Mimicking and analyzing the tumor microenvironment.

The tumor microenvironment (TME) is increasingly appreciated to play a decisive role in cancer development and response to therapy in all solid tumors. Hypoxia, acidosis, high interstitial pressure, nutrient-poor conditions, and high cellular heterogeneity of the TME arise from interactions between cancer cells and their environment. These properties, in turn, play key roles in the aggressiveness and therapy resistance of the disease, through complex reciprocal interactions between the cancer cell genotype and phenotype, and the physicochemical and cellular environment.

NHE1 regulation in NAFLD in vitro contributes to hepatocyte injury and HSC crosstalk.

Non-alcoholic fatty liver disease (NAFLD) is the fastest-growing cause of liver-associated death globally. Whole-body knockout (KO) of Na+/H+ exchanger 1 (NHE1, SLC9A1) was previously proposed to protect against high-fat diet-induced liver damage; however, mechanistic insight was lacking. The aim of the present work was to address this question in vitro to determine how NHE1, specifically in hepatocytes, impacts lipid overload-induced inflammation, fibrosis, and hepatocyte-hepatic stellate cell (HSC) crosstalk.

Adaptation to an acid microenvironment promotes pancreatic cancer organoid growth and drug resistance.

Harsh environments in poorly perfused tumor regions may select for traits driving cancer aggressiveness. Here, we investigated whether tumor acidosis interacts with driver mutations to exacerbate cancer hallmarks. We adapted mouse organoids from normal pancreatic duct (mN10) and early pancreatic cancer (mP4, KRAS-G12D mutation, ± p53 knockout) from extracellular pH 7.

How protons pave the way to aggressive cancers.

Cancers undergo sequential changes to proton (H) concentration and sensing that are consequences of the disease and facilitate its further progression. The impact of protonation state on protein activity can arise from alterations to amino acids or their titration. Indeed, many cancer-initiating mutations influence pH balance, regulation or sensing in a manner that enables growth and invasion outside normal constraints as part of oncogenic transformation.

The prolactin receptor scaffolds Janus kinase 2 via co-structure formation with phosphoinositide-4,5-bisphosphate.

Class 1 cytokine receptors transmit signals through the membrane by a single transmembrane helix to an intrinsically disordered cytoplasmic domain that lacks kinase activity. While specific binding to phosphoinositides has been reported for the prolactin receptor (PRLR), the role of lipids in PRLR signaling is unclear. Using an integrative approach combining nuclear magnetic resonance spectroscopy, cellular signaling experiments, computational modeling, and simulation, we demonstrate co-structure formation of the disordered intracellular domain of the human PRLR, the membrane constituent phosphoinositide-4,5-bisphosphate (PI(4,5)P) and the FERM-SH2 domain of the Janus kinase 2 (JAK2).

Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal β-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells.

Chronic acidosis rewires cancer cell metabolism through PPARα signaling.

The mechanisms linking tumor microenvironment acidosis to disease progression are not understood. Here, we used mammary, pancreatic, and colon cancer cells to show that adaptation to growth at an extracellular pH (pH ) mimicking acidic tumor niches is associated with upregulated net acid extrusion capacity and elevated intracellular pH at physiological pH , but not at acidic pH . Using metabolic profiling, shotgun lipidomics, imaging and biochemical analyses, we show that the acid adaptation-induced phenotype is characterized by a shift toward oxidative metabolism, increased lipid droplet-, triacylglycerol-, peroxisome content and mitochondrial hyperfusion.

Crosstalk between tumor acidosis, p53 and extracellular matrix regulates pancreatic cancer aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with minimal treatment options and a global rise in prevalence. PDAC is characterized by frequent driver mutations including KRAS and TP53 (p53), and a dense, acidic tumor microenvironment (TME). The relation between genotype and TME in PDAC development is unknown.

Acid Adaptation Promotes TRPC1 Plasma Membrane Localization Leading to Pancreatic Ductal Adenocarcinoma Cell Proliferation and Migration through Ca Entry and Interaction with PI3K/CaM.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a low overall survival rate of less than 10% and limited therapeutic options. Fluctuations in tumor microenvironment pH are a hallmark of PDAC development and progression. Many ion channels are bona fide cellular sensors of changes in pH.

The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca-Independent Manner.

Dysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21 expression.

Metabolic reprogramming by driver mutation-tumor microenvironment interplay in pancreatic cancer: new therapeutic targets.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers globally with a mortality rate exceeding 95% and very limited therapeutic options. A hallmark of PDAC is its acidic tumor microenvironment, further characterized by excessive fibrosis and depletion of oxygen and nutrients due to poor vascularity. The combination of PDAC driver mutations and adaptation to this hostile environment drives extensive metabolic reprogramming of the cancer cells toward non-canonical metabolic pathways and increases reliance on scavenging mechanisms such as autophagy and macropinocytosis.

The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells.

The gene, coding for the Naβ4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing Naβ4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts.

Dynamic Na/H exchanger 1 (NHE1) - calmodulin complexes of varying stoichiometry and structure regulate Ca-dependent NHE1 activation.

Calmodulin (CaM) engages in Ca-dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na/H-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails.

Putting Warburg to work: how imaging of tumour acidosis could help predict metastatic potential in breast cancer.

Solid tumours are often highly acidic compared to normal tissue, and tumour extracellular acidosis contributes to multiple aspects of cancer progression. Now, Anemone et al. in this issue of the British Journal of Cancer provide in vivo evidence that the degree to which various breast cancer cell lines acidify their environment correlates with their ability to metastasise to the lungs.

Cancer Cell Acid Adaptation Gene Expression Response Is Correlated to Tumor-Specific Tissue Expression Profiles and Patient Survival.

The acidic pH of the tumor microenvironment plays a critical role in driving cancer development toward a more aggressive phenotype, but the underlying mechanisms are unclear. To this end, phenotypic and genotypic changes induced by adaptation of cancer cells to chronic acidosis have been studied. However, the generality of acid adaptation patterns across cell models and their correlation to the molecular phenotypes and aggressiveness of human cancers are essentially unknown.

The γ-hydroxybutyric acid (GHB) analogue NCS-382 is a substrate for both monocarboxylate transporters subtypes 1 and 4.

The small-molecule ligand (E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is an analogue of γ-hydroxybutyric acid (GHB) and is widely used for probing the brain-specific GHB high-affinity binding sites. To reach these, brain uptake is imperative, and it is therefore important to understand the molecular mechanisms of NCS-382 transport in order to direct in vivo studies. In this study, we hypothesized that NCS-382 is a substrate for the monocarboxylate transporter subtype 1 (MCT1) which is known to mediate blood-brain barrier (BBB) permeation of GHB.

Annual Meeting of the International Society of Cancer Metabolism (ISCaM): Metabolic Adaptations and Targets in Cancer.

The metabolism of cancer cells differs from that of their normal counterparts in a spectrum of attributes, including imbalances in diverse metabolic arms and pathways, metabolic plasticity and extent of adaptive responses, levels, and activities of metabolic enzymes and their upstream regulators and abnormal fluxes of metabolic intermediates and products. These attributes endow cancer cells with the ability to survive stressors of the tumor microenvironment and enable them to landscape and exploit the host terrain, thereby facilitating cancer progression and therapy resistance. Understanding the molecular and physiological principles of cancer metabolism is one of the key prerequisites for the development of better anticancer treatments.

Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures.

Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes.

HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503.

The HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR.

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells.

Precise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pH, pH) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na/H exchanger NHE1 and Na, HCO transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells.

Trafficking, localization and degradation of the Na,HCO co-transporter NBCn1 in kidney and breast epithelial cells.

The Na+;HCO3 co-transporter NBCn1 (SLC4A7) is a major regulator of intracellular pH yet its trafficking and turnover are essentially unstudied. Here, we used MDCK-II and MCF-7 cells to investigate these processes in epithelial cells. GFP-NBCn1 membrane localization was abolished by truncation of the full NBCn1 C-terminal tail (C-tail) yet did not require the C-terminal PDZ-binding motif (ETSL).

The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms.

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H and CO , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na /H exchanger NHE1, the Na - HCO3- cotransporter NBCn1, and the lactate-H cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively.

Roles of pH and the Na/H exchanger NHE1 in cancer: From cell biology and animal models to an emerging translational perspective?

Acidosis is characteristic of the solid tumor microenvironment. Tumor cells, because they are highly proliferative and anabolic, have greatly elevated metabolic acid production. To sustain a normal cytosolic pH homeostasis they therefore need to either extrude excess protons or to neutralize them by importing HCO, in both cases causing extracellular acidification in the poorly perfused tissue microenvironment.

Oncogenic p95HER2 regulates Na+-HCO3- cotransporter NBCn1 mRNA stability in breast cancer cells via 3'UTR-dependent processes.

The Na-HCO cotransporter NBCn1 (SLC4A7) is up-regulated in breast cancer, important for tumor growth, and a single nucleotide polymorphism (SNP), rs4973768, in its 3' untranslated region (3'UTR) correlates with increased breast cancer risk. We previously demonstrated that NBCn1 expression and promoter activity are strongly increased in breast cancer cells expressing a constitutively active oncogenic human epidermal growth factor receptor 2 (HER2) (p95HER2). Here, we address the roles of p95HER2 in regulating NBCn1 expression via post-transcriptional mechanisms.