Low expression of PGC-1ß and other mitochondrial biogenesis modulators in melanoma is associated with growth arrest and the induction of an immunosuppressive gene expression program dependent on MEK and IRF-1.

Mitochondria are specialized metabolic and immune organelles that have important roles in tumor progression, metastasis, and response to chemotherapy and immunotherapy. Mitochondrial biogenesis and functions are under the control of the peroxisome-proliferator activated receptor-gamma (PGC-1) transcriptional coactivators. Recent research unveiled the role of PGC-1α in bolstering mitochondrial oxidative functions and in the suppression of metastasis in melanoma, but the role of PGC-1s in tumor immunology remains elusive. Herein, we show that low PGC-1s expression in human melanoma tumors is associated with increased expression of a repertoire of immunosuppressive (CD73, PD-L2, Galectin-9) and pro-inflammatory (IL-8, TNF, IL-1ß) transcripts, and that experimental depletion of PGC-1ß recapitulates this signature in human melanoma cell lines. The depletion of PGC-1ß reduces the expression of HSPA9, impairs mitochondrial activity, and leads to cell cycle arrest. Using pharmacological and gene silencing approaches, we further show that MEK1/2 and IRF-1 mediate the observed immune transcriptional response. Overall, this research suggests that mitochondrial biogenesis modulators can modulate tumor progression, immune evasion, and response to therapeutics through transcriptional control of immune pathways.

Translational and HIF-1α-Dependent Metabolic Reprogramming Underpin Metabolic Plasticity and Responses to Kinase Inhibitors and Biguanides.

There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.

A salicylic acid derivative extends the lifespan of Caenorhabditis elegans by activating autophagy and the mitochondrial unfolded protein response.

Plant extracts containing salicylates are probably the most ancient remedies to reduce fever and ease aches of all kind. Recently, it has been shown that salicylates activate adenosine monophosphate-activated kinase (AMPK), which is now considered as a promising target to slow down aging and prevent age-related diseases in humans. Beneficial effects of AMPK activation on lifespan have been discovered in the model organism Caenorhabditis elegans (C. elegans). Indeed, salicylic acid and acetylsalicylic acid extend lifespan in worms by activating AMPK and the forkhead transcription factor DAF-16/FOXO. Here, we investigated whether another salicylic acid derivative 5-octanoyl salicylic acid (C8-SA), developed as a controlled skin exfoliating ingredient, had similar properties using C. elegans as a model. We show that C8-SA increases lifespan of C. elegans and that a variety of pathways and genes are required for C8-SA-mediated lifespan extension. C8-SA activates AMPK and inhibits TOR both in nematodes and in primary human keratinocytes. We also show that C8-SA can induce both autophagy and the mitochondrial unfolded protein response (UPRmit ) in nematodes. This induction of both processes is fully required for lifespan extension in the worm. In addition, we found that the activation of autophagy by C8-SA fails to occur in worms with compromised UPRmit , suggesting a mechanistic link between these two processes. Mutants that are defective in the mitochondrial unfolded protein response exhibit constitutive high autophagy levels. Taken together, these data therefore suggest that C8-SA positively impacts longevity in worms through induction of autophagy and the UPRmit .

Alternative polyadenylation confers Pten mRNAs stability and resistance to microRNAs.

Fine regulation of the phosphatase and tensin homologue (PTEN) phosphatase dosage is critical for homeostasis and tumour suppression. The 3'-untranslated region (3'-UTR) of Pten mRNA was extensively linked to post-transcriptional regulation by microRNAs (miRNAs). In spite of this critical regulatory role, alternative 3'-UTRs of Pten have not been systematically characterized. Here, we reveal an important diversity of Pten mRNA isoforms generated by alternative polyadenylation sites. Several 3'-UTRs are co-expressed and their relative expression is dynamically regulated. In spite of encoding multiple validated miRNA-binding sites, longer isoforms are largely refractory to miRNA-mediated silencing, are more stable and contribute to the bulk of PTEN protein and signalling functions. Taken together, our results warrant a mechanistic re-interpretation of the post-transcriptional mechanisms involving Pten mRNAs and raise concerns on how miRNA-binding sites are being validated.

Interplay between ShcA Signaling and PGC-1α Triggers Targetable Metabolic Vulnerabilities in Breast Cancer.

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose. ShcA signaling simultaneously increased the metabolic rate and flexibility of breast cancer cells by inducing the PGC-1α transcriptional coactivator, a central regulator of mitochondrial metabolism. Breast tumors that engaged ShcA signaling were critically dependent on PGC-1α to support their increased metabolic rate. PGC-1α deletion drastically delayed breast tumor onset in an orthotopic mouse model, highlighting a key role for PGC-1α in tumor initiation. Conversely, reduced ShcA signaling impaired both the metabolic rate and flexibility of breast cancer cells, rendering them reliant on mitochondrial oxidative phosphorylation. This metabolic reprogramming exposed a targetable metabolic vulnerability, leading to a sensitization of breast tumors to inhibitors of mitochondrial complex I (biguanides). Genetic inhibition of ShcA signaling in the Polyoma virus middle T (MT) breast cancer mouse model sensitized mammary tumors to biguanides during the earliest stages of breast cancer progression. Tumor initiation and growth were selectively and severely impaired in MT/ShcA-deficient animals. These data demonstrate that metabolic reprogramming is a key component of ShcA signaling and serves an unappreciated yet vital role during breast cancer initiation and progression. These data further unravel a novel interplay between ShcA and PGC-1α in the coordination of metabolic reprogramming and demonstrate the sensitivity of breast tumors to drugs targeting oxidative phosphorylation.Significance: This study uncovers a previously unrecognized mechanism that links aberrant RTK signaling with metabolic perturbations in breast cancer and exposes metabolic vulnerabilities that can be targeted by inhibitors of oxidative phosphorylation. Cancer Res; 78(17); 4826-38. ©2018 AACR.

Deciphering the Dichotomous Effects of PGC-1α on Tumorigenesis and Metastasis.

Metabolic reprogramming confers cancer cells the ability to grow and survive under nutrient-depleted or stressful microenvironments. The amplification of oncogenes, the loss of tumor suppressors, as well as context- and lineage-specific determinants can converge and profoundly affect the metabolic status of cancer cells. Cumulating evidences suggest that highly glycolytic cells under the influence of oncogenes such as BRAF, or evolving in hypoxic microenvironments, will promote metastasis through modulation of multiple steps of tumorigenesis such as the epithelial-to-mesenchymal transition (EMT). On the contrary, increased reliance on mitochondrial respiration is associated with hyperplasic rather than metastatic disease. The PGC-1α transcriptional coactivator, a master regulator of mitochondrial biogenesis, has recently been shown to exert antimetastatic effects in cancer, notably through inhibition of EMT. Besides, PGC-1α has the opposite role in specific cancer subtypes, in which it appears to provide growth advantages. Thus, the regulation and role of PGC-1α in cancer is not univocal, and its use as a prognostic marker appears limited given its highly dynamic nature and its multifaceted regulation by transcriptional and posttranslational mechanisms. Herein, we expose key oncogenic and lineage-specific modules that finely regulate PGC-1α to promote or dampen the metastatic process. We propose a unifying model based on the systematic analysis of its controversial implication in cancer from cell proliferation to EMT and metastasis. This short review will provide a good understanding of current challenges associated with the study of PGC-1α.

RSK Regulates PFK-2 Activity to Promote Metabolic Rewiring in Melanoma.

Metabolic reprogramming is a hallmark of cancer that includes increased glucose uptake and accelerated aerobic glycolysis. This phenotype is required to fulfill anabolic demands associated with aberrant cell proliferation and is often mediated by oncogenic drivers such as activated BRAF. In this study, we show that the MAPK-activated p90 ribosomal S6 kinase (RSK) is necessary to maintain glycolytic metabolism in BRAF-mutated melanoma cells. RSK directly phosphorylated the regulatory domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), an enzyme that catalyzes the synthesis of fructose-2,6-bisphosphate during glycolysis. Inhibition of RSK reduced PFKFB2 activity and glycolytic flux in melanoma cells, suggesting an important role for RSK in BRAF-mediated metabolic rewiring. Consistent with this, expression of a phosphorylation-deficient mutant of PFKFB2 decreased aerobic glycolysis and reduced the growth of melanoma in mice. Together, these results indicate that RSK-mediated phosphorylation of PFKFB2 plays a key role in the metabolism and growth of BRAF-mutated melanomas.Significance: RSK promotes glycolytic metabolism and the growth of BRAF-mutated melanoma by driving phosphorylation of an important glycolytic enzyme. Cancer Res; 78(9); 2191-204. ©2018 AACR.

Metabolomics Analyses of Cancer Cells in Controlled Microenvironments.

The tumor microenvironment is a complex and heterogeneous milieu in which cancer cells undergo metabolic reprogramming to fuel their growth. Cancer cell lines grown in vitro using traditional culture methods represent key experimental models to gain a mechanistic understanding of tumor biology. This protocol describes the use of gas chromatography-mass spectrometry (GC-MS) to assess metabolic changes in cancer cells grown under varied levels of oxygen and nutrients that may better mimic the tumor microenvironment. Intracellular metabolite changes, metabolite uptake and release, as well as stable isotope ((13)C) tracer analyses are done in a single experimental setup to provide an integrated understanding of metabolic adaptation. Overall, this chapter describes some essential tools and methods to perform comprehensive metabolomics analyses.

ERRα mediates metabolic adaptations driving lapatinib resistance in breast cancer.

Despite the initial benefits of treating HER2-amplified breast cancer patients with the tyrosine kinase inhibitor lapatinib, resistance inevitably develops. Here we report that lapatinib induces the degradation of the nuclear receptor ERRα, a master regulator of cellular metabolism, and that the expression of ERRα is restored in lapatinib-resistant breast cancer cells through reactivation of mTOR signalling. Re-expression of ERRα in resistant cells triggers metabolic adaptations favouring mitochondrial energy metabolism through increased glutamine metabolism, as well as ROS detoxification required for cell survival under therapeutic stress conditions. An ERRα inverse agonist counteracts these metabolic adaptations and overcomes lapatinib resistance in a HER2-induced mammary tumour mouse model. This work reveals a molecular mechanism by which ERRα-induced metabolic reprogramming promotes survival of lapatinib-resistant cancer cells and demonstrates the potential of ERRα inhibition as an effective adjuvant therapy in poor outcome HER2-positive breast cancer.

nanoCAGE reveals 5' UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs.

The diversity of MTOR-regulated mRNA translation remains unresolved. Whereas ribosome-profiling suggested that MTOR almost exclusively stimulates translation of the TOP (terminal oligopyrimidine motif) and TOP-like mRNAs, polysome-profiling indicated that MTOR also modulates translation of mRNAs without the 5' TOP motif (non-TOP mRNAs). We demonstrate that in ribosome-profiling studies, detection of MTOR-dependent changes in non-TOP mRNA translation was obscured by low sensitivity and methodology biases. Transcription start site profiling using nano-cap analysis of gene expression (nanoCAGE) revealed that not only do many MTOR-sensitive mRNAs lack the 5' TOP motif but that 5' UTR features distinguish two functionally and translationally distinct subsets of MTOR-sensitive mRNAs: (1) mRNAs with short 5' UTRs enriched for mitochondrial functions, which require EIF4E but are less EIF4A1-sensitive; and (2) long 5' UTR mRNAs encoding proliferation- and survival-promoting proteins, which are both EIF4E- and EIF4A1-sensitive. Selective inhibition of translation of mRNAs harboring long 5' UTRs via EIF4A1 suppression leads to sustained expression of proteins involved in respiration but concomitant loss of those protecting mitochondrial structural integrity, resulting in apoptosis. Conversely, simultaneous suppression of translation of both long and short 5' UTR mRNAs by MTOR inhibitors results in metabolic dormancy and a predominantly cytostatic effect. Thus, 5' UTR features define different modes of MTOR-sensitive translation of functionally distinct subsets of mRNAs, which may explain the diverse impact of MTOR and EIF4A inhibitors on neoplastic cells.

The PGC-1α/ERRα Axis Represses One-Carbon Metabolism and Promotes Sensitivity to Anti-folate Therapy in Breast Cancer.

Reprogramming of cellular metabolism plays a central role in fueling malignant transformation, and AMPK and the PGC-1α/ERRα axis are key regulators of this process. The intersection of gene-expression and binding-event datasets for breast cancer cells shows that activation of AMPK significantly increases the expression of PGC-1α/ERRα and promotes the binding of ERRα to its cognate sites. Unexpectedly, the data also reveal that ERRα, in concert with PGC-1α, negatively regulates the expression of several one-carbon metabolism genes, resulting in substantial perturbations in purine biosynthesis. This PGC-1α/ERRα-mediated repression of one-carbon metabolism promotes the sensitivity of breast cancer cells and tumors to the anti-folate drug methotrexate. These data implicate the PGC-1α/ERRα axis as a core regulatory node of folate cycle metabolism and further suggest that activators of AMPK could be used to modulate this pathway in cancer.

mTOR coordinates protein synthesis, mitochondrial activity and proliferation.

Protein synthesis is one of the most energy consuming processes in the cell. The mammalian/mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that integrates a multitude of extracellular signals and intracellular cues to drive growth and proliferation. mTOR activity is altered in numerous pathological conditions, including metabolic syndrome and cancer. In addition to its well-established role in regulating mRNA translation, emerging studies indicate that mTOR modulates mitochondrial functions. In mammals, mTOR coordinates energy consumption by the mRNA translation machinery and mitochondrial energy production by stimulating synthesis of nucleus-encoded mitochondria-related proteins including TFAM, mitochondrial ribosomal proteins and components of complexes I and V. In this review, we highlight findings that link mTOR, mRNA translation and mitochondrial functions.

Serine deprivation enhances antineoplastic activity of biguanides.

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

Metformin directly acts on mitochondria to alter cellular bioenergetics.

BACKGROUND: Metformin is widely used in the treatment of diabetes, and there is interest in 'repurposing' the drug for cancer prevention or treatment. However, the mechanism underlying the metabolic effects of metformin remains poorly understood. METHODS: We performed respirometry and stable isotope tracer analyses on cells and isolated mitochondria to investigate the impact of metformin on mitochondrial functions. RESULTS: We show that metformin decreases mitochondrial respiration, causing an increase in the fraction of mitochondrial respiration devoted to uncoupling reactions. Thus, cells treated with metformin become energetically inefficient, and display increased aerobic glycolysis and reduced glucose metabolism through the citric acid cycle. Conflicting prior studies proposed mitochondrial complex I or various cytosolic targets for metformin action, but we show that the compound limits respiration and citric acid cycle activity in isolated mitochondria, indicating that at least for these effects, the mitochondrion is the primary target. Finally, we demonstrate that cancer cells exposed to metformin display a greater compensatory increase in aerobic glycolysis than nontransformed cells, highlighting their metabolic vulnerability. Prevention of this compensatory metabolic event in cancer cells significantly impairs survival. CONCLUSIONS: Together, these results demonstrate that metformin directly acts on mitochondria to limit respiration and that the sensitivity of cells to metformin is dependent on their ability to cope with energetic stress.

Stable isotope tracer analysis in isolated mitochondria from mammalian systems.

Mitochondria are a focal point in metabolism, given that they play fundamental roles in catabolic, as well as anabolic reactions. Alterations in mitochondrial functions are often studied in whole cells, and metabolomics experiments using 13C-labeled substrates, coupled with mass isotopomer distribution analyses, represent a powerful approach to study global changes in cellular metabolic activities. However, little is known regarding the assessment of metabolic activities in isolated mitochondria using this technology. Studies on isolated mitochondria permit the evaluation of whether changes in cellular metabolic activities are due to modifications in the intrinsic properties of the mitochondria. Here, we present a streamlined approach to accurately determine 13C, as well as 12C enrichments in isolated mitochondria from mammalian tissues or cultured cells by GC/MS. We demonstrate the relevance of this experimental approach by assessing the effects of drugs perturbing mitochondrial functions on the mass isotopomer enrichment of metabolic intermediates. Furthermore, we investigate 13C and 12C enrichments in mitochondria isolated from cancer cells given the emerging role of metabolic alterations in supporting tumor growth. This original method will provide a very sensitive tool to perform metabolomics studies on isolated mitochondria.

PGC-1α supports glutamine metabolism in breast cancer.

BACKGROUND: Glutamine metabolism is a central metabolic pathway in cancer. Recently, reductive carboxylation of glutamine for lipogenesis has been shown to constitute a key anabolic route in cancer cells. However, little is known regarding central regulators of the various glutamine metabolic pathways in cancer cells. METHODS: The impact of PGC-1α and ERRα on glutamine enzyme expression was assessed in ERBB2+ breast cancer cell lines with quantitative RT-PCR, chromatin immunoprecipitation, and immunoblotting experiments. Glutamine flux was quantified using 13C-labeled glutamine and GC/MS analyses. Functional assays for lipogenesis were performed using 14C-labeled glutamine. The expression of glutamine metabolism genes in breast cancer patients was determined by bioinformatics analyses using The Cancer Genome Atlas. RESULTS: We show that the transcriptional coactivator PGC-1α, along with the transcription factor ERRα, is a positive regulator of the expression of glutamine metabolism genes in ERBB2+ breast cancer. Indeed, ERBB2+ breast cancer cells with increased expression of PGC-1α display elevated expression of glutamine metabolism genes. Furthermore, ERBB2+ breast cancer cells with reduced expression of PGC-1α or when treated with C29, a pharmacological inhibitor of ERRα, exhibit diminished expression of glutamine metabolism genes. The biological relevance of the control of glutamine metabolism genes by the PGC-1α/ERRα axis is demonstrated by consequent regulation of glutamine flux through the citric acid cycle. PGC-1α and ERRα regulate both the canonical citric acid cycle (forward) and the reductive carboxylation (reverse) fluxes; the latter can be used to support de novo lipogenesis reactions, most notably in hypoxic conditions. Importantly, murine and human ERBB2+ cells lines display a significant dependence on glutamine availability for their growth. Finally, we show that PGC-1α expression is positively correlated with that of the glutamine pathway in ERBB2+ breast cancer patients, and high expression of this pathway is associated with reduced patient survival. CONCLUSIONS: These data reveal that the PGC-1α/ERRα axis is a central regulator of glutamine metabolism in ERBB2+ breast cancer. This novel regulatory link, as well as the marked reduction in patient survival time associated with increased glutamine pathway gene expression, suggests that targeting glutamine metabolism may have therapeutic potential in the treatment of ERBB2+ breast cancer.

mTORC1 controls mitochondrial activity and biogenesis through 4E-BP-dependent translational regulation.

mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

The proinflammatory actions of angiotensin II are dependent on p65 phosphorylation by the IkappaB kinase complex.

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-kappaB. We describe here a novel mode of NF-kappaB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKbeta subunit of the IKK complex by overexpression of a dominant negative version of IKKbeta (IKKbeta K44A) or silencing RNA technology demonstrated that the IKKbeta subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IkappaBalpha phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.