Dysregulated cholesterol homeostasis results in resistance to ferroptosis increasing tumorigenicity and metastasis in cancer.

Hypercholesterolemia and dyslipidemia are associated with an increased risk for many cancer types and with poor outcomes in patients with established disease. Whereas the mechanisms by which this occurs are multifactorial we determine that chronic exposure of cells to 27-hydroxycholesterol (27HC), an abundant circulating cholesterol metabolite, selects for cells that exhibit increased cellular uptake and/or lipid biosynthesis. These cells exhibit substantially increased tumorigenic and metastatic capacity. Notably, the metabolic stress imposed upon cells by the accumulated lipids requires sustained expression of GPX4, a negative regulator of ferroptotic cell death. We show that resistance to ferroptosis is a feature of metastatic cells and further demonstrate that GPX4 knockdown attenuates the enhanced tumorigenic and metastatic activity of 27HC resistant cells. These findings highlight the general importance of ferroptosis in tumor growth and metastasis and suggest that dyslipidemia/hypercholesterolemia impacts cancer pathogenesis by selecting for cells that are resistant to ferroptotic cell death.

The Dysregulated Pharmacology of Clinically Relevant ESR1 Mutants is Normalized by Ligand-activated WT Receptor.

The estrogen receptor (ER/ESR1) is expressed in a majority of breast cancers and drugs that inhibit ER signaling are the cornerstone of breast cancer pharmacotherapy. Currently, aromatase inhibitors are the frontline endocrine interventions of choice although their durability in metastatic disease is limited by activating point mutations within the ligand-binding domain of ESR1 that permit ligand-independent activation of the receptor. It has been suggested that the most commonly occurring ESR1 mutations would likely compromise the clinical activity of selective estrogen receptor downregulators and selective estrogen receptor modulators (SERMs) when used as second-line therapies. It was unclear, however, how these mutations, which are likely coexpressed in cells with ERWT, may impact response to ER ligands in a clinically meaningful manner. To address this issue, we dissected the molecular mechanism(s) underlying ESR1-mutant pharmacology in models relevant to metastatic disease. These studies revealed that the response of ESR1 mutations to ligands was dictated primarily by the relative coexpression of ERWT in cells. Specifically, dysregulated pharmacology was only evident in cells in which the mutants were overexpressed relative to ligand-activated ERWT; a finding that highlights the role of allelism in determining ER-mutant pharmacology. Importantly, we demonstrated that the antagonist activity of the SERM, lasofoxifene, was not impacted by mutant status; a finding that has led to its clinical evaluation as a treatment for patients with advanced ER-positive breast cancer whose tumors harbor ESR1 mutations.

Inhibition of ERRα Prevents Mitochondrial Pyruvate Uptake Exposing NADPH-Generating Pathways as Targetable Vulnerabilities in Breast Cancer.

Most cancer cells exhibit metabolic flexibility, enabling them to withstand fluctuations in intratumoral concentrations of glucose (and other nutrients) and changes in oxygen availability. While these adaptive responses make it difficult to achieve clinically useful anti-tumor responses when targeting a single metabolic pathway, they can also serve as targetable metabolic vulnerabilities that can be therapeutically exploited. Previously, we demonstrated that inhibition of estrogen-related receptor alpha (ERRα) significantly disrupts mitochondrial metabolism and that this results in substantial antitumor activity in animal models of breast cancer. Here we show that ERRα inhibition interferes with pyruvate entry into mitochondria by inhibiting the expression of mitochondrial pyruvate carrier 1 (MPC1). This results in a dramatic increase in the reliance of cells on glutamine oxidation and the pentose phosphate pathway to maintain nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis. In this manner, ERRα inhibition increases the efficacy of glutaminase and glucose-6-phosphate dehydrogenase inhibitors, a finding that has clinical significance.

CDK4/6 Therapeutic Intervention and Viable Alternative to Taxanes in CRPC.

Resistance to second-generation androgen receptor (AR) antagonists and CYP17 inhibitors in patients with castration-resistant prostate cancer (CRPC) develops rapidly through reactivation of the androgen signaling axis and has been attributed to AR overexpression, production of constitutively active AR splice variants, or the selection for AR mutants with altered ligand-binding specificity. It has been established that androgens induce cell-cycle progression, in part, through upregulation of cyclin D1 (CCND1) expression and subsequent activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Thus, the efficacy of the newly described CDK4/6 inhibitors (G1T28 and G1T38), docetaxel and enzalutamide, was evaluated as single agents in clinically relevant in vitro and in vivo models of hormone-sensitive and treatment-resistant prostate cancer. CDK4/6 inhibition (CDK4/6i) was as effective as docetaxel in animal models of treatment-resistant CRPC but exhibited significantly less toxicity. The in vivo effects were durable and importantly were observed in prostate cancer cells expressing wild-type AR, AR mutants, and those that have lost AR expression. CDK4/6i was also effective in prostate tumor models expressing the AR-V7 variant or the AR F876L mutation, both of which are associated with treatment resistance. Furthermore, CDK4/6i was effective in prostate cancer models where AR expression was lost. It is concluded that CDK4/6 inhibitors are a viable alternative to taxanes as therapeutic interventions in endocrine therapy-refractory CRPC.Implications: The preclinical efficacy of CDK4/6 monotherapy observed here suggests the need for near-term clinical studies of these agents in advanced prostate cancer. Mol Cancer Res; 15(6); 660-9. ©2017 AACR.

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.

ERRα-Regulated Lactate Metabolism Contributes to Resistance to Targeted Therapies in Breast Cancer.

Imaging studies in animals and in humans have indicated that the oxygenation and nutritional status of solid tumors is dynamic. Furthermore, the extremely low level of glucose within tumors, while reflecting its rapid uptake and metabolism, also suggests that cancer cells must rely on other energy sources in some circumstances. Here, we find that some breast cancer cells can switch to utilizing lactate as a primary source of energy, allowing them to survive glucose deprivation for extended periods, and that this activity confers resistance to PI3K/mTOR inhibitors. The nuclear receptor, estrogen-related receptor alpha (ERRα), was shown to regulate the expression of genes required for lactate utilization, and isotopomer analysis revealed that genetic or pharmacological inhibition of ERRα activity compromised lactate oxidation. Importantly, ERRα antagonists increased the in vitro and in vivo efficacy of PI3K/mTOR inhibitors, highlighting the potential clinical utility of this drug combination.

Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice.

Pregnancy promotes physiological adaptations throughout the body, mediated by the female sex hormones progesterone and estrogen. Changes in the metabolic properties of skeletal muscle enable the female body to cope with the physiological challenges of pregnancy and may also be linked to the development of insulin resistance. We conducted global microarray, proteomic, and metabolic analyses to study the role of the progesterone receptor and its transcriptional regulator, smoothelin-like protein 1 (SMTNL1) in the adaptation of skeletal muscle to pregnancy. We demonstrate that pregnancy promotes fiber-type changes from an oxidative to glycolytic isoform in skeletal muscle. This phenomenon is regulated through an interaction between SMTNL1 and progesterone receptor, which alters the expression of contractile and metabolic proteins. smtnl1(-/-) mice are metabolically less efficient and show impaired glucose tolerance. Pregnancy antagonizes these effects by inducing metabolic activity and increasing glucose tolerance. Our results suggest that SMTNL1 has a role in mediating the actions of steroid hormones to promote fiber switching in skeletal muscle during pregnancy. Our findings also bear on the management of gestational diabetes that develops as a complication of pregnancy in ~4% of women.

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.

Copper signaling axis as a target for prostate cancer therapeutics.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.

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

UNLABELLED: 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 (17ß-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.

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.

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 11ß 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.

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.

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.

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.

Unexpected novel relational links uncovered by extensive developmental profiling of nuclear receptor expression.

Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily.

Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells.

Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic/severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34(+)CD38(-)lin(-) cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.

Thyroid hormone receptor expression in the obligatory paedomorphic salamander Necturus maculosus.

Amphibian metamorphosis is under the strict control of thyroid hormones (TH). These hormones induce metamorphosis by controlling gene expression through binding to thyroid hormone receptors (TRs). Necturus maculosus is considered to be an obligatory paedomorphic Amphibian since metamorphosis never occurs spontaneously and cannot be induced by pharmacological means. Since metamorphosis depends on the acquisition of response of tadpole tissues to thyroid hormone, we aimed to determine TR gene expression patterns in Necturus maculosus as well as the expression of two TH-related genes: Cytosolic Thyroid Hormone-Binding Protein (CTHBP)-M2-pyruvate kinase, a gene encoding a cytosolic TH binding protein and stromelysin 3, a direct TH target gene in Xenopus laevis. Tissue samples were obtained from specimens of Necturus maculosus. We performed in situ hybridization using non-cross-hybridizing RNA probes obtained from the cloned Necturus TRalpha and TRbeta genes. We found clear expression of Necturus TRalpha gene in several tissues including the central nervous system, epithelial cells of digestive and urinary organs, as well as myocardium and skeletal muscle. TRbeta was also expressed in the brain. In other tissues, hybridization signals were too low to draw reliable conclusions about their precise distribution. In addition, we observed that the expression of CTHBP and ST3 is largely distinct from that of TRs. The fact that we observed a clear expression of TRalpha and TRbeta which are evolutionary conserved, suggests that Necturus tissues express TRs. Our results thus indicate that, in contrast to previously held hypotheses, Necturus tissues are TH responsive.

Pedomorphosis revisited: thyroid hormone receptors are functional in Necturus maculosus.

Heterochrony, a difference in developmental timing, is a central concept in modern evolutionary biology. An example is pedomorphosis, retention of juvenile characteristics in sexually mature adults, a phenomenon largely represented in salamanders. The mudpuppy (Necturus maculosus) is an obligate pedomorphic amphibian, never undergoing metamorphosis. Thyroid hormone induces tissue transformation in metamorphosing species and this action is mediated by nuclear thyroid hormone (TH) receptors (TRs). The absence of metamorphosis in Necturus has been attributed to a resistance to TH action as treatment with exogenous TH fails to induce transformation. The failure to metamorphose could be due to the lack of TR expression in target tissues, or to a loss of TR function. Toward understanding the molecular basis for the failure of Necturus tissues to respond to TH, and the ultimate cause for the expression of the obligate pedomorphic life history, we characterized the structure, function, and expression of TR genes in Necturus. Strikingly, we found that Necturus TRalpha and TRbeta genes encode fully functional TR proteins. These TRs bind both DNA and TH and can transactivate target genes in response to TH. Both TRalpha and TRbeta are expressed in various tissues. TH treatment in vivo induced expression in the gill of some but not all genes known to be activated by TH in anuran larvae, caused whole organism metabolic effects, but induced no external morphological changes in adults or larvae. Thus, Necturus possesses fully functional TRs and its tissues are not generally resistant to the actions of TH. Rather, the absence of metamorphosis may be due to the loss of TH-dependent control of key genes required for tissue transformation.

Coactivation of liver receptor homologue-1 by peroxisome proliferator-activated receptor gamma coactivator-1alpha 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.