S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation through GATA‐3/caspase‐14 pathway in A431 cells

S100A7 is expressed in many squamous cell carcinomas (SCCs), such as SCC of the skin, and well‐differentiated SCC always displays stronger staining of this protein. A431 cells, an epidermal cancer cell line, were selected as a cell model to investigate the roles and mechanism of S100A7 in SCC of the skin. In this study, we demonstrated that the overexpression of S100A7 in A431 cells significantly promoted cell proliferation in vitro and tumor growth in vivo, whereas it suppressed the expression of GATA‐3, caspase‐14 and three squamous differentiation markers, keratin‐1, TG‐1 and involucrin. Conversely, the overexpression of caspase‐14 not only significantly decreased cell proliferation and delayed tumor growth but also markedly induced the expression of three squamous differentiation markers, whereas S100A7 and GATA‐3 were not influenced. Further evidence showed that silencing GATA‐3 greatly inhibited the expression of caspase‐14 and three differentiation markers, while the expression of S100A7 was not changed; contrary results were obtained when overexpressing GATA‐3. Importantly, restoring the expression of GATA‐3 and caspase‐14 in A431‐S100A7 cells could bypass the ability of S100A7 to increase cell viability and repress squamous differentiation. These data suggested that S100A7 expression in SCC may play an important role in the maintenance of SCC cell dedifferentiation, at least in SCC of the skin.     

T‐cadherin loss induces an invasive phenotype in human keratinocytes and squamous cell carcinoma (SCC) cells in vitro and is associated with malignant transformation of cutaneous SCC in vivo

Background Cadherins play important roles in controlling keratinocyte growth, differentiation and survival. Atypical glycosylphosphatidylinositol‐anchored T‐cadherin (T‐cad) is highly expressed in the basal keratinocyte layer of skin. The role of T‐cad in keratinocyte biology and pathology is unclear. Objectives To define the role of T‐cad in the pathogenesis of cutaneous squamous cell carcinoma (SCC) through gain‐of‐function and loss‐of‐function studies in vitro and through examination of T‐cad expression patterns in human cutaneous SCC specimens in relation to histological classification of degree of tumour differentiation. Methods In vitro studies employed lentiviral‐mediated overexpression/silencing of T‐cad in normal human keratinocyte (HaCaT) and SCC (A431) cell lines, monolayer and multicellular spheroid culture models, cell morphology analyses and assays of random motility and invasion. Immunohistochemistry was performed on skin specimens from patients with actinic keratosis, Bowen disease or SCC. Results In vitro, silencing of T‐cad induced a morphologically elongated and disorganized cell phenotype, increased random motility and markedly enhanced invasive potential. Overexpression of T‐cad induced a morphologically spread and compact cell phenotype and blunted invasive potential. In vivo, regional loss of T‐cad expression was more frequent and prominent in SCC classified as moderately‐to‐poorly differentiated than in SCC classified as well differentiated. However, in both categories aberrant and/or absence of T‐cad expression was associated with histological features of a potentially more malignant and invasive phenotype of cutaneous SCC. Conclusions T‐cad is a controlling determinant of SCC phenotype and invasive behaviour and its loss is associated with the process of malignant transformation from noninvasive to invasive SCC.     

Prostaglandin D2 production in FM55 melanoma cells is regulated by α‐melanocyte‐stimulating hormone and is not related to melanin production

Please cite this paper as: Prostaglandin D₂ production in FM55 melanoma cells is regulated by α‐melanocyte‐stimulating hormone and is not related to melanin production. Experimental Dermatology 2010; 19 : 751–753. Abstract: This study shows that prostaglandins in human FM55 melanoma cells and epidermal melanocytes are produced by COX‐1. Prostaglandin production in FM55 melanoma cells was unrelated to that of melanin suggesting that the two processes can occur independently. α‐Melanocyte‐stimulating hormone, which had no effect on melanin production in FM55 cells, stimulated PGD₂ production in these cells without affecting PGE₂. While cAMP pathways may be involved in regulating PGD₂ production, our results suggest that α‐MSH acts independently of cAMP, possibly by regulating the activity of lipocalin‐type PGD synthase. This α‐MSH‐mediated effect may be associated with its role as an immune modulator.     

Matrix metalloproteinase‐7 activates heparin‐binding epidermal growth factor‐like growth factor in cutaneous squamous cell carcinoma

Background Tumour‐specific expression of matrix metalloproteinase (MMP)‐7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB). Objectives To examine the potential role of MMP‐7 in shedding of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) in RDEB‐associated and sporadic SCCs. Methods Tissue microarrays of RDEB‐associated SCC (n = 20), non‐EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP‐7, CD44 variant 3 (CD44v3) and HB‐EGF. Shedding of HB‐EGF was studied in vitro using two cutaneous SCC cell lines. Results Immunohistochemical analysis showed that HB‐EGF was absent in tumour cells when MMP‐7 and CD44v3 colocalized, and that the absence of HB‐EGF was more pronounced in RDEB‐associated SCCs than in non‐EB SCCs. The loss of HB‐EGF in MMP‐7–CD44v3 double‐positive areas was interpreted to indicate shedding and activation of HB‐EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP‐7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB‐EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. Conclusions These findings provide evidence for the role of MMP‐7 in promoting the growth of cutaneous SCCs by shedding HB‐EGF, and identify EGFR signalling as a potential therapeutic target in RDEB‐associated SCC and unresectable sporadic cutaneous SCC.     

1α,25-Dihydroxy Vitamin D3 Modulation of HLA-DR mRNA Induced by Gamma-Interferon in Cultured Epithelial Tumor Cell Lines

Using three cultured epithelial tumor cell lines, we investigated and analyzed the effects of gamma-interferon (γ-IFN) and 1α,25-dihydroxy vitamin D₃ (1,25-(OH)₂D₃) on the levels of HLA-DR (α) mRNA and HLA-DR (β) mRNA by Northern blot analysis. After treatment with γ-IFN alone, the levels of the mRNA increased. Treatment with both γ-IFN and 1,25-(OH)₂D₃ at the same time resulted in a significant decrease in the levels of mRNA in K-TL-1, IK-TL-2, and M-TL cells as compared to those induced by γ-IFN alone.     

Photodynamic therapy with topical methyl‐ and hexylaminolevulinate for prophylaxis and treatment of UV‐induced SCC in hairless mice

Please cite this paper as: Photodynamic therapy with topical methyl‐ and hexylaminolevulinate for prophylaxis and treatment of UV‐induced SCC in hairless mice. Experimental Dermatology 2010; 19 : e166–e172. Abstract Background: Hexyl aminolevulinate (HAL) is a long‐chained 5‐aminolevulinic acid‐ester that has been proposed as a novel photosensitizing agent to methyl aminolevulinate (MAL) in topical photodynamic therapy (PDT). The more lipophilic HAL, may improve treatment outcome for non‐melanoma skin cancer. Objective: To compare the prophylactic and therapeutic effects of HAL‐ and MAL‐PDT for ultraviolet‐induced squamous cell carcinomas (SCCs) in hairless mice. Methods: Mice (n = 249) were irradiated with solar UV‐radiation (UVR) until SCC occurred. Before any skin changes developed, two prophylactic PDT treatments were given, using creams of HAL (2%, 6%, 20%) or MAL (20%) followed by illumination (632 nm, Aktilite, Photocure). Two therapeutic PDT‐treatments were given by randomization to the first developed SCC of 1 mm. Primary end‐points were time to first SCC of 1 mm and complete SCC clearance. Secondary end‐points were time to SCC‐recurrence, PpIX fluorescence and skin reactions to PDT. Results: The median time to first SCC was significantly longer for mice treated with prophylactic HAL‐PDT (2%, 6% and 20% HAL, 264 days) and MAL‐PDT (20% MAL, 269 days) than mice exposed to UVR (186 days) and UVR + placebo‐PDT (199 days) (P < 0.0001). The therapeutic efficacy of HAL‐ and MAL‐PDT showed cure rates of 23–61.5% (P = 0.11). Similar PpIX fluorescence intensity and severity of clinical reactions were seen for HAL‐ and MAL‐groups, although mice developed more intense hyper‐pigmentation when treated with 20% MAL‐PDT compared with 2% HAL‐PDT. Conclusions: PDT with HAL (2%, 6% and 20%) and MAL (20%) is equally effective to prevent and treat UV‐induced SCC in hairless mice.     

Peroxisome proliferator‐activated receptor γ agonists reduce cell proliferation and viability and increase apoptosis in systemic sclerosis fibroblasts

Background No study has evaluated the effect of the peroxisome proliferator‐activated receptor γ (PPARγ) agonists on cell viability, proliferation and apoptosis in cultured systemic sclerosis (SSc) fibroblasts. Objectives The effects of two pure PPARγ agonists (rosiglitazone and pioglitazone) in cultured SSc fibroblasts were evaluated and compared with effects in normal fibroblasts. Methods The study included evaluation of cell viability and proliferation (based on the cleavage of tetrazolium salts and measurement of absorbance of the cell proliferation reagent WST‐1), and determination of cell apoptosis (by means of the Hoechst dye uptake). Results Rosiglitazone or pioglitazone (20 μmol L^?1) significantly reduced cell proliferation (cell count of 75% and 83% compared with baseline, respectively, after 2 h) and cell viability (absorbance reductions of 25% and 22% compared with baseline, respectively, after 2 h), and increased apoptosis (apoptotic cell percentages 9·9% and 8·6%, respectively, after 48 h of incubation) in SSc fibroblasts, whereas they did not present a significant influence on control fibroblasts. Conclusions The effects of rosiglitazone or pioglitazone shown on SSc fibroblasts raise the hypothesis of a therapeutic role for PPARγ agonists in patients affected by SSc.     

Changes of the activities of enzymes involved in prostaglandin synthesis in rat skin during development and aging

Summary The developmental changes of enzymes involved in prostaglandin (PG) synthesis were investigated in rat skin from birth to 1.5 years old. In all stages of development, the activities of PG-synthesizing enzymes were found in 100,000xg supernatants of homogenates of rat skin, and PGD₂ was the major PG among those formed from PGH₂ in the presence of 1 m glutathione (GSH). The PGD synthetase activity in rat skin at birth was 2.14 nmol/min per mg protein, increasing to the highest level (3.69 nmol/min per mg protein) at 3 weeks after birth and then gradually decreasing up to 1.5 years old. The activities of PGE₂ and PGF₂ synthetases in rat skin were almost unchanged during development and aging. In contrast, the activity of GSH-S-transferase was at its lowest level at birth and gradually increased, reaching a plateau at 3 weeks after birth and remaining relatively constant during the development. The increase of PGD synthetase activity in 3-week-old rats was mainly due to the increase of specific activity of PGD synthetase in the epidermis, which was separated from the dermis by heat treatment (55° C, 30s). Immunohis-tochemical study, using (rat spleen PGD synthetase)-specific antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, increased in the epidermis in 3-week-old rats. These results suggest that a change of PGD₂ synthetase activity during aging of the skin is closely related to the development of ATPase⁺ Langerhans cells in the epidermis.     

Properties of γ-Glutamyl Transpeptidase in Squamous Cell Carcinoma

γ-Glutamyl transpeptidase (GGT) was extracted from squamous cell carcinoma tissues of human skin (SCC) by Triton X-100 and bromelain treatment, and some of its biochemical properties were compared with those of GGT extracted from eccrine gland-rich tissue and normal kidney. GGT activity significantly increased in SCC, but there was no definitive differences in enzymological properties between GGT of SCC and normal tissue enzyme. However, GGT of SCC was distinguishable from those of normal tissues by isoelectric point, electrophoretic mobility, and sensitivity to neuraminidase treatment. These results indicate that GGT of SCC has some variant properties which may be related to skin carcinogenesis.     

Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations

Please cite this paper as: Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations. Experimental Dermatology 2010; 19 : e222–e227. Abstract: Non‐melanoma skin cancer represents the most frequent human cancer entity. Activation of the PI3K/AKT signalling pathway has been reported both in squamous cell carcinoma (SCC) of the skin and in basal cell carcinoma (BCC). In many cancers, including SCC of the head and neck, the oesophagus and the penis, activation of this pathway is mediated by oncogenic PIK3CA and AKT1 mutations. We therefore screened 61 non‐melanoma skin cancer samples (30 SCC and 31 BCC) for the presence of activating PIK3CA and AKT1 mutations. PIK3CA hotspot mutations were analysed using a highly sensitive SNaPshot assay, and exon 4 of AKT1 was sequenced directly. In addition, immunohistochemistry was performed for phosphorylated AKT protein. Immunohistochemical expression of pAkt was observed both in SCC and in BCC samples, indicating an activation of the PI3K/AKT pathway. Although SCC showed higher expression levels than BCC, this difference was not significant. However, none of the 61 non‐melanoma skin cancer samples revealed any PIK3CA and AKT1 hotspot mutations at the investigated loci. We conclude that PIK3CA and AKT1 hotspot mutations do not contribute to the activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer. The distinct PIK3CA mutation spectrum between SCC of the skin and SCC of other tissues may reflect the different carcinogens which are involved into the mutagenesis of these cancers. PIK3CA and AKT1 hotspot mutations are obviously not caused by UV light exposure, the main risk factor in non‐melanoma skin cancer.     

Chemokine Receptor CCR3 Expression in Malignant Cutaneous Tumors

Background

Chemokines and their receptors are important players in tumorigenesis by facilitating tumor proliferation and metastasis. Little is known about the possible function of chemokine receptors in relation to the development and progression of malignant cutaneous tumors.

Objective

The aim of this study was to determine the chemokine receptor CCR3 expression pattern and the protein expression level in selected malignant cutaneous tumors.

Methods

Four types of cell lines (G361, A431, SK-MEL-2, SK-MEL-24) were analyzed, using Western blotting, for the expression of CCR3 protein. Immunohistochemical staining for CCR3 was done on 36 skin cancer tissue samples that included 16 squamous cell carcinomas (SCCs), 16 basal cell carcinomas (BCCs), 16 malignant melanomas (MMs) and 6 normal tissue samples.

Results

Western blot analysis showed that CCR3 protein was more expressed in the MM cell lines (G361, SK-MEL-2,SK-MEL-24) than that in the SCC cell line (A431), and the immunohistochemical analysis showed that CCR3 protein was overexpressed in MM and SCC, it was mildly expressed in BCC and it was hardly expressed in normal tissue.

Conclusion

This study demonstrated via immunochemistry that CCR3 was more expressed in MM, followed by SCC and BCC. The existence of CCR3 protein may enhance the tumorigenic potential of malignant cutaneous tumors.     

Various peroxisome proliferator‐activated receptor (PPAR)‐γ agonists differently induce differentiation of cultured human keratinocytes

Peroxisome proliferator‐activated receptors (PPARs) are potentially useful for the treatment of skin diseases, because they stimulate keratinocyte differentiation, exert anti‐inflammatory effects and improve barrier function. We examined five PPAR‐γ agonists, including four thiazolidinediones (ciglitazone, troglitazone, rosiglitazone and pioglitazone) and an angiotensin‐II receptor blocker (telmisartan), for their ability to upregulate filaggrin and loricrin expression at both mRNA and protein levels in cultured normal human keratinocytes (NHKs). Troglitazone, rosiglitazone, pioglitazone and telmisartan significantly increased filaggrin expression at both mRNA and protein levels in calcium‐induced differentiated NHKs. Rosiglitazone and pioglitazone, but not troglitazone nor telmisartan, also significantly increased loricrin expression at both mRNA and protein levels in differentiated NHKs. These effects were not found in undifferentiated NHKs nor differentiated NHKs treated with ciglitazone. This study revealed differential effects of various PPAR‐γ agonists on epidermal differentiation, and the most potent of those are rosiglitazone and pioglitazone.     

Altered gene expression in squamous cell carcinoma arising from congenital unilateral linear porokeratosis

Congenital unilateral linear porokeratosis (CULP) is a rare disorder of keratinization that shares clinical and molecular similarities with psoriasis. It also has an increased risk for malignant transformation to cutaneous squamous cell carcinoma (SCC). We investigated the expression of psoriasin, human beta‐defensin‐2, cathelicidin antimicrobial peptide/LL‐37, e‐cadherin, involucrin, p16^INK4a, p53, cyclin D1 and microchromosome maintenance protein 7 in healthy skin and in lesions of psoriasis, CULP and SCC from the same patient. p16^INK4a was overexpressed in CULP but not in the subsequent SCC. Psoriasin was overexpressed in psoriasis, CULP and SCC compared with healthy skin. Speculatively, p16^INK4a and psoriasin could be involved in the pathogenesis of CULP. Moreover, psoriasin may play a role in the malignant transformation of CULP to SCC.     

Serum Th1/Th2 and macrophage lineage cytokines in leprosy; correlation with circulating CD4+ CD25highFoxP3+ T‐regs cells

Not only macrophages, T‐helper (Th)1 and Th2, but also CD4⁺ CD25^highFoxP3⁺ regulatory T cells (T‐regs) are involved in immune response to Mycobacterium leprae. We aimed to evaluate serum interleukin (IL)‐1β and IL‐12p70 (macrophage cytokines), interferon‐γ (IFN‐γ) (Th1 cytokine), IL‐4 (Th2 cytokine) and circulating CD4⁺ CD25^highFoxP3⁺ T‐regs, in untreated leprosy patients. Forty three patients and 40 controls were assessed for the mentioned cytokines using ELISA. Patients were assessed for circulating T‐regs using flow cytometry. Patients were subgrouped into tuberculoid (TT), pure neural leprosy (PNL), borderline cases, lepromatous (LL), type 1 reactional leprosy (RL1) and erythema nodosum leprosum (ENL). Serum IL‐12p70, IFN‐γ and IL‐4 were significantly higher in patients versus controls (P < 0.05). Serum IL‐4 was highest in LL and lowest in RL1 (P = 0.003). Serum IL‐1β levels was significantly higher in multibacillary versus paucibacillary patients (P = 0.006). Significantly higher T‐regs levels was detected in TT, RL1 and PNL, while the lowest levels in ENL(P < 0.001), with significant differences versus controls (P < 0.05). FoxP3 expression% was significantly lower in PNL than other patients and controls (P < 0.05). T‐regs/T‐effs was lowest in ENL(P < 0.05). IFN‐γ correlated positively with T‐regs but negatively with IL‐1β (P = 0.041&0.046 respectively), which correlated positively with T‐effs%( P = 0.05). IL‐4 correlated positively with T‐regs FoxP3 expression% (P = 0.009). We concluded that: Circulating T‐regs were increased in TT, RL1 and PNL patients, known of relatively high cell‐mediated immunity. This finding was supported by low FoxP3 expression (in PNL) and correlation between T‐regs count and IFN‐γ level. Overproduction of IL‐4 in LL may infer liability to develop ENL, with disease progression and immune hyperactivation, marked by deficient T‐regs and increased T‐regs FoxP3 expression%. IL‐1β probably has a pro‐inflammatory role in multibacillary patients as correlated with T‐effs%.