Stephan Singer

DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

Yes-Associated Protein Up-regulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma

BACKGROUND & AIMS Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

Protumorigenic overexpression of stathmin/Op18 by gain-of-function mutation in p53 in human hepatocarcinogenesis.

The microtubule (MT)‐destabilizing protein stathmin/Op18 has previously been described to be negatively regulated by p53 and to be highly expressed in several tumor entities. However, little is known about its expression profile, functional or therapeutic relevance, and regulation in human hepatocarcinogenesis. Here we demonstrate cytoplasmic overexpression of stathmin in premalignant lesions (dysplastic nodules; DNs) and hepatocellular carcinomas (HCCs), which significantly correlated with tumor progression, proliferation, and activation of other protumorigenic factors (e.g., nuclear p53). Inhibition of stathmin expression by gene‐specific short interfering RNA (siRNA) was associated with a significant reduction of MT‐dependent cellular functions such as tumor cell viability, proliferation, migration, and increased apoptosis in HCC cells. Loss of stathmin expression increased responsiveness of tumor cells to the treatment with cytostatic drugs targeting MT‐stability (paclitaxel, vinblastine) and to DNA cross‐linking agents (cisplatin). Surprisingly, inducible expression of p53wt in p53‐negative HCC cells as well as a reduction of p53wt by siRNA in p53wt‐positive cells did not alter stathmin expression. However, stathmin was down‐regulated after siRNA‐based reduction of p53mut/Y220C and p53mut/R213Q expression in different tumor cell types. Conclusion: Our results demonstrate that overexpression of stathmin is an early protumorigenic event in human hepatocarcinogenesis, and its up‐regulation can be mediated by gain‐of‐function mutations in p53. Thus, stathmin represents a potential therapeutic target, for example, by increasing responsiveness of tumor cells to treatment with chemotherapeutic agents after reduction of stathmin bioactivity. (HEPATOLOGY 2007.)

Hepatic NF-κB essential modulator deficiency prevents obesity-induced insulin resistance but synergizes with high-fat feeding in tumorigenesis

Development of obesity-associated insulin resistance and diabetes mellitus type 2 has been linked to activation of proinflammatory pathways in the liver, leading to impaired insulin signal transduction. To further define the role of hepatic NF-κB activation in this process, we have analyzed glucose metabolism in mice with liver-specific inactivation of the NF-κB essential modulator gene (NEMOL-KO mice) exposed to a high-fat diet (HFD). These animals are protected from the development of obesity-associated insulin resistance, highlighting the importance of hepatic NF-κB activation in this context. However, hepatic NEMO deficiency synergizes with HFD in the development of liver steatosis as a consequence of decreased peroxisome proliferator-activated receptor (PPAR-α) and increased PPAR-γ expression. Steatosis interacts with increased inflammation, causing elevated apoptosis in the livers of these mice under HFD. These changes result in liver tumorigenesis of NEMOL-KO mice under normal diet, a process that is largely aggravated when these mice are exposed to HFD. These data directly demonstrate the interaction of hepatic inflammation, dietary composition, and metabolism in the development of liver tumorigenesis.

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.)

The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells

Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells.

High-Fat Intake During Pregnancy and Lactation Exacerbates High-Fat Diet-Induced Complications in Male Offspring in Mice

Altered fetal environments, such as a high-fat milieu, induce metabolic abnormalities in offspring. Different postnatal environments reveal the predisposition for adult diseases that occur during the fetal period. This study investigates the ability of a maternal high-fat diet (HFD) to program metabolic responses to HFD reexposure in offspring after consuming normal chow for 23 weeks after weaning. Wild-type CD1 females were fed a HFD (H) or control (C) chow during pregnancy and lactation. At 26 weeks of age, offspring were either reexposed (H-C-H) or newly exposed (C-C-H) to the HFD for 19 weeks. Body weight was measured weekly, and glucose and insulin tolerance were measured after 10 and 18 weeks on the HFD. The metabolic profile of offspring on a HFD or C diet during pregnancy and lactation and weaned onto a low-fat diet was similar at 26 weeks. H-C-H offspring gained more weight and developed larger adipocytes after being reintroduced to the HFD later in life than C-C-H. H-C-H mice were glucose and insulin intolerant and showed reduced gene expression of cox6a2 and atp5i in muscle, indicating mitochondrial dysfunction. In adipocytes, the expression of slc2a4, srebf1, and adipoq genes was decreased in H-C-H mice compared with C-C-C, indicating insulin resistance. H-C-H showed extensive hepatosteatosis, accompanied by increased gene expression for cd36 and serpin1, compared with C-C-H. Perinatal exposure to a HFD programs a more deleterious response to a HFD challenge later in life even after an interval of normal diet in mice.

Spatiotemporal variation of mammalian protein complex stoichiometries

BackgroundRecent large-scale studies revealed cell-type specific proteomes. However, protein complexes, the basic functional modules of a cell, have been so far mostly considered as static entities with well-defined structures. The co-expression of their members has not been systematically charted at the protein level.ResultsWe used measurements of protein abundance across 11 cell types and five temporal states to analyze the co-expression and the compositional variations of 182 well-characterized protein complexes. We show that although the abundance of protein complex members is generally co-regulated, a considerable fraction of all investigated protein complexes is subject to stoichiometric changes. Compositional variation is most frequently seen in complexes involved in chromatin regulation and cellular transport, and often involves paralog switching as a mechanism for the regulation of complex stoichiometry. We demonstrate that compositional signatures of variable protein complexes have discriminative power beyond individual cell states and can distinguish cancer cells from healthy ones.ConclusionsOur work demonstrates that many protein complexes contain variable members that cause distinct stoichometries and functionally fine-tune complexes spatiotemporally. Only a fraction of these compositional variations is mediated by changes in transcription and other mechanisms regulating protein abundance contribute to determine protein complex stoichiometries. Our work highlights the superior power of proteome profiles to study protein complexes and their variants across cell states.

Depletion of the transcriptional coactivators megakaryoblastic leukaemia 1 and 2 abolishes hepatocellular carcinoma xenograft growth by inducing oncogene-induced senescence

Megakaryoblastic leukaemia 1 and 2 (MKL1/2) are coactivators of the transcription factor serum response factor (SRF). Here, we provide evidence that depletion of MKL1 and 2 abolishes hepatocellular carcinoma (HCC) xenograft growth. Loss of the tumour suppressor deleted in liver cancer 1 (DLC1) and the subsequent activation of RhoA were prerequisites for MKL1/2 knockdown‐mediated growth arrest. We identified oncogene‐induced senescence as the molecular mechanism underlying the anti‐proliferative effect of MKL1/2 knockdown. MKL1/2 depletion resulted in Ras activation, elevated p16 expression and hypophosphorylation of the retinoblastoma (Rb) protein in DLC1‐deficient HCC cells. Interestingly, reconstitution of HuH7 HCC cells with DLC1 also induced senescence. Evaluation of the therapeutic efficacy of MKL1/2 knockdown in vivo revealed that systemic treatment of nude mice bearing HuH7 tumour xenografts with MKL1/2 siRNAs complexed with polyethylenimine (PEI) completely abolished tumour growth. The regression of the xenografts was associated with senescence. Importantly, PEI‐complexed MKL1 siRNA alone was sufficient for complete abrogation of HCC xenograft growth. Thus, MKL1/2 represent promising novel therapeutic targets for the treatment of HCCs characterized by DLC1 loss.