Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F₁/F_o-ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI (i.e. NADH dehydrogenase) is far less efficient, with dramatic oversynthesis of numerous proteins, particularly in the matrix-exposed N and Q domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol-cytochrome c reductase Rieske iron-sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.

Oncogenic Kras mutations are one of the most common alterations in non‐small cell lung cancer and are associated with poor response to treatment and reduced survival. Driver oncogenes, such as Kras are now appreciated for their ability to promote tumor growth via up‐regulation of anabolic pathways. Therefore, we wanted to identify metabolic vulnerabilities in Kras‐mutant lung cancer. Using the Kras ^LSL‐G12D lung cancer model, we show that mutant Kras drives a lipogenic gene‐expression program. Stable‐isotope analysis reveals that mutant Kras promotes de novo fatty acid synthesis in vitro and in vivo. The importance of fatty acid synthesis in Kras‐induced tumorigenesis was evident by decreased tumor formation in Kras ^LSL‐G12D mice after treatment with a fatty acid synthesis inhibitor. Importantly, with gain and loss of function models of mutant Kras, we demonstrate that mutant Kras potentiates the growth inhibitory effects of several fatty acid synthesis inhibitors. These studies highlight the potential to target mutant Kras tumors by taking advantage of the lipogenic phenotype induced by mutant Kras.—Singh, A., Ruiz, C., Bhalla, K., Haley, J. A., Li, Q. K., Acquaah‐Mensah, G., Montal, E., Sudini, K. R., Skoulidis, F., Wistuba, I. I., Papadimitrakopoulou, V., Heymach, J. V., Boros, L. G., Gabrielson, E., Carretero, J., Wong, K.‐K., Haley, J.D., Biswal, S., Girnun, G.D. De novo lipogenesis represents a therapeutic target in mutant Kras non‐small cell lung cancer. FASEB J. 32, 7018–7027 (2018). www.fasebj.org

Metabolic reprogramming is a key feature of malignant cells. While glucose is one of the primary substrates for malignant cells, cancer cells also display a remarkable metabolic flexibility. Depending on nutrient availability and requirements, cancer cells will utilize alternative fuel sources to maintain the TCA cycle for bioenergetic and biosynthetic requirements. Lactate was typically viewed as a passive byproduct of cancer cells. However, studies now show that lactate is an important substrate for the TCA cycle in breast, lung, and pancreatic cancer. Metabolic analysis of colorectal cancer (CRC) cells was performed using a combination of bioenergetic analysis and 13C stable isotope tracing. We show here that CRC cells use lactate to fuel the TCA cycle and promote growth especially under nutrient-deprived conditions. This was mediated in part by maintaining cellular bioenergetics. Therefore targeting the ability of cancer cells to utilize lactate via the TCA cycle would have a significant therapeutic benefit. Phosphoenolpyruvate carboxykinase (PEPCK) is an important cataplerotic enzyme that promotes TCA cycle activity in CRC cells. Treatment of CRC cells with low micromolar doses of a PEPCK inhibitor (PEPCKi) developed for diabetes decreased cell proliferation and utilization of lactate by the TCA cycle in vitro and in vivo. Mechanistically, we observed that the PEPCKi increased nutrient stress as determined by decreased cellular bioenergetics including decreased respiration, ATP levels, and increased AMPK activation. 13C stable isotope tracing showed that the PEPCKi decreased the incorporation of lactate into the TCA cycle. These studies highlight lactate as an important substrate for CRC and the use of PEPCKi as a therapeutic approach to target lactate utilization in CRC cells.

Background The capacity of cancer cells to undergo epithelial mesenchymal trans-differentiation has been implicated as a factor driving metastasis, through the acquisition of enhanced migratory/invasive cell programs and the engagement of anti-apoptotic mechanisms promoting drug and radiation resistance. Our aim was to define molecular signaling changes associated with mesenchymal trans-differentiation in two KRas mutant NSCLC models. We focused on central transcription and epigenetic regulators predicted to be important for mesenchymal cell survival. Experimental design We have modeled trans-differentiation and cancer stemness in inducible isogenic mutant KRas H358 and A549 non-small cell lung cell backgrounds. We employed large-scale quantitative phospho-proteomic, proteomic, protein-protein interaction, RNA-Seq and network function prediction approaches to dissect the molecular events associated with the establishment and maintenance of the mesenchymal state. Results Gene set enrichment and pathway prediction indicated BMI1, KDM5B, RUNX2, MYC/MAX, NFkB, LEF1, and HIF1 target networks were significantly enriched in the trans-differentiation of H358 and A549 NSCLC models. Physical overlaps between multiple networks implicate NR4A1 as an overlapping control between TCF and NFkB pathways. Enrichment correlations also indicated marked decrease in cell cycling, which occurred early in the EMT process. RNA abundance time course studies also indicated early expression of epigenetic and chromatin regulators, including CITED4, RUNX3, CMBX1 and SIRT4. Conclusions Multiple transcription and epigenetic pathways where altered between epithelial and mesenchymal tumor cell states, notably the polycomb repressive complex-1, HP1g and BAF/Swi-Snf. Network analysis suggests redundancy in the activation and inhibition of pathway regulators, notably factors controlling epithelial cell state.