Transforming Growth Factor-ß and Axl Induce CXCL5 and Neutrophil Recruitment in Hepatocellular Carcinoma.

Transforming growth factor (TGF)-ß suppresses early hepatocellular carcinoma (HCC) development but triggers pro-oncogenic abilities at later stages. Recent data suggest that the receptor tyrosine kinase Axl causes a TGF-ß switch toward dedifferentiation and invasion of HCC cells. Here, we analyzed two human cellular HCC models with opposing phenotypes in response to TGF-ß. Both HCC models showed reduced proliferation and clonogenic growth behavior following TGF-ß stimulation, although they exhibited differences in chemosensitivity and migratory abilities, suggesting that HCC cells evade traits of anti-oncogenic TGF-ß. Transcriptome profiling revealed differential regulation of the chemokine CXCL5, which positively correlated with TGF-ß expression in HCC patients. The expression and secretion of CXCL5 was dependent on Axl expression, suggesting that CXCL5 is a TGF-ß target gene collaborating with Axl signaling. Loss of either TGF-ß or Axl signaling abrogated CXCL5-dependent attraction of neutrophils. In mice, tumor formation of transplanted HCC cells relied on CXCL5 expression. In HCC patients, high levels of Axl and CXCL5 correlated with advanced tumor stages, recruitment of neutrophils into HCC tissue, and reduced survival. Conclusion: The synergy of TGF-ß and Axl induces CXCL5 secretion, causing the infiltration of neutrophils into HCC tissue. Intervention with TGF-ß/Axl/CXCL5 signaling may be an effective therapeutic strategy to combat HCC progression in TGF-ß-positive patients.

Object-based neglect in number processing.

Recent evidence suggests that neglect patients seem to have particular problems representing relatively smaller numbers corresponding to the left part of the mental number line. However, while this indicates space-based neglect for representational number space little is known about whether and --if so --how object-based neglect influences number processing.To evaluate influences of object-based neglect in numerical cognition, a group of neglect patients and two control groups had to compare two-digit numbers to an internally represented standard. Conceptualizing two-digit numbers as objects of which the left part (i.e., the tens digit should be specifically neglected) we were able to evaluate object-based neglect for number magnitude processing.Object-based neglect was indicated by a larger unit-decade compatibility effect actually reflecting impaired processing of the leftward tens digits. Additionally, faster processing of within- as compared to between-decade items provided further evidence suggesting particular difficulties in integrating tens and units into the place-value structure of the Arabic number system.In summary, the present study indicates that, in addition to the spatial representation of number magnitude, also the processing of place-value information of multi-digit numbers seems specifically impaired in neglect patients.

Impairments of the mental number line for two-digit numbers in neglect.

Humans represent numbers along a left-to-right oriented Mental Number Line (MNL). Neglect patients seem to neglect the left part of the MNL, namely the smaller numbers within a given numerical interval. However, until now all studies examining numerical representation have focussed on single-digit numbers or two-digit numbers smaller than 50. In this study, the full range of two-digit numbers was assessed in neglect patients and two control groups. Participants were presented with number triplets (e.g., 10_13_18) and asked whether or not the central number is also the arithmetical middle of the interval. The factors manipulated were decade crossing (e.g., 22_25_28 vs 25_28_31), distance to the arithmetical middle (e.g., 18_19_32 vs 18_24_32), and, most importantly, whether the central number was smaller or larger than the arithmetical middle (e.g., 11_12_19 vs 11_18_19). Neglect patients differed from controls in that they benefited less when the middle number was smaller than the arithmetical middle of the interval. Neglect patients thus seem to have particular problems when accessing the left side of numerical intervals, also when adjusted to two-digit numbers. Such an impaired magnitude representation in neglect seems to have detrimental effects on two-digit number processing as the helpful spatial metric of magnitude cannot be properly activated.

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells.

Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl--1, HLF-Axl--2, SNU449-Axl--1, SNU449-Axl--2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl- clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region. However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl--1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl--1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

Cyclin-dependent kinase 5 stabilizes hypoxia-inducible factor-1α: a novel approach for inhibiting angiogenesis in hepatocellular carcinoma.

We recently introduced CDK5 as target in HCC, regulating DNA damage response. Based on this and on our previous knowledge about vascular effects of CDK5, we investigated the role of CDK5 in angiogenesis in HCC, one of the most vascularized tumors. We put a special focus on the transcription factor HIF-1α, a master regulator of tumor angiogenesis.The interaction of CDK5 with HIF-1α was tested by Western blot, PCR, reporter gene assay, immunohistochemistry, kinase assay, co-immunoprecipitation, mass spectrometry, and mutation studies. In vivo, different murine HCC models, were either induced by diethylnitrosamine or subcutaneous injection of HUH7 or HepG2 cells. The correlation of vascular density and CDK5 was assessed by immunostaining of a microarray of liver tissues from HCC patients.Inhibition of CDK5 in endothelial or HCC cells reduced HIF-1α levels in vitro and in vivo, and transcription of HIF-1α target genes (VEGFA, VEGFR1, EphrinA1). Mass spectrometry and site directed mutagenesis revealed a stabilizing phosphorylation of HIF-1α at Ser687 by CDK5. Vascular density was decreased in murine HCC models by CDK5 inhibition.In conclusion, inhibiting CDK5 is a multi-modal systemic approach to treat HCC, hitting angiogenesis, as well as the tumor cells themselves.

Novel inhibitors of cyclin-dependent kinases combat hepatocellular carcinoma without inducing chemoresistance.

Treatment options for hepatocellular carcinoma using chemotherapeutics at intermediate and advanced stages of disease are limited as patients most rapidly escape from therapy and succumb to disease progression. Mechanisms of the hepatic xenobiotic metabolism are mostly involved in providing chemoresistance to therapeutic compounds. Given the fact that the aberrant activation of cyclin-dependent kinases (CDK) is frequently observed in hepatocellular carcinomas, we focused on the efficacy of the novel compounds BA-12 and BP-14 that antagonize CDK1/2/5/7 and CDK9. Inhibition of those CDKs in human hepatocellular carcinoma cell lines reduced the clonogenicity by arresting cells in S-G2 and G2-M phase of the cell cycle and inducing apoptosis. In contrast, primary human hepatocytes failed to show cytotoxicity and apoptosis. No loss of chemosensitivity was observed in hepatocellular carcinoma cells after long-term exposure to inhibitors. In vivo, treatment of xenografted human hepatocellular carcinomas with BA-12 or BP-14 effectively repressed tumor formation. Moreover, BA-12 or BP-14 significantly diminished diethylnitrosamine (DEN)-induced hepatoma development in mice. These data show that BA-12 or BP-14 exhibit strong antitumorigenic effects in the absence of chemoresistance, resulting in a superior efficacy compared with currently used chemotherapeutics in hepatocellular carcinomas.

TGF-ß in epithelial to mesenchymal transition and metastasis of liver carcinoma.

Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) represent the majority of hepatic malignancies and are among the most frequent causes of cancer deaths worldwide with a rising incidence in western countries. Upon progression of liver cancer, the epithelial to mesenchymal transition (EMT) is considered a key process that drives intrahepatic metastasis. EMT is the transformation of epithelial cells to a mesenchymal phenotype exacerbating motility and invasiveness of various epithelial cell types. In this review we focus on EMT in hepatic fibrosis, HCC and CCC that is governed by the transforming growth factor (TGF)-ß signaling. This cytokine has been shown to play diverse and conflicting roles in malignant development, acting as a tumor-suppressor in early cancerogenesis but enhancing tumor dissemination in later stages of tumor progression. Importantly, TGF-ß can induce EMT in a variety of cancers including HCC and CCC, even though the complex molecular mechanisms underlying this process are not yet fully understood. We aim at collecting recent findings on the impact of TGF-ß-induced EMT in liver carcinoma progression and at discussing new insights on promising drugable targets for future therapeutic approaches against CCC and HCC.