Bone morphogenetic protein antagonist noggin promotes skin tumorigenesis via stimulation of the Wnt and Shh signaling pathways.

Bone morphogenetic proteins (BMPs) play pivotal roles in the regulation of skin development. To study the role of BMPs in skin tumorigenesis, BMP antagonist noggin was used to generate keratin 14-targeted transgenic mice. In contrast to wild-type mice, transgenic mice developed spontaneous hair follicle-derived tumors, which resemble human trichofolliculoma. Global gene expression profiles revealed that in contrast to anagen hair follicles of wild-type mice, tumors of transgenic mice showed stage-dependent increases in the expression of genes encoding the selected components of Wnt and Shh pathways. Specifically, expression of the Wnt ligands increased at the initiation stage of tumor formation, whereas expression of the Wnt antagonist and tumor suppressor Wnt inhibitory factor-1 decreased, as compared with fully developed tumors. In contrast, expression of the components of Shh pathway increased in fully developed tumors, as compared with the tumor placodes. Consistent with the expression data, pharmacological treatment of transgenic mice with Wnt and Shh antagonists resulted in the stage-dependent inhibition of tumor initiation, and progression, respectively. Furthermore, BMP signaling stimulated Wnt inhibitory factor-1 expression and promoter activity in cultured tumor cells and HaCaT keratinocytes, as well as inhibited Shh expression, as compared with the corresponding controls. Thus, tumor suppressor activity of the BMPs in skin epithelium depends on the local concentrations of noggin and is mediated at least in part via stage-dependent antagonizing of Wnt and Shh signaling pathways.

Telomere-based DNA damage responses: a new approach to melanoma.

Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11-base DNA oligonucleotide homologous to the telomere 3' overhang sequence (T-oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM-AN line, whereas normal human melanocytes exposed to the same or higher T-oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T-oligo effects. When MM-AN cells were briefly exposed to T-oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85-95% compared with control mice. Similarly, T-oligos administered intralesionally or systemically selectively inhibited the growth of previously established MM-AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T-oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down-regulation of the inhibitor of apoptosis protein livin/ML-IAP. We suggest that T-oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T-oligos may provide a novel therapeutic approach to melanoma.

Role of cytoplasmic dynein in perinuclear aggregation of phagocytosed melanosomes and supranuclear melanin cap formation in human keratinocytes.

Cytoplasmic dynein is a microtubule-associated motor molecule involved in the retrograde transport of membrane-bound organelles. To determine whether the supranuclear melanin cap of transferred, phagocytosed melanosomes in keratinocytes is associated with cytoplasmic dynein, we performed immunofluorescent confocal microscopy on human keratinocytes in situ. We identified the intermediate chain of cytoplasmic dynein by immunoblotting and examined its distribution by confocal microscopy in relation to microtubules and melano-phagolysosomes in vitro. We also used antisense and sense oligonucleotides of the cytoplasmic dynein heavy chain 1 (Dyh1) and time-lapse and microscopy. The intermediate chain of cytoplasmic dynein was identified in extracts of human foreskin epidermis and in isolated human keratinocytes. The intermediate chain localized with the perinuclear melano-phagolysosomal aggregates in vitro and the supranuclear melanin cap in situ. Antisense oligonucleotides directed towards Dyh1 resulted in dispersal of the keratinocyte perinuclear melano-phagolysosomal aggregates after 24 to 48 h, whereas cells treated with diluent or sense oligonucleotides maintained tight perinuclear aggregates. Taken together, these findings indicate that in human keratinocytes, the retrograde microtubule motor cytoplasmic dynein mediates the perinuclear aggregation of phagocytosed melanosomes, participates in the formation of the supranuclear melanin cap or "microparasol" and serves as a mechanism to help protect the nucleus from ultraviolet-induced DNA damage.

Role of zyxin in differential cell spreading and proliferation of melanoma cells and melanocytes.

Cell spreading, proliferation, and survival are modulated by focal adhesions linking extracellular matrix proteins, integrins, and the cytoskeleton. Zyxin is a focal-adhesion-associated phosphoprotein with one domain involved in the control of actin assembly and three protein-protein adapter domains implicated in the regulation of cell growth and differentiation. We characterized zyxin expression in normal human melanocytes and six melanoma cell lines in relation to cell spreading, growth, and differentiation using Western immunoblotting techniques, image analysis, flow cytometry, and confocal microscopy. We found that zyxin, focal adhesion kinase, and paxillin were significantly upregulated in melanoma cells compared to melanocytes. Zyxin expression directly related to cell spreading and proliferation and inversely related to differentiation, whereas focal adhesion kinase correlated only to cell spreading and paxillin did not significantly correlate with any of the parameters. Treatment of melanoma cells with 12-O-tetradecanoylphorbol-13-acetate downregulated zyxin expression, inhibited cell spreading and proliferation, and promoted differentiation. In contrast, 12-O-tetradecanoylphorbol-13-acetate, a mitogen for melanocytes, induced upregulation of zyxin expression in melanocytes. These findings are consistent with a role of zyxin in modulation of cell spreading, proliferation, and differentiation. Therapies directed at the downregulation of this focal adhesion phosphoprotein in melanoma cells implicate a new approach for controlling melanoma cell growth.

Cutaneous clear cell squamous cell carcinoma in situ : clinical, histological and immunohistochemical characterization.

BACKGROUND: Clear cell squamous cell carcinoma in situ (SCCIS) has not been defined in the literature with respect to its clinical, histological, and immunohistochemical features. METHODS: Nearly 1500 consecutive cases of SCCIS were assessed for percent clear cell change. The clinical features of all SCCIS with >or=10% clear cell change, including age, sex and site distribution, were compared with classical SCCIS using chi-square analysis. PAS special staining and immunohistochemical analysis with 11 cell markers were performed to characterize the clear cell of origin. RESULTS: Eighty SCCIS cases with a spectrum of clear cell change of >or=10% were identified. Six cases with >or=80% clear cells were defined as clear cell SCCIS. The clinical features of the cases did not vary significantly from classical SCCIS. Antibodies labeling outer root sheath (ORS) cells also labeled clear cells in the cases and included K8.12 (labeling CK13 and CK16), cellular retinoic acid binding protein II, CAM 5.2 and CK15. Antibodies that did not label ORS cells, but did label eccrine glands (CK7 and CK18) or sebocytes (EMA), also did not label the cases. CONCLUSION: Clear cell change in SCCIS is part of a spectrum which displays ORS differentiation.

Requirement of dynactin p150(Glued) subunit for the functional integrity of the keratinocyte microparasol.

The keratinocyte microparasol, composed of a perinuclear microtubular/melano-phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150(Glued) is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150(Glued) into human keratinocytes cultured with 0.5 mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150(Glued) subunit. The knockdown decreased p150(Glued) colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150(Glued) plays an important role in the functional integrity of the keratinocyte microparasol.

Macrophage-rich epithelioid angiosarcoma mimicking malignant melanoma.

BACKGROUND: Cutaneous epithelioid angiosarcoma is a type of cutaneous angiosarcoma and usually arise both on the head or neck of the elderly. CASE REPORT: An 86-year-old male with an epithelioid angiosarcoma of the scalp that mimicked malignant melanoma. RESULTS: A large irregular dark grey-blue plaque with an adjacent speckled tan nodule was suggestive of a primary cutaneous malignant melanoma with adjacent in-transit metastasis. Both had a well-circumscribed growth pattern and were composed of numerous large epithelioid cells with scattered severe atypia and mitoses. The tumor was positive for S-100 protein and vimentin and negative for low- and high-molecular weight cytokeratins. However, at high power, the epithelioid cells with severe atypia were negative for S-100 protein, and abundant large epithelioid macrophages were responsible for the S-100 protein positivity. The malignant tumor cells were negative for HMB-45, positive for CD31 and Factor VIII-related antigen, and focally positive for CD34. A focus of infiltrative, classical angiosarcoma with irregular vascular channels lined with plump, anaplastic endothelial cells was then found deep to the epithelioid tumor. CONCLUSIONS: Macrophage-rich epithelioid angiosarcoma demonstrates abundant S-100 protein-positive epithelioid macrophages. This subset of epithelioid angiosarcoma may mimic malignant melanoma and may present as a pitfall in diagnosis.

Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody.

A neutralizing monoclonal antibody (MAb) 26-2F to human angiogenin, a potent inducer of neovascularization, has been shown previously to prevent or delay the appearance of angiogenin-secreting human colon, fibrosarcoma and lung tumor cell xenografts implanted subcutaneously (s.c.) into athymic mice. In an analogous model system, we report here that the antibody also prevents the establishment of PC-3 androgen-independent human prostate cancer tumors in, on average, 40% of treated mice (p < 0.0001, survivor analysis). Intriguingly, combining MAb 26-2F together with cisplatin and suramin, 2 therapeutic agents that together showed little antitumor activity in the aforementioned model, resulted in an even greater degree of protection (71% protected, p = 0.009 compared to antibody treatment alone). This protective effect persisted several weeks after cessation of treatment. Additionally, prophylactic systemic administration of MAb 26-2F dramatically reduced by 50% the formation of spontaneous regional metastasis originating from primary growth in the prostate gland of PC-3M cells, highly metastatic variants of PC-3. Protection from metastasis was still significant when treatment with MAb 26-2F was delayed until after the primary tumor was well established. The antibody is not directly cytotoxic to either cell type, both of which secrete angiogenin in vitro and when growing as tumors in vivo, but changes the pattern of vascularity in primary tumors growing orthotopically. These findings, together with the observation that angiogenin protein and mRNA are apparently overexpressed in cancerous vs. normal human prostate tissues, demonstrate that angiogenin antagonism represents a promising new approach for preventing progression and metastasis of clinical prostate cancer.