A reverse pH gradient is a hallmark of cancer metabolism, manifested by extracellular acidosis and intracellular alkalization. While consequences of extracellular acidosis are known, the roles of intracellular alkalization are incompletely understood. By reconstructing and integrating enzymatic pH-dependent activity profiles into cell-specific genome-scale metabolic models, we develop a computational methodology that explores how intracellular pH (pHi) can modulate metabolism. We show that in silico, alkaline pHi maximizes cancer cell proliferation coupled to increased glycolysis and adaptation to hypoxia (i.e., the Warburg effect), whereas acidic pHi disables these adaptations and compromises tumor cell growth. We then systematically identify metabolic targets (GAPDH and GPI) with predicted amplified anti-cancer effects at acidic pHi, forming a novel therapeutic strategy. Experimental testing of this strategy in breast cancer cells reveals that it is particularly effective against aggressive phenotypes. Hence, this study suggests essential roles of pHi in cancer metabolism and provides a conceptual and computational framework for exploring pHi roles in other biomedical domains.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.

The harsh microenvironment of ductal carcinoma in situ (DCIS) exerts strong evolutionary selection pressures on cancer cells. We hypothesize that the poor metabolic conditions near the ductal center foment the emergence of a Warburg Effect (WE) phenotype, wherein cells rapidly ferment glucose to lactic acid, even in normoxia. To test this hypothesis, we subjected low-glycolytic breast cancer cells to different microenvironmental selection pressures using combinations of hypoxia, acidosis, low glucose, and starvation for many months and isolated single clones for metabolic and transcriptomic profiling. The two harshest conditions selected for constitutively expressed WE phenotypes. RNA sequencing analysis of WE clones identified the transcription factor KLF4 as potential inducer of the WE phenotype. In stained DCIS samples, KLF4 expression was enriched in the area with the harshest microenvironmental conditions. We simulated in vivo DCIS phenotypic evolution using a mathematical model calibrated from the in vitro results. The WE phenotype emerged in the poor metabolic conditions near the necrotic core. We propose that harsh microenvironments within DCIS select for a WE phenotype through constitutive transcriptional reprogramming, thus conferring a survival advantage and facilitating further growth and invasion.

Protein repeats are considered hotspots of protein evolution, associated with acquisition of new functions and novel phenotypic traits, including disease. Paradoxically, however, repeats are often strongly conserved through long spans of evolution. To resolve this conundrum, it is necessary to directly compare paralogous (horizontal) evolution of repeats within proteins with their orthologous (vertical) evolution through speciation. Here we develop a rigorous methodology to identify highly periodic repeats with significant sequence similarity, for which evolutionary rates and selection (dN/dS) can be estimated, and systematically characterize their evolution. We show that horizontal evolution of repeats is markedly accelerated compared with their divergence from orthologues in closely related species. This observation is universal across the diversity of life forms and implies a biphasic evolutionary regime whereby new copies experience rapid functional divergence under combined effects of strongly relaxed purifying selection and positive selection, followed by fixation and conservation of each individual repeat.