Proton export upregulates aerobic glycolysis

Introduction: Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect.” It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell.

Results: To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden.

Conclusions: Therefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.

https://doi.org/10.1186/s12915-022-01340-0

The Warburg Effect: protons suck

Aerobic glycolysis (the Warburg Effect) is a hallmark of cancer and is associated with local invasion and metastasis. This metabolic phenotype results in acidification of the microenvironment in solid tumors, which is responsible for many of the known sequelae. For example, systemic buffer therapy directly and specifically increases extracellular tumor pH and reduces spontaneous and experimental metastasis in vivo. Further, extracellular acidosis can be a potent inhibitor of anti-tumor immunity. Removal of glycolytically-derived acids requires the activity of proton transporting mechanisms, such as NHE, V-ATPase and CA-IX. While it is commonly believed that these transporters are responding to the demands imposed by increased glycolytic flux, an alternative hypothesis states that glycolytic flux is increased to satisfy the demand driven by these transporters. Hence, expression of transporters that drive extracellular acidosis would induce a more glycolytic phenotype. To test this, we have transfected lowly glycolytic and non-metastatic MCF-7 breast adenocarcinoma cells with two different proton transporters.

In the first model, we used the yeast plasma membrane proton ATPase 1 (PMA1); a Type 1 P-type ATPase. PMA1 was stably over-expressed in MCF7 cell line and over-expression of PMA1 transformed the cells into a more aggressive phenotype with increased glucose consumption, lactate production, and proton production. Phenotypically, the transfected clones had elevated rates of migration and invasion in vitro and increased metastasis in vivo. This experimental evidence supports the hypothesis that proton export alone could drive the Warburg phenotype. Hence, in this system, glucose is metabolized to replenish either the ATP expended or the exported hydrogen-ions.

In the second model we used CA-IX; an exofacial carbonic anhydrase that is highly expressed in invasive cancers and materially participates in acidifying the microenvironment. CA-IX is upregulated in multiple cancer types with minimal expression in normal tissue, and is therefore an attractive and biologically relevant therapeutic target. In vitro, we have observed

that many cells expressing a glycolytic phenotype have elevated CA-IX expression, leading us to postulate that these phenotypes are coupled. To test this, we stably overexpressed CA-IX in MCF7 cells and again this resulted in a stable, glycolytic phenotype with increased glucose consumption, lactate production, and proton production observed using assay kits and live cell metabolic phenotyping with the Seahorse Extracellular Flux Analyzer. Through this study we have shown that acidosis leads to a more aggressive behavior and that CA-IX expression alone can induce a glycolytic phenotype. Hence, we hypothesise this is due to the enzymatic activity of CA-IX “sucking in” glucose to replenish intracellular H+ that are exported by this mechanism.

https://doi.org/10.1158/1538-7445.AM2017-5429

Proton export drives the Warburg Effect

Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect”. It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H⁺ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H⁺ equivalents from the cell. To test this hypothesis, we stably transfected lowly-glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton exporting systems: either PMA1 (yeast H⁺-ATPase) or CAIX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Therefore, cancer cells with increased H⁺ export increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards a Warburg phenotype.

Proton export takes charge of proliferation

Increased intracellular pH (pHi) has emerged as a metabolic adaptation that is linked to increased cancer anabolism, but evidence supporting a direct role for pHi changes in cancer growth has been lacking.² The monocarboxylate transporter (MCT) family, including MCT1 and MCT4, consists of enzymes that mediate proton-linked cellular transport of monocarboxylates, such as lactate and pyruvate, across the plasma membrane; they are upregulated in a number of cancers.^3,4 Such enhanced expression has been considered a compensatory adaptation to Warburg metabolism to mitigate cellular toxicity by increasing lactate export.² The investigators uncovered that simply increasing pHi via an MCT4-dependent mechanism reprograms carbon metabolism and drives growth in normal and malignant myeloid cells; however, it is dispensable for normal hematopoietic stem cells (HSCs), pointing to MCT4 inhibition as a promising metabolic intervention to limit AML proliferation.

https://doi.org/10.1182/blood.2021014237

Proton Transport Inhibitors as Potentially Selective Anticancer Drugs

Different research groups have recently described a proton [H⁺]-related mechanism underlying the initiation and progression of the neoplastic process in which all cancer cells and tissues, regardless of their origin and genetic background, have a pivotal energetic and homeostatic disturbance of their metabolism that is completely different from all normal tissues: an aberrant regulation of hydrogen ion dynamics leading to a reversal of the pH gradient in cancer cells and tissues (∆pH_i to ∆pH_e) as compared to normal tissue pH gradients. This basic specific abnormality of the relationship between the intracellular and the extracellular proton dynamics, a phenomenon that is increasingly considered to be one of the most differential hallmarks of cancer, has led to the formation of a unifying thermodynamic view of cancer research that embraces cancer fields from etiopathogenesis, cancer cell metabolism, multiple drug resistance (MDR), neovascularization and the metastatatic process to selective apoptosis, cancer chemotherapy and even the spontaneous regression of cancer (SRC). This reversed proton gradient is driven by a series of proton export mechanisms that underlie the initiation and progression of the neoplastic process. This means that therapeutic targeting of the transporters that are active in cancer cells could be selective for malignancy and is likely to open new pathways towards the development of more effective and less toxic therapeutic measures for all malignant diseases. Here we review the transporters involved in driving the reversed proton gradient and their specific inhibitors.

https://ar.iiarjournals.org/content/29/6/2127