Fritz W Aberger

Abstract: It has been known for many years that cooperative interactions between oncogenes (e.g. RAS, MYC, BCL2) can fuel cancer growth (1-5), but the restricted druggability of many of those interacting cancer genes has hampered translation of combined targeting to medical cancer therapy. The identification and characterization of cooperative cancer signaling pathways amenable to medical therapy is therefore a crucial step towards the establishment of efficient targeted combination treatments urgently needed to improve cancer therapy. Here we review recent findings of our group and colleagues on the molecular mechanisms of cooperative Hedgehog/GLI and Epidermal Growth Factor Receptor (EGFR) signaling, two clinically relevant oncogenic pathways involved in the development of many human malignancies. We also discuss the possible implications of these findings for the design of a therapeutic regimen relying on combined targeting of key effectors of both pathways.

Abstract: Inhibition of Hedgehog (HH)/GLI signalling in cancer is a promising therapeutic approach. Interactions between HH/GLI and other oncogenic pathways affect the strength and tumourigenicity of HH/GLI. Cooperation of HH/GLI with epidermal growth factor receptor (EGFR) signalling promotes transformation and cancer cell proliferation in vitro. However, the in vivo relevance of HH-EGFR signal integration and the critical downstream mediators are largely undefined. In this report we show that genetic and pharmacologic inhibition of EGFR signalling reduces tumour growth in mouse models of HH/GLI driven basal cell carcinoma (BCC). We describe HH-EGFR cooperation response genes including SOX2, SOX9, JUN, CXCR4 and FGF19 that are synergistically activated by HH-EGFR signal integration and required for in vivo growth of BCC cells and tumour-initiating pancreatic cancer cells. The data validate EGFR signalling as drug target in HH/GLI driven cancers and shed light on the molecular processes controlled by HH-EGFR signal cooperation, providing new therapeutic strategies based on combined targeting of HH-EGFR signalling and selected downstream target genes.

Abstract: The highly conserved Hedgehog/GLI signaling pathway regulates multiple aspects of embryonic development and plays a decisive role in tissue homeostasis and the hematopoietic system by controlling cell fate decisions, stem cell self-renewal, and activation. Loss of negative control of Hedgehog signaling contributes to tumor pathogenesis and progression. In the classical view of canonical Hedgehog signaling, Hedgehog ligand binding to its receptor Patched culminates in the activation of the key pathway activator Smoothened, followed by activation of the GLI transcription factors. Its essential function and druggability render Smoothened well suited to therapeutic intervention. However, recent evidence suggests a critical role of Smoothened-independent regulation of GLI activity by several other signaling pathways including the PI3K/AKT and RAS/RAF/MEK/ERK axes. In addition, the contribution of canonical Hedgehog signaling via Patched and Smoothened to normal and malignant hematopoiesis has been the subject of recent controversies. In this review, we discuss the current understanding and controversial findings of canonical and noncanonical GLI activation in hematological malignancies in light of the current therapeutic strategies targeting the Hedgehog pathway.

Abstract: CYLD is a deubiquitination enzyme that regulates different cellular processes, such as cell proliferation and cell survival. Mutation and loss of heterozygosity of the CYLD gene causes development of cylindromatosis, a benign tumour originating from the skin. Our study shows that CYLD expression is dramatically downregulated in basal cell carcinoma (BCC), the most common cancer in humans. Reduced CYLD expression in basal cell carcinoma was mediated by GLI1-dependent activation of the transcriptional repressor Snail. Inhibition of GLI1 restored the CYLD expression-mediated Snail signaling pathway, and caused a significant delay in the G1 to S phase transition, as well as proliferation. Our data suggest that GLI1-mediated suppression of CYLD has a significant role in basal cell carcinoma progression.

Abstract: Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptch(flox/flox)ERT2(+/-) knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca(2+) signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling.

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Abstract: The Hedgehog (Hh) pathway regulates cell proliferation and survival and contributes to tumorigenesis. We investigated the expression and function of this pathway in B-cell chronic lymphocytic leukemia (CLL) cells and in healthy B lymphocytes. Profiling of cognate Hh pathway members revealed reduced expression of two key Hh signaling effectors, Smoothened (SMOH) and GLI, in CLL cells, whereas transcription levels of other investigated members resembled normal B-lymphocyte levels. Examining the functional role of SMOH and GLI in cell survival, we found that CLL cells were hardly sensitive toward specific SMOH inhibition, but showed an unspecific decline in cell viability in response to high concentrations of the SMOH antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT61 reduced expression of the target gene Patched and preferentially decreased the viability of malignant cells. Specific RNA interference knockdown experiments in a CLL-derived cell line confirmed the autonomous role of GLI in malignant cell survival. GANT61-induced apoptosis in primary leukemic cells was partly attenuated by protective stromal cells, but not soluble sonic hedgehog ligand. In summary, our data show a downregulation of the classical Hh pathway in CLL and suggest an intrinsic SMOH-independent role of GLI in the ex vivo survival of CLL cells.

Abstract: ABSTRACT: BACKGROUND: The GLI transcription factors, mediators of the hedgehog signal bind with high affinity to the consensus sequence GACCACCCA. The affinity of variant single substitutions in GLI binding sites has been measured systematically, but the affinities of the variant binding sites appears low compared to the frequency of occurrence of variant sites in known GLI target gene promoters. RESULTS: We quantified transcriptional activation by GLI using PTCH1 promoter based luciferase reporters containing all single substitutions of the GLI consensus binding site. As expected variants with very low affinity did not activate the reporter. Many lower affinity binding sequences are, however, functional in the presence of moderate GLI concentration. Using two natural non-consensus GLI site promoters we showed that substitution of the variant sequences by consensus leads to comparable activity. CONCLUSIONS: Variant GLI binding sites with relatively low affinity can within natural promoters lead to strong transcriptional activation. This may facilitate the identification of additional direct GLI target genes.

Abstract: Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC.

Abstract: Although deregulated Hedgehog signalling and elevated Gli transcription factor expression are known to promote the development of basal cell carcinoma (BCC), little is known about molecular mechanisms driving the development of specific growth pattern subtypes. Using gene array analysis, we have previously observed that over-expression of GLI1 in human keratinocytes promotes increased expression of the neuronal differentiation markers ARC and ULK1. We asked whether neuronal differentiation is a characteristic of BCC and whether there is any correlation with BCC subtype. Using RT-PCR and immunohistochemistry, we confirmed that the neuronal markers ARC, beta-tubulin III, GAP-43 and Neurofilament are expressed in human BCC but not in normal epidermis. Moreover, we found that expression of these neuronal differentiation markers showed strong correlation to BCC subtype, with more aggressive infiltrative and morphoeic BCC showing low levels or lack of expression compared to nodular, superficial and micronodular subtypes. Primary human keratinocytes retrovirally expressing GLI1(-) and GLI2(-) showed elevated levels of beta-tubulin III and ARC but not Neurofilament or GAP-43, suggesting that beta-tubulin III and Arc may be early targets of aberrant Gli expression in BCC, whereas expression of Neurofilament and GAP-43 are either later, downstream targets or under control of alternative pathways. We propose that neuronal differentiation is a feature of BCC and that expression of these markers is in part due to aberrant Hedgehog signalling. Moreover, we suggest that correlation between loss of expression of neuronal markers in infiltrative and morphoeic BCC subtypes reflects dedifferentiation of more aggressive BCC subtypes.

Abstract: The Hedgehog (Hh) pathway is a main regulation cascade in embryonic differentiation. It is also present in adult tissues and unusual expression has been associated with formation of benign and malignant lesions. We examined the presence of the Hedgehog pathway in normal and pathological human colon tissue. Components investigated include Sonic (Shh), Indian (Ihh), and Desert Hedgehog (Dhh), Gli1, Gli2, Gli3, and Patched (Ptch). Pathological tissue samples comprised 23 benign and 20 malignant lesions of human colon. The influence of the Hedgehog pathway on differentiation and proliferation has been investigated by analyzing the effect of the pathway inhibitor Cyclopamine on human colon cancer cell lines HT29 and CaCo2. In normal colon, we detected expression of Shh and Dhh within the lining epithelium and Patched, Gli1, and Gli2 along the whole crypts. Within all benign lesions, positive staining of Shh, Dhh, Gli1, Gli2, and Ptch was detected. Expression of Shh and Dhh was restricted to single cell aggregates. Malignant lesions also displayed focal staining pattern for Shh and Dhh but to a much lesser extent. We conclude that Hedgehog signaling is involved rather in constant differentiation and renewing of the colonic lining epithelium than in cancer formation, growth, or proliferation.

Abstract: BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. OBJECTIVE: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA). METHODS: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. RESULTS: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. CONCLUSIONS: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

Abstract: Hedgehog (HH) signaling in the epidermis is primarily mediated by the zinc finger transcription factors GLI1 and GLI2. Exquisite regulation of HH/GLI signaling is crucial for proper specification of the epidermal lineage and development of its derivatives, whereas dysregulation of HH/GLI signaling disrupts tissue homeostasis and causes basal cell carcinoma (BCC). Similarly, bone morphogenetic proteins (BMPs) and activins have been described as key signaling factors in the complex regulation of epidermal fate decisions, although their precise interplay with HH/GLI is largely elusive. Here we show that, in human epidermal cells, expression of the activin/BMP antagonist follistatin (FST) is predominantly up-regulated by the HH effector GLI2. Consistently, we found strong FST expression in the outer root sheath of human hair follicles and BCC. Detailed promoter analysis showed that two sequences with homology to the GLI consensus binding site are required for GLI2-mediated activation. Interestingly, activation of the FST promoter is highly GLI2-specific, because neither GLI1 nor GLI3 can significantly increase FST transcription. GLI2 specificity requires the presence of a 518-bp fragment in the proximal FST promoter region. On the protein level, sequences C-terminal to the zinc finger are responsible for GLI2-specific activation of FST transcription, pointing to the existence of GLI-interacting cofactors that modulate GLI target specificity. Our results reveal a key role of GLI2 in activation of the activin/BMP antagonist FST in response to HH signaling and provide new evidence for a regulatory interaction between HH and activin/BMP signaling in hair follicle development and BCC.

Abstract: Mutations in the Hedgehog (Hh) receptor Patched (Ptch) are responsible for a variety of tumors, which show ligand-independent stimulation of the Hh/Ptch signaling cascade. Cyclopamine is an alkaloid of the corn lily Veratrum californicum, which blocks activity of the pathway by inhibition of Smoothened (Smo), the signal transduction partner of Ptch. This results in growth inhibition of Hh/Ptch-dependent tumor cells in vitro, of subcutaneous xenografts as well as of precancerous lesions in Ptch(+/-) mice. However, the evidence that treatment with cyclopamine is an effective anti-cancer therapy against full-blown tumors is sparse. Here, we have investigated the responsiveness of full-blown Hh/Ptch-associated rhabdomyosarcoma (RMS) to this drug. Hh pathway activity and proliferation of cultured primary RMS cells was inhibited by cyclopamine. Hh signaling was also partially suppressed by the drug in RMS in vivo, but cyclopamine treatment did not result in stable disease or tumor regression. It also did not affect proliferation, apoptosis or the differentiation status of RMS. This was in contrast to anti-proliferative effects on tumor growth caused by doxorubicin, an anthracycline routinely used in therapy of human RMS. In summary, our data indicate that there must be additional factors that render full-blown Hh/Ptch-associated RMS insensitive against anti-proliferative effects of cyclopamine in vivo.

Abstract: Basal cell carcinoma (BCC) of the skin is a highly compact, non-metastatic epithelial tumour type that may arise from the aberrant propagation of epidermal or progenitor stem cell (SC) populations. Increased expression of GLI1 is a common feature of BCC and is linked to the induction of epidermal SC markers in immortalized N/Tert-1 keratinocytes. Here, we demonstrate that GLI1 over-expression is linked to additional SC characteristics in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is associated with repressed extracellular signal-regulated kinase (ERK) activity. Colony formation and repressed ERK activity remain evident when EGFR is increased exogenously to the basal levels in GLI1 cells revealing that ERK is additionally inhibited downstream of the receptor. Exposure to epidermal growth factor (EGF) to increase ERK activity and promote migration negates GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and E-cadherin protein expression this is not associated with epithelial-mesenchymal transition (EMT), and GLI1 actually represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated morphology of EGF/GLI1 keratinocytes stems from their being 'stretched' due to migrating cells displaying inefficient cell-cell detachment and impaired tail retraction. Taken together, these data suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype. Finally, ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not routinely co-operate with ERK to induce the formation of this common skin tumour.

Abstract: Efficient manipulation of Hedgehog (HH)/GLI signaling activity is crucial to the analysis of molecular events underlying HH/GLI-regulated cell fate determination and tumor growth. In this article, we describe the use of retroviral expression systems as a valuable tool to activate or repress Hh-pathway activity in a broad spectrum of mammalian cells-including human cells-either by forced expression of the major Hedgehog-effectors GLI1 and GLI2 or by expression of the short-hairpin RNAs-targeting GLI mRNAs. We focus on two distinct retroviral systems that allow efficient and sustainable expression of GLI proteins in primary cells and cell lines of human origin: (i) a Moloney Murine Leukemia Virus-based and (ii) an HIV-derived lentivirus expression system, which allows transduction of both dividing and quiescent cells.

Abstract: During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms (C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3-4 years of age) and five old (68-72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

Abstract: Hedgehog (HH)/GLI signaling plays a critical role in epidermal development and basal cell carcinoma. Here, we provide evidence that epidermal growth factor receptor (EGFR) signaling modulates the target gene expression profile of GLI transcription factors in epidermal cells. Using expression profiling and quantitative reverse transcriptase PCR, we identified a set of 19 genes whose transcription is synergistically induced by GLI1 and parallel EGF treatment. Promoter studies of a subset of GLI/EGF-regulated genes, including the genes encoding interleukin-1 antagonist IL1R2, Jagged 2, cyclin D1, S100A7, and S100A9, suggest convergence of EGFR and HH/GLI signaling at the level of promoters of selected direct GLI target genes. Inhibition of EGFR and MEK/ERK but not of phosphatidylinositol 3-kinase/AKT abrogated synergistic activation of GLI/EGF target genes, showing that EGFR can signal via RAF/MEK/ERK to cooperate with GLI proteins in selective target gene regulation. Coexpression of the GLI/EGF target IL1R2, EGFR, and activated ERK1/2 in human anagen hair follicles argues for a cooperative role of EGFR and HH/GLI signaling in specifying the fate of outer root sheath (ORS) cells. We also show that EGF treatment neutralizes GLI-mediated induction of epidermal stem cell marker expression and provide evidence that EGFR signaling is essential for GLI-induced cell cycle progression in epidermal cells. The results suggest that EGFR signaling modulates GLI target gene profiles which may play an important regulatory role in ORS specification, hair growth, and possibly cancer.

Abstract: We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Abstract: The GLI transcription factors mediate the hedgehog signal in development and carcinogenesis. Basal cell carcinoma can be caused by overexpression of either GLI1 or GLI2. Though GLI1 and GLI2 have identical or very similar DNA binding specificities, some of their activities are overlapping, some are clearly distinct. We analyzed target gene specificities of GLI1 and constitutively active GLI2 (GLI2DeltaN) by global expression profiling in an inducible, well-characterized HaCaT keratinocyte expression system. Four hundred fifty-six genes up- or downregulated at least twofold were identified. GLI target gene profiles correlated well with the biological activities of these transcription factors in hair follicles and basal cell carcinoma. Upregulation of largely overlapping sets of target genes was effected by both factors, repression occurred predominantly in response to GLI2. Also, significant quantitative differences in response to GLI1 and GLI2DeltaN were found for a small number of activated genes. Since we have not detected a putative processed GLI2 repressor, these results point to specific but indirect target gene repression by GLI2DeltaN via preferential activation of one or more negative regulators.

Abstract: The current concept of tumourigenesis holds that cancer results from the progressive acquisition of mutations that endow affected cells with selective growth advantages by activating multiple processes including intrinsic mitogenic and pro-survival pathways. Constitutive activation of the Hedgehog (HH)/GLI signalling cascade has recently been implicated in the growth of a number of human malignancies ranging from semi-malignant tumours of the skin to highly aggressive cancers of the brain, lung, pancreas and prostate. This review focuses on the role of the GLI zinc finger transcription factors, which mediate Hedgehog signalling at the distal end of the pathway. We summarise recent data on the mechanisms by which latent GLI proteins are activated in response to stimulation of Hedgehog signalling. Based on the identification of a growing number of direct GLI target genes, we propose that HH-driven tumourigenesis relies on multiple cellular processes such as promotion of G1/S phase progression, enhancement of cell survival by providing anti-apoptotic cues, increase in metastatic potential of Hedgehog responsive cells, and activation of potential tumour stem cells. In view of the critical role of GLI genes in Hedgehog-associated cancers, strategies that aim at interfering with GLI function are likely to represent efficient approaches in future targeted cancer therapy.

Abstract: Sonic hedgehog (Hh) signaling plays a key role in epidermal development and skin cancer. Mutational inactivation of the tumor suppressor gene patched (PTCH) leads to constitutive activation of the Hh signaling pathway, resulting in activation of target gene transcription by the zinc finger transcription factors GLI1 and GLI2. Recent experiments in mice point to GLI2 as the key mediator of Hh signaling in skin. We have concentrated on the identification of candidate mediators of GLI2 function in the human epidermis. We show here that the forkhead/winged-helix domain transcription factor FOXE1 is likely to be a direct GLI2 target gene. The kinetics of FOXE1 induction are similar to the known direct target PTCH, and a 2.5 kb upstream fragment containing five GLI-binding sites activates transcription in a reporter assay. We show by in situ hybridization that FOXE1 is expressed in the outer root sheath of the hair follicle, where murine Gli2 is also expressed. FOXE1 expression is also found in basal keratinocytes of the human epidermis and basal cell carcinoma (BCC). These data point to a putative role of FOXE1 in mediating Hh signaling in the human epidermis downstream of GLI2.

Abstract: In stratified epidermis, activation of the Hh/Gli signal transduction pathway has been implicated in the control of cell proliferation and tumorigenesis. The zinc-finger transcription factor Gli2 has been identified as critical mediator of the Hh signal at the distal end of the pathway, but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies such as basal cell carcinoma are still unclear. Here, we provide evidence for a role of human GLI2 in antagonizing contact inhibition and epidermal differentiation. We show by gene expression profiling that activation of the GLI2 oncogene in human keratinocytes activates the transcription of a number of genes involved in cell cycle progression such as E2F1, CCND1, CDC2 and CDC45L, while it represses genes associated with epidermal differentiation. Analysis of the proliferative effect of GLI2 revealed that GLI2 is able to induce G1-S phase progression in contact-inhibited keratinocytes. Detailed time-course experiments identified E2F1 as early transcriptional target of GLI2. Further, we show that GLI2 expression in human keratinocytes results in a marked downregulation of epidermal differentiation markers. The data suggest a role for GLI2 in Hh-induced epidermal neoplasia by opposing epithelial cell cycle arrest signals and epidermal differentiation.

Abstract: Sonic hedgehog (Shh) binds to its receptor patched (PTCH), leading to the activation and repression of target genes via the GLI family of zinc-finger transcription factors. Deregulation of the Shh pathway is associated with basal cell carcinoma (BCC) due to upregulation of GLI1 and GLI2. We recently demonstrated a positive feedback loop between GLI1 and GLI2, which revealed that GLI1 may be a direct target of GLI2. Using band shift and luciferase reporter assays, we now show that GLI2 binds the GLI-binding consensus sequence in the GLI1 promoter. These data suggest that GLI2 directly activates GLI1 and that retrovirally expressed GLI2 induces expression of endogenous GLI1 in human primary keratinocytes. Finally, using in situ hybridization, we show that GLI2 is expressed in the interfollicular epidermis and the outer root sheath of hair follicles in normal skin as well as in BCC tumor islands. These results suggest an important role for GLI2 in regulating epidermal proliferation and skin tumorigenesis.

Abstract: Aberrant activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of basal cell carcinoma (BCC). The zinc finger transcription factors GLI1 and GLI2 are considered mediators of the HH signal in epidermal cells, although their tumorigenic nature and their relative contribution to tumorigenesis are only poorly understood. To shed light on the respective role of these transcription factors in epidermal neoplasia, we screened for genes preferentially regulated either by GLI1 or GLI2 in human epidermal cells. We show here that expression of the key antiapoptotic factor BCL2 is predominantly activated by GLI2 compared with GLI1. Detailed promoter analysis and gel shift assays identified three GLI binding sites in the human BCL2 cis-regulatory region. We found that one of these binding sites is critical for conferring GLI2-specific activation of the human BCL2 promoter and that the selective induction of BCL2 expression depends on the zinc finger DNA binding domain of GLI2. In vivo, GLI2 and BCL2 were coexpressed in the outer root sheath of hair follicles and BCC and in plasma cells that infiltrated BCC tumor islands. On the basis of the latter observation, we analyzed plasma cell-derived tumors and found strong expression of GLI2 and BCL2 in neoplastic cells of plasmacytoma patients, implicating HH/GLI signaling in the development of plasma cell-derived malignancies. The results reveal a central role for GLI2 in activating the prosurvival factor BCL2, which may represent an important mechanism in the development or maintenance of cancers associated with inappropriate HH signaling.

Abstract: Proteins of the suppressors of cytokine signaling (SOCS) family have important functions as negative regulators of cytokine signaling. We show here that SOCS-1 expression can be induced in the human epithelial lung cell line A549 by IL-4 and IL-13. Analysis of reporter gene constructs under control of the SOCS-1 promoter provides evidence that IL-4- and IL-13-induced up-regulation is dependent on three IFN-gamma-activated sequence motifs of the sequence TTC(N)(4)GAA, which is known for binding STAT6. The three motifs are situated close to each other approximately 600 bp upstream of the transcriptional initiation site. When mutations were inserted into all three IFN-gamma-activated sequence motifs at the same time, IL-4-IL-13-induced luciferase activity was abrogated. With single and double mutants, promoter activity was diminished in comparison with the wild-type promoter. STAT6 is therefore required for IL-4-IL-13-dependent SOCS-1 expression in A549 cells, and the three identified binding motifs cooperate to induce maximal transcription. EMSAs conducted with nuclear extracts of IL-4- and IL-13-stimulated A549 cells showed that STAT6 was able to bind to each of the three binding motifs. Finally, cotransfection of a SOCS-1 expression vector inhibited activation of SOCS-1 promoter luciferase constructs. Thus, SOCS-1 is able to autoregulate its expression via a negative feedback loop.

Abstract: The photosensitizer protoporphyrin IX, endogenously accumulated from the precursor aminolevulinic acid, is a successful agent in photodynamic tumor therapy. In spite of encouraging clinical results, the basic mechanisms leading to cell death are not fully understood. We therefore set out to analyse the alteration of the gene expression pattern in the squamous cell carcinoma cell line A-431 after photodynamic treatment with endogenous protoporphyrin IX. Radioactively labelled cDNAs from untreated and treated cells were hybridized onto UniGene cDNA array filters containing lysed bacterial colonies with inserts representing approximately 32000 different human transcripts. Differentially expressed genes were identified and verified on sub-arrays containing only the candidate genes. We found increased expression of heat shock protein 70 and of the immediate early genes p55-c-fos and c-jun, may be due to oxidative stress and increased levels of intracellular calcium after photoactivation of protoporphyrin IX. Increased expression of heme oxygenase-1 following dark incubation was not further increased by irradiation and therefore probably caused by the need for heme degradation. Presumably heat shock protein 70 and heme oxygenase-1 serve for cell protection. Though similar results have been found for photodynamic treatment with external porphyrin-based photosensitizers, this is the first report about induction of the genes described above by (photoactivated) endogenous protoporphyrin IX.

Abstract: Transgenic mouse models have provided evidence that activation of the zinc-finger transcription factor GLI1 by Hedgehog (Hh)-signalling is a key step in the initiation of the tumorigenic programme leading to Basal Cell Carcinoma (BCC). However, the downstream events underlying Hh/GLI-induced BCC development are still obscure. Using in vitro model systems to analyse the effect of Hh/GLI-signalling in human keratinocytes, we identified a positive feedback mechanism involving the zinc finger transcription factors GLI1 and GLI2. Expression of GLI1 in human keratinocytes induced the transcriptional activator isoforms GLI2alpha and GLI2beta. Both isoforms were also shown to be expressed at elevated levels in 21 BCCs compared to normal skin. Detailed time course experiments monitoring the transcriptional response of keratinocytes either to GLI1 or to GLI2 suggest that GLI1 is a direct target of GLI2, while activation of GLI2 by GLI1 is likely to be indirect. Furthermore, expression of either GLI2 or GLI1 led to an increase in DNA-synthesis in confluent human keratinocytes. Taken together, these results suggest an important role of the positive GLI1-GLI2 feedback loop in Hh-mediated epidermal cell proliferation.

Abstract: A sensitive, specific, reproducible, robust, and cost-effective customized cDNA array system based on established nylon membrane technology has been developed for convenient multisample expression profiling for several hundred genes of choice. The genes represented are easily adjusted (depending on the availability of corresponding cDNAs) and the method is accordingly readily applicable to a wide variety of systems. Here we have focused on the expression profiles for interferon-alpha2a, the most widely used interferon for the treatment of viral hepatitis and malignancies, in primary cells (peripheral blood mononuclear cells, T cells, and dendritic cells) and cell lines (Kit255, HT1080, HepG2, and HuH7). Of 150 genes studied, only six were consistently induced in all cell types and donors, whereas 74 genes were induced in at least one cell type. IRF-7 was identified as the only gene exclusively induced in the hematopoietic cells. No gene was exclusively induced in the nonhematopoietic cell lines. In T cells 12, and in dendritic cells, 25 genes were induced in all donors whereas 45 and 42 genes, respectively, were induced in at least one donor. The data suggest that signaling through IFN-alpha2 can be substantially modulated to yield significant cell-type and donor-specific qualitative and quantitative differences in gene expression in response to this cytokine under highly standardized conditions.

Abstract: The combination of high and low density cDNA filter array technology potentially permits both the identification of subsets of induced genes and convenient and rapid multisample expression profiling of such subsets under a variety of conditions. The JAK/STAT1 pathway for IFN-gamma signaling in human cells has been well characterized, but the extent and importance of additional pathways remain to be established. Here, using high-density filter arrays of the RZPD UniGene set, we identified 18 novel IFN-gamma-inducible genes. Expression profiling was carried out using low-density arrays representing both novel and known IFN-gamma-inducible genes. Initial experiments failed to detect evidence for any novel non-JAK-dependent pathways in cells expressing a kinase-dead JAK2. The data, however, validated the potential of the combined methods in establishing rapid and convenient expression profiling of several hundred genes in response to any ligand of choice.

Abstract: To characterize the formation of the dopaminergic system in the developing zebrafish CNS, we cloned cDNAs encoding tyrosine hydroxylase (th), an enzyme in dopamine synthesis, and the dopamine transporter (dat), a membrane transport protein which terminates dopamine action by re-uptake. Dopaminergic neurons are first detected between 18 and 19 h post-fertilization in a cluster of cells in the ventral diencephalon. Subsequently, th and dat detection identifies dopaminergic neurons in the olfactory bulb, the pretectum, the retina and the locus coeruleus. Neurons expressing th but not dat are adrenergic or noradrenergic, and are found in the locus coeruleus, the medulla, the likely analog of the carotid body, and precursors of the enteric and sympathetic nervous system.

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