Project description:Autophagy is a catabolic membrane trafficking process involved in degradation of cellular constituents through lysosomes, which maintains cell and tissue homeostasis. While much attention has been focused on autophagic turnover of cytoplasmic materials, little is known regarding the role of autophagy in degrading nuclear components. Here we report that autophagy machinery mediates degradation of nuclear lamina in mammalian cells, a process we term laminophagy. The autophagy protein LC3 is present in the nucleus and directly interacts with the nuclear lamina protein Lamin B1, and associates with lamin-associated domains (LADs) on chromatin. This interaction does not downregulate Lamin B1 during starvation, but mediates nuclear lamina degradation upon tumorigenic insults, such as by oncogenic Ras. Laminophagy is achieved by nucleus-to-cytosol transport that delivers Lamin B1 to lysosome for degradation. Inhibiting autophagy or LC3-Lamin B1 interaction prevents oncogenic Ras-induced Lamin B1 loss and delays oncogene-induced cell cycle arrest. Our study unveils a role of autophagy in degrading nuclear materials, and suggests laminophagy as a guarding mechanism protecting cells from tumorigenesis.
Project description:Although abnormal nuclear structure is an important criterion for cancer diagnostics, remarkably little is known about its relationship to tumor development. Here we report that loss of lamin B1, a determinant of nuclear architecture, plays a key role in lung cancer. We found that lamin B1 levels were reduced in lung cancer patients. Lamin B1 silencing in lung epithelial cells promoted epithelial-mesenchymal transition, cell migration, tumor growth and metastasis. Mechanistically, we show that lamin B1 recruits the polycomb repressive complex 2 (PRC2) to alter the H3K27me3 landscape and repress genes involved in cell migration and signaling. In particular, epigenetic derepression of the RET proto-oncogene by loss of PRC2 recruitment, and activation of the RET/p38 signaling axis, play a crucial role in mediating the malignant phenotype upon lamin B1 disruption. Importantly, loss of a single lamin B1 allele induced spontaneous lung tumor formation and RET activation. Thus, lamin B1 acts as a tumor suppressor in lung cancer, linking aberrant nuclear structure and epigenetic patterning with malignancy.
Project description:To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in 3-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends upon specific ECM-interacting network hubs. Blockade of one such hub, the b1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression. There are 3 experiments in this study: (1a) a time course of human epidermal tissue transformed by oncogenic ER:H-Ras and IkBaM comprising days 0, 5, 20, and 35 post Ras activation using 4OHT; (1b) a matched time course of adjacent mouse stromal tissue during tumor progression; (2) Arrays comparing 4OHT-induced, Raf-1:ER/IkBaM transformed epidermal tissue and -4OHT controls; and (3) Arrays comparing Ras:ER/IkBaM grafts co-treated with 4OHT and either IgG control antibody or an anti-b1 integrin blocking antibody, P5D2 for 30 days. All arrays were done in biologic duplicate. Epidermal tissue co-expressing ER:H-RasG12V (ER:Ras) and IkBaM was regenerated on female scid/scid mouse recipients. Grafts were allowed to heal for at least 3 weeks before Ras activation via daily i.p. injections of 730ug of 4OHT (in 110ul of a corn oil/ethanol mixture). Duplicate grafts were harvested after 0, 5, 20, and 35 days of 4OHT treatment. Laser capture microdissection was utilized to independently isolate epithelial cells of the basal most layers and adajacent stromal tissue for each time point. RNA was subjected to one round of T7-based linear amplification (Ambion Message Amp II enhanced kit) and hybridized to either HG-U133A 2.0 or MG-430A affymetrix oligonucleotide arrays. Arrays from this experiment are labeled Ras and Stroma, respectively. The second experment was performed on Raf-1:ER/IkBaM expressing grafts with and without 4OHT treament for 30 days. The third experiment was done comparing ER:Ras/IkBaM grafts concomitantly treated with 4OHT and 1.5mg/week of either IgG control antibody or a mouse monoclonal blocking antibody against b1 integrin for 30 days. For experiments 2 and 3, HG-U133A 2.0 GeneChips were used; 4OHT treatment and RNA isolation/amplification was performed as above.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Senescence is a stress responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina, lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for H3K9me3. LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3, thus promoting SAHF formation, which is inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genomewide redistribution: firstly through the spatial re-organization of chromatin and, secondly, through gene repression. ChIP-seq for Lamin B1 in Growing and Ras Induced Senescence
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.