Abrogating GPT2 in triple-negative breast cancer inhibits tumor growth and promotes autophagy.

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.

Alterations of mTOR signaling impact metabolic stress resistance in colorectal carcinomas with BRAF and KRAS mutations.

Metabolic reprogramming is as a hallmark of cancer, and several studies have reported that BRAF and KRAS tumors may be accompanied by a deregulation of cellular metabolism. We investigated how BRAFV600E and KRASG12V affect cell metabolism, stress resistance and signaling in colorectal carcinoma cells driven by these mutations. KRASG12V expressing cells are characterized by the induction of glycolysis, accumulation of lactic acid and sensitivity to glycolytic inhibition. Notably mathematical modelling confirmed the critical role of MCT1 designating the survival of KRASG12V cells. Carcinoma cells harboring BRAFV600E remain resistant towards alterations of glucose supply or application of signaling or metabolic inhibitors. Altogether these data demonstrate that an oncogene-specific decoupling of mTOR from AMPK or AKT signaling accounts for alterations of resistance mechanisms and metabolic phenotypes. Indeed the inhibition of mTOR in BRAFV600E cells counteracts the metabolic predisposition and demonstrates mTOR as a potential target in BRAFV600E-driven colorectal carcinomas.

The answer's in the tail: MYC mRNA has a metabolic sensor that supports cancer chemoresistance.

Our recent study shows that the translation of MYC mRNA (mRNA) is controlled by its 3' untranslated region (UTR) in a process that depends on the availability of glutamine and levels of cellular nucleotides. Metabolic feedback protects cells from a lethal glutamine addiction and may compromise metabolic targeting of glutamine metabolism.

The MYC mRNA 3'-UTR couples RNA polymerase II function to glutamine and ribonucleotide levels.

Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.

A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer.

In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

Sortase A mediated site-specific immobilization for identification of protein interactions in affinity purification-mass spectrometry experiments.

Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein-protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C-terminal linker region. As a result of this site-directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC-based pull-down experiments and demonstrate the suitability of the approach.