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 CE (PKCE), a member of the novel PKC subfamily, is linked to the development of androgen-independent prostate cancer. PKCE expression levels, as determined by immunohistochemistry of human prostate cancer tissue microarrays, correlated with the aggressiveness of prostate cancer. The mechanism by which PKCE 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 PKCE. The interaction of PKCE 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 PKCE. Localization of PKCE with Stat3 was confirmed by double immunofluorescence staining. The interaction of PKCE with Stat3 was PKCE isoform specific. Inhibition of PKCE protein expression in DU145 cells using specific PKCE small interfering RNA (a) inhibited Stat3Ser727 phosphorylation, (b) decreased both Stat3 DNAbinding and transcriptional activity, and (c) decreased DU145 cell invasion. These results indicate that PKCE activation is essential for constitutive activation of Stat3 and prostate cancer progression. [Cancer Res 2007;67(18):8828-38]

Prostate cancer (PCa) is the second leading cause of cancerrelated 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 Mmol/L, inhibited in both cultured PCa cells and DU145 xenografts (a) the expression of protein kinase CE (PKCE), 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-KB, 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 PKCE, a predictive biomarker of PCa aggressiveness. PL may be a novel agent for therapy of hormone-refractory PCa. [Cancer Res 2008;68(21):9024-32]

Chronic exposure to UV radiation (UVR), especially in the UVA (315-400 nm) and UVB (280 -315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C ⑀ (PKC⑀), a member of the phospholipiddependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKC⑀ transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8-and 18-fold PKC⑀ protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m 2 three times weekly) induced irreparable skin damage in high PKC⑀-overexpressing mouse line 215. However, the PKC⑀ transgenic mouse line 224, when exposed to UVR (2 kJ/m 2 three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKC⑀ transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor ␣ (TNF␣) and other growth stimulatory cytokines, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. The role of TNF␣ in UVR-induced cutaneous damage was evaluated using PKC⑀ transgenic mice deficient in TNF␣. UVR treatment three times weekly for 13 weeks at 2 kJ/m 2 induced severe cutaneous damage in PKC⑀ transgenic mice (line 215), which was partially prevented in PKC⑀-transgenic TNF␣-knockout mice. Taken together, the results indicate that PKC⑀ signals UVR-induced TNF␣ release that is linked, at least in part, to the photosensitivity of PKC⑀ transgenic mice.

Protein kinase C epsilon (PKCε), a novel calcium-independent PKC isoform, has been shown to be a transforming oncogene. PKCε-mediated oncogenic activity is linked to its ability to promote cell survival. However, the mechanisms by which PKCε signals cell survival remain elusive. We found that signal transducers and activators of transcription 3 (Stat3), which is constitutively activated in a wide variety of human cancers, is a protein partner of PKCε. Stat3 has two conserved amino acid (Tyr705 and Ser727) residues, which are phosphorylated during Stat3 activation. PKCε interacts with Stat3α isoform which has Ser727 and not with Stat3β isoform which lacks Ser727. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was initially observed during induction of squamous cell carcinomas and in prostate cancer. Now we present that: 1) PKCε physically interacts with Stat3α isoform in various human cancer cells: skin melanomas (MeWo and WM266-4), gliomas (T98G and MO59K), bladder (RT-4 and UM-UC-3), colon (Caco-2), lung (H1650), pancreatic (PANC-1), and breast (MCF-7 and MDA:MB-231). 2) Inhibition of PKCε expression using specific siRNA inhibits Stat3Ser727 phosphorylation, Stat3-DNA binding, Stat3-regulated gene expression as well as cell invasion. 3) PKCε mediates Stat3Ser727 phosphorylation via integration with the MAPK cascade (RAF-1, MEK1/2, and ERK1/2). The results indicate that PKCε-mediated Stat3Ser727 phosphorylation is essential for constitutive activation of Stat3 and cell invasion in various human cancers.

Preclinical 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/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 antiinflammatory 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. ©2010 AACR.