[Nonautonomous effects of oncogenic YAP in hepatocarcinogenesis]. / Nichtautonome Effekte des Onkogens YAP in der Hepatokarzinogenese.

BACKGROUND: The transcriptional coactivator yes-associated protein (YAP) is a strong oncogene in liver cancer development. OBJECTIVES: To investigate if and how YAP-induced paracrine-acting factors are regulated in hepatocytes and liver cancer cells. MATERIAL AND METHODS: Transcriptome analysis and proteomics of murine wildtype and YAP-transgenic hepatocytes were performed to identify paracrine-acting proteins. Molecular and biochemical techniques were used to examine the mechanisms of YAP-dependent gene regulation. Gene expression data from HCC (hepatocellular carcinoma) patients was evaluated. RESULTS: Several YAP-dependent, secreted factors (e. g. CXCL10, GDF15, PDGFB) were identified. YAP regulates these factors through transcription factors of the TEAD (TEA domain) protein family. Moreover, the dysregulation of the YAP-target genes is often associated with poor HCC patient prognosis. CONCLUSIONS: YAP induces the expression of paracrine-acting factors that may affect the tumor microenvironment and therefore support carcinogenesis. This multicellular network could allow the development of novel and specific perturbation approaches.

Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress.

CD44, a large family of transmembrane glycoproteins, plays decisive roles in physiological and pathological conditions. CD44 isoforms are involved in several signaling pathways essential for life such as growth factor-induced signaling by EGF, HGF or VEGF. CD44 is also the main hyaluronan (HA) receptor and as such is involved in HA-dependent processes. To allow a genetic dissection of CD44 functions in homeostasis and disease, we generated a Cd44 floxed allele allowing tissue- and time-specific inactivation of all CD44 isoforms in vivo. As a proof of principle, we inactivated Cd44 in the skin epidermis using the K14Cre allele. Although the skin of such Cd44Δker mutants appeared morphologically normal, epidermal stiffness was reduced, wound healing delayed and TPA induced epidermal thickening decreased. These phenotypes might be caused by cell autonomous defects in differentiation and HA production as well as impaired adhesion and migration on HA by Cd44Δker keratinocytes. These findings support the usefulness of the conditional Cd44 allele in unraveling essential physiological and pathological functions of CD44 isoforms.

Patch-Based Nonlinear Image Registration for Gigapixel Whole Slide Images.

OBJECTIVE: Image registration of whole slide histology images allows the fusion of fine-grained information-like different immunohistochemical stains-from neighboring tissue slides. Traditionally, pathologists fuse this information by looking subsequently at one slide at a time. If the slides are digitized and accurately aligned at cell level, automatic analysis can be used to ease the pathologist's work. However, the size of those images exceeds the memory capacity of regular computers. METHODS: We address the challenge to combine a global motion model that takes the physical cutting process of the tissue into account with image data that is not simultaneously globally available. Typical approaches either reduce the amount of data to be processed or partition the data into smaller chunks to be processed separately. Our novel method first registers the complete images on a low resolution with a nonlinear deformation model and later refines this result on patches by using a second nonlinear registration on each patch. Finally, the deformations computed on all patches are combined by interpolation to form one globally smooth nonlinear deformation. The NGF distance measure is used to handle multistain images. RESULTS: The method is applied to ten whole slide image pairs of human lung cancer data. The alignment of 85 corresponding structures is measured by comparing manual segmentations from neighboring slides. Their offset improves significantly, by at least 15%, compared to the low-resolution nonlinear registration. CONCLUSION/SIGNIFICANCE: The proposed method significantly improves the accuracy of multistain registration which allows us to compare different antibodies at cell level.

miR-615-5p is restrictedly expressed in cirrhotic and cancerous liver tissues and its overexpression alleviates the tumorigenic effects in hepatocellular carcinoma.

microRNAs aberrant behavior in heptocellular carcinoma (HCC) plays a major role in HCC pathogenesis. miR-615-5p expression has never been evaluated in HCC. We showed that miR-615-5p was preferentially expressed in HCC, cirrhotic liver tissues and HCC cell lines, but undetected in normal livers. Forced miR-615-5p expression in HCC cell lines led to significant decrease in cell growth and migration. In-silico predication revealed insulin-like growth factor-II (IGF-II) as a potential downstream target for miR-615-5p. Forcing the expression of miR-615-5p showed downregulation of IGF-II mRNA, as well as inhibition of the luciferase activity in a luciferase reporter vector harboring the IGF-II-3'UTR target sequence. miR-615-5p acts as tumor-suppressor in HCC through targeting IGF-II.

The transcriptional coactivators megakaryoblastic leukemia 1/2 mediate the effects of loss of the tumor suppressor deleted in liver cancer 1.

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. Here, we show that the transcriptional coactivators Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack DLC1. Moreover, DLC1 loss and MKL1 nuclear localization correlate in primary human hepatocellular carcinoma. Nuclear accumulation of MKL1 in DLC1-deficient cancer cells is accomplished by activation of the RhoA/actin signaling pathway and concomitant impairment of MKL1 phosphorylation, which results in constitutive activation of MKL1/2 target genes. We provide evidence that MKL1/2 mediates cancerous transformation in DLC1-deficient hepatocellular and mammary carcinoma cells. Depletion of MKL1/2 suppresses cell migration, cell proliferation and anchorage-independent cell growth induced by DLC1 loss.

Signaling networks in human hepatocarcinogenesis--novel aspects and therapeutic options.

Hepatocellular carcinoma (HCC) represents one of the most common human malignancies with poor prognosis. Because therapeutic strategies are insufficient for most HCC patients, there is a great need to determine the central molecular mechanisms and pathways in order to derive novel targets for systemic therapy. There is vast evidence that not only the dysregulation of distinct signaling cascades, but also their interactions at different levels, affect tumor cell function. Through these interactions, the effects of pathways can be increased, and even new tumor-supporting qualities acquired that further facilitate HCC progression. Although several approaches for the modulation of these relevant pathways are under development, future therapeutic strategies should take into account that oncogenic stimuli cannot be understood in a monodimensional manner. In order to avoid escape mechanisms during therapy, strategies based on comprehensive knowledge of the interactive regulatory network in hepatocarcinogenesis are necessary.

[Molecular mechanisms of progression in human hepatocarcinogenesis]. / Molekulare Progressionsmechanismen der humanen Hepatokarzinogenese.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors worldwide with poor prognosis. Based on high-throughput screening technology, we and others have identified factors and pathways that are pivotal for tumor progression including transcription factors and microtubule-interacting proteins. In addition, aberrant activation of the IGF signalling pathway is frequently observed in HCCs which is predominantly based on high level expression of its ligand IGF-II. Because protumorigenic effects of IGF-II such as proliferation, anti-apoptosis, and migration are transmitted through its receptor IGF-1R, selective inhibition of this tyrosine kinase by small molecule compounds might reduce IGF-II-driven tumor growth. Indeed, administration of IGF-1R-selective inhibitors reduces IGF-II-induced effects and was associated with a significant reduction of tumor growth in a xenograft transplantation model. In conclusion, the IGF-II/IGF-1R signalling pathway is critically involved in the regulation of tumor growth and tumor cell dissemination, representing a promising therapeutic target structure in the treatment of HCC.

Proinflammatory cytokines cause FAT10 upregulation in cancers of liver and colon.

The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.

Large-scale N-terminal deletions but not point mutations stabilize beta-catenin in small bowel carcinomas, suggesting divergent molecular pathways of small and large intestinal carcinogenesis.

Small intestinal adenocarcinoma is rare and its molecular pathogenesis is incompletely understood. Stabilization of beta-catenin, a mediator of wnt/wingless signalling, can be detected in 50% of sporadic carcinomas but, in contrast to colorectal cancer, this finding can not be explained by the inactivation of adenomatous polyposis coli (APC). In order to elucidate the molecular background of beta-catenin stabilization in small intestinal adenocarcinoma, we investigated 20 non-familial adenomatous polyposis coli (FAP)-associated tumours, including five microsatellite-unstable carcinomas for beta-catenin alterations, by immunohistochemistry, western blot analysis and sequence analysis on the RNA and DNA levels. Nuclear accumulation of beta-catenin was found in 50% of carcinomas. In 30%, nuclear stabilization was restricted to tumour cells at the invasion front, while 20% of tumours displayed intense homogeneous nuclear stabilization throughout all areas. Large deletions and insertions in the beta-catenin gene (CTNNB1) resulting in a partial or complete in-frame loss of exons 3 and 4 on the RNA-transcript level were found in the latter, exclusively microsatellite-stable carcinomas. The mutations resulted in the stabilization of aberrant beta-catenin lacking large parts of N-terminal protein domains. No point mutations in CTNNB1 were observed. Our data show that large intragenic CTNNB1 mutations stabilize beta-catenin in small intestinal adenocarcinomas and influence the subcellular distribution of the protein. In contrast to colon carcinomas, neither APC nor CTNNB1 point mutations seem to play a significant role in carcinogenesis, indicating divergent mechanisms of wnt/wingless control in the small and the large intestine.

[Molecular pathogenesis of hepatocellular carcinoma: new therapeutic approaches and predictive pathology]. / Molekulare Pathogenese des hepatozellulären Karzinoms : Ansatzpunkte für neue Therapien und prädiktive Pathologie.

Hepatocellular carcinoma is one of the most prevalent malignancies worldwide and its incidence is increasing. Multimodal strategies directed towards this carcinoma include primary (e.g. immunisation) and secondary (e.g. antiviral therapy) prevention, surgical approaches, novel specific systemic therapies (targeted therapy), and the treatment of comorbidity (cirrhosis). New molecular approaches are currently under development. These tackle several specific targets, with pathology being challenged in many aspects: experimental evaluation, the development of valid tumor-relevant diagnostic tests as well as morphological evaluation in the context of clinical studies, and finally in routine diagnosis.

[Reduced expression of the E3-ubiquitin ligase seven in absentia homologue (SIAH)-1 in human hepatocellular carcinoma]. / Reduzierte Expression der E3-ubiquitin Ligase seven in absentia homologue (SIAH)-1 in Zellen des humanen hepatozellulären Karzinoms.

SIAH-1 (seven in absentia homologue-1) is an E3-ubiquitin ligase that facilitates labelling and subsequent proteasomal degradation of different proteins like transcription factors (e.g. c-myb) and coactivators (e.g. beta-catenin). Here we show that SIAH-1 expression is frequently reduced in human hepatocarcinogenesis. However, further reduction of SIAH-1 bioavailability by gene-specific siRNA (RNAinterference) in HCC cell lines resulted in significantly decreased tumor cell viability. Therefore we conclude that distinct SIAH-1 levels mediate pro-tumorigenic effects in HCC cells and that further SIAH-1 inhibition may represent a new therapeutic strategy in the treatment of human hepatocellular carcinoma.

[Insulin-like growth factor (IGF)-signalling pathway components are potential therapeutic targets in the treatment of human hepatocellular carcinoma]. / Insulin-like growth factor (IGF)-Signalwegskomponenten sind potentielle therapeutische Zielstrukturen bei der Behandlung des hepatozellulären Karzinoms.

The ligand insulin-like growth factor (IGF)-II is highly overexpressed in human hepatocellular carcinoma (HCC) and promotes tumour cell growth. Thus, this signalling axis is a prime target for potential anti-cancer therapies. In this context, gene-specific siRNA against IGF-signalling components as well as IGF1R selective receptor tyrosine kinase (RTK)-inhibitors (tyrphostins) may therefore offer new therapeutic options since both small interfering RNAs (siRNA) and small inhibitory molecules significantly reduce IGFIR signalling in HCC cell lines. However, since highly specific inhibition by siRNA is currently not applicable in the treatment of cancer, selective RTK-inhibitors represent the most promising approach for future therapeutic strategies.

Podocalyxin-like protein 1 expression in primary hepatic tumours and tumour-like lesions.

AIMS: The differential diagnosis of benign hepatic lesions and well-differentiated hepatocellular carcinomas can be a challenge, especially in small biopsy specimens. Recently, novel proteins expressed by the neovasculature in hepatocellular carcinoma (HCC) have been identified. The aim of this study was to compare the expression of podocalyxin-like protein 1 (PODXL1), a CD34-related sialomucin, in HCC and benign liver tumours or tumour-like lesions. METHODS AND RESULTS: Vascular marker expression was examined using tissue microarrays as well as standard paraffin sections from formalin fixed paraffin-embedded liver tissue samples. Expression of PODXL1 was compared with anti-CD34, CD31 and von Willebrand factor VIII staining by immunohistochemistry. PODXL1 is expressed in tumour-associated microvasculature endothelial cells in HCC, as well as in capillarized sinusoidal endothelium of focal nodular hyperplasia (FNH) and hepatic adenoma. Expression in cirrhotic nodules correlates with CD34 and highlights endothelium in the inflow area. In dysplastic nodules CD34 and PODXL1 are not or only focally expressed. CONCLUSIONS: Expression patterns of CD34 and PODXL1 are almost identical in primary hepatic tumours and tumour-like lesions. The presence of CD34+ and PODXL1+ sinusoidal endothelial cells aids in the diagnosis of HCC. Sinusoidal expression of PODXL1 is also seen in a less diffuse pattern in FNH and adenoma.

Dysregulation of growth factor signaling in human hepatocellular carcinoma.

Dysregulation of pleiotropic growth factors, receptors and their downstream signaling pathway components represent a central protumorigenic principle in human hepatocarcinogenesis. Especially the Insulin-like Growth Factor/IGF-1 receptor (IGF/IGF-1R), Hepatocyte Growth Factor (HGF/MET), Wingless (Wnt/beta-catenin/FZD), Transforming Growth Factor alpha/Epidermal Growth Factor receptor (TGFalpha/EGFR) and Transforming Growth Factor beta (TGFbeta/TbetaR) pathways contribute to proliferation, antiapoptosis and invasive behavior of tumor cells. This review focuses on the relevant alterations in these pathways identified in human human hepatocellular carcinomas (HCCs). Resultant functional effects are modulated by multiple cross-talks between the different signaling pathways and additional tumor-relevant factors, such as cyclooxygenase-2 and p53. Several specific strategies are currently under development such as receptor kinase inhibitors, neutralizing antibodies and antagonistic proteins, which may improve the systemic treatment of human HCCs.

Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade--results of an explorative CGH meta-analysis.

All available comparative genomic hybridisation (CGH) analyses (n=31, until 12/2003) of human hepatocellular carcinomas (HCCs; n=785) and premalignant dysplastic nodules (DNs; n=30) were compiled and correlated with clinical and histological parameters. The most prominent amplifications of genomic material were present in 1q (57.1%), 8q (46.6%), 6p (22.3%), and 17q (22.2%), while losses were most prevalent in 8p (38%), 16q (35.9%), 4q (34.3%), 17p (32.1%), and 13q (26.2%). Deletions of 4q, 16q, 13q, and 8p positively correlated with hepatitis B virus aetiology, while losses of 8p were more frequently found in hepatitis C virus-negative cases. In poorly differentiated HCCs, 13q and 4q were significantly under-represented. Moreover, gains of 1q were positively correlated with the occurrence of all other high-frequency alterations in HCCs. In DNs, amplifications were most frequently present in 1q and 8q, while deletions occurred in 8p, 17p, 5p, 13q, 14q, and 16q. In conclusion, aetiology and dedifferentiation correlate with specific genomic alterations in human HCCs. Gains of 1q appear to be rather early events that may predispose to further chromosomal abnormalities. Thus, explorative CGH meta-analysis generates novel and testable hypotheses regarding the cause and functional significance of genomic alterations in human HCCs.

[Insulin-like growth factor (IGF)-II in human hepatocarcinogenesis--a potential therapeutic target?]. / Insulin-like growth factor (IGF)-II in der humanen Hepatokarzinogenese--eine potentielle therapeutische Zielstruktur?

Several studies have examined the expression profiles of human hepatocellular carcinomas (HCCs) using high density microarray technology, but subtyping with potential mechanistic and therapeutic impact has not been achieved so far. Here we have analysed the expression pattern of human HCCs and HCC cell lines in comparison to normal liver. A characteristic of one group of HCCs and all HCC cell lines was overexpression of insulin-like growth factor (IGF)-II. Moreover, IGF-II expression was mutually exclusive to induction of several IFN-related genes. In vitro, treatment of HCC cells with IFNgamma leads to a strong reduction of IGF-II expression. Equally, specific reduction of IGF-II was achieved using RNAinterference in HCC cells. Therefore, IGF-II may represent an excellent target for IFNgamma-treatment and specific siRNA-mediated therapeutic intervention.

The designer cytokine hyper-IL-6 mediates growth inhibition and GM-CSF-dependent rejection of B16 melanoma cells.

The low immunogenic B16 melanoma cell line was transfected with a mammalian expression vector containing the complementary DNA for a sIL-6R/IL-6 fusion protein, termed Hyper-IL-6 (H-IL-6), which was shown to have biological activities at 100-1000-fold lower concentrations than IL-6 in combination with sIL-6R. The secreted p84 glycoprotein was detected in the supernatant of transfected cells and was fully active on BAF3/gp130 cells, which respond to IL-6/sIL-6R but not to IL-6 alone. Administration of recombinant H-IL-6 to C57BL/6 mice resulted in a prolonged acute phase protein gene expression indicating long systemic persistence of the fusion protein. Transfected B16 cells (B16/H-IL6 cells) showed morphological alterations in combination with a dramatic growth inhibition in vitro. Subcutaneous injection in C57BL/6 mice resulted in an almost complete rejection of B16/H-IL6 cells. This effect was partially abolished in FVB/BL/6 mice transgenic for a GM-CSF receptor antagonist, indicating a GM-CSF-dependent rejection of H-IL-6 transfected B16 cells. These results demonstrate that the anti-tumor effect of cytokines like IL-6 which are secreted by transfected melanoma cells at least in part depends on GM-CSF activity.

Up- and down-regulation of granulocyte/macrophage-colony stimulating factor activity in murine skin increase susceptibility to skin carcinogenesis by independent mechanisms.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumorigenesis is complex. On the one hand, GM-CSF can promote tumor cell growth, survival, and even metastasis. On the other hand, it can stimulate tumor cell rejection. In skin, it is early expressed after topic application of tumor-promoting agents and therefore may be responsible for changes that correlate with skin tumor promotion (e.g., epidermal hyperproliferation and inflammation). To analyze GM-CSF function in skin tumorigenesis, we generated transgenic mice epidermally overexpressing either GM-CSF or a GM-CSF antagonist. Both types of transgenic mice exhibited significantly increased numbers of benign tumors in a two-step skin carcinogenesis experiment using 7',12'-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-CSF displayed a significantly elevated carcinoma burden following a single-step carcinogenesis protocol consisting of tumor initiation only. Therefore, endogenous promotion is responsible for elevated tumor development in GM-CSF-overexpressing mice. In antagonist transgenic animals, an increased tumorigenicity of modified B16 tumor cells after cutaneous transplantation as compared with nontransgenic or GM-CSF transgenic mice was observed. Thus, the antitumor activity leading to the repression of tumor cell growth in control mice is GM-CSF dependent and is compromised in mice expressing the antagonist. We suggest that both, up-regulation and down-regulation of GM-CSF activity in skin, increase the incidence and growth of tumors via two independent mechanisms: endogenous tumor promotion in the case of increased GM-CSF activity and compromised tumor cell rejection in the case of decreased GM-CSF activity.

Keratinocyte-derived granulocyte-macrophage colony stimulating factor accelerates wound healing: Stimulation of keratinocyte proliferation, granulation tissue formation, and vascularization.

Chronic, nonhealing wounds represent a major clinical challenge to practically all disciplines in modern medicine including dermatology, oncology, surgery, and hematology. In skin wounds, granulocyte-macrophage colony stimulating factor (GM-CSF) is secreted by keratinocytes shortly after injury and mediates epidermal cell proliferation in an autocrine manner. Many other cells involved in wound healing including macrophages, lymphocytes, fibroblasts, endothelial cells, and dendritic cells synthesize GM-CSF and/or are targets of this cytokine. Therefore, GM-CSF is a pleiotropic cytokine evoking complex processes during wound repair. Despite this complexity and the scarcity of mechanistic understanding GM-CSF has been employed in trials of clinical treatment of skin wounds with some success. In this study, we evaluated a transgenic mouse model in order to analyze the effects of an excess of keratinocyte-derived GM-CSF on excisional wound healing in the skin. Transgenic mice constitutively overexpressing GM-CSF in the basal layer of the epidermis displayed accelerated reepithelialization of full-thickness skin wounds. In the early stages of wound repair, transgenic mice exhibited significantly higher numbers of proliferating keratinocytes at the wound edges and increased formation of granulation tissue with enhanced neovascularization. As a potential mechanism of these beneficial changes, we identified the differential temporal regulation of cytokines such as transforming growth factor-beta, a known angiogenetic factor, interferon-gamma, a proinflammatory cytokine, and interleukin 6, an essential factor for reepithelialization, in transgenic mice versus controls. We propose that the beneficial effects observed in GM-CSF transgenics are due not only to direct GM-CSF action but in addition to indirect processes via the induction of secondary cytokines.