Targeting Oncogenic Wnt/ß-Catenin Signaling in Adrenocortical Carcinoma Disrupts ECM Expression and Impairs Tumor Growth.

Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/ß-catenin signaling are frequently observed, the ß-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/ß-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/ß-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/ß-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/ß-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/ß-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/ß-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/ß-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this ß-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/ß-catenin inhibitor. These results show promise for the further clinical development of Wnt/ß-catenin inhibitors in ACC and unveil a novel Wnt/ß-catenin-regulated transcriptome.

Senescence-induced immune remodeling facilitates metastatic adrenal cancer in a sex-dimorphic manner.

Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer.

Phospholipase Cε insufficiency causes ascending aortic aneurysm and dissection.

Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.

Potential role of intermittent functioning of baroreflexes in the etiology of hypertension in spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is a genetic model of primary hypertension with an etiology that includes sympathetic overdrive. To elucidate the neurogenic mechanisms underlying the pathophysiology of this model, we analyzed the dynamic baroreflex response to spontaneous fluctuations in arterial pressure in conscious SHRs, as well as in the Wistar-Kyoto (WKY), the Dahl salt-sensitive, the Dahl salt-resistant, and the Sprague-Dawley rat. Observations revealed the existence of long intermittent periods (lasting up to several minutes) of engagement and disengagement of baroreflex control of heart rate. Analysis of these intermittent periods revealed a predictive relationship between increased mean arterial pressure and progressive baroreflex disengagement that was present in the SHR and WKY strains but absent in others. This relationship yielded the hypothesis that a lower proportion of engagement versus disengagement of the baroreflex in SHR compared with WKY contributes to the hypertension (or increased blood pressure) in SHR compared with WKY. Results of experiments using sinoaortic baroreceptor denervation were consistent with the hypothesis that dysfunction of the baroreflex contributes to the etiology of hypertension in the SHR. Thus, this study provides experimental evidence for the roles of the baroreflex in long-term arterial pressure regulation and in the etiology of primary hypertension in this animal model.

Impaired Myocardial Energetics Causes Mechanical Dysfunction in Decompensated Failing Hearts.

Cardiac mechanical function is supported by ATP hydrolysis, which provides the chemical-free energy to drive the molecular processes underlying cardiac pumping. Physiological rates of myocardial ATP consumption require the heart to resynthesize its entire ATP pool several times per minute. In the failing heart, cardiomyocyte metabolic dysfunction leads to a reduction in the capacity for ATP synthesis and associated free energy to drive cellular processes. Yet it remains unclear if and how metabolic/energetic dysfunction that occurs during heart failure affects mechanical function of the heart. We hypothesize that changes in phosphate metabolite concentrations (ATP, ADP, inorganic phosphate) that are associated with decompensation and failure have direct roles in impeding contractile function of the myocardium in heart failure, contributing to the whole-body phenotype. To test this hypothesis, a transverse aortic constriction (TAC) rat model of pressure overload, hypertrophy, and decompensation was used to assess relationships between metrics of whole-organ pump function and myocardial energetic state. A multiscale computational model of cardiac mechanoenergetic coupling was used to identify and quantify the contribution of metabolic dysfunction to observed mechanical dysfunction. Results show an overall reduction in capacity for oxidative ATP synthesis fueled by either fatty acid or carbohydrate substrates as well as a reduction in total levels of adenine nucleotides and creatine in myocardium from TAC animals compared to sham-operated controls. Changes in phosphate metabolite levels in the TAC rats are correlated with impaired mechanical function, consistent with the overall hypothesis. Furthermore, computational analysis of myocardial metabolism and contractile dynamics predicts that increased levels of inorganic phosphate in TAC compared to control animals kinetically impair the myosin ATPase crossbridge cycle in decompensated hypertrophy/heart failure.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models.

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca2+ channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mdx mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca2+ channels may represent a promising approach to treat DMD and related muscle diseases.

A genetic mouse model of severe iron deficiency anemia reveals tissue-specific transcriptional stress responses and cardiac remodeling.

Iron is a micronutrient fundamental for life. Iron homeostasis in mammals requires sustained postnatal intestinal iron absorption that maintains intracellular iron concentrations for central and systemic metabolism as well as for erythropoiesis and oxygen transport. More than 1 billion people worldwide suffer from iron deficiency anemia (IDA), a state of systemic iron insufficiency that limits the production of red blood cells and leads to tissue hypoxia and intracellular iron stress. Despite this tremendous public health concern, very few genetic models of IDA are available to study its progression. Here we developed and characterized a novel genetic mouse model of IDA. We found that tamoxifen-inducible deletion of the mammalian iron exporter ferroportin exclusively in intestinal epithelial cells leads to loss of intestinal iron absorption. Ferroportin ablation yielded a robust phenotype of progressive IDA that develops in as little as 3 months following disruption of intestinal iron absorption. We noted that, at end-stage IDA, tissue-specific transcriptional stress responses occur in which the heart shows little to no hypoxic and iron stress compared with other peripheral organs. However, morphometric and echocardiographic analysis revealed massive cardiac hypertrophy and chamber dilation, albeit with increased cardiac output at very low basal heart rates. We propose that our intestine-specific ferroportin knockout mouse model of end-stage IDA could be used in future studies to investigate IDA progression and cell-specific responses to hypoxic and iron stress.

Fibro-Adipogenic Remodeling of the Diaphragm in Obesity-Associated Respiratory Dysfunction.

Respiratory dysfunction is a common complication of obesity, conferring cardiovascular morbidity and increased mortality and often necessitating mechanical ventilatory support. While impaired lung expansion in the setting of increased adipose mass and reduced central response to hypercapnia have been implicated as pathophysiological drivers, the impact of obesity on respiratory muscles-in particular, the diaphragm-has not been investigated in detail. Here, we demonstrate that chronic high-fat diet (HFD) feeding impairs diaphragm muscle function, as assessed in vivo by ultrasonography and ex vivo by measurement of contractile force. During an HFD time course, progressive adipose tissue expansion and collagen deposition within the diaphragm parallel contractile deficits. Moreover, intradiaphragmatic fibro-adipogenic progenitors (FAPs) proliferate with long-term HFD feeding while giving rise to adipocytes and type I collagen-depositing fibroblasts. Thrombospondin 1 (THBS1), a circulating adipokine, increases with obesity and induces FAP proliferation. These findings suggest a novel role for FAP-mediated fibro-adipogenic diaphragm remodeling in obesity-associated respiratory dysfunction.

In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P.

Mutations that cause defects in levels of the signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P(2)] lead to profound neurodegeneration in mice. Moreover, mutations in human FIG4 predicted to lower PI(3,5)P(2) levels underlie Charcot-Marie-Tooth type 4J neuropathy and are present in selected cases of amyotrophic lateral sclerosis. In yeast and mammals, PI(3,5)P(2) is generated by a protein complex that includes the lipid kinase Fab1/Pikfyve, the scaffolding protein Vac14, and the lipid phosphatase Fig4. Fibroblasts cultured from Vac14(-/-) and Fig4(-/-) mouse mutants have a 50% reduction in the levels of PI(3,5)P(2), suggesting that there may be PIKfyve-independent pathways that generate this lipid. Here, we characterize a Pikfyve gene-trap mouse (Pikfyve(ß-geo/ß-geo)), a hypomorph with ~10% of the normal level of Pikfyve protein. shRNA silencing of the residual Pikfyve transcript in fibroblasts demonstrated that Pikfyve is required to generate all of the PI(3,5)P(2) pool. Surprisingly, Pikfyve also is responsible for nearly all of the phosphatidylinositol-5-phosphate (PI5P) pool. We show that PI5P is generated directly from PI(3,5)P(2), likely via 3'-phosphatase activity. Analysis of tissues from the Pikfyve(ß-geo/ß-geo) mouse mutants reveals that Pikfyve is critical in neural tissues, heart, lung, kidney, thymus, and spleen. Thus, PI(3,5)P(2) and PI5P have major roles in multiple organs. Understanding the regulation of these lipids may provide insights into therapies for multiple diseases.

Structure-activity studies of RFamide-related peptide-1 identify a functional receptor antagonist and novel cardiac myocyte signaling pathway involved in contractile performance.

Human RFamide-related peptide-1 (hRFRP-1, MPHSFANLPLRF-NH(2)) binds to neuropeptide FF receptor 2 (NPFF(2)R) to dramatically diminish cardiovascular performance. hRFRP-1 and its signaling pathway may provide targets to address cardiac dysfunction. Here, structure-activity relationship, transcript, Ca(2+) transient, and phospholabeling data indicate the presence of a hRFRP-1 pathway in cardiomyocytes. Alanyl-substituted and N-terminal truncated analogues identified that R(11) was essential for activity, hRFRP-1((8-12)) mimicked hRFRP-1, and [A(11)]hRFRP-1((8-12)) antagonized the effect of hRFRP-1 in cellular and integrated cardiac performance. RFRP and NPFF(2)R transcripts were amplified from cardiomyocytes and heart. Maintenance of the Ca(2+) transient when hRFRP-1 impaired myocyte shortening indicated the myofilament was its primary downstream target. Enhanced myofilament protein phosphorylation detected after hRFRP-1 treatment but absent in [A(11)]hRFRP-1((8-12))-treated cells was consistent with this result. Protein kinase C (PKC) but not PKA inhibitor diminished the influence of hRFRP-1 on the Ca(2+) transient. Molecules targeting this pathway may help address cardiovascular disease.