Alona Zilberberg

The EDD E3 ubiquitin ligase ubiquitinates and up-regulates β-catenin

The effector protein of the canonical Wnt pathway is β-catenin, which is regulated by the ubiquitin system. This study shows that the E3 ubiquitin ligase EDD ubiquitinates β-catenin, leading to up-regulation of β-catenins expression levels and activity. Thus the results demonstrate a role for the ubiquitin system in up-regulation of the Wnt pathway.

Restoration of APC Gene Function in Colorectal Cancer Cells by Aminoglycoside- and Macrolide-Induced Read-through of Premature Termination Codons

Adenomatous polyposis coli (APC) is a multifunctional tumour suppressor protein that negatively regulates the Wnt signalling pathway. The APC gene is ubiquitously expressed in tissues and organs, including the large intestine and central nervous system. The majority of patients with sporadic and hereditary colorectal cancer have mutations in the gene encoding APC. Approximately 30% of these mutations are single nucleotide changes that result in premature stop codons (nonsense mutations). A potential therapeutic approach for treatment of this subset of patients is the use of aminoglycosides and macrolides that induce nonsense mutation read-through and restore levels of full-length protein. We have used reporter plasmids and colorectal cancer cell lines to demonstrate that several aminoglycosides and tylosin, a member of the macrolide family, induced read-through of nonsense mutations in the APC gene. In xenograft experiments and in the ApcMin/+ mouse model, these compounds ameliorated the tumorigenic clinical symptoms caused by nonsense mutations in the APC gene.

Melanoma miRNA trafficking controls tumour primary niche formation

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

The armadillo repeat domain of the APC tumor suppressor protein interacts with Striatin family members.

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor protein that negatively regulates the Wnt signaling pathway. The APC gene is ubiquitously expressed in various tissues, especially throughout the large intestine and central nervous system. Mutations in the gene encoding APC have been found in most colorectal cancers and in other types of cancer. The APC gene product is a large multidomain protein that interacts with a variety of proteins, many of which bind to the well conserved armadillo repeat domain of APC. Through its binding partners, APC affects a large number of important cellular processes, including cell-cell adhesion, cell migration, organization of the actin and microtubule cytoskeletons, spindle formation and chromosome segregation. The molecular mechanisms that control these diverse APC functions are only partly understood. Here we describe the identification of an additional APC armadillo repeat binding partner - the Striatin protein. The Striatin family members are multidomain molecules that are mainly neuronal and are thought to function as scaffolds. We have found that Striatin is expressed in epithelial cells and co-localizes with APC in the epithelial tight junction compartment and in neurite tips of PC12 cells. The junctional localization of APC and Striatin is actin-dependent. Depletion of APC or Striatin affected the localization of the tight junction protein ZO-1 and altered the organization of F-actin. These results raise the possibility that the contribution of APC to cell-cell adhesion may be through interaction with Striatin in the tight junction compartment of epithelial cells.

PAX6 Regulates Melanogenesis in the Retinal Pigmented Epithelium through Feed-Forward Regulatory Interactions with MITF

During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of Pax6 in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of Mitf and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.

CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APCMin/CD24 double knockout transgenic mice

Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APCMin/+ mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation‐associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by β‐catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis.

The metabolic regulator PGC-1α links hepatitis C virus infection to hepatic insulin resistance

BACKGROUND & AIMS Chronic hepatitis C virus (HCV) infection is strongly associated with insulin resistance and diabetes mellitus. Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) is a transcriptional co-activator involved in the initiation of gluconeogenesis in the liver. Increased hepatic expression of PGC-1α has been implicated in insulin resistance. We investigated whether modulation of PGC-1α levels following HCV infection underlies HCV-associated hepatic insulin resistance. METHODS HCV genomes were expressed in hepatoma cells followed by analysis of PGC-1α and gluconeogenesis levels. RESULTS PGC-1α was robustly induced in HCV infected cells. PGC-1α induction was accompanied by an elevated expression of the gluconeogenic gene glucose-6 phosphatase (G6Pase) and increased glucose production. The induction of gluconeogenesis is HCV dependent, since interferon treatment abolishes PGC-1α and G6Pase elevation and decreases glucose output. Moreover, PGC-1α knockdown resulted in a significant reduction of G6Pase levels in HCV full length replicon cells, emphasizing the central role of PGC-1α in the exaggerated gluconeogenic response observed in HCV patients. Treatment of HCV replicon cells with the antioxidant N-acetylcysteine resulted in reduction of PGC-1α levels, suggesting that HCV-induced oxidative stress promoted PGC-1α upregulation. Finally, both PGC-1α and G6Pase RNA levels were significantly elevated in liver samples of HCV infected patients, highlighting the clinical relevance of these results. CONCLUSIONS PGC-1α is robustly induced following HCV infection, resulting in an upregulated gluconeogenic response, thereby linking HCV infection to hepatic insulin resistance. Our results suggest that PGC-1α is a potential molecular target for the treatment of HCV-associated insulin resistance.

14-3-3 and β-catenin are secreted on extracellular vesicles to activate the oncogenic Wnt pathway

Aberrant activation of the canonical Wnt signal transduction pathway is involved in a large number of human diseases. β‐catenin, the key effector protein of the canonical Wnt pathway, functions in the nucleus with T‐cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Here we show that members of the 14‐3‐3 protein family bind disheveled‐2 (Dvl‐2) and glycogen synthase‐3β (GSK‐3β) to attenuate the interaction between GSK‐3β and β‐catenin. Importantly, 14‐3‐3 and β‐catenin form “bleb‐like” structures and are secreted via extracellular vesicles to induce Wnt signaling activity in target cells. Our data suggest a novel way of transducing the oncogenic Wnt signal in which β‐catenin is regulated by 14‐3‐3ζ through the formation of “oncosomes” that contain both the 14‐3‐3 and β‐catenin proteins.

Abstract 1338: Knockdown of CD24 prevents colorectal polyp formation in APCmin/CD24 double knockout transgenic mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Previous studies in our lab have shown that CD24 is a potential oncogene in the colon. CD24, a mucin-like glycoprotein, is overexpressed in 90% of colorectal cancer (CRC) tumors at a fairly early stage in the multistep process of CR carcinogenesis (Sagiv et al., Gastroenterology, 2006). We have also shown that CD24 downregulation by shRNA interference (Sagiv et al., Can Res, 2008) or anti-CD24 mAb targeting (Shapira et al., Gastroenterology, 2011) retarded tumorigenicity of human CRC cell lines and/or reduced tumor volume in nude mice. The APCmin/+ is a popular animal model for studies of human colon carcinogenesis and the molecular changes associated with neoplasia in this system have been partially characterized. These mice have a germline nonsense mutation at codon 850 of the APC gene and spontaneously develop hundreds of polyps, mostly in the small intestine at the age of 10-12 weeks. Aim: To validate the importance of CD24 in intestinal carcinogenesis using a transgenic mouse model with a double knockout (KO) of CD24−/+ /CD24+/− and APCMin/+ alleles. Methods: We generated the CD24−/+/APCMin/+ double KO by crossing the mice with each of the single gene knockouts, in order to obtain both CD24+/−/APCMin/+ and CD24−/+ APCMin/+ genotypes. The double KO mice were followed up thrice a week and sacrificed at 12 weeks of age. Tumors from the entire small and large intestine were counted and verified by histological analysis Results: There was almost a complete absence of polyps in the small intestine in APCMin/+/CD24+/− double heterozygotes compared to that of the parental APCMin/+, CD24+/+ animals: 273±55.4 vs. 6±1.7 polyps, respectively (P=0.0001). APCMin/+and CD24−/+ homozygotes showed a striking lack of polyps (P< 0.0001). This reduction occurred in all sections of the small intestine. Histophatological analysis confirmed the absence of malignant lesions in the double KO mice as compared to the parental mice. Conclusions: 1. A deficiency of only one CD24 allele is sufficient to suppress almost completely polyp formation in the APCMin/+ mice that normally develop hundreds of polyps. 2. Although the mechanism/s underlying the pathophysiologic role of CD24 in the development of CRC are still unknown, these studies suggest that CD24 plays a critical role in the proliferation and progression of CRC and is a potential important target for the prevention and treatment of intestinal neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1338. doi:1538-7445.AM2012-1338