MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

The hallmarks of cancer.

Recent data have expanded the concept that inflammation is a critical component of tumour progression. Many cancers arise from sites of infection, chronic irritation and inflammation. It is now becoming clear that the tumour microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. In addition, tumour cells have co-opted some of the signalling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. These insights are fostering new anti-inflammatory therapeutic approaches to cancer development.

Inflammation and cancer

In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed “the Warburg effect.” Aerobic glycolysis is an inefficient way to generate adenosine 5′-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.

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Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation

Microarrays can measure the expression of thousands of genes to identify changes in expression between different biological states. Methods are needed to determine the significance of these changes while accounting for the enormous number of genes. We describe a method, Significance Analysis of Microarrays (SAM), that assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, the false discovery rate (FDR). When the transcriptional response of human cells to ionizing radiation was measured by microarrays, SAM identified 34 genes that changed at least 1.5-fold with an estimated FDR of 12%, compared with FDRs of 60 and 84% by using conventional methods of analysis. Of the 34 genes, 19 were involved in cell cycle regulation and 3 in apoptosis. Surprisingly, four nucleotide excision repair genes were induced, suggesting that this repair pathway for UV-damaged DNA might play a previously unrecognized role in repairing DNA damaged by ionizing radiation.

/pdf/significance-analysis-of-microarrays-applied-to-the-ionizing-10j4beasgw.pdf

Significance analysis of microarrays applied to the ionizing radiation response

A strong growth inhibition is observed when the human p53 tumor suppressor gene product is expressed in the fission yeastSchizosaccharomyces pombe. This growth inhibition is specific for wild-type p53; mutant alleles of p53 derived from human tumors show a greatly decreased ability to inhibit growth. These data suggest that there may be a p53-responsive pathway inS. pombe. To identify elements in this pathway genetically, we isolated a mutant yeast strain in which the growth inhibitory activity of p53 is largely suppressed. In addition, the activity of p53 as a transcription factor is also decreased in this strain. The suppression of p53 activity is not due to a decrease in p53 expression or a failure of p53 to localize to the nucleus. This p53 suppressor mutation is in a novelS. pombe gene with homology to thioredoxin reductase genes, and has been namedtrr1. Strains with a mutation of, or deletion in,trr1 are sensitive to oxidizing agents, suggesting that thetrr1 suppressor mutation causes partial loss oftrr1 function. Since oxidizing agents are able to suppress p53 activity in vitro, thistrr1 mutation may affect the activity of p53 in fission yeast by increasing the oxidation state of the tumor suppressor.

A mutation in a thioredoxin reductase homolog suppresses p53-induced growth inhibition in the fission yeast Schizosaccharomyces pombe

The present invention relates to methods and compositions for the elucidation of mammalian gene function. Specifically, the present invention relates to methods and compositions for improved mammalian complementation screening, functional inactivation of specific essential or non-essential mammalian genes, and identification of mammalian genes which are modulated in response to specific stimuli. In particular, the compositions of the present invention include, but are not limited to, replication-deficient retroviral vectors, libraries comprising such vectors, retroviral particles produced by such vectors in conjunction with retroviral packaging cell lines, integrated provirus sequences derived from the retroviral particles of the invention and circularized provirus sequences which have been excised from the integrated provirus sequences of the invention. The compositions of the present invention further include novel retroviral packaging cell lines.

Mammalian viral vectors and their uses

Effects of ketoconazole on sterol biosynthesis by Trypanosomacruzi epimastigotes

Senescence is a mechanism that limits cellular lifespan and constitutes a barrier against cellular immortalization. To identify new senescence regulatory genes that might play a role in tumorigenesis, we have designed and performed a large-scale antisense-based genetic screen in primary mouse embryo fibroblasts (MEFs). Out of this screen, we have identified five different genes through which loss of function partially bypasses senescence. These genes belong to very different biochemical families: csn2 (component of the Cop9 signalosome), aldose reductase (a metabolic enzyme) and brf1 (subunit of the RNA polymerase II complex), S-adenosyl homocysteine hydrolase and Bub1. Inactivation, at least partial, of these genes confers resistance to both p53- and p16INK4a-induced proliferation arrest. Furthermore, such inactivation inhibits p53 but not E2F1 transcriptional activity and impairs DNA-damage-induced transcription of p21. Since the aim of the screen was to identify new regulators of tumorigenesis, we have tested their inactivation in human tumors. We have found, either by northern blot or quantitative reverse transcriptase–PCR analysis, that the expression of three genes, Csn2, Aldose reductase and Brf1, is lost at different ratios in tumors of different origins. These genes are located at common positions of loss of heterogeneity (15q21.2, 7q35 and 14q32.33); therefore,we have measured genomic losses of these specific genes in different tumors. We have found that Csn2 and Brf1 also show genomic losses of one allele in different tumors. Our data suggest that the three genes identified in the genome-wide loss-of-function genetic screen are putative tumor suppressors located at 15q21.2; 7q35 and 14q32.33.

Cellular senescence bypass screen identifies new putative tumor suppressor genes

We have previously described the isolation of mcs2-75, a mutation obtained as an allele-specific suppressor of a dominant allele of cdc2. mcs2 was cloned and determined to be an essential gene, the product of which shares homology with the cyclin family of proteins. In contrast to the behavior of some, but not all cyclins, the mcs2 protein is constant in its abundance and localization throughout the cell cycle. A kinase activity that co-precipitates with mcs2 can be detected when myelin basic protein (MBP) is provided as an exogenous substrate. This kinase activity is constant throughout the cell cycle. mcs2 does not appear to associate with the cdc2 protein kinase or an antigenically related kinase. Finally, a protein kinase termed csk1 (cyclin suppressing kinase) was isolated as a high copy suppressor of an mcs2 mutation. csk1 is not essential, however, the level of kinase activity that co-precipitates with mcs2 is reduced approximately 3-fold in strains harboring a csk1 null allele. Therefore, csk1 may encode a protein kinase physically associated with mcs2 or alternatively may function as an upstream activator of the mcs2-associated kinase.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1993.tb05817.x

David Beach

Papers

A mutation in a thioredoxin reductase homolog suppresses p53-induced growth inhibition in the fission yeast Schizosaccharomyces pombe

Mammalian viral vectors and their uses

Effects of ketoconazole on sterol biosynthesis by Trypanosomacruzi epimastigotes

Cellular senescence bypass screen identifies new putative tumor suppressor genes

Characterization of the fission yeast mcs2 cyclin and its associated protein kinase activity.