A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming.

Cancer-associated fibroblasts (CAFs) are key regulators of tumorigenesis and promising targets for next-generation therapies. We discovered that cancer cell-derived activin A reprograms fibroblasts into pro-tumorigenic CAFs. Mechanistically, this occurs via Smad2-mediated transcriptional regulation of the formin mDia2, which directly promotes filopodia formation and cell migration. mDia2 also induces expression of CAF marker genes through prevention of p53 nuclear accumulation, resulting in the production of a pro-tumorigenic matrisome and secretome. The translational relevance of this finding is reflected by activin A overexpression in tumor cells and of mDia2 in the stroma of skin cancer and other malignancies and the correlation of high activin A/mDia2 levels with poor patient survival. Blockade of this signaling axis using inhibitors of activin, activin receptors, or mDia2 suppressed cancer cell malignancy and squamous carcinogenesis in 3D organotypic cultures, ex vivo, and in vivo, providing a rationale for pharmacological inhibition of activin A-mDia2 signaling in stratified cancer patients.

Activin promotes skin carcinogenesis by attraction and reprogramming of macrophages.

Activin has emerged as an important player in different types of cancer, but the underlying mechanisms are largely unknown. We show here that activin overexpression is an early event in murine and human skin tumorigenesis. This is functionally important, since activin promoted skin tumorigenesis in mice induced by the human papillomavirus 8 oncogenes. This was accompanied by depletion of epidermal γδ T cells and accumulation of regulatory T cells. Most importantly, activin increased the number of skin macrophages via attraction of blood monocytes, which was prevented by depletion of CCR2-positive monocytes. Gene expression profiling of macrophages from pre-tumorigenic skin and bioinformatics analysis demonstrated that activin induces a gene expression pattern in skin macrophages that resembles the phenotype of tumor-associated macrophages in different malignancies, thereby promoting angiogenesis, cell migration and proteolysis. The functional relevance of this finding was demonstrated by antibody-mediated depletion of macrophages, which strongly suppressed activin-induced skin tumor formation. These results demonstrate that activin induces skin carcinogenesis via attraction and reprogramming of macrophages and identify novel activin targets involved in tumor formation.

Src is activated by the nuclear receptor peroxisome proliferator-activated receptor ß/δ in ultraviolet radiation-induced skin cancer.

Although non-melanoma skin cancer (NMSC) is the most common human cancer and its incidence continues to rise worldwide, the mechanisms underlying its development remain incompletely understood. Here, we unveil a cascade of events involving peroxisome proliferator-activated receptor (PPAR) ß/δ and the oncogene Src, which promotes the development of ultraviolet (UV)-induced skin cancer in mice. UV-induced PPARß/δ activity, which directly stimulated Src expression, increased Src kinase activity and enhanced the EGFR/Erk1/2 signalling pathway, resulting in increased epithelial-to-mesenchymal transition (EMT) marker expression. Consistent with these observations, PPARß/δ-null mice developed fewer and smaller skin tumours, and a PPARß/δ antagonist prevented UV-dependent Src stimulation. Furthermore, the expression of PPARß/δ positively correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma (SCC), and critically, linear models applied to several human epithelial cancers revealed an interaction between PPARß/δ and SRC and TGFß1 transcriptional levels. Taken together, these observations motivate the future evaluation of PPARß/δ modulators to attenuate the development of several epithelial cancers.

Mast cells are dispensable for normal and activin-promoted wound healing and skin carcinogenesis.

The growth and differentiation factor activin A is a key regulator of tissue repair, inflammation, fibrosis, and tumorigenesis. However, the cellular targets, which mediate the different activin functions, are still largely unknown. In this study, we show that activin increases the number of mature mast cells in mouse skin in vivo. To determine the relevance of this finding for wound healing and skin carcinogenesis, we mated activin transgenic mice with CreMaster mice, which are characterized by Cre recombinase-mediated mast cell eradication. Using single- and double-mutant mice, we show that loss of mast cells neither affected the stimulatory effect of overexpressed activin on granulation tissue formation and reepithelialization of skin wounds nor its protumorigenic activity in a model of chemically induced skin carcinogenesis. Furthermore, mast cell deficiency did not alter wounding-induced inflammation and new tissue formation or chemically induced angiogenesis and tumorigenesis in mice with normal activin levels. These findings reveal that mast cells are not major targets of activin during wound healing and skin cancer development and also argue against nonredundant functions of mast cells in wound healing and skin carcinogenesis in general.

The bright and the dark sides of activin in wound healing and cancer.

Activin was initially described as a protein that stimulates release of follicle stimulating hormone from the pituitary, and it is well known for its important roles in different reproductive functions. In recent years, this multifunctional factor has attracted the attention of researchers in other fields, as new functions of activin in angiogenesis, inflammation, immunity, fibrosis and cancer have been discovered. Studies from our laboratory have identified activin as a crucial regulator of wound healing and skin carcinogenesis. On the one hand, it strongly accelerates the healing process of skin wounds but, on the other hand, it enhances scar formation and the susceptibility to skin tumorigenesis. Finally, results from several laboratories have revealed that activin enhances tumour formation and/or progression in some other organs, in particular through its effect on the tumour microenvironment, and that it also promotes cancer-induced bone disruption and muscle wasting. These findings provide the basis for the use of activin or its downstream targets for the improvement of impaired wound healing, and of activin antagonists for the prevention and treatment of fibrosis and of malignant tumours that overexpress activin. Here, we summarize the previously described roles of activin in wound healing and scar formation and discuss functional studies that revealed different functions of activin in the pathogenesis of cancer. The relevance of these findings for clinical applications will be highlighted.

Activin enhances skin tumourigenesis and malignant progression by inducing a pro-tumourigenic immune cell response.

Activin is an important orchestrator of wound repair, but its potential role in skin carcinogenesis has not been addressed. Here we show using different types of genetically modified mice that enhanced levels of activin in the skin promote skin tumour formation and their malignant progression through induction of a pro-tumourigenic microenvironment. This includes accumulation of tumour-promoting Langerhans cells and regulatory T cells in the epidermis. Furthermore, activin inhibits proliferation of tumour-suppressive epidermal γδ T cells, resulting in their progressive loss during tumour promotion. An increase in activin expression was also found in human cutaneous basal and squamous cell carcinomas when compared with control tissue. These findings highlight the parallels between wound healing and cancer, and suggest inhibition of activin action as a promising strategy for the treatment of cancers overexpressing this factor.

An important role of lymphatic vessel activation in limiting acute inflammation.

In contrast to the established role of blood vessel remodeling in inflammation, the biologic function of the lymphatic vasculature in acute inflammation has remained less explored. We studied 2 established models of acute cutaneous inflammation, namely, oxazolone-induced delayed-type hypersensitivity reactions and ultraviolet B irradiation, in keratin 14-vascular endothelial growth factor (VEGF)-C and keratin 14-VEGF-D transgenic mice. These mice have an expanded network of cutaneous lymphatic vessels. Transgenic delivery of the lymphangiogenic factors VEGF-C and the VEGFR-3 specific ligand mouse VEGF-D significantly limited acute skin inflammation in both experimental models, with a strong reduction of dermal edema. Expression of VEGFR-3 by lymphatic endothelium was strongly down-regulated at the mRNA and protein level in acutely inflamed skin, and no VEGFR-3 expression was detectable on inflamed blood vessels and dermal macrophages. There was no major change of the inflammatory cell infiltrate or the composition of the inflammatory cytokine milieu in the inflamed skin of VEGF-C or VEGF-D transgenic mice. However, the increased network of lymphatic vessels in these mice significantly enhanced lymphatic drainage from the ear skin. These results provide evidence that specific lymphatic vessel activation limits acute skin inflammation via promotion of lymph flow from the skin and reduction of edema formation.

Human leukocyte transmigration across Galalpha(1,3)Gal-negative porcine endothelium is regulated by human CD18 and CD99.

BACKGROUND: In pig-to-human xenotransplantation cross-species receptor interactions mediate cellular infiltration and rejection of porcine grafts. However, the mechanisms responsible for recruitment of human leukocyte subsets across porcine endothelial cells (EC) remain largely unknown. Here, we investigated the role of CD99, CD18, and Galalpha(1,3)Gal (Gal) in this process. METHODS: Adhesion and transmigration of human peripheral blood mononuclear cell (PBMC) subsets on Gal and Gal porcine EC (pEC) and on human EC was analyzed using a two-compartment system separated by a permeable membrane. The mechanisms of human PBMC recruitment to pEC were investigated by blocking cell surface receptors and by differentially measuring adhesion and transendothelial migration (TEM). RESULTS: Blocking of CD18, but not CD99, decreased human PBMC adhesion on pEC, whereas blocking of CD18 or CD99 strongly reduced the subsequent human PBMC TEM across pEC. The inhibitory effect of CD99 blockade was slightly stronger across pEC as compared with human EC. A critical role for Gal in TEM of human monocytes, B, natural killer (NK), NK/T, and T cells was excluded by evaluating TEM across pEC derived from Gal and Gal pigs. CONCLUSIONS: CD99 and CD18, but not Gal, play a critical role in human monocyte and lymphocyte TEM across pEC, and their respective porcine ligands may serve as targets to specifically inhibit human leukocyte recruitment in pig-to-human xenotransplantation.

Keratinocyte-derived follistatin regulates epidermal homeostasis and wound repair.

Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound.

Betacellulin regulates hair follicle development and hair cycle induction and enhances angiogenesis in wounded skin.

Betacellulin (BTC) belongs to the EGF family, whose members play important roles in skin morphogenesis, homeostasis, and repair. However, the role of BTC in skin biology is still unknown. We employed transgenic mice overexpressing BTC ubiquitously to study its role in skin physiology. Immunohistochemistry revealed increased levels of BTC especially in the hair follicles and in the epidermis of transgenic animals. Expression of key markers of epithelial differentiation was unaltered, but keratinocyte proliferation was significantly increased. At post-natal day 1 (P1), transgenic mice displayed a significant retardation of hair follicle morphogenesis. At P17, when most follicles in control mice had initiated hair follicle cycling and had already entered into their first late catagen or telogen phase, all follicles of transgenic mice were still at the mid- to late catagen phases, indicating retarded initiation of hair follicle cycling. Healing of full-thickness excisional wounds and bursting strength of incisional wounds were similar in control and transgenic mice. However, an increase in the area covered by blood vessels at the wound site was detected in transgenic animals. These results provide evidence for a role of BTC in the regulation of epidermal homeostasis, hair follicle morphogenesis and cycling, and wound angiogenesis.

Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes.

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.

Endothelial cells derived from pigs lacking Gal alpha(1,3)Gal: no reduction of human leukocyte adhesion and natural killer cell cytotoxicity.

BACKGROUND: The expression of galactose-alpha(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal-/- pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal-/- and Gal+/+ pigs, and (2) the susceptibility of such Gal-/- porcine endothelial cells to human natural killer (NK) cell cytotoxicity. METHODS: Primary porcine aortic endothelial cells (PAEC) were isolated from Gal-/- (PAEC-Gal-/-) and Gal (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays. RESULTS: Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal-/- cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal-/- and PAEC-Gal+/+ was detected. Tumor-necrosis factor-alpha stimulation of PAEC-Gal-/- and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal-/- expression on PAEC-Gal cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2-activated NK cells. CONCLUSIONS: Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.