Thomas Longerich

A complex secretory program orchestrated by the inflammasome controls paracrine senescence

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15INK4b and p21CIP1. Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

Posttranscriptional destabilization of the liver‐specific long noncoding RNA HULC by the IGF2 mRNA‐binding protein 1 (IGF2BP1)

Selected long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis. Although the cellular functions of these transcripts can be diverse, many lncRNAs regulate gene expression. In contrast, factors that control the expression of lncRNAs remain largely unknown. Here we investigated the impact of RNA binding proteins on the expression of the liver cancer‐associated lncRNA HULC (highly up‐regulated in liver cancer). First, we validated the strong up‐regulation of HULC in human hepatocellular carcinoma. To elucidate posttranscriptional regulatory mechanisms governing HULC expression, we applied an RNA affinity purification approach to identify specific protein interaction partners and potential regulators. This method identified the family of IGF2BPs (IGF2 mRNA‐binding proteins) as specific binding partners of HULC. Depletion of IGF2BP1, also known as IMP1, but not of IGF2BP2 or IGF2BP3, led to an increased HULC half‐life and higher steady‐state expression levels, indicating a posttranscriptional regulatory mechanism. Importantly, HULC represents the first IGF2BP substrate that is destabilized. To elucidate the mechanism by which IGF2BP1 destabilizes HULC, the CNOT1 protein was identified as a novel interaction partner of IGF2BP1. CNOT1 is the scaffold of the human CCR4‐NOT deadenylase complex, a major component of the cytoplasmic RNA decay machinery. Indeed, depletion of CNOT1 increased HULC half‐life and expression. Thus, IGF2BP1 acts as an adaptor protein that recruits the CCR4‐NOT complex and thereby initiates the degradation of the lncRNA HULC. Conclusion: Our findings provide important insights into the regulation of lncRNA expression and identify a novel function for IGF2BP1 in RNA metabolism. (Hepatology 2013;58:1703–1712)

S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver carcinogenesis

The nuclear factor‐kappaB (NF‐κB) signaling pathway has been recently shown to participate in inflammation‐induced cancer progression. Here, we describe a detailed analysis of the NF‐κB–dependent gene regulatory network in the well‐established Mdr2 knockout mouse model of inflammation‐associated liver carcinogenesis. Expression profiling of NF‐κB–deficient and NF‐κB–proficient hepatocellular carcinoma (HCC) revealed a comprehensive list of known and novel putative NF‐κB target genes, including S100a8 and S100a9. We detected increased co‐expression of S100A8 and S100A9 proteins in mouse HCC cells, in human HCC tissue, and in the HCC cell line Hep3B on ectopic RelA expression. Finally, we found a synergistic function for S100A8 and S100A9 in Hep3B cells resulting in a significant induction of reactive oxygen species (ROS), accompanied by enhanced cell survival. Conclusion: We identified S100A8 and S100A9 as novel NF‐κB target genes in HCC cells during inflammation‐associated liver carcinogenesis and provide experimental evidence that increased co‐expression of both proteins supports malignant progression by activation of ROS‐dependent signaling pathways and protection from cell death. (HEPATOLOGY 2009.)

Methylome analysis and integrative profiling of human HCCs identify novel protumorigenic factors

To identify new tumor‐suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome‐wide methylation profiling and vertical integration with array‐based comparative genomic hybridization (aCGH), as well as expression data from a cohort of well‐characterized human hepatocellular carcinomas (HCCs). Bisulfite‐converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy‐number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor‐suppressor gene (period homolog 3 [PER3], insulin‐like growth‐factor–binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down‐regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5‐aza‐dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor‐suppressive function for PER3 and IGFALS in vitro. Conclusion: The present study illustrates that vertical integration of methylation data with high‐resolution genomic and transcriptomic data facilitates the identification of new tumor‐suppressor gene candidates in human HCC. (HEPATOLOGY 2012;56:1817–1827)

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.)

A Direct In Vivo RNAi Screen Identifies MKK4 as a Key Regulator of Liver Regeneration

The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.

Epithelial-to-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma and Pancreatic Tumor Cell Lines: The Role of Neutrophils and Neutrophil-Derived Elastase

Pancreatic ductal adenocarcinoma (PDAC) is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT) is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN) and the tumor cell transition, biopsies of patients with PDAC (n = 115) were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and—by implication—to tumor progression is possible.

Receptor for advanced glycation endproducts (RAGE) is a key regulator of oval cell activation and inflammation‐associated liver carcinogenesis in mice

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor and member of the immunoglobulin superfamily. RAGE is mainly involved in tissue damage and chronic inflammatory disorders, sustaining the inflammatory response upon engagement with damage‐associated molecular pattern molecules (DAMPs) such as S100 proteins and high‐mobility group box 1 (HMGB1). Enhanced expression of RAGE and its ligands has been demonstrated in distinct tumors and several studies support its crucial role in tumor progression and metastasis by still unknown mechanisms. Here we show that RAGE supports hepatocellular carcinoma (HCC) formation in the Mdr2−/− mouse model, a prototype model of inflammation‐driven HCC formation, which mimics the human pathology. Mdr2−/− Rage−/− (dKO) mice developed smaller and fewer HCCs than Mdr2−/− mice. Interestingly, although in preneoplastic Mdr2−/− livers RAGE ablation did not affect the onset of inflammation, premalignant dKO livers showed reduced liver damage and fibrosis, in association with decreased oval cell activation. Oval cells expressed high RAGE levels and displayed reduced proliferation upon RAGE silencing. Moreover, stimulation of oval cells with HMGB1 promoted an ERK1/2‐Cyclin D1‐dependent oval cell proliferation in vitro. Finally, genetic and pharmacologic blockade of RAGE signaling impaired oval cell activation in an independent mouse model of oval cell activation, the choline deficient ethionine‐supplemented dietary regime. Conclusion: Our data identified a novel function of RAGE in regulating oval cell activation and tumor development in inflammation‐associated liver carcinogenesis. (Hepatology 2013)

Down‐regulation of tumor suppressor a kinase anchor protein 12 in human hepatocarcinogenesis by epigenetic mechanisms

The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down‐regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12β promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5‐aza‐2′deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA‐mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR‐183 and miR‐186 are up‐regulated in CL and DN and are able to target AKAP12. Conclusion: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis. (HEPATOLOGY 2010;.)