Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. ...However, the underlying mechanism by which MYC promotes FAO remains poorly understood.
We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC.
We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients.
We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.
•The Hepatitis B virus HBx protein elevates cytosolic Ca2+ levels in primary human hepatocytes.•HBx-mediated elevation of cytosolic Ca2+ is controlled by store-operated Ca2+ entry ...mechanisms.•HBx-mediated elevation of cytosolic Ca2+ requires functional mitochondria.•HBx elevates mitochondrial Ca2+ levels.•HBx elevation of mitochondrial Ca2+ is linked to elevation of cytosolic Ca2+.
Worldwide, approximately 240 million people are chronically infected with the hepatitis B virus (HBV); chronic HBV infection is associated with the development of life-threatening liver diseases. The HBV HBx protein alters hepatocyte physiology to promote HBV replication. We previously reported that HBx modulates calcium signaling to stimulate HBV replication in human hepatoblastoma HepG2 cells and primary rat hepatocytes. Whether HBx modulates calcium signaling in a primary human hepatocyte, the natural site of an HBV infection, has not been determined. Here, we report the effect of HBx on calcium signaling in primary human hepatocytes and show that HBx modulates calcium signaling via enhanced calcium entry through store-operated calcium channels and elevated mitochondrial calcium, similar to HBx effects in HepG2 cells and primary rat hepatocytes. In addition to demonstrating that HBV and HBx affect calcium signaling in human hepatocytes, these studies also show that HBV and HBx regulation of calcium signaling is identical in primary human and rat hepatocytes, further validating the use of cultured primary rat hepatocytes for HBV studies.
Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism ...underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.
Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is ...associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the β-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.
During the first phase of the COVID‐19 pandemic, Istat performed the quick survey “Situation and perspectives of Italian enterprises during the COVID‐19 health emergency,” with the aim of assessing ...the economic situation and the specific actions adopted by businesses to reduce the economic impacts of the emergency. To ensure the continuity in the information flow and to analyze the temporal evolution of the observed phenomena, the survey has been repeated in three different waves. The outcomes of each wave was released just after 2 months from the launch of the survey. The present work analyses the characteristics of the sampling strategy and describes the complexity of the data editing process, in the case of a survey planned to produce estimates able to ensure an acceptable level of accuracy in the maximum timeliness.
As the leading risk factor for the development of liver cancer, chronic infection with hepatitis B virus (HBV) represents a significant global health concern. Although an effective HBV vaccine ...exists, at least 240 million people are chronically infected with HBV worldwide. Therapeutic options for the treatment of chronic HBV remain limited, and none achieve an absolute cure. To develop novel therapeutic targets, a better understanding of the complex network of virus-host interactions is needed. Because of the central metabolic role of the liver, we assessed the metabolic impact of HBV infection as a means to identify viral dependency factors and metabolic pathways that could serve as novel points of therapeutic intervention.
Primary rat hepatocytes were infected with a control adenovirus, an adenovirus expressing a greater-than-unit-length copy of the HBV genome, or an adenovirus expressing the HBV X protein (HBx). A panel of 369 metabolites was analyzed for HBV- or HBx-induced changes 24 and 48 h post infection. Pathway analysis was used to identify key metabolic pathways altered in the presence of HBV or HBx expression, and these findings were further supported through integration of publically available gene expression data.
We observed distinct changes to multiple metabolites in the context of HBV replication or HBx expression. Interestingly, a panel of 7 metabolites (maltotriose, maltose, myristate 14:0, arachidate 20:0, 3-hydroxybutyrate BHBA, myo-inositol, and 2-palmitoylglycerol 16,0) were altered by both HBV and HBx at both time points. In addition, incorporation of data from a transcriptome-based dataset allowed us to identify metabolic pathways, including long chain fatty acid metabolism, glycolysis, and glycogen metabolism, that were significantly altered by HBV and HBx.
Because the liver is a central regulator of metabolic processes, it is important to understand how HBV replication and HBV protein expression affects the metabolic function of hepatocytes. Through analysis of a broad panel of metabolites we investigated this metabolic impact. The results of these studies have defined metabolic consequences of an HBV infection of hepatocytes and will help to lay the groundwork for novel research directions and, potentially, development of novel anti-HBV therapeutics.
•First global analysis of HBV-mediated changes in the primary hepatocyte metabolome•First global analysis of HBx-mediated changes in the primary hepatocyte metabolome•HBV and HBx alter primary hepatocyte lipid, long-chain fatty acid metabolism.•HBV and HBx alter primary hepatocyte glycolysis.•HBV and HBx alter primary hepatocyte glycogen metabolism.
HMG-CoA reductase inhibitors (statins) are commonly prescribed for lipid lowering to treat hypercholesterolemia. Although they are well tolerated, their pharmacokinetic interactions with other drugs ...can lead to some adverse clinical consequences. The avenue of interaction has been asserted to be CYP3A4 because most (or all) known interactions are with CYP3A4 inhibitors, and statin oxidative metabolism is mediated by CYP3A4 as well as other CYP enzymes. However, these same drugs that exert a clinical pharmacokinetic effect on statin disposition are generally also P-gp substrates/inhibitors; hence, this transporter may be, or may contribute to, the mechanism of interaction.
This study shows directly, as well as quantifies, the inhibition of P-gp-mediated transport of a fluorescent marker substrate.
Lovastatin and simvastatin are very potent and effective inhibitors of P-gp transport with IC50's of 26 and 9 microM, respectively, for the human enzyme. Atorvastatin is also an effective P-gp inhibitor, but at higher concentrations. Uniquely, pravastatin, whose functional groups render it an inferior inhibitor of P-gp in the whole cell, had no effect in this assay. This result is consistent with known clinical interactions. The effect of these statins on ATP consumption by P-gp was also assessed, and the Km results were congruent with the IC50 observations.
Therefore, the clinical interactions of statins with other drugs may be due, in part or all, to inhibition of P-gp transport.
Acetyl-CoA is a vitally important and versatile metabolite used for many cellular processes including fatty acid synthesis, ATP production, and protein acetylation. Recent studies have shown that ...cancer cells upregulate acetyl-CoA synthetase 2 (ACSS2), an enzyme that converts acetate to acetyl-CoA, in response to stresses such as low nutrient availability and hypoxia. Stressed cancer cells use ACSS2 as a means to exploit acetate as an alternative nutrient source. Genetic depletion of ACSS2 in tumors inhibits the growth of a wide variety of cancers. However, there are no studies on the use of an ACSS2 inhibitor to block tumor growth. In this study, we synthesized a small-molecule inhibitor that acts as a transition-state mimetic to block ACSS2 activity
and
. Pharmacologic inhibition of ACSS2 as a single agent impaired breast tumor growth. Collectively, our findings suggest that targeting ACSS2 may be an effective therapeutic approach for the treatment of patients with breast cancer. SIGNIFICANCE: These findings suggest that targeting acetate metabolism through ACSS2 inhibitors has the potential to safely and effectively treat a wide range of patients with cancer.
Chronic infection with hepatitis B virus (HBV) remains a major worldwide health concern and is the leading cause of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is the only regulatory ...protein encoded in the HBV genome; HBx stimulates HBV replication in vivo and in vitro. HBx also regulates cytosolic Ca
signaling, and altered Ca
signaling is associated with the development of many diseases, including HCC. Importantly, many HBx functions, including HBx modulation of cell proliferation, apoptosis, and transcription pathways, have been linked to changes in cytosolic Ca
signaling. Additionally, several stages of HBV replication, including capsid formation and activation of the HBV polymerase, are dependent on intracellular Ca
. Consequently, defining the molecular mechanism that underlies HBV and HBx modulation of cytosolic Ca
levels is important for understanding HBV pathogenesis and the role of HBx in HBV replication. Here, we describe a single-cell Ca
-imaging protocol that we use to investigate HBV and HBx effects on the level of cytosolic Ca
. We specifically outline two methods that we use to evaluate HBV and HBx regulation of cytosolic Ca
levels in cultured primary hepatocytes. This protocol can also be adapted for use in liver cell lines.
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