Michaela Bissinger

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

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. (HEPATOLOGY 2008.)

Coordinated expression of stathmin family members by far upstream sequence element-binding protein-1 increases motility in non-small cell lung cancer.

Dynamic instability of the microtubule network modulates processes such as cell division and motility, as well as cellular morphology. Overexpression of the microtubule-destabilizing phosphoprotein stathmin is frequent in human malignancies and represents a promising therapeutic target. Although stathmin inhibition gives rise to antineoplastic effects, additional and functionally redundant microtubule-interacting proteins may attenuate the efficiency of this therapeutic approach. We have systematically analyzed the expression and potential protumorigenic effects of stathmin family members in human non-small cell lung cancer (NSCLC). Both stathmin and stathmin-like 3 (SCLIP) were overexpressed in adenocarcinoma as well as squamous cell carcinoma (SCC) tissues and induced tumor cell proliferation, migration, and matrix invasion in respective cell lines. Accordingly, reduced stathmin and SCLIP levels affected cell morphology and were associated with a less malignant phenotype. Combined inhibition of both factors caused additive effects on tumor cell motility, indicating partial functional redundancy. Because stathmin and SCLIP expression significantly correlated in NSCLC tissues, we searched for common upstream regulators and identified the far upstream sequence element-binding protein-1 (FBP-1) as a pivotal inducer of several stathmin family members. Our results indicate that the coordinated overexpression of microtubule-destabilizing factors by FBP-1 is a critical step to facilitate microtubule dynamics and subsequently increases proliferation and motility of tumor cells.

Overexpression of far upstream element binding proteins: A mechanism regulating proliferation and migration in liver cancer cells

Microtubule‐dependent effects are partly regulated by factors that coordinate polymer dynamics such as the microtubule‐destabilizing protein stathmin (oncoprotein 18). In cancer cells, increased microtubule turnover affects cell morphology and cellular processes that rely on microtubule dynamics such as mitosis and migration. However, the molecular mechanisms deregulating modifiers of microtubule activity in human hepatocarcinogenesis are poorly understood. Based on profiling data of human hepatocellular carcinoma (HCC), we identified far upstream element binding proteins (FBPs) as significantly coregulated with stathmin. Coordinated overexpression of two FBP family members (FBP‐1 and FBP‐2) in >70% of all analyzed human HCCs significantly correlated with poor patient survival. In vitro, FBP‐1 predominantly induced tumor cell proliferation, while FBP‐2 primarily supported migration in different HCC cell lines. Surprisingly, reduction of FBP‐2 levels was associated with elevated FBP‐1 expression, suggesting a regulatory interplay of FBP family members that functionally discriminate between cell division and mobility. Expression of FBP‐1 correlated with stathmin expression in HCC tissues and inhibition of FBP‐1 but not of FBP‐2 drastically reduced stathmin at the transcript and protein levels. In contrast, further overexpression of FBP‐1 did not affect stathmin bioavailability. Accordingly, analyzing nuclear and cytoplasmic areas of HCC cells revealed that reduced FBP‐1 levels affected cell morphology and were associated with a less malignant phenotype. Conclusion: The coordinated activation of FBP‐1 and FBP‐2 represents a novel and frequent pro‐tumorigenic mechanism promoting proliferation (tumor growth) and motility (dissemination) of human liver cancer cells. FBPs promote tumor‐relevant functions by at least partly employing the microtubule‐destabilizing factor stathmin and represent a new potential target structure for HCC treatment. (HEPATOLOGY 2009.)

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.

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.

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

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. (Hepatology 2016;63:813–826)

AP-1-Controlled Hepatocyte Growth Factor Activation Promotes Keratinocyte Migration via CEACAM1 and Urokinase Plasminogen Activator/ Urokinase Plasminogen Receptor

Keratinocyte migration is essential for the rapid closure of the epidermis in the process of wound healing. Mesenchymal cell-derived hepatocyte growth factor (HGF) is a central regulator of this process. However, the molecular mechanisms and relevant genes that facilitate this cellular response are still poorly defined. We used heterologous cocultures combining primary human keratinocytes and genetically modified murine fibroblasts to identify key factors mediating HGF-induced epidermal cell migration. The absence of c-Jun activity in fibroblasts completely abolished the expression of HGF in these cells and consequently altered the behavior of keratinocytes. Time-resolved expression series of keratinocytes stimulated with HGF disclosed target genes regulating HGF-dependent motility. In addition to well-established HGF-dependent wound healing-associated genes, carcinoembryogenic antigen-related cell adhesion molecule (CEACAM)-1 and the urokinase plasminogen activator (uPA)/uPA-receptor (uPAR) pathway were identified as possible mediators in HGF-induced keratinocyte migration. The functional relevance of CEACAM-1 and uPA/uPAR on epidermal cell motility was demonstrated using the HaCaT cell culture model. In conclusion, the distinct spatiotemporal regulation of genes by HGF is essential for proper epidermal cell migration in cutaneous wound healing.

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

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. (Hepatology 2014;60:1241–1250)

A Systems Biology Study on NFκB Signaling in Primary Mouse Hepatocytes.

The cytokine tumor necrosis factor-alpha (TNFα) is one of the key factors during the priming phase of liver regeneration as well as in hepatocarcinogenesis. TNFα activates the nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) signaling pathway and contributes to the conversion of quiescent hepatocytes to activated hepatocytes that are able to proliferate in response to growth factor stimulation. Different mathematical models have been previously established for TNFα/NFκB signaling in the context of tumor cells. Combining these mathematical models with time-resolved measurements of expression and phosphorylation of TNFα/NFκB pathway constituents in primary mouse hepatocytes revealed that an additional phosphorylation step of the NFκB isoform p65 has to be considered in the mathematical model in order to sufficiently describe the dynamics of pathway activation in the primary cells. Also, we addressed the role of basal protein turnover by experimentally measuring the degradation rate of pivotal players in the absence of TNFα and including this information in the model. To elucidate the impact of variations in the protein degradation rates on TNFα/NFκB signaling on the overall dynamic behavior we used global sensitivity analysis that accounts for parameter uncertainties and showed that degradation and translation of p65 had a major impact on the amplitude and the integral of p65 phosphorylation. Finally, our mathematical model of TNFα/NFκB signaling was able to predict the time-course of the complex formation of p65 and of the inhibitor of NFκB (IκB) in primary mouse hepatocytes, which was experimentally verified. Hence, we here present a mathematical model for TNFα/NFκB signaling in primary mouse hepatocytes that provides an important basis to quantitatively disentangle the complex interplay of multiple factors in liver regeneration and tumorigenesis.

A20/TNFAIP3 Discriminates Tumor Necrosis Factor (TNF)-Induced NF-κB from JNK Pathway Activation in Hepatocytes

In the liver tumor necrosis factor (TNF)-induced signaling critically regulates the immune response of non-parenchymal cells as well as proliferation and apoptosis of hepatocytes via activation of the NF-κB and JNK pathways. Especially, the induction of negative feedback regulators, such as IκBα and A20 is responsible for the dynamic and time-restricted response of these important pathways. However, the precise mechanisms responsible for different TNF-induced phenotypes under physiological stimulation conditions are not completely understood so far. In addition, it is not known if varying TNF concentrations may differentially affect the desensitization properties of both pathways. By using computational modeling, we first showed that TNF-induced activation and downstream signaling is qualitatively comparable between primary mouse hepatocytes and immortalized hepatocellular carcinoma (HCC) cells. In order to define physiologically relevant TNF levels, which allow for an adjustable and dynamic NF-κB/JNK pathway response in parenchymal liver cells, a range of cytokine concentrations was defined that led to gradual pathway responses in HCC cells (1–5 ng/ml). Repeated stimulations with low (1 ng/ml), medium (2.5 ng/ml) and high (5 ng/ml) TNF amounts demonstrated that JNK signaling was still active at cytokine concentrations, which led to dampened NF-κB signaling illustrating differential pathway responsiveness depending on TNF input dynamics. SiRNA-mediated inhibition of the negative feedback regulator A20 (syn. TNFAIP3) or its overexpression did not significantly affect the NF-κB response. In contrast, A20 silencing increased the JNK response, while its overexpression dampened JNK phosphorylation. In addition, the A20 knockdown sensitized hepatocellular cells to TNF-induced cleavage and activity of the effector caspase-3. In conclusion, a mathematical model-based approach shows that the TNF-induced pathway responses are qualitatively comparable in primary and immortalized mouse hepatocytes. The cytokine amount defines the pathway responsiveness under repeated treatment conditions with NF-κB signaling being dampened ‘earlier’ than JNK. A20 appears to be the molecular switch discriminating between NF-κB and JNK signaling when stimulating with varying physiological cytokine concentrations.