Lung cancer cells and their sensitivity/resistance to cisplatin chemotherapy: Role of microRNAs and upstream mediators

Cisplatin (CP) is a well-known chemotherapeutic agent with excellent clinical effects. The anti-tumor activity of CP has been demonstrated in different cancers such as breast, cervical, reproductive, lung, brain, and prostate cancers. However, resistance of cancer cells to CP chemotherapy has led to its failure in eradication of cancer cells, and subsequent death of patients with cancer. Fortunately, much effort has been put to identify molecular pathways and mechanisms involved in CP resistance/sensitivity. It seems that microRNAs (miRs) are promising candidates in mediating CP resistance/sensitivity, since they participate in different biological aspects of cells such as proliferation, migration, angiogenesis, and differentiation. In this review, we focus on miRs and their regulation in CP chemotherapy of lung cancer, as the most malignant tumor worldwide. Oncogenic miRs trigger CP resistance in lung cancer cells via targeting various pathways such as Wnt/β-catenin, Rab6, CASP2, PTEN, and Apaf-1. In contrast, onco-suppressor miRs inhibit oncogene pathways such as STAT3 to suppress CP resistance. These topics are discussed to determine the role of miRs in CP resistance/sensitivity. We also describe the upstream modulators of miRs such as lncRNAs, circRNAs, NF-κB, SOX2 and TRIM65 and their association with CP resistance/sensitivity in lung cancer cells. Finally, the effect of anti-tumor plant-derived natural compounds on miR expression during CP sensitivity of lung cancer cells is discussed.     

Micro RNA 145 targets the insulin receptor substrate-1 and inhibits the growth of colon cancer cells

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3'-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3'-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.     

Prenatal Exposure to TCDD Triggers Significant Modulation of microRNA Expression Profile in the Thymus That Affects Consequent Gene Expression

Background

MicroRNAs (miRs) are a class of small RNAs that regulate gene expression. There are over 700 miRs encoded in the mouse genome and modulate most of the cellular pathways and functions by controlling gene expression. However, there is not much known about the pathophysiological role of miRs. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), an environmental contaminant is well known to induce severe toxicity (acute and chronic) with long-term effects. Also, in utero exposure of fetus to TCDD has been shown to cause thymic atrophy and alterations in T cell differentiation. It is also relevant to understand “the fetal basis of adult disease” hypothesis, which proposes that prenatal exposure to certain forms of nutritional and environmental stress can cause increased susceptibility to clinical disorders later in life. In the current study, therefore, we investigated the effects of prenatal exposure to TCDD on miR profile in fetal thymocytes and searched for their possible role in causing thymic atrophy and alterations in the expression of apoptotic genes.

Methodology/Principal Findings

miR arrays of fetal thymocytes post exposure to TCDD and vehicle were performed. Of the 608 mouse miRs screened, 78 miRs were altered more than 1.5 fold and 28 miRs were changed more than 2 fold in fetal thymocytes post-TCDD exposure when compared to vehicle controls. We validated the expression of several of the miRs using RT-PCR. Furthermore, several of the miRs that were downregulated contained highly complementary sequence to the 3′-UTR region of AhR, CYP1A1, Fas and FasL. Also, the Ingenuity Pathway Analysis software and database was used to analyze the 78 miRs that exhibited significant expression changes and revealed that as many as 15 pathways may be affected.

Conclusions/Significance

These studies revealed that TCDD-mediated alterations in miR expression may be involved in the regulation of its toxicity including cancer, hepatic injury, apoptosis, and cellular development.     

Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered,Phospholipase D (PLD)-mediated Cancer Cell Invasion

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of four microRNAs (miRs) mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitant with high expression of miR-203, -887, and -3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are, therefore, “tumor-suppressor-like” miRs. The combination miR-887+miR-3619 abolished >90% of PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 cells have low miR expression, high levels of PLD1/2, and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We determined that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas vimentin dampens expression of the miRs in post-EMT invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.     

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.     

Plant flavonoids in cancer chemoprevention: role in genome stability

Carcinogenesis is a multistage process that involves a series of events comprising of genetic and epigenetic changes leading to the initiation, promotion and progression of cancer. Chemoprevention is referred to as the use of nontoxic natural compounds, synthetic chemicals or their combinations to intervene in multistage carcinogenesis. Chemoprevention through diet modification, i.e., increased consumption of plant-based food, has emerged as a most promising and potentially cost-effective approach to reducing the risk of cancer. Flavonoids are naturally occurring polyphenols that are ubiquitous in plant-based food such as fruits, vegetables and teas as well as in most medicinal plants. Over 10,000 flavonoids have been characterized over the last few decades. Flavonoids comprise of several subclasses including flavonols, flavan-3-ols, anthocyanins, flavanones, flavones, isoflavones and proanthocyanidins. This review describes the most efficacious plant flavonoids, including luteolin, epigallocatechin gallate, quercetin, apigenin and chrysin; their hormetic effects; and the molecular basis of how these flavonoids contribute to the chemoprevention with a focus on protection against DNA damage caused by various carcinogenic factors. The present knowledge on the role of flavonoids in chemoprevention can be used in developing effective dietary strategies and natural health products targeted for cancer chemoprevention.     

Dysregulation of apoptotic signaling in cancer: molecular mechanisms and therapeutic opportunities

Apoptosis is a tightly regulated cell suicide program that plays an essential role in the maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Defects in this native defense mechanism promote malignant transformation and frequently confer chemoresistance to transformed cells. Indeed, the evasion of apoptosis has been recognized as a hallmark of cancer. Given that multiple mechanisms function at many levels to orchestrate the regulation of apoptosis, a multitude of opportunities for apoptotic dysregulation are present within the intricate signaling network of cell. Several of the molecular mechanisms by which cancer cells are protected from apoptosis have been elucidated. These advances have facilitated the development of novel apoptosis-inducing agents that have demonstrated single-agent activity against various types of cancers cells and/or sensitized resistant cancer cells to conventional cytotoxic therapies. Herein, we will highlight several of the central modes of apoptotic dysregulation found in cancer. We will also discuss several therapeutic strategies that aim to reestablish the apoptotic response, and thereby eradicate cancer cells, including those that demonstrate resistance to traditional therapies.     

Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modified PAMAM dendrimers into lung cancer cells

RNAi-based gene therapy has been recently considered as a promising approach against cancer. Targeted delivery of drug, gene or therapeutic RNAi-based systems to tumor cells is one of the important issues in order to reduce side effects on normal cells. Several strategies have been developed to improve the safety and selectivity of cancer treatments including antibodies, peptides and recently aptamers with various attractive characteristics including higher target specificity, affinity and reduced toxicity. Here we described a novel targeted delivery platform comprising modified PAMAM with 10-bromodecanoic acid (10C) and 10C-PEG for improvement of transfection efficiency, AS1411 aptamer for targeting nucleolin ligand on target cancer cells and shRNA plasmid for specific knockdown of Bcl-xL protein. Modified vector could significantly improve the transfection efficiency even after covalent or non-covalent aptamer binding compared to the non-targeted vector in A549 cells. The results of gene silencing and apoptosis assay indicated that our targeted shRNA delivery system could efficiently down-regulate the Bcl-xL expression up to 25% and induce 14% late apoptosis in target cancer cells with strong cell selectivity. This study proposed a novel targeted non-viral system for shRNA-mediated gene-silencing in cancer cells.     

MicroRNA function and neurotrophin BDNF

MicroRNAs (miRs), endogenous small RNAs, regulate gene expression through repression of translational activity after binding to target mRNAs. miRs are involved in various cellular processes including differentiation, metabolism, and apoptosis. Furthermore, possible involvement of miRs in neuronal function have been proposed. For example, miR-132 is closely related to neuronal outgrowth while miR-134 plays a role in postsynaptic regulation, suggesting that brain-specific miRs are critical for synaptic plasticity. On the other hand, numerous studies indicate that BDNF (brain-derived neurotrophic factor), one of the neurotrophins, is essential for a variety of neuronal aspects such as cell differentiation, survival, and synaptic plasticity in the central nervous system (CNS). Interestingly, recent studies, including ours, suggest that BDNF exerts its beneficial effects on CNS neurons via up-regulation of miR-132. Here, we present a broad overview of the current knowledge concerning the association between neurotrophins and various miRs.     

The Role of Exosomes in Pancreatic Cancer Microenvironment

Exosomes are nanovesicles shed by cells as a means of communication with other cells. Exosomes contain mRNAs, microRNAs (miRs) and functional proteins. In the present paper, we develop a mathematical model of tumor–immune interaction by means of exosomes shed by pancreatic cancer cells and dendritic cells. Cancer cells’ exosomes contain miRs that promote their proliferation and that inhibit immune response by dendritic cells, and by CD4+ and CD8+ T cells. Dendritic cells release exosomes with proteins that induce apoptosis of cancer cells and that block regulatory T cells. Simulations of the model show how the size of the pancreatic cancer can be determined by measurement of specific miRs (miR-21 and miR-203 in the case of pancreatic cancer), suggesting these miRs as biomarkers for cancer.     

MicroRNA expression profiling of endocrine sensitive and resistant breast cancer cell lines

BackgroundMicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion.MethodsMicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR.ResultsEach cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50–60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes.ConclusionsThese data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.     

Microtubule assembly dynamics: an attractive target for anticancer drugs

Microtubules, composed of alphabeta tubulin dimers, are dynamic polymers of eukaryotic cells. They play important roles in various cellular functions including mitosis. Microtubules exhibit differential dynamic behaviors during different phases of the cell cycle. Inhibition of the microtubule assembly dynamics causes cell cycle arrest leading to apoptosis; thus, qualifying them as important drug targets for treating several diseases including cancer, neuronal, fungal, and parasitic diseases. Although several microtubule-targeted drugs are successfully being used in cancer chemotherapy, the development of resistance against these drugs and their inherent toxicities warrant the development of new agents with improved efficacy. Several antimicrotubule agents are currently being evaluated for their possible uses in cancer chemotherapy. Benomyl, griseofulvin, and sulfonamides have been used as antifungal and antibacterial drugs. Recent reports have shown that these drugs have potent antitumor potential. These agents are shown to inhibit proliferation of different types of tumor cells and induce apoptosis by targeting microtubule assembly dynamics. However, unlike vincas and taxanes, which inhibit cancer cell proliferation in nanomolar concentration range, these agents act in micromolar range and are considered to have limited toxicities. Here, we suggest that these drugs may have a significant use in cancer chemotherapy when used in combination with other anticancer drugs.     

GSK3 and miRNA in neural tissue: From brain development to neurodegenerative diseases

MicroRNAs (miRs) are small RNAs modulating gene expression and creating intricate regulatory networks that are dysregulated in many pathological states, including neurodegenerative disorders. In silico analyses denote a multifunctional kinase glycogen synthase kinase-3 (GSK3) as a putative target of numerous miRs identified in neural tissue. GSK3 is engaged in almost all aspects of neuronal development and functioning. Moreover, there is an autoregulatory feedback between GSK3 and miRNAs as the kinase can influence biogenesis of miRs. Members of the miR-GSK3 axes might thus represent convenient therapeutic targets in neuropathologies that display its abnormal regulation. This review summarizes the present knowledge about direct interactions of GSK3 and miRs in brain, and their putative roles in pathogenesis of neurodegenerative and neuropsychiatric disorders. This article is part of a Special Issue entitled: GSK-3 and related kinases in cancer, neurological and other disorders edited by James McCubrey, Agnieszka Gizak and Dariusz Rakus.