Project description:Up to 41% of hepatocellular carcinomas (HCCs) result from activating mutations in the CTNNB1 gene encoding β-catenin. β-catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC-associated CTNNB1 mutations stabilize the β-catenin protein, leading to nuclear and/or cytoplasmic localization of β-catenin and downstream activation of Wnt target genes. In patient HCC samples, β-catenin nuclear and cytoplasmic localization are typically patchy, even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in β-catenin activation are not well understood. To define mechanisms of β-catenin-driven HCC initiation, we generated a Cre-lox system that enabled switching on activated β-catenin in 1) a small number of hepatocytes in early development; or 2) the majority of hepatocytes in later development or adulthood. We discovered that switching on activated β-catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/β-catenin signaling heterogeneity later in hepatocarcinogenesis, we performed RNA-seq analysis of zebrafish β-catenin-driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre-lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered, indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single-cell level, 2.9% to 15.2% of hepatocytes from zebrafish β-catenin-driven HCC expressed two or more of the Wnt target genes axin2, mtor, glula, myca, and wif1, indicating focal activation of Wnt signaling in established tumors. Thus, heterogeneous β-catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis.
Project description:Up to 41% of hepatocellular carcinomas (HCCs) result from activating mutations in the CTNNB1 gene encoding β-catenin. β-catenin has dual cellular functions as a component of the Wnt signaling pathway and adherens junctions. HCC-associated CTNNB1 mutations stabilize the β-catenin protein, leading to nuclear and/or cytoplasmic localization of β-catenin and downstream activation of Wnt target genes. In patient HCC samples, β-catenin nuclear and cytoplasmic localization are typically patchy, even among HCC with highly active CTNNB1 mutations. The functional and clinical relevance of this heterogeneity in β-catenin activation are not well understood. To define mechanisms of β-catenin-driven HCC initiation, we generated a Cre-lox system that enabled switching on activated β-catenin in 1) a small number of hepatocytes in early development; or 2) the majority of hepatocytes in later development or adulthood. We discovered that switching on activated β-catenin in a subset of larval hepatocytes was sufficient to drive HCC initiation. To determine the role of Wnt/β-catenin signaling heterogeneity later in hepatocarcinogenesis, we performed RNA-seq analysis of zebrafish β-catenin-driven HCC. Ingenuity Pathway Analysis of differentially expressed genes in the Cre-lox HCC model revealed that “Cancer” and “Liver Tumor” categories were significantly altered, indicating transcriptional similarities with human HCC and other vertebrate HCC models. At the single-cell level, 2.9% to 15.2% of hepatocytes from zebrafish β-catenin-driven HCC expressed two or more of the Wnt target genes axin2, mtor, glula, myca, and wif1, indicating focal activation of Wnt signaling in established tumors. Thus, heterogeneous β-catenin activation drives HCC initiation and persists throughout hepatocarcinogenesis.
Project description:Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. β-catenin is widely thought to be a major oncogene in HCC based on the frequency of mutations associated with aberrant Wnt signaling in HCC patients. Challenging this model, our data reveal that β-catenin nuclear accumulation is restricted to the late stage of the disease. Until then, β-catenin is primarily located at the plasma membrane in complex with multiple cadherin family members where it drives tumor cell survival by enhancing the signaling of growth factor receptors such as EGFR. Therefore, our study reveals the evolving nature of β-catenin in HCC to establish it as a compound tumor promoter during the progression of the disease.
Project description:The importance of unanchored Ub in innate immunity has been shown only for a limited number of unanchored Ub-interactors. We investigated what additional cellular factors interact with unanchored Ub and whether unanchored Ub plays a broader role in innate immunity. To identify unanchored Ub-interacting factors from murine lungs, we used His-tagged recombinant poly-Ub chains as bait. These chains were mixed with lung tissue lysates and protein complexes were isolated with Ni-NTA beads. Sample elutions were subjected to mass spectrometry (LC-MSMS) analysis.
Project description:Wnt signals control three functions of intestinal crypts: maintenance of Lgr5 stem cells, proliferation of transit-amplifying daughters and formation of Paneth cells. Here, we study how the Wnt effector β-catenin/Tcf4 cooperates with the Wnt-activated transcription factor Ascl2 to control a stem cell transcription program. DNA elements that are co-occupied and synergistically regulated by Ascl2 and Tcf4 specifically map to stem cell genes. In vitro, Tcf4-/- mini-guts are rescued by Ascl2 expression, while Ascl2-/- organoids are rescued by Wnt signaling. A direct auto-activatory loop leads to an on/off expression pattern of Ascl2 with a threshold that depends on the previous state. Wnt/R-spondin1 activates this loop. This mechanism interprets Wnt levels in crypts and translates this continuous signal into a discrete Ascl2 âonâ or âoffâ decision. In turn Ascl2, together with β-catenin/Tcf, activates stem cell genes. Thus, Ascl2 forms a transcriptional 'stemness switch' that is both Wnt-responsive and Wnt-dependent Examination of Tcf4, B-catenin and Ascl2 DNA occupancy in murine intestinal organoids and human colorectal cancer cell lines *** Original raw files unavailable due to loss during backup ***
Project description:Hepatocellular carcinoma (HCC) is one of the most aggressive tumors and the treatment outcome of this disease is improved when the cancer is diagnosed at early stages. This requires biomarkers allowing for an accurate and early tumor diagnosis. To identify potential markers for such applications we performed a label-free proteome analysis using samples collected from 19 patients. We analyzed the data considering events known to take place in early events of HCC development such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). We expected that such analysis can lead to the identification of potential biomarkers for early-stage HCC diagnosis. 31 proteins functionally linked to downstream interactors of Wnt/b-catenin and parts of RTKs´-activated pathways Ras and JAK/STAT were selected for verification in a larger and independent cohort (n= 31) using selected (multiple) reaction monitoring (SRM/MRM).