PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells.

UNLABELLED: Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. CONCLUSION: These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.

Overexpression of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) supports growth of hepatocellular carcinoma.

UNLABELLED: The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. CONCLUSION: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression.

Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs.

Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of "antioxidant-capacity" biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

Nuclear accumulation of seven in absentia homologue-2 supports motility and proliferation of liver cancer cells.

Stability of many tumor-relevant proteins is partly mediated by E3 ligases, which determine substrate specificity within the ubiquitin system. Recent data demonstrated that increased nuclear expression of the E3 ligase seven in absentia homologue (SIAH)-1 in human hepatocarcinogenesis supports tumor cell proliferation and migration. To define whether closely related SIAH-2 synergizes with protumorigenic SIAH-1, we systematically analyzed expression, localization and functional relevance of SIAH-2 in human hepatocellular carcinoma (HCC). Nuclear accumulation of SIAH-2 is detectable in more than 60% of all HCCs and correlates with tumor progression, cell proliferation and distant metastasis. An inverse correlation between nuclear SIAH-1 and SIAH-2 was detected, suggesting independent mechanisms for nuclear enrichment. Inhibition of nuclear SIAH-2 by RNAi in HCC cell lines reduced proliferation as well as lateral tumor cell motility and transmigration; however, combined knock down of both SIAH-1 and SIAH-2 did not further amplify biological effects compared to single gene inhibition. Reduction of SIAH-2 expression sensitizes HCC cells to the treatment with different cytostatic drugs, demonstrating that SIAH-2-targeting approaches may increase the response of HCC cells to conventional chemotherapy. Together, these data show that SIAH-2--as described for SIAH-1--accumulates in nuclei of HCC cells where it supports tumor growth and tumor cell dissemination. Because the nuclear pattern of SIAH-2 differs in HCC tissues from the SIAH-1 pattern and because the inactivation of SIAH-2 is not compensated by SIAH-1, the specific inhibition of SIAH-2 (especially in combination with other drugs) represents a promising therapeutic strategy for HCC.

FoxO proteins mediate hypoxic induction of connective tissue growth factor in endothelial cells.

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

Nuclear expression of the ubiquitin ligase seven in absentia homolog (SIAH)-1 induces proliferation and migration of liver cancer cells.

BACKGROUND & AIMS: Differential expression of tumor-relevant proteins based on aberrant proteasomal degradation may contribute to human (hepato)carcinogenesis. Recently, we identified the E3 ubiquitin ligase seven in absentia homolog (SIAH)-1 as frequently dysregulated in human hepatocellular carcinoma (HCC). We therefore systematically analyzed the expression, functional relevance, as well as possible downstream effectors of SIAH-1 in human liver carcinogenesis. METHODS: SIAH-1 expression was analyzed at the transcript and protein levels in human hepatocarcinogenesis and in HCC cells. Proliferation, apoptosis, and migration of different HCC cell lines were examined after siRNA-mediated inhibition of SIAH-1. In order to identify downstream effectors that mediate SIAH-1 effects, correlative analyses of protein expression profiles were performed. RESULTS: In HCC tissues both reduction of cytoplasmic SIAH-1 and especially its nuclear accumulation positively correlated with HCC progression. RNA interference revealed that nuclear expression of SIAH-1 predominantly supported HCC cell proliferation and migration while only moderately affecting anti-apoptosis. In de-differentiated human HCCs, nuclear SIAH-1 accumulation significantly correlated with the expression of the transcription factor far-upstream element (FUSE)-binding protein (FBP)-3. In vitro, SIAH-1 positively and indirectly regulated FBP-3 which itself primarily supported HCC cell proliferation. Indeed, high level expression of FBP-3 in human HCCs significantly correlated with reduced overall survival of patients. CONCLUSIONS: Nuclear accumulation of the E3 ubiquitin ligase SIAH-1 supports different pro-tumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis. It promotes HCC cell proliferation by at least partly employing the transcription factor FBP-3. Therefore, interference with SIAH-1 activity represents a promising approach to suppress HCC growth.

Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4.

Incubation of microvascular endothelial cells with combretastatin A-4 phosphate (CA-4P), a microtubule-destabilizing compound that preferentially targets tumor vessels, altered cell morphology and induced scattering of Golgi stacks. Concomitantly, CA-4P up-regulated connective tissue growth factor (CTGF/CCN2), a pleiotropic factor with antiangiogenic properties. In contrast to the effects of other microtubule-targeting agents such as colchicine or nocodazole, up-regulation of CTGF was only detectable in sparse cells, which were not embedded in a cell monolayer. Furthermore, CA-4P induced CTGF expression in endothelial cells, forming tube-like structures on basement membrane gels. Up-regulation of CTGF by CA-4P was dependent on Rho kinase signaling and was increased when p42/44 mitogen-activated protein kinase was inhibited. Additionally, FoxO transcription factors were identified as potent regulators of CTGF expression in endothelial cells. Activation of FoxO transcription factors by inhibition of phosphatidylinositol 3-kinase/AKT signaling resulted in a synergistic increase in CA-4P-mediated CTGF induction. CA-4P-mediated expression of CTGF was thus potentiated by the inhibition of kinase pathways, which are targets of novel antineoplastic drugs. Up-regulation of CTGF by low concentrations of CA-4P may thus occur in newly formed tumor vessels and contribute to the microvessel destabilization and antiangiogenic effects of CA-4P observed in vivo.

Autocrine insulin-like growth factor-II stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis.

UNLABELLED: The protumorigenic insulin-like growth factor (IGF)-II is highly expressed in a significant fraction of human hepatocellular carcinomas (HCC). However, a functional dissection that clarifies the contribution of IGF-II-binding receptors in tumor progression and a respective molecular characterization of IGF-II signaling has not been performed. Therefore, expression of IGF-II and its receptors IGF-receptor type I (IGF-IR) and insulin receptor (IR) was efficiently blocked using small interfering RNA (siRNA) in HCC cells. Despite functional IR-signaling, oncogenic IGF-II effects such as tumor cell viability, proliferation, and anti-apoptosis were solely transmitted by IGF-IR. Although IGF-II signaling was previously not described in the context of HCC cell migration, the IGF-II-dependent expression profile displayed a high percentage of genes involved in cell motility and adhesion. Indeed, IGF-II overexpression promoted HCC cell migration, especially in synergy with hepatocyte growth factor (HGF). The therapeutic relevance of IGF-II/IGF-IR signaling was tested in vitro and in a murine xenograft transplantation model using the IGF-IR inhibitor picropodophyllin (PPP). IGF-IR inhibition by small molecule treatment efficiently reduced IGF-II-dependent signaling and all protumorigenic properties of the IGF-II/IGF-IR pathway. CONCLUSION: In human HCC cells, IGF-IR but not IR is involved in oncogenic IGF-II signaling. Autocrine stimulation of IGF-II induces HCC motility by integration of paracrine signals for full malignant competence. Thus, activation of IGF-II/IGF-IR signaling is likely a progression switch selected by function that promotes tumor cell dissemination and aggressive tumor behavior.

Inhibition of Rho kinases increases directional motility of microvascular endothelial cells.

Rho kinases are major regulators of actin cytoskeletal organization and cell motility. Depending on the model system, inhibitors of Rho kinases (ROCK) have been reported to increase or decrease endothelial cell migration. In the present study we investigated the effect of Rho kinase inhibitors on microvascular endothelial cell migration with a special focus on the isoform ROCK2. Migration of microvascular endothelial cells was analyzed in a wound-healing, a spheroid-on-collagen migration assay and in cells embedded in collagen-1 gels. The non-selective Rho kinase inhibitor H1152 was compared to the selective ROCK2 inhibitor SLX2119 and to siRNA knock down. Non-selective inhibition of Rho kinases decreased cell-spanning F-actin fibers, loosened cell-cell contacts visualized by VE cadherin staining, and reduced cell-matrix interactions as shown by reduced Hic-5 expression in focal contacts. Rho kinase inhibitors facilitated directed migration of endothelial cells away from spheroids on fibronectin-coated plates and in collagen-1 gels. By contrast, migration of firmly attached endothelial cells, resembling intact vessels, was not promoted by Rho kinase inhibition. Selective inhibition of ROCK2 mimicked the cytoskeletal effects of H1152 and also increased cell motility, although to a lesser extent. In summary, Rho kinase inhibition enhanced the migration and cytoskeletal restructuring preferentially in freshly attached endothelial cells. ROCK2 may be a potential target to manipulate endothelial cell migration after vessel injury.

Hic-5 as a regulator of endothelial cell morphology and connective tissue growth factor gene expression.

The functional role of the LIM-domain protein Hic-5 was investigated in microvascular endothelial cells using a siRNA approach. Knock down of Hic-5 reduced endothelial cell spreading and impaired structural organization of the cells on basement membrane extracts. Furthermore, Hic-5 was involved in the regulation of the multifunctional protein connective tissue growth factor (CTGF, CCN2). Upon Hic-5 down-regulation, induction of CTGF by lysophosphatidic acid or colchicine was reduced. Inhibition of CTGF expression was even more pronounced in cells treated with transforming growth factor beta and inhibitors of histone deacetylases. Treatment of endothelial cells with Hic-5 siRNA reduced CTGF promoter activity. Mutation analyses of the promoter revealed transcription factors binding to the basic control element as part of the proposed Hic-5-modulated transcription complex. Further analyses showed down-regulation of Hic-5 protein upon overnight treatment with inhibitors of histone deacetylases. These data suggest that the reduced expression of Hic-5 may contribute to the anti-angiogenic effects of histone deacetylase inhibitors.

Cell type-specific regulation of CCN2 protein expression by PI3K-AKT-FoxO signaling.

The biological activity of connective tissue growth factor (CTGF, CCN2) is regulated at the level of intracellular signaling leading to gene expression, and by its extracellular interaction partners which determine the functional outcome of CCN2 action. In this overview, we summarize the data which provide evidence that one of the major signaling pathways, phosphatidylinositol-3 kinase (PI3K)-AKT signaling, shows a remarkable cell type-dependence in terms of regulation of CCN2 expression. In smooth muscle cells, fibroblasts, and epithelial cells, inhibition of this pathway either reduced CCN2 expression or was not involved in CCN2 gene expression depending on the stimulus used. In microvascular endothelial cells by contrast, activation of PI3K-AKT signaling was inversely related to CCN2 expression. Upregulation of CCN2 upon inhibition of PI3K-AKT was also observed in primary cultures of human endothelial cells (HUVEC) exposed to laminar flow in an in vitro flow-through system. In different types of endothelial cells, FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CCN2 gene expression. In HUVEC, we observed a correlation between enhanced nuclear localization of FoxO1 and increased synthesis of CCN2 protein in areas of non-uniform shear stress. These data indicate that FoxO proteins are key regulators of CCN2 gene expression which determine the effect of PI3K-AKT activation in terms of CCN2 regulation. Short summary Phosphatidylinositol-3 kinase (PI3K)-AKT signaling shows a remarkable cell type-dependence in terms of regulation of CCN2 expression. In endothelial cells activation of PI3K - AKT signaling was inversely related to CCN2 expression. FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CCN2 gene expression.