Potential role of the ABCG2-Q141K polymorphism in type 2 diabetes

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease and variations in multispecific membrane transporter functions may affect T2DM development, complications or treatment. In this work we have analyzed the potential effects of a major polymorphism, the Q141K variant of the ABCG2 transporter in T2DM. The ABCG2 protein is a multispecific xeno- and endobiotic transporter, affecting drug metabolism and playing a key role in uric acid extrusion. The ABCG2-Q141K variant, with reduced expression level and function, is present in 15–35% of individuals, depending on the genetic background of the population, and has been shown to significantly affect gout development. Several other diseases, including hypertension, chronic renal failure, and T2DM have also been reported to be associated with high serum uric acid levels, suggesting that ABCG2 may also play a role in these conditions. In this work we have compared relatively small cohorts (n = 203) of T2DM patients (n = 99) and healthy (n = 104) individuals regarding the major laboratory indicators of T2DM and determined the presence of the SNP rs2231142 (C421A), resulting the ABCG2-Q141K protein variant. We found significantly higher blood glucose and HbA1c levels in the T2DM patients carrying the ABCG2-Q141K variant. These findings may emphasize the potential metabolic role of ABCG2 in T2DM and indicate that further research should explore how prevention and treatment of this disease may be affected by the frequent polymorphism of ABCG2.

Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons

ABCG2 is a membrane transporter protein that has been associated with multidrug resistance phenotype and tumor development. Additionally, it is expressed in various stem cells, providing cellular protection against endobiotics and xenobiotics. In this study, we designed artificial mirtrons to regulate ABCG2 expression posttranscriptionally. Applying EGFP as a host gene, we could achieve efficient silencing not only in luciferase reporter systems but also at the ABCG2 protein level. Moreover, we observed important new sequential-functional features of the designed mirtrons. Mismatch at the first position of the mirtron-derived small RNA resulted in better silencing than full complementarity, while the investigated middle and 3′ mismatches did not enhance silencing. These latter small RNAs operated most probably via non-seed specific translational inhibition in luciferase assays. Additionally, we found that a mismatch in the first position has not, but a second mismatch in the third position has abolished target mRNA decay. Besides, one nucleotide mismatch in the seed region did not impair efficient silencing at the protein level, providing the possibility to silence targets carrying single nucleotide polymorphisms or mutations. Taken together, we believe that apart from establishing an efficient ABCG2 silencing system, our designing pipeline and results on sequential-functional features are beneficial for developing artificial mirtrons for other targets.

Dihydrotestosterone induces chemo-resistance of triple-negative breast MDA-MB-231 cancer cells towards doxorubicin independent of ABCG2 and miR-328-3p

Background: Androgens potentially have an important role in the biology of breast cancer, particularly triple-negative breast cancer (TNBC). Androgen receptor (AR) may offer a novel therapeutic strategy including the use of microRNA (miRNA) molecules. We have previously shown that AR agonist, dihydrotestosterone (DHT), increases the expression of miR-328-3p in the TNBC MDA-MB-231 cells. One target of the latter miRNA is ATP-binding cassette subfamily G member 2 (ABCG2), which modulates the chemo-response of cancer cells by pumping out xenobiotics. Objective: Using MDA-MB-231 cells as a model system for TNBC, we hypothesized that DHT would induce cell sensitivity towards doxorubicin via increasing levels of miR-328-3p and, consequently, reducing ABCG2 levels. Methods: Chemo-response of cells towards doxorubicin, tamoxifen, and mitoxantrone was evaluated using cell viability MTT assay. Cells were transfected with both miR-328-3p mimic or antisense molecules. Real-time PCR was utilized to assess RNA levels and immunoblotting was performed to investigate levels of ABCG2 protein. PCR arrays were used to assess changes in the expression of drug response regulatory genes. Results: Contrary to our hypothesis, treating MDA-MB-231 cells with DHT, no effect towards tamoxifen or mitoxantrone and increased cell resistance towards doxorubicin were noted, concomitant with decreased expression of ABCG2. This under-expression of ABCG2 was also found in MCF-7 and MDA-MB-453 cells treated with DHT. Although miR-328-3p decreased ABCG2 mRNA and protein levels, the miRNA did not alter the chemo-response of cells towards doxorubicin and did not affect DHT-induced chemo-resistance. AR activation slightly decreased the expression of 5 genes, including insulin-like growth factor 1 receptor, that may explain the mechanism of DHT-induced chemo-resistance of cells. Conclusion: DHT regulates chemo-response via a mechanism independent of ABCG2 and miR-328-3p.

POS0352 ALLELIC VARIANTS IN THE ABCG2 GENE CAN LEAD TO HYPERURICEMIA AND GOUT

Background:The level of uric acid is largely determined by the functions of urate transporters, which are located in the kidney and intestine. The ABCG2 protein is the major excretor of uric acid and its dysfunction may lead to the development of hyperuricemia and gout.Objectives:The aim of our study was to detect the occurrence and frequency of allelic variants in the ABCG2 gene that can lead to impaired function of the ABCG2 protein and to the development of hyperuricemia and gout.Methods:We examined allelic variants of ABCG2 using PCR amplification and Sanger sequencing of all coding regions and exon-intron boundaries in 359 patients with primary hyperuricemia and gout.Results:We found a rare in-frame deletion p.K360del and 15 missense variants, two of which were common (p.V12M, p.Q141K) and 13 were very rare (p.M71V, p.G74D, p.M131I, p.R147W, p.T153M, p.I242T, p.R236X, p.F373C, p.T421A, p.T434M, p.S476P, p.S572R, p.D620N). The p.R236X variant leads to a premature stop codon. The p.V12M variant probably has a protective effect against gout (minor allele frequency – MAF – in our cohort = 0,025 / MAF in the European population = 0,061), while the p.Q141K variant increases the risk of gout (MAF in our cohort = 0,213 / MAF in the European population = 0,094) (1). As for the rare variants, the p.R147W, p.T153M, p.F373C, p.T434M, p.S476P and p.S572R according to functional analyzes reduce the function of the ABCG2 protein (2). Based on in silico prediction, the impact on reduced function is expected for variants p.M71V, p.G74D, p.M131I, p.R147W, p.I242T, p.F373C, p.T434M, p.S476P and p.S572R.Conclusion:Our data suggest that the common variant p.Q141K and most of the rare variants in the ABCG2 gene affect the function of the ABCG2 urate transporter and are a genetic risk factor for hyperuricemia and gout.References:[1]Stiburkova B, et al. Functional non-synonymous variants of ABCG2 and gout risk. Rheumatology (Oxford). 2017 Nov 1; 56(11):1982-1992.[2]Toyoda Y, et al. Functional characterization of clinically-relevant rare variants in ABCG2 identified in a gout and hyperuricemia cohort. Cells. 2019 Apr 18;8(4).Acknowledgements:This study was supported by the project for conceptual development of research organization 00023728 (Institute of Rheumatology) and RVO VFN64165.Disclosure of Interests:None declared

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.

ABCG2 Protein Levels and Association to Response to First-Line Irinotecan-Based Therapy for Patients with Metastatic Colorectal Cancer

In this study we investigated the use of cancer cell protein expression of ABCG2 to predict efficacy of systemic first-line irinotecan containing therapy in patients with metastatic colorectal cancer (mCRC). From a Danish national cohort, we identified 119 mCRC patients treated with irinotecan containing therapy in first-line setting. Among these, 108 were eligible for analyses. Immunohistochemistry (IHC) analyses were performed on the primary tumor tissue in order to classify samples as high or low presence of ABCG2 protein. Data were then associated with patient outcome (objective response (OR), progression free survival (PFS) and overall survival (OS)). ABCG2 protein expression in the basolateral membrane was high (score 3+) in 33% of the patients. Exploratory analyses revealed a significant interaction between ABCG2 score, adjuvant treatment and OR (p = 0.041) in the 101 patients with evaluable disease. Patients with low ABCG2 (score 0–2) and no prior adjuvant therapy had a significantly higher odds ratio of 5.6 (Confidence Interval (CI) 1.68–18.7; p = 0.005) for obtaining OR. In contrast, no significant associations between ABCG2 expression and PFS or OS were found. These results suggest that measurement of the ABCG2 drug efflux pump might be used to select patients with mCRC for irinotecan treatment. However, additional studies are warranted before conclusions regarding a clinical use can be made. Moreover, patients with high ABCG2 immunoreactivity could be candidates for specific ABCG2 inhibition treatment in combination with irinotecan.

Cellular Processing of the ABCG2 Transporter—Potential Effects on Gout and Drug Metabolism

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking.

Functional Characterization of Clinically-Relevant Rare Variants in ABCG2 Identified in a Gout and Hyperuricemia Cohort

ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein.

The study of drug resistance properties of ABCG2 (ATP-binding cassette G2) in contact with thymoquinone, gallic acid, and hesperetin antioxidants

Introduction: ATP-binding cassette (ABC) transporters are a group of intra membrane proteins that play key roles in the transmission and exchange of vital compounds on both sides of the membrane. These proteins can specially transport anti-cancer drugs out of cancer cells. ABCG2 is a member of this family that is extremely expressed in many cancers. This study, aims to evaluate the binding affinity of three antioxidants thymoquinone (TQ), gallic acid (GA), and hesperetin (HP) to ABCG2 compared with an anti-cancer drug, mitoxantrone (Mit), to export cells. Methods: The PDB file of ABCG2 was obtained from the protein data bank server (http://www.rcsb.org) with ID: 5NJ3. After 200 stages of molecular docking running on ABCG2 protein in AutoDock v.4.2 software, the amino acids involved in the binding site of each compound were identified using the LigPlot+ software. Results: HP had the lowest (-6.36 kcal/mol) and GA had the highest (-3.93 kcal/mol) binding energy in comparison with Mit (-0.06 kcal/mol) for binding to ABCG2. Effective concentration required to perform the reaction between ABCG2 was higher in GA (1.31 mM) than TQ (42.69 μM) and HP (21.74 μM). GA, HP, and TQ formed 17, 18, and 22 hydrogen and hydrophobic bonds at the binding site of ABCG2. Conclusion: It seems that GA has the lowest affinity to make contact with ABCG2 binding site. So, GA tends to remain in the cell but TQ and HP tend to leave the cell easily via ABCG2 transporter.