Rachid Safi

Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP

The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 Å of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1s control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.

Coactivation of Liver Receptor Homologue-1 by Peroxisome Proliferator-Activated Receptor γ Coactivator-1α on Aromatase Promoter II and Its Inhibition by Activated Retinoid X Receptor Suggest a Novel Target for Breast-Specific Antiestrogen Therapy

Aromatase inhibitors target the production of estrogen in breast adipose tissue, but in doing so, also decrease estrogen formation in bone and other sites, giving rise to deleterious side effects, such as bone loss and arthralgia. Thus, it would be clinically useful to selectively inhibit aromatase production in breast. In this regard, we have determined that the orphan nuclear receptor liver receptor homologue-1 (LRH-1) is a specific transcriptional activator of aromatase gene expression in human breast preadipocytes but not in other tissues of postmenopausal women. In this study, we show that the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is a physiologically relevant modulator of LRH-1, and that its transcriptional activity can be inhibited effectively using receptor-interacting peptide antagonists that prevent PGC-1alpha recruitment. Interestingly, we note that all of these peptides also interact in an agonist-dependent manner with retinoid X receptor alpha (RXRalpha), suggesting that these two receptors may compete for limiting cofactors within target cells. In support of this hypothesis, we show that 9-cis-retinoic acid, acting through RXR, inhibits both the basal and PGC-1alpha-induced transcriptional activity of LRH-1. The importance of this finding was confirmed by showing that LRH-1-dependent, PGC-1alpha-stimulated regulation of aromatase gene expression in primary human breast preadipocytes was effectively suppressed by RXR agonists. We infer from these data that LRH-1 is a bona fide target whose inhibition would selectively block aromatase expression in breast, while sparing other sites of expression.

Inhibition of Aldehyde Dehydrogenase Expands Hematopoietic Stem Cells with Radioprotective Capacity

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34−c‐kit+Sca‐1+lineage− (34−KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34−KSL cells. Treatment of bone marrow (BM) 34−KSL cells with DEAB caused a fourfold increase in 4‐week competitive repopulating units, verifying the amplification of short‐term HSCs (ST‐HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB‐mediated expansion of ST‐HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST‐HSCs for transplantation purposes. STEM CELLS 2010;28:523–534

Identification of Ligand-Selective Peptide Antagonists of the Mineralocorticoid Receptor Using Phage Display

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. The glucocorticoid cortisol binds the MR with equivalent affinity to that of the mineralocorticoids aldosterone and deoxycorticosterone. In nonepithelial tissues, including the myocardium, which do not express the cortisol-inactivating enzyme 11β hydroxysteroid dehydrogenase 2, cortisol has been implicated in the activation of MR. The mechanisms for ligand- and tissue-specific actions of the MR are undefined. Over the past decade, it has become clear that coregulator proteins are critical for nuclear receptor-mediated gene expression. A subset of these coregulators may confer specificity to MR-mediated responses. To evaluate whether different physiological ligands can induce distinct MR conformations that underlie differential coregulator recruitment and ligand-specific gene regulation, we utilized phage display technology to screen 10(8) 19mer peptides for their interaction with the MR in the presence of agonist ligands. We identified ligand-selective MR-interacting peptides that acted as potent antagonists of MR-mediated transactivation. This represents a novel mechanism of MR antagonism that may be manipulated in the rational design of a ligand- or tissue-selective MR modulator to treat diseases like heart failure without side effects such as hyperkalemia.

Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer

Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS‐mediated death. Inflammatory breast cancer (IBC) is a distinct, advanced BC subtype characterized by high rates of residual disease and recurrence despite advances in multimodality treatment. Using a cellular model of IBC, we identified an oxidative stress response (OSR) signature in surviving IBC cells after administration of an acute dose of an ROS inducer. Metagene analysis of patient samples revealed significantly higher OSR scores in IBC tumor samples compared to normal or non‐IBC tissues, which may contribute to the poor response of IBC tumors to common treatment strategies, which often rely heavily on ROS induction. To combat this adaptation, we utilized a potent redox modulator, the FDA‐approved small molecule Disulfiram (DSF), alone and in combination with copper. DSF forms a complex with copper (DSF‐Cu) increasing intracellular copper concentration both in vitro and in vivo, bypassing the need for membrane transporters. DSF‐Cu antagonized NFκB signaling, aldehyde dehydrogenase activity and antioxidant levels, inducing oxidative stress‐mediated apoptosis in multiple IBC cellular models. In vivo, DSF‐Cu significantly inhibited tumor growth without significant toxicity, causing apoptosis only in tumor cells. These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS‐inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemo‐ and/or radio‐sensitivity for advanced BC subtypes where therapeutic resistance is an impediment to durable responses to current standard of care.

Delineation of a FOXA1/ERα/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer

Tamoxifen, a selective estrogen receptor (ER) modulator (SERM), remains a frontline clinical therapy for patients with ERα-positive breast cancer. However, the relatively rapid development of resistance to this drug in the metastatic setting remains an impediment to a durable response. Although drug resistance likely arises by many different mechanisms, the consensus is that most of the implicated pathways facilitate the outgrowth of a subpopulation of cancer cells that can either recognize tamoxifen as an agonist or bypass the regulatory control of ERα. Notable in this regard is the observation here and in other studies that expression of anterior gradient homology 2 (AGR2), a known proto-oncogene and disulfide isomerase, was induced by both estrogen (17β-estradiol, E2) and 4-hydroxytamoxifen (4OHT) in breast cancer cells. The importance of AGR2 expression is highlighted here by the observation that (i) its knockdown inhibited the growth of both tamoxifen-sensitive and -resistant breast cancer cells and (ii) its increased expression enhanced the growth of ERα-positive tumors in vivo and increased the migratory capacity of breast cancer cells in vitro. Interestingly, as with most ERα target genes, the expression of AGR2 in all breast cancer cells examined requires the transcription factor FOXA1. However, in tamoxifen-resistant cells, the expression of AGR2 occurs in a constitutive manner, requiring FOXA1, but loses its dependence on ER. Taken together, these data define the importance of AGR2 in breast cancer cell growth and highlight a mechanism where changes in FOXA1 activity obviate the need for ER in the regulation of this gene. Implications: These findings reveal the transcriptional interplay between FOXA1 and ERα in controlling AGR2 during the transition from therapy-sensitive to -resistant breast cancer and implicate AGR2 as a relevant therapeutic target. Mol Cancer Res; 12(12); 1829–39. ©2014 AACR.

Pharmacological Manipulation of the RAR/RXR Signaling Pathway Maintains the Repopulating Capacity of Hematopoietic Stem Cells in Culture

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However, RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study, we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506, a selective RXR modulator, inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters, suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly, using combinatorial peptide phage display, we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation, and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture.

CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662-73. ©2017 AACR.

Purification and characterization of recombinant human Mineralocorticoid Receptor

The mineralocorticoid receptor (MR) plays a critical role in the maintenance of electrolyte homeostasis and blood pressure via direct effects on the distal nephron and the cardiovascular system. The MR also has an important role in the pathology of cardiovascular disease, particularly heart failure, and is therefore an attractive therapeutic target. However, renal side effects limit its use in the clinic. Previous studies of MR molecular pharmacology have been performed on its isolated ligand-binding domain (LBD); however, current evidence suggests that nuclear receptor LBDs behave differently in isolation, than in the context of the full-length receptor. To date, technical issues have precluded production of full-length MR, thereby preventing molecular and structural studies of the MR LBD in its natural context. Here, we describe expression and purification of full-length human MR (hMR). hMR was expressed in Sf9 insect cells with an N-terminal biotinylated (bt)-tag, and stabilised by addition of ligand. bt-hMR exhibited ligand-binding and transactivation properties similar to that of the native protein. Affinity purification using an avidin matrix yielded approximately 120mug MR protein from 0.5lt Sf9 culture, and the receptor was purified bound to either aldosterone or cortisol. Recombinant hMR had a molecular weight of 110-130kDa, bound an MR DNA response element in vitro and interacted with a known co-regulator, PGC-1alpha, in GST pull-down assays, indicating its functional activity. Availability of this reagent will now enable analysis of MR structure and ligand interactions in the context of the full-length receptor, a prerequisite for future development of ligand-selective MR antagonists for the treatment of cardiovascular disease.

Smoothelin-like 1 protein is a bifunctional regulator of the progesterone receptor during pregnancy

During pregnancy, uterine smooth muscle (USM) coordinately adapts its contractile phenotype in order to accommodate the developing fetus and then prepare for delivery. Herein we show that SMTNL1 plays a major role in pregnancy to promote adaptive responses in USM and that this process is specifically mediated through interactions of SMTNL1 with the steroid hormone receptor PR-B. In vitro and in vivo SMTNL1 selectively binds PR and not other steroid hormone receptors. The physiological relationship between the two proteins was also established in global gene expression and transcriptional reporter studies in pregnant smtnl1−/− mice and by RNA interference in progesterone-sensitive cell lines. We show that the contraction-associated and progestin-sensitive genes (oxytocin receptor, connexin 43, and cyclooxygenase-2) and prolactins are down-regulated in pregnant smtnl1−/− mice. We suggest that SMTNL1 is a bifunctional co-regulator of PR-B signaling and thus provides a molecular mechanism whereby PR-B is targeted to alter gene expression patterns within USM cells to coordinately promote alterations in USM function during pregnancy.