Tumor-Extrinsic Axl Expression Shapes an Inflammatory Microenvironment Independent of Tumor Cell Promoting Axl Signaling in Hepatocellular Carcinoma.

The activation of the receptor tyrosine kinase Axl by Gas6 is a major driver of tumorigenesis. Despite recent insights, tumor cell-intrinsic and -extrinsic Axl functions are poorly understood in hepatocellular carcinoma (HCC). Thus, we analyzed the cell-specific aspects of Axl in liver cancer cells and in the tumor microenvironment. We show that tumor-intrinsic Axl expression decreased the survival of mice and elevated the number of pulmonary metastases in a model of resection-based tumor recurrence. Axl expression increased the invasion of hepatospheres by the activation of Akt signaling and a partial epithelial-to-mesenchymal transition (EMT). However, the liver tumor burden of Axl+/+ mice induced by diethylnitrosamine plus carbon tetrachloride was reduced compared to systemic Axl-/- mice. Tumors of Axl+/+ mice were highly infiltrated with cytotoxic cells, suggesting a key immune-modulatory role of Axl. Interestingly, hepatocyte-specific Axl deficiency did not alter T cell infiltration, indicating that these changes are independent of tumor cell-intrinsic Axl. In this context, we observed an upregulation of multiple chemokines in Axl+/+ compared to Axl-/- tumors, correlating with HCC patient data. In line with this, Axl is associated with a cytotoxic immune signature in HCC patients. Together these data show that tumor-intrinsic Axl expression fosters progression, while tumor-extrinsic Axl expression shapes an inflammatory microenvironment.

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.

c-Met Signaling Is Essential for Mouse Adult Liver Progenitor Cells Expansion After Transforming Growth Factor-ß-Induced Epithelial-Mesenchymal Transition and Regulates Cell Phenotypic Switch.

Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulates HPCs biology. Here, we characterize the TGF-ß-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-ß triggers a partial EMT in oval cells based on coexpression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicates that TGF-ß-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-ß-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-ß and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. Stem Cells 2019;37:1108-1118.

Loss of SR-BI Down-Regulates MITF and Suppresses Extracellular Vesicle Release in Human Melanoma.

Melanoma is a skin tumor with a high tendency for metastasis and thus is one of the deadliest cancers worldwide. Here, we investigated the expression of the scavenger receptor class B type 1 (SR-BI), a high-density lipoprotein (HDL) receptor, and tested for its role in melanoma pigmentation as well as extracellular vesicle release. We first analyzed the expression of SR-BI in patient samples and found a strong correlation with MITF expression as well as with the melanin synthesis pathway. Hence, we asked whether SR-BI could also play a role for the secretory pathway in metastatic melanoma cells. Interestingly, gain- and loss-of-function of SR-BI revealed regulation of the proto-oncogene MET. In line, SR-BI knockdown reduced expression of the small GTPase RABB22A, the ESCRT-II protein VPS25, and SNAP25, a member of the SNARE complex. Accordingly, reduced overall extracellular vesicle generation was detected upon loss of SR-BI. In summary, SR-BI expression in human melanoma enhances the formation and transport of extracellular vesicles, thereby contributing to the metastatic phenotype. Therapeutic targeting of SR-BI would not only interfere with cholesterol uptake, but also with the secretory pathway, therefore suppressing a key hallmark of the metastatic program.

Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins: a new hope for laboratory animals?

Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.

Dynamics of Axl Receptor Shedding in Hepatocellular Carcinoma and Its Implication for Theranostics.

Signaling of the receptor tyrosine kinase Axl and its ligand Gas6 is crucially involved in the development of liver fibrosis and hepatocellular carcinoma (HCC) by activation of hepatic stellate cells and modulation of hepatocyte differentiation. Shedding of Axl's ectodomain leads to the release of soluble Axl (sAxl), which is increased in advanced fibrosis and in early-to-late stage HCC in the presence and absence of cirrhosis. Here, we focus on the dynamics of Axl receptor shedding and delineate possible scenarios how Axl signaling might act as driver of fibrosis progression and HCC development. Based on experimental and clinical data, we discuss the consequences of modifying Axl signaling by sAxl cleavage, as well as cellular strategies to escape from antagonizing effects of Axl shedding by the involvement of the hepatic microenvironment. We emphasize a correlation between free Gas6 and free sAxl levels favoring abundant Gas6/Axl signaling in advanced fibrosis and HCC. The raised scenario provides a solid basis for theranostics allowing the use of sAxl as an accurate diagnostic biomarker of liver cirrhosis and HCC, as well as Axl receptor signaling for therapeutic intervention in stratified HCC patients.

Epithelial to mesenchymal transition-related proteins ZEB1, ß-catenin, and ß-tubulin-III in idiopathic pulmonary fibrosis.

Epithelial to mesenchymal transition has been suggested as a relevant contributor to pulmonary fibrosis, but how and where this complex process is triggered in idiopathic pulmonary fibrosis is not fully understood. Beta-tubulin-III (Tubß3), ZEB1, and ß-catenin are partially under the negative control of miR-200, a family of micro-RNAs playing a major role in epithelial to mesenchymal transition, that are reduced in experimental lung fibrosis and idiopathic pulmonary fibrosis. We wonder whether in situ expression of these proteins is increased in idiopathic pulmonary fibrosis, to better understand the significance of miR-200 feedback loop and epithelial to mesenchymal transition. We investigated the immunohistochemical and immunofluorescent expression and precise location of ZEB1, Tubß3, and ß-catenin in tissue samples from 34 idiopathic pulmonary fibrosis cases and 21 controls (5 normal lungs and 16 other interstitial lung diseases). In 100% idiopathic pulmonary fibrosis samples, the three proteins were concurrently expressed in fibroblastic foci, as well in damaged epithelial cells overlying these lesions and in pericytes within neo-angiogenesis areas. These results were also confirmed by immunofluorescence assay. In controls the abnormal expression of the three proteins was absent or limited. This is the first study that relates concurrent expression of Tubß3, ZEB1, and ß-catenin to abnormal epithelial and myofibroblast differentiation in idiopathic pulmonary fibrosis, providing indirect but robust evidence of miR-200 deregulation and epithelial to mesenchymal transition activation in idiopathic pulmonary fibrosis. The abnormal expression and localization of these proteins in bronchiolar fibro-proliferative lesions are unique for idiopathic pulmonary fibrosis, and might represent a disease-specific marker in challenging lung biopsies.

Transforming Growth Factor-ß Drives the Transendothelial Migration of Hepatocellular Carcinoma Cells.

The entry of malignant hepatocytes into blood vessels is a key step in the dissemination and metastasis of hepatocellular carcinoma (HCC). The identification of molecular mechanisms involved in the transmigration of malignant hepatocytes through the endothelial barrier is of high relevance for therapeutic intervention and metastasis prevention. In this study, we employed a model of hepatocellular transmigration that mimics vascular invasion using hepatic sinusoidal endothelial cells and malignant hepatocytes evincing a mesenchymal-like, invasive phenotype by transforming growth factor (TGF)-ß. Labelling of respective cell populations with various stable isotopes and subsequent mass spectrometry analyses allowed the "real-time" detection of molecular changes in both transmigrating hepatocytes and endothelial cells. Interestingly, the proteome profiling revealed 36 and 559 regulated proteins in hepatocytes and endothelial cells, respectively, indicating significant changes during active transmigration that mostly depends on cell-cell interaction rather than on TGF-ß alone. Importantly, matching these in vitro findings with HCC patient data revealed a panel of common molecular alterations including peroxiredoxin-3, epoxide hydrolase, transgelin-2 and collectin 12 that are clinically relevant for the patient's survival. We conclude that hepatocellular plasticity induced by TGF-ß is crucially involved in blood vessel invasion of HCC cells.

Role of epithelial to mesenchymal transition in hepatocellular carcinoma.

The epithelial to mesenchymal transition (EMT) is a multistep biological process whereby epithelial cells change in plasticity by transient de-differentiation into a mesenchymal phenotype. EMT and its reversal, mesenchymal to epithelial transition (MET), essentially occur during embryogenetic morphogenesis and have been increasingly described in fibrosis and cancer during the last decade. In carcinoma progression, EMT plays a crucial role in early steps of metastasis when cells lose cell-cell contacts due to ablation of E-cadherin and acquire increased motility to spread into surrounding or distant tissues. Epithelial plasticity has become a hot issue in hepatocellular carcinoma (HCC), as strong inducers of EMT such as transforming growth factor-ß are able to orchestrate both fibrogenesis and carcinogenesis, showing rising cytokine levels in cirrhosis and late stage HCC. In this review, we consider the significance of EMT-MET in malignant hepatocytes as well as changes in the plasticity of hepatic stellate cells for cellular heterogeneity of HCC, and further aim at explaining the current limiting insights into EMT by snapshot analyses of HCC tissues. Recent advances in the identification of clinically relevant mechanisms that impinge on important EMT-transcription factors, as well as on miRNAs causing EMT signatures and HCC progression are highlighted. In addition, we draw particular attention to framing EMT in the context of potential clinical relevance for HCC patients. We conclude that some aspects of EMT are still elusive and further studies are required to better link the clinical management of HCC with biomarkers and targeted therapies related to EMT.

Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche.

BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in human hepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.

Axl activates autocrine transforming growth factor-ß signaling in hepatocellular carcinoma.

UNLABELLED: In hepatocellular carcinoma (HCC), intrahepatic metastasis frequently correlates with epithelial to mesenchymal transition (EMT) of malignant hepatocytes. Several mechanisms have been identified to be essentially involved in hepatocellular EMT, among them transforming growth factor (TGF)-ß signaling. Here we show the up-regulation and activation of the receptor tyrosine kinase Axl in EMT-transformed hepatoma cells. Knockdown of Axl expression resulted in abrogation of invasive and transendothelial migratory abilities of mesenchymal HCC cells in vitro and Axl overexpression-induced metastatic colonization of epithelial hepatoma cells in vivo. Importantly, Axl knockdown severely impaired resistance to TGF-ß-mediated growth inhibition. Analysis of the Axl interactome revealed binding of Axl to 14-3-3ζ, which is essentially required for Axl-mediated cell invasion, transendothelial migration, and resistance against TGF-ß. Axl/14-3-3ζ signaling caused phosphorylation of Smad3 linker region (Smad3L) at Ser213, resulting in the up-regulation of tumor-progressive TGF-ß target genes such as PAI1, MMP9, and Snail as well as augmented TGF-ß1 secretion in mesenchymal HCC cells. Accordingly, high Axl expression in HCC patient samples correlated with elevated vessel invasion of HCC cells, higher risk of tumor recurrence after liver transplantation, strong phosphorylation of Smad3L, and lower survival. In addition, elevated expression of both Axl and 14-3-3ζ showed strongly reduced survival of HCC patients. CONCLUSION: Our data suggest that Axl/14-3-3ζ signaling is central for TGF-ß-mediated HCC progression and a promising target for HCC therapy.

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis.

UNLABELLED: Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr(-/-), a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-)), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-);Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2(-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-);Mdr2(-/-) mice had a pronounced down-regulation of hepatoprotective genes Hnf6, Egfr, and Igf-1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-)) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild-type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-);Mdr2(-/-) mice displayed a significant decrease in tumor incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. CONCLUSION: GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.

The rationale for targeting TGF-ß in chronic liver diseases.

BACKGROUND: Transforming growth factor (TGF)-ß is a pluripotent cytokine that displays several tissue-specific biological activities. In the liver, TGF-ß is considered a fundamental molecule, controlling organ size and growth by limiting hepatocyte proliferation. It is involved in fibrogenesis and, therefore, in worsening liver damage, as well as in triggering the development of hepatocellular carcinoma (HCC). TGF-ß is known to act as an oncosuppressor and also as a tumour promoter in HCC, but its role is still unclear. DESIGN: In this review, we discuss the potential role of TGF-ß in regulating the tumoural progression of HCC, and therefore the rationale for targeting this molecule in patients with HCC. RESULTS: A considerable amount of experimental preclinical evidence suggests that TGF-ß is a promising druggable target in patients with HCC. To support this hypothesis, a phase II clinical trial is currently ongoing using a TGF-ß pathway inhibitor, and results will soon be available. CONCLUSIONS: The identification of new TGF-ß related biomarkers will help to select those patients most likely to benefit from therapy aimed at inhibiting the TGF-ß pathway. New formulations that may provide a more controlled and sustained delivery of the drug will improve the therapeutic success of such treatments.

Multicenter analysis of soluble Axl reveals diagnostic value for very early stage hepatocellular carcinoma.

If diagnosed at early stages, patients with hepatocellular carcinoma (HCC) can receive curative therapies, whereas therapeutic options at later stages are very limited. Here, we addressed the potential of soluble Axl (sAxl) as a biomarker of early HCC by analyzing levels of sAxl in 311 HCC and 237 control serum samples from centers in Europe and China. Serum concentrations of sAxl were significantly increased in HCC (18.575 ng/mL) as compared to healthy (13.388 ng/mL) or cirrhotic (12.169 ng/mL) controls. Receiver operating characteristic curve analysis of sAxl in very early stage HCC patients (BCLC 0) showed an area under the curve (AUC) of 0.848, with a sensitivity of 76.9% and a specificity of 69.2%. α-Fetoprotein (AFP)-negative HCC patients displayed an AUC of 0.803, with sensitivity and specificity of 73% and 70.8%. Combination of sAxl and AFP improved diagnostic accuracy to 0.936 in very early HCC patients and to 0.937 in all HCC. Differential diagnosis of very early HCC versus liver cirrhosis showed a combined performance for sAxl and AFP of 0.901 with a sensitivity of 88.5% and a specificity of 76.7%. Furthermore, sAxl levels failed to be elevated in primary ovarian, colorectal and breast carcinomas as well as in secondary hepatic malignancies derived from colon. In summary, sAxl outperforms AFP in detecting very early HCC as compared to healthy or cirrhotic controls and shows high diagnostic accuracy for AFP-negative patients. sAxl is specific for HCC and suggested as a biomarker for routine clinical use.

Neuropilin-2 induced by transforming growth factor-ß augments migration of hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the third most lethal cancer worldwide. The epithelial to mesenchymal transition (EMT) describes the transformation of well-differentiated epithelial cells to a de-differentiated phenotype and plays a central role in the invasion and intrahepatic metastasis of HCC cells. Modulation of the transforming growth factor-ß (TGF-ß) signaling is known to induce various tumor-promoting and EMT-inducing pathways in HCC. The meta-analysis of a panel of EMT gene expression studies revealed that neuropilin 2 (NRP2) is significantly upregulated in cells that have undergone EMT induced by TGF-ß. In this study we assessed the functional role of NRP2 in epithelial and mesenchymal-like HCC cells and focused on the molecular interplay between NRP2 and TGF-ß/Smad signaling. METHODS: NRP2 expression was analyzed in human HCC cell lines and tissue arrays comprising 133 HCC samples. Cell migration was examined by wound healing and Transwell assays in the presence and absence of siRNA against NRP2. NRP2 and TGF-ß signaling were analyzed by Western blotting and confocal immunofluorescence microscopy. RESULTS: We show that NRP2 is particularly expressed in HCC cell lines with a dedifferentiated, mesenchymal-like phenotype. NRP2 expression is upregulated by the canonical TGF-ß/Smad signaling while NRP2 expression has no impact on TGF-ß signaling in HCC cells. Reduced expression of NRP2 by knock-down or inhibition of TGF-ß signaling resulted in diminished cell migration independently of each other, suggesting that NRP2 fails to collaborate with TGF-ß signaling in cell movement. In accordance with these data, elevated levels of NRP2 correlated with a higher tumor grade and less differentiation in a large collection of human HCC specimens. CONCLUSIONS: These data suggest that NRP2 associates with a less differentiated, mesenchymal-like HCC phenotype and that NRP2 plays an important role in tumor cell migration upon TGF-ß-dependent HCC progression.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans.

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

Inhibition of tumour spheroid-induced prometastatic intravasation gates in the lymph endothelial cell barrier by carbamazepine: drug testing in a 3D model.

Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment. Nevertheless, the effect of carbamazepine on the inhibition of CCID formation as an early step of breast cancer metastasis was significant and substantial (~30 %) and achieved at concentrations that are found in the plasma of carbamazepine-treated adults (40-60 µM). The fact that carbamazepine is a drug approved by the US Food and Drug Administration facilitates a "from-bench-to-bedside" perspective. Therefore, the here presented data should undergo scrutiny in vivo.

PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition.

The extracellular matrix protein Laminin B1 (LamB1) regulates tumor cell migration and invasion. Carcinoma cells acquire invasive properties by epithelial to mesenchymal transition (EMT), which is a fundamental step in dissemination of metastatic cells from the primary tumor. Recently, we showed that enhanced translation of LamB1 upon EMT of malignant hepatocytes is mediated by an internal ribosome entry site (IRES). We demonstrated that the IRES transacting factor La binds the minimal IRES motif and positively modulates IRES activity of LamB1. Here, we show that platelet-derived growth factor (PDGF) enhances IRES activity of LamB1 by the increasing cytoplasmic localization of La during EMT. Accordingly, cells expressing dominant negative PDGF receptor display reduced cytoplasmic accumulation of La and show no elevation of IRES activity or endogenous LamB1 levels after stimulation with PDGF. Furthermore, La-mediated regulation of LamB1 IRES activity predominantly depends on MAPK/ERK signaling downstream of PDGF. Notably, LamB1 expression is not significantly downregulated by the impairment of the translation initiation factor eIF4E. In vivo, knockdown of La associated with decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT.

La enhances IRES-mediated translation of laminin B1 during malignant epithelial to mesenchymal transition.

The majority of transcripts that harbor an internal ribosome entry site (IRES) are involved in cancer development via corresponding proteins. A crucial event in tumor progression referred to as epithelial to mesenchymal transition (EMT) allows carcinoma cells to acquire invasive properties. The translational activation of the extracellular matrix component laminin B1 (LamB1) during EMT has been recently reported suggesting an IRES-mediated mechanism. In this study, the IRES activity of LamB1 was determined by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1. Furthermore, no other LamB1 mRNA species arising from alternative transcription start sites or polyadenylation signals were detected that account for its translational control. Mapping of the LamB1 5'-untranslated region (UTR) revealed the minimal LamB1 IRES motif between -293 and -1 upstream of the start codon. Notably, RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction and its regulation during EMT were confirmed by ribonucleoprotein immunoprecipitation. In addition, La was able to positively modulate LamB1 IRES translation. In summary, these data indicate that the LamB1 IRES is activated by binding to La which leads to translational upregulation during hepatocellular EMT.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.