Revisiting the Warburg Effect with Focus on Lactate.

Rewired metabolism is acknowledged as one of the drivers of tumor growth. As a result, aerobic glycolysis, or the Warburg effect, is a feature of many cancers. Increased glucose uptake and glycolysis provide intermediates for anabolic reactions necessary for cancer cell proliferation while contributing sufficient energy. However, the accompanying increased lactate production, seemingly wasting glucose carbon, was originally explained only by the need to regenerate NAD+ for successive rounds of glycolysis by the lactate dehydrogenase (LDH) reaction in the cytosol. After the discovery of a mitochondrial LDH isoform, lactate oxidation entered the picture, and lactate was recognized as an important oxidative fuel. It has also been revealed that lactate serves a variety of signaling functions and helps cells adapt to the new environment. Here, we discuss recent findings on lactate metabolism and signaling in cancer while attempting to explain why the Warburg effect is adopted by cancer cells.

Dynamic regulation of mitochondrial pyruvate metabolism is necessary for orthotopic pancreatic tumor growth.

BACKGROUND: Pyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1-4) and two reactivating phosphatases (PDP1, 2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. METHODS: To isolate the function of the serine-dependent regulation of PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293, and 300 or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. RESULTS: In this family of cells with non-phosphorylatable PDHA1, we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. CONCLUSIONS: We found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had a very modest reduction of growth in vitro, but failed to grow as tumors indicating that dynamic PDC adaptation to microenvironmental conditions is necessary to support pancreatic cancer growth in vivo.

Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.

Quantitative Proteomics Reveal Peroxiredoxin Perturbation Upon Persistent Lymphocytic Choriomeningitis Virus Infection in Human Cells.

Experimental data indicate that during persistent infection, lymphocytic choriomeningitis virus (LCMV) may both directly or indirectly modulate regulatory cellular processes and alter cellular functions that are not critical for survival, but are essential for cell homeostasis. In order to shed more light on these processes, two-dimensional differential in-gel electrophoresis (2D-DIGE) and MALDI-TOF tandem mass spectrometry were used to determine the proteome response of the HeLa cell line to persistent LCMV infection. Quantitative analysis revealed 24 differentially abundant proteins. Functional analysis showed that LCMV-responsive proteins were primarily involved in metabolism, stress, and the defense response. Among identified proteins, we discovered significant changes for peroxiredoxins, a family of antioxidant enzymes. Decreased amount of these antioxidant proteins correlated with elevation of reactive oxygen species (ROS) in infected cells. Increased levels of ROS were accompanied by changes in the pattern of telomere restriction fragments (TRFs) in infected cells and mediated activation of hypoxia-inducible transcription factor-1 (HIF-1) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. Moreover, treatment with antioxidants resulted in reduced levels of viral nucleoprotein, indicating a connection between ROS-dependent signaling and viral replication.

Microenvironmental control of glucose metabolism in tumors by regulation of pyruvate dehydrogenase.

During malignant progression cancer cells undergo a series of changes, which promote their survival, invasiveness and metastatic process. One of them is a change in glucose metabolism. Unlike normal cells, which mostly rely on the tricarboxylic acid cycle (TCA), many cancer types rely on glycolysis. Pyruvate dehydrogenase complex (PDC) is the gatekeeper enzyme between these two pathways and is responsible for converting pyruvate to acetyl-CoA, which can then be processed further in the TCA cycle. Its activity is regulated by PDP (pyruvate dehydrogenase phosphatases) and PDHK (pyruvate dehydrogenase kinases). Pyruvate dehydrogenase kinase exists in 4 tissue specific isoforms (PDHK1-4), the activities of which are regulated by different factors, including hormones, hypoxia and nutrients. PDHK1 and PDHK3 are active in the hypoxic tumor microenvironment and inhibit PDC, resulting in a decrease of mitochondrial function and activation of the glycolytic pathway. High PDHK1/3 expression is associated with worse prognosis in patients, which makes them a promising target for cancer therapy. However, a better understanding of PDC's enzymatic regulation in vivo and of the mechanisms of PDHK-mediated malignant progression is necessary for the design of better PDHK inhibitors and the selection of patients most likely to benefit from such inhibitors.

Carbonic Anhydrase IX-Mouse versus Human.

In contrast to human carbonic anhydrase IX (hCA IX) that has been extensively studied with respect to its molecular and functional properties as well as regulation and expression, the mouse ortholog has been investigated primarily in relation to tissue distribution and characterization of CA IX-deficient mice. Thus, no data describing transcriptional regulation and functional properties of the mouse CA IX (mCA IX) have been published so far, despite its evident potential as a biomarker/target in pre-clinical animal models of tumor hypoxia. Here, we investigated for the first time, the transcriptional regulation of the Car9 gene with a detailed description of its promoter. Moreover, we performed a functional analysis of the mCA IX protein focused on pH regulation, cell-cell adhesion, and migration. Finally, we revealed an absence of a soluble extracellular form of mCA IX and provided the first experimental evidence of mCA IX presence in exosomes. In conclusion, though the protein characteristics of hCA IX and mCA IX are highly similar, and the transcription of both genes is predominantly governed by hypoxia, some attributes of transcriptional regulation are specific for either human or mouse and as such, could result in different tissue expression and data interpretation.

Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours.

Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs.