Cancer Research AACR Membership Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 8828, September 15, 2007. doi: 10.1158/0008-5472.CAN-07-1604
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aziz, M. H.
Right arrow Articles by Verma, A. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aziz, M. H.
Right arrow Articles by Verma, A. K.

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Protein Kinase C{varepsilon} Interacts with Signal Transducers and Activators of Transcription 3 (Stat3), Phosphorylates Stat3Ser727, and Regulates Its Constitutive Activation in Prostate Cancer

Moammir H. Aziz1, Herbert T. Manoharan1, Dawn R. Church2, Nancy E. Dreckschmidt1, Weixiong Zhong3, Terry D. Oberley3, George Wilding2 and Ajit K. Verma1

Departments of 1 Human Oncology and 2 Medicine, School of Medicine and Public Health, University of Wisconsin and 3 Department of Pathology and Laboratory Medicine, Veterans Administration Hospital, Madison, Wisconsin

Requests for reprints: Ajit K. Verma, Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792. Phone: 608-263-9136; Fax: 608-262-6654; E-mail: akverma{at}facstaff.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is the most common type of cancer in men and ranks second only to lung cancer in cancer-related deaths. The management of locally advanced prostate cancer is difficult because the cancer often becomes hormone insensitive and unresponsive to current chemotherapeutic agents. Knowledge about the regulatory molecules involved in the transformation to androgen-independent prostate cancer is essential for the rational design of agents to prevent and treat prostate cancer. Protein kinase C{varepsilon} (PKC{varepsilon}), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKC{varepsilon} expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKC{varepsilon} mediates progression to prostate cancer remains elusive. We present here for the first time that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, including prostate cancer, interacts with PKC{varepsilon}. The interaction of PKC{varepsilon} with Stat3 was observed in human prostate cancer, human prostate cancer cell lines (LNCaP, DU145, PC3, and CW22rv1), and prostate cancer that developed in transgenic adenocarcinoma of mouse prostate mice. In reciprocal immunoprecipitation/blotting experiments, prostatic Stat3 coimmunoprecipitated with PKC{varepsilon}. Localization of PKC{varepsilon} with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKC{varepsilon} with Stat3 was PKC{varepsilon} isoform specific. Inhibition of PKC{varepsilon} protein expression in DU145 cells using specific PKC{varepsilon} small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNA-binding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKC{varepsilon} activation is essential for constitutive activation of Stat3 and prostate cancer progression. [Cancer Res 2007;67(18):8828–38]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is the second leading cause of cancer-related deaths in men (1). The risk of prostate cancer increases rapidly after age 50, with two thirds of all prostate cancer cases found in men after age 50. Prostate cancer first manifests as an androgen-dependent disease and can be treated with androgen deprivation therapy. Despite the initial success of androgen ablation therapy, prostate cancer progresses from androgen dependent to androgen independent. The hormone-refractory invasive prostate cancer is the end stage and accounts for the majority of prostate cancer patient deaths (2–4). Defining the molecular mechanisms linked to the transition of androgen-dependent prostate cancer to an androgen-independent prostate cancer is essential for planning strategies in the prevention and treatment of prostate cancer. Both protein kinase C{varepsilon} (PKC{varepsilon}) and signal transducers and activators of transcription 3 (Stat3) have been shown to play roles in the development of androgen-independent prostate cancer (5, 6).

Stats comprise a family of six [Stat1 ({alpha} and ß isoforms), Stat2 and Stat3 ({alpha} and ß isoforms), Stat4, Stat5 ({alpha} and ß isoforms), and Stat6] latent transcription factors, which reside in the cytoplasm and are encoded by seven distinct genes (7). Stat activation is linked to cell proliferation, differentiation, apoptosis, embryogenesis, and immune responses (8–11). Stats exhibit functional divergence in their roles in oncogenesis. Stat3 and Stat5 promote cell survival, whereas Stat1 has been associated with growth-inhibitory effects (12, 13). Constitutively activated Stats, particularly Stat3, are found in several human cancers [e.g., head and neck squamous cell carcinoma (SCC), breast, ovary, prostate, and lung; refs. 14–18]. Because naturally occurring mutations of Stat3 have not been observed, constitutive activation of Stat3 seems to be mediated by aberrant growth factor signaling (7, 8). Tyrosine phosphorylation of Stat3 (Tyr705) is mediated by a wide variety of polypeptides and is essential for Stat3 dimerization and nuclear translocation. Stat3 also has a conserved Ser727 residue, which is a target for phosphorylation (19). Evidence indicates that cooperation of both tyrosine and serine phosphorylations is necessary for full activation of Stat3 (20). The identity of the protein kinase responsible for Stat3Ser727 phosphorylation in prostate cancer is unknown.

PKC is a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; ref. 21). PKC represents a large family of phosphatidylserine (PS)-dependent serine/threonine kinases (22–25). Based on structural similarities and cofactor dependency, 11 PKC isoforms have been classified into three subfamilies: classic (cPKC), novel (nPKC), and atypical (aPKC) isoforms. The cPKCs ({alpha}, ßI, ßII, and {gamma}) are dependent on PS, diacylglycerol (DAG), and Ca2+. The nPKCs ({delta}, {varepsilon}, {eta}, and {theta}) retain responsiveness to DAG and PS but do not require Ca2+ for full activation. The aPKCs ({lambda} and {varsigma}) only require PS for their activation (22). PKC isoforms exhibit functional specificity in their signals to oncogenesis (22). PKC{varepsilon} participates in the regulation of diverse cellular functions, including gene expression, neoplastic transformation, cell adhesion, mitogenicity, and cellular motility. Overexpression of PKC{varepsilon} in rodent fibroblasts led to increase in growth rates, anchorage independence, and tumor formation in nude mice. Additionally, PKC{varepsilon} overexpression transformed nontumorigenic rat colonic epithelial cells (reviewed in ref. 22). PKC{varepsilon} is a transforming oncogene (26) and a predictive biomarker of various human cancers (27), including prostate cancer (5). We found PKC{varepsilon} is linked to the development of SCC elicited either by the 7,12-dimethylbenz(a)anthracene-TPA protocol (28–30) or by repeated UV radiation (UVR) exposures (31, 32). PKC{varepsilon} is also overexpressed in human prostate cancer (33). Overexpression of PKC{varepsilon} transformed androgen-dependent LNCaP tumor cells to androgen-independent cells (5). The transformation of androgen-dependent LNCaP cells to an androgen-independent variant was associated with increased cell proliferation and resistance to apoptosis (34).

We and others have shown, using both in vivo experimental animal models and human cancer–derived cell lines, that PKC{varepsilon}-mediated oncogenic activity is linked to its ability to promote cell survival (16, 26, 34). However, the mechanisms by which PKC{varepsilon} signals cell survival remain elusive. We present here for the first time in prostate cancer that Stat3, which is constitutively activated in a wide variety of human cancers, is a protein partner of PKC{varepsilon}. PKC{varepsilon} interacts with Stat3, phosphorylates Stat3Ser727, and increases both DNA-binding and transcriptional activity of Stat3. PKC{varepsilon}-mediated Stat3Ser727 phosphorylation seems to be essential for constitutive activation of Stat3 and prostate cancer cell invasion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals, antibodies, and assay kits. The antibodies and sources of the antibodies used in this study were as follows: PKC{varepsilon}, Stat3, phosphorylated Stat3Tyr705 (pStat3Tyr705), phosphatidylinositol 3-kinase (PI3K; p85), PI3K (p110), p21, p27, Bcl-2, Bcl-xL, survivin, cyclooxygenase-2 (COX-2), ß-actin, donkey anti-goat immunoglobulin (IgG)-FITC for PKC{varepsilon}, and donkey anti-rabbit IgG-rhodamine for Stat3 (Santa Cruz Biotechnology); phosphorylated AKT (pAKT; Ser473), pAKT (Thr308), AKT, phosphorylated Janus-activated kinase (pJAK) 1 (Tyr1022/1023), and pJAK2 (Tyr1007/1008; Cell Signaling Technology); and phosphorylated Stat3Ser727 (pStat3Ser727; BD Biosciences). Blocking peptides for PKC{varepsilon} and Stat3 were obtained from Santa Cruz Biotechnology. Immunocomplex PKC assay kit was from Biotrak assay system (Amersham Biosciences). Double-stranded Stat3 consensus DNA-binding motif 5'-GATCCTTCTGGGAATTCCTAGATC was obtained from Santa Cruz Biotechnology. PKC{varepsilon} small interfering RNA (siRNA) and siRNA transfection reagents were purchased from Dharmacon, Inc. Dual-Glo luciferase reporter assay kit was purchased from Promega Corp. Cell Invasion Assay kit was purchased from Chemicon International. Plasmid pLucTKS3 was obtained from Dr. James Turkson (University of Central Florida, Orlando, FL).

Human specimens. Prostate cancer specimens were obtained at radical prostatectomy. The University of Wisconsin Human Subjects Committee approved the use of these human prostate specimens. Tissue microarrays were obtained from Folio Biosciences.4 Tissue microarrays were produced by relocating tissue from conventional histologic paraffin blocks (Folio Biosciences). This was done using a needle to biopsy a standard histology section and placing the core into an array on a recipient paraffin block. Optimal sectioning of arrays was obtained with ~5-µm sections. Using this technology, each tissue is treated in an identical manner to avoid slide-to-slide variations in the conventional sections. The tissue microarrays allowed the entire cohort to be analyzed in one batch on a single slide. Specificities of the tissue microarrays were as follows: fixative, formalin; core size, 1.5 mm; section thickness, 5 µm; control quality, pathology of every 10th slide confirmed by board-certified pathologist associated with the company; >95% tissue core retention; and validation, validated for immunohistochemistry.

Animals. Transgenic adenocarcinoma of mouse prostate (TRAMP) mice were obtained both from Dr. Norman Greenberg (Fred Hutchinson Cancer Research Center, Seattle, WA; ref. 35) and from The Jackson Laboratory and screened for the presence of the SV40 large T antigen gene by PCR as detailed on The Jackson Laboratory Web site.5 FVB/N mice were obtained from Harlan Sprague Dawley. The mice used in our experiments were either C57BL/6 or [C57BL/6XFVB] F1. Animal care and handling was conducted in accordance with established human guidelines and protocol approved by the University of Wisconsin, School of Medicine and Public Health Animal Care Committee.

Cell lines. Human prostate cell lines LNCaP, DU145, PC3, CW22rv1, and RWPE-1 were obtained from the American Type Culture Collection.

Histology. Prostate specimens were fixed for 1 h in 10% neutral buffered formalin, transferred to PBS (pH 7.4), and then embedded in paraffin. Sections (4 µm thick) of each specimen were cut for immunohistochemical study.

Western blot analysis. Indicated prostate specimens were homogenized in immunoprecipitation lysis buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/L MgCl2, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 200 mmol/L Na3VO4, 200 mmol/L NaF, 1 mmol/L EGTA]. The homogenate was centrifuged at 14,000 x g for 30 min at 4°C. Whole-cell lysate (25 µg) was fractionated on 10% or 15% Tris-glycine SDS-polyacrylamide gels for Western blot analysis as described before (16).

Determination of PKC{varepsilon} and Stat3 localization by immunofluorescence staining. Paraffin-fixed prostate samples and prostate cancer cell line DU145 were used to determine the localization of PKC{varepsilon} and Stat3. Tissue sections (5 µm thick) of prostate samples were cut to determine the localization of PKC{varepsilon} and Stat3 as described before (16).

PKC{varepsilon} immunocomplex kinase assay. Prostate tissue was placed in 0.5 mL of immunoprecipitation lysis buffer, homogenized using a glass Teflon tissue homogenizer, agitated for 30 min at 4°C, and centrifuged at 14,000 rpm in a microcentrifuge for 15 min at 4°C. The clear supernatant was used for immunoprecipitation using polyclonal antibody to PKC{varepsilon}. PKC{varepsilon} immunoprecipitate (25 µL) was assayed for kinase activity as described before (28).

PKC{varepsilon} siRNA transfection. Eighty percent confluent prostate cancer DU145 cells were starved by incubation for 18 to 24 h before assay in the serum-free medium. The transfection was done as per the manufacturer's instructions (Dharmacon). A set of four pooled nontargeting siRNAs (2 µmol/L/100-mm Petri dish) was used as a control. A set of four PKC{varepsilon}-specific siRNA (2 µmol/L/100-mm Petri dish) was used to silence PKC{varepsilon}. After 48 h of siRNA transfection, the cells were harvested and used for Western blot analysis, electrophoretic mobility shift assay (EMSA), and luciferase assay.

Electrophoretic mobility shift assay. Indicated amount of protein extract was incubated in a final volume of 20 µL of 10 mmol/L HEPES (pH 7.9), 80 mmol/L NaCl, 10% glycerol, 1 mmol/L DTT, 1 mmol/L EDTA, and 100 µg/mL poly(deoxyinosinic-deoxycytidylic acid) for 15 min. A {gamma}-32P–radiolabeled double-stranded Stat3 consensus binding motif 5'-GATCCTTCTGGGAATTCCTAGATC (Santa Cruz Biotechnology) probe was then added and incubated for 20 min at room temperature. The protein-DNA complexes were resolved on a 4.5% nondenaturing polyacrylamide gel containing 2.5% glycerol in 0.25x Tris-borate EDTA at room temperature, and gels were dried and autoradiographed. Stat3 DNA-binding activities were determined.

Luciferase assay. To determine the influence of PKC{varepsilon} on Stat3-associated gene expression, a sensitive Dual-Glo luciferase reporter assay using plasmids pLucTKS3 and pTKRenillaLuc was used (pGEM-luc vector, Promega).

Cell invasion assay. The cell invasion was assayed using a 24-well Collagen-Based Cell Invasion Assay kit as per the manufacturer's instructions. Briefly, DU145 cells at 80% confluency were serum starved for 18 to 24 h before the assay. Cells were harvested and the pellet was gently resuspended in serum-free medium. In the upper chamber, 0.5 x 106 cells per well were plated in triplicates and incubated for 2 h at 37°C in a humidified incubator with 5% CO2 before PKC{varepsilon} siRNA transfection. Both the insert and the holding well were subjected to the same medium composition with the exception of serum. The insert contained no serum, whereas the lower well contained 10% fetal bovine serum that served as a chemoattractant. PKC{varepsilon} siRNAs were obtained from Dharmacon. The transfection of siRNA was done as per the manufacturer's instructions. A set of four pooled nontargeting siRNAs (described earlier) was used as a control. A set of four PKC{varepsilon} siRNA (described earlier) was used to silence PKC{varepsilon}. Forty-eight hours after siRNA transfection, the cell invasion assay was done as per the manufacturer's instructions. The cells in the insert were removed by wiping gently with a cotton swab. Migrated cells sticking to the bottom side of the insert were stained with Cell Stain. Invading cells on the bottom side of the membrane were photographed using a light inverted microscopy (Nikon Eclipse TS 100) at x20 magnification. In addition, the number of cells migrated to the bottom side was estimated by colorimetric measurements at 560 nm according to assay instructions. Mean ± SE was calculated from three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC{varepsilon} and Stat3 Expression and Their Colocalization in Human Prostate Cancer
Expression levels of PKC{varepsilon} and Stat3 and their in vivo colocalization were analyzed in human prostate cancer (Fig. 1 ). In the analysis illustrated in Fig. 1, prostate tumor specimens were obtained after prostatectomy and fixed in 10% neutral buffered formalin. Sections (5 µm thick) were cut for immunohistochemical staining of PKC{varepsilon} and Stat3. Small samples of prostate cancer were examined in a blinded fashion by two pathologists (T.D.O. and W.Z.) without previous knowledge of Gleason score and classified according to both Gleason pattern and WHO criteria (36, 37). Expression levels of PKC{varepsilon} in prostate are shown in Fig. 1A. Low levels of PKC{varepsilon} expression were detected in the cytoplasm of benign prostate epithelial cells. Atrophic epithelium showed trace to moderate cytoplasmic labeling for PKC{varepsilon}. Areas of prostate intraepithelial neoplasia showed light cytoplasmic (Fig. 1A, double arrow) and nuclear (Fig. 1A, arrowhead) expression levels of PKC{varepsilon}. Cytoplasm of the well-differentiated prostate adenocarcinoma showed only trace levels of PKC{varepsilon} staining. Cytoplasm of moderately differentiated prostate carcinoma showed moderate to strong expression levels of PKC{varepsilon} labeling. Poorly differentiated prostate adenocarcinoma showed moderate cytoplasmic and nuclear (arrow) PKC{varepsilon} expression levels (Fig. 1A). Immunohistochemical staining of human prostate carcinoma specimens showed increased nuclear staining of Stat3 in prostate cancer specimens compared with benign prostate specimens (Fig. 1B). Benign prostate epithelium showed low level of cytoplasmic Stat3. Moderately to poorly differentiated prostate adenocarcinoma showed moderate cytoplasmic and intense nuclear staining (Fig. 1B). Inclusion of appropriate blocking peptide before immunostaining of human SCC specimens completely prevented PKC{varepsilon} and Stat3 staining, indicating the immunostaining was specific (Fig. 1C).


Figure 1
View larger version (84K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. PKC{varepsilon} and Stat3 expression and colocalization in human prostate cancer. A, immunohistochemical staining for PKC{varepsilon} expression. Shown are benign prostate; prostate gland showing atrophic epithelium; area of gland showing prostate intraepithelial neoplasia (double arrow), light cytoplasmic staining (arrowhead), and nuclear staining; malignant glands of well-differentiated prostate adenocarcinoma (Gleason pattern 2); moderately differentiated prostate carcinoma (Gleason patterns 3–4); and poorly differentiated prostate adenocarcinoma (Gleason pattern 5; arrow pointing to nuclear staining). Magnification, x20. B, immunohistochemical staining for Stat3 expression. Normal prostate epithelium showing low level of cytoplasmic Stat3 staining. Arrow, moderately differentiated prostate carcinoma (Gleason pattern 4) to poorly differentiated prostate adenocarcinoma (Gleason pattern 5) show moderate cytoplasmic and intense nuclear staining. Magnification, x40. Gleason patterns were scored according to standard criteria (36). Bar, 50 µm. C, specificity of PKC{varepsilon} and Stat3 antibodies. Inclusion of PKC{varepsilon} or Stat3 blocking peptide before immunostaining of human SCC specimens completely prevented Stat3 staining, indicating that PKC{varepsilon} and Stat3 immunostaining were specific. D, colocalization of PKC{varepsilon} and Stat3. Immunofluorescence images of human prostate cancer illustrate localization of PKC{varepsilon} (green) and Stat3 (red). White arrows, colocalization is indicated by yellow fluorescence. Magnification, x20. Bar, 25 µm.

 
The localization of PKC{varepsilon} with Stat3 was determined by double immunofluorescence staining. In this experiment (Fig. 1D), 5-µm-thick sections from paraffin-fixed human prostate cancer specimen were used. The sections were incubated with a mixture of PKC{varepsilon} (goat polyclonal, 1:50) and Stat3 (rabbit polyclonal, 1:50) primary antibodies. The sections were subsequently incubated with the mixture of two secondary antibodies (donkey anti-goat IgG-FITC for PKC{varepsilon} and donkey anti-rabbit IgG-rhodamine for Stat3). The stained sections were examined using an Olympus microscope. PKC{varepsilon} and Stat3 localization are indicated by the presence of green and red fluorescence, respectively. The yellow fluorescence indicates colocalization of both PKC{varepsilon} and Stat3 (Fig. 1D). Merged images clearly illustrate significant colocalization of PKC{varepsilon} and Stat3 (Fig. 1D, white arrow).

In a more detailed analysis, PKC{varepsilon} and Stat3 levels were determined by immunohistochemistry of human prostate tissue microarrays prepared from paraffin-fixed benign, hyperplastic, and carcinoma (grade 1–4) specimens (Table 1 ). For each section, staining was assessed as weak or faint (+), moderate or intermediate (++), and strong or intensive (+++) staining when present in >50% of total cells evaluated in each tissue core. As compared with benign prostate tissue, PKC{varepsilon} expression level in prostate cancer grade 2 to 4 was significantly (P < 0.001) increased. The level of expression of PKC{varepsilon} seemed to correlate with aggressiveness of prostate cancer (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. PKC{varepsilon} and Stat3 correlate with the aggressiveness of human prostate cancer

 
Stat3 staining was moderate to strong in human prostate cancer specimens compared with benign prostate specimens, which exhibited very weak staining (Table 1). Cytoplasmic as well as nuclear Stat3 staining was found in prostate cancer tissue specimens. Lower-grade or well-differentiated human prostate cancer specimens had more cytoplasmic Stat3 staining, whereas higher-grade or poorly differentiated human prostate cancer specimens showed more nuclear staining. As compared with benign prostate tissue, Stat3 expression level in prostate cancer grade 2 to 4 was significantly (P < 0.001) elevated (Table 1).

Expression Levels of PKC{varepsilon} and Stat3 and Their Related Signaling Components and Characterization of PKC{varepsilon} Interaction with Stat3 in Prostate Cancer from TRAMP Mice
To precisely determine the modulation of PKC{varepsilon}, Stat3, and related signaling components in prostate cancer, we used prostate cancer from TRAMP mice. The TRAMP mice spontaneously develop progressive prostate cancer that is invasive and capable of metastatic spread to distant sites, primarily in pelvic lymph nodes and the lungs (35, 38, 39). It has been reported that TRAMP mice castrated at 12 weeks of age develop androgen-independent prostate cancer (38). In addition, TRAMP mice develop spontaneous mutations in the androgen receptor (AR; ref. 39). Thus, prostate cancer in TRAMP mice closely mimics clinical prostate cancer with respect to progression, androgen independence, and biochemical profiles (35, 38, 39). The mouse genetic background makes a difference in prostate cancer progression (39). Prostate cancer progression in TRAMP mice on a mixed C57BL/6XFVB background is different from that of a C57BL/6 background. The C57BL/6 TRAMP mice can survive longer (~52 weeks) than [C57BL/6 TRAMP x FVB] F1 mice (~32 weeks; ref. 35). In addition, prostate cancer in C57BL/6 TRAMP mice often invades into seminal vesicles. Like the human prostate cancer disease, prostate cancer in TRAMP mice is also multifocal and heterogenous. In the experiments described here, both C57BL/6 and [C57BL/6 TRAMP x FVB] F1 male TRAMP mice were used.

PKC isoforms and Stat3 expression levels. In this experiment (Fig. 2 ), [C57BL/6 TRAMP x FVB] F1 mice (n = 13) were sacrificed at 26 weeks of age and prostate cancer was excised. A portion of the prostate cancer specimen was fixed in formalin, whereas another part was homogenized in lysis buffer to extract total protein for analysis of protein expression levels of PKC isoforms, Stat3, pStat3Tyr705, and pStat3Ser727 as well as PKC{varepsilon} activity and Stat3 DNA-binding activity. Normal prostate tissue specimens from nontransgenic mice were processed in parallel.


Figure 2
View larger version (87K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. PKC{varepsilon} and Stat3 expression in prostate cancer from TRAMP mice. A, immunohistochemical staining for PKC{varepsilon} and Stat3 expression. WT and TRAMP [C57BL/6XFVB] F1 male mice (n = 13) were sacrificed at 26 wk of age and normal prostate and prostate cancer were excised. Prostate specimens were fixed in formalin for immunohistochemical study. White arrows, expression of PKC{varepsilon} and Stat3 in a typical prostate cancer specimen. Specificity of PKC{varepsilon} and Stat3 antibodies was determined using respective blocking peptides (BP). Magnification, x20. Bar, 25 µm. B and C, PKC isoforms and Stat3 expression levels. The normal prostate from nontransgenic and prostate cancer from male TRAMP mice [C57BL/6 TRAMP x FVB] F1 (26 wk old; n = 3) were homogenized in lysis buffer to extract proteins for Western blot analysis. The protein extracts (25 µg protein) were immunoblotted for analyses of PKC isoforms (B) and pStat3Tyr705, pStat3Ser727, and Stat3 (C). D, PKC{varepsilon} activity. Top, normal prostate from 42-week-old WT mice and prostate cancer from age-matched TRAMP mice (C57BL/6) were excised and total protein extract was prepared in immunoprecipitation lysis buffer for PKC activity using an immunocomplex kinase assay as described in Materials and Methods; bottom, Stat3 DNA-binding activities. Normal prostate from 42-week-old WT mice and prostate cancer from age-matched TRAMP mice (C57BL/6) was excised. Prostate tissues and DU145 cells were homogenized in the lysis buffer to extract total proteins. The whole-cell extract (50 µg protein) was subjected to EMSA as described in Materials and Methods using a {gamma}-32P–lableled oligonucleotide probe containing the Stat3-binding motif following the manufacturer's protocol (Santa Cruz Biotechnology). Lane 1, oligo only; lane 2, DU145; lane 3, normal prostate sample from nontransgenic mice; lane 4, prostate cancer sample from TRAMP mice; lane 5, prostate cancer sample + 200x excess cold oligo.

 
Typical PKC{varepsilon} and Stat3 immunostaining results are illustrated in Fig. 2A. Increased protein expression levels of PKC{varepsilon} in prostate cancer (Fig. 2A) correlated with increased PKC{varepsilon} activity (Fig. 2D). In addition, constitutively activated Stat3 in prostate cancer (Fig. 2A) seems to have increased DNA-binding activity (Fig. 2D). We also determined the level of expression of various PKC isoforms in prostate cancer from TRAMP mice. Western blot analysis of PKC isoforms in the normal prostate from nontransgenic mice and prostate cancer from 26-week-old TRAMP mice is shown in Fig. 2B. PKC{gamma}, PKC{theta}, and PKC{lambda} were not detected in either normal or prostate cancer samples. PKC{varepsilon} expression was elevated in prostate cancer with reciprocal decreases in the expression levels of PKC{delta}, PKCßII, PKC{eta}, and PKC{zeta}. PKC{alpha} and PKCßI remain unaltered in prostate cancer (Fig. 2B). It is noteworthy that these results are consistent with the expression of PKC isoforms in human prostate adenocarcinomas as reported by Cornford et al. (33). In human prostate cancer specimens, the level of PKC{varepsilon} was elevated, whereas the levels of expression of PKC{delta} and PKCßII were decreased or undetected (33).

Western blot analysis revealed increased expression levels of pStat3Tyr705, pStat3Ser727, and Stat3ß in prostate cancer from 26-week-old TRAMP mice compared with normal prostate from age-matched nontransgenic mice (Fig. 2C).

Expression level of cytokines and Stat3 signaling components in prostate cancer from TRAMP mice. Several cancer-promoting agents induce the release of proinflammatory cytokines [interleukin (IL)-1, IL-6, IL-7, IL-15, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-{alpha} (TNF-{alpha})], chemotactic cytokines (IL-1 and TNF-{alpha}), and cytokines regulating immunity (IL-10, IL-12, and IL-18; ref. 40). Overwhelming evidence indicates a relationship between cytokines and the development and progression of many cancers. Elevated serum levels of specific cytokines have been reported in prostate cancer specimens and in prostate cancer cell lines (41, 42). Several reports indicate high concentrations of IL-6 in men with hormone-refractory prostate cancer compared with normal controls (43, 44). We also found elevated levels of IL-6 in prostate cancer from [C57BL/6 TRAMP x FVB] F1 mice (Fig. 3A ). Increased IL-6 expression levels accompanied increased expression of pJAK1 and pJAK2 (Fig. 3B).


Figure 3
View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Expression level of cytokines and Stat3 signaling components in prostate cancer from TRAMP mice. A, analysis of cytokines. Normal prostate from WT and prostate cancer from TRAMP mice [C57BL/6 TRAMP x FVB] F1 were excised at 26 wk of age. Whole tissue lysate was prepared for analysis of cytokines by Linco Diagnostics. Columns, mean of determinations from samples from four separate mice; bars, SE. B, pJAK expression levels. C and D, expression of proliferative, cell cycle, and antiapoptotic proteins. Normal prostate from WT and prostate cancer from TRAMP mice [C57BL/6 TRAMP x FVB] F1 (n = 2) were excised at 26 wk of age. Prostate tissue protein extracts (25 µg) were subjected to 10% or 15% Tris-glycine SDS-PAGE followed by immunoblot analysis using antibodies specific for the indicated proteins. Columns, mean of three samples; bars, SE.

 
To find clues of the kinase cascade(s) involved with increased IL-6 expression in prostate cancer, we examined the expression of various signaling kinases and found increased expression of PI3K (p85), PI3K (p110), phosphorylated AKTSer473, phosphorylated AKTThr308, and AKT (Fig. 3C).

Activation of the PKC{varepsilon} and Stat3 pathway has been shown to inhibit apoptosis and promote survival of prostate cancer cells (7, 13, 34). We determined the expression of genes associated with apoptosis (Bcl-2, Bcl-xL, and survivin), cell cycle regulation (p21 and p27), and proliferation (COX-2) in prostate cancer from TRAMP mice. As shown in Fig. 3D, increased expression of PKC{varepsilon} and Stat3 in prostate cancer from TRAMP mice accompanied decreased expression of p21 and p27 and increased expression of Bcl-xL, Bcl-2, survivin, and COX-2.

PKC{varepsilon} Interacts with Stat3 in Prostate Cancer
To determine whether PKC{varepsilon} associates with Stat3, reciprocal immunoprecipitation/blotting experiments were done using prostate tissue specimens from wild-type (WT) and TRAMP mice (Fig. 4A ) and human prostate cancer cell lines (Fig. 4B). In these experiments, the protein extract was immunoprecipitated with antibodies against PKC{varepsilon} or Stat3. The immunoprecipitated samples were subjected to immunoblot analysis using antibodies against PKC{varepsilon}, Stat3, pStat3Tyr705, and pStat3Ser727. The results of PKC{varepsilon} interaction with Stat3 in prostate tissue are shown in Fig. 4A. Stat3, pStat3Tyr705, and pStat3Ser727 coimmunoprecipitated with PKC{varepsilon}. In the reciprocal immunoprecipitation/blotting experiments, PKC{varepsilon} coimmunoprecipitated with Stat3 (Fig. 4A). PKC{varepsilon} interaction with pStat3Ser727 was more dramatic in prostate cancer from TRAMP mice than normal prostate tissue from WT mice. PKC{varepsilon} interaction with Stat3 in human prostate cancer cell lines is shown in Fig. 4B. Analysis of steady-state expression levels of PKC{varepsilon} revealed increased PKC{varepsilon} expression level in DU145 cells (Fig. 4B, top). PKC{varepsilon} interaction with Stat3 was observed in LNCaP, DU145, PC3, and CW22rv1 cell lines. However, PKC{varepsilon} interaction with Stat3 was not detectable in RWPE-1 cell line. The co-localization of PKC{varepsilon} with Stat3 in prostate cancer from TRAMP mice (Fig. 4C, left) and in DU145 cells (Fig. 4C, right) was confirmed by double immunofluorescence staining.


Figure 4
View larger version (62K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. PKC{varepsilon} interacts with Stat3. A, WT and TRAMP [C57BL/6] mice (n = 3) were sacrificed at 42 wk and the prostate tissues were homogenized in immunoprecipitation (IP) lysis buffer as described in Materials and Methods. The homogenate was centrifuged at 14,000 x g for 30 min at 4°C. The whole-cell lysates were pooled (50 µg protein) and used for immunoprecipitation with the indicated antibodies. Immunoprecipitated samples were analyzed by Western blot (IB) using the indicated antibodies. B, association of PKC{varepsilon} with Stat3 in human prostate cancer cell lines LNCaP, DU145, PC3, CW22rv1, and RWPE-1. Whole-cell lysate (50 µg protein) from the human prostate cancer cell lines was used for immunoprecipitation with the indicated antibodies. The immunoprecipitated samples were analyzed by Western blot using the indicated antibodies. C, double immunofluorescence localization of PKC{varepsilon} and Stat3 in normal mouse prostate, prostate cancer (PCa) from TRAMP [C57BL/6], and DU145 cells. Localization of PKC{varepsilon} and Stat3 is shown by green and red fluorescence, respectively. Arrows, colocalization of PKC{varepsilon} with Stat3 is shown by yellow fluorescence. Magnification, x20 magnification. Bar, 25 µm. D, PKC isoform interaction with Stat3. WT and TRAMP [C57BL/6] mice were sacrificed at age 42 wk and the tissue extracts were prepared for reciprocal immunoprecipitation/blotting experiments using antibodies specific for PKC{varepsilon} and Stat3.

 
The interaction of Stat3 with other PKC isoforms was also determined. In this experiment (Fig. 4D), prostate cancer protein extract from TRAMP mice was immunoprecipitated with Stat3 antibody. The immunoprecipitated samples were subjected to immunoblot analysis using antibodies against various PKC isoforms. PKC{varepsilon} coimmunoprecipitated with Stat3. However, other PKC isoforms showed little ({alpha} and ßI) or no (ßII, {gamma}, {delta}, {lambda}, {zeta}, {eta}, and {theta}) association with Stat3 (Fig. 4D).

PKC{varepsilon} Mediates Stat3Ser727 Phosphorylation and Transcriptional Activity of Stat3
To determine whether PKC{varepsilon} is linked to Stat3Ser727 phosphorylation and whether this phosphorylation is essential for the DNA-binding and transcriptional activity of Stat3, we used siRNAs to silence PKC{varepsilon} in DU145 cells (Fig. 5A and B ). A pool of four specific siRNA oligonucleotides directed against PKC{varepsilon} mRNA was transfected into DU145 cells to inhibit PKC{varepsilon} synthesis (Fig. 5A). Inhibition of PKC{varepsilon} expression resulted in dramatic inhibition of Stat3Ser727 phosphorylation but minimally inhibited Stat3Tyr705 phosphorylation and total Stat3 levels (Fig. 5A). Furthermore, siRNA-mediated inhibition of PKC{varepsilon} inhibited Stat3 DNA-binding activity as determined by EMSA (Fig. 5B, left). In addition, siRNA-mediated PKC{varepsilon} deficiency in DU145 cells inhibited Stat3-regulated gene expression as determined using the luciferase reporter gene assay (Fig. 5C, right).


Figure 5
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. PKC{varepsilon} is linked to phosphorylation of Stat3Ser727, Stat3 DNA-binding activity, Stat3 transcriptional activity, and prostate cancer progression. A, PKC{varepsilon} and Stat3 expression. DU145 cells were untransfected (lane 1) or transfected with nontargeting siRNA (lane 2) or PKC{varepsilon}-specific siRNA (lane 3), and whole-cell lysates were prepared as described in Materials and Methods. The protein extracts (25 µg protein) were immunoblotted and indicated protein expression levels were detected with the appropriate antibodies. ß-Actin was used as a control for gel loading variations. The quantification of proteins (normalized to ß-actin) was done as described in Materials and Methods. Columns, mean of two independent experiments; bars, SE. B, EMSA. DU145 total cells were suspended in buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MagCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L PMSF]. After 15 min of incubation on ice, the cells were pelleted and resuspended in buffer B [20 mmol/L HEPES (pH 7.9), 20 mmol/L NaF, 1.5 mmol/L MgCl2, 1 mmol/L Na3VO4, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 420 mmol/L NaCl, 20% glycerol, 1 µg/mL leupeptin, 1 µg/mL aprotinin]. The samples were then centrifuged and the clear supernatant was used for EMSA as described in Materials and Methods. Lane 1, control; lane 2, nontargeting; lane 3, PKC{varepsilon} siRNA. C, Stat3 transcriptional activity. DU145 cells were transfected with Stat3 reporter plasmid, pLucTKS3, and pLucRenillaLuc with or without PKC{varepsilon}-specific siRNA (Dharmacon) using LipofectAMINE. Cells were lysed in passive lysis buffer and firefly luciferase (pLucTKS3) and Renilla luciferase activities (transfection control) were measured using a luciferase assay system (Promega) as outlined in the manufacturer's instructions. Luciferase activities were normalized to total protein. Columns, mean of triplicate assays; bars, SE. D, DU145 cell invasion. DU145 cells were transfected with PKC{varepsilon} siRNA and cell invasion was determined as described in Materials and Methods. Top, photographs of invading cells. Magnification, x20. Bottom, the number of invading cells was estimated by colorimetric measurements at 560 nm according to assay instructions (Chemicon International). Columns, mean of three separate wells; bars, SE.

 
PKC{varepsilon} Is Linked to DU145 Cell Invasion
The possibility was explored whether PKC{varepsilon} is linked to prostate cancer progression. In this experiment (Fig. 5D), PKC{varepsilon} expression was inhibited using PKC{varepsilon} siRNA. PKC{varepsilon} inhibition, which accompanies inhibition of Stat3Ser727 phosphorylation and Stat3 DNA-binding and transcriptional activities, decreased the invasive ability of DU145 cells (Fig. 5D, top). siRNA-mediated inhibition of DU145 cells invasion was dependent on the dose of siRNA (Fig. 5D, bottom).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here that both PKC{varepsilon} and constitutively activated Stat3 are overexpressed in prostate cancer and their expression levels correlate with prostate cancer aggressiveness. PKC{varepsilon} overexpression in prostate cancer accompanied (a) increased in the levels of IL-6 and pJAK1 and pJAK2, (b) increased phosphorylation of PI3K and AKT, (c) decreased expression of cyclin-dependent protein kinase inhibitors (p21 and p27), and (d) up-regulation of antiapoptotic (Bcl-2, Bcl-xL, and survivin) and proliferative (COX-2) markers. PKC{varepsilon} interacts with Stat3 and phosphorylates Stat3Ser727. PKC{varepsilon}-mediated Stat3Ser727 phosphorylation correlated with Stat3 DNA-binding and transcriptional activities as well as prostate cancer progression.

PKC{varepsilon} was overexpressed in human prostate cancer and prostate cancer developed either in C57BL/6 or [C57BL/6 TRAMP x FVB] F1 TRAMP mice (Figs. 1 and 2). The fact that PKC{varepsilon} expression is significantly elevated in prostate cancer and correlates with prostate cancer aggressiveness (Figs. 1 and 2; Table 1) implies that PKC{varepsilon} is probably linked to the maintenance of androgen-independent prostate cancer. In this context, the pioneering work of Terrian and his associates on the role of PKC{varepsilon} in prostate carcinogenesis, using prostate cancer–derived cell lines, is noteworthy (5, 34, 45). In their reports, PKC{varepsilon} overexpression transformed androgen-dependent LNCaP tumor cells to androgen-independent cells (5). The transformation of androgen-dependent LNCaP cells to an androgen-independent variant was associated with increased cell proliferation and resistance to apoptosis. Antisense experiments established that endogenous PKC{varepsilon} plays an important role in regulating the growth and survival of androgen-independent prostate cancer cells, suggesting that PKC{varepsilon} expression may be sufficient to maintain prostate cancer growth and survival after androgen ablation (5).

PKC{varepsilon}, which is overexpressed in prostate cancer, seems to be constitutively active. This conclusion is supported by two lines of evidence. First, increased PKC{varepsilon} expression correlated with PKC{varepsilon} activity in prostate cancer from TRAMP mice (Fig. 2), and second, in an immunohistochemical analysis, PKC{varepsilon} was observed to translocate to both plasma and nuclear membranes (Figs. 1 and 2). The mechanism by which PKC{varepsilon} remained constitutively active during progression of prostate cancer can be speculated. The generally accepted paradigm for PKC activation (22) consists of two major biological events. The initial event is a well-ordered sequential phosphorylation followed by allosteric activation mediated by two lipid signals. Priming phosphorylation on the activation loop residue threonine by phospholipid-dependent kinase 1 initiates autophosphorylation on the turn motif threonine residue and hydrophobic motif serine residues. Most PKC in unstimulated cells is in the triple-phosphorylated mature form. The mature, phosphorylated species are released to the cytosol and the pseudosubstrate gains access to the active site, thus maintaining the enzyme in an autoinhibited state (22). As the mature PKC positions near the membrane for rapid access to DAG, the translocation of the mature enzyme from the cytosolic soluble fraction to particulate plasma membrane fraction is mediated by the binding of a lipid second messenger. Subsequent association with the membrane allows PKC to carry out phosphorylation of target substrates. The constitutive activation of PKC{varepsilon} is perhaps due to dysregulation of PKC{varepsilon} synthesis, activation, and/or degradation.

Constitutively activated PKC{varepsilon} regulates activation of the signaling network linked to cell survival essential for the maintenance of androgen-independent prostate cancer (5). As observed in prostate cancer from TRAMP mice, PKC{varepsilon} expression accompanied up-regulation of phosphorylated PI3K and AKT (Fig. 3), major components of the cell survival pathway (Fig. 6 ). Consistent with these findings, using CWR22 xenografts, it was shown by proteomic analysis that the association of PKC{varepsilon} with Bax may neutralize apoptotic signals propagated through the mitochondrial death signaling pathway. In addition, integrin signaling links PKC{varepsilon} to the protein kinase B/AKT survival pathway in recurrent prostate cancer cells (34). Our results (Fig. 3), using the in vivo TRAMP mouse model, are consistent with these findings (34). PKC{varepsilon} overexpression, which correlates with prostate cancer aggressiveness (Table 1), accompanied an increase in proteins that modulate apoptosis (survivin, Bcl-2, and Bcl-xL) and cell survival (Stat3; Fig. 3).


Figure 6
View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Proposed model illustrating how PKC{varepsilon} is linked to constitutive activation of Stat3 in prostate cancer. Both PKC{varepsilon} and Stat3 are constitutively activated in prostate cancer. Stat3 is activated by phosphorylation at both Tyr705 and Ser727 residues. PKC{varepsilon} may mediate, through activation of the PI3K-AKT pathway, induction of cytokines (e.g., IL-6). IL-6, either via autocrine or paracrine mechanism and through the JAK-Stat pathway, phosphorylates Stat3Tyr705. PKC{varepsilon} either directly or indirectly via association with other protein kinases [e.g., Raf1, MAPK/ERK 1/2 (MEK1/2), ERK1/2, and p38MAPK] phosphorylates Stat3Ser727. Constitutive activation of PKC{varepsilon} and Stat3 correlates with the aggressiveness of prostate cancer. This model assumes that PKC{varepsilon} and Stat3, the proteins with oncogenic traits, are essential components of development and maintenance of androgen-independent prostate cancer.

 
Several lines of evidence support the link between PKC{varepsilon} and Stat3. First, both PKC{varepsilon} and Stat3 are overexpressed in prostate cancer (Figs. 1 and 2; Table 1). Second, using reciprocal immunoprecipitation experiments, it was observed that PKC{varepsilon} associates with Stat3 (Figs. 4 and 5). Finally, double immunofluorescence studies confirm their colocalization (Figs. 1 and 4). PKC{varepsilon} depletion prevents PKC{varepsilon} association with Stat3 and Stat3Ser727 phosphorylation. Together, these biochemical, immunologic, and enzymology studies indicate that PKC{varepsilon} interacts with Stat3 and is linked to Stat3Ser727 phosphorylation.

Depending on the cellular context, Stat3 is a substrate for several protein kinases (46). Stat3 is an in vitro substrate for mitogen-activated protein kinase (MAPK) and/or extracellular signal-regulated kinases (ERK; ref. 47). Pioneering research from Dr. Zigang Dong's laboratory (Hormel Institute, University of Minnesota, Austin, MN) has shown that UV-induced Stat1 and Stat3 (Ser727) phosphorylation involves the integration of multiple kinase pathways (48, 49). Recently, we have reported that PKC{varepsilon} interacts with Stat3 in mouse skin. PKC{varepsilon} interaction with Stat3 was increased after exposure of skin with UVR (16). We also observed in vivo using PKC{varepsilon} knockout mice and in vitro in an immunocomplex kinase assay that PKC{varepsilon} phosphorylated Stat3 at Ser727 residue, implying that PKC{varepsilon} is a Stat3Ser727 kinase (16). Our results (Figs. 4 and 5) indicate that PKC{varepsilon} is also an initial signal that is linked to phosphorylation of Stat3Ser727 in prostate cancer. Dependent on the cellular context and stimulus, Stat3 may be a direct or indirect substrate of PKC{varepsilon}.

The functional specificity of each PKC isoform is determined in part by the differential localization of the isozyme-specific RACKS. RACKS, which anchor activated PKC isozymes in close proximity to its selective substrates, are not PKC substrates. Proteins that interact with PKC include proteins localizing PKC to a specific intracellular site (e.g., RACKS and RICKS), protein kinases (e.g., Raf1, Btk, S6 kinase, PKD, PDK1, PKF, and Fyn), substrate proteins (e.g., MARCKS and STICKS), and other proteins with unique functions (25, 50–54). Several proteins have specifically been shown to interact with PKC{varepsilon} (51, 54). Our results (Figs. 4 and 5) indicate that PKC{varepsilon} interacts with Stat3 and phosphorylates Stat3Ser727. PKC{varepsilon}-mediated phosphorylation of Stat3Ser727 may be an essential component of constitutive activation of Stat3 in a wide variety of human cancers (14–18).

The mechanism by which Stat3 is constitutively activated in prostate cancer can be possibly explained as shown in Fig. 6. Stat3 activation, which involves dimerization, nuclear translocation, DNA binding, and transactivation of transcription, requires phosphorylation of both Tyr705 and Ser727 (8–12). Stat3Tyr705 phosphorylation is mediated by a wide variety of growth factors (e.g., IL-6). IL-6 has been characterized as a prostate exocrine gene product that interacts with its receptors, activates AR, and regulates prostate cancer cell proliferation. IL-6 signaling is mediated through JAK, Stat3, as well as MAPK, which induces AR-mediated gene activation in prostate cancer (12, 13). JAK-Stat is the classic pathway that has been shown to mediate cellular responses to a variety of cytokines, including IL-6. In response to IL-6, Stat3 is transiently associated with gp130 and subsequently phosphorylated by JAKs on Tyr705 of Stat3. Stat3 represents an important regulator of IL-6–targeted gene expression. PKC{varepsilon}-mediated Stat3 Ser727 phosphorylation is essential for both optimal DNA-binding and transcriptional activities of Stat3 (Fig. 6).

In summary, PKC{varepsilon} and constitutively activated Stat3 are key components in the mechanisms involved in transition of androgen-dependent prostate cancer to androgen-independent prostate cancer (45). We have made the novel observation that PKC{varepsilon} associates with Stat3 and regulates Stat3 activation (Figs. 1, 4, and 5). PKC{varepsilon} activation transduces multiple signals involving inhibition of apoptotic pathways and promotion of cell survival pathways (Figs. 3 and 6). PKC{varepsilon}-mediated cell survival pathway involves constitutive activation of Stat3. PKC{varepsilon} is an initial signal that regulates activation of Stat3. PKC{varepsilon} and Stat3, the proteins with oncogenic traits, should be explored as potential targets for prevention of androgen-independent prostate cancer.


   Acknowledgments
 
Grant support: Department of Defense grant W81XWH and NIH grant CA35368 (A.K. Verma).

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 David B. Pierce for expert technical assistance.


   Footnotes
 
4 http://www.foliobio.com Back

5 http://jaxmice.jax.org/pubcgi/protocols/protocols.sh?objtype=protocol&protocol_id=188 Back

Received 5/ 2/07. Revised 6/27/07. Accepted 7/10/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30.[Abstract/Free Full Text]
  2. Edwards J, Bartlett JM. The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 2. Androgen-receptor cofactors and bypass pathways. BJU Int 2005;95:1327–35.[CrossRef][Medline]
  3. Zhou J, Scholes J, Hsieh JT. Characterization of a novel negative regulator (DOC-2/DAB2) of c-Src in normal prostatic epithelium and cancer. J Biol Chem 2003;278:6936–41.[Abstract/Free Full Text]
  4. Miyamoto H, Altuwaijri S, Cai Y, et al. Inhibition of the Akt, cyclooxygenase-2, and matrix metalloproteinase-9 pathways in combination with androgen deprivation therapy: potential therapeutic approaches for prostate cancer. Mol Carcinog 2005;44:1–10.[CrossRef][Medline]
  5. Wu D, Foreman TL, Gregory CW, et al. Protein kinase C{varepsilon} has the potential to advance the recurrence of human prostate cancer. Cancer Res 2002;62:2423–9.[Abstract/Free Full Text]
  6. Huang HF, Murphy TF, Shu P, et al. Stable expression of constitutively-activated STAT3 in benign prostatic epithelial cells changes their phenotype to that resembling malignant cells. Mol Cancer 2005;4:2.[CrossRef][Medline]
  7. Klampfer L. Signal transducers and activators of transcription (STATs): novel targets of chemopreventive and chemotherapeutic drugs. Curr Cancer Drug Targets 2006;6:107–21.[CrossRef][Medline]
  8. Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in inflammation and cancer. Eur J Cancer 2005;41:2502–12.[CrossRef][Medline]
  9. Kortylewski M, Jove R, Yu H. Targeting STAT3 affects melanoma on multiple fronts. Cancer Metastasis Rev 2005;24:315–27.[CrossRef][Medline]
  10. Nikitakis NG, Siavash H, Sauk JJ. Targeting the STAT pathway in head and neck cancer: recent advances and future prospects. Curr Cancer Drug Targets 2004;4:637–51.[CrossRef][Medline]
  11. Vinkemeier U. Getting the message across, STAT! Design principles of a molecular signaling circuit. J Cell Biol 2004;167:197–201.[Abstract/Free Full Text]
  12. Stephanou A, Latchman DS. Opposing actions of STAT-1 and STAT-3. Growth Factors 2005;23:177–82.[CrossRef][Medline]
  13. Akira S. Roles of STAT3 defined by tissue-specific gene targeting. Oncogene 2000;19:2607–11.[CrossRef][Medline]
  14. Chan KS, Sano S, Kiguchi K, et al. Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis. J Clin Invest 2004;114:720–8.[CrossRef][Medline]
  15. Alvarez JV, Febbo PG, Ramaswamy S, et al. Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res 2005;65:5054–62.[Abstract/Free Full Text]
  16. Aziz MH, Manoharan HT, Verma AK. Protein kinase C{varepsilon}, which sensitizes skin to sun's UV radiation-induced cutaneous damage and development of squamous cell carcinomas, associates with Stat3. Cancer Res 2007;67:1385–94.[Abstract/Free Full Text]
  17. Rivat C, Rodrigues S, Bruyneel E, et al. Implication of STAT3 signaling in human colonic cancer cells during intestinal trefoil factor 3 (TFF3)—and vascular endothelial growth factor-mediated cellular invasion and tumor growth. Cancer Res 2005;65:195–202.[Abstract/Free Full Text]
  18. Kobielak A, Fuchs E. Links between {alpha}-catenin, NF-{kappa}B, and squamous cell carcinoma in skin. Proc Natl Acad Sci U S A 2006;103:2322–7.[Abstract/Free Full Text]
  19. Decker T, Kovarik P. Serine phosphorylation of STATs. Oncogene 2000;19:2628–37.[CrossRef][Medline]
  20. Li L, Shaw PE. A STAT3 dimer formed by inter-chain disulphide bridging during oxidative stress. Biochem Biophys Res Commun 2004;322:1005–11.[CrossRef][Medline]
  21. Kazanietz MG. Eyes wide shut: protein kinase C isozymes are not the only receptors for the phorbol ester tumor promoters. Mol Carcinog 2000;28:5–11.[CrossRef][Medline]
  22. Griner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. Nat Rev Cancer 2007;7:281–94.[CrossRef][Medline]
  23. Mellor H, Parker PJ. The extended protein kinase C superfamily. Biochem J 1998;332:281–92.[Medline]
  24. Newton AC. Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev 2001;101:2353–64.[CrossRef][Medline]
  25. Mochly-Rosen D, Kauvar LM. Modulating protein kinase C signal transduction. Adv Pharmacol 1998;44:91–145.[Medline]
  26. Basu A, Sivaprasad U. Protein kinase C{varepsilon} makes the life and death decision. Cell Signal 2007;19:1633–42.[CrossRef][Medline]
  27. Pan Q, Bao LW, Kleer CG, et al. Protein kinase C{varepsilon} is a predictive biomarker of aggressive breast cancer and a validated target for RNA interference anticancer therapy. Cancer Res 2005;65:8366–71.[Abstract/Free Full Text]
  28. Reddig PJ, Dreckschmidt NE, Zou J, et al. Transgenic mice overexpressing protein kinase C{varepsilon} in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion. Cancer Res 2000;60:595–602.[Abstract/Free Full Text]
  29. Wheeler DL, Ness KJ, Oberley TD, et al. Protein kinase C{varepsilon} is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-{alpha} ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase C{varepsilon} transgenic mice. Cancer Res 2003;63:6547–55.[Abstract/Free Full Text]
  30. Jansen AP, Verwiebe EG, Dreckschmidt NE, et al. Protein kinase C-{varepsilon} transgenic mice: a unique model for metastatic squamous cell carcinoma. Cancer Res 2001;61:808–12.[Abstract/Free Full Text]
  31. Wheeler DL, Martin KE, Ness KJ, et al. Protein kinase C{varepsilon} is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas. Cancer Res 2004;64:7756–65.[Abstract/Free Full Text]
  32. Wheeler DL, Li Y, Verma AK. Protein kinase C{varepsilon} signals ultraviolet light-induced cutaneous damage and development of squamous cell carcinoma possibly through induction of specific cytokines in a paracrine mechanism. Photochem Photobiol 2005;81:9–18.[CrossRef][Medline]
  33. Cornford P, Evans J, Dodson A, et al. Protein kinase C isoenzyme patterns characteristically modulated in early prostate cancer. Am J Pathol 1999;154:137–44.[Abstract/Free Full Text]
  34. Wu D, Thakore CU, Wescott GG, et al. Integrin signaling links protein kinase C{varepsilon} to the protein kinase B/Akt survival pathway in recurrent prostate cancer cells. Oncogene 2004;23:8659–72.[CrossRef][Medline]
  35. Greenberg NM, DeMayo F, Finegold MJ, et al. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A 1995;92:3439–43.[Abstract/Free Full Text]
  36. Bostwick DG. Neoplasms of the prostate. In: Bostwick DG, Eble JN, editors. Urologic surgical pathology. St. Louis: Mosby-Year Book, Inc.; 1997. p. 359.
  37. Eble JN, Sauter G, Epstein JI, Sesterhenn IA, editors. World Health Organization classification of tumours: pathology and genetics of tumours of the urinary system and male genital organs. Lyon: IARC Press; 2004. p. 161.
  38. Eng MH, Charles LG, Ross BD, et al. Early castration reduces prostatic carcinogenesis in transgenic mice. Urology 1999;54:1112–9.[CrossRef][Medline]
  39. Kaplan-Lefko PJ, Chen TM, Ittmann MM, et al. Pathobiology of autochthonous prostate cancer in a pre-clinical transgenic mouse model. Prostate 2003;55:219–37.[CrossRef][Medline]
  40. Corsini E, Galli CL. Cytokines and irritant contact dermatitis. Toxicol Lett 1998;102–103:277–82.
  41. Perambakam SM, Srivastava R, Peace DJ. Distinct cytokine patterns exist in peripheral blood mononuclear cell cultures of patients with prostate cancer. Clin Immunol 2005;117:94–9.[CrossRef][Medline]
  42. Uehara H, Troncoso P, Johnston D, et al. Expression of interleukin-8 gene in radical prostatectomy specimens is associated with advanced pathologic stage. Prostate 2005;64:40–9.[CrossRef][Medline]
  43. Tan D, Wu X, Hou M, et al. Interleukin-6 polymorphism is associated with more aggressive prostate cancer. J Urol 2005;174:753–6.[CrossRef][Medline]
  44. Culig Z, Steiner H, Bartsch G, et al. Interleukin-6 regulation of prostate cancer cell growth. J Cell Biochem 2005;95:497–505.[CrossRef][Medline]
  45. Wu D, Terrian DM. Regulation of caveolin-1 expression and secretion by a protein kinase C{varepsilon} signaling pathway in human prostate cancer cells. J Biol Chem 2002;277:40449–55.[Abstract/Free Full Text]
  46. Jain N, Zhang T, Kee WH, et al. Protein kinase C{delta} associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J Biol Chem 1999;274:24392–400.[Abstract/Free Full Text]
  47. Xuan YT, Guo Y, Zhu Y, et al. Role of the protein kinase C-{varepsilon}-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation 2005;112:1971–8.[Abstract/Free Full Text]
  48. Zhang Y, Cho YY, Petersen BL, et al. Ataxia telangiectasia mutated proteins, MAPKs, and RSK2 are involved in the phosphorylation of STAT3. J Biol Chem 2003;278:12650–9.[Abstract/Free Full Text]
  49. Zhang Y, Liu G, Dong Z. MSK1 and JNKs mediate phosphorylation of STAT3 in UVA-irradiated mouse epidermal JB6 cells. J Biol Chem 2001;276:42534–42.[Abstract/Free Full Text]
  50. Poole AW, Pula G, Hers I, et al. PKC-interacting proteins: from function to pharmacology. Trends Pharmacol Sci 2004;25:528–35.[CrossRef][Medline]
  51. Prekeris R, Hernandez RM, Mayhew MW, et al. Molecular analysis of the interactions between protein kinase C-{varepsilon} and filamentous actin. J Biol Chem 1998;273:26790–8.[Abstract/Free Full Text]
  52. Vallentin A, Mochly-Rosen D. RBCK1, a protein kinase CßI (PKCßI)-interacting protein, regulates PKCß-dependent function. J Biol Chem 2007;282:1650–7.[Abstract/Free Full Text]
  53. Kashiwagi M, Ohba M, Watanabe H, et al. PKC{eta} associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes. Oncogene 2000;19:6334–41.[CrossRef][Medline]
  54. Edmondson RD, Vondriska TM, Biederman KJ, et al. Protein kinase C{varepsilon} signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS. Mol Cell Proteomics 2002;1:421–33.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
P. Gandellini, M. Folini, N. Longoni, M. Pennati, M. Binda, M. Colecchia, R. Salvioni, R. Supino, R. Moretti, P. Limonta, et al.
miR-205 Exerts Tumor-Suppressive Functions in Human Prostate through Down-regulation of Protein Kinase C{varepsilon}
Cancer Res., March 15, 2009; 69(6): 2287 - 2295.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. K. Dissanayake, P. B. Olkhanud, M. P. O'Connell, A. Carter, A. D. French, T. C. Camilli, C. D. Emeche, K. J. Hewitt, D. T. Rosenthal, P. D. Leotlela, et al.
Wnt5A Regulates Expression of Tumor-Associated Antigens in Melanoma via Changes in Signal Transducers and Activators of Transcription 3 Phosphorylation
Cancer Res., December 15, 2008; 68(24): 10205 - 10214.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. H. Aziz, N. E. Dreckschmidt, and A. K. Verma
Plumbagin, a Medicinal Plant-Derived Naphthoquinone, Is a Novel Inhibitor of the Growth and Invasion of Hormone-Refractory Prostate Cancer
Cancer Res., November 1, 2008; 68(21): 9024 - 9032.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. R. Qin, H.-J. Kim, J.-Y. Kim, E. M. Hurt, G. J. Klarmann, B. T. Kawasaki, M. A. Duhagon Serrat, and W. L. Farrar
Activation of Signal Transducer and Activator of Transcription 3 through a Phosphomimetic Serine 727 Promotes Prostate Tumorigenesis Independent of Tyrosine 705 Phosphorylation
Cancer Res., October 1, 2008; 68(19): 7736 - 7741.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Chen, F. Deng, S. V. Singh, and Q. J. Wang
Protein Kinase D3 (PKD3) Contributes to Prostate Cancer Cell Growth and Survival Through a PKC{varepsilon}/PKD3 Pathway Downstream of Akt and ERK 1/2
Cancer Res., May 15, 2008; 68(10): 3844 - 3853.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aziz, M. H.
Right arrow Articles by Verma, A. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aziz, M. H.
Right arrow Articles by Verma, A. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online