Perspective on this Article from A Randomized, Double-Blind, Placebo-Controlled Phase 3 Skin Cancer Prevention Study of α-Difluoromethylornithine in Subjects with Previous History of Skin Cancer

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.

We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present several novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m(2)/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention.

AbstractPreclinical studies have shown that the inhibition of ornithine decarboxylase (ODC) by α-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/m2/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/m2/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. Cancer Prev Res; 3(1); 35–47

Abstract Protein kinase C epsilon (PKCε) is among the six PKC isoforms (α, δ, ε, η, μ, ζ) expressed in both mouse and human skin. We have reported that epidermal PKCε levels dictate the susceptibility of PKCε transgenic (TG) mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposures to ultraviolet radiation (UVR) or initiation with 7,12-dimethylbenz[a]anthracene and tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Histologically, SCC in TG mice, like human SCC, is poorly differentiated and metastatic. To find clues about the mechanism by which PKCε may impart susceptibility to UVR-induced development of SCC, we compared the effects of UVR treatment of TG mice with their wildtype (WT) littermates on hair follicle putative stem cells (HSCs). HSCs in the mouse hair follicle are known to be the precursor cells for SCC in the mouse skin (Mol Carcinog. 46: 579-84, 2007). The cell surface markers CD34 and α6-integrin mark mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. In this experiment, TG and WT mice were exposed to UVR (2kJ/m2, 3x weekly i.e, Monday, Wednesday, Friday) emitted by Kodacel-filtered FS-40 sun lamps. At 24 hr post last UVR (1, 2 or 4) exposures, mice were sacrificed and the dorsal skin removed for keratinocyte isolation. Keratinocytes were incubated for 30 minutes in the dark at 4oC with FITC-conjugated rat anti-human α6-integrin antibody at 10 μl per 106 cells and PE-conjugated rat anti-mouse CD34 antibody at 2 μg per 106 cells (FITC-α6-integrin and PE-CD34 antibodies; BD Biosciences). Flow cytometric analysis was done using a BD Biosciences FACS Calibur flow cytometer using a 488 nm laser as excitation. For both untreated and treated mice, the percent of double positive cells was higher in the TG than in WT mice. Both acute and chronic UVR treatment increased (2-fold) the number of double positive cell in both TG and WT mice. To examine the rate of proliferation of bulge region stem cells, a 5-bromo-2′-deoxyuridine labeling (BrdU) experiment was performed. Three-day old neonatal mice were injected twice daily for three days with 50 mg/kg of BrdU in PBS. At 8 weeks of age mice were sacrificed and the dorsal skin harvested for keratinocytes. The cells were stained for α6-integrin and CD34, fixed then stained for BrdU. In the WT mice, the percent of double positive cells maintaining BrdU label was 28.4 + 0.6% compared to 4.0 + 0.06% for the TG mice, an approximately 7-fold decrease in the TG mice. Similar results were obtained in a repeat experiment, indicating that the double positive cells in the TG mice cycle at a faster rate. This may contribute to the sensitivity of these transgenic animals to UVR-induced development of squamous cell carcinomas (Support: NIH grants CA102431 and CA35368 T32ES007015). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5151.

Abstract 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 Plumbago zeylanica have been used in Indian medicine for more than 2,500 years for treatments of various ailments. PL is also present in black walnut and other various medicinal plants. In Ayurveda, this herb is considered one of the most potent medicines used for various skin ailments, like leucoderma, psoriasis, leprosy, and scabies. We present here that topical application of non-toxic doses (100-500 nmoles) of PL to skin elicits dose-dependent inhibition of ultraviolet radiation (UVR)-induced cutaneous damage and development of squamous cell carcinomas (SCC). In this experiment, FVB/N mice were exposed to UVR (2 kJ/m2) three times weekly from a bank of six kodacel-filtered FS40 sunlamps (approximately 60% UVB and 40% UVA). The vehicle acetone or the indicated doses of PL in 0.2 ml acetone were applied to the dorsal skin 15 min after UVR exposure. Carcinoma incidence in mice treated with vehicle, 100, 200 or 500 nmoles PL, at 44 weeks post UVR, were 86%, 80% (p=0.67), 53% (p=0.06) and 7% (p=0.0025), respectively. Both vehicle and PL treated mice gained weight and did not exhibit any sign of toxicity during the entire period of the experiment. The molecular mechanisms associated with inhibition of UVR-induced development of SCC involved both induction of apoptosis and inhibition of cell proliferation. Specific findings are that PL treatment increased UVR-induced expression of fas-associated death domain (FADD) protein, Bax, p21, p27 and PARP cleavage. PL treatment also inhibited cell survival by decreasing the expression of pERK1/2, p85 PI3K, pAKT(ser473), Bcl2, and BclXL. Plumbagin also inhibited constitutive activation of transcription factors Stat3, NF-kB and AP-1. In summary, PL has been shown to exert both anticancer and antiproliferative activities in animal models as well as in cell culture. For example, PL, fed in the diet (200 ppm), significantly inhibits azoxymethane-induced intestinal tumors in rat. PL has chemotherapeutic potential as an anti-cancer agent. PL inhibits ectopic growth of breast cancer cells MDA-MB-231, nonsmall cell lung cancer cells A549, melanoma A375-52 cells and prostate cancer DU145 cells in nude mice (J Biol Chem 2006,281:17023, Cancer Res. 2008, 68:9024). We have now shown that PL inhibits the induction of SCC elicited by UVR, the most ubiquitous environmental carcinogen. Taken together, we conclude that PL may be explored as a naturally occurring agent in the prevention and treatment of human cancers (Support: NIH grants CA102431, CA35368 and T32ES007015) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1881.

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.

Abstract Pancreatic cancer (PaC) is one of the most fatal of all cancers and is ranked as the fourth most common cause of cancer related deaths among both men and women in the United States. It is estimated that 42,407 cases of PaC will be diagnosed in the United States alone in 2009 and 35,240 cancer related deaths are projected. Chemotherapy and radiation therapy are the widely practical major treatment modalities for the disease. Systemic toxicity caused by high doses of current chemotherapeutic drugs in PaC patients is of great concern, as it limits their use in patients. Chemotherapy through natural agents has proved successful for the prevention of a variety of cancer types. Plumbagin (PL) is a quinoid constituent isolated from the root of Plumbago zeylanica L. The root of this plant has been used in Indian system of medicine for more than 2500 years for the treatment of various ailments. A recent study demonstrates that PL treatment induces apoptosis of PaC cells. In this study, we investigated the chemopreventive potential of PL and its associated mechanism against PaC. To test the effect of PL in vitro condition, we have used PaC PANC1 and BxPC3 cells which were treated with PL treatment (5, 10, 15, or 20 M) for 24 h. We observed that PL treatment to PaC cells resulted in dose and time-dependent decrease in cell viability. The IC50 of PL in PANC1 and BxPC3 cells was observed 10 M after 24 h treatment. PL treatment at 15 M arrested cell cycle in G0/G1 phase in PANC1 (82.11%) and BxPC3 (68.93%) cells after 24 h post-treatment. To determine whether PL treatment inhibits the cell invasion of PaC cells, we performed in vitro chemoinvasion assay. PL treatment at doses of 15 and 20 M depicted 55% and 70 % inhibition of PANC1 cells invasion. To establish molecular mechanism associated with PL chemoprevention, we used western blot analysis to show that PL treatment inhibits the phosphorylation of pNFkBSer552, pStat3Ser727, and pSTAT3Tyr705 in PaC cells. PL treatment also inhibited DNA-binding of Stat3 and NF-kB in PaC cells as assessed by electro mobility shift assay. We further evaluated the effect of PL on NF-kB and Stat3 downstream target genes in PaC cells. Results demonstrated that PL treatment of PaC cells inhibited the expression of cyclin D1 and VEGF. To establish the chemopreventive potential of PL in PaC, we ectopically implanted 2.5×106 PaC PANC1 in SCID mice. Three days after implantation, PL treatment (2mg/kg body weight, i.p. five days a week) was given up to 9 weeks. Control mice were treated with PBS. PL treatment showed significant (P<0.01) inhibition in tumor growth (50%), and tumor weight (50%), when compared with control animals. Taken together, these results indicate that PL is an effective natural agent which could be use against PaC chemoprevention and treatment (Support: R&D Funds, Department of Human Oncology, and UW Madison, WI). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-424.

Abstract Pancreatic adenocarcinoma is one of the most fatal of all cancers and is ranked as the fourth most common cause of cancer related deaths among both men and women in the United States. It is estimated that 42,407 cases of pancreatic cancer (PaC) will be diagnosed in the United States alone in 2009 and 35,240 cancer related deaths are projected. The systemic toxicity caused by high doses of current chemotherapeutic drugs in PaC patients is of great concern, as it limits their use in patients. Since most of the chemotherapeutic agents generally target a specific pathway, they most often fail to inhibit growth of PaC in preclinical and clinical settings. Thus, there is a need to develop new novel non-toxic therapeutic agents which can either target the multiple oncogenic signaling pathways or target major pathways at multiple level. α-Mangostin a novel dietary natural agent isolated from the pericarp of Mangosteen fruit (Garcinia mangostana) has been shown to have anti-inflammatory, anti-carcinogenic and cardioprotective activities. However, no study exists examining the effects of α-Mangostin in the prevention and/or treatment of PaC. In this study, we present that α-Mangostin treatment of PaC PANC1 and BxPC3 cells results in dose-dependent decreases in cells viability and arrests PaC cells in G0/G1 phase of cell cycle. To understand the molecular mechanism of α-Mangostin for the inhibition of cell proliferation, we target the multiple signaling molecules which are aberrantly expressed and involved in survival, proliferation, initiation, development and chemoresistance of PaC cells. It has been reported that Sonic hedgehog (Shh) interacts with activated K-Ras and cooperates in initiation and maintenance of PaC. Western blot analysis showed that α-Mangostin treatment significantly inhibited the constitutive expression of K-Ras, Shh and transcription factor GLI-1 proteins in dose- and time-dependent manner in PaC cells suggesting that α-Mangostin targets Ras and Shh signalings molecules. Since NF-kB and Stat3 are another molecular targets in PaC, therefore, we next examined the effect of α-Mangostin in these transcription factors. We observed that α-Mangostin treatment of PaC cells not only inhibits the constitutive expression of of NF-kB and Stat3 proteins but also inhibits their phosphorylation. α-Mangostin treatment also elicited the inhibition of DNA-binding of Stat3 and NF-kB, suggesting inhibition in translocation of these transcription factors into the nucleus by α-Mangostin. Taken together, these results indicate that α-Mangostin is a novel multi-targeting dietary agent which could be developed as a natural dietary non-toxic agent for the prevention and treatment of PaC. (Support: R&D Funds, Department of Human Oncology, UW Madison, Madison, WI). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3800.

Protein kinase C (PKC)epsilon overexpression in FVB/N transgenic mice sensitized skin to UVR-induced development of squamous cell carcinomas and suppressed formation of sunburn cells, which are DNA-damaged keratinocytes undergoing apoptosis. Here, we elucidated the mechanisms associated with the inhibition of UVR-induced appearance of sunburn cells in PKCepsilon transgenic mice. We found that the inhibition of UVR-induced sunburn cell formation in PKCepsilon transgenic mice may be the result of the inhibition of the expression of Fas, Fas ligand, and the mammalian death adaptor protein termed Fas-associated with death domain (FADD). The adaptor protein FADD is the key component of the death-inducing signaling complex of both Fas and tumor necrosis factor receptor 1. A decreased expression of epidermal FADD was observed after a single UVR exposure. However, a complete loss of FADD expression was found after four (Monday, Wednesday, Friday, and Monday) repeated UVR exposures. FADD transmits apoptotic signals from death receptors to the downstream initiator caspase-8 and connects to the mitochondrial intrinsic apoptotic signal transduction pathway by the cleavage of Bid, a Bcl-2 family member. PKCepsilon-mediated loss of FADD expression inhibited UVR signals to the activation of both extrinsic and intrinsic apoptotic pathways.

1609 Protein kinase C (PKC), a family of eleven (α, βI, βII, γ, δ, ϵ, η, θ, λ, ζ, μ) phospholipid-dependent serine/threonine protein kinases, exhibits functional diversity in its signals to a variety of cellular functions including cell proliferation, differentiation, apoptosis and oncogenesis. PKCϵ, a member of the novel PKC sub-family, is a transforming oncoprotein when overexpressed either in fibroblasts or epithelial cells. PKCϵ is overexpressed and constitutively activated in various human cancers including breast, head and neck, prostate, brain, skin, and bladder. PKCϵ expression levels correlate with the aggressiveness of both prostate and breast cancer. It is also noteworthy that PKCϵ overexpression is sufficient to transform the androgen-dependent prostate cancer cell line LNCaP into an androgen-independent variant, which rapidly initiates growth in both intact and castrated male nude mice. Inhibition of PKCϵ expression, using specific siRNA, inhibits cancer cell growth, motility, and invasion. These results indicate that PKCϵ activation constitutes an initial signal in cancer growth and progression. However, the mechanisms by which PKCϵ mediates initial oncogenic activity remain elusive. We found that PKCϵ interacts with Stat3, which is constitutively activated in a wide variety of human cancers. Stat3 regulates several genes linked to embryogenesis, cell proliferation, differentiation, apoptosisand tumorigenesis. Stat3 has two conserved amino acid residues, Tyrosine 705 and Serine 727, both phosphorylated during Stat3 activation. PKCϵ interacts with Stat3, phosphorylates Stat3Ser727, and regulates its constitutive activation. PKCϵ-Stat3 interaction was observed in various human cancer cell lines. We also investigated whether PKCϵ directly interacts with Stat3 or via intermediate protein kinases to phosphorylate Stat3Ser727. In an immunocomplex kinase assay, purified bacterially expressed recombinant PKCϵ phosphorylated purified recombinant GST-Stat3 at Ser727. These results indicate that Stat3 can be a direct substrate for PKCϵ. However, we also found that PKCϵ may interact with other protein kinases. In reciprocal immunoprecipitation/blotting experiments using whole cell lysates from prostate cancer, PKCϵ and Stat3 were found to associate with Raf-1, MEK1/2, and ERK1/2. Inhibition of PKCϵ in DU145 cells using PKCϵ specific siRNA inhibited the phosphorylation of Raf-1, MEK1/2, ERK1/2 and Stat3. Thus, it appears that Stat3Ser727 phosphorylation is mediated by a protein kinase complex which involves PKCϵ-Raf-1-MEK1/2-ERK1/2. PKCϵ mediates oncogenesis through interaction and activation of Stat3. These results indicate that PKCϵ is a master switch in the induction and progression of human cancer. PKCϵ is a potential diagnostic and prognostic marker of cancer and a possible molecular target for the prevention and treatment. (Support: DOD-W81XWH and NIH-CA35368).

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.

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

118 Prostate cancer (PCa) is the second leading cause of cancer deaths in men. We now present that Protein kinase C ϵ (PKCϵ) and signals transducers and activators of transcription 3 (Stat3), two widely documented proteins with oncogenic traits, interact with each other in their signals to the development of PCa. Both PKCϵ and activated Stat3 are overexpressed in human PCa. The expression levels of PKCϵ, as determined by immunohistochemistry of human PCa tissue microarrays, correlated with the aggressiveness of PCa. To investigate the significance of the upregulation of PKCϵ and Stat3 in the induction and progression of PCa, we employed TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice, which spontaneously develop PCa. Both PKCϵ protein and activity levels were increased while Stat3 was constitutively activated in PCa specimens from TRAMP mice. Further analyses of Stat3-associated upstream and downstream signaling components revealed elevated expression of IL-6, Jak1 and Jak2, several cell cycle (p21 and p27) and anti-apoptotic (survivin, Bcl-2, Bcl-xL) regulatory proteins. A more detailed kinetic study with TRAMP mice (13, 26, 39, and 42 weeks old) elicited a parallel increase in the level of expression of PKCϵ and phosphorylated Stat3 in prostate tissue starting in 26-week old TRAMP mice. To further explore the PKCϵ signal transduction pathways to activation of Stat3, the interaction of PKCϵ with Stat3 was determined via reciprocal immunoprecipitation/blotting experiments. We found that prostate Stat3 co-immunoprecipitated with PKCϵ. PKCϵ-associated Stat3 was phosphorylated at both tyrosine 705 and serine 727. Association of PKCϵ with Stat3, as determined by double immunofluorescence staining, was observed both in the cytoplasm and nucleus of PCa specimens. Similar association of PKCϵ with Stat3 was observed in human prostate cancer cell lines. (LNCaP, PC-3, DU145) Inhibition of PKCϵ using specific siRNA in DU145 cells prevented Stat3Ser727 phosphorylation. Also, in an immunocomplex kinase assay, PKCϵ phosphorylated Stat3 at serine 727 residue. Taken together, these results indicated for the first time that PKCϵ associates with Stat3 in PCa and PKCϵ is a Stat3Ser727 kinase. PKCϵ and Stat3 may be potential molecular targets for the prevention and treatment of human PCa (Supported by NIH grants CA102431 and CA35368).

Proc Amer Assoc Cancer Res, Volume 47, 2006 339 Prostate cancer (PCa) is the most common type of cancer in men and ranks second to lung cancer in cancer-related deaths. PCa first manifests as an androgen-dependent (AD) disease and can be treated with androgen-deprivation therapy. Despite the initial success of androgen ablation therapy, PCa progresses from AD to androgen-independent (AI). The hormone refractory invasive PCa is the end stage and accounts for the majority of PCa patient deaths. Defining the molecular mechanisms linking the transition of AD PCa to a hormone refractory PCa is essential in planning strategies in the management of PCa. We determined that Protein kinase Ce (PKCe) may be linked in vivo to the progression of human AI PCa and should be explored as a molecular target for the prevention and therapy of hormone refractory PCa. PKCe is among the PKC isoforms expressed in human and mouse prostate tissue. PKCe levels were determined by immunohistochemistry of human prostate tissue microarrays prepared from paraffin-fixed benign, hyperplastic and carcimona (Grades I-IV) specimens ([Table][1]). Results showed that PKCe was located predominantly in the cytoplasm of both benign (non-neoplastic) and malignant (neoplastic) epithelial cells. PKCe was uniformly distributed in cancer cells. PKCe was more densely located in the apical (luminal) side of the cytoplasm in normal epithelial cells. In addition, some cancer cells showed slight dense staining for PKCe in the nuclear membrane. Stromal cells were also positive for PKCe, but the intensity was lower than that in epithelial cells. The results of the level of the expression of PKCe are shown in the [table][1]. The level of expression of PKCe appear to correlate with the aggressiveness of PCa. Also, evidence from in vitro studies have demonstrated that PKCe, as compared to AD prostate cancer cell lines, is overexporessed in AI cell lines DU145 and PC3. Also, PKCe has also shown to transform the AD human prostate cancer cell line LNCaP to an AI variant, which forms tumors in castrated male nude mice. PKCe may be a molecular marker of AI PCa. [Table][1]: PKCe expression in human prostate specimens. The level of expression is denoted by the sign +. One + is the lowest and four + is the highest. ![Figure][2] [1]: #F1 [2]: pending:yes