High levels of type VII collagen expression in recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma keratinocytes increases PI3K and MAPK signalling, cell migration and invasion.

BACKGROUND: Epidermolysis bullosa is a group of inherited skin fragility diseases varying in severity from mild scarring to infant mortality. Great efforts are being undertaken to develop therapeutic strategies to treat the more pernicious forms of this disease, particularly those associated with recessive, loss-of-function mutations. In such cases significant effort is directed toward delivering recombinant protein at levels sufficient to demonstrate clinical benefit. Recessive dystrophic epidermolysis bullosa (RDEB) predisposes patients to a high incidence of life-threatening cutaneous squamous cell carcinoma (cSCC). Mutations in the gene encoding type VII collagen, COL7A1, are the sole cause of this disease and conflicting reports concerning type VII collagen and COL7A1 in carcinogenesis exist. OBJECTIVES: To investigate potential oncogenic effects of expressing recombinant type VII collagen in patient cells. METHODS: We used retroviral transduction to introduce type VII collagen into keratinocytes derived from patients with and without RDEB. RESULTS: Retroviral expression of type VII collagen in cSCC keratinocytes established from patients with RDEB resulted in increased cell adhesion, migration and invasion coupled with a concurrent increase in PI3K and MAPK signalling. CONCLUSIONS: Our data suggest caution when formulating strategies where delivery of type VII collagen is likely to exceed levels seen under normal physiological conditions in a patient group with a higher inherent risk of developing skin cancer.

Infrared laser pulse triggers increased singlet oxygen production in tumour cells.

Photodynamic therapy (PDT) is a technique developed to treat the ever-increasing global incidence of cancer. This technique utilises singlet oxygen ((1)O2) generation via a laser excited photosensitiser (PS) to kill cancer cells. However, prolonged sensitivity to intensive light (6-8 weeks for lung cancer), relatively low tissue penetration by activating light (630 nm up to 4 mm), and the cost of PS administration can limit progressive PDT applications. The development of quantum-dot laser diodes emitting in the highest absorption region (1268 nm) of triplet oxygen ((3)O2) presents the possibility of inducing apoptosis in tumour cells through direct (3)O2 → (1)O2 transition. Here we demonstrate that a single laser pulse triggers dose-dependent (1)O2 generation in both normal keratinocytes and tumour cells and show that tumour cells yield the highest (1)O2 far beyond the initial laser pulse exposure. Our modelling and experimental results support the development of direct infrared (IR) laser-induced tumour treatment as a promising approach in tumour PDT.

Mutations in AEC syndrome skin reveal a role for p63 in basement membrane adhesion, skin barrier integrity and hair follicle biology.

BACKGROUND: AEC (ankyloblepharon-ectodermal defects-clefting) syndrome is an autosomal dominant ectodermal dysplasia disorder caused by mutations in the transcription factor p63. Clinically, the skin is dry and often fragile; other features can include partial eyelid fusion (ankyloblepharon), hypodontia, orofacial clefting, sparse hair or alopecia, and nail dystrophy. OBJECTIVES: To investigate how p63 gene mutations affect gene and protein expression in AEC syndrome skin. METHODS: We performed microarray analysis on samples of intact and eroded AEC syndrome skin compared with control skin. Changes were verified by quantitative real-time reverse transcription-polymerase chain reaction and, for basal keratinocyte-associated genes, by immunohistochemistry and analysis of microdissected skin. RESULTS: We identified significant upregulation of six genes and downregulation of 69 genes in AEC syndrome skin, with the main changes in genes implicated in epidermal adhesion, skin barrier formation and hair follicle biology. There was reduced expression of genes encoding the basement membrane proteins FRAS1 and collagen VII, as well as the skin barrier-associated small proline-rich proteins 1A and 4, late cornified envelope protein 5A, hornerin, and lipid transporters including ALOX15B. Reduced expression of the hair-associated keratins 25, 27, 31, 33B, 34, 35, 81 and 85 was also noted. We also confirmed similar alterations in gene expression for 26 of the 75 genes in eroded AEC scalp skin. CONCLUSIONS: This study identifies specific changes in skin structural biology and signalling pathways that result from mutant p63 and provides new molecular insight into the AEC syndrome phenotype.

Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma.

Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture. Candidate drivers of cSCC development were derived by first defining a set of in vitro cancer genes and then comparing their expression in a range of clinical data sets containing normal skin, cSCC and the benign hyper-proliferative condition psoriasis. A small interfering RNA (siRNA) screen of the resulting 21 upregulated genes has yielded targets capable of reducing xenograft tumor volume in vivo. Small-molecule inhibitors for one target, Polo-like kinase-1 (PLK1), are already in clinical trials for other malignancies, and our data show efficacy in cSCC. Another target, C20orf20, is identified as being overexpressed in cSCC, and siRNA-mediated knockdown induces apoptosis in vitro and reduces tumor growth in vivo. Thus, our approach has shown established and uncharacterized drivers of tumorigenesis with potent efficacy as therapeutic targets for the treatment of cSCC.

Unusual molecular findings in Kindler syndrome.

Kindler syndrome (KS) is a rare inherited skin disorder with blistering and poikiloderma as its main clinical features. It is caused by loss-of-function mutations in the C20orf42 (KIND1) gene which encodes kindlin-1, an actin cytoskeleton-focal contact-associated protein which is predominantly expressed in keratinocytes. We investigated the molecular basis of KS in a 16-year-old Indian boy who had additional clinical findings, including scleroatrophic changes of the hands and feet, pseudoainhum and early onset of squamous cell carcinoma on his foot. Immunostaining for kindlin-1 in the patient's skin was completely absent and sequencing of C20orf42 (KIND1) genomic DNA showed a homozygous splice-site mutation at the -6 position, IVS9-6T-->A. Amplification and sequencing of cDNA from the skin revealed aberrant splicing with either deletion of exon 10 or deletion of exons 9, 10 and 11, both of which involve loss of the pleckstrin homology domain of kindlin-1 that is thought to play a role in cytoskeletal attachment and integrin-mediated cell signalling. Pathogenic splice-site mutations at the -6 position are unusual and have rarely been reported for any genetic disorder. Collectively, these findings extend the spectrum of clinical and molecular abnormalities in this rare genodermatosis.

[Effects of endocrine peptides on proliferation, migration and differentiation of human endothelial cells]. / Effets des peptides endocrines sur la prolifération, la migration et la différenciation des cellules endothéliales humaines.

AIMS: We aimed to evaluate the effects of several peptides (substance P, VIP, neuropeptide Y, bombesin, glucagon and somatostatin) on the proliferation, migration and differentiation of human endothelial cells and their modulation by an anti-angiogenic factor, endostatin. METHODS: Human endothelial cells (HUVEC) were isolated from umbilical veins. Their proliferation was measured by the incorporation of tritiated thymidine. Their migration was evaluated by using an haptotactic assay performed in Boyden chambers, after metabolic labeling of HUVEC through (35) S-methionin. Differentiation was evaluated as the capacity for HUVEC to form capillaries. RESULTS: Endothelial cell proliferation was increased by neuropeptide Y, bombesin and glucagon. Somatostatin induced a significant decrease in basal and stimulated endothelial cell proliferation. The migration of HUVEC increased in the presence of substance P, VIP, neuropeptide Y, bombesin, glucagon and somatostatin. The number of capillaries was increased by substance P and VIP and decreased by neuropeptide Y, bombesin and somatostatin. Endostatin induced a significant decrease in endothelial cell proliferation in the basal state and after stimulation by neuropeptide Y and bombesin. Endostatin had no additive effect on the anti-proliferative action of somatostatin. CONCLUSIONS: Our results suggest a role for endocrine peptides in the regulation of tumor angiogenesis. The potent anti-angiogenic effect of somatostatin may promote new therapeutic strategies.

Anti-beta4 integrin antibodies enhance migratory and invasive abilities of human colon adenocarcinoma cells and their MMP-2 expression.

Integrin-mediated adhesion of cells to extracellular matrix proteins has been shown to activate various intracellular signaling events. In the present study, we demonstrate that the addition of a monoclonal antibody raised against the beta4 integrin subunit in the culture medium of a clone derived from the colon adenocarcinoma cell line LoVo specifically results in stimulation of cell migration and invasion through reconstituted basement membrane matrices. Moreover, an increase in MMP-2 activity is observed. Conversely, monoclonal anti-alpha6 and anti-beta1 have no effect on MMP-2 expression. The s. c. co-injection of adenocarcinoma cells with antibodies raised against the beta4 integrin subunit to immunosuppressed newborn rats gives rise to tumors displaying altered and disorganized peri-tumoral basement membranes compared with tumors obtained when cells are injected with adenocarcinoma cells alone. Higher metastatic capacity of cells results when they are co-injected with antibodies to the beta4 integrin subunit. Our results suggest that the beta4 subunit of alpha6beta4 integrin, a laminin receptor in colon adenocarcinoma, may be responsible for the specific signals which stimulate cell motility, expression of MMP-2 and tumor invasion.

Site-specific epithelial-mesenchymal interactions in digestive neuroendocrine tumors. An experimental in vivo and in vitro study.

Little is known about the functional interactions between digestive neuroendocrine tumor cells and their stromal microenvironment. The focus of our study is whether mesenchymal cells modulate peptide expression, cell proliferation, and invasiveness in digestive neuroendocrine tumor cells. We designed an experimental in vivo and in vitro study using the mouse enteroendocrine cell line STC-1. In vivo, STC-1 cells were injected subcutaneously in 18 immunosuppressed newborn rats. At day 21, all animals presented poorly differentiated neuroendocrine tumors with lung metastases. Subcutaneous tumors were usually limited by a capsule containing basement membrane components and myofibroblasts that presented a low mitotic index. Lung tumors were devoid of capsule and poor in myofibroblasts, and their mitotic index was high. The profile of peptide expression in STC-1 tumors was different from that of cultured STC-1 cells. In vitro, STC-1 cells were cultured with fibroblasts of different origins, including dermis, lung, digestive tract, and liver. Based on their origin, myofibroblasts differentially modulated hormone synthesis, proliferation, spreading, and adhesion of STC-1 cells. In conclusion, our results show that site-specific functional interactions between mesenchymal and neuroendocrine cells may contribute to modulating the behavior of digestive neuroendocrine tumors, depending on their growth site.

[Integrins and metalloproteinases: an efficient collaboration in the invasive process]. / Intégrins et métalloprotéinases: une collaboration efficace dans le processus invasif.

During the invasive process, tumor cells must move through the extracellular matrix. They have to adhere to the extracellular matrix components, then proteolyse them and migrate on their fragments. This implicates integrins and proteinases, namely metalloproteinases. Numerous experiments which had been performed on various models, namely malignant melanomas proved that integrins have a major role in the transduction of signals from the outside to the inside of the cells, such signals enhancing the expression of the metalloproteinases or, in the contrary, inhibiting it. The modifications of this expression are dependent of extracellular matrix components and may be induced by the linking of specific antibodies to integrins. In some instances, the integrins localized on the tumor cell surface may act as receptors for extracellular matrix proteins and metalloproteinases at once, that may give to tumor cells an higher efficiency in the invasive process. Such mechanisms may result in interesting clinical perspectives for the control of metalloproteinases regulation in pathological processes.

Loss of epithelial differentiation markers and acquisition of vimentin expression after xenograft with laminin-1 enhance migratory and invasive abilities of human colon cancer cells LoVo C5.

Clone C5 of the human colon adenocarcinoma LoVo cell line was subcutaneously injected with or without exogenous laminin-1 (EHS laminin) into immunosuppressed newborn rats. Cultures were initiated from lung metastases obtained with or without laminin-1 and gave rise to the C5 sublines LM and M4, respectively. The LM subline was mainly composed of spreading cells whereas most C5 and M4 cells remained round and aggregated. The mesenchymal marker vimentin was expressed by very rare C5 and M4 cells (< 1%), and by many LM cells (about 35%). On the opposite, the epithelial markers villin and dipeptidylpeptidase IV were well expressed by C5 cells but not by LM cells. In in vitro migration and invasion assays, LM cells migrated and invaded basement membrane extract twice as much as the parental C5 clone and the M4 subline, probably in association with vimentin-expressing cells, because invasion of basement membrane extract Matrigel by LM cells gave rise to 100% vimentin-positive cells (sublines LM 22, LM 23 and LM 24). When subcutaneously injected, C5 cells induced tumors limited by an interrupted but well organized basement membrane, whereas LM cells induced tumor masses, occasionally limited by a very irregular basement membrane, as observed when C5 cells were injected with laminin-1. Gelatin zymographic analysis clearly showed an increased expression of matrix metalloproteinase-2 by LM cells. Our results suggest a specific role of laminin-1 on the in vivo proliferation of highly invasive vimentin-expressing colon carcinoma cells. This proliferation may result from the initial interaction of C5 cells with large amounts of laminin-1, leading to a selection of vimentin-expressing cells during the metastatic cascade.