Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies

SummaryBackgroundRetinoblastoma is the childhood retinal cancer that defined tumour-suppressor genes. Previous work shows that mutation of both alleles of the RB1 retinoblastoma suppressor gene initiates disease. We aimed to characterise non-familial retinoblastoma tumours with no detectable RB1 mutations.MethodsOf 1068 unilateral non-familial retinoblastoma tumours, we compared those with no evidence of RB1 mutations (RB1+/+) with tumours carrying a mutation in both alleles (RB1−/−). We analysed genomic copy number, RB1 gene expression and protein function, retinal gene expression, histological features, and clinical data.FindingsNo RB1 mutations (RB1+/+) were reported in 29 (2·7%) of 1068 unilateral retinoblastoma tumours. 15 of the 29 RB1+/+ tumours had high-level MYCN oncogene amplification (28–121 copies; RB1+/+MYCNA), whereas none of 93 RB1−/− primary tumours tested showed MYCN amplification (p<0·0001). RB1+/+MYCNA tumours expressed functional RB1 protein, had fewer overall genomic copy-number changes in genes characteristic of retinoblastoma than did RB1−/− tumours, and showed distinct aggressive histological features. MYCN amplification was the sole copy-number change in one RB1+/+MYCNA retinoblastoma. One additional MYCNA tumour was discovered after the initial frequencies were determined, and this is included in further analyses. Median age at diagnosis of the 17 children with RB1+/+MYCNA tumours was 4·5 months (IQR 3·5–10), compared with 24 months (15–37) for 79 children with non-familial unilateral RB1−/− retinoblastoma.InterpretationAmplification of the MYCN oncogene might initiate retinoblastoma in the presence of non-mutated RB1 genes. These unilateral RB1+/+MYCNA retinoblastomas are characterised by distinct histological features, only a few of the genomic copy-number changes that are characteristic of retinoblastoma, and very early age of diagnosis.FundingNational Cancer Institute–National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa

Critical Role of the Rb Family in Myoblast Survival and Fusion

The tumor suppressor Rb is thought to control cell proliferation, survival and differentiation. We recently showed that differentiating Rb-deficient mouse myoblasts can fuse to form short myotubes that quickly collapse through a mechanism involving autophagy, and that autophagy inhibitors or hypoxia could rescue the defect leading to long, twitching myotubes. Here we determined the contribution of pRb relatives, p107 and p130, to this process. We show that chronic or acute inactivation of Rb plus p107 or p130 increased myoblast cell death and reduced myotube formation relative to Rb loss alone. Treatment with autophagy antagonists or hypoxia extended survival of double-knockout myotubes, which appeared indistinguishable from control fibers. In contrast, triple mutations in Rb, p107 and p130, led to substantial increase in myoblast death and to elongated bi-nuclear myocytes, which seem to derive from nuclear duplication, as opposed to cell fusion. Under hypoxia, some rare, abnormally thin triple knockout myotubes survived and twitched. Thus, mutation of p107 or p130 reduces survival of Rb-deficient myoblasts during differentiation but does not preclude myoblast fusion or necessitate myotube degeneration, whereas combined inactivation of the entire Rb family produces a distinct phenotype, with drastically impaired myoblast fusion and survival

Lunatic Fringe Deficiency Cooperates with the Met/Caveolin Gene Amplicon to Induce Basal-Like Breast Cancer

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC

Conservation of Complex Nuclear Localization Signals Utilizing Classical and Non-Classical Nuclear Import Pathways in LANA Homologs of KSHV and RFHV

ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin β1 in a RanGTP-sensitive manner and serves as an NLS in the importin β1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/β-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency

A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes

Lunatic Fringe Deficiency Cooperates with the Met/Caveolin Gene Amplicon to Induce Basal-like Breast Cancer

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Progression to Metastasis of Solid Cancer

Metastatic dissemination of cancer cells, their colonization at distal sites, and ultimate disruption of tissue physiology are the root causes of most deaths from solid cancers, particularly in tumor types where the primary lesion can be easily dissected and discarded [...