Gratien G. Prefontaine

Distinct roles for aryl hydrocarbon receptor nuclear translocator and ah receptor in estrogen-mediated signaling in human cancer cell lines.

The activated AHR/ARNT complex (AHRC) regulates the expression of target genes upon exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Importantly, evidence has shown that TCDD represses estrogen receptor (ER) target gene activation through the AHRC. Our data indicates that AHR and ARNT act independently from each other at non-dioxin response element sites. Therefore, we sought to determine the specific functions of AHR and ARNT in estrogen-dependent signaling in human MCF7 breast cancer and human ECC-1 endometrial carcinoma cells. Knockdown of AHR with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated repression of estrogen-regulated transcription, suggesting that AHR represses ER function independently of ARNT. This theory is supported by the ability of the selective AHR modulator 3′,4′-dimethoxy-α-naphthoflavone (DiMNF) to repress estrogen-inducible transcription. Furthermore, basal and estrogen-activated transcription of the genes encoding cathepsin-D and pS2 are down-regulated in MCF7 cells but up-regulated in ECC-1 cells in response to loss of ARNT. These responses are mirrored at the protein level with cathepsin-D. Furthermore, knock-down of ARNT led to opposite but corresponding changes in estrogen-stimulated proliferation in both MCF7 and ECC-1 cells. We have obtained experimental evidence demonstrating a dioxin-dependent repressor function for AHR and a dioxin-independent co-activator/co-repressor function for ARNT in estrogen signalling. These results provide us with further insight into the mechanisms of transcription factor crosstalk and putative therapeutic targets in estrogen-positive cancers.

The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Family of Proteins: Transcriptional Modifiers with Multi-Functional Protein Interfaces

The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of transcription factors mediates cellular responses to a variety of internal and external stimuli. As functional transcription factors, these proteins act as bHLH-PAS heterodimers and can be further sub-classified into sensory/activated subunits and regulatory or ARNT-like proteins. This class of proteins act as master regulators of the bHLH-PAS superfamily of transcription factors that mediate circadian rhythm gene programs, innate and adaptive immune responses, oxygen-sensing mechanisms and compensate for deleterious environmental exposures. Some contribute to the etiology of human pathologies including cancer because of their effects on cell growth and metabolism. We will review the canonical roles of ARNT and ARNT-like proteins with an emphasis on coactivator selectivity and recruitment. We will also discuss recent advances in our understanding of noncanonical DNA-binding independent or off-target roles of ARNT that are uncoupled from its classic heterodimeric bHLH-PAS binding partners. Understanding the DNA binding-independent functions of ARNT may identify novel therapeutic options for the treatment of a large spectrum of disease states.

Epigenetic Characterization of the Growth Hormone Gene Identifies SmcHD1 as a Regulator of Autosomal Gene Clusters

Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin β cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS & SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes.

The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.

Epigenetic events regulating monoallelic gene expression

Abstract In mammals, generally it is assumed that the genes inherited from each parent are expressed to similar levels. However, it is now apparent that in non-sex chromosomes, 6–10% of genes are selected for monoallelic expression. Monoallelic expression or allelic exclusion is established either in an imprinted (parent-of-origin) or a stochastic manner. The stochastic model explains random selection while the imprinted model describes parent-of-origin specific selection of alleles for expression. Allelic exclusion occurs during X chromosome inactivation, parent-of-origin expression of imprinted genes and stochastic monoallelic expression of cell surface molecules, clustered protocadherin (PCDH) genes. Mis-regulation or loss of allelic exclusion contributes to developmental diseases. Epigenetic mechanisms are fundamental players that determine this type of expression despite a homogenous genetic background. DNA methylation and histone modifications are two mediators of the epigenetic phenomena. The majority of DNA methylation is found on cytosines of the CpG dinucleotide in mammals. Several covalent modifications of histones change the electrostatic forces between DNA and histones modifying gene expression. Long-range chromatin interactions organize chromatin into transcriptionally permissive and prohibitive regions leading to simultaneous regulation of gene expression and repression. Non-coding RNAs (ncRNAs) are also players in regulating gene expression. Together, these epigenetic mechanisms fine-tune gene expression levels essential for normal development and survival. In this review, first we discuss what is known about monoallelic gene expression. Then, we focus on the molecular mechanisms that regulate expression of three monoallelically expressed gene classes: the X-linked genes, selected imprinted genes and PCDH genes.

A TRIP230-Retinoblastoma Protein Complex Regulates Hypoxia-Inducible Factor-1α-Mediated Transcription and Cancer Cell Invasion

Localized hypoxia in solid tumors activates transcriptional programs that promote the metastatic transformation of cells. Like hypoxia-inducible hyper-vascularization, loss of the retinoblastoma protein (Rb) is a trait common to advanced stages of tumor progression in many metastatic cancers. However, no link between the role of Rb and hypoxia-driven metastatic processes has been established. We demonstrated that Rb is a key mediator of the hypoxic response mediated by HIF1α/β, the master regulator of the hypoxia response, and its essential co-activator, the thyroid hormone receptor/retinoblastoma-interacting protein (TRIP230). Furthermore, loss of Rb unmasks the full co-activation potential of TRIP230. Using small inhibitory RNA approaches in vivo, we established that Rb attenuates the normal physiological response to hypoxia by HIF1α. Notably, loss of Rb results in hypoxia-dependent biochemical changes that promote acquisition of an invasive phenotype in MCF7 breast cancer cells. In addition, Rb is present in HIF1α-ARNT/HIF1β transcriptional complexes associated with TRIP230 as determined by co-immuno-precipitation, GST-pull-down and ChIP assays. These results demonstrate that Rb is a negative modulator of hypoxia-regulated transcription by virtue of its direct effects on the HIF1 complex. This work represents the first link between the functional ablation of Rb in tumor cells and HIF1α-dependent transcriptional activation and invasion.

Localized, Macromolecular Transport for Thin, Adherent, Single Cells Via an Automated, Single Cell Electroporation Biomanipulator

Single cell electroporation (SCE), via microcapillary, is an effective method for molecular, transmembrane transport used to gain insight on cell processes with minimal preparation. Although possessing great potential, SCE is difficult to execute and the technology spans broad fields within cell biology and engineering. The technical complexities, the focus and expertise demanded during manual operation, and the lack of an automated SCE platform limit the widespread use of this technique, thus the potential of SCE has not been realized. In this study, an automated biomanipulator for SCE is presented. Our system is capable of delivering molecules into the cytoplasm of extremely thin cellular features of adherent cells. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation, leaving only the focus of the experimental design to the operator. Each sequence of SCE including cell and SCE site localization, tip-membrane contact detection, and SCE has been automated. Positions of low-contrast cells are localized and “SCE sites” for microcapillary tip placement are determined using machine vision. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of automated SCE of “thin” cell features less than 10 μm in thickness. Finally, SCE events are anticipated using visual feedback, while monitoring fluorescing dye entering the cytoplasm of a cell. The execution is demonstrated by inserting a combination of a fluorescing dye and a reporter gene into NIH/3T3 fibroblast cells.

ChIP-re-ChIP: Co-occupancy Analysis by Sequential Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) exploits the specific interactions between DNA and DNA-associated proteins. It can be used to examine a wide range of experimental parameters. A number of proteins bound at the same genomic location can identify a multi-protein chromatin complex where several proteins work together to regulate gene transcription or chromatin configuration. In many instances, this can be achieved using sequential ChIP; or simply, ChIP-re-ChIP. Whether it is for the examination of specific transcriptional or epigenetic regulators, or for the identification of cistromes, the ability to perform a sequential ChIP adds a higher level of power and definition to these analyses. In this chapter, we describe a simple and reliable method for the sequential ChIP assay.

Abstract 2921: The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells

The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies. Citation Format: Mandeep K. Takhar, Mark P. Labrecque, Kevin J. Tam, Anne Haegert, Robert H. Bell, Manuel Altamirano-Dimas, Colin C. Collins, Gratien G. Prefontaine, Michael E. Cox, Kevin L. Bennewith, Timothy V. Beischlag. The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2921.

Abstract 2922: Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumour that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22rV1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that promote cell invasion and neuroendocrine differentiation. Gene ontology analysis of the hypoxia-inducible genes sensitive to loss of Rb revealed that a significant portion of these genes are involved in neuroendocrine differentiation (NED), specifically ENO2, KISS1R and HTR5A. ENO2 is the bonafide marker of neuroendocrine differentiation and it9s presence is a signature of late stage castrate resistant prostate cancer. Furthermore, we have functional evidence KISS1R is linked to intracellular calcium mobilization in 22RV1 cells. We have demonstrated that increased expression of HIF-regulated genes in response to loss of Rb activates Akt and ERK signaling pathways and promotes neuroendocrine differentiation and invasion. Inhibition of these signaling pathways significantly decreased actin polymerization in LNCaP cells. For the first time, we have established a direct link between hypoxic tumour environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumours to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. Citation Format: Mark Labrecque, Mandeep Takhar, Rebecca J. Nason, Stephanie Santacruz, Kevin Tam, Shabnam Massah, Anne Haegert, Robert Bell, Manuel Altamirano-Dimas, Colin Collins, Frank Lee, Gratien Prefontaine, Michael Cox, Timothy Beischlag. Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2922.