Microcephaly gene links trithorax and REST/NRSF to control neural stem cell proliferation and differentiation.

Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335 null mice are embryonically lethal, and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that ZNF335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.

Skin Metabolite, Farnesyl Pyrophosphate, Regulates Epidermal Response to Inflammation, Oxidative Stress, and Migration.

Skin produces cholesterol and a wide array of sterols and non-sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT-1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co-transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits ß-catenin and, unlike glucocorticoids, recruits co-repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. J. Cell. Physiol. 231: 2452-2463, 2016. © 2016 Wiley Periodicals, Inc.

Fas Activated Serine-Threonine Kinase Domains 2 (FASTKD2) mediates apoptosis of breast and prostate cancer cells through its novel FAST2 domain.

BACKGROUND: Expression of NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. This occurs through binding of NRIF3 or its 30 amino acid Death Domain-1 (DD1) region to the transcriptional repressor, DIF-1 (DD1 Interacting Factor-1). DIF-1 acts in a wide variety of breast cancer cells but not other cell types to repress the pro-apoptotic gene, FASTKD2. Expression of NRIF3 or DD1 inactivates the DIF-1 repressor leading to rapid derepression of FASTKD2, which initiates apoptosis within 5-8 h of expression. Although FASTKD2 is an inner mitochondrial membrane protein, it does not require mitochondrial localization to initiate apoptosis. METHODS: Androgen dependent LNCaP cells as well as two androgen independent LNCaP cell lines (LNCaP-AI and LNCaP-abl) were studied and LNCaP-AI cells were engineered to conditionally express DD1 or the inactive DD1-S28A with 4-hydroxytamoxifen. Apoptosis was assessed by TUNEL assay. FASTKD2 is related to 4 other proteins encoded in the human genome (FASTKD1, 3, 4, 5). All contain a poorly conserved putative bipartite kinase domain designated as FAST1_FAST2. We examined whether expression of any of the other FASTKD isoforms leads to apoptosis and sought to identify the region of FASTKD2 necessary to initiate the apoptotic pathway. RESULTS: Of the FASTKD1-5 isoforms only expression of FASTKD2 leads to apoptosis. Although, the NRIF3/DD1/DIF-1 pathway does not mediate apoptosis of a wide variety of non-breast cancer cell lines, because of certain similarities and gene signatures between breast and prostate cancer we explored whether the NRIF3/DD1/DIF-1/FASTKD2 pathway mediates apoptosis of prostate cancer cells. We found that the pathway leads to apoptosis in LNCaP cells, including the two androgen-independent LNCaP cell lines that are generally resistant to apoptosis. Lastly, we identified that FASTKD2-mediated apoptosis is initiated by the 81 amino acid FAST2 region. CONCLUSIONS: The NRIF3/DIF-1/FASTKD2 pathway acts as a "death switch" in breast and prostate cancer cells. Deciphering how this pathway is regulated and how FASTKD2 initiates the apoptotic response will allow for the development of therapeutic agents for the treatment of androgen-independent prostate cancer or Tamoxifen-unresponsive Estrogen Receptor negative tumors as well as metastatic breast or prostate cancer.

Farnesyl pyrophosphate inhibits epithelialization and wound healing through the glucocorticoid receptor.

Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.

Nuclear hormone receptor coregulator: role in hormone action, metabolism, growth, and development.

Nuclear hormone receptor coregulator (NRC) (also referred to as activating signal cointegrator-2, thyroid hormone receptor-binding protein, peroxisome proliferator activating receptor-interacting protein, and 250-kDa receptor associated protein) belongs to a growing class of nuclear cofactors widely known as coregulators or coactivators that are necessary for transcriptional activation of target genes. The NRC gene is also amplified and overexpressed in breast, colon, and lung cancers. NRC is a 2063-amino acid protein that harbors a potent N-terminal activation domain (AD1) and a second more centrally located activation domain (AD2) that is rich in Glu and Pro. Near AD2 is a receptor-interacting domain containing an LxxLL motif (LxxLL-1), which interacts with a wide variety of ligand-bound nuclear hormone receptors with high affinity. A second LxxLL motif (LxxLL-2) located in the C-terminal region of NRC is more restricted in its nuclear hormone receptor specificity. The intrinsic activation potential of NRC is regulated by a C-terminal serine, threonine, leucine-regulatory domain. The potential role of NRC as a cointegrator is suggested by its ability to enhance transcriptional activation of a wide variety of transcription factors and from its in vivo association with a number of known transcriptional regulators including CBP/p300. Recent studies in mice indicate that deletion of both NRC alleles leads to embryonic lethality resulting from general growth retardation coupled with developmental defects in the heart, liver, brain, and placenta. NRC(-/-) mouse embryo fibroblasts spontaneously undergo apoptosis, indicating the importance of NRC as a prosurvival and antiapoptotic gene. Studies with 129S6 NRC(+/-) mice indicate that NRC is a pleiotropic regulator that is involved in growth, development, reproduction, metabolism, and wound healing.

The nuclear receptor interacting factor-3 transcriptional coregulator mediates rapid apoptosis in breast cancer cells through direct and bystander-mediated events.

We previously reported that amino acids 20 to 50 of nuclear receptor interacting factor-3 mediates rapid apoptosis in breast cancer cell lines but not in cells derived from other tissues. We refer to this short region as death domain-1 (DD1). Small interfering RNA studies indicated that DD1-mediated apoptosis is caspase-2 dependent. In this study, we examined DD1-mediated apoptosis in more detail and generated stable caspase-2 knockdown breast cancer cells. These cells are resistant to DD1-mediated apoptosis. Time-lapse movies suggested that DD1-mediated apoptosis also leads to a "bystander effect." We found that within 5 h of DD1 expression, breast cancer cells release a factor(s) into the medium that leads to apoptosis of naive breast cancer cells or DD1-resistant cells (e.g., HeLa). The DD1-expressing caspase-2 knockdown cells also release a factor(s) that kills other cells, indicating that this effect is not dependent on the apoptogenic process. The bystander effect seems dependent on the production of reactive oxygen species (ROS). These and other studies indicate that DD1 expression in breast cancer cells leads to at least two death signals: one involving the rapid production of ROS and/or other soluble factors that directly or indirectly leads to a bystander effect and a second caspase-2-dependent process that leads to apoptosis in cells in which DD1 is expressed.

Nuclear receptor coregulator (NRC): mapping of the dimerization domain, activation of p53 and STAT-2, and identification of the activation domain AD2 necessary for nuclear receptor signaling.

Nuclear receptor coregulator (NRC) is a 250-kDa nuclear protein involved in transcriptional activation of nuclear hormone receptors, nuclear factor-kappaB, c-Jun, c-Fos, and cAMP response element-binding protein. NRC is organized into a modular structure consisting of two activation domains (AD1 and AD2), two nuclear hormone receptor-interacting motifs, LxxLL-1 and LxxLL-2, and a C-terminal regulatory region rich in serines, threonines, and leucines. The LxxLL-1 motif of NRC binds to a broad spectrum of nuclear hormone receptors with high affinity whereas LxxLL-2 interacts with a very limited number of receptors. In this study we present further evidence that NRC can act as a dimer and have identified a dimerization region of 146 amino acids including LxxLL-1. Mutation of the core LxxLL-1 motif, however, indicates that it is not involved in the dimerization of NRC. AD2, just C-terminal of LxxLL-1, was found to play a central role in ligand-dependent activation by nuclear receptors even though AD1 exhibits more potent intrinsic activity. Thus, a short region of approximately 300 amino acids including and flanking LxxLL-1 plays an important role in NRC dimerization and nuclear receptor binding and transcriptional activation. In addition, consistent with its role as a cointegrator for transcriptional activation, NRC also functions as a coactivator for signal transducer and activator of transcription 2 (STAT-2) and p53. Activation of p53 by NRC appears to involve a novel mechanism where NRC interacts indirectly with p53 through Trap80, a member of the mediator complex, which binds NRC interacting factor-1 (NIF-1), which interacts with and potentiates the effect of NRC.

Farnesyl pyrophosphate is a novel transcriptional activator for a subset of nuclear hormone receptors.

In silico docking of a chemical library with the ligand-binding domain of thyroid hormone nuclear receptor-beta (TRbeta) suggested that farnesyl pyrophosphate (FPP), a key intermediate in cholesterol synthesis and protein farnesylation, might function as an agonist. Surprisingly, addition of FPP to cells activated TR as well as the classical steroid hormone receptors but not peroxisome proliferative-activating receptors, farnesoid X receptor, liver X receptor, or several orphan nuclear receptors the ligands of which are unknown. FPP enhanced receptor-coactivator binding in vitro and in vivo, and elevation of FPP levels in cells by squalene synthetase or farnesyl transferase inhibitors leads to activation. The FPP effect was blocked by selective receptor antagonists, and in silico docking with 143 nuclear receptor ligand-binding domain structures revealed that FPP only docked with the agonist conformation of those receptors activated by FPP. Our results suggest that certain nuclear receptors maintain a common structural feature that may reflect an action of FPP on an ancient nuclear receptor or that FPP could function as a ligand for one of the many orphan nuclear receptors the ligands of which have not yet been identified. This finding also has potential interesting implications that may, in part, explain the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells.

The NRIF3 family of transcriptional coregulators induces rapid and profound apoptosis in breast cancer cells.

Many anticancer drugs kill cancer cells by inducing apoptosis. Despite the progress in understanding apoptosis, how to harness the cellular death machinery to selectively deliver tumor-specific cytotoxicity (while minimizing damage to other cells) remains an important challenge. We report here that expression of the NRIF3 family of transcriptional coregulators in a variety of breast cancer cell lines induces rapid and profound apoptosis (nearly 100% cell death within 24 h). A novel death domain (DD1) was mapped to a short 30-amino-acid region common to all members of the NRIF3 family. Mechanistic studies showed that DD1-induced apoptosis occurs through a novel caspase 2-mediated pathway that involves mitochondrial membrane permeabilization but does not require other caspases. Interestingly, the cytotoxicity of NRIF3 and DD1 appears to be cell type specific, as they selectively kill breast cancer or related cells but not other examined cells of different origins. Our study demonstrates the feasibility of selectively inducing cytotoxicity in a specific cancer and suggests that breast cancer cells contain a novel "death switch" that can be specifically triggered by NRIF3 or DD1. Strategies utilizing NRIF3 and/or DD1 and/or targeting this death switch may lead to the development of novel and more selective therapeutics against breast cancer.

NRC-interacting factor 1 is a novel cotransducer that interacts with and regulates the activity of the nuclear hormone receptor coactivator NRC.

We previously reported the cloning and characterization of a novel nuclear hormone receptor transcriptional coactivator, which we refer to as NRC. NRC is a 2,063-amino-acid nuclear protein which contains a potent N-terminal activation domain and several C-terminal modules which interact with CBP and ligand-bound nuclear hormone receptors as well as c-Fos and c-Jun. In this study we sought to clone and identify novel factors that interact with NRC to modulate its transcriptional activity. Here we describe the cloning and characterization of a novel protein we refer to as NIF-1 (NRC-interacting factor 1). NIF-1 was cloned from rat pituitary and human cell lines and was found to interact in vivo and in vitro with NRC. NIF-1 is a 1,342-amino-acid nuclear protein containing a number of conserved domains, including six Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif. Zinc fingers 1 to 3 are potential DNA-binding BED finger domains recently proposed to play a role in altering local chromatin architecture. We mapped the interaction domains of NRC and NIF-1. Although NIF-1 does not directly interact with nuclear receptors, it markedly enhances ligand-dependent transcriptional activation by nuclear hormone receptors in vivo as well as activation by c-Fos and c-Jun. These results, and the finding that NIF-1 interacts with NRC in vivo, suggest that NIF-1 functions to regulate transcriptional activation through NRC. We suggest that NIF-1, and factors which associate with coactivators but not receptors, be referred to as cotransducers, which act in vivo either as part of a coactivator complex or downstream of a coactivator complex to modulate transcriptional activity. Our findings suggest that NIF-1 may be a functional component of an NRC complex and acts as a regulator or cotransducer of NRC function.

The nuclear hormone receptor coactivator NRC is a pleiotropic modulator affecting growth, development, apoptosis, reproduction, and wound repair.

Nuclear hormone receptor coregulator (NRC) is a 2,063-amino-acid coregulator of nuclear hormone receptors and other transcription factors (e.g., c-Fos, c-Jun, and NF-kappaB). We and others have generated C57BL/6-129S6 hybrid (C57/129) NRC(+/-) mice that appear outwardly normal and grow and reproduce. In contrast, homozygous deletion of the NRC gene is embryonic lethal. NRC(-/-) embryos are always smaller than NRC(+/+) embryos, and NRC(-/-) embryos die between 8.5 and 12.5 days postcoitus (dpc), suggesting that NRC has a pleotrophic effect on growth. To study this, we derived mouse embryonic fibroblasts (MEFs) from 12.5-dpc embryos, which revealed that NRC(-/-) MEFs exhibit a high rate of apoptosis. Furthermore, a small interfering RNA that targets mouse NRC leads to enhanced apoptosis of wild-type MEFs. The finding that C57/129 NRC(+/-) mice exhibit no apparent phenotype prompted us to develop 129S6 NRC(+/-) mice, since the phenotype(s) of certain gene deletions may be strain dependent. In contrast with C57/129 NRC(+/-) females, 20% of 129S6 NRC(+/-) females are infertile while 80% are hypofertile. The 129S6 NRC(+/-) males produce offspring when crossed with wild-type 129S6 females, although fertility is reduced. The 129S6 NRC(+/-) mice tend to be stunted in their growth compared with their wild-type littermates and exhibit increased postnatal mortality. Lastly, both C57/129 NRC(+/-) and 129S6 NRC(+/-) mice exhibit a spontaneous wound healing defect, indicating that NRC plays an important role in that process. Our findings reveal that NRC is a coregulator that controls many cellular and physiologic processes ranging from growth and development to reproduction and wound repair.

Functional evidence for retinoid X receptor (RXR) as a nonsilent partner in the thyroid hormone receptor/RXR heterodimer.

Many members of the thyroid hormone/retinoid receptor subfamily (type II nuclear receptors) function as heterodimers with the retinoid X receptor (RXR). In heterodimers which are referred to as permissive, such as peroxisome proliferator activated receptor/RXR, both partners can bind cognate ligands and elicit ligand-dependent transactivation. In contrast, the thyroid hormone receptor (TR)/RXR heterodimer is believed to be nonpermissive, where RXR is thought to be incapable of ligand binding and is often referred to as a silent partner. In this report, we used a sensitive derepression assay system that we developed previously to reexamine the TR/RXR interrelationship. We provide functional evidence suggesting that in a TR/RXR heterodimer, the RXR component can bind its ligand in vivo. Ligand binding by RXR does not appear to directly activate the TR/RXR heterodimer; instead, it leads to a (at least transient or dynamic) dissociation of a cellular inhibitor(s)/corepressor(s) from its TR partner and thus may serve to modulate unliganded TR-mediated repression and/or liganded TR-mediated activation. Our results argue against the current silent-partner model for RXR in the TR/RXR heterodimer and reveal an unexpected aspect of cross regulation between TR and RXR.

The N-Terminal A/B domain of the thyroid hormone receptor-beta2 isoform influences ligand-dependent recruitment of coactivators to the ligand-binding domain.

Thyroid hormone receptors (TRs), expressed as TRalpha1, TRbeta1, and TRbeta2 isoforms, are members of the steroid hormone nuclear receptor gene superfamily, which comprises ligand-dependent transcription factors. The TR isoforms differ primarily in their N-terminal (A/B) domains, suggesting that the A/B regions mediate distinct transcriptional activation functions in a cell type-dependent or promoter-specific fashion. The nuclear receptor ligand-binding domain (LBD) undergoes a conformational change upon ligand binding that results in the recruitment of coactivators to the LBD. For glucocorticoid receptor and estrogen receptor-alpha, the same coactivator can contact both the LBD and A/B domains, thus leading to enhanced transcriptional activation. Very little is known regarding the role of the A/B domains of the TR isoforms. The A/B domain of TRbeta2 exhibits higher ligand-independent transcriptional activity than the A/B regions of TRalpha1 or TRbeta1. Thus, we examined the role of the A/B domain and the LBD of rat TRbeta2 in integrating the transcriptional activation function of the A/B and LBD domains by different coactivators. Both domains are essential for a productive functional interaction with cAMP response element-binding protein (CREB)-binding protein (CBP), and we found that CBP binds to the A/B domain of TRbeta2 in vitro. In contrast, steroid receptor coactivator-1a (SRC-1a) interacts strongly with the LBD but not the A/B domain. The coactivator NRC (nuclear receptor coactivator) interacts primarily with the LBD, although a weak interaction with the A/B domain further enhances ligand-dependent binding with TRbeta2. Our studies document the interplay between the A/B domain and the LBD of TRbeta2 in recruiting different coactivators to the receptor. Because NRC and SRC-1a bind CBP, and CBP enhances ligand-dependent activity, our studies suggest a model in which coactivator recruitment of NRC (or SRC-1a) occurs primarily through the LBD whereas the complex is further stabilized through an interaction of CBP with the N terminus of TRbeta2.

PSF-TFE3 oncoprotein in papillary renal cell carcinoma inactivates TFE3 and p53 through cytoplasmic sequestration.

Papillary renal cell carcinomas are associated with chromosomal translocations involving the helix-loop-helix leucine-zipper region of the TFE3 gene on the X chromosome. These translocations lead to the expression of TFE3 chimeras of PRCC, RCC17, NonO and PSF (PTB-associated splicing factor). In this study, we explored the role of PSF-TFE3 fusion protein in mediating cell transformation. Unlike wild-type TFE3 or PSF, which are nuclear proteins, PSF-TFE3 is not a nuclear protein and is targeted to the endosomal compartment. Although PSF-TFE3 has no effect on the nuclear localization of wild-type PSF, it sequesters wild-type TFE3 as well as p53 in the extranuclear compartment leading to functionally null p53 and TFE3 cells. In UOK-145 papillary renal carcinoma cells, which endogenously express PSF-TFE3, siRNA complementary to the PSF-TFE3 fusion junction leads to a reduction in PSF-TFE3 and redistribution of endogenous TFE3 and p53 from the cytoplasmic compartment to the nucleus. Our results indicate that PSF-TFE3 acts through a novel mechanism, and exports TFE3, p53 and possibly other factors from the nucleus to the cytoplasm for degradation leading to the transformed phenotype. Thus, PSF-TFE3 is a promising target for the treatment for a subset of renal cell carcinomas.

Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis.

The growth of human breast tumor cells is regulated through signaling involving cell surface growth factor receptors and nuclear receptors of the steroid/thyroid/retinoid receptor gene family. Retinoic acid receptors (RARs), members of the steroid/thyroid hormone receptor gene family, are ligand-dependent transcription factors, which have in vitro and in vivo growth inhibitory activity against breast cancer cells. RAR-agonists inhibit the proliferation of many human breast cancer cell lines, particularly those whose growth is stimulated by estradiol (E2) or growth factors. Additionally, RAR-agonists and synthetic retinoids such as Ferentinide have been shown to induce apoptosis in malignant breast cells but not normal breast cells. To better define the genes involved in RAR-mediated growth inhibition of breast cancer cells, we used oligonucleotide microarray analysis to create a database of genes that are potentially regulated by RAR-agonists in breast cancer cells. We found that PDCD4 (programmed cell death 4), a tumor suppressor gene presently being evaluated as a target for chemoprevention, was induced about three-fold by the RARalpha-selective agonist Am580, in T-47D breast cancer cells. RAR pan-agonists and Am580, but not retinoid X receptors (RXR)-agonists, stimulate the expression of PDCD4 in a wide variety of retinoid-inhibited breast cancer cell lines. RAR-agonists did not induce PDCD4 expression in breast cancer cell lines, which were not growth inhibited by retinoids. We also observed that antiestrogen and the HER-2/neu antagonist, Herceptin (Trastuzumab), also induced PDCD4 expression in T-47D cells, suggesting that PDCD4 may play a central role in growth inhibition in breast cancer cells. Transient overexpression of PDCD4 in T-47D (ER+, RAR+) and MDA-MB-231 (ER-, RAR-) cells resulted in apoptotic death, suggesting a role for PDCD4 in mediating apoptosis in breast cancer cells. PDCD4 protein expression has previously been reported in small ductal epithelium of normal breast. To date, there has been no report of induction of PDCD4 expression by RAR-agonists, antiestrogen or HER2/neu antagonist in breast cancer cells and its potential role in apoptosis in these cells.

RAR agonists stimulate SOX9 gene expression in breast cancer cell lines: evidence for a role in retinoid-mediated growth inhibition.

Retinoic acid receptors (RARs) are ligand-dependent transcription factors which are members of the steroid/thyroid hormone receptor gene family. RAR-agonists inhibit the proliferation of many human breast cancer cell lines, particularly those whose growth is stimulated by estradiol (E2) or growth factors. PCR-amplified subtractive hybridization was used to identify candidate retinoid-regulated genes that may be involved in growth inhibition. One candidate gene identified was SOX9, a member of the high mobility group (HMG) box gene family of transcription factors. SOX9 gene expression is rapidly stimulated by RAR-agonists in T-47D cells and other retinoid-inhibited breast cancer cell lines. In support of this finding, a database search indicates that SOX9 is expressed as an EST in breast tumor cells. SOX9 is known to be expressed in chondrocytes where it regulates the transcription of type II collagen and in testes where it plays a role in male sexual differentiation. RAR pan-agonists and the RARalpha-selective agonist Am580, but not RXR agonists, stimulate the expression of SOX9 in a wide variety of retinoid-inhibited breast cancer cell lines. RAR-agonists did not stimulate SOX9 in breast cancer cell lines which were not growth inhibited by retinoids. Expression of SOX9 in T-47D cells leads to cycle changes similar to those found with RAR-agonists while expression of a dominant negative form of SOX9 blocks RA-mediated cell cycle changes, suggesting a role for SOX9 in retinoid-mediated growth inhibition.

A novel cell lysis approach reveals that caspase-2 rapidly translocates from the nucleus to the cytoplasm in response to apoptotic stimuli.

Unlike other caspases, caspase-2 appears to be a nuclear protein although immunocytochemical studies have suggested that it may also be localized to the cytosol and golgi. Where and how caspase-2 is activated in response to apoptotic signals is not clear. Earlier immunocytochemistry studies suggest that caspase-2 is activated in the nucleus and through cleavage of BID leads to increased mitochondrial permeability. More recent studies using bimolecular fluorescence complementation found that caspase-2 oligomerization that leads to activation only occurs in the cytoplasm. Thus, apoptotic signals may lead to activation of caspase-2 which may already reside in the cytoplasm or lead to release of nuclear caspase-2 to the extra-nuclear cytoplasmic compartment. It has not been possible to study release of nuclear caspase-2 to the cytoplasm by cell fractionation studies since cell lysis is known to release nuclear caspase-2 to the extra-nuclear fraction. This is similar to what is known about unliganded nuclear estrogen receptor-α (ERα ) when cells are disrupted. In this study we found that pre-treatment of cells with N-ethylmaleimide (NEM), which alkylates cysteine thiol groups in proteins, completely prevents redistribution of caspase-2 and ERα from the nucleus to the extra-nuclear fraction when cells are lysed. Using this approach we provide evidence that apoptotic signals rapidly leads to a shift of caspase-2 from the nucleus to the extra-nuclear fraction, which precedes the detection of apoptosis. These findings are consistent with a model where apoptotic signals lead to a rapid shift of caspase-2 from the nucleus to the cytoplasm where activation occurs.

A novel transcription complex that selectively modulates apoptosis of breast cancer cells through regulation of FASTKD2.

We previously reported that expression of NRIF3 (nuclear receptor interacting factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (also known as interferon regulatory factor-2 binding protein 2 [IRF-2BP2]), the cellular target of NRIF3, was identified as a transcriptional repressor, and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF-2BP1 and EAP1 (enhanced at puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF-2BP1, and EAP1 occurs through the conserved C4 zinc fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the death domain (DD1) region of NRIF3. The DIF-1 complex was found to repress FASTKD2, a putative proapoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown, while expression of FASTKD2 leads to apoptosis of both breast and nonbreast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer.

Nuclear receptor engineering based on novel structure activity relationships revealed by farnesyl pyrophosphate.

Nuclear receptors (NRs) comprise the second largest protein family targeted by currently available drugs, acting via specific ligand interactions within the ligand binding domain (LBD). Recently, farnesyl pyrophosphate (FPP) was shown to be a unique promiscuous NR ligand, activating a subset of NR family members and inhibiting wound healing in skin. The current study aimed at visualizing the unique basis of FPP interaction with multiple receptors in order to identify general structure-activity relationships that operate across the NR family. Docking of FPP to the 3D structures of the LBDs of a diverse set of NRs consistently revealed an electrostatic FPP pyrophosphate contact with an NR arginine conserved in the NR family, a hydrophobic farnesyl contact with NR helix-12 and a ligand binding pocket volume between 300 and 430 Å(3) as the minimal requirements for FPP activation of any NR. Lack of any of these structural features appears to render a given NR resistant to FPP activation. We used these structure-activity relationships to rationally design and successfully engineer several mutant human estrogen receptors that retain responsiveness to estradiol but no longer respond to FPP.

Identification of a novel pathway that selectively modulates apoptosis of breast cancer cells.

Expression of the nuclear receptor interacting factor 3 (NRIF3) coregulator in a wide variety of breast cancer cells selectively leads to rapid caspase-2-dependent apoptotic cell death. A novel death domain (DD1) was mapped to a 30-amino acid region of NRIF3. Because the cytotoxicity of NRIF3 and DD1 seems to be cell type-specific, these studies suggest that breast cancer cells contain a novel "death switch" that can be specifically modulated by NRIF3 or DD1. Using an MCF-7 cell cDNA library in a yeast two-hybrid screen, we cloned a factor that mediates apoptosis by DD1 and refer to this factor as DD1-interacting factor-1 (DIF-1). DIF-1 is a transcriptional repressor that mediates its effect through SirT1, and this repression is attenuated by the binding of NRIF3/DD1. DIF-1 expression rescues breast cancer cells from NRIF3/DD1-induced apoptosis. Small interfering RNA (siRNA) knockdown of DIF-1 selectively leads to apoptosis of breast cancer cells, further suggesting that DIF-1 plays a key role in NRIF3/DD1-mediated apoptosis. A protein kinase A inhibitor (H89) also elicits apoptosis of breast cancer cells but not of the other cell types examined, and DIF-1 also protects these cells from H89-mediated apoptosis. In addition, H89 incubation results in a rapid increase in NRIF3 levels and siRNA knockdown of NRIF3 protects breast cancer cells from H89-mediated apoptosis. Our results indicate that DIF-1 plays a key role in breast cancer cell survival and further characterizing this pathway may provide important insights into developing novel therapies to selectively target breast cancer cells for apoptosis.