Radon Biomonitoring and microRNA in Lung Cancer.

Radon is the number one cause of lung cancer in non-smokers. microRNA expression in human bronchial epithelium cells is altered by radon, with particular reference to upregulation of miR-16, miR-15, miR-23, miR-19, miR-125, and downregulation of let-7, miR-194, miR-373, miR-124, miR-146, miR-369, and miR-652. These alterations alter cell cycle, oxidative stress, inflammation, oncogene suppression, and malignant transformation. Also DNA methylation is altered as a consequence of miR-29 modification induced by radon. Indeed miR-29 targets DNA methyltransferases causing inhibition of CpG sites methylation. Massive microRNA dysregulation occurs in the lung due to radon expose and is functionally related with the resulting lung damage. However, in humans this massive lung microRNA alterations only barely reflect onto blood microRNAs. Indeed, blood miR-19 was not found altered in radon-exposed subjects. Thus, microRNAs are massively dysregulated in experimental models of radon lung carcinogenesis. In humans these events are initially adaptive being aimed at inhibiting neoplastic transformation. Only in case of long-term exposure to radon, microRNA alterations lead towards cancer development. Accordingly, it is difficult in human to establish a microRNA signature reflecting radon exposure. Additional studies are required to understand the role of microRNAs in pathogenesis of radon-induced lung cancer.

Resistance to cancer chemotherapeutic drugs is determined by pivotal microRNA regulators.

Chemo-resistance, which is the main obstacle in cancer therapy, is caused by the onset of drug-resistant cells in the heterogeneous cell population in cancer tissues. MicroRNAs regulate gene expression at the post-transcriptional level, and they are involved in many different biological processes, including cell proliferation, differentiation, metabolism, stress response, and apoptosis. The aberrant expression of microRNAs plays a major pathogenic role from the early stages of the carcinogenesis process. Recently, microRNAs have been reported to play an important role in inducing resistance to anti-cancer drugs. Specific microRNA alterations occur selectively in cancer cells, rendering these cells resistant to various chemotherapeutic agents. For example, resistance to 5-fluorouracil is mediated by alterations in miR-21, miR-27a/b, and miR-155; the sensitivity to Docetaxel is influenced by miR-98, miR-192, miR-194, miR-200b, miR-212, and miR-424; and the resistance to Cisplatin is mediated by miR-let-7, miR-15, miR-16 miR-21 and miR-214. Chemo-resistant cancer cells are characterized by altered functions in enzymes that are involved in microRNA maturation, primarily including Dicer, as demonstrated in ovarian cancer, oral squamous cell carcinoma, breast cancer and cervical cancer. Based on the evidence reviewed in this paper, various strategies have been developed to artificially re-establish microRNA expression in resistant cells, thus restoring chemo-sensitivity. These strategies employ synthetic analogs, anti-microRNA oligonucleotides, locked nucleic acid, microRNA sponges, drugs that inhibit DNA methylation or histone deacetylation, and the introduction of microRNA mimics. The ability to modulate microRNA expression is a promising strategy for overcoming the problem of drug resistance in cancer treatment.

Extracellular MicroRNA in liquid biopsy: applicability in cancer diagnosis and prevention.

One of the goals of contemporary cancer research is the development of new markers that facilitate earlier and non-invasive diagnosis. MicroRNAs are non-coding RNA molecules that regulate gene expression; studies have shown that their expression levels are altered in cancer. Recently, extra-cellular microRNAs have been detected in biological fluids and studied as possible cancer markers that can be detected by noninvasive procedures. In this review, we analyze the current understanding of extracellular miRNAs based on clinical studies to establish their possible use for the prevention of the most common tumors. Despite discrepancies among different studies of the same cancers, panels of specific extracellular microRNAs are emerging as a new tool for the secondary (selection of high-risk individuals to undergo screening) and tertiary (relapse) prevention of cancer.

Autoregulation of the mechanistic target of rapamycin (mTOR) complex 2 integrity is controlled by an ATP-dependent mechanism.

Nutrients are essential for living organisms because they fuel biological processes in cells. Cells monitor nutrient abundance and coordinate a ratio of anabolic and catabolic reactions. Mechanistic target of rapamycin (mTOR) signaling is the essential nutrient-sensing pathway that controls anabolic processes in cells. The central component of this pathway is mTOR, a highly conserved and essential protein kinase that exists in two distinct functional complexes. The nutrient-sensitive mTOR complex 1 (mTORC1) controls cell growth and cell size by phosphorylation of the regulators of protein synthesis S6K1 and 4EBP1, whereas its second complex, mTORC2, regulates cell proliferation by functioning as the regulatory kinase of Akt and other members of the AGC kinase family. The regulation of mTORC2 remains poorly characterized. Our study shows that the cellular ATP balance controls a basal kinase activity of mTORC2 that maintains the integrity of mTORC2 and phosphorylation of Akt on the turn motif Thr-450 site. We found that mTOR stabilizes SIN1 by phosphorylation of its hydrophobic and conserved Ser-260 site to maintain the integrity of mTORC2. The optimal kinase activity of mTORC2 requires a concentration of ATP above 1.2 mM and makes this kinase complex highly sensitive to ATP depletion. We found that not amino acid but glucose deprivation of cells or acute ATP depletion prevented the mTOR-dependent phosphorylation of SIN1 on Ser-260 and Akt on Thr-450. In a low glucose medium, the cells carrying a substitution of SIN1 with its phosphomimetic mutant show an increased rate of cell proliferation related to a higher abundance of mTORC2 and phosphorylation of Akt. Thus, the homeostatic ATP sensor mTOR controls the integrity of mTORC2 and phosphorylation of Akt on the turn motif site.

Isolation of the mTOR complexes by affinity purification.

The mammalian Target Of Rapamycin (mTOR) protein is a central component of the essential and highly conserved signaling pathway that emerged as a critical effector in regulation of cell physiology. Biochemical studies defined mTOR as the protein kinase that exists at least in two distinct complexes. The first complex has been characterized as the nutrient-sensitive mTOR complex 1 that controls cell growth and cell size by regulating protein synthesis and autophagy. The second complex of mTOR has been defined as the component of growth factor signaling that functions as a major regulatory kinase of Akt/PKB. Here, we provide the detailed methods how to purify the functional complexes of mTOR by affinity purification. In the first part, we describe the purification of the distinct mTOR complexes by immunoprecipitation. Purification of the soluble mTOR complexes is explained in the second part of this chapter.