Protein kinase C epsilon (PKCε), a novel PKC isoform, is overexpressed in prostate cancer (PCa) and correlates with disease aggressiveness. However, the functional contribution of PKCε to development or progression of PCa remained to be determined. Here we present the first in vivo genetic evidence that PKCε is essential for both the development and metastasis of PCa in the transgenic mouse model of prostate adenocarcinoma (TRAMP). Heterozygous or homozygous genetic deletions of PKCε in FVB/N TRAMP inhibited PCa development and metastasis as analyzed by positron emission tomography/computed tomography, tumor weight determinations, and histopathology. We also examined biomarkers associated with tumor progression in this model, including markers of survival, proliferation, angiogenesis, inflammation, and metastatic progression. To find clues about the genes regulated by PKCε and linked to the Stat3 signaling pathway, we carried out focused PCR arrays of JAK/STAT signaling in excised PCa tissues from PKCε wild-type and nullizygous TRAMP mice. Notably, PKCε loss was associated with significant downregulation of proliferative and metastatic genes C/EBPβ (CCAAT/enhancer binding protein β), CRP (C-reactive protein), CMK, EGFR (epidermal growth factor receptor), CD64, Jun B, and gp130. Taken together, our findings offer the first genetic evidence of the role of PKCε in PCa development and metastasis. PKCε may be potential target for prevention and/or treatment of PCa. Cancer Res; 71(6); 2318–27. ©2011 AACR.
Prostate cancer (PCa) is the most common type of cancer in American men and ranks second to lung cancer in cancer-related deaths. The American Cancer Society has estimated that 217,730 new cases will be diagnosed and 32,050 deaths will occur in the United States alone in the year of 2010 (1). The lack of effective therapies for advanced PCa is related to a large extent to the poor understanding for the molecular mechanisms underlying the progression of the disease toward invasion and metastasis (2). Therefore, defining the novel molecular targets linked to PCa progression and metastasis will improve the planning strategies for the prevention and treatment of PCa.
PKC is a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate. It represents a large family of phosphatidylserine (PS)-dependent serine/threonine kinases (3–7). On the basis of structural similarities and cofactor dependence, 11 PKC isoforms have been classified into 3 subfamilies: the classic (cPKC), the novel (nPKC), and the atypical (aPKC). The cPKCs (α, βI, βII, γ) are dependent on PS, diacylglycerol (DAG), and Ca2+. The nPKCs (δ, ε, η, and θ) retain responsiveness to DAG and PS but do not require Ca2+ for full activation. The aPKCs (λ and ζ) require only PS for their activation (3–5). PKC epsilon (PKCε) is involved in the regulation of diverse cellular functions including gene expression, neoplastic transformation, cell adhesion, mitogenicity, and cell invasion (8, 9).
PKCε has been considered to be the hallmark of PCa development. Evidence suggests that overexpression of PKCε is sufficient to promote conversion of androgen-dependent LNCaP cells to androgen-independent (AI) variant, which rapidly initiates tumor growth in vivo in both intact and castrated athymic nude mice (10). A recent study has shown that overexpression of PKCε protected LNCaP cells against apoptotic stimuli via inducing phosphorylation of Bad at Ser112 residue (11). It has been shown that integrin signaling links PKCε to the PKB/AKT survival pathway in recurrent PCa cells (12). Proteomic analysis of PCa CWR22 cells xenografts show that association of PKCε with Bax may neutralize apoptotic signals propagated through the mitochondrial death signaling pathway (13). We and others have previously shown that PKCε level correlates with the aggressiveness of human PCa. Also, PKCε is overexpressed in PCa spontaneously developed in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, an autochthonous transgenic model that mimics to the human disease (14). We have also shown that PKCε is a protein partner of transcription factor Stat3. PKCε associates with Stat3 and this association increases with the progression of the diseases in TRAMP mice and in human PCa. Taken together, all these findings suggest that PKCε is an oncogene and is involved in PCa development and aggressiveness and in the emergence of AI PCa (14). However, the role of PKCε in PCa development and progression in intact mouse model remains elusive. We present here for the first time that genetic loss of PKCε in TRAMP mice prevents development and metastasis of PCa, possibly via downregulation of proliferative and metastatic genes.
Materials and Methods
Monoclonal or polyclonal antibodies specific for Bcl-xL, β-actin, COX-2, gp130, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), PKCε, PKCα, PKCδ, PKCβII, PKCθ, PKCγ, PKCλ, PKCζ PKCε, PI3K85, PI3K110, PCNA (proliferating cell nuclear antigen), total Stat3, total Stat5, and VEGF were purchased from Santa Cruz Biotechnology. Blocking peptides for gp130 and Stat3 antibodies and mouse IgG were also procured from Santa Cruz Biotechnology. Monoclonal antibodies specific for pStat3Tyr705, and pStat3Ser727 were obtained from BD Biosciences. Antibodies specific to pAKTSer308, pAKTSer473, and total AKT were purchased from Cell Signaling Technology.
FVB-TRAMP mice were provided by Dr. Barbara Foster, Roswell Park Cancer Institute, Buffalo, NY. PKCε KO mice were generated and provided by Dr. Michael Leitges, Max-Plank-Institut Fur Immunoliologie, and Freiburg, Germany. To create the bigenic mice, TRAMP mice (TG/WT//+/+) were crossbred with heterozygous (Het) null PKCε (WT/WT//−/+). TG/WT//−/+ and WT/WT//−/+ mice were obtained and crossbred to generate the TRAMP-PKCε WT (WT), TRAMP-PKCε Het (Het), and TG//PKCε KO (KO) mice. A total of 21 mice containing TG (n = 7), Het (n = 7), and KO (n = 7) were used for the study. After 18 weeks, all the mice of TG, Het, and KO groups were euthanized and their blood was collected for serum preparation.
Two animals from each group (TG, Het, and KO) were used for positron emission tomography (PET)/computed tomography (CT). We utilize a radioiodinated phospholipid ether analogue, 124I-NM404. Following injection with 124I-NM404, animals were anesthetized with isoflurane and scanned at desired time intervals by hybrid micro-CT/PET (100-μm spatial CT resolution, 40 million PET counts, filtered back projection image reconstruction).
Prostate, lung, kidney, liver, and lymph nodes were excised and processed for histology as described previously (15).
IL-6 ELISA assay
Interleukin 6 (IL-6) levels were determined from mouse serum by using specific ELISA kit for mouse IL-6 (eBioscience, Inc.). All of the procedures were followed by the manufacturer's protocol.
Tissues from PCa and brain were excised and whole tissue lysates were prepared. In brief, part of the PCa and brain tissues of TG, Het, and KO mice were homogenized in lysis buffer [50 mmol/L HEPES, 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 μmol/L Na3VO4, 200 μmol/L NaF, and 1 mmol/L EGTA (final pH 7.5)]. The homogenate was centrifuged at 14,000 × g for 30 minutes at 4°C. Supernatants were collected and stored at −80°C until further use. Protein was estimated using Bio-Rad protein assay kit as per the manufacturer's protocol. Forty micrograms of tissue lysate were fractionated on 10% criterion precast SDS-polyacrylamide gel (Bio-Rad Laboratories). The protein was transferred to 0.45 μm Hybond-P polyvinylidene difluoride transfer membrane (Amersham Life Sciences). The membrane was then incubated with the indicated antibody followed by a horseradish peroxidase secondary antibody (Thermo Scientific), and the detection signal was developed with Amersham's enhanced chemiluminescence reagent using FOTO/Analyst Luminary Work Station (Fotodyne Inc.). The Western blots were quantitated by densitometric analysis by using Totallab Nonlinear Dynamic Image analysis software (Nonlinear USA, Inc.).
Electrophoretic mobility shift assay
Nuclear extracts were prepared from prostate tissues from TG, Het, and KO mice in a high-salt buffer [20 mmol/L HEPES (pH 7.9), 20 mmol/L NaF, 1 mmol/L Na3P2O7, 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, and 1 μg/mL aprotinin]. The samples were then centrifuged and harvested. Protein was estimated by using protein assay kit (Bio-Rad) as per the manufacturer's protocol. Stat3 DNA binding activity was determined in individual mouse nuclear lysates of each group. In brief, nuclear extracts (10 μg) were 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 minutes. A P32-radiolabeled double-stranded Stat3 consensus binding motif 5′-GATCCTTCTGGGAATTCCTAGATC-3′ (Santa Cruz Biotechnology) probe was then added and incubated for 20 minutes at room temperature. The protein–DNA complexes were resolved on a 4.5% nondenaturing polyacrylamide gel containing 2.5% glycerol in 0.25× Tris-borate EDTA at room temperature, and gels were dried and autoradiographed. Stat3 DNA binding activities were determined.
To determine the expression of Stat3, IL-6 receptor (IL-6R), and PCNA proteins in prostatic intraepithelial neoplasia (PIN) and moderately and poorly differentiated (PD) adenocarcinoma of each mouse of TG and KO, we conducted immunohistochemistry in paraffin-embedded sections (4-μm thickness). In brief, sections were deparafffinized by placing the slides at 60°C for 2 hours followed by 3 changes of xylenes for 10 minutes each. Slides were placed in 0.3% methanol/hydrogen peroxide for 20 minutes for quenching endogenous peroxidase. Slides were rehydrated in one change of absolute, 95%, 75%, and 50% ethanol and distilled water. Antigen retrieval was carried out by incubating samples at 116°C for 15 seconds in the declocking chamber by using a Tris–urea solution (pH 9.5). After antigen retrieval, tissues slides were incubated with 2.5% normal horse serum (R.T.U. Vectastain Universal Elite ABC Kit; Vector Laboratories) for 20 minutes to block nonspecific binding of the antibodies. Subsequently, the slides were incubated over night with a mixture of Stat3 (1:50), IL-6R (1:50), and PCNA (1:50) dilution in normal antibody diluents (Scy Tek # ABB-125) in a humidified chamber. Blocking peptides of Stat3 and IL-6 antibodies were used to determine the specific immunoreactivity of these antibodies. We further confirmed the specificity of immunostaining of PCNA, IL-6, and Stat3 by using IgG antibody (served as a negative control). The mixture of antibodies was decanted, and the slides were washed thrice in TBS (pH 7.4). The slides were incubated with appropriate secondary antibodies for 30 minutes at room temperature. Slides were rinsed with TBS for 5 minutes and ABC reagent (Vector kit) was applied for 30 minutes. Immunoreactive complexes were detected using 3,3′-diaminobenzidine substrate (Thermo Scientific) and counter stained by using hematoxylin (Fischer Scientific) for nuclear staining. Finally, slides were mounted with cover slip by using mounting medium. All sections were visualized under a Zeiss-Axiophot DM HT microscope and images were captured with an attached camera. Dr. Weixiong Zhong, MD, a certified pathologist, examined all the slides. For the quantification of Stat3- and PCNA-positive staining cells, 5 random areas were selected from each slide of TG and KO mice. The number of cells showing positive labeling and the total number of cells counted were recorded. An average percentage was then calculated on the basis of the total number of positive staining cells from each set of 5 fields counted. Results are expressed as mean ± SEM.
Quantitative real-time PCR array
Changes in expression of genes involved in the JAK/STAT signaling pathway was determined using real-time PCR (RT-PCR) arrays. In brief, excised PCa tissue from TG and KO mice were snap frozen and stored at −80°C. Total RNA was isolated using TRIzol reagent (Invitrogen) as per the manufacturer's instruction. RNA concentration and purity were checked by UV spectrophotometry, and RNA integrity was determined by electrophoresis. The first cDNA strand was synthesized using RT2 First Strand Kit (SA Biosciences). The cDNA from either TG or KO PCa tissues was then applied to the RT2 Profiler PCR Array for the JAK/STAT pathway (SA Biosciences) according to manufacturer's instructions. The RT-PCR reactions were run on the MyIQ (Bio-Rad) and the results (Ct), normalized to β-actin (ΔCt), were determined using iQ5 software (Bio-Rad). Fold expression was determined as, where ΔΔCt is ΔCt(KO) − ΔCt(TG).
Student's t test was carried out to determine the significance. P < 0.05 was considered as significant.
Generation and characterization of PKCε-deleted TRAMP mice
As shown in Figure 1A, TG, Het, and KO mice were generated by cross-breeding 6 to 7 weeks old homozygous TRAMP with PKCε Het mice (Fig. 1A). Both TRAMP and PKCε KO mice were on FVB/N background. No PKCε expression was observed in either prostate or brain excised tissues of KO mice [Fig. 1B and C (i and ii)]. A significant decrease in protein levels of PKCε was observed in the prostate of Het mice compared with TG mice (Fig. 1B). To determine whether deletion of PKCε has any compensatory effect in TRAMP mice, we carried out immunoblot analysis of PKC isoforms in prostate and brain tissue lysates of TG, Het, and KO mice. Results indicate no change in the expression of other PKC isoforms in brain tissue of Het and KO mice [Fig. 1C (i and ii)], suggesting no compensatory effects in PKCε-deleted TRAMP mice.
Deletion of PKCε in TRAMP mice inhibits PCa development and metastasis
Accumulating evidence now indicates that PKCε is an oncogene which plays a vital role in the development of various types of human cancers including the prostate. Molecular genetic evidence of the role of PKCε in PCa development in an intact mouse model still remains obscure. In this study, we explored the possibility whether PKCε deletion in TRAMP mice inhibits the development and metastasis of PCa. A total of 21 mice (TG, n = 7; Het, n = 7; and KO, n = 7) were used in this study. In our first experiment, we carried out micro-PET/CT imaging, using a tumor selective radiopharmaceutical agent 124I-NM404 (16), of two 16 weeks old mice from each of TG and KO mice [Fig. 2A (i) and B (i)]. Results illustrated a lack of focal uptake of 124I-NM404 in KO mice [Fig 2B (i and ii)] compared with TG mice [Fig. 2A (i and ii)]. TG mice showed metastasis in proximal lymph node as evident by uptake of 124I-NM404 [Fig. 2A (i and ii)]. However, no metastasis was observed in KO mice, suggesting the role of PKCε in the development and metastasis of PCa. All of the remaining mice from each group were sacrificed at the same age (18 weeks). Their bloods were collected from retro-orbital plexus for serum isolation. PCa tissues were excised and parts of the tissues were used in preparation of whole tissue lysates, nuclear lysates, RNA isolation, and histology sectioning as described in Material and Methods. Parts of the tissues excised from the kidneys, brains, livers, lungs, and lymph nodes were fixed in 10% buffered formalin and used for histopathology. Deletion of PKCε in TRAMP mice, or even one allele deletion, shows significant (P < 0.01) reduction in growth of PCa in all of the Het and KO mice (Fig. 2C and D). All TG mice developed 1 or 2 large-sized prostate tumors (Fig. 2C), whereas Het and KO mice had only a single small-sized tumor (Fig. 2C). One of the TG mice also showed grossly visible metastases in a local lymph node, both lungs, and the left kidney, which were confirmed by light microscopy (Fig. 2E and F). In addition, microscopic metastases were identified in another 3 TG mice. No metastasis was identified in any of the Het and KO mice (Fig. 2G and H). Hematoxylin and eosin (H&E)-stained tissue sections showed that all the grossly visible tumors were PD carcinomas characterized by solid sheets of large polymorphic cancer cells, with a high nucleus-to-cytoplasm ratio, frequent apoptosis, central necrosis, and neuroendocrine differentiation [Fig. 3D (i) and E (i)]. Some of the Het and KO mice showed small foci of PD carcinoma at the microscopic level only. In addition, all the mice showed PIN characterized by epithelial cell proliferation with enlarged hyperchromatic nuclei and nuclear stratification in papillary and cribriform structures [Fig. 3D (i) and E (i)].
Deletion of PKCε in TRAMP mice inhibits Stat3 activation
Aberrant activation of Stat3 has been linked to the progression of PCa metastasis (17–19). A study suggests that overexpression of Stat3 in normal prostate epithelial cells leads to conversion of malignant phenotype (20). To determine whether PKCε deletion in TRAMP mice inhibits Stat3 activation, we carried out immunoblot analysis in excised tissue PCa lysates of TG, Het, and KO mice. Results illustrated significant inhibition of both Ser727 and Tyr705 phosphorylation of Stat3 in both Het and KO mice (Fig. 3A). A significant decrease in DNA binding activity of Stat3 was observed in Het and KO mice [Fig. 3B (i and ii)]. Immunohistochemistry results show a significant decrease in intensity of nuclear staining of Stat3 in PIN and PD PCa of KO mice [Fig. 3C and E (ii)] compared with those in TG mice [Fig. 3D (ii)]. Specificity of Stat3 staining in TG PCa tumor tissue was confirmed using blocking peptide of Stat3 (Fig. 3F).
Deletion of PKCε inhibits serum IL-6 and IL-6R gp130 expression
We have previously shown elevated levels of IL-6 in TRAMP mice compared with nontransgenic mice at the same age (14). To determine whether deletion of PKCε in TRAMP mice decreases serum IL-6 levels, we carried out specific ELISA for mouse IL-6 and observed a significant (P < 0.05) decrease in serum IL-6 levels in Het and KO mice compared with TG mice (Fig. 4H), suggesting that PKCε might be initiator of the IL-6/Stat3 signaling pathway. IL-6R (gp130) is one of the downstream target genes of Stat3. Immunohistochemistry results showed a significant decrease in the protein levels of IL-6R in KO mice compared with TG mice (Fig. 4A). Weak immunostaining of IL-6R was observed in benign epithelial cells and moderate staining in PD carcinoma cells of both TG and KO mice [Fig. 4E (i)], although there is strong immunostaining of IL-6R in PIN of TG mice and only moderate staining in PIN of KO mice [Fig. 4D (i)]. Specific immunoreactivity of IL-6R was confirmed by using blocking peptide of IL-6R antibody [Fig. 4F (i)].
Deletion of PKCε inhibits markers of proliferation, antiapoptosis, and metastasis
Evidence from published studies, including our laboratory, suggests modulation of various apoptotic and proliferative biomarkers in PCa in both humans and TRAMP mice during the progression and metastasis (14, 21). To determine possible changes in biomarkers involved in apoptosis, proliferation, and metastasis of PCa in PKCε-deleted TRAMP mice, we conducted immunoblot analysis of selected biomarkers in PCa tissue lysates of TG, Het, and KO mice. We observed a decrease in the protein levels of Bcl-xL, COX-2, cyclin D1, and VEGF in Het and KO mice (Fig. 4A and B). We have previously shown overexpression of PI3K/AKT in PCa tissues of TRAMP mice compared with nontransgenic prostate tissues at the same age (14). We observed a decrease in the protein levels of regulatory subunit (p85β) of PI3K in Het and KO mice but no change was observed in the protein levels of catalytic isoform (p110β) of PI3K (Fig. 4B), pAKTSer473, or pAKTSer308 (Fig. 4C). We also carried out immunohistochemistry of PCNA in PCa tissues of TG and KO mice and observed a significant decrease in the nuclear levels of PCNA in PIN and PD PCa of KO mice [Fig. 4E (ii)–G].
Quantitative PCR array identifies decreased transcripts of genes implicated in PCa development and metastasis
To further define the role of PKCε in modulating the other genes associated with JAK/STAT3 signaling and involved in PCa development and metastasis, we carried out quantitative RT-PCR (qRT-PCR) array of genes associated with JAK/STAT signaling pathway on total RNA isolated from PCa tissues of TG and KO mice. This array contained 84 genes related to JAK/STAT family members, the receptors that activate them, nuclear cofactor and coactivators associated with the Stat proteins, Stat-inducible genes, and negative regulators. A significant decrease in the mRNA expression of CCAAT/enhancer binding protein β (C/EBPβ; 2.5-fold), C-reactive protein (CRP; 4.34-fold), epidermal growth factor receptor (EGFR; 4.54-fold), gp130 (2.5-fold), Jun B (2.5-fold), and Stat3 (2.85-fold) was observed in KO mice compared with TG mice (Table 1). These results indicate that PKCε directly and/or indirectly regulates the expression of Stat3 and Stat3 downstream target genes involved in PCa development and metastasis.
In this study, we for the first time investigated PKCε link between the development and metastasis of PCa in transgenic mouse model (TRAMP). We used a molecular genetic approach to generate bigenic TRAMP mice, which were also either wild-type, heterozygous null, or homozygous null for PKCε. Deletion of PKCε did not affect other isoforms of PKC in Het or KO mice, suggesting no compensatory effect of PKCε deletion in TRAMP mice. All of the PKCε-deleted TRAMP mice were healthy and we did not observe any gross pathologic changes in any organs of these animals.
TRAMP PCa closely mimics human PCa, as they both spontaneously develop progressive PCa. TRAMP PCa is invasive and capable of metastasis to distant sites, primarily to pelvic lymph nodes and to the lungs (22). Mouse genetic background affects the development of TRAMP PCa. TRAMP mice on FVB/N background more readily develop PIN compared with TRAMP mice on C57Black/B16 background (22). In our study, we observed that deletion of PKCε in TRAMP mice prevents tumor development, which was evident by a lack of focal uptake of 124I-NM404, a selective tumor agent [Fig. 2B (ii)]. This was followed by a significant (P < 0.01) decrease in prostate tumor weight in Het and KO animals (Fig. 2C and D). Metastasis of PCa to distant organs is the main cause of morbidity and mortality in PCa patients. Importantly, we observed the metastasis of PCa to lungs, lymph nodes, and kidneys of the 70% TG animals, which was evident by the uptake of 124I-NM404 into thelymph nodes. Histopathologic examinations of lungs, lymph nodes, and kidneys of TG mice further confirm the metastasis of PCa. However, no metastasis was detected in any of the animals, even in the 2 mice from each Het and KO groups that had PCa. Taken together, these results strongly support that loss of PKCε inhibits both primary PCa and metastasis.
PCa is heterogeneous in nature, which involves activation of multiple signaling pathways. Constitutive activation of the transcription factor Stat3 has been shown to be a marker of poor prognosis in human PCa. Activation of Stat3 has been linked to the PCa development and metastasis through the induction of various genes responsible for tumor cell proliferation, cell survival, and carcinogenesis (17–19). In our previous studies, we have shown that PKCε activates and physically interacts with Stat3 in various types of human cancer cells including the prostate (23). In this study, loss of PKCε in TRAMP mice inhibited activation of Stat3, which was manifested by significant inhibition of both pStat3Ser727 and pStat3Tyr705 protein levels and decreased Stat3-DNA binding activity. Decrease in Stat3 nuclear staining was observed in PIN and PD adenocarcinoma of KO mice. These results are in accordance with our previously published reports, in which we have shown the interaction of PKCε with Stat3 during development of skin carcinoma in PKCε transgenic mice (24). These data further proves that PKCε may be an initiator for activation of Stat3 during PCa development. IL-6 is involved in activation of Stat3, which phosphorylates tyrosine residue of Stat3. Higher level of serum IL-6 has been associated with larger PCa burden, especially bone metastases (25, 26). In our study, KO mice showed a significant (P < 0.05) decrease in serum IL-6 and its receptor gp130, which indicates that PKCε may have a direct or indirect role in potentiating the autocrine loop of IL-6 signaling in PCa. However, further detail investigation is warranted to understand the molecular mechanism of PKCε-mediated IL-6 signaling during the PCa development and metastasis. We further investigated various downstream target genes of Stat3 which have a role in the PCa inflammation, proliferation, angiogenesis, and metastasis (COX-2, cyclin D1, VEGF, Bcl-xL, IL-6R) which were significantly inhibited in KO mice. These data further provide evidence that PKCε is linked to activation of Stat3. PCNA is a nuclear protein, which has been known as a cell proliferative marker, and a decrease in the expression of PCNA reflects cell-cycle arrest in G1/S phase (27). We observed a significant decrease in the expression of PCNA in KO mice. These data correspond to the previous study, which has shown that knockdown of PKCε in PCa LNCaP cells arrested the cell cycle in G1/S phase (10). We also conducted focused qRT-PCR arrays of genes involved in the JAK/STAT signaling pathway in TG and KO PCa tissues to determine the role of PKCε in JAK/STAT signaling. A significant decrease in mRNA expression of CEBPβ, CRP, EGFR, gp130, Jun B, and Stat3 was observed in KO mice compared with TG mice (Table 1). These results further provide clues about the role of PKCε in activation of Stat3 and other signaling molecules linked to the PCa development and metastasis. However, a detailed investigation is warranted to find out the cross-talk of PKCε with JAK/STAT signaling pathway–associated molecules.
In summary, deletion of PKCε in FVB/N TRAMP mice inhibits PCa development and metastasis. We have previously reported that Stat3 is a protein partner of PKCε (14, 23). PKCε interacts with Stat3 and phosphorylates Stat3Ser727, essential for Stat3 activation and nuclear localization (14). In consistent with previous findings, deletion of PKCε in FVB/N TRAMP mice downregulated prostatic Stat3 activation and Stat3-regulated gene expressions. We conclude that PKCε and its downstream protein partner Stat3 constitute essential components of the signal transduction pathways involved in PCa development and metastasis. Therefore, we suggest that targeting PKCε is a novel approach for prevention or treatment of PCa.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This study was supported by DOD grant (W81XWH), NIH grant CA35368, and UWCCC Cancer Center Support grant 2 P30 CA014520-34 for small animal imaging facility.
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.
- Received November 17, 2010.
- Revision received January 21, 2011.
- Accepted January 26, 2011.
- ©2011 American Association for Cancer Research.