Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Roles of IkappaB kinase alpha in centrosome duplication and skin carcinogenesis

IκB kinase α (IKKα) is one kinase subunit of the IKK complex that is responsible for NF-κB activation. Previous studies have shown that IKKα determines mouse keratinocyte terminal differentiation independent of the NF-κB pathway. Accumulating evidence suggests that IKKα functions as a tumor suppressor in skin carcinogenesis; however, the downstream pathways mediating this function are largely unknown. By using primary cultured keratinocytes, we found that Ikkα-/- cells developed aneuploidy and underwent spontaneous immortalization and transformation while wild type cells underwent terminal differentiation in the same culture condition. Using proteomic analysis we identified nucleophosmin (NPM), a centrosome duplication regulator, as an IKKα substrate. We further demonstrated that IKKα interacted with NPM and colocalized with NPM on the centrosome, suggesting that NPM is a physiological substrate of IKKα. Loss of IKKα reduced centrosome-bound NPM and promoted abnormal centrosome amplification, which contributed to aneuploidy development. Detailed analysis revealed that ablation of IKKα target site serine-125 of NPM induced destabilization of NPM hexamers, disrupted NPM association with centrosomes, and resulted in abnormal centrosome amplification. Re-introduction of IKKα rescued the defect in Ikkα-/- keratinocytes. Thus, IKKα is required for maintaining proper centrosome duplication by phosphorylating NPM. ^ UV is the major etiological agent for human skin cancer and UV-induced mouse skin carcinogenesis is one of the most relevant experimental models for human skin carcinogenesis. Thus, we further evaluated IKKα function in UV-induced skin carcinogenesis in Ikkα+/- mice. We demonstrated that IKKα is also critical in UV skin carcinogenesis, as evidenced by increased tumor multiplicity and reduced tumor latency in Ikkα+/- mice after chronic UVB treatment. Reduced expression of IKKα decreased UV-induced apoptosis and promoted accumulation of P53 mutations in the epidermis. This indicates that IKKα is critical for UV-induced apoptosis in vivo and thus prevents mutation accumulation that is important for tumor development. ^ Together, these findings uncover previously unknown in vivo functions of IKKα in centrosome duplication and apoptosis, thus providing a possible mechanism of how loss of IKKα may contribute to skin carcinogenesis. ^

A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

PLASMACYTOID DENDRITIC CELLS SENSE SKIN INJURY AND PROMOTE WOUND HEALING THROUGH TYPE I INTERFERONS

Plasmacytoid dendritic cells (pDCs) are a rare population of circulating cells, which selectively express intracellular Toll-like receptors (TLR)-7 and TLR-9 and have the capacity to produce large amounts of type I IFNs (IFN-a/b) in response to viruses or host derived nucleic acid containing complexes. pDCs are normally absent in skin but accumulate in the skin of psoriasis patients where their chronic activation to produce IFN-a/b drives the disease formation. Whether pDCs and their activation to produce IFN-a/b play a functional role in healthy skin is unknown. Here we show that pDCs are rapidly and transiently recruited into healthy human and mouse skin upon epidermal injury. Infiltrating pDCs were found to sense nucleic acids in wounded skin via TLRs, leading to the production of IFN-a/b. The production of IFN-a/b was paralleled by a short lived expression of cathelicidins, which form complexes with extracellular nucleic acids and activated pDCs to produce IFN-a/b in vitro. In vivo, cathelicidins were sufficient but not necessary for the induction of IFN-a/b in wounded skin, suggesting redundancy of this pathway. Depletion of pDCs or inhibition of IFN-a/bR signaling significantly impaired the inflammatory response and delayed re-epithelialization of skin wounds. Thus we uncover a novel role of pDCs in sensing skin injury via TLR mediated recognition of nucleic acids and demonstrate their involvement in the early inflammatory process and wound healing response through the production of IFN-a/b.

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

Nuclear localization of HBD-1 in human keratinocytes.

OBJECTIVE: Human defensins and cathelicidins are a family of cationic antimicrobial peptides (AMPs), which play multiple roles in both innate and adaptive immune systems. They have direct antimicrobial activity against several microorganisms including burn pathogens. The majority of components of innate and adaptive immunity either express naturally occurring defensins or are otherwise chemoattracted or functionally affected by them. They also enhance adaptive immunity and wound healing and alter antibody production. All mechanisms to explain multiple functions of AMPs are not clearly understood. Prior studies to localize defensins in normal and burned skin using deconvolution fluorescence scanning microscopy indicate localization of defensins in the nucleus, perinuclear regions, and cytoplasm. The objective of this study is to further confirm the identification of HBD-1 in the nucleus by deconvolution microscopic studies involving image reconstruction and wire frame modeling. RESULTS: Our study demonstrated the presence of intranuclear HBD-1 in keratinocytes throughout the stratum spinosum by costaining with the nuclear probe DAPI. In addition, HBD-1 sequence does show some homology with known cationic nuclear localization signal sequences. CONCLUSION: To our knowledge, this is the first report to localize HBD-1 in the nuclear region, suggesting a role for this peptide in gene expression and providing new data that may help determine mechanisms of defensin functions.

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Population and molecular evolution studies on the Melanocortin 1 receptor locus implicate its role in human pigmentation variation

Human pigmentation is a complex trait with the observed variation caused by the varied production of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) by the melanocytes. The melanocortin 1 receptor (MC1R), a G protein-coupled receptor expressed in the melanocytes, is a regulator eu- and phaeomelanin synthesis, and MC1R mutations causing skin and coat color changes are known in many mammals. To understand the role of MC1R in human pigmentation variation, I have sequenced the MC1R gene in 121 individuals sampled from world populations. In addition, I have sequenced the MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon to study the evolution of MC1R and to infer the ancestral human MC1R sequence. The ancestral MC1R sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. To further evaluate the role of MC1R variants in human pigmentation variation, I have combined these molecular evolution and population studies with functional assays on MC1R variants and primate MC1Rs. ^

HUMAN PROPHASE CHROMOSOME UNIQUE BAND SEQUENCES; DEFINITION, CHARACTERIZATION, AND APPLICATION (CYTOGENETICS, IMAGE PROCESSING)

Extensive experience with the analysis of human prophase chromosomes and studies into the complexity of prophase GTG-banding patterns have suggested that at least some prophase chromosomal segments can be accurately identified and characterized independently of the morphology of the chromosome as a whole. In this dissertation the feasibility of identifying and analyzing specified prophase chromosome segments was thus investigated as an alternative approach to prophase chromosome analysis based on whole chromosome recognition. Through the use of prophase idiograms at the 850-band-stage (FRANCKE, 1981) and a comparison system based on the calculation of cross-correlation coefficients between idiogram profiles, we have demonstrated that it is possible to divide the 24 human prophase idiograms into a set of 94 unique band sequences. Each unique band sequence has a banding pattern that is recognizable and distinct from any other non-homologous chromosome portion.^ Using chromosomes 11p and 16 thru 22 to demonstrate unique band sequence integrity at the chromosome level, we found that prophase chromosome banding pattern variation can be compensated for and that a set of unique band sequences very similar to those at the idiogram level can be identified on actual chromosomes.^ The use of a unique band sequence approach in prophase chromosome analysis is expected to increase efficiency and sensitivity through more effective use of available banding information. The use of a unique band sequence approach to prophase chromosome analysis is discussed both at the routine level by cytogeneticists and at an image processing level with a semi-automated approach to prophase chromosome analysis. ^

IN VITRO INDUCTION OF XENOIMMUNE RESPONSES TO LIPOSOMES CONTAINING HUMAN COLON TUMOR ANTIGENS

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl(,2) to a dried lipid film. Cytoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposomes also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposomes were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens.^ The adjuvant effect of liposomes was shown to be mediated primarily by tumor antigens exposed on the outer surface of liposomes. Trypsinization of the liposomes which eliminated 96% of the surface protein reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes of increasing protein concentration showed that while 10 (mu)g protein was threshold, 100 (mu)g protein induced maximal responses. In addition, membrane fluidity studies revealed that rigid liposomes were significantly (P < 0.05) more efficacious than fluid liposomes in inducing cytotoxicity.^ The induction of the primary response required the presence of nonadherent splenocytes bearing the Thy-1, Lyt-1, and Lyt-2 surface markers. The role of a Lyt-123 subpopulation was suggested by the inability of both the Lyt-1 and Lyt-2 depleted populations to completely restore the cytolytic levels to normal. In addition, the interaction of I-A('+) spleen adherent cells with liposomes for at least 8 hours was required to generate maximal cytotoxic activity. The phenotype of the cytotoxic effector was Thy-1('+), Lyt-2('+), and I-A('d-).^ Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select specific immune responses, as well as to identify colon tumor-specific determinants.^

The role of Akt activation in mouse skin tumor promotion

The importance of IGF-1/IGF-1R signaling is evident in human cancers including breast, colon, prostate, and lung which have been shown to overexpress IGF-1. Also, serum levels of IGF-1 have been identified as a risk factor for these cancers. IGF-1 has been primarily shown to mediate its mitogenic effects through signaling pathways such as MAPK and PI3K/Akt. In this regard, BK5.IGF-1 transgenic mice were generated and these mice displayed hyperplasia and hyperkeratosis in the epidermis. In addition, these mice were also found to have elevated MAPK, PI3K, and Akt activities. Furthermore, overexpression of IGF-1 in epidermis can act as a tumor promoter. BK5.IGF-1 transgenic mice developed papillomas after initiation with DMBA without further treatment with a tumor promoter such as TPA. Previous data has also shown that inhibition of the PI3K/Akt signaling pathway by the inhibitor LY294002 was able to reduce the number of tumors formed by IGF-1 mediated tumor promotion. The current studies presented demonstrate that Akt may be the critical effector molecule in IGF-1/IGF-1R mediated tumor promotion. We have found that inhibition of PI3K/Akt by LY294002 inhibits cell cycle components, particularly those associated with G1 to S phase transition including Cyclin D1, Cyclin E, E2F1, and E2F4, that are elevated in epidermis of BK5.IGF-1 transgenic mice. We have also demonstrated that Akt activation may be a central theme in early tumor promotion. In this regard, treatment with diverse tumor promoters such as TPA, okadaic acid, chrysarobin, and UVB was shown to activate epidermal Akt and its downstream signaling pathways after a single treatment. Furthermore, overexpression of Akt targeted to the basal cells of the epidermis led to hyperplasia and increased labeling index as determined by BrdU staining. These mice also had constitutively elevated levels of cell cycle components, particularly Cyclin D1, Cyclin E, E2F1, E2F4, and Mdm-2. These mice developed skin tumors following initiation with DMBA and were hypersensitive to the tumor promoting effects of TPA. Collectively, these studies provide evidence that Akt activation plays an important role in the process of mouse skin tumor promotion. ^

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

The role of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) in normal human prostate epithelial cells and prostate cancer development

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial (NHP) cells. Western blotting with multiple NHP cell strains and prostate cancer (PCa) cell lines reveals that the expression of 15-LOX2 is lost in all PCa cell lines, accompanied by decreased enzymatic activity. 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 are mostly excluded from the nucleus. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor arachidonic acid-metabolizing activity. Interestingly, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. When orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Finally, cultured NHP cells lose the expression of putative stem/progenitor cell markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-LOX2 plays a role in replicative senescence of NHP cells: (1) promoter activity and the mRNA and protein levels of 15-LOX2 and its splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner; (2) PCa cells stably expressing 15-LOX2 or 15-LOX2sv-b show a passage-related senescence-like phenotype; (3) enforced expression of 15-LOX2 or 15-LOX2sv-b in young NHP cells induce partial cell-cycle arrest and senescence-like phenotypes. Together, these results suggest that 15-LOX2 suppress prostate tumor development and do not necessarily depend on arachidonic acid-metabolizing activity and nuclear localization. Also, 15-LOX2 may serve as an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence. ^

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.