Jody Filkowski

Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin

Many chemotherapy regiments are successfully used to treat breast cancer; however, often breast cancer cells develop drug resistance that usually leads to a relapse and worsening of prognosis. We have shown recently that epigenetic changes such as DNA methylation and histone modifications play an important role in breast cancer cell resistance to chemotherapeutic agents. Another mechanism of gene expression control is mediated via the function of small regulatory RNA, particularly microRNA (miRNA); its role in cancer cell drug resistance still remains unexplored. In the present study, we investigated the role of miRNA in the resistance of human MCF-7 breast adenocarcinoma cells to doxorubicin (DOX). Here, we for the first time show that DOX-resistant MCF-7 cells (MCF-7/DOX) exhibit a considerable dysregulation of the miRNAome profile and altered expression of miRNA processing enzymes Dicer and Argonaute 2. The mechanistic link of miRNAome deregulation and the multidrug-resistant phenotype of MCF-7/DOX cells was evidenced by a remarkable correlation between specific miRNA expression and corresponding changes in protein levels of their targets, specifically those ones that have a documented role in cancer drug resistance. Furthermore, we show that microRNA-451 regulates the expression of multidrug resistance 1 gene. More importantly, transfection of the MCF-7/DOX-resistant cells with microRNA-451 resulted in the increased sensitivity of cells to DOX, indicating that correction of altered expression of miRNA may have significant implications for therapeutic strategies aiming to overcome cancer cell resistance. [Mol Cancer Ther 2008;7(7):2152–9]

Pathogen-induced systemic plant signal triggers DNA rearrangements

Plant genome stability is known to be affected by various abiotic environmental conditions, but little is known about the effect of pathogens. For example, exposure of maize plants to barley stripe mosaic virus seems to activate transposable elements and to cause mutations in the non-infected progeny of infected plants. The induction by barley stripe mosaic virus of an inherited effect may mean that the virus has a non-cell-autonomous influence on genome stability. Infection with Peronospora parasitica results in an increase in the frequency of somatic recombination in Arabidopsis thaliana; however, it is unclear whether effects on recombination require the presence of the pathogen or represent a systemic plant response. It is also not clear whether the changes in the frequency of somatic recombination can be inherited. Here we report a threefold increase in homologous recombination frequency in both infected and non-infected tissue of tobacco plants infected with either tobacco mosaic virus or oilseed rape mosaic virus. These results indicate the existence of a systemic recombination signal that also results in an increased frequency of meiotic and/or inherited late somatic recombination.

Alterations of microRNAs and their targets are associated with acquired resistance of MCF‐7 breast cancer cells to cisplatin

Cancer cells that develop resistance to chemotherapeutic agents are a major clinical obstacle in the successful treatment of breast cancer. Acquired cancer chemoresistance is a multifactorial phenomenon, involving various mechanisms and processes. Recent studies suggest that chemoresistance may be linked to drug‐induced dysregulation of microRNA function. Furthermore, mounting evidence indicates the existence of similarities between drug‐resistant and metastatic cancer cells in terms of resistance to apoptosis and enhanced invasiveness. We studied the role of miRNA alterations in the acquisition of cisplatin‐resistant phenotype in MCF‐7 human breast adenocarcinoma cells. We identified a total of 103 miRNAs that were overexpressed or underexpressed (46 upregulated and 57 downregulated) in MCF‐7 cells resistant to cisplatin. These differentially expressed miRNAs are involved in the control of cell signaling, cell survival, DNA methylation and invasiveness. The most significantly dysregulated miRNAs were miR‐146a, miR‐10a, miR‐221/222, miR‐345, miR‐200b and miR‐200c. Furthermore, we demonstrated that miR‐345 and miR‐7 target the human multidrug resistance‐associated protein 1. These results suggest that dysregulated miRNA expression may underlie the abnormal functioning of critical cellular processes associated with the cisplatin‐resistant phenotype.

Hypomethylation and genome instability in the germline of exposed parents and their progeny is associated with altered miRNA expression

Recent studies suggest that transgenerational genome instability may be epigenetic in nature and mediated via altered DNA methylation and microRNAome. Here, we investigated the nature and mechanisms underlying the disruption of DNA methylation and microRNA expression status in the germline and progeny of exposed parents. We have found that paternal irradiation leads to upregulation of the miR-29 family in the exposed male germline, which causes decreased expression of de novo methyltransferase, DNA methyltransferase 3a, and profound hypomethylation of long interspersed nuclear elements 1 (LINE1) and short interspersed nuclear elements B2 (SINE B2). Epigenetic changes in the male germline further resulted in deleterious effects in the somatic thymus tissue from the progeny of exposed animals, including hypomethylation of LINE1 and SINE B2. Hypomethylation of LINE1 and SINE B2 in the thymus tissue from the progeny was associated with a significant decrease in the levels of lymphoid-specific helicase (LSH) that is crucial for the maintenance of methylation and silencing of repetitive elements. Furthermore, we noted a significant upregulation of miR-468 that targets LSH and leads to its decreased expression in thymus in the progeny of exposed parents. We suggest that miR-468-mediated suppression of LSH leads to aberrant methylation of LINE1 and SINE B2. In summary, altered microRNAome and hypomethylation of retroelements constitute deleterious effects that may significantly influence genome stability of the parental germline and consequently cause genome instability in the progeny.

Double-Strand Break Repair in Plants Is Developmentally Regulated

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional β-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 × 10−8) being only 30% of the recombination rate at 2 dpg (9.14 × 10−8). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.

microRNAome changes in bystander three-dimensional human tissue models suggest priming of apoptotic pathways

The radiation-induced bystander effect (RIBE) is a phenomenon whereby unexposed cells exhibit molecular symptoms of stress exposure when adjacent or nearby cells are traversed by ionizing radiation (IR). Recent data suggest that RIBE may be epigenetically mediated by microRNAs (miRNAs), which are small regulatory molecules that target messenger RNA transcripts for translational inhibition. Here, we analyzed microRNAome changes in bystander tissues after α-particle microbeam irradiation of three-dimensional artificial human tissues using miRNA microarrays. Our results indicate that IR leads to a deregulation of miRNA expression in bystander tissues. We report that major bystander end points, including apoptosis, cell cycle deregulation and DNA hypomethylation, may be mediated by altered expression of miRNAs. Specifically, c-MYC-mediated upregulation of the miR-17 family was associated with decreased levels of E2F1 and RB1, suggesting a switch to a proliferative state in bystander tissues, while priming these cells for impending death signals. Upregulation of the miR-29 family resulted in decreased levels of its targets DNMT3a and MCL1, consequently affecting DNA methylation and apoptosis. Altered expression of miR-16 led to changes in expression of BCL2, suggesting modulation of apoptosis. Thus, our data clearly show that miRNAs play a profound role in the manifestation of late RIBE end points. In summary, this study creates a roadmap for understanding the role of microRNAome in RIBE and for developing novel RIBE biomarkers.

Sex and tissue-specific differences in low-dose radiation-induced oncogenic signaling

Purpose: The possible adverse health effects of low-dose radiation (LDR) exposure constitute a growing concern. Clinically and environmentally relevant exposures occur predominantly under chronic conditions, notwithstanding that most studies of LDR effects have been performed using a single acute exposure. Sex- and tissue-specificity of the LDR-induced changes have not been considered before. We investigated LDR-related expression patterns in muscle, liver and spleen of male and female mice subjected to acute and chronic LDR exposure. Genes involved in oncogenic signaling were of specific interest, as radiation is a well-known carcinogen. Materials and methods: We analyzed the expression pattern of genes coding for growth factors and growth-factor receptors, cytoplasmic serine/threonine protein kinases, G-proteins and nuclear DNA-binding proteins, and other important components of oncogenic signaling. Results: We found sex- and tissue-specific changes in the expression of Ras superfamily members (Nras, Rab2, Rab34, Vav2), protein kinase C (PKC) isoforms (PKCβ, PKCμ), AP-1 factor components (Jun, JunB and FosB), Wnt signaling pathway members as well as in a variety of other cellular proto-oncogenes and oncogenes. Importantly, Western blot analysis of JunB, PKCμ and Rab2 proteins supported the transcriptomic data. Conclusions: Substantially different protein levels were observed in all three tissues (muscle, spleen and liver) of acutely and chronically irradiated female and male animals. Based on the obtained data and available literature, we discuss several possible mechanisms that may contribute to radiation-induced carcinogenesis in various tissues of males and females. From our results we could identify the genes that may serve as sex- and tissue-specific biomarkers of the LDR exposure.

High and low dose radiation effects on mammary adenocarcinoma cells – an epigenetic connection

The successful treatment of cancer, including breast cancer, depends largely on radiation therapy and proper diagnostics. The effect of ionizing radiation on cells and tissues depends on the radiation dose and energy level, but there is insufficient evidence concerning how tumor cells respond to the low and high doses of radiation that are often used in medical diagnostic and treatment modalities. The purpose of this study was to investigate radiation-induced gene expression changes in the MCF-7 breast adenocarcinoma cell line. Using microarray technology tools, we were able to screen the differential gene expressions profiles between various radiation doses applied to MCF-7 cells. Here, we report the substantial alteration in the expression level of genes after high-dose treatment. In contrast, no dramatic gene expression alterations were noticed after the application of low and medium doses of radiation. In response to a high radiation dose, MCF-7 cells exhibited down-regulation of biological pathways such as cell cycle, DNA replication, and DNA repair and activation of the p53 pathway. Similar dose-dependent responses were seen on the epigenetic level, which was tested by a microRNA expression analysis. MicroRNA analysis showed dose-dependent radiation-induced microRNA expression alterations that were associated with cell cycle arrest and cell death. An increased rate of apoptosis was determined by an Annexin V assay. The results of this study showed that high doses of radiation affect gene expression genetically and epigenetically, leading to alterations in cell cycle, DNA replication, and apoptosis.

A dual role of miR-22 modulated by RelA/p65 in resensitizing fulvestrant-resistant breast cancer cells to fulvestrant by targeting FOXP1 and HDAC4 and constitutive acetylation of p53 at Lys382

Antiestrogen resistance is a major challenge encountered during the treatment of estrogen receptor alpha positive (ERα+) breast cancer. A better understanding of signaling pathways and downstream transcription factors and their targets may identify key molecules that can overcome antiestrogen resistance in breast cancer. An aberrant expression of miR-22 has been demonstrated in breast cancer; however, its contribution to breast cancer resistance to fulvestrant, an antiestrogen drug, remains unknown. In this study, we demonstrated a moderate elevation in miR-22 expression in the 182R-6 fulvestrant-resistant breast cancer line we used as a model system, and this elevation was positively correlated with the expression of the miRNA biogenesis enzymes AGO2 and Dicer. The level of phosphorylated HER2/neu at Tyr877 was also upregulated in these cells, whereas the level of RelA/p65 phosphorylated at Ser536 (p-p65) was downregulated. Knockdown of HER2/neu led to an induction of p-p65 and a reduction in miR-22 levels. Luciferase assays identified two NF-κB binding motifs in the miR-22 promoter that contributed to transcriptional repression of miR-22. Activation of RelA/p65, triggered by LPS, attenuated miR-22 expression, but this expression was restored by sc-514, a selective IKKβ inhibitor. Inhibition of miR-22 suppressed cell proliferation, induced apoptosis and caused cell cycle S-phase arrest, whereas enhancing expression of p21Cip1/Waf1 and p27Kip1. Surprisingly, ectopic expression of miR-22 also suppressed cell proliferation, induced apoptosis, caused S-phase arrest, and promoted the expression of p21Cip1/Waf1 and p27Kip1. Ectopic overexpression of miR-22 repressed the expression of FOXP1 and HDAC4, leading to a marked induction of acetylation of HDAC4 target histones. Conversely, inhibition of miR-22 promoted the expression of both FOXP1 and HDAC4, without the expected attenuation of histone acetylation. Instead, p53 acetylation at lysine 382 was unexpectedly upregulated. Taken together, our findings demonstrated, for the first time, that HER2 activation dephosphorylates RelA/p65 at Ser536. This dephosphoryalted p65 may be pivotal in transactivation of miR-22. Both increased and decreased miR-22 expression cause resensitization of fulvestrant-resistant breast cancer cells to fulvestrant. HER2/NF-κB (p65)/miR-22/HDAC4/p21 and HER2/NF-κB (p65)/miR-22/Ac-p53/p21 signaling circuits may therefore confer this dual role on miR-22 through constitutive transactivation of p21.

Role of Epigenetic Changes in Radiation-Induced Genome Instability

Ionizing radiation (IR) is an important diagnostic and treatment modality, yet it is also a potent genotoxic agent that causes genome instability and carcinogenesis. While modern cancer radiation therapy has led to increased patient survival rates, the risk of radiation treatment-related complications is becoming a growing problem as radiation poses a threat to the exposed individuals and their progeny. Radiation-induced genome instability, which manifests as an elevated mutation rate (both delayed and non-targeted), chromosomal aberrations and changes in gene expression, has been well-documented in directly exposed cells and organisms. However, it has also been observed in distant, naive, out-of-field, ‘bystander’ cells and their progeny. Enigmatically, this increased instability is even observed in the pre-conceptually exposed progeny of animals, including humans. The mechanisms by which these distal effects arise remain obscure and, recently, have been proposed to be epigenetic in nature.