Roles of acid-extruding ion transporters in regulation of breast cancer cell growth in a 3-dimensional microenvironment.

BACKGROUND: The 3-dimensional (3D) microenvironment of breast carcinomas is characterized by profoundly altered pH homeostasis, reflecting increased metabolic acid production and a confined extracellular space characterized by poor diffusion, yet the relative contributions of specific pH-regulatory transporters to 3D growth are poorly understood. The aim of this work was to determine how 3D spheroid growth of breast cancer cells impacts the expression and spatial organization of major acid extruding proteins, and how these proteins in turn are required for spheroid growth. METHODS: MCF-7 (Luminal-A) and MDA-MB-231 (Triple-negative) human breast cancer cells were grown as ~700-950 µm diameter spheroids, which were subjected to Western blotting for relevant transporters (2- and 3D growth), quantitative immunohistochemical analysis, and spheroid growth assays. Individual transporter contributions were assessed (i) pharmacologically, (ii) by stable shRNA- and transient siRNA-mediated knockdown, and (iii) by CRISPR/Cas9 knockout. RESULTS: In MCF-7 spheroids, expression of the lactate-H(+) cotransporter MCT1 (SLC16A1) increased from the spheroid periphery to its core, the Na(+),HCO3 (-) cotransporter NBCn1 (SLC4A7) was most highly expressed at the periphery, and the Na(+)/H(+) exchanger NHE1 (SLC9A1) and MCT4 (SLC16A3) were evenly distributed. A similar pattern was seen in MDA-MB-231 spheroids, except that these cells do not express MCT1. The relative total expression of NBCn1 and NHE1 was decreased in 3D compared to 2D, while that of MCT1 and MCT4 was unaltered. Inhibition of MCT1 (AR-C155858) attenuated MCF-7 spheroid growth and this was exacerbated by addition of S0859, an inhibitor of Na(+),HCO3 (-) cotransporters and MCTs. The pharmacological data was recapitulated by stable knockdown of MCT1 or NBCn1, whereas knockdown of MCT4 had no effect. CRISPR/Cas9 knockout of NHE1, but neither partial NHE1 knockdown nor the NHE1 inhibitor cariporide, inhibited MCF-7 spheroid growth. In contrast, growth of MDA-MB-231 spheroids was inhibited by stable or transient NHE1 knockdown and by NHE1 knockout, but not by knockdown of NBCn1 or MCT4. CONCLUSIONS: This work demonstrates the distinct expression and localization patterns of four major acid-extruding transporters in 3D spheroids of human breast cancer cells and reveals that 3D growth is dependent on these transporters in a cell type-dependent manner, with potentially important implications for breast cancer therapy.

Glycosylation of solute carriers: mechanisms and functional consequences.

Solute carriers (SLCs) are one of the largest groups of multi-spanning membrane proteins in mammals and include ubiquitously expressed proteins as well as proteins with highly restricted tissue expression. A vast number of studies have addressed the function and organization of SLCs as well as their posttranslational regulation, but only relatively little is known about the role of SLC glycosylation. Glycosylation is one of the most abundant posttranslational modifications of animal proteins and through recent advances in our understanding of protein-glycan interactions, the functional roles of SLC glycosylation are slowly emerging. The purpose of this review is to provide a concise overview of the aspects of glycobiology most relevant to SLCs, to discuss the roles of glycosylation in the regulation and function of SLCs, and to outline the major open questions in this field, which can now be addressed given major technical advances in this and related fields of study in recent years.

A glycogene mutation map for discovery of diseases of glycosylation.

Glycosylation of proteins and lipids involves over 200 known glycosyltransferases (GTs), and deleterious defects in many of the genes encoding these enzymes cause disorders collectively classified as congenital disorders of glycosylation (CDGs). Most known CDGs are caused by defects in glycogenes that affect glycosylation globally. Many GTs are members of homologous isoenzyme families and deficiencies in individual isoenzymes may not affect glycosylation globally. In line with this, there appears to be an underrepresentation of disease-causing glycogenes among these larger isoenzyme homologous families. However, genome-wide association studies have identified such isoenzyme genes as candidates for different diseases, but validation is not straightforward without biomarkers. Large-scale whole-exome sequencing (WES) provides access to mutations in, for example, GT genes in populations, which can be used to predict and/or analyze functional deleterious mutations. Here, we constructed a draft of a functional mutational map of glycogenes, GlyMAP, from WES of a rather homogenous population of 2000 Danes. We cataloged all missense mutations and used prediction algorithms, manual inspection and in case of carbohydrate-active enzymes family GT27 experimental analysis of mutations to map deleterious mutations. GlyMAP (http://glymap.glycomics.ku.dk) provides a first global view of the genetic stability of the glycogenome and should serve as a tool for discovery of novel CDGs.

Low density lipoprotein receptor class A repeats are O-glycosylated in linker regions.

The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the "SimpleCell" O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position -1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.

The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality.

Heterotaxy is a disorder of left-right body patterning, or laterality, that is associated with major congenital heart disease. The aetiology and mechanisms underlying most cases of human heterotaxy are poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer, where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals. The mechanism that specifies these two cilia types remains unknown. Here we show that the N-acetylgalactosamine-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking the ciliopathy primary ciliary dyskinesia. Together our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the left-right organizer to determine laterality, and reveal a novel mechanism for human heterotaxy.

Genetic ablation of aquaporin-2 in the mouse connecting tubules results in defective renal water handling.

Body water balance is regulated via the water channel aquaporin-2 (AQP2), which is expressed in the renal connecting tubule (CNT) and collecting duct (CD). The relative roles of AQP2 in the CNT and CD are not fully understood. To study the role of AQP2 in the CNT we generated a mouse model with CNT-specific AQP2 deletion (AQP2-CNT-knockout (KO)). Confocal laser scanning microscopy and immunogold electron microscopy demonstrated an absence of AQP2 in the CNT of AQP2-CNT-KO mice. Twenty-four hour urine output was significantly increased (KO: 3.0 ± 0.3 ml (20 g body weight (BW))(-1); wild-type (WT): 1.9 ± 0.3 ml (20 g BW)(-1)) and urine osmolality decreased (KO: 1179 ± 107 mosmol kg(-1); WT: 1790 ± 146 mosmol kg(-1)) in AQP2-CNT-KO mice compared with controls. After 24 h water restriction, urine osmolality was still significantly lower in AQP2-CNT-KO mice (KO: 2087 ± 169 mosmol kg(-1); WT: 2678 ± 144 mosmol kg(-1)). A significant difference in urine osmolality between groups before desmopressin (dDAVP) (KO: 873 ± 129 mosmol kg(-1); WT: 1387 ± 163 mosmol kg(-1)) was not apparent 2 h after injection, with urine osmolality increased significantly in both groups (KO: 2944 ± 41 mosmol kg(-1); WT: 3133 ± 66 mosmol kg(-1)). Cortical kidney fractions from AQP2-CNT-KO mice had significantly reduced AQP2, with no compensatory changes in sodium potassium chloride cotransporter (NKCC2), AQP3 or AQP4. Lithium chloride treatment increased urine volume and decreased osmolality in both WT and AQP2-CNT-KO mice. After 8 days of treatment, the AQP2-CNT-KO mice still had a significantly higher urine volume and lower urine osmolality, suggesting that the CNT does not play a significant role in the pathology of lithium-induced nephrogenic diabetes insipidus. Our studies indicate that the CNT plays a role in regulating body water balance under basal conditions, but not for maximal concentration of the urine during antidiuresis.

Pharmacokinetics of trefoil peptides and their stability in gastrointestinal contents.

Trefoil factor family (TFF) peptides are considered promising for therapeutic use in gastrointestinal diseases, and there is a need to explore the fate of injected TFF and the stability of the peptides in the gastrointestinal tract. We studied the pharmacokinetics of intravenously (i.v.) administered hTFF2 in mice and rats and of hTFF3 administered i.v., intramuscularly, intraperitoneally, and subcutaneously in mice, and estimated by ELISA the decay of the peptides added to rat and human gastrointestinal contents. We found that i.v. injected hTFF2 and hTFF3 were cleared from the circulation within 2-3h, exhibiting comparable pharmacokinetic profiles. In contents from the rat stomach, hTFF levels remained unchanged for up to 6 days. In the small and large intestine of rats, the hTFF levels decreased markedly after 4 and 1h, respectively. In small intestinal contents from humans, the levels remained stable for more than 24h. We conclude that systemically administered hTFF2 and hTFF3 are rapidly eliminated from the circulation and that the stability of hTFF2 and hTFF3 in GI contents appeared higher in the gastric and small intestinal milieu than in the large intestine and feces, suggesting a higher stability toward gastric acid and digestive enzymes than toward microbial degradation.