Norbert E. Fusenig

c-Jun and JunB Antagonistically Control Cytokine-Regulated Mesenchymal–Epidermal Interaction in Skin

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes

An in vitro carcinogenesis model of human skin keratinocytes has been developed based on the spontaneously immortalized keratinocyte cell line HaCaT. Immortalization, the initial stage in human carcinogenesis in vitro, was induced by ultraviolet‐type mutations in the p53 gene followed by further genetic alterations leading to the loss of senescence genes, in particular on chromosome 3p. Despite multiple genetic changes, the HaCaT cell line sustained its genomic balance up to high passage levels and maintained a non‐tumorigenic phenotype. Tumorigenic transformation was induced by ras oncogene transfection but also by culture stress and elevated temperature, resulting in benign and malignant tumorigenic clones. Malignant conversion was associated with the loss of a copy of chromosome 15, leading to a decrease in thrombospondin‐1 (TSP‐1) expression. Heat‐induced malignant conversion was associated with a gain of material on chromosome 11, including the cyclin D1 gene. The microenvironment plays a major role in tumorigenic transformation and the control of malignant cells. Overexpression of platelet‐derived growth factor in HaCaT cells caused mesenchyme activation and formation of benign tumors. Halting tumor angiogenesis completely prevented invasion of malignant cells and induced a benign tumor phenotype. Transfer of a normal chromosome 15 or TSP‐1 transfection into a skin carcinoma line resulted in tumor suppression due to TSP‐1–blocked tumor vascularization. Because of the reduced TSP‐1 expression, blood vessels infiltrated the tumor, and it expanded. Progression to more aggressive tumor phenotypes required the in vivo environment and was caused by selection of a subpopulation and further genetic modifications. The improved autonomous growth of these cells was associated with new expression of granulocyte colony‐stimulating factor and granulocyte‐macrophage colony‐stimulating factor, which acted in an autocrine manner to stimulate proliferation and migration. With this in vitro skin carcinogenesis model we were able to demonstrate multiple stages in the transformation process that were associated with different genetic and phenotypic characteristics. In addition, we documented that modulation of the tumor stroma plays an important and decisive role in tumor development and progression. From this we hypothesize that the growth restraints of the microenvironment are increasingly lost with advancing stages of carcinogenesis but can be restored by modulation of the tumor stroma. Mol. Carcinog. 23:144–158, 1998.

Volumetric computed tomography (VCT): a new technology for noninvasive, high-resolution monitoring of tumor angiogenesis.

Volumetric computed tomography (VCT) is a technology in which area detectors are used for imaging large volumes of a subject with isotropic imaging resolution. We are experimenting with a prototype VCT scanner that uses flat-panel X-ray detectors and is designed for high-resolution three-dimensional (3D) imaging. Using this technique, we have demonstrated microangiography of xeno-transplanted skin squamous cell carcinomas in nude mice. VCT shows the vessel architecture of tumors and animals with greater detail and plasticity than has previously been achieved, and is superior to contrast-enhanced magnetic resonance (MR) angiography. VCT and MR images correlate well for larger tumor vessels, which are tracked from their origin on 3D reconstructions of VCT images. When compared with histology, small tumor vessels with a diameter as small as 50 μm were clearly visualized. Furthermore, imaging small vessel networks inside the tumor tissue improved discrimination of vital and necrotic regions. Thus, VCT substantially improves imaging of vascularization in tumors and offers a promising tool for preclinical studies of tumor angiogenesis and antiangiogenic therapies.

Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.

Mouse epidermal cell cultures: II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin

Abstract In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10 6 viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%. The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.

Autocrine Growth Regulation by Granulocyte Colony-Stimulating Factor and Granulocyte Macrophage Colony-Stimulating Factor in Human Gliomas with Tumor Progression

Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) and/or their receptors are increasingly detected in solid human tumors, although little is known about their function in tumor growth and invasion. We analyzed RNA and protein expression of both factors and their receptors in 22 human gliomas (WHO grade II, III, and IV) and derived cell cultures. G-CSF, GM-CSF, and/or their receptors were expressed in all tumors and derived cell cultures, but coexpression of both factors and receptors was almost exclusively found in grade IV glioblastomas and thus correlated with advanced tumor stage. The functional significance of G-CSF and GM-CSF as regulators for glioma cells was demonstrated by 1) stimulation of proliferation and migration in tumor cells expressing one or both receptors by the corresponding factor; 2) inhibition of growth and migration of glioma cells expressing G-CSF, GM-CSF, and their receptors by neutralizing antibodies to both factors. These results indicate a significant role for both factors in the autocrine regulation of growth and migration in late-stage malignant gliomas and suggest a shift from paracrine to autocrine regulation with tumor progression. The implication of G-CSF and GM-CSF in glioblastoma growth regulation could make these factors further prognostic indicators and raises questions concerning their use in cancer therapy.

Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermo- epidermal junction in organotypic co-culture

Besides medical application as composite skin grafts, in vitro constructed skin equivalents (SEs) or organotypic co-cultures represent valuable tools for cutaneous biology. Major drawbacks of conventional models, employing collagen hydrogels as dermal equivalents (DEs), are a rather poor stability and limited life span, restricting studies to early phases of skin regeneration. Here we present an improved stabilised in vitro model actually providing the basis for skin-like homeostasis. Keratinocytes were grown on dermal equivalents (DEs) reinforced by modified hyaluronic acid fibres (Hyalograft-3D) and colonised with skin fibroblasts, producing genuine dermis-type matrix. These SEs developed a superior epidermal architecture with regular differentiation and ultrastructure, which occurred also faster than in SEs based on collagen-DEs. Critical aspects of differentiation, still unbalanced in early stages, were perfectly re-normalised, most strikingly the co-expression of keratins K1/K10 and downregulation of regeneration-associated keratins such as K16. The restriction of integrin and K15 distribution as well as keratinocyte proliferation to the basal layer underlined the restored tissue polarity, while the drop of growth rates towards physiological levels implied finally accomplishment of homeostasis. This correlated to faster basement membrane (BM) formation and ultrastructurally defined dermo-epidermal junction including abundant anchoring fibrils for strong tissue connection. Whereas the fibroblasts in the scaffold initially secreted a typical provisional regenerative matrix (fibronectin, tenascin), with time collagens of mature dermis (type I and III) were accumulating giving rise to an in vivo-like matrix with regularly organised bundles of striated collagen fibrils. In contrast to the more catabolic state in conventional DEs, the de novo reconstruction of genuine dermal tissue seemed to be a key element for maintaining prolonged normal keratinocyte proliferation (followed up to 8 wks), fulfilling the criteria of tissue-homeostasis, and possibly providing a stem cell niche.

Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

The human keratinocyte cell line HaCaT expresses essentially all epidermal differentiation markers but exhibits deficiencies in tissue organization as surface transplants in nude mice and even more so in organotypic co-cultures with fibroblasts. Whereas tissue differentiation by normal keratinocytes (NEKs) is regulated by stromal interactions, this mechanism is impaired in HaCaT cells. This regulatory process is initiated by interleukin-1 (IL-1) release in keratinocytes, which induces expression of keratinocyte growth factor (KGF/FGF-7) and granulocyte macrophage-colony stimulating factor (GM-CSF) in fibroblasts. Production and release of IL-1 is very low and, consequently, expression of the fibroblast-derived growth factors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GMCSF, respectively, is inefficient to improve stratification and differentiation by HaCaT cells due to the low expression of their cognate receptors. More importantly, expression and release of the autocrine keratinocyte growth factor TGF-α is dramatically decreased in HaCaT cells. Addition of TGF- α or EGF stimulated HaCaT cell proliferation but, even more effectively, suppressed apoptosis, thus facilitating the formation of a regularly stratified epithelium. Furthermore, TGF-α enhanced the expression of the receptors for KGF and GM-CSF so that addition of these growth factors, or of their inducer IL-1, further improved epidermal tissue differentiation leading to in vitro skin equivalents comparable with cultures of NEKs. Thus, supplementing TGF-α normalized epidermal tissue regeneration by immortal HaCaT keratinocytes and their interaction with stromal cells so that regular skin equivalents are produced as standardized in vitro models.

Critical role for NF-κB-induced JunB in VEGF regulation and tumor angiogenesis

Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP‐1 transcription factor is considered as facilitator of hypoxia‐induced VEGF expression through interaction with hypoxia‐inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP‐1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP‐1 subunit junB is a target gene of hypoxia‐induced signaling via NF‐κB. Loss of JunB in various cell types results in severely impaired hypoxia‐induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB‐deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB−/− teratocarcinomas because of severely impaired paracrine‐acting VEGF and the subsequent inability to efficiently recruit host‐derived vessels.

E-cadherin Suppression Accelerates Squamous Cell Carcinoma Progression in Three-Dimensional, Human Tissue Constructs

We studied the link between loss of E-cadherin-mediated adhesion and acquisition of malignant properties in three-dimensional, human tissue constructs that mimicked the initial stages of squamous cell cancer progression. Suppression of E-cadherin expression in early-stage, skin-derived tumor cells (HaCaT-II-4) was induced by cytoplasmic sequestration of beta-catenin upon stable expression of a dominant-negative E-cadherin fusion protein (H-2Kd-Ecad). In monolayer cultures, expression of H-2Kd-Ecad resulted in decreased levels of E-cadherin, redistribution of beta-catenin to the cytoplasm, and complete loss of intercellular adhesion when compared with control II-4 cells. This was accompanied by a 7-fold decrease in beta-catenin-mediated transcription and a 12-fold increase in cell migration. In three-dimensional constructs, E-cadherin-deficient tissues showed disruption of architecture, loss of adherens junctional proteins from cell contacts, and focal tumor cell invasion. Invasion was linked to activation of matrix metalloproteinase (MMP)-mediated degradation of basement membrane in H-2Kd-Ecad-expressing tissue constructs that was blocked by MMP inhibition (GM6001). Quantitative reverse transcription-PCR showed a 2.5-fold increase in MMP-2 and an 8-fold increase in MMP-9 in cells expressing the H-2Kd-Ecad fusion protein when compared with controls, and gel zymography showed increased MMP protein levels. Following surface transplantation of three-dimensional tissues, suppression of E-cadherin expression greatly accelerated tumorigenesis in vivo by inducing a switch to high-grade carcinomas that resulted in a 5-fold increase in tumor size after 4 weeks. Suppression of E-cadherin expression and loss of its function fundamentally modified squamous cell carcinoma progression by activating a highly invasive, aggressive tumor phenotype, whereas maintenance of E-cadherin prevented invasion in vitro and limited tumor progression in vivo.