[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.
[Show abstract][Hide abstract] ABSTRACT: In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.
[Show abstract][Hide abstract] ABSTRACT: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleo-cytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here, we identified Cellular Apoptosis Susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r = 0.463; p<0.01) supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- vs. p53 +/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner.Conclusion: Our study indicates that the CAS/imp-α1 transport cycle is linked to XIAP and required to maintain tumor cell survival in HCC. Moreover, we found that CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis. (Hepatology 2014)
[Show abstract][Hide abstract] ABSTRACT: In recent years, it has become clear that splicing factors play a direct role in cancer development. We showed previously that splicing factors SRSF1, SRSF6, and hnRNP A2/B1 are up-regulated in several cancers and can act as oncogenes when up-regulated. Here we examined the role of splicing factors hnRNP A1/A1b and hnRNP A2/B1 in hepatocellular carcinoma (HCC). We show that the splicing factors hnRNP A1 and hnRNP A2 are up-regulated in HCC tumors derived from inflammation-induced liver cancer mouse model. Overexpression of hnRNP A1 or hnRNP A2, but not the splicing isoform hnRNP B1, induced tumor formation of immortalized liver progenitor cells, while knockdown of these proteins inhibited anchorage-independent growth and tumor growth of human liver cancer cell lines. In addition, we found that cells overexpressing hnRNP A2 showed constitutive activation of the Ras-MAPK-ERK pathway. In contrast, knockdown of hnRNP A2 inhibited the Ras-MAPK-ERK pathway and prevented ERK1/2 activation by EGF. Moreover, we found that hnRNP A2 regulates the splicing of A-Raf, reducing the production of a short dominant-negative isoform of A-Raf and elevating the full-length A-Raf transcript. Taken together, our data suggest that hnRNP A2 up-regulation in HCC induces an alternative splicing switch that down-regulates a dominant-negative isoform of A-Raf, leading to activation of the Raf-MEK-ERK pathway and cellular transformation.
[Show abstract][Hide abstract] ABSTRACT: A broad in vivo screen of the effects of specific gene inhibition on the antitumour activity of immune cells in mice bearing melanomas has revealed potential targets for cancer therapy. See Article p.52
[Show abstract][Hide abstract] ABSTRACT: Acute liver failure remains a critical clinical condition with high mortality rates, and increased apoptosis of hepatocytes represents a key event in the cause of liver failure. Alpha-1-Antitrypsin (AAT) is synthesized and secreted mainly by hepatocytes, and plasma purified AAT is used for augmentation therapy in patients with AAT deficiency. Because AAT therapy exerts anti-inflammatory and immune modulatory activities in various experimental models and it was recently suggested that AAT exerts anti-apoptotic activities, we aimed to explore whether administration of AAT may represent a therapeutic strategy to treat acute liver failure in mice. Well established preclinical models of acute liver failure such as the Jo2 FAS/CD95 activating model and models of Acetaminophen and α-Amanitin poisoning were used. Therapeutic effects of AAT were evaluated by monitoring animal survival, histopathological changes, measurement of caspase activity and serum cytokine levels. Systemic treatment with AAT significantly decreased Jo2-induced liver cell apoptosis and prolonged survival of mice. Native and oxidized (lacking elastase inhibitory activity) forms of AAT were equally effective in preventing acute liver injury and showed direct inhibition of active caspase-3 and -8 in liver homogenates and in a cell-free system, in vitro. Concomitantly, mice treated with AAT showed significantly lower serum levels of TNF-α, which also paralleled with reduced activity of ADAM17 (TACE). Noticeably, the increased survival and a reduction of apoptotic hepatocytes were also observed in the α-Amanitin and Acetaminophen-induced liver injury mouse models. Conclusions: Our data suggest that systemic administration of AAT can be as a promising therapy to treat acute liver failure and clinical studies to explore this treatment in humans should be initiated. (Hepatology 2014;).
[Show abstract][Hide abstract] ABSTRACT: Immunotherapy of solid tumors is often hampered by the low frequency of tumor-specific T cells elicited by current vaccination strategies. Here we describe a prime-boost vaccination protocol based on the administration of antigen conjugated to PLGA microspheres followed by booster vaccination with Listeria monocytogenes vectors, which rapidly generates potent immune responses within two weeks. Compared with conventional vaccination with antigen-pulsed dendritic cells, the use of PLGA microspheres resulted in immune responses of significantly higher magnitude, which could be further enhanced via co-injection of Toll-like receptor 3 agonists. In an immunocompetent model of subcutaneous hepatocellular carcinoma, PLGA/Listeria vaccination resulted in complete remission of established tumors and prolonged survival. To further test the efficacy of the novel vaccination for the treatment of solid tumors, we developed an orthotopic liver cancer model based on the injection of transposon-flanked plasmids expressing oncogenes and model antigens. In this transgenic mouse model of liver cancer, PLGA/Listeria vaccination resulted in eradication of liver tumors, long-term survival of animals and establishment of stable cancer-specific memory CD8(+) T-cell populations. Therefore, combined PLGA/Listeria vaccination holds promise as a novel immunotherapeutic option for the treatment of solid cancers and as a means to boost the therapeutic efficacy of established cancer vaccines. This article is protected by copyright. All rights reserved.
European Journal of Immunology 12/2013; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mouse Double Minute homolog 4 (MDM4) gene upregulation often occurs in human hepatocellular carcinoma (HCC), but the molecular mechanisms responsible for its induction remain poorly understood. Here, we investigated the role of the phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of Rapamycin (PI3K/AKT/mTOR) axis in the regulation of MDM4 levels in HCC. The activity of MDM4 and the PI3K/AKT/mTOR pathway was modulated in human HCC cell lines via silencing and overexpression experiments. Expression of main pathway components was analyzed in an AKT mouse model and human HCCs. MDM4 inhibition resulted in growth restraint of HCC cell lines both in vitro and in vivo. Inhibition of the PI3K-AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells and reactivated p53-dependent transcription. De-ubiquitination by ubiquitin-specific protease 2a and AKT-mediated phosphorylation protected MDM4 from proteasomal degradation and increased AKT protein stability. The eukaryotic elongation factor 1A2 (EEF1A2) was identified as an upstream inducer of PI3K supporting MDM4 stabilization. Also, we detected MDM4 protein upregulation in an AKT mouse model and a strong correlation between the expression of EEF1A2, activated/phosphorylated AKT, and MDM4 in human HCC (each rho>.8, P<.001). Noticeably, a strong activation of this cascade was associated with shorter patients' survival. Conclusions: The EEF1A2/PI3K/AKT/mTOR axis promotes the protumorigenic stabilization of the MDM4 protooncogene in human HCC via a post-transcriptional mechanism. The activation level of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis significantly influences the survival probability of HCC patients in vivo and may thus represent a promising molecular target. (Hepatology 2013;).
[Show abstract][Hide abstract] ABSTRACT: Data from different laboratories and theoretical considerations challenged our current view on anticancer immunity. Immune cells are capable of destroying cancer cells under in vitro and in vivo conditions. Therefore, cellular immunity is considered to control cancers through mechanisms that kill cancers. Yet, therapeutic anticancer immune responses rarely delete cancers, but rather establish a life with stable disease. This raises the question of whether killing is the sole mechanism by which immune therapy attacks cancers. Here, we propose additional mechanisms involved in controlling disseminated cancer cells. We suggest that, besides cancer eradication by cytotoxic lymphocytes, other modes of action are operative. We showed that T helper-1 cells impair cancer growth by driving cancers into senescence. Such immune cells establish cytokine-producing walls around the tumors. When producing interferon-γ and tumor necrosis factor, tumor surveillance keeps the cancer cells in a permanently non-proliferating state. Simultaneously, antiangiogenic chemokines cut their connections to the surrounding tissues. In mice, this strategy significantly reduced the tumor burden and prolonged the life of cancer-bearing animals. As human cancers also undergo senescence, our data raise the question of whether therapy should focus on tumor eradication, or whether efficient cancer control is a more realistic and better tolerated goal.
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing regulators have emerged as new players in cancer development, modulating the activities of many tumor suppressors and oncogenes and regulating signaling pathways. However, little is known about the mechanisms by which these oncogenic splicing factors lead to cellular transformation. We have shown previously that the splicing factor SRSF1 (SF2/ASF) is a proto-oncogene which is amplified in breast cancer and transforms immortal cells when overexpressed. In this study we performed a structure-function analysis of SRSF1 and found that the RNA recognition motif 1 domain (RRM1) is required for its oncogenic activity. Deletion of RRM1 eliminated the splicing activity of SRSF1 on some of its endogenous targets. Moreover, we found that SRSF1 elevates the expression of B-Raf and activates the MEK-ERK pathway, and that RRM1 is required for this activation as well. B-Raf-MEK-ERK activation by SRSF1 contributes to transformation and pharmacological inhibition of MEK1 inhibits SRSF1-mediated transformation. In conclusion, the RRM1 of SRSF1 is both required, and when tethered to the RS domain, also sufficient to activate the Raf-MEK-ERK pathway and to promote cellular transformation.
[Show abstract][Hide abstract] ABSTRACT: Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease.
The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages.
An accumulation of MDSC was found in mice with HCC irrespectively of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL-6, IL-1 and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF over-expression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib.
Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.
Journal of Hepatology 06/2013; · 9.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 p15(INK4b) and p21(CIP1). 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.
[Show abstract][Hide abstract] ABSTRACT: The incidence of cholangiocellular carcinoma (CCC) is increasing worldwide. Using a transgenic mouse model, we found that expression of the intracellular domain of Notch 1 (NICD) in mouse livers results in the formation of intrahepatic CCCs. These tumors display features of bipotential hepatic progenitor cells, indicating that intrahepatic CCC can originate from this cell type. We show that human and mouse CCCs are characterized by high expression of the cyclin E protein and identified the cyclin E gene as a direct transcriptional target of the Notch signaling pathway. Intriguingly, blocking γ-secretase activity in human CCC xenotransplants results in downregulation of cyclin E expression, induction of apoptosis, and tumor remission in vivo.
[Show abstract][Hide abstract] ABSTRACT: Complete surgical tumor resection (R0) for treatment of intrahepatic cholangiocarcinoma (ICC) is potentially curative, but the prognosis remains dismal due to frequent tumor recurrence and metastasis after surgery. Adjuvant therapies may improve the outcome, but clinical studies for an adjuvant approach are difficult and time consuming for rare tumor entities. Therefore, animal models reflecting the clinical situation are urgently needed to investigate novel adjuvant therapies. To establish a mouse model of resectable cholangiocarcinoma including the most frequent genetic alterations of human ICC, we electroporated sleeping beauty-based oncogenic transposon plasmids into the left liver lobe of mice. KRas-activation in combination with p53-knockout in hepatocytes resulted in formation of a single ICC nodule within 3-5 weeks. Lineage tracing analyses confirmed the development of ICC by transdifferentiation of hepatocytes. Histologic examination demonstrated that no extrahepatic metastases were detectable during primary tumor progression. However, formation of tumor satellites close to the primary tumor and vascular invasion could be observed indicating early invasion into normal tissue adjacent to the tumor. After R0-resection of the primary tumor, we were able to prolong median survival thereby observing tumor stage-dependent local recurrence, peritoneal carcinomatosis and lung metastasis. Adjuvant gemcitabine chemotherapy after R0-resection significantly improved median survival of treated animals. Conclusion: we have developed a murine model of single, R0-resectable ICC with favorable characteristics for the study of recurrence patterns and mechanisms of metastasis after resection. This model holds great promise for preclinical evaluation of novel multimodal or adjuvant therapies to prevent recurrence and metastasis after R0-resection. (HEPATOLOGY 2013.).
[Show abstract][Hide abstract] ABSTRACT: 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.