Androgen Receptor and Its Splicing Variant 7 Expression in Peripheral Blood Mononuclear Cells and in Circulating Tumor Cells in Metastatic Castration-Resistant Prostate Cancer.

Androgen receptor (AR) signaling remains crucial in castration-resistant prostate cancer (CRPC). Since it is also essential in immune cells, we studied whether the expression of AR full-length (ARFL) and its splicing variant ARV7 in peripheral blood mononuclear cells (PBMC) predicts systemic treatment response in mCRPC in comparison with circulating-tumor cells (CTC). We measured ARFL and ARV7 mRNA in PBMC and CTC from patients prior to receiving abiraterone (AA), enzalutamide (E), or taxanes by a pre-amplification plus quantitative reverse-transcription PCR. They were also tested in LNCaP-ARV7-transfected and in 22RV1 docetaxel-resistant (22RV1DR) cells. We studied 171 PBMC from 136 patients and from 24 non-cancer controls, and 47 CTC from 22 patients. High PBMC ARV7 levels correlated with worse AA/E and better taxane response. In taxane-treated patients high PBMC ARFL also correlated with longer progression-free survival (PFS). High ARV7 and ARFL expression were independently associated with better biochemical-PFS. Conversely, high CTC ARV7 and ARFL correlated with shorter radiological-PFS and overall survival, respectively. High ARV7 in 22RV1DR and LNCaP-ARV7 cells correlated with taxane resistance. In conclusion, ARFL and ARV7 at PBMC or CTC have a different predictive role in the taxane response, suggesting a potential influence of the AR pathway from PBMC in such response modulation.

Ultraviolet radiation-induced differential microRNA expression in the skin of hairless SKH1 mice, a widely used mouse model for dermatology research.

Cutaneous squamous cell carcinoma (cSCC) is the most common type of non-melanoma skin cancer that can metastasize. The major etiological factor associated with cSCC is Ultraviolet radiation (UVR) with a limited understanding of its molecular mechanism. It was hypothesized that there is a direct effect of UVR on modulation of microRNAs (miRNAs), a novel class of short noncoding RNAs which affects translation and stability of mRNAs. To test the hypothesis, the dorsal skin of the SKH1 mice (6-7 week old) was exposed to acute and chronic doses of UVR. In miRNA array profiling, we found differential expression (log fold change>1) of miR-25-5p between untreated and acute UVR treated (4kJ/m2) SKH1 mice skin. However, differential expression (>1 log fold) of miR-144-3p, miR-33-5p, miR-32-5p, miR-1983, miR-136-5p, miR-142-3p, miR-376a-3p, miR-142-5p, miR-3968, and miR-29b-3p was observed between untreated and chronically UVR treated mice skin. Differentially expressed selected miRNAs (miR-32-5p, miR-33-5p, miR-144-3p, and miR-376a-3p) were further validated in real time PCR using miRNA specific primers. Web based data mining, for the prediction of potential miRNA associated gene pathways in miRBase database revealed a link with important pathways (PI3K-Akt, MAPK, Wnt, transcriptional misregulation, and other oncogenic pathway) associated with cSCC. Furthermore, findings of PI3K-Akt pathway genes affected due to chronic UVR were confirmed using cDNA array.

Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer.

PKCε is a transforming oncogene and a predictive biomarker of various human cancers. However, a precise in vivo link of PKCε to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice (PKCε-CKO) using cre-lox technology. Homozygous PKCε(LoxP/LoxP) mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostate, the PKCε(LoxP/LoxP) mice were bred to probasin cre (PB-Cre4+) mice which express cre specifically in the prostate epithelium of postnatal mice. Western blot and immunohistochemical analyses showed reduced levels of PKCε specifically in the prostate of PKCε-CKO mice. Histopathological analyses of prostate from both PKCε(LoxP/LoxP) and prostate PKCε-CKO mice showed normal pathology. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMPC1, 2×106) were implanted in the prostate of PKCε-CKO mice. Mice were sacrificed at 6th week post-implantation. Results demonstrated a significant (P<0.05) decrease in the growth of TRAMPC1 cells-derived xenograft tumors in PKCε-CKO mice compared to wild type. To determine a link of PKCε to ultraviolet radiation (UVR) exposure-induced epidermal Stat3 phosphorylation, PKCε(LoxP/LoxP) mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of UVR-induced Stat3 phosphorylation. In summary, our novel PKCε(LoxP/LoxP) mice will be useful for defining the link of PKCε to various cancers in specific organ, tissue, or cells.

Genetic deletion of TNFα inhibits ultraviolet radiation-induced development of cutaneous squamous cell carcinomas in PKCε transgenic mice via inhibition of cell survival signals.

Protein kinase C epsilon (PKCε), a Ca(2+)-independent phospholipid-dependent serine/threonine kinase, is among the six PKC isoforms (α, δ, ε, η, µ, ζ) expressed in both mouse and human skin. Epidermal PKCε level dictates the susceptibility of PKCε transgenic (TG) mice to the development of cutaneous squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by using the DMBA initiation-TPA (12-O-tetradecanoylphorbol-13-acetate) tumor promotion protocol (Wheeler,D.L. et al. (2004) Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas. Cancer Res., 64, 7756-7765). Histologically, SCC in TG mice, like human SCC, is poorly differentiated and metastatic. Our earlier studies to elucidate mechanisms of PKCε-mediated development of SCC, using either DMBA-TPA or UVR, indicated elevated release of cytokine TNFα. To determine whether TNFα is essential for the development of SCC in TG mice, we generated PKCε transgenic mice/TNFα-knockout (TG/TNFαKO) by crossbreeding TNFαKO with TG mice. We now present that deletion of TNFα in TG mice inhibited the development of SCC either by repeated UVR exposures or by the DMBA-TPA protocol. TG mice deficient in TNFα elicited both increase in SCC latency and decrease in SCC incidence. Inhibition of UVR-induced SCC development in TG/TNFαKO was accompanied by inhibition of (i) the expression levels of TNFα receptors TNFRI and TNFRII and cell proliferation marker ornithine decarboxylase and metastatic markers MMP7 and MMP9, (ii) the activation of transcription factors Stat3 and NF-kB and (iii) proliferation of hair follicle stem cells and epidermal hyperplasia. The results presented here provide the first genetic evidence that TNFα is linked to PKCε-mediated sensitivity to DMBA-TPA or UVR-induced development of cutaneous SCC.

Topically applied Hsp90 inhibitor 17AAG inhibits UVR-induced cutaneous squamous cell carcinomas.

We present here that heat-shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG), when topically applied to mouse skin, inhibits UVR-induced development of cutaneous squamous cell carcinoma (SCC). In these experiments, DMSO:acetone (1:40 v/v) solution of 17AAG (500 nmol) was applied topically to mouse skin in conjunction with each UVR exposure (1.8 kJ m(-2)). The UVR source was Kodacel-filtered FS-40 sun lamps (approximately 60% UVB and 40% UVA). In independent experiments with three separate mouse lines (SKH-1 hairless mice, wild-type FVB, and protein kinase C epsilon (PKCɛ)-overexpressing transgenic FVB mice), 17AAG treatment increased the latency and decreased both the incidence and multiplicity of UVR-induced SCC. Topical 17AAG alone or in conjunction with UVR treatments elicited neither skin nor systemic toxicity. 17AAG-caused inhibition of SCC induction was accompanied by a decrease in UVR-induced (1) hyperplasia, (2) Hsp90ß-PKCɛ interaction, and (3) expression levels of Hsp90ß, Stat3, pStat3Ser727, pStat3Tyr705, pAktSer473, and matrix metalloproteinase (MMP). The results presented here indicate that topical Hsp90 inhibitor 17AAG is effective in prevention of UVR-induced epidermal hyperplasia and SCC. One may conclude from the preclinical data presented here that topical 17AAG may be useful for prevention of UVR-induced inflammation and cutaneous SCC either developed in UVR-exposed or organ transplant population.

Protein Kinase C ε , Which Is Linked to Ultraviolet Radiation-Induced Development of Squamous Cell Carcinomas, Stimulates Rapid Turnover of Adult Hair Follicle Stem Cells.

To find clues about the mechanism by which kinase C epsilon (PKC ε ) may impart susceptibility to ultraviolet radiation (UVR)-induced development of cutaneous squamous cell carcinomas (SCC), we compared PKC ε transgenic (TG) mice and their wild-type (WT) littermates for (1) the effects of UVR exposures on percent of putative hair follicle stem cells (HSCs) and (2) HSCs proliferation. The percent of double HSCs (CD34+ and α 6-integrin or CD34+/CD49f+) in the isolated keratinocytes were determined by flow cytometric analysis. Both single and chronic UVR treatments (1.8 kJ/m(2)) resulted in an increase in the frequency of double positive HSCs in PKC ε TG mice as compared to their WT littermates. To determine the rate of proliferation of bulge region stem cells, a 5-bromo-2'-deoxyuridine labeling (BrdU) experiment was performed. In the WT mice, the percent of double positive HSCs retaining BrdU label was 28.4 ± 0.6% compared to 4.0 ± 0.06% for the TG mice, an approximately 7-fold decrease. A comparison of gene expression profiles of FACS sorted double positive HSCs showed increased expression of Pes1, Rad21, Tfdp1 and Cks1b genes in TG mice compared to WT mice. Also, PKC ε over expression in mice increased the clonogenicity of isolated keratinocytes, a property commonly ascribed to stem cells.

Plumbagin, a medicinal plant (Plumbago zeylanica)-derived 1,4-naphthoquinone, inhibits growth and metastasis of human prostate cancer PC-3M-luciferase cells in an orthotopic xenograft mouse model.

We present here first time that Plumbagin (PL), a medicinal plant-derived 1,4-naphthoquinone, inhibits the growth and metastasis of human prostate cancer (PCa) cells in an orthotopic xenograft mouse model. In this study, human PCa PC-3M-luciferase cells (2 × 10(6)) were injected into the prostate of athymic nude mice. Three days post cell implantation, mice were treated with PL (2 mg/kg body wt. i.p. five days in a week) for 8 weeks. Growth and metastasis of PC-3M-luciferase cells was examined weekly by bioluminescence imaging of live mice. PL-treatment significantly (p = 0.0008) inhibited the growth of orthotopic xenograft tumors. Results demonstrated a significant inhibition of metastasis into liver (p = 0.037), but inhibition of metastasis into the lungs (p = 0.60) and lymph nodes (p = 0.27) was not observed to be significant. These results were further confirmed by histopathology of these organs. Results of histopathology demonstrated a significant inhibition of metastasis into lymph nodes (p = 0.034) and lungs (p = 0.028), and a trend to significance in liver (p = 0.075). None of the mice in the PL-treatment group showed PCa metastasis into the liver, but these mice had small metastasis foci into the lymph nodes and lungs. However, control mice had large metastatic foci into the lymph nodes, lungs, and liver. PL-caused inhibition of the growth and metastasis of PC-3M cells accompanies inhibition of the expression of: 1) PKCε, pStat3Tyr705, and pStat3Ser727, 2) Stat3 downstream target genes (survivin and Bcl(xL)), 3) proliferative markers Ki-67 and PCNA, 4) metastatic marker MMP9, MMP2, and uPA, and 5) angiogenesis markers CD31 and VEGF. Taken together, these results suggest that PL inhibits tumor growth and metastasis of human PCa PC3-M-luciferase cells, which could be used as a therapeutic agent for the prevention and treatment of human PCa.

Plumbagin inhibits prostate cancer development in TRAMP mice via targeting PKCε, Stat3 and neuroendocrine markers.

Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al. (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024-9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2 mg/kg body weight i.p. in 0.2 ml phosphate-buffered saline, 5 days a week) to FVB-TRAMP resulted in a significant (P < 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKCε), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), isolated from Plumbago zeylanica, inhibits ultraviolet radiation-induced development of squamous cell carcinomas.

Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone, was isolated from the roots of the Plumbago zeylanica L. (also known as Chitrak). The roots of P. zeylanica L. have been used in Indian medicine for >2500 years as an anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective agent. We present here that topical application of non-toxic doses (100-500 nmol) of PL to skin elicits dose-dependent inhibition of ultraviolet radiation (UVR)-induced development of squamous cell carcinomas (SCC). In this experiment, FVB/N mice were exposed to UVR (2 kJ/m(2)) three times weekly from a bank of six Kodacel-filtered FS40 sunlamps (∼ 60% UVB and 40% UVA). Carcinoma incidence in mice treated with vehicle, 100, 200 or 500 nmol PL, at 44 weeks post-UVR, were 86, 80 (P = 0.67), 53 (P = 0.12) and 7% (P = 0.0075), respectively. Both vehicle and PL-treated mice gained weight and did not exhibit any signs of toxicity during the entire period of the experiment. Molecular mechanisms associated with inhibition of UVR-induced development of SCC involved induction of apoptosis and inhibition of cell proliferation. Specific findings are that PL treatment (i) inhibited UVR-induced DNA binding of activating protein-1, nuclear factor-kappaB, Stat3 transcription factors and Stat3-regulated molecules (cdc25A and Survivin); (ii) inhibited protein levels of pERK1/2, PI3K85, pAKTSer473, Bcl(2), BclxL, proliferating cell nuclear antigen and cell cycle inhibitory proteins p27 and p21 and (iii) increased UVR-induced Fas-associated death domain expression, poly (ADP-ribose) polymerase protein cleavage and Bax/Bcl(2) ratio. Taken together, our findings suggest that PL may be a novel agent for the prevention of skin cancer.

Genetic ablation of PKC epsilon inhibits prostate cancer development and metastasis in transgenic mouse model of prostate adenocarcinoma.

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/EBPß (CCAAT/enhancer binding protein ß), 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.

A randomized, double-blind, placebo-controlled phase 3 skin cancer prevention study of {alpha}-difluoromethylornithine in subjects with previous history of skin cancer.

Preclinical studies have shown that the inhibition of ornithine decarboxylase (ODC) by alpha-difluoromethylornithine (DFMO) and resultant decreases in tissue concentrations of polyamines (putrescine and spermidine) prevents neoplastic developments in many tissue types. Clinical studies of oral DFMO at 500 mg/m(2)/day revealed it to be safe and tolerable and resulted in significant inhibition of phorbol ester-induced skin ODC activity. Two hundred and ninety-one participants (mean age, 61 years; 60% male) with a history of prior nonmelanoma skin cancer (NMSC; mean, 4.5 skin cancers) were randomized to oral DFMO (500 mg/m(2)/day) or placebo for 4 to 5 years. There was a trend toward a history of more prior skin cancers in subjects randomized to placebo, but all other characteristics including sunscreen and nonsteroidal anti-inflammatory drug use were evenly distributed. Evaluation of 1,200 person-years of follow-up revealed a new NMSC rate of 0.5 events/person/year. The primary end point, new NMSCs, was not significantly different between subjects taking DFMO and placebo (260 versus 363 cancers, P = 0.069, two-sample t test). Evaluation of basal cell (BCC) and squamous cell cancers separately revealed very little difference in squamous cell cancer between treatment groups but a significant difference in new BCC (DFMO, 163 cancers; placebo, 243 cancers; expressed as event rate of 0.28 BCC/person/year versus 0.40 BCC/person/year, P = 0.03). Compliance with DFMO was >90% and it seemed to be well tolerated with evidence of mild ototoxicity as measured by serial audiometric examination when compared with placebo subjects. The analysis of normal skin biopsies revealed a significant (P < 0.05) decrease in 12-0-tetradecanoylphorbol-13-acetate-induced ODC activity (month 24, 36, and 48) and putrescine concentration (month 24 and 36 only) in DFMO subjects. Subjects with a history of skin cancer taking daily DFMO had an insignificant reduction (P = 0.069) in new NMSC that was predominantly due to a marked reduction in new BCC. Based on these data, the potential of DFMO, alone or in combination, to prevent skin cancers should be explored further.

PKCepsilon overexpression, irrespective of genetic background, sensitizes skin to UVR-induced development of squamous-cell carcinomas.

Chronic exposure to UVR is the major etiologic factor in the development of human skin cancers including squamous-cell carcinoma (SCC). We have previously shown that protein Kinase C epsilon (PKCepsilon) transgenic mice on FVB/N background, which overexpress PKCepsilon protein approximately eightfold over endogenous levels in epidermis, exhibit about threefold more sensitivity than wild-type littermates to UVR-induced development of SCC. To determine whether it is PKCepsilon and not the mouse genetic background that determines susceptibility to UVR carcinogenesis, we cross-bred PKCepsilon FVB/N transgenic mice with SKH-1 hairless mice to generate PKCepsilon-overexpressing SKH-1 hairless mice. To evaluate the susceptibility of PKCepsilon SKH-1 hairless transgenic mice to UVR carcinogenesis, the mice were exposed to UVR (1-2 KJ m(-2)) three times weekly from a bank of six kodacel-filtered FS40 sunlamps. As compared with the wild-type hairless mice, PKCepsilon overexpression in SKH-1 hairless mice decreased the latency (12 weeks), whereas it increased the incidence (twofold) and multiplicity (fourfold) of SCC. The SKH hairless transgenic mice were observed to be as sensitive as FVB/N transgenic mice to UVR-induced development of SCC and expression of proliferative markers (proliferating cell nuclear antigen, signal transducers and activators of transcription 3, and extracellular signal-regulated kinase 1/2). The results indicate that PKCepsilon level dictates susceptibility, irrespective of genetic background, to UVR carcinogenesis.

Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Hormone-refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. We present here that plumbagin (PL), a quinoid constituent isolated from the root of the medicinal plant Plumbago zeylanica L., may be a potential novel agent in the control of hormone-refractory PCa. Specific observations are the findings that PL inhibited PCa cell invasion and selectively induced apoptosis in PCa cells but not in immortalized nontumorigenic prostate epithelial RWPE-1 cells. In addition, i.p. administration of PL (2 mg/kg body weight), beginning 3 days after ectopic implantation of hormone-refractory DU145 PCa cells, delayed tumor growth by 3 weeks and reduced both tumor weight and volume by 90%. Discontinuation of PL treatment in PL-treated mice for as long as 4 weeks did not result in progression of tumor growth. PL, at concentrations as low as 5 micromol/L, inhibited in both cultured PCa cells and DU145 xenografts (a) the expression of protein kinase Cepsilon (PKCepsilon), phosphatidylinositol 3-kinase, phosphorylated AKT, phosphorylated Janus-activated kinase-2, and phosphorylated signal transducer and activator of transcription 3 (Stat3); (b) the DNA-binding activity of transcription factors activator protein-1, nuclear factor-kappaB, and Stat3; and (c) Bcl-xL, cdc25A, and cyclooxygenase-2 expression. The results indicate for the first time, using both in vitro and in vivo preclinical models, that PL inhibits the growth and invasion of PCa. PL inhibits multiple molecular targets including PKCepsilon, a predictive biomarker of PCa aggressiveness. PL may be a novel agent for therapy of hormone-refractory PCa.

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

Protein kinase Cepsilon interacts with signal transducers and activators of transcription 3 (Stat3), phosphorylates Stat3Ser727, and regulates its constitutive activation in prostate cancer.

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 Cepsilon (PKCepsilon), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCepsilon expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCepsilon 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 PKCepsilon. The interaction of PKCepsilon 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 PKCepsilon. Localization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCepsilon with Stat3 was PKCepsilon isoform specific. Inhibition of PKCepsilon protein expression in DU145 cells using specific PKCepsilon 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 PKCepsilon activation is essential for constitutive activation of Stat3 and prostate cancer progression.

Protein kinase Cepsilon interacts with Stat3 and regulates its activation that is essential for the development of skin cancer.

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)-dependent serine/threonine protein kinases. At least six PKC isoforms (alpha, delta, epsilon, eta, micro, and zeta) are expressed in epidermis. PKC is a major intracellular receptor for 12-O-tetradecanoylphorbol-13-acetate (TPA) and is also activated by a variety of stress factors including ultraviolet radiation (UVR). PKC isozymes (alpha, delta, epsilon, and eta), exhibit specificities to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform, is linked to the development of squamous cell carcinoma (SCC) elicited either by the 7,12-Dimethylbenzanthracene (DMBA)-TPA protocol or by repeated exposures to UVR. PKCepsilon overexpressing transgenic mice, when treated either with TPA or exposed to UVR, elicit similar responses such as inhibition of apoptosis, promotion of cell survival, and development of SCC. PKCepsilon overexpression increases Stat3 activation after either TPA treatment or UVR exposure. Both PKCepsilon and signal transducers and activators of transcription-3 (Stat3) are implicated in the development of SCC. However, the link between PKCepsilon and Stat3 remains elusive. We found that PKCepsilon interacts with Stat3. PKCepsilon interaction with Stat3 was dependent upon UVR treatment. In reciprocal immunoprecipitation/blotting experiments, Stat3 coimmunoprecipitated with PKCepsilon. Colocalization of PKCepsilon with Stat3 was confirmed by double immunofluorescence staining. PKCepsilon interaction with Stat3 was PKCepsilon isoform specific and was not observed with other protein kinases. As observed in vitro with immunocomplex kinase assay with immunopurified PKCepsilon and Stat3, PKCepsilon phosphorylated Stat3 at the serine 727 residue. PKCepsilon depletion prevented Stat3Ser727 phosphorylation, Stat3 DNA binding, and transcriptional activity. The results presented indicate that PKCepsilon mediates Stat3 activation.

Protein kinase C epsilon, which sensitizes skin to sun's UV radiation-induced cutaneous damage and development of squamous cell carcinomas, associates with Stat3.

Chronic exposure to UV radiation (UVR) is the major etiologic factor in the development of human skin cancers including squamous cell carcinoma (SCC). We have shown that protein kinase C(epsilon) (PKC(epsilon)), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is an endogenous photosensitizer. PKC(epsilon) is among the six isoforms (alpha, delta, epsilon, eta, mu, and zeta) expressed in both mouse and human skin. PKC(epsilon) transgenic mice, which overexpress PKC(epsilon) in the basal epidermal cells and cells of the hair follicle, are highly sensitive to UVR-induced cutaneous damage and development of SCC. We now present that PKC(epsilon)-overexpressing, but not PKC(delta)-overexpressing, transgenic mice, when exposed to a single (4 kJ/m(2)) or repeated (four doses, 2 kJ/m(2)/dose, thrice weekly) UVR, emitted by Kodacel-filtered FS-40 sun lamps, elicit constitutive phosphorylation of signal transducers and activators of transcription 3 (Stat3) at both Tyr705 and Ser727 residues. UVR-induced phosphorylation of Stat3 accompanied increased expression of Stat3-regulated genes (c-myc, cyclin D1, cdc25A, and COX-2). In reciprocal immunoprecipitation/blotting experiments, phosphorylated Stat3 co-immunoprecipitated with PKC(epsilon). As observed in vivo using PKC(epsilon) knockout mice and in vitro in an immunocomplex kinase assay, PKC(epsilon) phosphorylated Stat3 at Ser727 residue. These results indicate for the first time that (a) PKC(epsilon) is a Stat3Ser727 kinase; (b) PKC(epsilon)-mediated phosphorylation of StatSer727 may be essential for transcriptional activity of Stat3; and (c) UVR-induced phosphorylation of Ser727 may be a key component of the mechanism by which PKC(epsilon) imparts sensitivity to UVR-induced development of SCC.

Protein kinase Cepsilon and development of squamous cell carcinoma, the nonmelanoma human skin cancer.

Protein kinase C (PKC) represents a large family of phosphatidylserine (PS)-dependent serine/threonine protein kinases. At least five PKC isoforms (alpha, delta, epsilon, eta, and zeta) are expressed in epidermal keratinocytes. PKC isoforms are differentially expressed in proliferative (basal layer) and nonproliferative compartments (spinous, granular, cornified layers), which exhibit divergence in their roles in the regulation of epidermal cell proliferation, differentiation, and apoptosis. Immunocytochemical localization of PKC isoforms indicate that PKCalpha is found in the membranes of suprabasal cells in the spinous and granular layers. PKCepsilon is mostly localized in the proliferative basal layers. PKCeta is localized exclusively in the granular layer. PKCdelta is detected throughout the epidermis. PKC isozymes exhibit specificities in their signals to the development of skin cancer. PKCepsilon, a calcium-insensitive PKC isoform mediates the induction of squamous cell carcinoma (SCC) elicited either by the DMBA-TPA protocol or by repeated exposures to ultraviolet radiation (UVR). PKCepsilon overexpression, which sensitizes skin to UVR-induced carcinogenesis, suppresses UVR-induced sunburn (apoptotic) cell formation, and enhances both UVR-induced levels of TNFalpha and hyperplasia. UVR-induced sunburn cell formation is mediated by Fas/Fas-L and TNFalpha NFR1 extrinsic apoptotic pathways. The death adaptor protein termed Fas-associated death domain (FADD) is a common adaptor protein for both of these apoptotic pathways. PKCepsilon inhibits UVR-induced expression of FADD leading to the inhibition of both apoptotic pathways. It appears that PKCepsilon sensitizes skin to the development of SCC by UVR by transducting signals, which inhibit apoptosis on one hand, and enhances proliferation of preneoplastic cells on the other hand.

Protein kinase C delta overexpressing transgenic mice are resistant to chemically but not to UV radiation-induced development of squamous cell carcinomas: a possible link to specific cytokines and cyclooxygenase-2.

Protein kinase C delta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the novel PKCs (delta, epsilon, and eta) expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 8-fold) PKCdelta protein in basal epidermal cells and cells of the hair follicle are resistant to the development of both skin papillomas and squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion protocol. We now present that PKCdelta overexpression in transgenic mice failed to suppress the induction of SCC developed by repeated exposures to UV radiation (UVR), the environmental carcinogen linked to the development of human SCC. Both TPA and UVR treatment of wild-type mice (a) increased the expression of proliferating cell nuclear antigen (PCNA) and apoptosis; (b) stimulated the expression of cytokines tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte CSF (G-CSF); and (c) increased cyclooxygenase-2 (COX-2) expression and expression of phosphorylated Akt (p-Akt), p38, extracellular signal-regulated kinase-1 (ERK1), and ERK2. PKCdelta overexpression in transgenic mice enhanced TPA-induced but not UVR-induced apoptosis and suppressed TPA-stimulated but not UVR-stimulated levels of cell PCNA, cytokines (TNF-alpha, G-CSF, and GM-CSF), and the expression of COX-2, p-Akt, and p38. The results indicate that UVR-mediated signal transduction pathway to the induction of SCC does not seem to be sensitive to PKCdelta overexpression. The proapoptotic activity of PKCdelta coupled with its ability to suppress TPA-induced expression of proinflammatory cytokines, COX-2 expression, and the phosphorylation of Akt and p38 may play roles in the suppression of TPA-promoted development of SCC.