MYC is a regulator of androgen receptor inhibition-induced metabolic requirements in prostate cancer.

Advanced prostate cancers are treated with therapies targeting the androgen receptor (AR) signaling pathway. While many tumors initially respond to AR inhibition, nearly all develop resistance. It is critical to understand how prostate tumor cells respond to AR inhibition in order to exploit therapy-induced phenotypes prior to the outgrowth of treatment-resistant disease. Here, we comprehensively characterize the effects of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. The metabolic response to AR inhibition is defined by reduced glycolysis, robust elongation of mitochondria, and increased reliance on mitochondrial oxidative metabolism. We establish DRP1 activity and MYC signaling as mediators of AR-blockade-induced metabolic phenotypes. Rescuing DRP1 phosphorylation after AR inhibition restores mitochondrial fission, while rescuing MYC restores glycolytic activity and prevents sensitivity to complex I inhibition. Our study provides insight into the regulation of treatment-induced metabolic phenotypes and vulnerabilities in prostate cancer.

Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease.

Alveolar epithelial type 2 cell (AEC2) dysfunction is implicated in the pathogenesis of adult and pediatric interstitial lung disease (ILD), including idiopathic pulmonary fibrosis (IPF); however, identification of disease-initiating mechanisms has been impeded by inability to access primary AEC2s early on. Here, we present a human in vitro model permitting investigation of epithelial-intrinsic events culminating in AEC2 dysfunction, using patient-specific induced pluripotent stem cells (iPSCs) carrying an AEC2-exclusive disease-associated variant (SFTPCI73T). Comparing syngeneic mutant versus gene-corrected iPSCs after differentiation into AEC2s (iAEC2s), we find that mutant iAEC2s accumulate large amounts of misprocessed and mistrafficked pro-SFTPC protein, similar to in vivo changes, resulting in diminished AEC2 progenitor capacity, perturbed proteostasis, altered bioenergetic programs, time-dependent metabolic reprogramming, and nuclear factor κB (NF-κB) pathway activation. Treatment of SFTPCI73T-expressing iAEC2s with hydroxychloroquine, a medication used in pediatric ILD, aggravates the observed perturbations. Thus, iAEC2s provide a patient-specific preclinical platform for modeling the epithelial-intrinsic dysfunction at ILD inception.

The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells.

An oral antiviral against SARS-CoV-2 that also attenuates inflammatory instigators of severe COVID-19 is not available to date. Herein, we show that the apoA-I mimetic peptide 4 F inhibits Spike mediated viral entry and has antiviral activity against SARS-CoV-2 in human lung epithelial Calu3 and Vero-E6 cells. In SARS-CoV-2 infected Calu3 cells, 4 F upregulated inducers of the interferon pathway such as MX-1 and Heme oxygenase 1 (HO-1) and downregulated mitochondrial reactive oxygen species (mito-ROS) and CD147, a host protein that mediates viral entry. 4 F also reduced associated cellular apoptosis and secretion of IL-6 in both SARS-CoV-2 infected Vero-E6 and Calu3 cells. Thus, 4 F attenuates in vitro SARS-CoV-2 replication, associated apoptosis in epithelial cells and secretion of IL-6, a major cytokine related to COVID-19 morbidity. Given established safety of 4 F in humans, clinical studies are warranted to establish 4 F as therapy for COVID-19.

Recruitment and remodeling of peridroplet mitochondria in human adipose tissue.

Beige adipocyte mitochondria contribute to thermogenesis by uncoupling and by ATP-consuming futile cycles. Since uncoupling may inhibit ATP synthesis, it is expected that expenditure through ATP synthesis is segregated to a disparate population of mitochondria. Recent studies in mouse brown adipocytes identified peridroplet mitochondria (PDM) as having greater ATP synthesis and pyruvate oxidation capacities, while cytoplasmic mitochondria have increased fatty acid oxidation and uncoupling capacities. However, the occurrence of PDM in humans and the processes that result in their expansion have not been elucidated. Here, we describe a novel high-throughput assay to quantify PDM that is successfully applied to white adipose tissue from mice and humans. Using this approach, we found that PDM content varies between white and brown fat in both species. We used adipose tissue from pheochromocytoma (Pheo) patients as a model of white adipose tissue browning, which is characterized by an increase in the capacity for energy expenditure. In contrast with control subjects, PDM content was robustly increased in the periadrenal fat of Pheo patients. Remarkably, bioenergetic changes associated with browning were primarily localized to PDM compared to cytoplasmic mitochondria (CM). PDM isolated from periadrenal fat of Pheo patients had increased ATP-linked respiration, Complex IV content and activity, and maximal respiratory capacity. We found similar changes in a mouse model of re-browning where PDM content in whitened brown adipose tissue was increased upon re-browning induced by decreased housing temperature. Taken together, this study demonstrates the existence of PDM as a separate functional entity in humans and that browning in both mice and humans is associated with a robust expansion of peri-droplet mitochondria characterized by increased ATP synthesis linked respiration.

Fgr kinase is required for proinflammatory macrophage activation during diet-induced obesity.

Proinflammatory macrophages are key in the development of obesity. In addition, reactive oxygen species (ROS), which activate the Fgr tyrosine kinase, also contribute to obesity. Here we show that ablation of Fgr impairs proinflammatory macrophage polarization while preventing high-fat diet (HFD)-induced obesity in mice. Systemic ablation of Fgr increases lipolysis and liver fatty acid oxidation, thereby avoiding steatosis. Knockout of Fgr in bone marrow (BM)-derived cells is sufficient to protect against insulin resistance and liver steatosis following HFD feeding, while the transfer of Fgr-expressing BM-derived cells reverts protection from HFD feeding in Fgr-deficient hosts. Scavenging of mitochondrial peroxides is sufficient to prevent Fgr activation in BM-derived cells and HFD-induced obesity. Moreover, Fgr expression is higher in proinflammatory macrophages and correlates with obesity traits in both mice and humans. Thus, our findings reveal the mitochondrial ROS-Fgr kinase as a key regulatory axis in proinflammatory adipose tissue macrophage activation, diet-induced obesity, insulin resistance and liver steatosis.

Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation.

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2-/- mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.

Mitochondria Bound to Lipid Droplets Have Unique Bioenergetics, Composition, and Dynamics that Support Lipid Droplet Expansion.

Mitochondria associate with lipid droplets (LDs) in fat-oxidizing tissues, but the functional role of these peridroplet mitochondria (PDM) is unknown. Microscopic observation of interscapular brown adipose tissue reveals that PDM have unique protein composition and cristae structure and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities; (2) PDM have reduced ß-oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and ß-oxidation; (3) PDM support LD expansion as Perilipin5-induced recruitment of mitochondria to LDs increases ATP synthase-dependent triacylglyceride synthesis; and (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis.

Cell cycle-related metabolism and mitochondrial dynamics in a replication-competent pancreatic beta-cell line.

Cell replication is a fundamental attribute of growth and repair in multicellular organisms. Pancreatic beta-cells in adults rarely enter cell cycle, hindering the capacity for regeneration in diabetes. Efforts to drive beta-cells into cell cycle have so far largely focused on regulatory molecules such as cyclins and cyclin-dependent kinases (CDKs). Investigations in cancer biology have uncovered that adaptive changes in metabolism, the mitochondrial network, and cellular Ca2+ are critical for permitting cells to progress through the cell cycle. Here, we investigated these parameters in the replication-competent beta-cell line INS 832/13. Cell cycle synchronization of this line permitted evaluation of cell metabolism, mitochondrial network, and cellular Ca2+ compartmentalization at key cell cycle stages. The mitochondrial network is interconnected and filamentous at G1/S but fragments during the S and G2/M phases, presumably to permit sorting to daughter cells. Pyruvate anaplerosis peaks at G1/S, consistent with generation of biomass for daughter cells, whereas mitochondrial Ca2+ and respiration increase during S and G2/M, consistent with increased energy requirements for DNA and lipid synthesis. This synchronization approach may be of value to investigators performing live cell imaging of Ca2+ or mitochondrial dynamics commonly undertaken in INS cell lines because without synchrony widely disparate data from cell to cell would be expected depending on position within cell cycle. Our findings also offer insight into why replicating beta-cells are relatively nonfunctional secreting insulin in response to glucose. They also provide guidance on metabolic requirements of beta-cells for the transition through the cell cycle that may complement the efforts currently restricted to manipulating cell cycle to drive beta-cells through cell cycle.

Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner.

Mitochondrial dysfunction is linked to apoptosis, aging, cancer, and a number of neurodegenerative and muscular disorders. The interplay between mitophagy and mitochondrial dynamics has been linked to the removal of dysfunctional mitochondria ensuring mitochondrial quality control. An open question is what role mitochondrial fission plays in the removal of mitochondria after mild and transient oxidative stress; conditions reported to result in moderately elevated reactive oxygen species (ROS) levels comparable to physical activity. Here we show that applying such conditions led to fragmentation of mitochondria and induction of mitophagy in mouse and human cells. These conditions increased ROS levels only slightly and neither triggered cell death nor led to a detectable induction of non-selective autophagy. Starvation led to hyperfusion of mitochondria, to high ROS levels, and to the induction of both non-selective autophagy and to a lesser extent to mitophagy. We conclude that moderate levels of ROS specifically trigger mitophagy but are insufficient to trigger non-selective autophagy. Expression of a dominant-negative variant of the fission factor DRP1 blocked mitophagy induction by mild oxidative stress as well as by starvation. Taken together, we demonstrate that in mammalian cells under mild oxidative stress a DRP1-dependent type of mitophagy is triggered while a concomitant induction of non-selective autophagy was not observed. We propose that these mild oxidative conditions resembling well physiological situations are thus very helpful for studying the molecular pathways governing the selective removal of dysfunctional mitochondria.