epidermal-growth-factor and Skin-Neoplasms

The field of cutaneous oncology is exploding with innovative treatment options, specifically in the field of targeted therapy. These advances offer new hope to select patients with high risk skin cancers. In part two of our series on targeted therapy for skin cancer, we focus our attention on squamous cell carcinoma. We begin with the epidermal growth factor receptor inhibitors and branch out into newer areas of active research.

Topics: Antineoplastic Agents; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Clinical Trials as Topic; Dermatology; Epidermal Growth Factor; Humans; Molecular Targeted Therapy; Skin Neoplasms

Targeting the vascular endothelial growth factor (VEGF) in combination with standard chemotherapy has recently proved successful in the treatment of different types of advanced cancer. The achievements of combinatorial anti-VEGF monoclonal antibody bevacizumab (BEV) renewed the confidence in targeted antiangiogenic approaches to constitute a complementary therapeutic modality in addition to surgery, radiotherapy and chemotherapy. While several second-generation multitargeted tyrosine kinase inhibitors show promise in defined tumor entities, these novel antiangiogenic compounds have yet to meet or exceed the efficacy of combinatorial BEV therapy in ongoing clinical trials. Current developments of targeted antiangiogenic agents include their use in the adjuvant setting and the combination of different antiangiogenesis inhibitors to take a more comprehensive approach in blocking tumor angiogenesis. The identification of surrogate markers that can monitor the activity and efficacy of antiangiogenic drugs in patients belongs to the most critical challenges to exploit the full potential of antiangiogenic therapies. The opportunities and obstacles in further development of growth factor- and growth factor receptor-targeted antiangiogenic approaches for advanced cancer, including malignant melanoma, will be discussed herein with particular reference to selected ongoing clinical trials.

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Colorectal Neoplasms; Combined Modality Therapy; Epidermal Growth Factor; Humans; Platelet-Derived Growth Factor; Receptor Protein-Tyrosine Kinases; Skin Neoplasms; Vascular Endothelial Growth Factor A

Topics: Epidermal Growth Factor; Genetic Predisposition to Disease; Humans; Melanoma; Polymorphism, Genetic; Skin Neoplasms

Transforming growth factor alpha (TGF alpha) is one of a group of structurally-related growth factors (the epidermal growth factor family of ligands) that interact with one or other members of the epidermal growth factor family of protein tyrosine kinase receptors (EGF-R's). A number of excellent reviews detailing our knowledge of this area have been recently published (Carpenter and Wahl, 1991; Derynck, 1992; Prigent and Lemoine, 1992). Rather than add to their number, this review focuses on new insights into the importance of TGF alpha and signaling through the EGF receptor considered in the context of the laboratory mouse. The new information has emerged from analysis of mutant mice generated either by classical gene targeting in embryonic stem (ES) cells or by accidents of nature. In addition to their intrinsic interest, these mice are proving invaluable in determining the importance of EGF receptor signaling in wound healing and as a contributing factor in the conversion of a normal cell into its tumorigenic counterpart.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Transformation, Neoplastic; Cocarcinogenesis; Epidermal Growth Factor; ErbB Receptors; Gene Targeting; Hair; Mice; Mice, Knockout; Mice, Mutant Strains; Multigene Family; Mutagenesis, Insertional; Papilloma; Recombination, Genetic; Signal Transduction; Skin Neoplasms; Transforming Growth Factor alpha; Vibrissae; Wound Healing

Transgenic animals have greatly enhanced our understanding of the contribution of various structural and regulatory components to epidermal biology. The expression of mutant versions of these components in the epidermis of transgenic mice has generated animal models of specific human skin diseases.. The expression of mutant keratin genes has produced animal models of epidermolysis bullosa simplex and epidermolytic hyperkeratosis and, in doing so, has focused attention on the genetics of keratins in these and other skin disorders. Similarly, the generation of mice overexpressing growth factors and/or oncogenes, exclusively in the epidermis, has identified the role of these factors in normal skin and produced models of disease states where the regulation of these factors is perturbed.. These models of keratin disorders and other diseases not only enable the determination of the cause of these disorders, but also allow evaluation of novel therapeutic techniques for the amelioration of these skin diseases.

Topics: Animals; Animals, Newborn; Epidermal Growth Factor; Gene Expression; Genes, Dominant; Genes, fos; Genes, ras; Humans; Keratins; Mice; Mice, Transgenic; Models, Biological; Mutation; Phenotype; Skin; Skin Diseases; Skin Neoplasms; Transforming Growth Factor alpha; Transforming Growth Factor beta

Cell growth is controlled by two types of genes, i.e., activating genes (oncogenes) and negative regulator genes (antioncogenes). Studies have shown that malignant transformation of a cell can result from either increased oncogene activity or decreased antioncogene activity. Current knowledge of genes relevant to dermatology is discussed.

Topics: Animals; Cell Transformation, Neoplastic; Epidermal Growth Factor; Genes, p53; Genes, Tumor Suppressor; Humans; Mice; Proto-Oncogenes; Psoriasis; Skin Neoplasms

Topics: Animals; Cytokines; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Humans; Melanoma; Peptides; Platelet-Derived Growth Factor; Skin Neoplasms; Transforming Growth Factors

Epidermal growth factor (EGF) may promote wound healing and decrease laser-induced postinflammatory hyperpigmentation (PIH).. To evaluate the effectiveness of an EGF-containing cream on PIH, post-laser erythema, and transepidermal water loss (TEWL) after 1,064-nm Q-Switched Nd: YAG laser treatment of Hori's nevus.. This is a split-face, double-blinded, randomized, controlled study conducted in 30 subjects with bilateral Hori's nevus. After laser treatment, participants were randomized to apply EGF cream on one facial side and placebo on the other side for 8 weeks. The incidence and intensity of PIH were assessed by photographs and melanin indexes (MIs) ratio at baseline, Week 2, Week 4, and Week 8. Post-laser erythema and TEWL were measured at baseline, Day 1, Day 3, and Day 7. Side effects and patient satisfaction score were evaluated.. The incidence of PIH was 26.7% in EGF group compared to 20% in placebo. The intensity of PIH was 0.057 (0.033-0.086) and 0.045 (0.027-0.076) in EGF and placebo group, respectively. There was no significant difference in both incidence (p = 0.5) and intensity of PIH (p = 0.145). Post-laser erythema was not statistically different between groups. EGF could alleviate TEWL better than placebo but without statistical significance. Patient satisfaction score was significantly higher in EGF group compared to placebo (p < 0.001).. The EGF-containing cream could not prevent PIH. It may reduce laser-induced skin barrier damage. Future studies in more subjects are needed.

Topics: Asian People; Epidermal Growth Factor; Erythema; Humans; Hyperpigmentation; Lasers, Solid-State; Nevus of Ota; Skin Neoplasms; Treatment Outcome

Dermatofibrosarcoma protuberans (DFSP) is a soft-tissue sarcoma characterized by a high risk of local infiltration. The identification of the COL1A1-PDGFB t(17;22) translocation activating the PDGF pathway led to the use of imatinib in unresectable DFSP, with a response rate of 36-80%. Pazopanib is a multitarget tyrosine kinase inhibitor approved for soft-tissue sarcomas. We conducted a phase II study of patients with unresectable DFSP to evaluate the efficacy and safety of pazopanib. Patients received 800 mg of pazopanib daily. The primary endpoint was the objective response rate defined as the reduction of the largest diameter of the tumor by ≥30% at 6 months or at surgery. A total of 23 patients, including one pretreated with imatinib, were enrolled. With a median follow-up of 6.2 months (interquartile range = 5.6-7.8 months), five patients (22%, 95% confidence interval = 7-22%) had a partial response to pazopanib. The best objective response rate was 30% (95% confidence interval = 13-53%) using Response Evaluation Criteria in Solid Tumors. One patient with metastatic DFSP previously treated with imatinib died after 2.4 months. Nine patients (39%) discontinued the treatment owing to adverse events. Pharmacodynamics analyses of tumor samples were conducted: the enrichment of EGF and the EGFR-associated gene panel was associated with resistance, suggesting that EGFR-targeted therapies could be a therapeutic option to explore in DFSP. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01059656.

Topics: Adult; Aged; Biomarkers, Tumor; Dermatofibrosarcoma; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Indazoles; Male; Middle Aged; Protein Kinase Inhibitors; Pyrimidines; Response Evaluation Criteria in Solid Tumors; Skin; Skin Neoplasms; Sulfonamides; Tumor Burden

Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Melanocytes; Melanoma; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Neoplasms; Zebrafish

Hotspot mutations of the oncogenes BRAF and NRas are the most common genetic alterations in cutaneous melanoma. Still, the nanoscale organization and signal coupling of these proteins remain incompletely understood, particularly upon expression of oncogenic NRas mutants. Here we employed single-molecule localization microscopy to study the nanoscale organization of NRas and BRAF at the plasma membrane (PM) of melanoma cells. NRas and BRAF resided in self-clusters that did not associate well in resting cells. In EGF-activated cells, NRas clusters became more diffused while overall protein levels at the PM increased; thus allowing enhanced association of NRas and BRAF and downstream signaling. In multiple melanoma cell lines, mutant NRas resided in more pronounced self-clusters relative to wild-type (WT) NRas yet associated more with the clustered and more abundant BRAF. In cells resistant to trametinib, a clinical MEK inhibitor (MEKi), a similar coclustering of NRas and BRAF was observed upon EGF activation. Strikingly, treatment of cells expressing mutant NRas with trametinib reversed the effect of mutant NRas expression by restoring the nonoverlapping self-clusters of NRas and BRAF and by reducing their PM levels and elevated pERK levels caused by mutant NRas. Our results indicate a new mechanism for signal regulation of NRas in melanoma through its nanoscale dynamic organization and a new mechanism for MEKi function in melanoma cells carrying NRas mutations but lacking MEK mutations. SIGNIFICANCE: Nanoscale dynamic organization of WT and mutant NRas relative to BRAF serves as a regulatory mechanism for NRas signaling and may be a viable therapeutic target for its sensitivity to MEKi.

Topics: Cell Line, Tumor; Cell Membrane; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; GTP Phosphohydrolases; Humans; MAP Kinase Kinase 1; Melanoma; Melanoma, Cutaneous Malignant; Membrane Proteins; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Signal Transduction; Single Molecule Imaging; Skin Neoplasms

The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.

Topics: Animals; Cell Line, Tumor; Epidermal Growth Factor; Gold; Humans; Low-Level Light Therapy; Male; Melanoma; Metal Nanoparticles; Mice, SCID; Multifunctional Nanoparticles; Oleic Acid; Proof of Concept Study; Skin Neoplasms; Xenograft Model Antitumor Assays

Topics: Antineoplastic Agents, Immunological; Cetuximab; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Melanoma; Middle Aged; Nevus, Pigmented; Skin Neoplasms

Malignant melanoma remains a challenge for clinical practice and novel therapeutic strategies are urgently needed. Herbacetin, a natural flavonoid compound that has multiple pharmacological activities, exerts anticancer effects on several human tumors. In this study, the anti-angiogenesis effect of Herbacetin in human malignant melanoma was investigated. The results indicated that Herbacetin treatment significantly suppressed tumor growth and angiogenesis of malignant melanoma both in vitro and in vivo. In melanoma A375 and Hs294T cells, Herbacetin treatment suppressed both EGF-induced and constitutive phosphorylation of EGFR, accelerated the internalization and degradation of EGFR, and subsequently suppressed the activation of the downstream kinases (AKT and ERK). Moreover, MMP9 was determined as a key angiogenic factor in Herbacetin treated melanoma cells. Knockdown of MMP9 suppressed the in vitro angiogenesis while overexpression of MMP9 in Herbacetin treated melanoma cells restored the angiogenesis ability. We concluded that Herbacetin suppressed melanoma angiogenesis through blocking EGFR-ERK/AKT-MMP9 signaling pathway and Herbacetin may be developed as a potential drug for melanoma treatment.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Melanoma; Melanoma, Cutaneous Malignant; Mice; Mice, Nude; Neovascularization, Pathologic; Skin Neoplasms; Xenograft Model Antitumor Assays

Carvedilol is reported to prevent cancers in humans and animal models. However, a molecular mechanism has yet to be established, and the extent to which other β-blockers are chemopreventive remains relatively unknown. A comparative pharmacological approach was utilized with the expectation that a mechanism of action could be devised. JB6 Cl 41-5a (JB6 P+) murine epidermal cells were used to elucidate the chemopreventative properties of β-blockers, as JB6 P+ cells recapitulate in vivo tumor promotion and chemoprevention. The initial hypothesis was that β-blockers that are GRK/β-arrestin biased agonists, like carvedilol, are chemopreventive. Sixteen β-blockers of different classes, isoproterenol, and HEAT HCl were individually co-administered with epidermal growth factor (EGF) to JB6 P+ cells to examine the chemopreventative properties of each ligand. Cytotoxicity was examined to ensure that the anti-transformation effects of each ligand were not due to cellular growth inhibition. Many of the examined β-blockers suppressed EGF-induced JB6 P+ cell transformation in a non-cytotoxic and concentration-dependent manner. However, the IC50 values are high for the most potent inhibitors (243, 326, and 431 nM for carvedilol, labetalol, and alprenolol, respectively) and there is no correlation between pharmacological properties and inhibition of transformation. Therefore, the role of α1- and β2-adrenergic receptors (AR) was examined by standard competition assays and shRNA targeting β2-ARs, the only β-AR expressed in JB6 P+ cells. The results reveal that pharmacological inhibition of α1- and β2-ARs and genetic knockdown of β2-ARs did not abrogate carvedilol-mediated inhibition of EGF-induced JB6 P+ cell transformation. Furthermore, topical administration of carvedilol protected mice from UV-induced skin damage, while genetic ablation of β2-ARs increased carvedilol-mediated effects. Therefore, the prevailing hypothesis that the chemopreventive property of carvedilol is mediated through β-ARs is not supported by this data.

Topics: Adrenergic beta-Antagonists; Alprenolol; Animals; Carvedilol; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Inhibitory Concentration 50; Labetalol; Ligands; Mice; Mice, Inbred C57BL; Receptors, Adrenergic; Receptors, Adrenergic, beta; RNA, Small Interfering; Signal Transduction; Skin; Skin Neoplasms; Ultraviolet Rays

Melanoma represents one of the most aggressive forms of cancer. With the rapid increases in the incidence of melanoma in the United States, Australia and Europe over the last decades, melanoma has been considered an epidemic cancer in these areas. The aim of our study was to evaluate the utility of osteoprotegerin (OPG), osteopontin (OPN), epidermal growth factor (EGF) and vascular endothelial growth factor VEGF for the diagnosis and prognosis of melanoma.. Overall, 322 individuals were assessed: 183 melanoma patients and 139 healthy individuals. Melanoma patients were divided into four subgroups according to the Breslow score. OPN, OPG, EGF, and VEGF were determined in each plasma sample.. The serum levels of the following biomarkers were statistically significantly higher in the melanoma group compared to the control group: OPG and, OPN (p<0.0001), EGF (p=0.0379). In the first stage, OPG (p=0.0236) and OPN (p=0.0327) showed a statistically significant increase. Concerning positive and negative sentinel node metastases a statistically significant change was observed in: OPN (p<0.0001), EGF (p=0.0114), VEGF (p=0.0114).. OPG and OPN are promising biomarkers of early-stage melanoma. EGF and VEGF appear to be prognostic biomarkers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Epidermal Growth Factor; Female; Humans; Lymphatic Metastasis; Male; Melanoma; Melanoma, Cutaneous Malignant; Middle Aged; Neoplasm Staging; Osteopontin; Osteoprotegerin; Prognosis; Skin Neoplasms; Vascular Endothelial Growth Factor A; Young Adult

Melanoma is a potentially lethal skin cancer with high mortality rate. Recently, the peptide-mediated transdermal delivery of small interference RNA (siRNA) emerges as a promising strategy to treat melanoma by inducing the apoptosis of tumor cells, but the related theoretical model describing the delivery of siRNA under the effect of SPACE-EGF, the growth inhibition of melanoma and the dynamic expanding of the bump on the skin due to the growth of melanoma has not been reported yet. In this article, a theoretical model is developed to describe the percutaneous siRNA delivery mediated by SPACE-EGF to melanoma and the growth inhibition of melanoma. The results present the spatial-temporal distribution of siRNA and the growth of melanoma under the inhibition of siRNA, which shows a good consistency with the experimental results. In addition, this model represents the uplift process of tumors on the skin surface. The model presented here is a useful tool to understand the whole process of the SPACE-EGF-mediated delivery of the siRNA to melanoma through skin, to predict the therapeutic effect, and to optimize the therapeutic strategy, providing valuable references for the treatment of melanoma.

Topics: Administration, Cutaneous; Animals; Cell Line, Tumor; Cell-Penetrating Peptides; Epidermal Growth Factor; Melanoma, Experimental; Mice, Inbred C57BL; Models, Biological; RNA, Small Interfering; Skin; Skin Neoplasms

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Skin Neoplasms

Activating mutations in neuroblastoma RAS viral oncogene homolog (NRAS) are frequent driver events in cutaneous melanoma. NRAS is a guanosine triphosphate-binding protein whose most well-characterized downstream effector is RAF, leading to activation of mitogen-activated protein kinase (MEK)-extracellular signal-regulated protein kinase 1/2 signaling. Although there are no Food and Drug Administration-approved targeted therapies for melanoma patients with a primary mutation in NRAS, one form of targeted therapy that has been explored is MEK inhibition. In clinical trials, MEK inhibitors have shown disappointing efficacy in mutant NRAS patients, the reasons for which are unclear. To explore the effects of MEK inhibitors in mutant NRAS melanoma, we used a high-throughput reverse-phase protein array platform to identify signaling alterations. Reverse-phase protein array analysis of phospho-proteomic changes in mutant NRAS melanoma in response to trametinib indicated a compensatory increase in v-akt murine thymoma viral oncogene homolog signaling and decreased expression of mitogen-inducible gene 6 (MIG6), a negative regulator of epidermal growth factor receptor/v-erb-b2 erythroblastic leukemia viral oncogene homolog receptors. MIG6 expression did not alter the growth or survival properties of mutant NRAS melanoma cells. Rather, we identified a role for MIG6 as a negative regulator of epidermal growth factor-induced signaling and cell migration and invasion. In MEK-inhibited cells, further depletion of MIG6 increased migration and invasion, whereas MIG6 expression decreased these properties. Therefore, a decrease in MIG6 may promote the migration and invasiveness of MEK-inhibited mutant NRAS melanoma, especially in response to epidermal growth factor stimulation.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Movement; Down-Regulation; Epidermal Growth Factor; GTP Phosphohydrolases; Humans; Immunohistochemistry; MAP Kinase Kinase 1; Melanoma; Membrane Proteins; Mutation; Skin Neoplasms; Tumor Suppressor Proteins

Cutaneous melanoma (CM) is a malignant skin cancer with the high incidence in whiteskinned populations. Host genetic factors (such as: genes in nucleotide excision repair system or cell proliferation regulation system) interacted with ultraviolet radiation are potential reasons for CM. Previous studies about associations between CM and the rs4444903 (+61A>G) in the Epidermal growth factor gene (EGF) or rs13181 (+35931 A>C) in the xeroderma pigmentosum group D gene (XPD) have produced inconsistent results. To clarify these associations, metaanalyses of available candidate case-control association studies about Caucasians were performed. Data of each study were gathered according to the "Quality-Evaluation Score" (Ver.1.0). Finally, the meta-analysis with 2167 cases/4211 controls showed that the EGF rs4444903 had no significant association with CM (p>0.05), while the analysis with 3,492 cases/5,381 controls indicated the A allele of XPD rs13181 was significantly associated with CM (odds ratio= 0.93, 95% CI: 0.87-0.99; p=0.019). These results are also supported with linkage disequilibrium (LD) structure analysis. The current meta-analyses results suggest that the XPD gene, but not the EGF gene, has contributed to CM susceptibility, and XPD is a possible drug target.

Topics: Epidermal Growth Factor; Humans; Melanoma; Polymorphism, Genetic; Skin Neoplasms; White People; Xeroderma Pigmentosum Group D Protein

Wnt10b is a signaling protein regulating skin development and homeostasis, and the expression of Wnt10b is restricted to epidermal keratinocytes in embryonic and postnatal skin. Recent studies indicate an elevated expression of Wnt10b in skin tumors. However, how Wnt10b regulates skin tumorigenesis remains largely unknown. Here we report that continuous expression of Wnt10b mediates transformation of epidermal keratinocytes through activating genes involved in EGF/MAPK signaling pathways. We first established a prolonged Wnt10b overexpression system in JB6P- cells to represent the elevated Wnt10b expression level in skin keratinocytes. Through expression assays and observations under phase-contrast microscopy, prolonged expression of Wnt10b activated Wnt/β-catenin pathway and induced morphological changes of cells showing longer protrusions and multilayer growth, indicating early-stage cell transformation. Wnt10b also increased cellular proliferation and migration according to BrdU incorporation and cell mobility assays. Furthermore, multi-doses of AdWnt10b treatment to JB6P- cells induced colony formation, stronger invasive ability in transwell system, and anchorage-independent growth in agar gel. In molecular level, AdWnt10b treatment induced increased transcriptional expressions of Egf, downstream Mapk pathway factors, and MMPs. Administration of Wnt antagonist DKK1 blocked the tumor promotion process induced by Wnt10b. Taken together, these findings clearly demonstrate that Wnt10b promotes epidermal keratinocyte transformation through induced Egf pathway.

Topics: Antimetabolites, Antineoplastic; Bromodeoxyuridine; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Intercellular Signaling Peptides and Proteins; Keratinocytes; Matrix Metalloproteinases; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins; Signal Transduction; Skin Neoplasms; Tumor Stem Cell Assay; Wnt Proteins

The identification of primary molecular targets of cancer-preventive phytochemicals is essential for a comprehensive understanding of their mechanism of action. In the present study, we investigated the chemopreventive effects and molecular targets of acacetin, a flavonoid found in Robinia p seudoacacia, also known as black locust. Acacetin treatment significantly suppressed epidermal growth factor (EGF)-induced cell transformation. Immunoblot analysis revealed that acacetin attenuated EGF-induced phosphorylation of Akt and p70(S6K), which are downstream effectors of phosphatidylinositol 3-kinase (PI3-K). An immunoprecipitation kinase assay of PI3-K and pull-down assay results demonstrated that acacetin substantially inhibits PI3-K activity by direct physical binding. Acacetin exhibited stronger inhibitory effects against anchorage-dependent and -independent cell growth in cells expressing higher PI3-K activity compared with those exhibiting relatively low PI3-K activity. Binding assay data combined with computational modeling suggest that acacetin binds in an adenosine triphosphate (ATP)-competitive manner with the p110α subunit of PI3-K and interacts with Val828, Glu826, Asp911, Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900 residues. Acacetin was also found to significantly reduce SK-MEL-28 tumor growth and Akt phosphorylation in vivo. Taken together, these results indicate that acacetin is an ATP-competitive PI3-K inhibitor and a promising agent for melanoma chemoprevention.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Cell Proliferation; Cell Transformation, Neoplastic; Class Ia Phosphatidylinositol 3-Kinase; Enzyme Inhibitors; Epidermal Growth Factor; Flavones; Mice; Mice, Nude; Molecular Docking Simulation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Skin Neoplasms; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.

Topics: Allosteric Regulation; Animals; Binding Sites; Carcinoma, Squamous Cell; Cell Line, Tumor; Crystallography, X-Ray; Cyclin-Dependent Kinases; Epidermal Growth Factor; Flavonoids; G1 Phase; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Neoplasm Transplantation; Protein Kinase Inhibitors; Retinoblastoma Protein; S Phase; Skin Neoplasms

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma, but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like (EGFL) repeats as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased Rho-associated kinase (ROCK) signaling concomitant with myosin light chain 2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL-expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNCEGFL exert a role in melanoma invasion by modulating ROCK signaling and cell migration.

Topics: Cardiac Myosins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Humans; Melanoma; Myosin Light Chains; Neoplasm Invasiveness; Peptide Fragments; Phosphorylation; rho-Associated Kinases; Signal Transduction; Skin Neoplasms; Tenascin

Alterations in epidermal growth factor (EGF) expression are known to be of prognostic relevance in human melanoma, but EGF-mediated effects on melanoma have not been extensively studied. As lymph node metastasis usually represents the first major step in melanoma progression, we were trying to identify a potential role of primary tumor-derived EGF in the mediation of melanoma lymph node metastases. Stable EGF knockdown (EGFkd) in EGF-high (M24met) and EGF-low (A375) expressing melanoma cells was generated. Only in EGF-high melanoma cells, EGFkd led to a significant reduction of lymph node metastasis and primary tumor lymphangiogenesis in vivo, as well as impairment of tumor cell migration in vitro. Moreover, EGF-induced sprouting of lymphatic but not of blood endothelial cells was abolished using supernatants of M24met EGFkd cells. In addition, M24met EGFkd tumors showed reduced vascular endothelial growth factor-C (VEGF-C) expression levels. Similarly, in human primary melanomas, a direct correlation between EGF/VEGF-C and EGF/Prox-1 expression levels was found. Finally, melanoma patients with lymph node micrometastases undergoing sentinel node biopsy were found to have significantly elevated EGF serum levels as compared with sentinel lymph node-negative patients. Our data indicate that tumor-derived EGF is important in mediating melanoma lymph node metastasis.

Topics: Animals; Biomarkers, Tumor; Cell Movement; Endothelial Cells; Epidermal Growth Factor; Female; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Lymphangiogenesis; Lymphatic Metastasis; Melanoma; Mice; Mice, SCID; Neoplasm Micrometastasis; Sentinel Lymph Node Biopsy; Skin Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor C

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Topics: Animals; Carcinoma, Squamous Cell; Caveolin 1; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Enzyme Activation; Epidermal Growth Factor; Gene Knockdown Techniques; Humans; Keratin-18; Keratinocytes; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Serum; Skin Neoplasms; Transcription Factor AP-1

Discerning lymphatics from blood vessels using lineage-specific markers has advanced our understanding of tumor lymphangiogenesis. Many studies have demonstrated that the newly formed lymphatics are the result of a dynamic and complex interplay between lymphatic endothelium, tumor cells, secreted growth factors, and inhibitors in the tumor microenvironment. Results provide evidence that lymphatics play an active role in cancer metastasis rather than once thought to be passively invaded by infiltrating tumor cells. The latest discovery of Bracher et al. (2012) further supports a dynamic role for lymphatics-mediated melanoma metastasis to sentinel lymph node prompted by tumor-derived epidermal growth factor.

Topics: Animals; Epidermal Growth Factor; Female; Humans; Lymphangiogenesis; Melanoma; Skin Neoplasms; Vascular Endothelial Growth Factor C

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2',3'-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner.

Topics: Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cyclic AMP; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Mice; Neurocalcin; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Skin Neoplasms; Snail Family Transcription Factors; Transcription Factors; Tumor Suppressor Proteins

In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas.. Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx.. Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis.. Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.

Topics: Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Cell Survival; Cetuximab; Drug Resistance, Neoplasm; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Gene Knockdown Techniques; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Pyrimidines; Receptor, Fibroblast Growth Factor, Type 3; Signal Transduction; Skin Neoplasms; Structure-Activity Relationship; Tumor Cells, Cultured

Our previous report demonstrated that RSK2 plays an important role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor mediated through the N-terminal kinase domain of RSK2 in JB6 Cl41 mouse skin epidermal cells in vitro. However, no direct evidence has been reported regarding the relationship of RSK2 activity and human skin cancer. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on epidermal growth factor-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2 but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of malignant melanoma cells. Moreover, RSK2(-/-) mouse embryonic fibroblast (MEF) showed enhanced sub-G(1) accumulation induced by UVB stimulation compared with RSK2(+/+) MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with ultraviolet. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth.

Topics: Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Humans; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Skin Neoplasms; Tissue Array Analysis; Ultraviolet Rays

The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation.

Topics: Animals; Blotting, Western; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Immunoenzyme Techniques; Immunoprecipitation; Melanoma; Mice; Mutation; p21-Activated Kinases; Phosphorylation; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Skin; Skin Neoplasms; Threonine; Tissue Array Analysis; Transcription Factor AP-1

RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.

Topics: Animals; Cell Proliferation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Knockdown Techniques; Guanine Nucleotide Exchange Factors; Hepatocyte Growth Factor; Humans; Male; Melanoma; Mice; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; ras Guanine Nucleotide Exchange Factors; Signal Transduction; Skin Neoplasms; Xenograft Model Antitumor Assays

Highly invasive A431-III cells, which are derived from parental A431-P cells, were originally isolated by three successive passages through a Boyden chamber using a Matrigel-coated membrane support. The greater invasion potential shown by A431-III cells was due to their increased ability to spread/migrate, which was associated with enhanced MMP activity. The tumor progression events evoked by A431-P cells compared to A431-III cells may help identify useful strategies for evaluating the epithelial-mesenchymal transition (EMT) and these cell lines could be a reliable model for evaluating tumor metastasis events. Using this approach, we evaluated the effects of luteolin and quercetin using the A431-P/A431-III EMT model. These flavonoids reversed cadherin switching, downregulated EMT markers, and nullified the invasion ability of A431-III cells. Overexpression of MMP-9 resulted in induction of the EMT in A431-P cells and this could be reversed by treating with luteolin or quercetin. Cotreatment of A431-P and A431-III cells with epidermal growth factor (EGF) plus luteolin or quercetin resulted in a more epithelial-like morphology, led to reduced levels of EGF-induced markers of EMT, and caused the restoration of cell-cell junctions. E-cadherin was decreased by EGF, but increased by luteolin and quercetin. Our results suggest that luteolin and quercetin are potentially beneficial agents that target and prevent the occurrence of EMT in epidermal carcinoma cells. These chemicals also have the ability to attenuate tumor progression in A431-III cells. Luteolin and quercetin show inherent potential as chemopreventive/antineoplastic agents and do this by abating tumor progression through a reversal of EMT.

Topics: Antioxidants; Cadherins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Flavonoids; Humans; Intercellular Junctions; Luteolin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phosphorylation; Quercetin; Skin Neoplasms

Dermatofibrosarcoma protuberans (DFSP) is an uncommon, locally aggressive, malignant cutaneous tumor that sparingly presents on the scalp. Dermatofibrosarcomas often result from the formation of a fusion oncogene on translocated or supernumerary ring chromosomes 17 and 22, causing the overexpression of PDGFRbeta driven by the COL1A1 promoter. Because of uncertainty surrounding appropriate treatment of aggressive scalp DFSP, the authors performed an extensive review of the available data from a MEDLINE (Ovid) search to describe the clinical presentation and treatment options for this rare tumor. Their search identified 39 different cases, including the illustrative case presented in this study. Adjuvant therapy for this malignant lesion is not universally established in the literature. In the present case, the authors successfully treated a locally invasive scalp DFSP with presurgical therapy that specifically inhibited the PDGFbeta receptor. Imatinib significantly shrank the DFSP tumor mass, reduced hypervascularity, reduced metabolic activity on PET scanning, and permitted a safe gross-total resection. Although wide excision and Mohs micrographic surgery remain the standard surgical treatments for DFSP, the authors illustrate that presurgical chemotherapeutic treatment by imatinib provides a critical adjunct to traditional therapy.

Topics: Antineoplastic Agents; Benzamides; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 22; Collagen Type I; Collagen Type I, alpha 1 Chain; Dermatofibrosarcoma; Epidermal Growth Factor; Humans; Imatinib Mesylate; Male; Middle Aged; Neurosurgical Procedures; Piperazines; Pyrimidines; Receptor, Platelet-Derived Growth Factor beta; Scalp; Skin Neoplasms; Treatment Outcome

Connexin43 (Cx43) is known to have tumor-suppressive effects, but the underlying mechanisms are still poorly understood. In keratinocytes, we previously showed that the COOH-terminal domain of Cx43 directly interacts with the tumor suppressor Cav-1. We now show that rat epidermal keratinocytes (REK) that are reduced in Cx43 present features of epithelial-to-mesenchymal transition and are more invasive than their control counterparts, whereas overexpression of Cx43 inhibited the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)- and epidermal growth factor (EGF)-induced invasive properties. Carbenoxolone did not alter the inhibitory effect of Cx43 against TPA- and EGF-induced cell invasion, indicating the involvement of a gap junctional intercellular communication-independent mechanism. Interestingly, the association of Cx43 with Cav-1 was found to be reduced after TPA and EGF treatment. Accordingly, the colocalization of Cx43 with Cav-1 was diminished in cells from a human epidermal squamous cell carcinoma, as well as in sections from human keratinocyte tumors, suggesting that Cx43/Cav-1 interaction plays a protective role against keratinocyte transformation. As opposed to cells that overexpress Cx43-GFP, invasion could be induced in rat epidermal keratinocytes that overexpressed a GFP-tagged truncated mutant of Cx43 (Delta244-GFP) that we previously showed not to interact with Cav-1, as well as in cells that overexpressed Cx43-GFP but were reduced in Cav-1. Our data show that Cx43 possesses tumor-suppressive properties in keratinocytes and provide the first evidence that the Cx43/Cav-1 interaction is altered in keratinocyte transformation processes, as well as in human keratinocyte tumors, and that this association might play a role in Cx43-mediated tumor suppression.

Topics: Animals; Anti-Ulcer Agents; Blotting, Western; Carbenoxolone; Carcinogens; Carcinoma, Squamous Cell; Caveolin 1; Cell Communication; Cell Proliferation; Connexin 43; Epidermal Cells; Epidermal Growth Factor; Epidermis; Epithelial Cells; Fluorescent Antibody Technique; Gap Junctions; Green Fluorescent Proteins; Humans; Immunoprecipitation; Keratinocytes; Mesoderm; Neoplasm Invasiveness; Rats; RNA, Small Interfering; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta1

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents.

Topics: Animals; Anticarcinogenic Agents; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Mice; Phosphatidylinositol 3-Kinases; Retinoblastoma Protein; Serine; Skin Neoplasms

We examined the anti-angiogenic effects of Ganoderma tsugae methanol extract (GTME) on human epidermoid carcinoma A-431 cells. Our data indicate that GTME inhibits the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in vitro and in vivo, and also inhibits the capillary tube formation of human umbilical vein endothelial cells (HUVECs). We also show that the suppression of VEGF expression by GTME can be restored by treatment with EGF. These results suggest that GTME inhibits VEGF expression via the suppression of EGFR expression, resulting in the downregulation of VEGF secretion from epidermoid carcinoma A-431 cells. These findings reveal a novel role for G. tsugae in inhibiting EGFR and VEGF expression, which are important for tumor angiogenesis and growth. Thus, GTME may provide a potential therapeutic approach for anti-tumor treatment.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Squamous Cell; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Drugs, Chinese Herbal; Endothelium, Vascular; Epidermal Growth Factor; ErbB Receptors; Ganoderma; Gene Expression Regulation, Neoplastic; Humans; Methanol; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Neovascularization, Pathologic; Neovascularization, Physiologic; Phytotherapy; Plant Extracts; Reishi; Skin Neoplasms; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The phenotypic switching called epithelial-to-mesenchymal transition is frequently associated with epithelial tumor cell progression from a comparatively benign to an aggressive, invasive malignancy. Coincident with the emergence of such cellular plasticity is an altered response to transforming growth factor-beta (TGF-beta) as well as epidermal growth factor (EGF) receptor amplification. TGF-beta in the tumor microenvironment promotes invasive traits largely through reprogramming gene expression, which paradoxically supports matrix-disruptive as well as stabilizing processes. ras-transformed HaCaT II-4 keratinocytes undergo phenotypic changes typical of epithelial-to-mesenchymal transition, acquire a collagenolytic phenotype, and effectively invade collagen type 1 gels as a consequence of TGF-beta1 + EGF stimulation in a three-dimensional physiologically relevant model system that monitors collagen remodeling. Enhanced collagen degradation was coupled to a significant increase in matrix metalloproteinase (MMP)-10 expression and involved a proteolytic axis composed of plasmin, MMP-10, and MMP-1. Neutralization of any one component in this cascade inhibited collagen gel lysis. Similarly, addition of plasminogen activator inhibitor type 1 (SERPINE1) blocked collagen degradation as well as the conversion of both proMMP-10 and proMMP-1 to their catalytically active forms. This study therefore identifies an important mechanism in TGF-beta1 + EGF-initiated collagen remodeling by transformed human keratinocytes and proposes a crucial upstream role for plasminogen activator inhibitor type 1-dependent regulation in this event.

Topics: Carcinoma, Squamous Cell; Cell Line, Transformed; Collagen Type I; Epidermal Growth Factor; Fibrinolysin; Humans; Keratinocytes; Matrix Metalloproteinase 1; Matrix Metalloproteinase 10; Plasminogen Activator Inhibitor 1; Skin Neoplasms; Transforming Growth Factor beta1

A single nucleotide polymorphism (61A>G) in the epidermal growth factor (EGF) gene has been implicated in both melanoma pathogenesis and increased melanoma risk. To further evaluate this association, we conducted a case-control study in a clinic-based Italian population.. Individuals with less than 10 (N = 127) or more than 100 (N = 128) benign nevi, and patients with cutaneous melanoma (N = 418) were investigated for the EGF +61A>G polymorphism, using an automated sequencing approach.. Overall, no difference in EGF genotype frequencies was observed among subjects with different number of nevi as well as when non-melanoma healthy controls were compared with the melanoma patients. However, a heterogeneous distribution of the frequencies of the G/G genotype was detected among cases and controls originating from North Italy (21.1 and 18.3%, respectively) vs. those from South Italy (12.6 and 17.1%, respectively).. Our findings further suggest that EGF +61A>G polymorphism may have a limited impact on predisposition and/or pathogenesis of melanoma and its prevalence may vary in different populations.

Topics: Adult; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; Italy; Male; Melanoma; Middle Aged; Polymorphism, Single Nucleotide; Prevalence; Risk Factors; Skin Neoplasms; Young Adult

The diagnosis of melanoma is becoming ever more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal coreceptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Cell Line, Tumor; Epidermal Growth Factor; Epigenesis, Genetic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Melanoma; Membrane Glycoproteins; Neoplasm Proteins; Neoplastic Stem Cells; Nodal Protein; Octamer Transcription Factor-3; Protein Kinases; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta

AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H(2)O(2)-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H(2)O(2) induce AMPK activation (Thr(172) phosphorylation) in cultured human skin keratinocytes. UV and H(2)O(2) also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK serves as a positive regulator for p38 and p53 (Ser(15)) phosphorylation induced by UV radiation and H(2)O(2) treatment. We also observed that AMPK serves as a negative feedback signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-apoptotic effect of AMPK on UV- or H(2)O(2)-treated cells. Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H(2)O(2)-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer.

Topics: Acetyl-CoA Carboxylase; Adenylate Kinase; AMP-Activated Protein Kinases; Apoptosis; Cell Line; Epidermal Growth Factor; Humans; Hydrogen Peroxide; Keratinocytes; Models, Biological; p38 Mitogen-Activated Protein Kinases; Skin; Skin Neoplasms; Tumor Suppressor Protein p53; Ultraviolet Rays

It has long been known that excessive mitotic activity due to H-Ras can block keratinocyte differentiation and cause skin cancer. It is not clear whether there are any innate surveillants that are able to ensure that keratinocytes undergo terminal differentiation, preventing the disease. IKKalpha induces keratinocyte terminal differentiation, and its downregulation promotes skin tumor development. However, its intrinsic function in skin cancer is unknown. Here, we found that mice with IKKalpha deletion in keratinocytes develop a thickened epidermis and spontaneous squamous cell-like carcinomas. Inactivation of epidermal growth factor receptor (EGFR) or reintroduction of IKKalpha inhibits excessive mitosis, induces terminal differentiation, and prevents skin cancer through repressing an EGFR-driven autocrine loop. Thus, IKKalpha serves as an innate surveillant.

Topics: ADAM Proteins; Amphiregulin; Animals; Animals, Newborn; EGF Family of Proteins; Epidermal Growth Factor; Epidermis; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation; Glycoproteins; Homeostasis; I-kappa B Kinase; Intercellular Signaling Peptides and Proteins; Keratinocytes; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Mice, Transgenic; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins p21(ras); Receptors, Tumor Necrosis Factor; Skin; Skin Neoplasms

Cripto-1 is an epidermal growth factor-Cripto/FRL1/Cryptic family member that plays a role in early embryogenesis as a coreceptor for Nodal and is overexpressed in human tumors. Here we report that in the two-stage mouse skin carcinogenesis model, Cripto-1 is highly up-regulated in tumor promoter-treated normal skin and in benign papillomas. Treatment of primary mouse keratinocytes with Cripto-1 stimulated proliferation and induced expression of keratin 8 but blocked induction of the normal epidermal differentiation marker keratin 1, changes that are hallmarks of tumor progression in squamous cancer. Chemical or genetic blockade of the transforming growth factor (TGF)-beta1 signaling pathway using the ALK5 kinase inhibitor SB431542 and dominant negative TGF-beta type II receptor, respectively, had similar effects on keratinocyte differentiation. Our results show that Cripto-1 could block TGF-beta1 receptor binding, phosphorylation of Smad2 and Smad3, TGF-beta-responsive luciferase reporter activity, and TGF-beta1-mediated senescence of keratinocytes. We suggest that inhibition of TGF-beta1 by Cripto-1 may play an important role in altering the differentiation state of keratinocytes and promoting outgrowth of squamous tumors in the mouse epidermis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Differentiation; DNA Replication; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Reporter; Keratinocytes; Membrane Glycoproteins; Mice; Neoplasm Proteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta1

Topics: Adenine; Epidermal Growth Factor; Guanine; Humans; Melanoma; Osmolar Concentration; Polymorphism, Genetic; Prognosis; Skin Neoplasms

Locally advanced skin cancers including squamous cell carcinoma (SCC) of the skin are increasing in incidence. Patients are often elderly with significant comorbidities and therapy can be difficult. New targeted therapies, such as treatment directed at the epidermal growth factor receptor (EGFR), may be effective and less toxic in these patients. However, before designing appropriate clinical trials it is necessary to characterize the expression and activation of targets such as the EGFR to evaluate the rationale of using EGFR inhibitors (EGFRIs) in the treatment of this type of cancer.. To characterize the expression and activation by phosphorylation of EGFR in SCC of the skin by quantitative Western blotting using the LiCor immunofluorescence detection system with validation by immunohistochemistry. Secondary objectives were to evaluate downstream targets of EGFR expression and activation in SCC of the skin and to examine the associations between EGFR, pathological features and clinical behaviour of these tumours.. Twenty-one mainly locally advanced skin SCCs collected in our institution and stored in our tissue bank over a 4-year period were used for the study.. Nine of 21 (43%) tumours expressed EGFR above background. Of those nine, five expressed phosphorylated EGFR. There was no correlation with downstream activation of canonical signalling pathways, pathological features or clinical behaviour.. EGFR is expressed in a minority of tumours and then is not always activated. These results show that, before designing a trial with a targeted agent such as an EGFRI in SCC of the skin, it is important to verify the presence of the appropriate target to maximize the best outcome.

Topics: Adolescent; Adult; Carcinoma, Squamous Cell; Child; Child, Preschool; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Male; Neoplasm Proteins; Phosphorylation; Retrospective Studies; Skin Neoplasms; Treatment Outcome

Topics: Adenine; Age Factors; Aged; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Follow-Up Studies; Gene Frequency; Genotype; Guanine; Heart Transplantation; Humans; Italy; Kidney Transplantation; Liver Transplantation; Male; Middle Aged; Organ Transplantation; Polymorphism, Single Nucleotide; Prognosis; Proportional Hazards Models; Risk Assessment; Risk Factors; Skin Neoplasms; Time Factors

In this study, we examined ErbB1 signaling in human basal and squamous cell carcinomas (BCC and SCC) of the skin in vivo. We used enzyme-linked immunosorbent assay, laser capture microdissection-coupled real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry to assess expression and activation levels of ErbB1 protein, ligands, and potential downstream effectors, in BCC and SCC tumors, stroma, and adjacent epidermis. Although total ErbB1 protein and mRNA were similar in cancerous and normal skin, we found that ErbB1 activation (phospho-Tyr(1068)) was greater in bulk SCC versus BCC or normal skin. In addition, three ErbB1 ligand transcripts (amphiregulin, heparin-binding epidermal growth factor-like growth factor, and transforming growth factor-alpha) were up-regulated in tumor cells of SCC but not BCC. Expression of these ligands was also increased in asymptomatic epidermis adjacent to both SCC and BCC, relative to normal skin. Interestingly, betacellulin transcript levels were inversely regulated compared with the other ligands. Consistently, downstream ErbB1 effectors (Erk1/2 and Akt) were activated in tumor cells of SCC but not of BCC and in adjacent epidermis of both BCC and SCC. These results demonstrate that ErbB1 signaling is hyperactive in tumor cells of SCC but not of BCC and in nearby asymptomatic epidermis of both tumor types. Our results suggest that targeting ErbB1 signaling might be of benefit in the treatment of SCC.

Topics: Amphiregulin; Animals; Betacellulin; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Skin; Skin Neoplasms; Transforming Growth Factor alpha

Squamous cell carcinoma of the skin (SCCS) is rarely encountered by medical oncologists owing to success of local therapies. When advanced SCCS requires systemic palliation, treatment with conventional chemotherapy, such as cisplatin, is often precluded by a patient's age or medical comorbidities. Cetuximab is a human and mouse chimeric antibody against epidermal growth factor receptor, a tyrosine kinase receptor richly expressed by SCCS cells, including lymph node metastases. This drug, approved for treatment of squamous cell carcinoma of the upper aerodigestive tract as well as colorectal cancer, is well tolerated. Toxic effects include acneiform rash and diarrhea. Preclinical data suggest that epidermal growth factor receptor is important in SCCS carcinogenesis.. Herein, we report 2 cases of elderly patients with extensive, in-transit recurrence of SCCS who have been treated with palliative cetuximab. The drug was well tolerated, with the exception of acneiform rash requiring dose reduction in 1 patient. Both patients had excellent responses to cetuximab: the first patient had complete response by week 16 of treatment and the second a near-complete response by week 12. In both cases, initial response to cetuximab was evident by week 4 of therapy.. To our knowledge, these are the first reported cases of cetuximab use in patients with SCCS. The encouraging responses justify the prospective study of cetuximab in SCCS.

Topics: Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cetuximab; Combined Modality Therapy; Epidermal Growth Factor; Face; Female; Humans; Male; Neoplasm Recurrence, Local; Nose; Scalp; Skin Neoplasms

Glycogen synthase kinase 3beta (GSK3beta) is a multifunctional serine/threonine kinase. We showed that the expression of GSK3beta was drastically down-regulated in human cutaneous squamous cell carcinomas and basal cell carcinomas. Due to its negative regulation of many oncogenic proteins, we hypothesized that GSK3beta may function as a tumor suppressor during the neoplastic transformation of epidermal cells. We tested this hypothesis using an in vitro model system, JB6 mouse epidermal cells. In response to epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the promotion-sensitive JB6 P+ cells initiate neoplastic transformation, whereas the promotion-resistant JB6 P- cells do not. JB6 P- cells expressed much higher levels of GSK3beta than JB6 P+ cells; JB7 cells, the transformed derivatives of JB6, had the least amount of GSK3beta. The activity of GSK3beta is negatively regulated by its phosphorylation at Ser9. EGF and TPA induced strong Ser9 phoshorylation in JB6 P+ cells, but phosphorylation was seen at a much lesser extent in JB6 P- cells. EGF and TPA-stimulated Ser9 phosphorylation was mediated by phosphoinositide-3-kinase (PI3K)/Akt and protein kinase C (PKC) pathways. Inhibition of GSK3beta activation significantly stimulated activator protein-1 (AP-1) activity. Overexpression of wild-type (WT) and S9A mutant GSK3beta in JB6 P+ cells suppressed EGF and TPA-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient (K85R) GSK3beta, in contrast, potentiated anchorage-independent growth and drastically enhanced in vivo tumorigenicity. Together, these results indicate that GSK3beta plays an important role in skin tumorigenesis.

Topics: Animals; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Enzyme Activation; Epidermal Growth Factor; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mice, Inbred BALB C; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Protein Kinase C; Signal Transduction; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transfection

An earlier study reported that a common polymorphism in the 5' untranslated region of the epidermal growth factor (EGF) gene is associated with increased risk for cutaneous malignant melanoma (MM) and Breslow thickness. Since then, several independent studies have reported conflicting results that have challenged this hypothesis. However, none of the previous studies examined survival as the primary outcome. We therefore sought to study the association between this polymorphism and survival. One hundred and thirty patients diagnosed with MM with a Breslow thickness of >1.5 mm were included in this study. In our collective, the G/G genotype represented a significant risk factor for both shorter disease-free period (hazard ratio of 2.246, 95% CI: 1.06-4.78, P=0.036) and overall MM-specific survival (hazard ratio of 3.8, 95% CI: 1.5-9.5, P=0.004) compared with the A/A genotype, while the heterozygous A/G genotype showed an intermediate risk. In the present study, we demonstrate for the first time that the EGF A61G polymorphism is associated with survival. Our data suggest that this polymorphism is a potential marker for disease severity that predicts earlier progression of MM.

Topics: 5' Untranslated Regions; Adult; Aged; Epidermal Growth Factor; Female; Genotype; Humans; Male; Melanoma; Middle Aged; Polymorphism, Genetic; Skin Neoplasms

Paxillin is a substrate of the Src tyrosine onco-kinase and is involved in cell transformation, cell spreading, migration, and cancer development mediated through the mitogen-activated protein kinase signaling cascades. Here, we showed that paxillin plays a key role in skin cell transformation induced by epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the mechanism of paxillin's role in cell transformation, we established a paxillin knockdown stably transfected cell line by introducing small interfering RNA-paxillin (si-paxillin). The si-paxillin cells displayed a dramatic suppression of cell proliferation and anchorage-independent cell transformation induced by EGF or TPA compared with si-mock control cells. In si-paxillin cells, decreased activator protein-1 (AP-1)-dependent luciferase activity corresponded with suppressed AP-1 DNA binding activity. Importantly, knockdown of paxillin inhibited EGF- or TPA-induced c-Jun phosphorylation at Ser(63) and Ser(73). Furthermore, total c-Jun protein level was dramatically decreased in si-paxillin cells and was dependent on serum deprivation time. The down-regulation of c-Jun was restored in si-paxillin cells by treatment with the proteasome inhibitor lactacystin but not by the lysosome inhibitor leupeptin. These results clearly provided evidence that paxillin regulates c-Jun protein level and plays a key role in cell transformation most likely through the regulation of c-Jun stability.

Topics: Animals; Base Sequence; Cell Growth Processes; Cell Line; Cell Transformation, Neoplastic; Down-Regulation; Epidermal Growth Factor; MAP Kinase Kinase 4; Mice; Molecular Sequence Data; Paxillin; Phosphorylation; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Serine; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection

The deregulation of the sonic hedgehog (shh) signaling pathway in epidermal keratinocytes is a primary event leading to the formation of basal cell carcinoma (BCC). The mechanisms by which this pathway exerts this effect remain largely undefined. We demonstrate that overexpression of shh in HaCaT keratinocytes grown in organotypic cultures induced a basal cell phenotype, as evidenced by their morphology, trans-epithelial staining of cytokeratin 14, and suprabasalar proliferation. Shh also induced keratinocyte infiltration into the underlying collagen matrix. Constitutive shh expression was associated with increased phosphorylation of the epidermal growth factor receptor (EGFR) as well as jnk and raf. Additionally, levels of c-jun and matrix metalloproteinase-9 (MMP-9) protein were elevated in shh-expressing cells. Inhibition of EGFR activity with either the tyrphostin, AG1478, or blocking receptor-ligand interaction with the monoclonal antibody, C-225, blocked matrix infiltration. In contrast, exogenously supplied EGF significantly augmented the invasiveness of the HaCaT cells. These observations provide insight into the impact of deregulated shh on epidermal homeostasis. The findings further suggest that an intact EGF signaling axis cooperates with shh and is a critical mediator of matrix invasion in a tumor type characterized by disrupted shh.

Topics: Carcinoma, Basal Cell; Cell Line; Epidermal Growth Factor; Extracellular Matrix; Gene Expression; Hedgehog Proteins; Humans; Keratinocytes; Organ Culture Techniques; Phenotype; Signal Transduction; Skin Neoplasms; Trans-Activators; Transfection

Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochemistry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immunoelectron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is heterogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such as an involvement in cell-cell adhesion and tumorigenesis and a potential value in diagnosis of BCC.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Humans; Immunoblotting; Keratinocytes; Microfilament Proteins; Microscopy, Confocal; Microscopy, Immunoelectron; Neoplasms, Glandular and Epithelial; Neuropeptides; Skin; Skin Neoplasms; Transfection

Melanomas are heterogeneous tumours, and differentiation from other melanocytic lesions may cause problems. It may be possible that the distribution and/or colocalization pattern of different markers in the lesions can enable a more accurate diagnosis of melanocytic tumours.. To test this hypothesis, melanocytic naevi, primary melanomas and metastases were investigated.. The distribution and colocalization of markers for proliferation, invasion, angiogenesis and motility of the tumour cells were investigated using antibodies directed against actin, cathepsin B (CatB), transforming growth factor-beta, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen/Ki-67 and basic fibroblast growth factor (FGF-2). In addition, melanoma markers (HMB-45 and Melan-A) and proteins unrelated to melanoma progression [epidermal growth factor (EGF) and cathepsin H] were investigated.. Malignant melanomas tended to express more markers of malignancy compared with melanocytic naevi, and the differences were statistically significant for EGF and actin immunoreactivity: melanocytic naevi displayed clear EGF labelling more often (60% vs. 5%) and melanomas showed more intense actin labelling (70% vs. 0%). HMB-45+ cells to a large extent also stained with antibodies to CatB but not to EGF or actin; EGF-, FGF-2- and VEGF-immunoreactive cells were predominantly HMB-45-. Similar combinations were observed in melanocytic naevi and in melanomas.. Labelling with EGF may improve the differential diagnosis of melanocytic neoplasias. However, we did not detect a clear-cut increase of markers of malignancy in melanoma. Cells expressing multiple malignancy markers were also found in some melanocytic naevi; this may confirm the dormant potential of melanocytic naevi for melanoma development.

Topics: Biomarkers, Tumor; Cell Movement; Cell Proliferation; Diagnosis, Differential; Epidermal Growth Factor; Humans; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Neovascularization, Pathologic; Nevus, Pigmented; Skin Neoplasms

In the present study, we have investigated the possible role of signal transducers and activators of transcription (STATs), particularly Stat3, in mouse skin tumor promotion and multistage carcinogenesis. Stat1, Stat3, and Stat5 were activated in mouse epidermis after treatment with different classes of tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and chrysarobin. In addition, Stat1, Stat3, and Stat5 were constitutively activated in skin tumors generated by the two-stage carcinogenesis regimen using 7,12-dimethylbenz(a)anthracene as initiator and TPA as promoter. Several approaches were used to examine the possible role of epidermal growth factor receptor (EGFR) in modulating Stat3 activity during tumor promotion. In primary cultures of mouse keratinocytes, addition of exogenous EGF led to activation of Stat3 as shown by an elevation in tyrosine phosphorylation and nuclear translocation. In epidermis of transgenic mice expressing transforming growth factor alpha under control of the keratin 14 promoter, Stat3 was constitutively activated. Abrogation of EGFR function in mouse epidermis using an EGFR kinase inhibitor or by overexpressing a dominant negative form of EGFR led to a reduction in Stat3 activation in response to TPA treatment. Immunoprecipitation analyses using lysates from TPA-treated epidermis and skin papillomas showed enhanced interaction between the EGFR and Stat3. Finally, Stat3 deficiency in mouse epidermis significantly reduced the proliferative response after TPA treatment. Collectively, the current results suggest that Stat3 activation may be a critical event during mouse skin tumor promotion, possibly through regulation of keratinocyte proliferation. In addition, Stat3 activation in tumor promoter-treated epidermis and in skin papillomas may occur, at least in part, via interaction with and phosphorylation by the EGFR. Finally, constitutive activation of Stat3 in both papillomas and squamous cell carcinomas suggest a role in both the development of autonomous growth and the progression of epithelial tumors in mouse skin.

Topics: Animals; Carcinogens; Cell Division; Cells, Cultured; DNA-Binding Proteins; Epidermal Growth Factor; Epidermis; ErbB Receptors; Female; Keratinocytes; Mice; Mice, Inbred SENCAR; Mice, Transgenic; Signal Transduction; Skin Neoplasms; STAT3 Transcription Factor; Tetradecanoylphorbol Acetate; Trans-Activators

Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-alpha, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-alpha into MLTK-alpha-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-alpha fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-alpha-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-alpha. Very importantly, MLTK-alpha-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-alpha stably transfected cells. These results are the first to indicate that MLTK-alpha plays a key role in neoplastic cell transformation and cancer development.

Topics: Animals; Base Sequence; Carcinogens; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Nude; Molecular Sequence Data; Phosphorylation; RNA, Small Interfering; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription Factors

The inheritance of a G allele in position 61 in the 5'UTR of the epidermal growth factor (EGF) gene has been reported to increase melanoma susceptibility, a finding we have investigated in this study. The most potent phenotypic risk factor for melanoma is the atypical mole syndrome (AMS) phenotype. Our hypothesis is that the AMS is genetically determined and that nevus genes are also low penetrance melanoma susceptibility genes. We report that the G allele frequencies were the same in 697 healthy women and 380 melanoma cases (OR 0.97, 95% CI 0.8-1.2 p=0.76). We therefore found no evidence that this polymorphism is a melanoma susceptibility gene. Furthermore, we found no evidence that the polymorphism controls the nevus phenotype (nevus number, number atypical nevi or AMS phenotype). We did find some evidence that the G allele may be associated with decreased tumor Breslow thickness (OR 0.5, 95% CI 0.3-0.9) for the A/A genotype versus A/G and G/G combined in tumors of thickness >3.5 vs < or =3.5 mm and may therefore act as a predictor of survival, although this finding is not in accord with the original report. This is the second study to find no association between EGF +61 and melanoma susceptibility.

Topics: 5' Untranslated Regions; Adult; Case-Control Studies; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Melanoma; Middle Aged; Nevus; Penetrance; Polymorphism, Genetic; Skin Neoplasms

A common single nucleotide polymorphism (SNP) in the 5' untranslated region (5'UTR) of the epidermal growth factor (EGF) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case-control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.

Topics: Case-Control Studies; Epidermal Growth Factor; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Melanoma; Melanosis; Nevus; Polymorphism, Genetic; Prognosis; Risk Factors; Skin Neoplasms

To investigate the function of c-Jun during skin development and skin tumor formation, we conditionally inactivated c-jun in the epidermis. Mice lacking c-jun in keratinocytes (c-jun(Deltaep)) develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun(Deltaep) mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, we show that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation.

Topics: Animals; Apoptosis; Carcinogens; Cell Division; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Eyelids; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, jun; Genetic Predisposition to Disease; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Models, Biological; Papilloma; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transgenes

Since Unna's Abtropfung hypothesis, the process of migration of nevus cells in the dermis remains unknown. To investigate its mechanisms, we studied the role of gelatinases in dermal nevus cells obtained from congenital pigmented nevi, which are major actors in the remodeling of basement membrane proteins. Our previous studies have shown that dermal nevus cells express pro-matrix metalloproteinase (MMP)-2 exclusively and cannot return to the dermis when seeded together with keratinocytes on top of the dermis in a skin reconstruction model. To examine why MMP-2 was not in its active form, we used Western blot to study the expression of members of the MMP-2 activation pathway (membrane type 1-MMP and tissue inhibitor of metalloproteinase-2), which proved to be normally expressed. To induce the dermal passage of nevus cells artificially, we also tried to activate gelatinases with phorbol-12-myristate-13-acetate and epidermal growth factor, using epidermis reconstructed with nevus cells. No migration in the dermis could be triggered. We conclude that the absence of active MMP-2 is due to a functional blockade of its activation pathway and may prevent dermal nevus cells from reaching the dermal compartment in skin reconstructs. Furthermore, our findings reinforce the concept that dermal nevus cells originating from congenital nevi are in a quiescent status.

Topics: Adolescent; Cell Line, Tumor; Cell Movement; Cells, Cultured; Child; Child, Preschool; Dermis; Enzyme Activation; Epidermal Growth Factor; Gelatinases; Humans; Infant; Infant, Newborn; Isoenzymes; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Melanoma; Nevus; Nevus, Pigmented; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tissue Inhibitor of Metalloproteinase-2

CMM is the most serious cutaneous malignancy and is increasing in frequency among most Caucasian populations, where the most important risk factor is exposure to UV light. Relatively little is known of the genetic factors that mediate susceptibility to and prognosis in sporadic CMM, although a number of genes have been implicated. A striking association between EGF polymorphism and Breslow thickness of invasive CMM has been reported. We have sought confirmation of this finding in an independent study of 159 patients and 310 controls using TaqMan fluorescence-based genotyping for EGF +61. In our study group, there were no significant differences in EGF genotype frequencies between patients and controls nor was EGF genotype associated with tumour growth phase, stage or mitotic count. However, correlation between EGF genotype and Breslow thickness showed a modestly significant increase in frequency of the EGF (G/G) genotype among tumours >3.5 mm thick (30.0% vs. 9.8%, p = 0.03). In summary, in our group, the EGF +61 polymorphism was not a risk factor for CMM susceptibility, but this polymorphism may play a role in disease progression.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Case-Control Studies; DNA; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Melanoma; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Skin Neoplasms; United Kingdom

Activation of telomerase, which stabilizes the telomere length of chromosomes, is crucial for the continued growth or progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumors. Because epidermal growth factor plays an important role during the tumorigenesis of epithelial tissue, we have now examined the role of epidermal growth factor signaling in regulating telomerase activity using HSC-1 human cutaneous squamous cell carcinoma cells. Treatment of HSC-1 cells with AG 1478, an inhibitor of the epidermal growth factor receptor, or with a neutralizing antibody to the epidermal growth factor receptor, significantly suppressed their telomerase activity, in association with inhibiting their growth. The suppression of telomerase activity was obvious at day 3 and was maximal at day 5 after treatment with AG 1478. The suppression of telomerase activity correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. The expression of c-Myc and of Sp1 proteins, transcription factors for hTERT, were also suppressed by AG 1478 in HSC-1 cells, but the expression of Ets-2 protein, another transcription factor, was not affected. The expression of Mad-1, a competitor of c-Myc, was increased. Inhibition of ERK, Src, or Akt suppressed telomerase activity in HSC-1 cells, but to a lesser extent than did treatment with AG 1478. Serum starvation suppressed telomerase activity, but addition of epidermal growth factor or transforming growth factor alpha did not increase it, indicating the involvement of other epidermal growth factor receptor ligands in the activation of telomerase in HSC-1 cells. These data indicate that blockade of the epidermal growth factor receptor might be effective in inhibiting telomerase activity of squamous cell carcinomas, which would lead to the suppression of tumor growth.

Topics: Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Quinazolines; Repressor Proteins; RNA, Messenger; Skin Neoplasms; Telomerase; Transforming Growth Factor alpha; Tyrphostins

We report 6 cases of pseudoepitheliomatous hyperplasia (PEH) mimicking squamous cell carcinoma in association with an atypical CD30+ dermal infiltrate. Three patients had lymphomatoid papulosis type A, and 3 patients had cutaneous CD30+ lymphoma. All 6 cases showed histologic evidence of PEH with keratinocyte atypia. In 4 cases there was significant atypia to prompt a diagnosis of squamous cell carcinoma. Three of these received treatment with wide local excision and 2 had been engrafted. Immunohistochemical staining for epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) showed similar expression in lesional and perilesional skin. Epidermal growth factor receptor (EGFR) expression by the proliferating epithelium was similar to that of the suprabasal adjacent normal epidermis. There was no aberrant expression of EGF, TGF-alpha, and EGFR by atypical lymphocytes. These cases demonstrate that PEH associated with CD30+ lymphoproliferative disease may closely resemble squamous cell carcinoma, thereby leading to inappropriate diagnosis and treatment.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Humans; Immunohistochemistry; Ki-1 Antigen; Lymphoma, T-Cell, Cutaneous; Lymphomatoid Papulosis; Male; Middle Aged; Skin; Skin Neoplasms; Transforming Growth Factors

A 17 kD heparin-binding protein (HBp17) has a biphasic dose-dependent effect on DNA synthesis in 3T3 cells. Maximal stimulation of DNA synthesis occurs at 8 ng/ml HBp17, but a half-maximal inhibition occurs at approximately 500 ng/ml. This inhibition can easily be reversed by addition of 400 pg/ml aFGF or 100 pg/ml bFGF, whereas EGF had no effect. This biphasic action of HBp17 was also seen in human umbilical vein endothelial cells (HUVEC), whereas it was not found in the malignant cell line, A431-AJC. The functional relationship between HBp17 and FGF is discussed.

Topics: 3T3 Cells; Animals; Carrier Proteins; Cell Division; DNA Replication; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Mice; Skin Neoplasms; Tumor Cells, Cultured; Umbilical Veins

The fruit juice of Morinda citrifolia (noni), a plant originally grown in the Hawaiian and Tahitian islands, has long been used by islanders to treat diseases, including cancer. Two novel glycosides, 6-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-glucopyranose and asperulosidic acid, extracted from the juice of noni fruits, were used to examine their effects on 12-O-tedtradecanoylphorbol-13-acetate (TPA)- and epidermal growth factor (EGF)-induced AP-1 transactivation and cell transformation in mouse epidermal JB6 cells. The results indicated that both compounds were effective in suppressing TPA- or EGF-induced cell transformation and associated AP-1 activity. TPA- or EGF-induced phosphorylation of c-Jun, but not extracellular signal-regulated kinases or p38 kinases, was also blocked by the compounds, indicating that c-Jun N-terminal kinases were critical in mediating TPA- or EGF-induced AP-1 activity and subsequent cell transformation in JB6 cells.

Topics: Animals; Anticarcinogenic Agents; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; DNA; Epidermal Growth Factor; Fruit; Glucosides; Glycosides; Mice; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Plants, Medicinal; Proto-Oncogene Proteins c-jun; Rubiaceae; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcriptional Activation

Pseudoepitheliomatous hyperplasia has occasionally been reported in cutaneous T-cell lymphoma (CTCL). This association raises the question of the relationship between epidermal hyperplasia and the lymphomatous infiltrate. Because epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) have been demonstrated to be involved in epidermal proliferation through binding to EGF receptor (EGFr), we tested the hypothesis that these cytokines could be secreted by lymphomatous cells, and induce the overlying pseudoepitheliomatous hyperplasia. The purposes of this study were: (i) to describe the clinical and immunohistological features of pseudoepitheliomatous hyperplasia; (ii) to determine its frequency in a large series of CTCLs; and (iii) to evaluate the expression of EGF, TGF-alpha and EGFr in CTCL with or without pseudoepitheliomatous hyperplasia. Eleven cases of CTCL with pseudoepitheliomatous hyperplasia were collected from a series of 353 cases of cutaneous lymphoma registered from 1990 to 1996. They consisted of eight of 28 (28.5%) CD30+ large T-cell lymphomas and three of 148 (2%) cases of mycosis fungoides. Epidermal expression of EGF, EGFr and TGF-alpha was stronger in CTCL than in control normal human skin. Lymphomatous T cells expressed EGF and TGF-alpha whereas no expression of these cytokines could be detected in cutaneous and nodal B-cell lymphomas, nor in a normal lymph node. In addition, epidermal expression of EGFr was stronger in CTCL with pseudoepitheliomatous hyperplasia than in control cases of CTCL without pseudoepitheliomatous hyperplasia, suggesting that these cytokines, in association with other factors, are probably involved in the epidermal hyperplasia observed in some cases of CTCL.

Topics: Epidermal Growth Factor; Humans; Hyperplasia; Immunohistochemistry; Immunophenotyping; Lymphoma, T-Cell, Cutaneous; Neoplasm Proteins; Skin Neoplasms; Transforming Growth Factor alpha

Recently we showed that the skin cancer preventive effect of silymarin involves inhibition of erbB1 activation. Here we assessed the effect of silymarin on cytoplasmic and nuclear signals employing human epidermoid carcinoma A431 cells. Silymarin treatment of cells resulted in a significant inhibition of mitogen-activated protein kinase (MAPK)/ERK1/2 activation only at lower doses, whereas higher doses activated MAPK/JNK1. These differential responses of silymarin were accompanied by its growth inhibitory and apoptotic cell death effects at low and high doses, respectively. Silymarin-caused growth inhibition was via both G2-M and G1 arrests due to a significant decrease in the kinase activity and protein levels of CDKs and cyclins. In other studies, only low doses of silymarin also showed an induction of Cip1/p21 and Kip1/p27. Together, these results identify distinct signaling pathways for the antiproliferative and apoptotic effects of silymarin and form a basis for developing strategies targeted to ERK and JNK pathways for the prevention and intervention of malignancies by silymarin.

Topics: Anticarcinogenic Agents; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cyclin-Dependent Kinases; Enzyme Activation; Epidermal Growth Factor; Humans; Signal Transduction; Silymarin; Skin Neoplasms; Tumor Cells, Cultured

We set out to investigate the interactions between malignant transformation of keratinocytes, presence of oncoproteins and immunosurveillance in squamous cell carcinoma (SCC) and in a preneoplastic lesion, actinic keratosis (AK).. Samples of SCC, AK and normal skin (NS) were subjected to quantitative analysis using the following antibodies: anti-p53, Ki67, OKT6, OK-DR, B7/BB1, anti-CD54, anti-CD11, OKT3, OKT4, OKT8; positivity for ras-p21, EGFr and bcl-2 was evaluated by semiquantitative analysis.. Oncoprotein alterations and increased keratinocyte proliferative activity were observed both in AK and SCC. The number of Langerhans cells (CD1a+ cells) was similar in the two lesions but lower in SCC compared to AK. The proportion of CD1a(+)-B7/BB1+ cells was slightly higher in AK and SCC than in NS. The Langerhans cells expressed the HLA-DR antigen in all groups. Values were highest in AK and NS, and quite low in SCC. Cytotoxic T lymphocytes were more numerous in SCC than in AK and NS. Interestingly, the total CD4/CD8 ratio was much lower in SCC than in AK and NS, which indicates an increase in the CD8+ subpopulation in samples of SCC. In the epithelia of SCC samples there were a considerable number of B7/BB1+ keratinocytes.. We suggest that alterations in the immunodefence mechanisms have an important role in the transformation of AK into SCC, and that these changes affect not only lymphocytes, but also professional (i.e., Langerhans cells) and non-professional (i.e., keratinocytes) antigen presenting cells.

Topics: Aged; Analysis of Variance; Biopsy, Needle; Carcinoma, Squamous Cell; CD3 Complex; CD4 Antigens; CD8 Antigens; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Female; HLA-DR Antigens; Humans; Immunohistochemistry; Intercellular Adhesion Molecule-1; Keratinocytes; Keratosis; Male; Middle Aged; Oncogene Proteins; Precancerous Conditions; Skin Neoplasms

Enhanced tyrosine kinase activity due to aberrant or overexpression of receptor and/or non-receptor tyrosine kinases has been implicated in a variety of human malignancies including cutaneous neoplasms. Epidermal growth factor receptor (EGFR)-mediated tyrosine phosphorylation may be a primary indicator of signal transduction regulating cell growth and proliferation. Recent studies have shown that skin tumor promoters such as phorbol ester and ultraviolet B radiation activate EGFR in mouse skin as well as in cell culture. Similarly, oxidative stress, which is implicated in skin tumor promotion, also activates EGFR-mediated cell signaling. Since this signaling pathway has been suggested to be involved in skin tumor promotion, its impairment by antioxidants may lead to an efficient way for skin cancer prevention and therapy. Recently, we showed that silymarin, a flavonoid antioxidant, affords exceptionally high to complete protection against several skin tumor promoters caused tumor promotion in mouse skin. Employing human epidermoid carcinoma cells A431 that contain overexpressed EGFR, in this study, we assessed whether the anti-skin tumor promoting effects of silymarin are due to its inhibitory effect on EGFR activation and down stream signaling pathway leading to perturbations in cell cycle progression. Treatment of cells with silymarin resulted in a significant inhibition of ligand-induced activation of EGFR with no change in its protein levels. Silymarin treatment also resulted in a significant decrease in tyrosine phosphorylation of Shc, an immediate downstream target of EGFR, but no change in the protein levels of Shc. The inhibition of EGFR activation by silymarin was associated with a highly significant to complete inhibition of EGFR intrinsic kinase activity. Cells treated with silymarin also showed a significant G2-M arrest in cell cycle progression, and a highly significant inhibition of DNA synthesis and cell growth in a dose-dependent manner. Taken together, these results suggest that skin cancer chemopreventive effects of silymarin are mediated via impairment of EGFR signaling which ultimately leads to perturbation in cell cycle progression resulting in the inhibition of proliferation and induction of growth arrest.

Topics: Animals; Antioxidants; Carcinogens; Cell Cycle; Cell Division; DNA; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Neoplasms, Radiation-Induced; Oxidative Stress; Receptor Protein-Tyrosine Kinases; Signal Transduction; Silymarin; Skin Neoplasms; Tumor Cells, Cultured; Ultraviolet Rays

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Amphiregulin; Animals; Betacellulin; Carcinogens; Carcinoma, Squamous Cell; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Ligands; Mice; Mice, Inbred SENCAR; Papilloma; RNA, Messenger; Skin; Skin Neoplasms; Transforming Growth Factor alpha; Up-Regulation

Although the nuclear vitamin D receptor (VDR) is involved in the control of keratinocyte proliferation and differentiation by its ligand 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], its role in epidermal physiology remains poorly understood. Because VDR abundance reflects cellular responsiveness to 1,25(OH)2D3, we investigated VDR expression in cultured human keratinocytes and identified cell anchorage and cytoskeletal integrity as essential requirements for the maintenance of VDR levels. Suspension culture rapidly suppressed VDR expression and 1,25(OH)2D3 responsiveness (as estimated by induction of 24-hydroxylase mRNA), due to decreased transcription of the VDR gene. Concomitantly, overt growth arrest with p21WAF1 induction and cyclin D1 and c-myc suppression occurred, together with induction of differentiation markers and retinoid X receptor alpha, the heterodimeric partner for VDR. Reattachment of suspended keratinocytes to fibronectin led to a rapid restoration of VDR expression, which could be blocked by RGD peptides or a blocking anti-beta1 integrin antibody. VDR expression was also reduced by disruption of the actin cytoskeleton with cytochalasin D. Malignant keratinocytes (SCC12B2 and A431), characterized by, anchorage-independent growth, displayed a profound resistance to suspension-induced suppression of VDR, cyclin D1, and c-myc. Taken together, our results associate VDR expression [and 1,25(OH)2D3 responsiveness] with cell adhesion and an organized cytoskeleton, which are also required for cell growth of primary cells.

Topics: Actins; Calcitriol; Cell Adhesion; Cell Differentiation; Cell Division; Child, Preschool; Cytoskeleton; Epidermal Growth Factor; Fibronectins; Humans; Infant; Infant, Newborn; Keratinocytes; Male; Receptors, Calcitriol; Skin Neoplasms; Skin Physiological Phenomena; Transforming Growth Factor beta

Wa-1 mutant mice possess a defect in the production of transforming growth factor-alpha (TGF-alpha) that leads to a phenotype characterized by wavy hair and curly whiskers. In light of recent evidence indicating the importance of TGF-alpha in epithelial tumorigenesis, this study characterizes the responsiveness of wa-1 mice to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). The responsiveness of wa-1 mice to TPA was compared with that of SENCAR and C57BL/6 mice, representing mouse lines highly sensitive and resistant to skin tumor promotion, respectively. Wa-1 mice were found to be very resistant to skin tumor promotion by TPA after initiation with 10 nmol DMBA, similar to C57BL/6 mice. TPA failed to induce a dramatic increase in TGF-alpha mRNA and protein in the skin of wa-1 mice, whereas TGF-alpha mRNA and protein were dramatically induced in the skin (both epidermis and dermis) of SENCAR and C57BL/6 mice. TPA treatment dramatically increased mRNA levels of two other EGF receptor ligands, amphiregulin and heparin binding-EGF, however, in the skin of all three mouse lines. Comparison of histologic changes in skin revealed that wa-1 mice exhibited only modest sustained epidermal hyperplasia after multiple treatments with TPA, similar in magnitude to that of C57BL/6 mice and significantly lower than that of SENCAR mice. The current data indicate that wa-1 mice are relatively resistant to TPA promotion. Possible mechanisms for this resistance are discussed.

Topics: Amphiregulin; Animals; Antineoplastic Agents; Carcinogens; EGF Family of Proteins; Epidermal Growth Factor; Female; Glycoproteins; Growth Substances; Heparin; Heparin-binding EGF-like Growth Factor; Hyperplasia; Intercellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred SENCAR; Mice, Mutant Strains; Neutrophils; RNA, Messenger; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

In recent work we showed that the EGF receptor (EGFr) was activated in tumor promoter treated mouse epidermis (Cell Growth & Differentiation, 6: 1447-1455, 1995). In the present study, we have investigated the possible role of other erbB family members in the process of tumor promotion. Both erbB2 and erbB3, but not erbB4, were expressed in cultured mouse keratinocytes and in mouse epidermis in vivo. In cultured mouse keratinocytes, EGF stimulated rapid tyrosine phosphorylation of erbB2 followed by a time-dependent degradation of erbB2 protein. Furthermore, an increase in erbB2:EGFr heterodimer formation was also induced by EGF. In contrast to the results with erbB2, EGF did not induce tyrosine phosphorylation, the degradation of erbB3, or erbB3:EGFr heterodimer formation in cultured keratinocytes. Further analyses revealed that c-src kinase activity was dramatically elevated in cultured mouse keratinocytes exposed to EGF. In mouse epidermis following multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), the phosphotyrosine content of erbB2 was significantly elevated in a dose-dependent manner. Concomittantly, erbB2:EGFr heterodimer formation and c-src kinase activity were also elevated in TPA-treated epidermis. Structure-activity relationships with several phorbol ester analogs showed that the elevated phosphorylation of erbB2 in mouse epidermis followed closely with tumor promoting ability. Activation of erbB2 and c-src kinase were also observed in the epidermis of TGF alpha transgenic mice where expression of human TGF alpha was targeted to basal keratinocytes with the human K14 promoter. Collectively, the current data suggest that the activation of erbB2 in phorbol ester treated skin can be explained solely by a mechanism involving elevation of EGFr ligands and activation of the EGFr. In addition, activation of c-src may be an important downstream effector in mouse keratinocytes both in vivo and in vitro, following activation of the EGFr, erbB2, or both.

Topics: Animals; Carcinogens; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; Epidermis; ErbB Receptors; Female; Keratinocytes; Mice; Mice, Transgenic; Phorbol Esters; Phosphorylation; Phosphotyrosine; Proto-Oncogene Proteins pp60(c-src); Receptor Aggregation; Receptor, ErbB-2; Signal Transduction; Skin Neoplasms; Transforming Growth Factor alpha

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12-dimethylbenz[a]anthracene-initiated, 12-O-tetradecanoylphorbol-13-acetate-promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor-beta1 (TGFbeta1) were strongly expressed in the suprabasal layers, whereas integrin alpha6beta4 was expressed only in basal cells and only moderate staining for transforming growth factor-alpha (TGFalpha) was seen. In hyperplastic skin, TGFalpha expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin-binding EGF-like growth factor (HB-EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap-junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of alpha6beta4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20-30 wk), focal areas of gamma-glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFbeta1 expression began to decrease. Cx26 and TGFalpha staining became patchier in some late-stage papillomas (30-40 wk), whereas suprabasal alpha6beta4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFalpha, HB-EGF, TGFbeta1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for alpha6beta4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antigens, Surface; Carcinogens; Connexin 26; Connexins; Cyclin D1; Cyclins; Epidermal Growth Factor; Female; gamma-Glutamyltransferase; Heparin-binding EGF-like Growth Factor; Integrin alpha6beta4; Integrins; Intercellular Signaling Peptides and Proteins; Keratins; Mice; Mice, Inbred SENCAR; Neoplasm Proteins; Oncogene Proteins; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26-41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers.

Topics: Amino Acid Sequence; Antibodies; Blotting, Western; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Infant; Infant, Newborn; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Scalp; Skin; Skin Neoplasms

Various types of tumors show aberrant expression and overexpression of epidermal growth factor (EGF) receptor and the degree of receptor expression correlates with a malignant phenotype in many epithelial tumors. However, in vitro evidence supporting the advantageous role of receptor overexpression is deficient. In this study, we compared the effects of exogenous EGF on the cell colony morphology in monolayer and collagen gel culture between HSC-1 squamous carcinoma cells overexpressing EGF receptor and their revertant subline cells. These cells formed coherent cell colonies under routine culture conditions, but addition of EGF induced dissociation of cell colonies within 24 h in the parent HSC-1 cells, though not in the subline cells. Since the colony dissociation apparently involved loss of cell-cell adhesion, we also studied the effects of EGF on E-cadherin expression and its function. Cell aggregation assays showed that EGF reduced E-cadherin function dose-dependently in the parent cells, but not in the subline cells. However, immunoblotting analysis and ELISA showed the absence of downregulation or degradation of E-cadherin. Instead, EGF tyrosine phosphorylated cadherin/catenin complex components including beta-catenin and increased the detergent solubility of E-cadherin in the parent cells. These results suggest that EGF modified the functional association between E-cadherin and actin filament through tyrosine phosphorylation of the cadherin/catenin complex and thereby made the adhesion molecule incompetent. Our results indicate that the ligand activation of overexpressed EGF receptor impairs E-cadherin-mediated cell-cell adhesion and causes dissociation of the squamous carcinoma cell colonies, which facilitates tumor cell invasion in vivo. This might be relevant to the advantageous role of EGF receptor overexpression in malignant phenotype of epithelial tumor cells.

Topics: beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Size; Collagen; Cytoskeletal Proteins; Detergents; Epidermal Growth Factor; ErbB Receptors; Humans; Octoxynol; Phosphorylation; Skin Neoplasms; Solubility; Trans-Activators; Tumor Cells, Cultured; Tyrosine

Some human neoplasms show aberrant expression or overexpression of epidermal growth factor (EGF) receptor, and the degree of the receptor expression is correlated with the malignant phenotype in certain epithelial tumors including squamous carcinoma cells. Since phenotypic transformation of cells could involve quantitative and qualitative alteration of integrin function, the effects of EGF on cell-matrix interactions were studied using HSC-1 cells, a human squamous carcinoma cell line showing EGF receptor overexpression. The EGF-treated HSC-1 cells interacted with matrix proteins differently from the untreated cells, as shown by cell adhesion and phagokinetic track assays. Among fibronectin, laminin, fibrinogen, and type I collagen, fibronectin was the most efficient substratum to promote untreated HSC-1 cell adhesion and migration. Pretreatment of the cells with 50 ng/ml EGF for 18 h selectively increased the number of spread cells and the size of the individual cell migration area on type I collagen by 250 and 400%, respectively. The same pretreatment diminished cell adhesion and migration on other substrata so that the EGF treatment converted type I collagen as the most efficient substratum for cell adhesion and migration of the HSC-1 cells. ELISA and immunoprecipitation studies showed that EGF up-regulated the expression of alpha 2 beta 1 integrin collagen receptor in a time- and dose-dependent manner by stimulating biosynthesis of alpha 2 subunit, but did not up-regulate those of the alpha 3 beta 1, alpha 5 beta 1, or alpha v beta 3 integrins. These results suggest that EGF preferentially enhances HSC-1 cell interaction with type I collagen, leading to the enhanced cellular migratory activity on the substratum, as a result of selective up-regulation of alpha 2 beta 1 integrin expression.

Topics: Antigens, CD; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Adhesion; Cell Movement; Collagen; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Integrins; Oligopeptides; Skin Neoplasms; Tumor Cells, Cultured; Up-Regulation

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of A431 cells, leading to a approximately 30% rise in cellular filamentous actin levels. EGF-induced actin polymerization depends upon EGF receptor (EGFR) tyrosine kinase activity, since the selective tyrosine kinase inhibitor AG213 abolishes EGF-induced actin polymerization. In accordance, confocal laser scanning microscopy shows that newly assembled actin filaments localize selectively to the tyrosine-phosphorylated EGFR in the plasma membrane, since actin polymerization is not observed at the internalized tyrosine-phosphorylated EGFR. Actin binding proteins (ABP's) are generally believed to regulate actin filament assembly. Ca2+ is known as one of the important regulatory factors for the activity of ABP's in vitro [15]. Therefore, we investigated the importance of the EGF-induced transient rise in [Ca2+]i for the regulation of actin polymerization in vivo. Continuous high [Ca2+]i in the millimolar range induces a prominent rise in cellular filamentous actin levels to approximately 50% over control cells. However, actin polymerization is unimpaired under conditions which effectively block the EGF-induced [Ca2+]i transient. These data demonstrate that EGF-induced actin polymerization localizes to the activated EGFR in the membrane skeleton, independent of the cytosolic free calcium transient.

Topics: Actins; Adenosine Triphosphate; Calcimycin; Calcium; Carcinoma, Squamous Cell; Catechols; Cell Line; Cell Membrane; Cytosol; Egtazic Acid; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Kinetics; Macromolecular Substances; Microscopy, Confocal; Nitriles; Phosphorylation; Skin Neoplasms; Time Factors; Tumor Cells, Cultured; Tyrphostins

Topics: Abdominal Neoplasms; Adenocarcinoma, Scirrhous; Adult; Carcinoembryonic Antigen; Epidermal Growth Factor; ErbB Receptors; Fatal Outcome; Humans; Male; Skin Neoplasms; Stomach Neoplasms; Umbilicus

Photodynamic therapy combines photosensitizers absorbing light in the red spectral region and irradiation with light of the corresponding wavelength. To analyse the influence of cell differentiation on susceptibility to photodynamic therapy, we compared the proliferation inhibition induced by photodynamic therapy on normal human keratinocytes, spontaneously transformed human keratinocyte cell line HaCat and on squamous cell carcinoma lines. Cells were irradiated with polychromatic methylene blue as well as the precursor of protoporphyrin, 5-aminolevulinic acid. When incubated with Photosan-3, normal human keratinocytes exhibited and ED50 about 10-fold lower than the other cell lines studied. When methylene blue and 5-aminolevulinic acid were used, photodynamic therapy had comparable effects on all cell types. Stimulation of normal human keratinocytes with either EGF-alpha or IFN-gamma resulted in an increase susceptibility to photodynamic therapy when 5-aminolevulinic acid was used. This effect was more pronounced in the case of EGF-alpha. Our experiments suggest that activation and differentiation are two important parameters determining susceptibility to photodynamic therapy.

Topics: Aminolevulinic Acid; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line, Transformed; Dose-Response Relationship, Drug; Epidermal Growth Factor; Hematoporphyrins; Humans; Interferon-gamma; Keratinocytes; Methylene Blue; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms; Tumor Cells, Cultured

Calcyclin is the product of a gene that is regulated in dependence of the cell cycle in fibroblasts in vitro. It has recently been proven to be a sialic acid-binding protein in vitro and in the case of mammalian tissues to bind specifically to annexin II, annexin VI, annexin XI, and glyceraldehyde-3-phosphate dehydrogenase in a Ca(2+)-dependent manner. Since calcyclin can be labelled without impairment of its binding activity, it can be employed as a histochemical tool to localize its accessible ligands. Concomitantly, immunohistochemical localization of calcyclin with a specific antibody is warranted. By using histochemical and immunohistochemical techniques, the expression of calcyclin and its accessible binding sites are demonstrated in serial sections of normal skin and benign, pre-cancerous and malignant tumors of the skin, namely in verruca vulgaris, papillary hidradenoma, syringoma, keratoacanthoma, Bowen's disease, squamous cell carcinoma, melanocytic naevi, primary and metastatic malignant melanoma and non-Hodgkin lymphoma (NHL) of the skin. Cytoplasmic and nuclear expression of calcyclin and its ligands is unexceptionally found in normal skin, epithelial tumors and benign melanocytic tumors. Presence of calcyclin and calcyclin-binding sites is detected in more than 80% of tumor cells in the epithelial lesions. In the group of melanomas and lymphomas heterogeneity is obvious. The application of annexin-specific antibodies raises evidence that members of this protein family co-localize with calcyclin in situ to some extent. These findings suggest that calcyclin and accessible calcyclin-binding molecules, like certain annexins, may be differentially regulated in melanomas and lymphomas in contrast to epithelial lesions with presently undefined biological implications.

Topics: Annexins; Calcium-Binding Proteins; Cell Cycle Proteins; Epidermal Growth Factor; Humans; Immunohistochemistry; Ligands; Precancerous Conditions; Reference Values; S100 Calcium Binding Protein A6; S100 Proteins; Skin; Skin Diseases; Skin Neoplasms; Tissue Distribution

To assess the effects of transforming growth factor alpha (TGF-alpha) on mammalian skin in vivo, we have targeted its expression to the epidermis of transgenic mice using a vector based on the human K1 (HK1) gene. Neonatal mice expressing the HK1.TGF-alpha transgene were often smaller than normal littermates and had precocious eyelid opening and wrinkled, scaly skin with diffuse alopecia. Juvenile transgenic mouse epidermis was uniformly hyperkeratotic, but this pattern was generally less pronounced in adult transgenic mice unless they expressed high levels of the HK1.TGF-alpha transgene. Spontaneous, squamous papillomas occurred at sites of wounding in adult mice expressing high levels of HK1.TGF-alpha; however, most were prone to regression. Immunoreactive TGF-alpha was 2-6 times higher in the epidermis of these HK1.TGF-alpha lines. Immunoreactive epidermal growth factor receptor had a normal pattern of expression in nonphenotypic adult epidermis, but a marked reduction in the receptor population was detected in hyperplastic newborn epidermis and phenotypic adult epidermis. Autoradiographic localization of 125I-epidermal growth factor showed a similar pattern of distribution, suggesting that the sites of increased TGF-alpha expression induced epidermal growth factor receptor down-regulation. These data demonstrate the in vivo effect of deregulated TGF-alpha expression on epidermal proliferation and differentiation and suggest a potential role for TGF-alpha in carcinogenesis and other hyperproliferative epidermal disorders.

Topics: Animals; Base Sequence; Cell Division; Epidermal Growth Factor; Epidermis; ErbB Receptors; Hyperplasia; Keratosis; Mice; Mice, Transgenic; Molecular Sequence Data; Papilloma; Skin Neoplasms; Transforming Growth Factor alpha

The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

Topics: Animals; Conjunctiva; Epidermal Growth Factor; Fibroma Virus, Rabbit; Genome, Viral; Growth Substances; Hyperplasia; Intercellular Signaling Peptides and Proteins; Mutagenesis, Insertional; Myxoma virus; Myxomatosis, Infectious; Peptides; Rabbits; Skin Neoplasms; Transforming Growth Factor alpha; Tumor Virus Infections; Vaccinia virus

Exogenous administration of epidermal growth factor (EGF) enhances tumor growth of H2T, a hamster pancreatic cancer that is inoculated in the cheek pouch. The effect of endogenous EGF on tumor growth, however, is not known. The main source of EGF in the body is the submandibular glands. This study examined the influence of submandibular gland resection (SMx) on H2T tumor growth in the cheek pouch and compared it to the growth in SC tissue. Bilateral SMx or sham operation was performed on male Syrian golden hamsters. In 30 hamsters (n = 15 for each operation group), 5 x 10(4) and 5 x 10(5) H2T cells were inoculated in the bilateral cheek pouches and intrascapular SC tissue, respectively (Exp. 1). In another 30 hamsters (n = 15 for each operation group), 5 x 10(5) and 1 x 10(6) H2T cells were inoculated at the same sites (Exp. 2). Two longest perpendicular diameters of the tumor were measured once a week for 12 wk (Exp. 1) or 8 wk (Exp. 2), and the tumor area was calculated. The tumor area in the cheek pouch became significantly smaller in the SMx group than the sham-operated group after the 8th wk (Exp. 1) or the 7th wk (Exp. 2). On the other hand, the tumor area in the SC tissue did not show any difference between groups through the experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cheek; Cricetinae; Epidermal Growth Factor; Male; Mesocricetus; Mouth Neoplasms; Neoplasm Transplantation; Pancreatic Neoplasms; Skin Neoplasms; Submandibular Gland

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.

Topics: Animals; Carcinoma, Squamous Cell; Cell Nucleus; Epidermal Growth Factor; Humans; Interphase; Liver; Liver Neoplasms, Experimental; Melanoma, Experimental; Mitosis; Mouth Neoplasms; Neoplasms, Experimental; Neutral Red; Rats; Skin Neoplasms; Tumor Cells, Cultured

Staurosporine is a potent but nonselective inhibitor of protein kinase C (PKC) and blocks responses to 12-O-tetradecanoylphorbol-13-acetate (TPA) in several cell types in vitro. In cultured primary mouse keratinocytes, however, staurosporine fails to inhibit TPA-mediated keratinocyte maturation and itself elicits responses that are similar to TPA (T. Sako et al., Cancer Res., 48: 4646-4650, 1988). After exposure to 10 nM staurosporine for 24 h, essentially all keratinocytes undergo morphological differentiation, whereas 160 nM TPA induces this response in about 50% of epidermal cells. These concentrations of staurosporine and TPA cause a 4-5-fold induction of epidermal transglutaminase activity and cornified envelopes, both markers of the terminal stage of keratinocyte differentiation. Staurosporine, but not TPA, also induces morphological and biochemical maturation in 2 neoplastic mouse keratinocyte cell lines, 308 and SP-1. The ability of staurosporine to elicit the same responses as TPA suggested that it may be functioning paradoxically as a PKC agonist in intact keratinocytes. In support of this hypothesis, staurosporine induces ornithine decarboxylase activity, inhibits 125I-labeled epidermal growth factor binding, and induces expression of c-fos mRNA. Down-regulation of PKC by pretreatment of primary keratinocytes with 60 nM bryostatin partially blocks staurosporine-mediated induction of cornified envelopes and inhibition of 125I-labeled epidermal growth factor binding, implicating PKC in these responses. The ability of staurosporine to mimic and/or enhance certain responses to TPA suggests that this agent is acting as a functional PKC agonist in cultured keratinocytes.

Topics: Alkaloids; Animals; Carcinogenicity Tests; Cell Differentiation; Dimethyl Sulfoxide; Enzyme Induction; Epidermal Growth Factor; Keratinocytes; Mice; Mice, Inbred BALB C; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA, Messenger; Skin Neoplasms; Staurosporine; Tetradecanoylphorbol Acetate; Transglutaminases; Tumor Cells, Cultured

The psoralen analogs 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP), in combination with ultraviolet light (UVA, 320-400 nm), are potent modulators of epidermal cell growth and differentiation and are commonly used in photochemotherapy of psoriasis and vitiligo. We have used KB cells, a human epithelial cell line, to examine the mechanism of action of these compounds. In KB cells, 8-MOP was found to bind to specific, saturable receptor sites. Binding of [3H]-8-MOP to its receptor was inhibited by TMP as well as psoralen. We found that binding of these analogs to the cells followed by UVA light treatment was associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding was temperature dependent, occurred immediately following UVA light exposure, and appeared to be due to a decrease in the number of EGF receptors. In KB cells, 125I-labeled EGF surface receptor binding is followed by its rapid internalization and degradation. We found that photoactivated psoralens also inhibited internalization of 125I-EGF, but had no apparent effect on EGF metabolism. These data indicate that the cell surface membrane may be an important target for the photoactivated psoralens. In addition, since photoactivated psoralens regulate cell proliferation, the interaction of these compounds with EGF receptor function may underlie their biological activity.

Topics: Binding, Competitive; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Methoxsalen; PUVA Therapy; Receptors, Cell Surface; Skin Neoplasms; Temperature; Trioxsalen

The presence of EGF-autocrine secretion was investigated in a cell line derived from a human squamous cell carcinoma (HSC-1). The cell line was found to secrete the epidermal growth factor (EGF)-like substance. Meanwhile, normal human and uninvolved and involved psoriatic epidermal cells did not show any evidence of EGF secretion in culture. Not was any evidence of EGF secretion observed in vitro in several malignant cell lines other than HSC-1 derived from human squamous cell carcinomas, adenocarcinoma and melanoma. The addition of EGF did not stimulate, but rather inhibited, the growth of HSC-1 cells in GIT medium as well as Dulbecco's modified essential medium with low concentrations of fetal calf serum (0.5-1%) in vitro. Overexpression of EGF receptors is known to occur in HSC-1. The results suggest that HSC-1 cells exhibit autocrine secretion of the EGF-like substance but not autostimulation in anchorage-dependent cell culture.

Topics: Carcinoma, Squamous Cell; Cell Division; Culture Media; Epidermal Growth Factor; Humans; Reference Values; Skin; Skin Neoplasms; Time Factors; Tumor Cells, Cultured

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin decreased 125I-labeled epidermal growth factor (EGF) binding in epidermal membrane preparations within 1 h while 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) gradually reduced binding with maximum inhibition at 15 h. Subsequently, 125I-EGF binding increased to approximately 200% of control in epidermal membrane preparations from both TPA- and chrysarobin-treated mice. A single application of TPA but not chrysarobin resulted in a rapid translocation of protein kinase C (PKC) to the membrane; however, treatment with both promoters ultimately led to a time-dependent loss of PKC activity in both membrane and cytosol fractions. The initial inhibition of 125I-EGF binding was sustained for at least 24 h after single and multiple treatments with both promoting agents. Acid washing restored EGF binding to control levels in membrane preparations obtained 24 h after a single application, whereas acid washing of membrane preparations obtained 24 h after a second application of TPA or chrysarobin increased binding (2.5-fold and 1.5-fold that of the control, respectively). The presence of increased amounts of ligands for the EGF receptor in tumor promoter-treated epidermis was initially confirmed in 125I-EGF binding competition experiments using NRK-49F cells. A single topical application of TPA or chrysarobin induced elevated levels of transforming growth factor-alpha (TGF-alpha) mRNA at 6 h or 15-24 h, respectively. Elevated levels of a TGF-alpha precursor (21 kDa) were subsequently observed in cytosol and membrane preparations after single and multiple applications of TPA or chrysarobin. These results suggest that repeated topical application of tumor promoters may lead to sustained loss of a negative-feedback mechanism involving phosphorylation at Thr-654 of the EGF receptor by PKC. The concomitant elevation of ligands, such as TGF-alpha, may provide a mechanism for sustained cell proliferation essential for skin tumor promotion.

Topics: Animals; Anthracenes; Binding, Competitive; Carcinogens; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Mice; Protein Kinase C; RNA, Messenger; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

Bryostatin 1, a macrocyclic lactone, functions like the phorbol esters biochemically in binding to and activating protein kinase C. Biologically, however, although it induces some phorbol ester responses such as mitogenesis in Swiss 3T3 cells, it paradoxically blocks the effects of the phorbol esters on differentiation in HL-60 promyelocytic leukemia cells and Friend erythroleukemia cells. Since the phorbol esters induce proliferation and terminal differentiation in distinct subpopulations of epidermal basal cells, we have now examined the action of bryostatin 1 in that system. Bryostatin 1 decreased epidermal growth factor binding and induced ornithine decarboxylase activity, the latter a marker of proliferation. The magnitude of the maximal induction of ornithine decarboxylase was less than for phorbol 12,13-dibutyrate. Bryostatin 1 only transiently caused the morphological change typical of phorbol ester treatment and did not induce transglutaminase or cornified envelope production, markers of the differentiative pathway. Combined treatment with bryostatin 1 and phorbol 12,13-dibutyrate gave similar results to treatment with bryostatin 1 alone, i.e., slight reduction to complete inhibition of phorbol ester action, depending on the response. The mechanism may reflect time dependent block of the protein kinase C pathway by bryostatin 1 in this system; although bryostatin 1 inhibited epidermal growth factor binding at short incubation times (1-2 h), by 4 h of incubation its inhibition was markedly reduced and it correspondingly blocked inhibition of epidermal growth factor binding by phorbol 12,13-dibutyrate. Since induction of terminal differentiation is proposed to be an essential component of phorbol ester mediated tumor promotion in skin, our findings suggest that bryostatin 1 may function as an inhibitor of phorbol ester promotion.

Topics: Animals; Bryostatins; Cells, Cultured; Cycloheximide; Dactinomycin; Enzyme Activation; Epidermal Growth Factor; Epidermis; Lactones; Macrolides; Mice; Ornithine Decarboxylase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transglutaminases

Several tumor cells secrete significantly increased amounts of the plasminogen activator urokinase, a trypsinlike serine protease, whose biological function in tumor biology is unclear. In this study we report that cells of the human epidermal tumor cell line CCL 20.2 express about 80,000 high-affinity urokinase receptors per cell that bind active as well as diisopropylfluorophosphate-treated high-molecular-weight (HMW) urokinase. Low-molecular-weight (LMW) urokinase is not bound to the receptor. Occupation of these receptors by active HMW urokinase stimulates cell proliferation independently in the presence of plasminogen in the culture medium. LMW urokinase has again no effect on cell proliferation. Calculated on a molar basis, this effect is about 28% of that of epidermal growth factor. Active HMW urokinase might therefore provide an autocrine receptor-mediated growth-promoting mechanism for tumor cells similar to those described for other growth factors.

Topics: Cell Division; Cell Line; Epidermal Growth Factor; Humans; Plasminogen; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Skin Neoplasms; Urokinase-Type Plasminogen Activator

Epidermal growth factor (EGF) induced the formation of thin sheetlike extensions (lamellipodia) and filamentous extensions at the edges of colonies of A431 cells. To determine the necessary processes for the induction of the morphological changes mediated by EGF, the effects of a variety of ions on these changes were examined. In a NaCl solution supplemented with CaCl2, MgCl2 and glucose, no EGF-induced morphological changes were observed. However, when the NaCl was replaced with LiCl, fingerlike extensions were formed, but sheetlike extensions were not. Addition of vanadate to the NaCl solution also induced fingerlike extensions in cells treated with EGF. In contrast, sheetlike lamellipodia were formed in EGF-treated cells by the addition of K+ or PO4(3-) to the NaCl solution or by the addition of PO4(3-) to the LiCl solution. These findings indicate that Li+, K+, PO4(3-) and vanadate are involved in the processes of EGF-induced morphological changes. Since vanadate and Li+ have been shown to inhibit phosphatases, an EGF-dependent phosphorylation step may play an important role in the induction of the morphological changes observed.

Topics: Carcinoma, Squamous Cell; Cell Line; Cytoplasm; Epidermal Growth Factor; Humans; Inositol; Lithium; Microscopy, Phase-Contrast; Phosphates; Potassium; Skin Neoplasms; Sodium; Vanadium

The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Humans; Mouth Neoplasms; Proto-Oncogenes; Receptors, Cell Surface; Skin Neoplasms

Cowden disease (CD) is a familial syndrome characterized by tumors of the skin, oral mucosa, breast, thyroid, and intestinal epithelium. Since the syndrome is inherited as an autosomal dominant, we examined a battery of gene markers in a family with CD to detect linkage between the CD gene and known marker genes. There was no positive evidence for linkage of a CD locus with any of the markers; other investigators can add to our data to confirm and extend these findings. Additionally, we measured epidermal growth factor (EGF) in body fluids from CD patients and controls to determine if elevated EGF levels might be responsible for the widespread epithelial proliferation in CD. EGF levels in saliva, serum, plasma, and urine were similar in CD patients and control subjects. Although alterations in growth factors or their receptors may play a role in CD, excess circulating EGF is not responsible for the manifestations of the syndrome.

Topics: Adult; Body Fluids; Epidermal Growth Factor; Female; Genetic Markers; Hamartoma; Humans; Lod Score; Male; Neoplasms, Multiple Primary; Skin Neoplasms

Terminal differentiation of normal and malignant keratinocytes is routinely determined by the ability of these cells to form cornified envelopes after incubation with a calcium ionophore. We have used the human squamous cell carcinoma, SqCC/Y1, to quantify cellular differentiation by the formation of detergent-insoluble protein. The methodology developed employs the metabolic labeling of detergent-insoluble cellular protein with [35S]methionine in the presence of a calcium ionophore. The ratio of filter-retainable radioactivity to that of total cellular protein was shown to be closely correlated to the results obtained by measuring the number of envelope-competent cells when cells were induced to enter a pathway of terminal differentiation in culture by serum deprivation or by treatment with hydrocortisone, and during the inhibition of maturation by either retinoic acid (RA) or epidermal growth factor (EGF). This way of measuring the degree of terminal differentiation of epidermal cells is a relatively simple one that readily allows the simultaneous measurement of multiple samples.

Topics: Blood; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Culture Media; Epidermal Growth Factor; Epidermis; Humans; Hydrocortisone; Membrane Proteins; Skin Neoplasms; Solubility; Tretinoin

After a chance observation that multiple cutaneous papillomas and squamous cell carcinomas occurred in 2 adult mice heterozygous for the repeated epilation gene Er, we surveyed a panel of 10 +/+ (wild type) and 30 Er/+ (heterozygous) mice from birth to over 2 years of age. Homozygous Er/Er mice could not be included since their defect is lethal at birth. Whereas no cutaneous tumors developed in the +/+ mice, 20 of the Er/+ mice, males and females, had developed 1-5 cutaneous papillomas and at least 1 cutaneous invasive squamous cell carcinoma by 2 years of age. No lesions were seen in mice younger than 6 months old. Although almost all Er/+ mice died with their tumor burden, no metastases have yet been proven histologically. The Er/+ mouse should serve as a useful model for the exploration of genetic factors in cutaneous squamous cell carcinomas in humans.

Topics: Alopecia; Animals; Antigens, Viral; Carcinoma, Squamous Cell; DNA, Neoplasm; DNA, Viral; Epidermal Growth Factor; Female; Genes, Lethal; Heterozygote; Male; Mice; Mice, Mutant Strains; Neoplasms, Multiple Primary; Papillomaviridae; Rodent Diseases; Skin Neoplasms

Specific binding of 125I-labeled epidermal growth factor (EGF) was measured in 62 skin tumors of different severity. Within a group of 28 benign tumors, 11 of 15 condylomata acuminata were receptor positive, whereas the investigated mesenchymal tumors and normal skin as a control were receptor negative. 6 of 18 basal cell epitheliomas bound EGF specifically. In the group of precancerous and malignant skin tumors, 7 of 8 squamous cell carcinomas had the highest number of EGF binding sites and a high affinity state, whereas 5 malignant melanomas were receptor negative. The clinical relevance of these findings is not yet clear due to the short follow-up of the patients.

Topics: Binding Sites; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Condylomata Acuminata; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Melanoma; Receptors, Cell Surface; Skin; Skin Diseases; Skin Neoplasms

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Child; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Humans; Receptors, Cell Surface; Skin Neoplasms

Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.

Topics: Calcimycin; Cell Division; Cell Line; Epidermal Growth Factor; Humans; Oncogenes; Protein Biosynthesis; Protein Kinase C; Protein Kinases; Proto-Oncogene Mas; Skin Neoplasms; Tetradecanoylphorbol Acetate

beta-All-trans-retinoic acid (RA) inhibited the anchorage-independent growth of JB6 cells induced by either mezerein or alpha-epidermal growth factor (alpha-EGF) (a purified fraction of epidermal growth factor). The inhibition was dose dependent for alpha-EGF as well as for RA. Mezerein-induced growth in soft agar was inhibited to a greater extent by RA than was alpha-EGF-induced growth in soft agar, at similar colony yields. The extent of inhibition of anchorage-dependent growth induced by RA was similar for nontransformed JB6 cells and for alpha-EGF-transformed cells, so that transformation was shown not to influence the sensitivity of cells to retinoid inhibition of anchorage-dependent growth. RA was as effective at inhibiting anchorage-independent growth when it was applied after promoter-induced transformation as when it was applied during promoter-induced transformation. Therefore, the antiproliferative effect of RA, without an additional antitransformation effect, was sufficient to account for the reduced colony yield. These results suggest that the antipromoting action of retinoids in JB6 cells may occur by limiting proliferation, the regulation of which may be coupled with the state of differentiation of cells.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Epidermal Growth Factor; Epidermis; Mice; Skin Neoplasms; Terpenes; Tretinoin

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.

Topics: ADP Ribose Transferases; Antibodies, Monoclonal; Bacterial Toxins; Cell Line; Cell Survival; Diltiazem; Drug Synergism; Epidermal Growth Factor; Exotoxins; Fluorescent Antibody Technique; Humans; Lysosomes; Nifedipine; Pseudomonas aeruginosa Exotoxin A; Receptors, Cell Surface; Receptors, Transferrin; Skin Neoplasms; Verapamil; Virulence Factors

Epidermal growth factor (EGF) interactions with primary epidermal cells in culture were examined in BALB/c and SENCAR mice, strains resistant and sensitive, respectively, to carcinogenesis by initiation-promotion. Using low (less than 0.1 mM) calcium growth conditions, which select for basal cells, and calcium-induced terminal differentiation, we determined the effects of retinoids, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), and cell density on binding of 125I-labeled EGF. Over the range tested, EGF binding increased with increasing cell density similarly in basal cells of both strains, which at similar densities bound similar amounts of EGF. Increasingly differentiated epidermal cells of both strains bound less EGF. Responses of basal and differentiating cells were dissimilar in several respects. Most notably, differentiating cells bound but failed to metabolize EGF. TPA treatment of basal cells from either strain led to a rapid, pronounced reduction in EGF binding, while treatment with retinoic acid, an antipromoter in vivo, increased binding. In contrast, EGF binding by differentiating cells was much less affected by TPA treatment, and retinoic acid had no effect or was slightly inhibitory, while combined treatment was more inhibitory than either alone. Given an adequate plating density, inclusion of 1 ng EGF per ml of media significantly enhanced growth of basal cells. Because of the similarities in binding patterns between the two strains, it seems unlikely that differential responses of BALB/c and SENCAR epidermal cells to EGF and to modulators of EGF binding are involved in the differences in susceptibility of these strains to carcinogenesis.

Topics: Animals; Animals, Newborn; Calcium; Cell Differentiation; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Mice, Inbred Strains; Phorbols; Receptors, Cell Surface; Skin; Skin Neoplasms; Skin Physiological Phenomena; Species Specificity; Tetradecanoylphorbol Acetate

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO#1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 X 10(6) sites/cell) and a low-affinity class (affinity constant = 4,880 nM; 7 X 10(7) sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [3H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [3H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.

Topics: Caenorhabditis elegans Proteins; Carrier Proteins; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Melanoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug; Skin Neoplasms; Tetradecanoylphorbol Acetate

SENCAR mice are markedly more susceptible to two-stage skin carcinogenesis than BALB/c mice. Studies were carried out to elucidate the basis for this sensitivity. It is not related to differences in the metabolism of polycyclic aromatic hydrocarbons (DiGiovanni et al., 1980) but appears to be determined by the target tissue, since when SENCAR skin was grafted onto nude mice they developed papillomas at a high frequency after initiation and promotion, whereas after grafting of BALB/c skin, no tumours developed. DNA repair capacity was studied in SENCAR and BALB/c epidermal cells in culture. Host cell reactivation, utilizing ultra-violet light-irradiated herpes simplex virus, was similar in cells of the two strains. SENCAR cells have a greater binding capacity for epidermal growth factor than BALB/c cells; however, the increased binding in response to retinoic acid and the rapid decrease after exposure to phorbol esters are similar in the two strains. Spontaneous expression of endogenous proviral DNA sequences for xenotropic-type C RNA viruses occurs more readily in BALB/c epidermal cells than in those of SENCAR. The frequency of spontaneous differentiation-resistant foci in vitro (Kulesz-Martin et al., 1980) is greater in SENCAR than in BALB/c epidermal cells. These results suggest that susceptibility for skin carcinogenesis in SENCAR mice is determined by the target tissue itself and has no clear relation to DNA excision repair, endogenous virus complement or epidermal growth factor receptors.

Topics: Animals; Calcium; Cell Differentiation; DNA Repair; Epidermal Growth Factor; Methylnitronitrosoguanidine; Mice; Mice, Mutant Strains; Neoplasms, Experimental; Skin Neoplasms; Virus Replication

Topics: Cell Line; Epidermal Growth Factor; Humans; Kinetics; Molecular Weight; Peptides; Receptors, Drug; Skin Neoplasms

High affinity binding of epidermal growth factor (EGF) was detected with mouse epidermal cells in primary culture and with 2 epidermal cell lines maintained in permanent culture. EGF binding was inhibited by low concentrations of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) and phorbol didecanoate (PDD), but not by the non-promoting phorbol esters 4-O-methyl TPA and 4 alpha-PDD.

Topics: Animals; Binding Sites; Cells, Cultured; Epidermal Growth Factor; Mice; Neoplasms, Experimental; Peptides; Phorbol Esters; Phorbols; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

Topics: Animals; Autoradiography; Bone Marrow Cells; Carcinoma, Squamous Cell; Cell Count; Cell Division; Cells, Cultured; Diffusion; DNA; Epidermal Cells; Epidermal Growth Factor; Epidermis; Growth Inhibitors; Humans; Interferons; Mice; Peptides; Psoriasis; Skin Neoplasms