Repurposing mebendazole against triple-negative breast cancer CNS metastasis.

PURPOSE: Triple-negative breast cancer (TNBC) often metastasizes to the central nervous system (CNS) and has the highest propensity among breast cancer subtypes to develop leptomeningeal disease (LMD). LMD is a spread of cancer into leptomeningeal space that speeds up the disease progression and severely aggravates the prognosis. LMD has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. METHODS: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, CNS metastasis was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging and immunohistochemistry. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. RESULTS: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced leptomeningeal spread, and extended survival in brain metastasis model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. CONCLUSIONS: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC CNS metastasis. Our findings are concordant with previous efforts involving MBZ and CNS pathology and support the drug's potential utility to slow down leptomeningeal spread.

Using SRM-MS to quantify nuclear protein abundance differences between adipose tissue depots of insulin-resistant mice.

Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.

Glucocorticoid signaling and the impact of high-fat diet on adipogenesis in vivo.

Our research used glucocorticoids as a medically relevant molecular probe to identify a previously unrecognized ADAMTS1-PTN-Wnt pathway. We elucidated the role of this pathway in regulating adipose precursor cell (APC) behavior to either proliferate or differentiate in response to systemic cues, such as elevated caloric intake. Further, our studies identified the non-muscle myosin protein MYH9 as a key target of this pathway to modulate adipogenesis in vivo. These findings enable strategies toward developing novel therapeutics for obesity and related metabolic disorders.

White adipocytes in subcutaneous fat depots require KLF15 for maintenance in preclinical models.

Healthy adipose tissue is essential for normal physiology. There are 2 broad types of adipose tissue depots: brown adipose tissue (BAT), which contains adipocytes poised to burn energy through thermogenesis, and white adipose tissue (WAT), which contains adipocytes that store lipids. However, within those types of adipose, adipocytes possess depot and cell-specific properties that have important implications. For example, the subcutaneous and visceral WAT confers divergent risk for metabolic disease. Further, within a depot, different adipocytes can have distinct properties; subcutaneous WAT can contain adipocytes with either white or brown-like (beige) adipocyte properties. However, the pathways that regulate and maintain this cell and depot-specificity are incompletely understood. Here, we found that the transcription factor KLF15 is required for maintaining white adipocyte properties selectively within the subcutaneous WAT. We revealed that deletion of Klf15 is sufficient to induce beige adipocyte properties and that KLF15's direct regulation of Adrb1 is a critical molecular mechanism for this process. We uncovered that this activity is cell autonomous but has systemic implications in mouse models and is conserved in primary human adipose cells. Our results elucidate a pathway for depot-specific maintenance of white adipocyte properties that could enable the development of therapies for obesity and associated diseases.

Repurposing mebendazole against triple-negative breast cancer leptomeningeal disease.

Purpose: Triple-negative breast cancer (TNBC) is an aggressive subtype that often metastasizes to the brain. Leptomeningeal disease (LMD), a devastating brain metastasis common in TNBC, has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. Methods: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, LMD was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. Results: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced tumor growth and extended survival in the LMD model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. Conclusions: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC LMD. Our findings are concordant with previous efforts involving MBZ and central nervous system pathology and further support the drug's potential utility as an alternative therapeutic for TNBC LMD.

Quantification of cell energetics in human subcutaneous adipose progenitor cells after target gene knockdown.

Pro-preadipocytes are adipocyte progenitor cells within subcutaneous adipose tissue that are conserved in human adipose tissue with distinct cellular energetics. Here, we detail a protocol to quantify cellular oxygen consumption rates of primary human cells harvested from adipose tissue. We describe steps for primary cell expansion, cell seeding, transfection, differentiation, and respirometry followed by Agilent Seahorse Analytics. The measurement of bioenergetic profiles and resulting data further expand our knowledge of the functional properties of primary cells isolated from adipose tissue. For complete details on the use and execution of this protocol, please refer to Chen et al. (2023).1.

Identification of an adipose tissue-resident pro-preadipocyte population.

Elucidating the transitional stages that define the pathway stem cells progress through during differentiation advances our understanding of biology and fosters the identification of therapeutic opportunities. However, distinguishing progenitor cells from other cell types and placing them in an epistatic pathway is challenging. This is exemplified in the adipocyte lineage, where the stromal vascular fraction (SVF) from adipose tissue is enriched for progenitor cells but also contains heterogeneous populations of cells. Single-cell RNA sequencing (scRNA-seq) has begun to facilitate the deconvolution of cell types in the SVF, and a hierarchical structure is emerging. Here, we use scRNA-seq to discover a population of CD31- CD45- cells in the SVF that are distinguished by a specific expression profile. Further, we place this population on an epistatic pathway upstream of the previously defined preadipocyte population. Finally, we discover functional properties of this population with broad implications, including revealing physiological mechanisms that regulate adipogenesis.

Circadian rhythm gene period 3 is an inhibitor of the adipocyte cell fate.

Glucocorticoids rapidly and robustly induce cell fate decisions in various multipotent cells, although the precise mechanisms of these important cellular events are not understood. Here we showed that glucocorticoids repressed Per3 expression and that this repression was critical for advancing mesenchymal stem cells to the adipocyte fate. Exogenous expression of Per3 inhibited adipogenesis, whereas knocking out Per3 enhanced that fate. Moreover, we found that PER3 formed a complex with PPARγ and inhibited PPARγ-mediated transcriptional activation via Pparγ response elements. Consistent with these findings, Per3 knock-out mice displayed alterations in body composition, with both increased adipose and decreased muscle tissue compared with wild-type mice. Our findings identify Per3 as potent mediator of cell fate that functions by altering the transcriptional activity of PPARγ.

Glucocorticoid regulation of the circadian clock modulates glucose homeostasis.

Circadian clock genes are regulated by glucocorticoids; however, whether this regulation is a direct or secondary effect and the physiological consequences of this regulation were unknown. Here, we identified glucocorticoid response elements (GREs) at multiple clock genes and showed that 3 were directly regulated by the glucocorticoid receptor. We determined that a GRE within the core clock gene Per2 was continuously occupied during rhythmic expression and essential for glucocorticoid regulation of that gene in vivo. We further demonstrated that mice with a genomic deletion spanning this GRE expressed elevated leptin levels and were protected from glucose intolerance and insulin resistance on glucocorticoid treatment but not from muscle wasting. We conclude that Per2 is an integral component of a particular glucocorticoid regulatory pathway and that glucocorticoid regulation of the peripheral clock is selectively required for some actions of glucocorticoids.

Conservation analysis predicts in vivo occupancy of glucocorticoid receptor-binding sequences at glucocorticoid-induced genes.

The glucocorticoid receptor (GR) interacts with specific GR-binding sequences (GBSs) at glucocorticoid response elements (GREs) to orchestrate transcriptional networks. Although the sequences of the GBSs are highly variable among different GREs, the precise sequence within an individual GRE is highly conserved. In this study, we examined whether sequence conservation of sites resembling GBSs is sufficient to predict GR occupancy of GREs at genes responsive to glucocorticoids. Indeed, we found that the level of conservation of these sites at genes up-regulated by glucocorticoids in mouse C3H10T1/2 mesenchymal stem-like cells correlated directly with the extent of occupancy by GR. In striking contrast, we failed to observe GR occupancy of GBSs at genes repressed by glucocorticoids, despite the occurrence of these sites at a frequency similar to that of the induced genes. Thus, GR occupancy of the GBS motif correlates with induction but not repression, and GBS conservation alone is sufficient to predict GR occupancy and GRE function at induced genes.

MYH9 facilitates autoregulation of adipose tissue depot development.

White adipose tissue not only serves as a reservoir for energy storage but also secretes a variety of hormonal signals and modulates systemic metabolism. A substantial amount of adipose tissue develops in early postnatal life, providing exceptional access to the formation of this important tissue. Although a number of factors have been identified that can modulate the differentiation of progenitor cells into mature adipocytes in cell-autonomous assays, it remains unclear which are connected to physiological extracellular inputs and are most relevant to tissue formation in vivo. Here, we elucidate that mature adipocytes themselves signal to adipose depot-resident progenitor cells to direct depot formation in early postnatal life and gate adipogenesis when the tissue matures. Our studies revealed that as the adipose depot matures, a signal generated in mature adipocytes is produced, converges on progenitor cells to regulate the cytoskeletal protein MYH9, and attenuates the rate of adipogenesis in vivo.

Agonist-independent interactions between beta-arrestins and mutant vasopressin type II receptors associated with nephrogenic syndrome of inappropriate antidiuresis.

Nephrogenic syndrome of inappropriate antidiuresis is a recently identified genetic disease first described in two unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. It was found that each infant had a different mutation of the vasopressin type II receptor (V2R) at codon 137 where arginine was converted to cysteine or leucine (R137C or R137L), resulting in constitutive signaling. Interestingly, a missense mutation at the same codon, converting arginine to histidine (R137H), leads to the opposite disease phenotype with a loss of the kidney's ability to concentrate urine resulting in nephrogenic diabetes insipidus. This mutation is associated with impaired signaling, although whether this is predominantly due to impaired trafficking to the plasma membrane, agonist-independent internalization, or G protein uncoupling is currently unclear. Using bioluminescence resonance energy transfer and confocal microscopy, we demonstrate that both V2R-R137C and V2R-R137L mutants interact with beta-arrestins in an agonist-independent manner resulting in dynamin-dependent internalization. This phenotype is similar to that observed for V2R-R137H, which is intriguing considering that it is accompanied by constitutive rather than impaired signaling. Consequently, it would seem that agonist-independent internalization per se is unlikely to be the major determinant of impaired V2R-R137H signaling. Our findings indicate that the V2R-R137C and V2R-R137L mutants traffic considerably more efficiently to the plasma membrane than V2R-R137H, identifying this as a potentially important mutation-dependent difference affecting V2R function.

A Novel Radioligand Reveals Tissue Specific Pharmacological Modulation of Glucocorticoid Receptor Expression with Positron Emission Tomography.

The complexity of glucocorticoid receptor (GR) signaling cannot be measured with direct tissue analysis in living subjects, which has stifled our understanding of GR's role in human physiology or disease and impeded the development of selective GR modulators. Herein, we report 18F-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]quinoline (18F-YJH08), a radioligand that enables noninvasive measurements of tissue autonomous GR expression levels in vivo with positron emission tomography (PET). YJH08 potently binds GR (Ki ∼ 0.4 nM) with ∼100-fold selectivity compared to nuclear hormone receptors in the same subfamily. 18F-YJH08 was prepared via Cu(OTf)2(py)4-mediated radiofluorination of an arylboronic acid pinacol ester with ∼12% decay corrected radiochemical yield from the starting 18F-fluoride ion. We applied treatment with the tissue-wide GR agonist dexamethasone and adrenalectomy and generated an adipocyte specific GR knockout mouse to show that 18F-YJH08 specifically binds GR in normal mouse tissues, including those for which aberrant GR expression is thought to drive severe diseases (e.g., brain, adipose tissue, kidneys). Remarkably, 18F-YJH08 PET also revealed that JG231, a potent and bioavailable HSP70 inhibitor, selectively degrades GR only in the adipose tissue of mice, a finding that foreshadows how GR targeted PET might be integrated into drug discovery to screen for selective GR modulation at the tissue level, beyond the historical screening that was performed at the transcriptional level. In summary, 18F-YJH08 enables a quantitative assessment of GR expression levels in real time among multiple tissues simultaneously, and this technology is a first step toward unraveling the daunting complexity of GR signaling and rationally engineering tissue specific therapeutic modulators in vivo.

Glucocorticoids influence on mesenchymal stem cells and implications for metabolic disease.

Metabolic disease is a well established major public health problem in the adult population. However, the origins of metabolic disease of adults can begin early in life. In addition, in recent years, there has been a disturbing increase in the number of children developing the full presentation of metabolic disease as a result of the increase in obesity in this population. Therefore, pediatricians and pediatric physician-scientists are essential both for instituting preventive measures and developing new therapies. This challenge has been met with a substantial increase in research into both the clinical and basic science of metabolism. A connection between glucocorticoids and the origins of metabolic disease is one enticing clue because of the clinical similarity between patients with glucocorticoid excess and those with metabolic disease. This perspective highlights one series of investigations that has advanced our understanding of the development of metabolic disease. In this work, a unifying link was found by investigating the role of glucocorticoids on cell fate and differentiation of mesenchymal stem cells. We conclude that elucidating the mechanisms by which glucocorticoids modulate cell fate decisions holds promise for developing new therapies and preventative measures.

A novel platform for isotype-specific testing of autoantibodies.

The objective of this study was to test if a novel platform could be used for isotype-specific autoantibody testing in humans. Further, we evaluated if testing with this novel platform enables earlier detection of insulin autoantibodies in individuals that have first-degree relatives with type-1 diabetes than currently used approaches. Longitudinal serum samples from participants were collected before and after they converted to become positive for insulin autoantibodies by the current standardly used assays. Using a novel plasmonic gold chip platform, we tested these samples for IgM isotype-specific autoantibodies. Serial serum samples from individuals without diabetes were also tested as a comparison control cohort. Our results demonstrate proof-of-concept that a plasmonic gold chip can specifically detect the IgM insulin autoantibody. Five out of the six individuals that converted to being positive for insulin autoantibodies by standard testing had significant IgM autoantibodies on the plasmonic chip platform. The plasmonic chip platform detected IgM autoantibodies earlier than standard testing by up to 4 years. Our results indicate that the plasmonic gold platform can specifically detect the IgM isotype autoantibodies and suggest that combining isotype-specific testing with currently used approaches enables earlier detection of insulin autoantibodies in individuals that have first-degree relatives with type 1 diabetes.

Nephrogenic syndrome of inappropriate antidiuresis.

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We describe two infants whose clinical and laboratory evaluations were consistent with the presence of SIADH, yet who had undetectable arginine vasopressin (AVP) levels. We hypothesized that they had gain-of-function mutations in the V2 vasopressin receptor (V2R). DNA sequencing of each patient's V2R gene (AVPR2) identified missense mutations in both, with resultant changes in codon 137 from arginine to cysteine or leucine. These novel mutations cause constitutive activation of the receptor and are the likely cause of the patients' SIADH-like clinical picture, which we have termed "nephrogenic syndrome of inappropriate antidiuresis."

A context-specific circadian clock in adipocyte precursor cells modulates adipogenesis.

The circadian clock is an intricate molecular network that paces a variety of physiological process to ~ 24 hour day/night cycles. Whereas the central circadian clock in the brain is primarily entrained by light signals, peripheral circadian clocks, which are in most cells in the body, receive cues not only from the central pacemaker but also endocrine and other systemic and tissue-specific signals. Prior studies have connected peripheral circadian clocks to metabolism, primarily with studies focused on the robust clock in the liver that responds to feeding/fasting cycles. Adipose tissue is also critical for metabolism and adipocytes have circadian clocks. Yet, the role of the circadian clock in adipocytes is poorly understood. Here we describe our studies that revealed components of the circadian clock in primary adipocyte precursor cells (APCs) in mice. We made the surprising discovery of a particularly prominent role for the circadian gene Period 3 (Per3) in the APC clock. Furthermore, we elucidated that Per3 directly regulates an output pathway of the APC clock to modulate the expression of the Kruppel-like factor 15 (Klf15) gene. Finally, we discovered that this clock-Klf15 pathway regulates adipogenesis in APCs. These finding have important implications for our understanding of adipose tissue biology and metabolism and, we speculate, will generate opportunities to develop novel therapeutic strategies based on the context-specific features of the circadian clock in APCs.

Identification of tumor-autonomous and indirect effects of vitamin D action that inhibit breast cancer growth and tumor progression.

Several epidemiological studies have found that low vitamin D levels are associated with worse prognosis and poorer outcomes in patients with breast cancer (BCa), although some studies have failed to find this association. In addition, prior research has found that BCa patients with vitamin D deficiency have a more aggressive molecular phenotype and worse prognostic biomarkers. As vitamin D deficiency is common in patients diagnosed with BCa, elucidating the cause of the association between poor outcomes and vitamin D deficiency promises to have a significant impact on improving care for patients with BCa including enabling the development of novel therapeutic approaches. Here we review our recent findings in this area, including our data revealing that reduction of the expression of the vitamin D receptor (Vdr) within BCa cells accelerates primary tumor growth and enables the development of metastases, demonstrating a tumor autonomous effect of vitamin D signaling to suppress BCa metastases. We believe that these findings are likely relevant to humans as we discovered evidence that a mechanism of VDR regulation identified in our mouse models is conserved in human BCa. In particular, we identified a negative correlation between serum 25(OH)D concentration and the level of expression of the tumor progression factor ID1 in primary tumors from patients with breast cancer.

Report of a hürthle cell neoplasm in a peripubertal girl.

Thyroid nodules are rare in children compared to adults. Although most thyroid nodules are benign, the risk of malignancy is greater in pediatric patients. Papillary and follicular cell tumors account for the majority of thyroid neoplasms; Hürthle cell tumors account for less than 5%. Despite being uncommon, malignant Hürthle cell tumors are potentially more aggressive than papillary and follicular cell tumors. Therefore, distinguishing between types of thyroid neoplasms in a timely fashion has implications for prognosis and therapy. We describe a 12-year-old peripubertal girl who presented with a large right-sided thyroid nodule that was subsequently diagnosed as a Hürthle cell adenoma. To our knowledge, she represents the youngest patient with a Hürthle cell neoplasm.

Cardiac troponin increases among runners in the Boston Marathon.

STUDY OBJECTIVE: Studies indicate that running a marathon can be associated with increases in serum cardiac troponin levels. The clinical significance of such increases remains unclear. We seek to determine the prevalence of troponin increases and epidemiologic factors associated with these increases in a large and heterogeneous cohort of marathon finishers. METHODS: Entrants in the 2002 Boston Marathon were recruited 1 to 2 days before the race. Data collected included demographic and training history, symptoms experienced during the run, and postrace troponin T and I levels. Simple descriptive statistics were performed to describe the prevalence of troponin increases and runner characteristics. RESULTS: Of 766 runners enrolled, 482 had blood analyzed at the finish line. In all, 34% were women, 20% were younger than 30 years, and 92% had run at least 1 previous marathon. Most runners (68%) had some degree of postrace troponin increase (troponin T > or = 0.01 ng/mL or troponin I > or = 0.1 ng/mL), and 55 (11%) had significant increases (troponin T > or = 0.075 ng/mL or troponin I > or = 0.5 ng/mL). Running inexperience (< 5 previous marathons) and young age (< 30 years) were associated with elevated troponins. These correlates were robust throughout a wide range of troponin thresholds considered. Health factors, family history, training, race performance, and symptoms were not associated with increases. CONCLUSION: Troponin increases were relatively common among marathon finishers and can reach levels typically diagnostic for acute myocardial infarction. Less marathon experience and younger age appeared to be associated with troponin increases, whereas race duration and the presence of traditional cardiovascular risk factors were not. Further work is needed to determine the clinical significance of these findings.