afimoxifene and Skin-Neoplasms

Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.

Topics: Animals; Cell Division; Cell Line, Tumor; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Estrogen Antagonists; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; MAP Kinase Signaling System; Melanoma; Mice; NIH 3T3 Cells; Phosphorylation; Proto-Oncogene Proteins B-raf; Receptors, Estrogen; Retinoblastoma Protein; Skin Neoplasms; Tamoxifen; Transduction, Genetic

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a variety of human malignancies and has been suggested to play an important role in carcinogenesis. Recently, our laboratory demonstrated that Stat3 is required for the development of skin tumors via two-stage carcinogenesis using skin-specific loss-of-function transgenic mice. To investigate further the role of Stat3 in each stage of chemical carcinogenesis in mouse skin, i.e. initiation and promotion stages, we generated inducible Stat3-deficient mice (K5.Cre-ER(T2) x Stat3(fl/fl)) that show epidermal-specific disruption of Stat3 following topical treatment with 4-hydroxytamoxifen (TM). The epidermis of inducible Stat3-deficient mice treated with TM showed a significant increase in apoptosis induced by 7,12-dimethylbenz[a]anthracene (DMBA) and reduced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate. In two-stage skin carcinogenesis assays, inducible Stat3-deficient mice treated with TM during the promotion stage showed a significant delay of tumor development and a significantly reduced number of tumors compared with control groups. Inducible Stat3-deficient mice treated with TM before initiation with DMBA also showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups. Finally, treatment of inducible Stat3-deficient mice that had existing skin tumors generated by the two-stage carcinogenesis protocol with TM (by intraperitoneal injection) led to inhibition of tumor growth compared with tumors formed in control groups. Collectively, these results directly demonstrate that Stat3 is required for skin tumor development during both the initiation and promotion stages of skin carcinogenesis in vivo.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; Immunohistochemistry; Mice; Mice, Transgenic; Polymerase Chain Reaction; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor; Tamoxifen; Tetradecanoylphorbol Acetate

Protein farnesyltransferase (FTase) is an enzyme responsible for posttranslational modification of proteins carrying a carboxy-terminal CaaX motif. Farnesylation allows substrates to interact with membranes and protein targets. Using gene-targeted mice, we report that FTase is essential for embryonic development, but dispensable for adult homeostasis. Six-month-old FTase-deficient mice display delayed wound healing and maturation defects in erythroid cells. Embryonic fibroblasts lacking FTase have a flat morphology and reduced motility and proliferation rates. Ablation of FTase in two ras oncogene-dependent tumor models has no significant consequences for tumor initiation. However, elimination of FTase during tumor progression had a limited but significant inhibitory effect. These results should help to better understand the role of protein farnesylation in normal tissues and in tumor development.

Topics: Alkyl and Aryl Transferases; Animals; Cell Proliferation; Embryo Loss; Embryo, Mammalian; Embryonic Development; Erythroid Cells; Estrogen Antagonists; Fibroblasts; Gene Expression; Homeostasis; Integrases; Liver; Lung; Mice; Mice, Knockout; Mutation; Neoplasms; ras Proteins; Skin Neoplasms; Spleen; Tamoxifen; Wound Healing

Mammalian epidermis is maintained by self-renewal of stem cells, but the underlying mechanisms are unknown. Deletion of Rac1, a Rho guanosine triphosphatase, in adult mouse epidermis stimulated stem cells to divide and undergo terminal differentiation, leading to failure to maintain the interfollicular epidermis, hair follicles, and sebaceous glands. Rac1 exerts its effects in the epidermis by negatively regulating c-Myc through p21-activated kinase 2 (PAK2) phosphorylation. We conclude that a pleiotropic regulator of cell adhesion and the cytoskeleton plays a critical role in controlling exit from the stem cell niche and propose that Rac and Myc represent a global stem cell regulatory axis.

Topics: Animals; Antigens, Differentiation; Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Epidermal Cells; Gene Deletion; Humans; Keratinocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuropeptides; p21-Activated Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; Skin Neoplasms; Stem Cells; Tamoxifen

The proto-oncogene c-myc is involved in the regulation of cell proliferation, differentiation, and apoptosis. In this study, we used an inducible transgenic mouse model in which c-Myc was targeted to the epidermis and, after activation, gave rise to hyperplastic and dysplastic skin lesions and to dermal angiogenesis, involving both vascular endothelial growth factor (VEGF) receptor-1 and VEGF receptor-2. After c-Myc activation, VEGF mRNA was expressed in postmitotic keratinocytes where it colocalized with transgene expression and areas of tissue hypoxia, suggesting a role of hypoxia in VEGF induction. In vitro, c-Myc activation alone was able to induce VEGF protein release and in conjunction with hypoxia, c-Myc activation further increased VEGF protein. Blocking VEGF signaling in vivo significantly reduced dermal angiogenesis, demonstrating the importance of VEGF as a mediating factor for the c-Myc-induced angiogenic phenotype.

Topics: Animals; Cell Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Indoles; Keratinocytes; Mice; Neovascularization, Physiologic; Oxindoles; Papilloma; Precancerous Conditions; Propionates; Proto-Oncogene Proteins c-myc; Pyrroles; RNA, Messenger; Skin; Skin Neoplasms; Tamoxifen; Transcription Factors; Transgenes; Vascular Endothelial Growth Factor A

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Blotting, Western; DNA Primers; Flow Cytometry; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Deletion; Genotype; Hydroxytestosterones; Immunohistochemistry; Integrases; Keratin-14; Keratinocytes; Keratins; Mice; Mice, Transgenic; Neoplasm Metastasis; Protein-Tyrosine Kinases; Receptors, Estrogen; Skin Neoplasms; Tamoxifen

The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis--changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER.

Topics: Animals; Cell Differentiation; Cell Division; Epidermis; Estrogen Antagonists; Gene Expression Regulation, Neoplastic; Hair Follicle; Humans; Hyperplasia; Keratinocytes; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Neovascularization, Pathologic; Papilloma; Phenotype; Proto-Oncogene Proteins c-myc; Skin Neoplasms; Tamoxifen

The literature concerning cytoskeletal changes and metastatic progression is unresolved, some studies suggesting a positive association between the ability of cells to organize their cytoskeleton and others suggesting an inverse correlation. In an attempt to learn more about cytoskeletal changes and the ability of melanoma cells to interact with extracellular matrix proteins we examined the effects of pharmacological manipulation of cell attachment and cell invasion through fibronectin on levels of F-actin and vimentin in a highly metastatic cutaneous melanoma cell line (A375-SM cells). Additionally, we examined whether any correlation existed between the levels of the cytoskeletal proteins and subpopulations of the cell line of varying invasive ability. We report that agents which reduced cell attachment to plastic and invasion through fibronectin in vitro (tamoxifen, N-desmethyltamoxifen and 17beta-oestradiol) caused increases in levels of F-actin and vimentin, whereas agents which did not affect attachment or invasion (4-hydroxytamoxifen and dihydrotestosterone) had little or no effect on the cytoskeletal proteins. In contrast, however, those cells which were most effective at invading through fibronectin were significantly better at acutely increasing their levels of F-actin and vimentin than less invasive cells. We speculate that the ability to rapidly and possibly reversibly alter the cytoskeleton might be associated with metastatically successful cells in vivo.

Topics: Actins; Antineoplastic Agents, Hormonal; Cell Adhesion; Cytoskeletal Proteins; Cytoskeleton; Dihydrotestosterone; Embryonal Carcinoma Stem Cells; Estradiol; Fibronectins; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Neoplastic Stem Cells; Skin Neoplasms; Tamoxifen; Tumor Cells, Cultured; Vimentin