Negative regulation of interferon lambda induced JAK-STAT signaling and development of patient-derived xenograft models from fresh human hepatocellular carcinoma biopsies

Type I and type III interferons (IFNs) act as the first line of defense against invading pathogens by inducing a fast and strong host response characterized by the expression of hundreds of interferon stimulated genes (ISGs). However, the magnitude and duration of cellular responses to viral and bacterial infections needs to be controlled properly to maintain tissue homeostasis. Ubiquitin specific peptidase 18 (USP18), suppressor of cytokine signaling 1 (SOCS1) and SOCS3 are the three known inducible negative regulators of the IFN-α induced signaling cascade. However, the role of USP18 on IFN-λ signaling is a matter of controversy. Furthermore, the physiological relevance of the relative contribution of SOCS1 and SOCS3 on in vitro IFN-λ signaling needs to be validated in vivo. Thus, we aimed to investigate the role of USP18, SOCS1 and SOCS3 on the IFN-λ induced signaling cascade both in vitro and in vivo. Based on experiments with USP18, SOCS1 and SOCS3 knockout cells, we demonstrated that USP18 is the major negative regulator of IFN-α induced JAK-STAT signaling whereas IFN-λ is negatively regulated by SOCS1. Furthermore, using USP18 and SOCS1 knockout mice, we confirmed USP18 and SOCS1 as physiological relevant negative regulators of IFN-α and IFN-λ, respectively. Importantly, we demonstrated that negative regulation of IFN-α was strong and immediate while that for IFN-λ was more subtle both in kinetics as well as magnitude. Taken together, our results suggest that the differences in negative regulations are the basis for the distinct kinetic properties of IFN-α and IFN-λ signaling reflecting their specific functions. IFN-α signaling provides a powerful and immediate defense system against systemic infections but has to be controlled tightly to maintain tissue homeostasis. Therefore a strong negative regulator like USP18 that completely shuts down the system is needed. By contrast, IFN-λ provides a continuous first line defense in mucosal epithelial cells that are constantly exposed to pathogens. Thus, SOCS1 facilitates a maintained but controlled IFN-λ signaling that allows fighting invading pathogens without loosing tissue homeostasis. Hepatocellular carcinoma (HCC) is the second deadliest cancer worldwide with yearly increasing incidence and unsatisfying treatment options. Thus, there is a clear need for new and more efficient drugs for the treatment of HCC. A major obstacle for the understanding of the pathogenesis of HCC is the lack of an efficient in vivo model that accurately reflects the broad spectrum of human HCC. Patient-derived xenograft (PDX) models gained a lot of interest in pre-clinical studies of anti-cancer drugs. Indeed, several HCC PDX models have been established in recent years. However, all these models are derived from resected HCC specimen and therefore limited to early stage disease. Patients with advanced stage HCC are not represented, although they would benefit most from new treatment options. Therefore, we aimed to generate HCC PDX models from fresh human HCC biopsies that cover all disease stages, with special interest in advanced stage HCC. We successfully established and passaged eleven HCC PDX mouse models from patients presenting with all major underlying liver diseases. The biopsies that successfully engrafted were representative of the spectrum of poorly differentiated HCCs, including both early and late-stage disease. Importantly, the PDX models recapitulated tumor morphology, differentiation grade and the expression pattern of known HCC markers. Finally, RNA sequencing analyses demonstrated that our PDX models maintained the transcriptomic profiles and expression of somatic mutations of their originating tumors over at least four generations. Taken together, these novel HCC PDX models do not only allow investigation of the biology of all stages of HCC but also the study of drug-induced resistance mechanisms and the development of new HCC therapies

USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients

Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

Type I (α and β) and type III (λ) IFNs are induced upon viral infection through host sensory pathways that activate IFN regulatory factors (IRFs) and nuclear factor κB. Secreted IFNs induce autocrine and paracrine signaling through the JAK-STAT pathway, leading to the transcriptional induction of hundreds of IFN-stimulated genes, among them sensory pathway components such as cGAS, STING, RIG-I, MDA5, and the transcription factor IRF7, which enhance the induction of IFN-αs and IFN-λs. This positive feedback loop enables a very rapid and strong host response that, at some point, has to be controlled by negative regulators to maintain tissue homeostasis. Type I IFN signaling is controlled by the inducible negative regulators suppressor of cytokine signaling 1 (SOCS1), SOCS3, and ubiquitin-specific peptidase 18 (USP18). The physiological role of these proteins in IFN-γ signaling has not been clarified. Here we used knockout cell lines and mice to show that IFN-λ signaling is regulated by SOCS1 but not by SOCS3 or USP18. These differences were the basis for the distinct kinetic properties of type I and III IFNs. We found that IFN-α signaling is transient and becomes refractory after hours, whereas IFN-λ provides a long-lasting IFN-stimulated gene induction

Closed versus open treatment of mandibular condylar process fractures: A meta-analysis of retrospective and prospective studies

INTRODUCTION: The treatment of fractures of the mandibular process remains controversial, although there is a trend towards open reduction and internal fixation. This study compared open and closed treatments and assessed the results with a meta-analysis. MATERIALS AND METHODS: A literature search of PubMed found eight studies that met the search criteria and were included in the meta-analysis. RESULTS: The studies increasingly suggest better results for open treatment, in terms of mouth opening, protrusion, laterotrusion, pain, and malocclusion. In the meta-analysis, the outcome was significantly better for laterotrusion and protrusion in patients treated by open reduction and internal fixation. CONCLUSION: Due to the different study protocols and lack of information on classification, follow-up time, and inclusion criteria, comparison of the studies remains difficult and further prospective, randomized studies should examine these issues

Hepatocellular Carcinoma Xenografts Established From Needle Biopsies Preserve the Characteristics of the Originating Tumors.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Treatment options for patients with advanced-stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient-derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma-c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor-associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development

YAP promotes proliferation, chemoresistance and angiogenesis in human cholangiocarcinoma

Cholangiocarcinoma (CC) is the second most prevalent liver tumor with resistance to chemotherapy and high mortality. The underlying molecular mechanisms that propagate CC are poorly understood. The YAP/Hippo pathway has been implicated in tumorigenesis, however, its role in CC is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Proliferation and colony formation of CC cells are increased upon YAP activation and impaired upon YAP downregulation. Likewise, CC xenografts show increased tumor size and growth when YAP is overexpressed in vivo. Moreover, YAP activation prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. Using microarray expression profiling of CC cells with overexpressed or downregulated YAP, respectively, we establish a CC YAP target gene signature demonstrating that YAP regulates genes involved in proliferation, anti-apoptotic mechanisms and angiogenesis. We show that MFAP5 is a direct transcriptional target in CC cells and that secreted MFAP5 protein promotes tube formation of human microvasculature endothelial cells. The expression of YAP target genes and TEADs, transcription factors mediating YAP signaling, correlates with MFAP5 expression in 176 cell lines derived from different tumors, suggesting that this mechanism is not restricted to CC. Moreover, we show that YAP promotes CC cell proliferation, resistance to apoptosis and angiogenesis via functionally interacting with TEAD transcription factors. Together, our data establish YAP as a key regulator of proliferation and anti-apoptotic mechanisms in CC. Additionally, this report provides first evidence that the YAP/Hippo regulates MFAP5 expression which in turn promotes angiogenesis

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors

The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature.; These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins