Inhibiting Notch Activity in Breast Cancer Stem Cells by Glucose Functionalized Nanoparticles Carrying γ-secretase Inhibitors.

Cancer stem cells (CSCs) are a challenge in cancer treatment due to their therapy resistance. We demonstrated that enhanced Notch signaling in breast cancer promotes self-renewal of CSCs that display high glycolytic activity and aggressive hormone-independent tumor growth in vivo. We took advantage of the glycolytic phenotype and the dependence on Notch activity of the CSCs and designed nanoparticles to target the CSCs. Mesoporous silica nanoparticles were functionalized with glucose moieties and loaded with a γ-secretase inhibitor, a potent interceptor of Notch signaling. Cancer cells and CSCs in vitro and in vivo efficiently internalized these particles, and particle uptake correlated with the glycolytic profile of the cells. Nanoparticle treatment of breast cancer transplants on chick embryo chorioallantoic membranes efficiently reduced the cancer stem cell population of the tumor. Our data reveal that specific CSC characteristics can be utilized in nanoparticle design to improve CSC-targeted drug delivery and therapy.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

Blocking Aerobic Glycolysis by Targeting Pyruvate Dehydrogenase Kinase in Combination with EGFR TKI and Ionizing Radiation Increases Therapeutic Effect in Non-Small Cell Lung Cancer Cells.

Increased glycolytic activity is a hallmark of cancer initiation and progression and is often observed in non-small cell lung cancer (NSCLC). Pyruvate dehydrogenase (PDH) complex acts as a gatekeeper between glycolysis and oxidative phosphorylation, and activation of PDH is known to inhibit glycolytic activity. As part of a standard therapeutic regimen, patients with NSCLC harboring oncogenic mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). Independent of good initial response, development of resistance to this therapy is inevitable. In the presented work, we propose that inhibition of glycolysis will add to the therapeutic effects and possibly prevent development of resistance against both EGFR TKIs and ionizing radiation in NSCLC. Analysis of transcriptome data from two independent NSCLC patient cohorts identified increased expression of pyruvate dehydrogenase kinase 1 (PDHK1) as well as upregulated expression of genes involved in glucose metabolism in tumors compared to normal tissue. We established in vitro models of development of resistance to EGFR TKIs to study metabolism and determine if targeting PDHK would prevent development of resistance to EGFR TKIs in NSCLC cells. The PDHK1 inhibitor dichloroacetate (DCA) in combination with EGFR TKIs and/or ionizing radiation was shown to increase the therapeutic effect in our NSCLC cell models. This mechanism was associated with redirected metabolism towards pyruvate oxidation and reduced lactate production, both in EGFR TKI sensitive and resistant NSCLC cells. Using DCA, the intracellular pool of pyruvate available for lactic fermentation becomes limited. Consequently, pyruvate is redirected to the mitochondria, and reinforces mitochondrial activity. Addition of DCA to cell culture deacidifies the extracellular microenvironment as less lactate is produced and excreted. In our study, we find that this redirection of metabolism adds to the therapeutic effect of EGFR TKI and ionizing radiation in NSCLC.

Epithelial to mesenchymal transition (EMT) is associated with attenuation of succinate dehydrogenase (SDH) in breast cancer through reduced expression of SDHC.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is a well-characterized process of cell plasticity that may involve metabolic rewiring. In cancer, EMT is associated with malignant progression, tumor heterogeneity, and therapy resistance. In this study, we investigated the role of succinate dehydrogenase (SDH) as a potential key regulator of EMT. METHODS: Associations between SDH subunits and EMT were explored in gene expression data from breast cancer patient cohorts, followed by in-depth studies of SDH suppression as a potential mediator of EMT in cultured cells. RESULTS: We found an overall inverse association between EMT and the SDH subunit C (SDHC) when analyzing gene expression in breast tumors. This was particularly evident in carcinomas of basal-like molecular subtype compared to non-basal-like tumors, and a low SDHC expression level tended to have a prognostic impact in those patients. Studies in cultured cells revealed that EMT was induced by SDH inhibition through SDHC CRISPR/Cas9 knockdown or by the enzymatic inhibitor malonate. Conversely, overexpression of EMT-promoting transcription factors TWIST and SNAI2 caused decreased levels of SDHB and C and reduced rates of SDH-linked mitochondrial respiration. Cells overexpressing TWIST had reduced mitochondrial mass, and the organelles were thinner and more fragmented compared to controls. CONCLUSIONS: Our findings suggest that downregulation of SDHC promotes EMT and that this is accompanied by structural remodeling of the mitochondrial organelles. This may confer survival benefits upon exposure to hostile microenvironment including oxidative stress and hypoxia during cancer progression.

Automated quantification and integrative analysis of 2D and 3D mitochondrial shape and network properties.

Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. "mitochondrial dynamics") are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells.

Regulation and quantification of cellular mitochondrial morphology and content.

Mitochondria play a key role in signal transduction, redox homeostasis and cell survival, which extends far beyond their classical functioning in ATP production and energy metabolism. In living cells, mitochondrial content ("mitochondrial mass") depends on the cell-controlled balance between mitochondrial biogenesis and degradation. These processes are intricately linked to changes in net mitochondrial morphology and spatiotemporal positioning ("mitochondrial dynamics"), which are governed by mitochondrial fusion, fission and motility. It is becoming increasingly clear that mitochondrial mass and dynamics, as well as its ultrastructure and volume, are mechanistically linked to mitochondrial function and the cell. This means that proper quantification of mitochondrial morphology and content is of prime importance in understanding mitochondrial and cellular physiology in health and disease. This review first presents how cellular mitochondrial content is regulated at the level of mitochondrial biogenesis, degradation and dynamics. Next we discuss how mitochondrial dynamics and content can be analyzed with a special emphasis on quantitative live-cell microscopy strategies.