Borja Belandia

Hey1, a Mediator of Notch Signaling, Is an Androgen Receptor Corepressor

ABSTRACT Hey1 is a member of the basic helix-loop-helix-Orange family of transcriptional repressors that mediate Notch signaling. Here we show that transcription from androgen-dependent target genes is inhibited by Hey1 and that expression of a constitutively active form of Notch is capable of repressing transactivation by the endogenous androgen receptor (AR). Our results indicate that Hey1 functions as a corepressor for AF1 in the AR, providing a mechanism for cross talk between Notch and androgen-signaling pathways. Hey1 colocalizes with AR in the epithelia of patients with benign prostatic hyperplasia, where it is found in both the cytoplasm and the nucleus. In marked contrast, we demonstrate that Hey1 is excluded from the nucleus in most human prostate cancers, raising the possibility that an abnormal Hey1 subcellular distribution may have a role in the aberrant hormonal responses observed in prostate cancer.

Functional Interaction between the p160 Coactivator Proteins and the Transcriptional Enhancer Factor Family of Transcription Factors

SRC1, initially identified as a nuclear receptor coactivator, was found to interact with a member of the transcriptional enhancer factor (TEF) family of transcription factors, TEF-4. The interaction, which occurs in both intact cells and in a cell-free system, is mediated by the highly conserved basichelix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain present in the N-terminal region of SRC1. Moreover, all three members of the p160 family of nuclear receptor coactivators, SRC1, TIF2, and RAC3, are able to potentiate transcription from a TEF response element in transient transfection experiments, and this activation requires the presence of the bHLH-PAS domain. These results suggest that the p160 proteins could be bona fidecoactivators of the TEF family of transcription factors.

The SWI/SNF Chromatin Remodeling Subunit BAF57 Is a Critical Regulator of Estrogen Receptor Function in Breast Cancer Cells

Estrogen receptors (ERs) play critical roles in both normal mammary gland development and in the formation and progression of breast tumors, constituting a major therapeutic target for breast cancer treatment. We have previously described that ER transcriptional activity is potentiated by BAF57, a core subunit of the mammalian SWI/SNF chromatin remodeling complex. Here we provide evidence demonstrating an important role for BAF57 as regulator of ER functions in breast cancer cells. Different experimental manipulations leading to the abrogation of BAF57 expression and/or function severely reduced the expression of various endogenous ER target genes and blocked estrogen-stimulated proliferation in ZR-75-1 breast cancer cells. Moreover, using a structure-function analysis, we have defined the protein domains required for the functional interaction between ERα and BAF57, including a key region within the hinge of ERα that is essential for BAF57 recruitment and its function on ER-mediated transcription. Interestingly, we found that BAF57 is an ER subtype-selective modulator that specifically regulates ERα-mediated transcription. Taken together, our results suggest that targeting BAF57 could represent a new way to effectively inhibit the action of ERα.

Thyroid Hormone Negatively Regulates the Transcriptional Activity of the β-Amyloid Precursor Protein Gene

The expression of the β-amyloid precursor protein (APP), which plays a key role in the development of Alzheimer’s disease, is regulated by a variety of cellular mediators in a cell-dependent manner. In the present study, we present evidence that thyroid hormones negatively regulate the expression of the APP gene in neuroblastoma cells. Transient transfection studies using plasmids that contain progressive deletions of the 5′ region of the gene demonstrate that triiodothyronine (T3), the more active form of the thyroid hormones, represses APP promoter activity by a mechanism that requires binding of the nuclear T3 receptor (TR) to a specific sequence located in the first exon. The unliganded receptor increases promoter activity, and T3 reverses that activity to basal levels. The repressive effect of T3 does not exhibit TR isoform specificity, and it is equally mediated by TRα and TRβ. Gel mobility shift assays using in vitro synthesized nuclear receptors and nuclear extracts led to the identification of a negative thyroid hormone response element, at nucleotide position +80/+96, that preferentially binds heterodimers of TR with the retinoid X receptor. Insertion of sequences containing this element confers negative regulation by T3 to a heterologous TK promoter, thus indicating the functionality of the element.

Thyroid Hormones Regulate β-Amyloid Gene Splicing and Protein Secretion in Neuroblastoma Cells1

The β-amyloid protein (Aβ), the major component of the senile plaques found in Alzheimer brains, derives from a larger β-amyloid precursor protein (APP). Alternative splicing of the APP gene yields three major APP messenger RNAs (mRNAs), which, in turn, give rise to the APP770, APP751, and APP695 protein isoforms. In this study we examined the effects of thyroid hormone on APP expression in N2a-β neuroblastoma cells. T3 caused a significant increase in the APP770 mRNA band, in detriment of the APP695 mRNA, which was proportionately reduced. In agreement with these results, T3 markedly altered the relative ratio of intracellular APP isoforms, increasing the amount of APP770 and causing an equivalent reduction of the immature APP695 isoform. In accordance with these results, the soluble APP695-derived form was specifically reduced in the culture medium obtained from T3-treated cells. In contrast, the increase in intracellular APP770 was not followed by an enhanced release of soluble derivatives of this isof...

Notch signaling: A potential therapeutic target in prostate cancer

The Notch pathway and the endocrine system constitute two key biological signaling mechanisms, responsible for cell-to-cell communication between adjacent cells and long-distance hormonal signals respectively. They play central roles during the development of higher eukaryotic organisms but they also take part in the regulation of many aspects of adult physiology and homeostasis. The contribution of defects in the normal transmission of hormone-dependent signals to the development of endocrine cancers has been widely analyzed and the knowledge derived from these studies has allowed us to develop many successful therapeutic strategies. However, in many cases these hormonal treatments become ineffective despite the fact that cancer cells maintain normal expression levels of wild-type hormone nuclear receptors. Less is known about the involvement of altered Notch signaling in the origin and progression of cancer, although there is clear evidence indicating that deregulation of Notch activity occurs in several types of tumors, including highly prevalent hormone-dependent types of cancer such as breast, ovarian and prostate cancer. This review will summarize accumulating data suggesting that Notch signaling plays a key role in the control of proliferation, differentiation and survival of prostate epithelial cells. Notch signals are required for normal prostate development and homeostasis, and abnormalities in Notch signaling may be critical during the development of prostate cancer. We will also discuss the possible oncogenic role for alterations in the crosstalk mechanisms between Notch and androgen-dependent signals during tumorigenesis in the prostate and how they could influence the outcome of anti-cancer hormonal treatments.

Repression of androgen receptor activity by HEYL, a third member of the hairy/enhancer-of-split-related family of notch effectors

The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer.

HEY1 Leu94Met gene polymorphism dramatically modifies its biological functions.

The hairy/enhancer-of-split related with YRPW motif 1 (HEY1) is a member of the basic-helix-loop-helix-Orange (bHLH-O) family of transcriptional repressors that mediate Notch signaling. Several cancer-related pathways also regulate HEY1 expression, and HEY1 itself acts as an indirect positive regulator of the p53 tumor suppressor protein and a negative regulator of androgen receptor activity. In this study we show how a naturally occurring non-synonymous polymorphism at codon 94 of HEY1, which results in a substitution of leucine by methionine (Leu94Met), converts HEY1 from an androgen receptor corepressor to an androgen receptor co-activator without affecting its intrinsic transcriptional repressive domains. The polymorphism Leu94Met also abolishes HEY1-mediated activation of p53 and suppresses the ability of HEY1 to induce p53-dependent cell-cycle arrest and aberrant cell differentiation in human osteosarcoma U2OS cells. Moreover, expression of HEY1, but not of the variant Leu94Met, confers sensitivity to p53-activating chemotherapeutic drugs on U2OS cells. In addition, we have identified motifs in HEY1 that are critical for the regulation of its subcellular localization and analysed how mutations in those motifs affect both HEY1 and HEY1-Leu94Met functions. These findings suggest that the polymorphism Leu94Met in HEY1 radically alters its biological activities and may affect oncogenic processes.

Nuclear receptor regulation gears up another Notch

In this perspective we describe examples of crosstalk between nuclear receptors (NRs) and Notch signaling by means of direct functional interactions between components of both pathways. This crosstalk may provide eukaryotic organisms with molecular mechanisms for the coordination of llong-distance endocrine signals with cell-to-cell juxtacrine communication.