aPKC regulate cell death through GSK-3beta in 3D epithelial morphogenesis J. Cell Sci., July 15, 2007; 120(14): 2309 - 2317. [Abstract] [Full Text] [PDF]

				K.-M. Bae, H. Wang, G. Jiang, M. G. Chen, L. Lu, and L. Xiao Protein Kinase C{varepsilon} Is Overexpressed in Primary Human Non-Small Cell Lung Cancers and Functionally Required for Proliferation of Non-Small Cell Lung Cancer Cells in a p21/Cip1-Dependent Manner Cancer Res., July 1, 2007; 67(13): 6053 - 6063. [Abstract] [Full Text] [PDF]

				M. Nanjundan, Y. Nakayama, K. W. Cheng, J. Lahad, J. Liu, K. Lu, W.-L. Kuo, K. Smith-McCune, D. Fishman, J. W. Gray, et al. Amplification of MDS1/EVI1 and EVI1, Located in the 3q26.2 Amplicon, Is Associated with Favorable Patient Prognosis in Ovarian Cancer Cancer Res., April 1, 2007; 67(7): 3074 - 3084. [Abstract] [Full Text] [PDF]

				Y. Pu, M. L. Peach, S. H. Garfield, S. Wincovitch, V. E. Marquez, and P. M. Blumberg Effects on Ligand Interaction and Membrane Translocation of the Positively Charged Arginine Residues Situated along the C1 Domain Binding Cleft in the Atypical Protein Kinase C Isoforms J. Biol. Chem., November 3, 2006; 281(44): 33773 - 33788. [Abstract] [Full Text] [PDF]

				I. Sarkaria, P. O-charoenrat, S. G. Talbot, P. G. Reddy, I. Ngai, E. Maghami, K. N. Patel, B. Lee, Y. Yonekawa, M. Dudas, et al. Squamous Cell Carcinoma Related Oncogene/DCUN1D1 Is Highly Conserved and Activated by Amplification in Squamous Cell Carcinomas Cancer Res., October 1, 2006; 66(19): 9437 - 9444. [Abstract] [Full Text] [PDF]

				E. Erdogan, T. Lamark, M. Stallings-Mann, Lee Jamieson, M. Pellechia, E. A. Thompson, T. Johansen, and A. P. Fields Aurothiomalate Inhibits Transformed Growth by Targeting the PB1 Domain of Protein Kinase C{iota} J. Biol. Chem., September 22, 2006; 281(38): 28450 - 28459. [Abstract] [Full Text] [PDF]

				B. A. Teicher Protein kinase C as a therapeutic target. Clin. Cancer Res., September 15, 2006; 12(18): 5336 - 5345. [Full Text] [PDF]

				L. Zhang, J. Huang, N. Yang, S. Liang, A. Barchetti, A. Giannakakis, M. G. Cadungog, A. O'Brien-Jenkins, M. Massobrio, K. F. Roby, et al. Integrative Genomic Analysis of Protein Kinase C (PKC) Family Identifies PKC{iota} as a Biomarker and Potential Oncogene in Ovarian Carcinoma. Cancer Res., May 1, 2006; 66(9): 4627 - 4635. [Abstract] [Full Text] [PDF]

				M. Stallings-Mann, L. Jamieson, R. P. Regala, C. Weems, N. R. Murray, and A. P. Fields A Novel Small-Molecule Inhibitor of Protein Kinase C{iota} Blocks Transformed Growth of Non-Small-Cell Lung Cancer Cells Cancer Res., February 1, 2006; 66(3): 1767 - 1774. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Regala, R. P. Articles by Fields, A. P.