Wait Times and Survival in Lung Cancer Patients across the Province of Quebec, Canada.

Lung cancer is the leading cause of cancer death worldwide, with a five-year survival of 22% in Canada. Guidelines recommend rapid evaluation of patients with suspected lung cancer, but the impact on survival remains unclear. We reviewed medical records of all patients with newly diagnosed lung cancer in four hospital networks across the province of Quebec, Canada, between 1 February and 30 April 2017. Patients were followed for 3 years. Wait times for diagnosis and treatment were collected, and survival analysis using a Cox regression model was conducted. We included 1309 patients, of whom 39% had stage IV non-small cell lung cancer (NSCLC). Median wait times were, in general, significantly shorter in patients with stage III-IV NSCLC or SCLC. Surgery was associated with delays compared to other types of treatments. Median survival was 12.9 (11.1-15.7) months. The multivariate survival model included age, female sex, performance status, histology and stage, treatment, and the time interval between diagnosis and treatment. Longer wait times had a slightly protective to neutral effect on survival, but this was not significant in the stage I-II NSCLC subgroup. Wait times for the diagnosis and treatment of lung cancer were generally within targets. The shorter wait times observed for advanced NSCLC and SCLC might indicate a tendency for clinicians to act quicker on sicker patients. This study did not demonstrate the detrimental effect of longer wait times on survival.

High bone resorption in adult aging transgenic mice overexpressing cbfa1/runx2 in cells of the osteoblastic lineage.

The runt family transcription factor core-binding factor alpha1 (Cbfa1) is essential for bone formation during development. Surprisingly, transgenic mice overexpressing Cbfa1 under the control of the 2.3-kb collagen type I promoter developed severe osteopenia that increased progressively with age and presented multiple fractures. Analysis of skeletally mature transgenic mice showed that osteoblast maturation was affected and that specifically in cortical bone, bone resorption as well as bone formation was increased, inducing high bone turnover rates and a decreased degree of mineralization. To understand the origin of the increased bone resorption, we developed bone marrow stromal cell cultures and reciprocal coculture of primary osteoblasts and spleen cells from wild-type or transgenic mice. We showed that transgenic cells of the osteoblastic lineage induced an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells, suggesting that primary osteoblasts as well as bone marrow stromal cells from transgenic mice have stronger osteoclastogenic properties than cells derived from wild-type animals. We investigated the candidate genes whose altered expression could trigger this increase in bone resorption, and we found that the expression of receptor activator of NF-kappaB ligand (RANKL) and collagenase 3, two factors involved in bone formation-resorption coupling, was markedly increased in transgenic cells. Our data thus suggest that overexpression of Cbfa1 in cells of the osteoblastic lineage does not necessarily induce a substantial increase in bone formation in the adult skeleton but has a positive effect on osteoclast differentiation in vitro and can also dramatically enhance bone resorption in vivo, possibly through increased RANKL expression.

Blockade of Metallothioneins 1 and 2 Increases Skeletal Muscle Mass and Strength.

Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo We found that concomitant abrogation of metallothioneins 1 and 2 results in activation of the Akt pathway and increases in myotube size, in type IIb fiber hypertrophy, and ultimately in muscle strength. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function was also observed in the setting of glucocorticoid addition, which is a strong atrophy-inducing stimulus. Given the blockade of atrophy and the preservation of strength in atrophy-inducing settings, these results suggest that blockade of metallothioneins 1 and 2 constitutes a promising approach for the treatment of conditions which result in muscle atrophy.

X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha.

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORalpha has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORalpha, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3beta-ol (cholesterol). Moreover, RORalpha transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORalpha could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

Selective estrogen receptor modulators with conformationally restricted side chains. Synthesis and structure-activity relationship of ERalpha-selective tetrahydroisoquinoline ligands.

We disclose herein the discovery of estrogen receptor alpha (ERalpha) selective estrogen receptor modulators (SERMs) of the tetrahydroisoquinoline series that incorporate novel conformationally restricted side chains as replacement of the aminoethoxy residue typical of SERMs. Molecular modeling studies used in conjunction with the X-ray crystal structure of the ERalpha ligand binding domain (LBD) with raloxifene (7) suggested a diazadecaline moiety as a viable mimic of the SERM side chain. On the basis of this knowledge, the piperidinylethoxy moiety of our lead compound 60 was replaced by a diazadecaline subunit, providing the novel tetrahydroisoquinoline 29. In addition to exhibiting a binding affinity to ERalpha and antagonistic properties in the estrogen response element and MCF-7 assays similar to those of the parent compound 60, ligand 29 showed a reduced agonist behavior in the MCF-7 assay in the absence of 17beta-estradiol. These data point toward the fact that 29 may have a potential for breast cancer prevention/treatment in vivo, a feature which is particularly attractive in the quest for safe alternatives to hormone replacement therapy. In a pharmacokinetic experiment carried out in rats, 29 displayed an interesting profile, with a bioavailability of 49%. We also disclose the X-ray crystal structure of 29 in complex with ERalpha-LBD, which reveals the preferred configurations of 29 at the two chiral centers and the details of its interactions with the receptor. Finally, our structure-activity relationship studies show that other analogues bearing constrained side chains retain potency and antagonist activity and that a 3-OH substituted phenyl D-ring increases the selectivity of a set of piperazinyl-containing ligands in favor of ERalpha over ERbeta.

Estrogen receptor modulators: identification and structure-activity relationships of potent ERalpha-selective tetrahydroisoquinoline ligands.

As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), tetrahydroisoquinoline derivative 27 was discovered by high throughput screening. Successive replacements of the p-F substituent of 27 by an aminoethoxy side chain and of the 1-H of the tetrahydroisoquinoline core by a 1-Me group provided analogues 19 and 20. These compounds showed potencies in a cell-based reporter gene assay (ERE assay) varying between 0.6 and 20 nM and displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line with IC(50)s in the range of 2-36 nM. The effect of N-phenyl substituents on the activity and pharmacokinetic properties of tetrahydroisoquinoline analogues was explored. As a result of this investigation, two potent derivatives bearing a p-F N-aryl group, 19c and 20c, were discovered as candidates suitable for further profiling. To gain insight into the ligand-receptor interaction, the X-ray crystallographic structure of the 1-H tetrahydroisoquinoline derivative (R)-18a in complex with ERalpha-ligand binding domain (LBD)(301)(-)(553)/C-->S triple mutant was solved to 2.28 A. An overlay of this X-ray crystal structure with that reported for the complex of ERalpha-LBD(301)(-)(553)/carboxymethylated C and raloxifene (5) shows that both compounds bind to the same cleft of the receptor and display comparable binding modes, with differences being observed in the conformation of their "D-ring" phenyl groups.

Estrogen Receptor α (ERα) and Estrogen Related Receptor α (ERRα) are both transcriptional regulators of the Runx2-I isoform.

Runx2 is a master regulator of bone development and has also been described as an oncogene. Estrogen Receptor α (ERα) and Estrogen Related Receptor α (ERRα), both implicated in bone metabolism and breast cancer, have been shown to share common transcriptional targets. Here, we show that ERα is a positive regulator of Runx2-I transcription. Moreover, ERRα can act as a transcriptional activator of Runx2-I in presence of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α). In contrast, ERRα behaves as a negative regulator of Runx2-I transcription in presence of PGC-1ß. ERα and ERRα cross-talk via a common estrogen receptor response element on the Runx2-I promoter. In addition, estrogen regulates PGC-1ß that in turn is able to modulate both ERα and ERRα transcriptional activity.

Genomic and proteomic profiling reveals reduced mitochondrial function and disruption of the neuromuscular junction driving rat sarcopenia.

Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia.

Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism.

Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

Follicle-stimulating hormone does not impact male bone mass in vivo or human male osteoclasts in vitro.

Bone loss in the elderly is mainly caused by osteoclast-induced bone resorption thought to be causally linked to the decline in estrogen and testosterone levels in females and males. Recently, involvement of follicle stimulating-hormone (FSH) in this process has been suggested to explain in part the etiology of the disease in females, whereas its role in males has never been examined. In this study, the direct impact of FSH on bone mass of 16-week-old C57BL/6J male mice by either daily intermittent application of 6 or 60 mug/kg of FSH or continuous delivery via miniosmotic pump of a dose of 6 mug/kg over the course of a month was assessed. Femoral peripheral quantitative computed tomographic and microcomputed tomographic analyses at 0, 2, and 4 weeks of FSH-treated mice did not reveal any differences in cancellous and cortical bone compared to sham-treated mice. FSH functionality was verified by demonstrating cAMP induction and activation of a cAMP-response element-containing reporter cell line by FSH. Furthermore, osteoclastogenesis from human mononuclear cell precursors and from RAW 264.7 cells was not affected by FSH (3, 10, 30 ng/mL) compared to control. No direct effect of FSH on gene regulation was observed by Affymetrix Gene Array on RAW 264.7 cells. Lastly, no expression of FSH receptor (FSHR) mRNA or FSHR was observed by quantitative polymerase chain reaction and Western blot in either human male osteoclasts or RAW 264.7 cells. These data show that FSH does not appear to modulate male bone mass regulation in vivo and does not act directly on osteoclastogenesis in vitro.

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism.

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).

Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A.

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo.

Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach.

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.

Evidence for ligand-independent transcriptional activation of the human estrogen-related receptor alpha (ERRalpha): crystal structure of ERRalpha ligand binding domain in complex with peroxisome proliferator-activated receptor coactivator-1alpha.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor alpha (ERRalpha, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERRalpha LBD, solved to a resolution of 2.5 A, and the first structure of a PGC-1alpha complex. The putative ligand binding pocket (LBP) of ERRalpha is almost completely occupied by side chains, in particular with the bulky side chain of Phe328 (corresponding to Ala272 in ERRgamma and Ala350 in estrogen receptor alpha). Therefore, a ligand of a size equivalent to more than approximately 4 carbon atoms could only bind in the LBP, if ERRalpha would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERRalpha. The interactions of PGC-1alpha with ERRalpha also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1alpha peptide containing this nuclear box motif from the L3 site binds ERRalpha LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1alpha are present.