Current Perspectives on the Role of Matrix Metalloproteinases in the Pathogenesis of Basal Cell Carcinoma.

Basal cell carcinoma (BCC) is the most common skin malignancy, which rarely metastasizes but has a great ability to infiltrate and invade the surrounding tissues. One of the molecular players involved in the metastatic process are matrix metalloproteinases (MMPs). MMPs are enzymes that can degrade various components of the extracellular matrix. In the skin, the expression of MMPs is increased in response to various stimuli, including ultraviolet (UV) radiation, one of the main factors involved in the development of BCC. By modulating various processes that are linked to tumor growth, such as invasion and angiogenesis, MMPs have been associated with UV-related carcinogenesis. The sources of MMPs are multiple, as they can be released by both neoplastic and tumor microenvironment cells. Inhibiting the action of MMPs could be a useful therapeutic option in BCC management. In this review that reunites the latest advances in this domain, we discuss the role of MMPs in the pathogenesis and evolution of BCC, as molecules involved in tumor aggressiveness and risk of recurrence, in order to offer a fresh and updated perspective on this field.

Matrix metalloproteinases in keratinocyte carcinomas.

The incidence of cutaneous keratinocyte-derived cancers is increasing globally. Basal cell carcinoma (BCC) is the most common malignancy worldwide, and cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. BCC can be classified into subtypes based on the histology, and these subtypes are classified further into low- and high-risk tumors. There is an increasing need to identify new therapeutic strategies for the treatment of unresectable and metastatic cSCC, and for aggressive BCC variants such as infiltrating, basosquamous or morpheaform BCCs. The most important risk factor for BCC and cSCC is solar UV radiation, which causes genetic and epigenetic alterations in keratinocytes. Similar gene mutations are noted already in sun-exposed normal skin emphasizing the role of the alterations in the tumor microenvironment in the progression of cSCC. Early events in cSCC progression are alterations in the composition of basement membrane and dermal extracellular matrix induced by influx of microbes, inflammatory cells and activated stromal fibroblasts. Activated fibroblasts promote inflammation and produce growth factors and proteolytic enzymes, including matrix metalloproteinases (MMPs). Transforming growth factor-ß produced by tumor cells and fibroblasts induces the expression of MMPs by cSCC cells and promotes their invasion. Fibroblast-derived keratinocyte growth factor suppresses the malignant phenotype of cSCC cells by inhibiting the expression of several MMPs. These findings emphasize the importance of interplay of tumor and stromal cells in the progression of cSCC and BCC and suggest tumor microenvironment as a therapeutic target in cSCC and aggressive subtypes of BCC.

Nicotinamide N-methyltransferase in nonmelanoma skin cancers.

BACKGROUND: Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) represent the most common forms of nonmelanoma skin cancers (NMSCs). Although successful treatment of these neoplasms is based on surgical excision, an increasing number of BCCs relapses and many SCCs display high rates of recurrence and metastasis. Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme, which was found to be upregulated in different solid tumours. However, there are no data regarding enzyme expression in NMSCs. The aim of this study was therefore to evaluate the potential involvement of NNMT in BCCs and SCCs. MATERIALS AND METHODS: Immunohistochemical analyses were carried out on 40 BCC cases and 39 SCC cases, to evaluate enzyme expression in tumour and surrounding healthy margins. Moreover, the relationship between NNMT intratumour levels and clinico-pathological parameters were explored. RESULTS: Nicotinamide N-methyltransferase was found to be overexpressed in BCCs compared with control tissues, while a significant enzyme downregulation was detected in SCCs with respect to corresponding healthy margins. In addition, NNMT levels were negatively related to aggressiveness of both BCCs (distinguishing between infiltrative and nodular tumours) and SCCs (considering head and neck forms and tumours of the extremities and trunk). CONCLUSIONS: These evidences seem to demonstrate that the different NNMT dysregulation detected in BCC and SCC may be the result of important biological traits distinctively characterizing these two forms within NMSCs. In addition, enzyme levels seem to be inversely correlated with tumour aggressiveness, thus suggesting the potential suitability of the enzyme as a prognostic biomarker for both neoplasms.

Transglutaminase 3 is expressed in basal cell carcinoma of the skin.

Transglutaminase 3 (TG3) belongs to a family of Ca2+-dependent enzymes which catalyse protein crosslinking. TG3 is important for proper development of the skin and hair shaft, and knock-out mice for the Tgm3 gene are sensitive to UVB-induced photodamage due to aberrations in cornified envelope formation. Loss of TG3 is reported in head and neck and oesophageal squamous cell carcinoma, yet, its expression in skin cancer has not been studied. The aim of the present study was to analyse the expression pattern of TG3 in skin cancer. TG3 expression was investigated based on immunohistochemical staining of a tissue micro-array of different types of skin cancer, as well as meta-analysis of public gene array data. Our findings demonstrated that TG3 is normally expressed in spinous/granular layers of the epidermis, but is absent in melanocytes as well as melanoma samples. As expected, its expression was absent in poorly differentiated squamous cell carcinoma of the skin. Surprisingly, we show that samples of basal cell carcinoma demonstrated strong staining for TG3 both in the cytoplasm and nucleus. Furthermore, at the mRNA level, the expression pattern of TGM3 was crucially altered in BCC, but not other types of skin cancer. These findings lead to new questions regarding TG3 involvement in basal cell carcinoma tumourigenesis. Moreover, the expression pattern of TG3 renders it a potential specific marker for basal cell carcinoma diagnosis.

The prognostic roles of cyclooxygenase-2 for patients with basal cell carcinoma.

Background: Cyclooxygenase-2 (Cox-2) is critical for tumor invasion, angiogenesis, and poor prognosis in many human cancers. It was reported to be an abnormal expression in many human malignancies, including basal cell carcinoma (BCC). However, the prognostic significance of cox-2 in BCC was still unclear. The aim of this study was to investigate the prognostic roles of cox-2 for patients with BCC. Methods: We detected the expression of cox-2 both at mRNA and protein level in tumor tissue and adjacent normal tissues from 180 patients with BCC by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Results: Cox-2 expression was significantly increased in BCC tissues compared with the adjacent normal cohorts (p < .001). Its expression was significantly associated with angiogenesis (p < .001) and depth of invasion (p < .001). Kaplan-Meier analysis suggested patients with high expression of cox-2 had a shorter overall survival rate than those with low expression (log rank test, p < .001). Conclusions: The expression of cox-2 was up-regulated in BCC and it could be used as a bio-marker for the prognosis of BCC patients with a high risk of recurrence.

N-Glycosylation in progression of skin cancer.

Skin cancer can be classified as cutaneous malignant melanoma, basal cell carcinoma, and squamous cell carcinoma. Due to the high level of morbidity and mortality, skin cancer has become a global public health issue worldwide while the pathogenesis of skin cancer is still unclear. It is necessary to further identify the pathogenesis of skin cancer and find candidate targets to diagnose and treat skin cancer. A variety of factors are known to be associated with skin cancer including N-glycosylation, which partly explained the malignant behaviors of skin cancer. In this review, we retrieved databases such as PubMed and Web of Science to elucidate its relationship between glycosylation and skin cancer. We summarized some key glycosyltransferases and proteins during the process of N-glycosylation related to skin cancer, which was helpful to unmask the additional mechanism of skin cancer and find some novel targets of skin cancer.

HMG-CoA reductase expression in human eyelid tissue and in a human meibomian gland epithelial cell line.

PURPOSE: 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of cholesterol production, has been found to contribute to lipid secretion from skin sebaceous glands and hair follicles. We assessed for HMGCR expression in human eyelid tissue and in immortalized human meibomian gland epithelial cells (HMGECs) using immunohistochemistry. METHODS: Full thickness human eyelid specimens in archival paraffin blocks were obtained. A section from each block was stained with hematoxylin and eosin and examined by an ocular pathologist for validation of tissue pathology. Immunohistochemistry was performed using rabbit anti-human HMGCR antibody on serial sections using the Ventana automated staining system. HMGCR expression was examined for in HMEGCs with fluorescence immunocytochemistry and confocal microscopy. RESULTS: Thirteen full thickness eyelid specimens met the inclusion criteria. All specimens contained meibomian glands, and 2 (15%) contained glands of Zeis, 3 (23%) pilosebaceous glands, 2 (15%), accessory lacrimal glands, and 2 (15%), glands of Moll, respectively. Immunohistochemistry showed HMGCR expression in meibocytes of meibomian glands and sebocytes of Zeis and pilosebaceous glands in all specimens. HMGCR expression was also evident in vascular endothelium. Immunofluorescence was positive for HMGCR expression on HMGEC cells. No labeling was seen for the negative Ig control. CONCLUSION: HMGCR was expressed in all eyelid sebaceous-type glands and in HMGECs, consistent with a role for cholesterol production in the genesis of tear film lipids. The observed expression also provides a rationale for using topical statins, inhibitors of HMGCR, as novel tear film lipid stabilizers in conditions such as blepharitis, where meibum production is aberrant.

Expression of n-MYC, NAMPT and SIRT1 in Basal Cell Carcinomas and their Cells of Origin.

Deregulated Hedgehog signalling is a driver of basal cell carcinomas. One effector of the Hedgehog pathway is n-MYC. c/n-MYC proteins, NAMPT and DBC1 are linked to SIRT1 in a positive feedback loop that may contribute to tumorigenesis of basal cell carcinoma. In 5 basal cell carcinoma types immunohistochemistry revealed n-MYC, NAMPT and SIRT1 expression. DBC1 was homogenously expressed in all epithelial cells. NAMPT, SIRT1 and c-MYC were expressed in the stratum basale of human and murine skin. In hair follicles NAMPT and SIRT1 were expressed together with c-MYC and n-MYC, except for the matrix, where n-MYC was strongly positive, but c-MYC expression was absent. Therefore, a common pathway connecting n-MYC, NAMPT and SIRT1 may be active in basal cell carcinomas and in their cells of origin. This pathway may contribute to the development of basal cell carcinomas. Targeting factors in the feedback loop may offer novel therapeutic options.

The involvement of EGFR, HER2 and HER3 in the basal cell carcinomas aggressiveness.

Basal cell carcinomas (BCCs) are the most common malignant skin tumors, with variable prognosis and recurrence rates, depending on histopathological subtypes. The study analyzed the immunoexpression of epidermal growth factor receptors (EGFRs) in 53 cases of nodular, adenoid and morpheaform BCCs in relation to clinico-pathological associated parameters. We found significant differences in the expression of EGFR, HER2 and HER3 reported to histological BCC types. The nodular type presented the weakest expression of EGFRs, while the morpheaform type had a high expression of all receptors and the adenoid type an increased expression only in case of EGFR and HER2. This study supports the involvement of EGFR, HER2 and HER3 in BCC aggressiveness of and in tumor differentiating towards different histological subtypes.

TdT expression in normal and neoplastic sebaceous cells.

AIMS: Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in immature, normal and neoplastic, lymphoid or haematopoietic cells and in neuroendocrine carcinomas, such as Merkel cell carcinoma and small-cell carcinoma. It has not yet been described in cells of epithelial origin. After observing TdT immunoreactivity in normal sebaceous glands, we analysed its spectrum of expression in cases of sebaceous cell hyperplasia (SGH) and sebaceous cell neoplasm. METHODS AND RESULTS: Twelve cases of SGH and three cases of other benign lesions, namely sebaceoma, sebaceous adenoma, and sebaceous naevus, along with four archived cases of sebaceous cell carcinoma (SC) were collected and stained with TdT antibody. In addition, tissue microarrays were constructed from 11 cases of basal cell carcinoma (BCC) and 10 cases of squamous cell carcinoma (SCC), which had nine evaluable cases each, and, after carcinoma type confirmation with immunostaining for epithelial membrane antigen, TdT immunohistochemistry was performed. All cases of SGH and sebaceous cell neoplasm were positive for TdT. The staining intensity was variable, being often weak to moderate in a significant proportion of cells, apart from one case of SC and the case of sebaceous naevus, which were only focally positive. No BCCs and only one SCC showed immunoreactivity. CONCLUSIONS: TdT protein can be found in cells of epithelial origin and specifically sebaceous cells, both benign and malignant. It can be hypothesized that this expression is due to sebaceous cell differentiation as a prelude to apoptosis and holocrine secretion. Additional studies are needed to further elucidate its biological role.

Basal subtype is predictive for response to cetuximab treatment in patient-derived xenografts of squamous cell head and neck cancer.

Cetuximab is the single targeted therapy approved for the treatment of head and neck cancer (HNSCC). Predictive biomarkers have not been established and patient stratification based on molecular tumor profiles has not been possible. Since EGFR pathway activation is pronounced in basal subtype, we hypothesized this activation could be a predictive signature for an EGFR directed treatment. From our patient-derived xenograft platform of HNSCC, 28 models were subjected to Affymetrix gene expression studies on HG U133+ 2.0. Based on the expression of 821 genes, the subtype of each of the 28 models was determined by integrating gene expression profiles through centroid-clustering with previously published gene expression data by Keck et al. The models were treated in groups of 5-6 animals with docetaxel, cetuximab, everolimus, cis- or carboplatin and 5-fluorouracil. Response was evaluated by comparing tumor volume at treatment initiation and after 3 weeks of treatment (RTV). Tumors distributed over the 3 signature-defined subtypes: 5 mesenchymal/inflamed phenotype (MS), 15 basal type (BA), 8 classical type (CL). Cluster analysis revealed a strong correlation between response to cetuximab and the basal subtype. RTV MS 3.32 vs. BA 0.78 (MS vs. BA, unpaired t-test, p 0.0002). Cetuximab responders were distributed as following: 1/5 in MS, 5/8 in CL and 13/15 in the BA group. Activity of classical chemotherapies did not differ between the subtypes. In conclusion basal subtype was associated with response to EGFR directed therapy in head and neck squamous cell cancer patient-derived xenografts.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

BACKGROUND:: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. OBJECTIVES:: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). METHODS:: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). RESULTS:: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. CONCLUSION:: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma

Abstract: Background: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

The activity and tissue distribution of thioredoxin reductase in basal cell carcinoma.

PURPOSE: Basal cell carcinoma (BCC) is the most prevalent cancer worldwide. Different mechanisms are proposed to be involved in its pathogenesis such as oxidative stress. Oxidative stress, which is the consequence of the disruption of redox balance in favor of oxidants, is involved in the initiation or progression of many tumors. Thioredoxin reductase (TrxR) is a key enzyme of the thioredoxin (Trx) system, containing Trx and TrxR and NADPH, which is one of the main cellular oxidoreductases with an essential role in cellular health and survival through providing and maintaining redox balance. Therefore, we aimed to study and compare the activity and tissue distribution of TrxR in tumoral tissue and its healthy margin in patients with BCC. METHODS: After biopsy and taking samples from 18 patients, TrxR activity was measured using a commercial kit and its tissue distribution was assessed immunohistochemically. RESULTS: Both the activity and tissue distribution of TrxR in tumoral tissues were significantly higher compared to their healthy margins. Regarding the tissue distribution, this significant increase in TrxR in tumoral tissues was documented based on both staining intensity and abundance of positive cells in immunohistochemistry. CONCLUSIONS: Based on these results, it is concluded that TrxR is involved in the pathogenesis of BCC; however, more investigations are required to clarify whether this increase is a consequence of BCC or it is an initiating mechanism.

Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer.

Nonmelanoma skin cancers (NMSC) are the most common human malignancies. IKKα is an essential protein for skin development and is also involved in the genesis and progression of NMSC, through mechanisms not fully understood. While different studies show that IKKα protects against skin cancer, others indicate that it promotes NMSC. To resolve this controversy we have generated two models of transgenic mice expressing the IKKα protein in the nucleus (N-IKKα mice) or the cytoplasm (C-IKKα mice) of keratinocytes. Chemical skin carcinogenesis experiments show that tumors developed by both types of transgenic mice exhibit histological and molecular characteristics that make them more prone to progression and invasion than those developed by Control mice. However, the mechanisms through which IKKα promotes skin tumors are different depending on its subcellular localization; while IKKα of cytoplasmic localization increases EGFR, MMP-9 and VEGF-A activities in tumors, nuclear IKKα causes tumor progression through regulation of c-Myc, Maspin and Integrin-α6 expression. Additionally, we have found that N-IKKα skin tumors mimic the characteristics associated to aggressive human skin tumors with high risk to metastasize. Our results show that IKKα has different non-overlapping roles in the nucleus or cytoplasm of keratinocytes, and provide new targets for intervention in human NMSC progression.

Erk1/2 activation in stromal fibroblasts from sporadic basal cell carcinomas.

BACKGROUND: Constitutive activation of the Erk pathway can lead to oncogenic transformation. However, the Erk pathway is not activated in human basal cell carcinomas (BCCs); although in animal models, this seems to be important. OBJECTIVE: To help understand the role of Erk activity in BCC formation. MATERIALS AND METHODS: The authors assayed the specific levels of phosphorylated Erk by immunohistochemistry in BCCs and normal skin biopsies. They have also analyzed Erk activation by immunoblot in fibroblasts isolated from BCC. RESULTS: By immunohistochemical analysis, the authors have observed that 10 of BCCs (56%) did not show phosphor-Erk staining in tumor masses and 7 (40%) showed a gradient staining exhibiting phospho-Erk only in the epidermal side of tumor masses. Remarkably, 15 BCC samples (83%) showed phospho-Erk accumulation in stroma. Six of the 9 independent cultures of dermal fibroblasts isolated from BCC maintained Erk activation "in vitro." CONCLUSION: The authors propose that there is a specific cell-type regulation of Erk activity in BCC, and this feature may be relevant during BCC formation. Stroma region from BCCs showed Erk activation and reduced proliferation. Conversely, Erk activation is barely detectable in proliferative BCCs.

The heme oxygenase/biliverdin reductase system in skin cancers.

The heme oxygenase/biliverdin reductase (HO/BVR) pathway enhances cell stress response by degrading excess heme or producing antioxidant and cytoprotective molecules. Recently, members of the HO/BVR system have been proposed as biomarkers for the early diagnosis of free radical-related diseases. In this study, the presence of both the inducible and constitutive HO isoforms (HO-1 and HO-2, respectively) and BVR was evaluated by immunohistochemistry in human skin cancer samples. Moderate/strong immunoreactivities against HO-1, HO-2 and BVR were detected in 100% of the nodular malignant melanoma samples, whereas in basal cell carcinoma specimens these figures were 62%, 88% and 60%, respectively, with a faint/moderate degree of expression. Faint/moderate HO-1, HO-2 and BVR immunoreactivities were detected in 33%, 66% and 100% of melanocytic nevi samples, respectively. In conclusion, HO-1 and HO-2 and BVR were expressed in the cytosols of skin cancer cells, whereas perilesional normal epidermis showed only faint staining, thus leading to the hypothesis that the HO/BVR system is activated in skin cancers.

Incidencia del cáncer de piel no melanoma en un cupo del Centro de Salud de Zaramaga / Incidence of non-melanoma skin cancer in a population of the Zaramaga Health Centre

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Involvement of metformin and AMPK in the radioresponse and prognosis of luminal versus basal-like breast cancer treated with radiotherapy.

Metformin is under evaluation as a potential anticancer agent. Expression of total and phospho(Thr172)-adenosine monophosphate-activated kinase-α (AMPKα and pAMPKα(Thr172) respectively), a main metformin target, was examined in radiotherapy treated breast cancers and metformin's ability to modulate Trx system expression and breast cancer radiosensitivity evaluated in vitro. AMPKα and pAMPKα(Thr172) expression was assessed using a discovery (n=166) and validation cohort (n=609). Metformin's role in regulating radioresponse, and Trx family expression, was examined via clonogenic assays and Western blots. Intracellular reactive oxygen species (ROS) levels, cell cycle progression and apoptosis were assessed by flow cytometry. High AMPKα expression associated with improved local recurrence-free (P=0.019), relapse-free (P=0.016) and breast cancer-specific survival (P=0.000065) and was, from multivariate analysis, an independent prognostic factor from the discovery cohort. From the validation cases AMPKα expression associated with relapse-free and breast cancer-specific survival in luminal breast cancers. Metformin substantially increased radiosensitivity, intracellular ROS levels and reduced Trx expression, in luminal breast cancer cells, but had little effect on basal phenotype cells. In conclusion, high AMPKα expression associates with improved prognosis, especially in luminal breast cancer. Metformin preferentially radiosensitises luminal breast cancer cells, potentially due to alterations to intracellular ROS levels via modulation of Trx family protein expression.