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Persistent organic pollutants (POPs) may alter tumor cells phenotype, possibly increasing malignancy, but there is a lack of studies investigating the mechanisms by which POPs may affect tumor cells. The ATP-Binding Cassette (ABC) transporter proteins are a widely studied component of drug resistance and tumor progression. We hypothesized that the levels of BDE-209 and TCDD detected in human serum can modulate the gene expression or activity of ATP-binding cassette (ABC) transporters in murine melanoma (B16-F1) cells. In this study, we observed an upregulation of the ABCB1 and ABCC4 (24 h) genes followed by an increased protein activity after BDE-209 15 day-exposure. We also observed that cells exposed to TCDD showed an upregulation of ABCB5, ABCC1 and ABCC4 genes (24 h) and change of protein activity after 15 days of exposure. These findings suggest that BDE-209 and TCDD can regulate the phenotype of B16-F1 cells by interfering with the expression and activity of ATP-binding cassette (ABC) transporters. This investigation revealed that environmental pollutants might intervene and modify cells’ resistance to chemotherapy and cancer prognosis.

Background Sulfoxaflor is a new insecticide for controlling against Aphis gossypii in the field. ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins and play an important role in the detoxification process. However, the potential role of ABC transporters in sulfoxaflor resistance in A. gossypii is unknown. Results In this study, an ABC transporter inhibitor, verapamil, dramatically increased the toxicity of sulfoxaflor in the resistant population with a synergistic ratio of 8.55. However, verapamil did not synergize sulfoxaflor toxicity in the susceptible population. The contents of ABC transporters were significantly increased in the Sul-R population. Based on RT-qPCR analysis, 10 of 23 ABC transcripts, ABCA1, ABCA2, ABCB1, ABCB5, ABCD1, ABCG7, ABCG16, ABCG26, ABCG27, and MRP7, were up-regulated in the Sul-R population compared to the Sus population. Meanwhile, inductive effects of ABCA1, ABCD1, ABCG7 and ABCG26 by sulfoxaflor were found in A. gossypii. Furthermore, knockdown of ABCA1 and ABCD1 using RNAi significantly increased the sulfoxaflor sensitivity in Sul-R aphids. Conclusion These results suggested that ABC transporters, especially the ABCA1 and ABCD1 genes, might be related with sulfoxaflor resistance in A. gossypii. This study will promote further work to validate the functional roles of these ABCs in sulfoxaflor resistance and might be helpful for the management of sulfoxaflor-resistant A. gossypii.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, incurable blistering skin disease caused by biallelic mutations in type VII collagen (C7). Advancements in treatment of RDEB have come from harnessing the immunomodulatory potential of mesenchymal stem cells (MSCs). Although human bone marrow-derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. Our work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgammanull (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Furthermore, ABCB5+ DSCs vs BM-MSCs cocultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of major histocompatibility complex class II and Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms among MSC populations could have broader regenerative medicine implications beyond RDEB treatment.

Multidrug resistance is a major factor causing the failure of cancer chemotherapy. Efflux pumps of the ATP-binding cassette (ABC) transporter family expel a large array of diverse anticancer drugs out of tumor cells thereby rendering them unresponsive to drug treatment. During the past 2 decades, we focused on three ABC transporters, i.e. P-glycoprotein (MDR1/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and ABCB5 with the aim to find natural products with activity against multidrug-resistant cells. We investigated more than 102 cytotoxic medicinal plants and more than 228 isolated cytotoxic phytochemicals with defined chemical structures from 16 countries in Africa, Near East, Asia, and Europe for their activity to kill multidrug-resistant cells. A synopsis of these results is given in this review article. ABCB1-, ABCG2- or ABCB5-expressing cells were moderately or strongly cross-resistant to a considerable portion of plant extracts or phytochemicals, making them less suitable for the treatment of multidrug resistant tumors. While another large portion of compounds inhibited sensitive and multidrug-resistant cells with similar efficacy, a small fraction of medicinal plants and phytochemicals (3–8%) suppressed multidrug-resistant cells with even much better efficacy as their drug-sensitive counterparts. This hypersensitivity phenomenon is termed “collateral sensitivity”. These compounds may be exquisitely suited to kill otherwise resistant and refractory tumors. Molecular modes of action of collateral sensitivity as well as possibilities for further drug development, chemical derivatization of natural lead compounds and rationale phytotherapy are discussed.

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro. Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G(2)/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

Purpose To identify factors associated with isolation yields of ATP-binding cassette (ABC) superfamily member B5 (ABCB5)-positive limbal stem cells (LSCs) from human cadaveric donor eyes. Methods Whole eye globes were obtained from the Saving Sight eye bank, Kansas City, MO and the CorneaGen eye bank, Seattle, WA. ABCB5-positive LSCs were sorted by flow cytometry upon anti-ABCB5 monoclonal antibody staining within one week after donor death. The yields of live limbal epithelial cells in their entirety and of isolated pure ABCB5-positive LSC subsets were correlated with variables contained in the eye donors’ medical information. Results The mean isolation yield of live limbal epithelial cells and ABCB5-positive LSCs per donor eye was (340,000 ± 160,000 and 2,608 ± 1,842 respectively, mean ± SD). Stepwise regression analysis showed that cardiac disease-related death was the strongest negative predictor of the ABCB5-positive LSC isolation yield (p = 0.01). While we observed a trend for an age-related decline in the yield of ABCB5-positive LSCs, a statistically significant association could not be established (2% decrease/year, p = 0.11). Additionally, despite a trend for decreased isolation yields of total live limbal epithelial cells isolated from single donors with a longer time between death and tissue processing (p = 0.04), this did not affect the yields of purified ABCB5-positive LSC, which was independent of increasing time between death and tissue processing (p = 0.50). Conclusions Our study identifies cardiac disease-related death as a donor variable significantly associated with lower ABCB5-positive LSC isolation yields.

Given its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.

Purpose: Our aim was to construct a signature that accurately predicted the prognostic and immune response of melanoma.Methods: First, the weighted co-expression network analysis (WGCNA) algorithm was used to identify the hub genes related to clinical phenotypes of melanoma in the cancer genome atlas (TCGA) database. Nest, the least absolute shrinkage and selection operator (LASSO) analysis was used to dimensionality reduction of these hub genes and constructed a prognostic signature to predict the prognosis and immunosuppressive response of melanoma.Result: Through in-depth analysis, we constructed a 5-mRNA prognostic signature and verified its prognostic value in internal (TCGA-SKCM, n = 452) and external independent datasets (GSE53118, n = 79). Based on this signature, the tumor immune microenvironment (TME) of melanoma was characterized, and the result was found that patients in the high-risk group had lower CD8 T cell infiltration and immune checkpoint expression (PD-1, PD-L1, CTLA4), as well as higher M0/M2 macrophage infiltration. Our results also found the risk score based on a 5-mRNA signature was significantly associated with tumor mutational burden (TMB) and tumor stem cell markers (CD20, CD38, ABCB5, CD44, etc.). Lastly, we built a nomogram for clinician prediction for the prognosis of patients with melanoma.Conclusion: Our findings indicated that the 5-mRNA signature has an important predictive value for the overall survival of melanoma. By analyzing the tumor immune microenvironment and tumor stem cell marker between different groups, a new method is provided for the stratified diagnosis and treatment of melanoma.

Summary Human induced pluripotent stem cells (hiPSCs) can generate a multiplicity of organoids, yet no compelling evidence currently exists as to whether or not these contain tissue-specific, holoclone-forming stem cells. Here, we show that a subpopulation of cells in a hiPSC-derived corneal epithelial cell sheet is positive for ABCB5 (ATP-binding cassette, sub-family B, member 5), a functional marker of adult corneal epithelial stem cells. These cells possess remarkable holoclone-forming capabilities, which can be suppressed by an antibody-mediated ABCB5 blockade. The cell sheets are generated from ABCB5+ hiPSCs that first emerge in 2D eye-like organoids around six weeks of differentiation and display corneal epithelial immunostaining characteristics and gene expression patterns, including sustained expression of ABCB5. The findings highlight the translational potential of ABCB5-enriched, hiPSC-derived corneal epithelial cell sheets to recover vision in stem cell-deficient human eyes and represent the first report of holoclone-forming stem cells being directly identified in an hiPSC-derived organoid., Graphical abstract, Highlights • Human iPS cell-derived corneal epithelia contain ABCB5-positive stem cells • The ABCB5-positive cells possess holoclone-forming capabilities • An antibody-mediated ABCB5 blockade suppresses holoclone formation • Holoclone-forming stem cells are present in a human iPS cell-derived tissue construct, Bioengineering; Cell biology; Stem cells research; Tissue engineering

Ionocytes are specialized cells in the epidermis of embryonic zebrafish (Danio rerio) that play important roles in ion homeostasis and have functional similarities to mammalian renal cells. Here, we examined whether these cells might also share another functional similarity with renal cells, which is the presence of efflux transporter activities useful for elimination of toxic small molecules. Xenobiotic transporters (XTs), including the ATP-Binding Cassette (ABC) family, are a major defense mechanism against diffusible toxic molecules in aquatic embryos, including zebrafish, but their activity in the ionocytes has not previously been studied. Using fluorescent small molecule substrates of XT, we observed that specific populations of ionocytes uptake and efflux fluorescent small molecules in a manner consistent with active transport. We specifically identified a P-gp/ABCB1 inhibitor-sensitive efflux activity in the H+-ATPase-rich (HR) ionocytes, and show that these cells exhibit enriched expression of the ABCB gene, abcb5. The results extend our understanding of the functional significance of zebrafish ionocytes and indicate that these cells could play an important role in protection of the fish embryo from harmful small molecules.

Tumor antigen-specific redirection of cytotoxic T cells (CTLs) or natural killer (NK) cells including chimeric antigen receptor (CAR-) and T cell receptor (TCR-) cell therapy is currently being evaluated in different tumor entities including melanoma. Expression of melanoma-specific antigen recognized by the respective CAR or TCR directly or presented by HLA molecules is an indispensable prerequisite for this innovative therapy. In this study, we investigated in 168 FFPE tumor specimens of patients with stage I-IV melanoma the protein expression of HER2, TRP2, ABCB5, gp100, p53, and GD2 by immunohistochemistry (IHC). These results were correlated with clinical parameters. Membrane expression of HER2 and GD2 was also investigated in ten melanoma cell lines by flow cytometry for which corresponding tumors were analyzed by IHC. Our results demonstrated that gp100 was the most frequently overexpressed protein (61%), followed by TRP2 (50%), GD2 (38%), p53 (37%), ABCB5 (17%), and HER2 (3%). TRP2 expression was higher in primary tumors compared to metastases (p = 0.005). Accordingly, TRP2 and ABCB5 expression was significantly associated with lower tumor thickness of the primary (p = 0.013 and p = 0.025). There was no association between protein expression levels and survival in advanced melanoma patients. Flow cytometric analysis revealed abundant surface expression of GD2 and HER2 in all melanoma cell lines. The discordant HER2 expression in situ and in vitro suggests a tissue culture associated induction. In summary, our data support the use of gp100 and GD2 as a potential target for developing engineered TCR- or CAR-cell therapies, respectively, against melanoma.

Breast cancer is the most common cancer in women in the world. The role of germline genetic variability in ATP-binding cassette (ABC) transporters in cancer chemoresistance and prognosis still needs to be elucidated. We used next-generation sequencing to assess associations of germline variants in coding and regulatory sequences of all human ABC genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105). A total of 43 prioritized variants associating with response or survival in the above testing phase were then analyzed by allelic discrimination in the large validation set (n = 802). Variants in ABCA4, ABCA9, ABCA12, ABCB5, ABCC5, ABCC8, ABCC11, and ABCD4 associated with response and variants in ABCA7, ABCA13, ABCC4, and ABCG8 with survival of the patients. No association passed a false discovery rate test, however, the rs17822931 (Gly180Arg) in ABCC11, associating with response, and the synonymous rs17548783 in ABCA13 (survival) have a strong support in the literature and are, thus, interesting for further research. Although replicated associations have not reached robust statistical significance, the role of ABC transporters in breast cancer should not be ruled out. Future research and careful validation of findings will be essential for assessment of genetic variation which was not in the focus of this study, e.g., non-coding sequences, copy numbers, and structural variations together with somatic mutations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland., Czech Medical Council [17-28470A]; Czech Ministry of Education, Youth and SportsMinistry of Education, Youth & Sports - Czech Republic [CZ.02.1.01/0.0/0.0/16_013/0001634]; Grant Agency of Charles University [UNCE/MED/006]; Grant Agency of the Czech RepublicGrant Agency of the Czech Republic [19-03063S]

ATP-binding cassette (ABC) transporters mediate multidrug resistance in cancer. In contrast to DNA single nucleotide polymorphisms in normal tissues, the role of mutations in tumors is unknown. Furthermore, the significance of their expression for prediction of chemoresistance and survival prognosis is still under debate. We investigated 18 tumors by RNA-sequencing. The mutation rate varied from 27,507 to 300885. In ABCB1, three hotspots with novel mutations were in transmembrane domains 3, 8, and 9. We also mined the cBioPortal database with 11,814 patients from 23 different tumor entities. We performed Kaplan-Meier survival analyses to investigate the effect of ABC transporter expression on survival rates of cancer patients. Novel mutations were also found in ABCA2, ABCA3, ABCB2, ABCB5, ABCC1-6, and ABCG2. Mining the cBioPortal database with 11,814 patients from 23 different tumor entities validated our results. Missense and in-frame mutations led to altered binding of anticancer drugs in molecular docking approaches. The ABCB1 nonsense mutation Q856* led to a truncated P-glycoprotein, which may sensitize tumors to anticancer drugs. The search for ABC transporter nonsense mutations represents a novel approach for precision medicine.. Low ABCB1 mRNA expression correlated with significantly longer survival in ovarian or kidney cancer and thymoma. In cancers of breast, kidney or lung, ABC transporter expression correlated with different tumor stages and human populations as further parameters to refine strategies for more individualized chemotherapy.

BACKGROUND Gastric cancer (GC) is the fourth most common cause of cancer-related deaths in the world and 5-year overall survival (OS) rate is less than 10%. So, it is urgent to identified novel diagnostic and prognostic biomarkers. METHODS Twelve GEO (gene expression omnibus) datasets were obtained from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between GC and normal tissues were screened and integrated using limma and RobustRankAggreg (RRA) packages in R software. Protein-protein interaction (PPI) network, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses for DEGs were conducted via STRING and DAVID, respectively. Moreover, Cox regression model was used to construct a gene prognosis signature. RESULTS Ten genes (COL1A1, CXCL8, COL3A1, SPP1, COL1A2, TIMP1, CXCL1, BGN, MMP3 and SERPINE1) were identified and might be highly related to GC. Further analysis showed high expression of CXCL8, COL3A1, CXCL1, MMP3 and SERPINE1, were significantly associated with late stage of GC. Lastly, we build a seven-gene prognosis signature (CYP19A1, SERPINE1, CGB5, CALCR, ASGR2, CYTL1 and ABCB5), which can give a good prediction of OS. CONCLUSIONS Our article screened out key genes highly associating with GC's developments and prognosis, and it is useful for researcher to further understand GC's molecular basis and direct the synthesis medicine of GC.

Background Carnosic acid (CA) is one of the main constituents in rosemary extract. It possesses valuable pharmacological properties, including anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer activities. Numerous in vitro and in vivo studies investigated the anticancer profile of CA and emphasized its potentiality for cancer treatment. Nevertheless, the role of multidrug-resistance (MDR) related mechanisms for CA's anticancer effect is not yet known. Purpose We investigated the cytotoxicity of CA against known mechanisms of anticancer drug resistance (P-gp, ABCB5, BCRP, EGFR and p53) and determined novel putative molecular factors associated with cellular response towards CA. Study design Cytotoxicity assays, bioinformatic analysis, flow cytometry and western blotting were performed to identify the mode of action of CA towards cancer cells. Methods The cytotoxicity to CA was assessed using the resazurin assays in cell lines expressing the mentioned resistance mechanisms. A pharmacogenomic characterization of the NCI 60 cell line panel was applied via COMPARE, hierarchical cluster and network analyses. Flow cytometry was used to detect cellular mode of death and ROS generation. Changes in proteins-related to apoptosis were determined by Western blotting. Results Cell lines expressing ABC transporters (P-gp, BCRP or ABCB5), mutant EGFR or p53 were not cross-resistant to CA compared to their parental counterparts. By pharmacogenomic approaches, we identified genes that belong to different functional groups (e.g. signal transduction, regulation of cytoskeleton and developmental regulatory system). These genes were predicted as molecular determinants that mediate CA tumor cellular responses. The top affected biofunctions included cellular development, cellular proliferation and cellular death and survival. The effect of CA-mediated apoptosis in leukemia cells, which were recognized as the most sensitive tumor type, was confirmed via flow cytometry and western blot analysis. Conclusion CA may provide a novel treatment option to target refractory tumors and to effectively cooperate with established chemotherapy. Using pharmacogenomic approaches and network pharmacology, the relationship between cancer complexity and multi-target potentials of CA was analyzed and many putative molecular determinants were identified. They could serve as novel targets for CA and further studies are needed to translate the possible implications to clinical cancer treatment.

During the process of metastasis, cancer cells dissociate from primary tumors, migrate to distal sites, and finally colonize, eventually leading to the formation of metastatic tumors. These cancer cells, defined circulating tumor cells (CTCs) spreading through the blood stream, may develop metastatic lesions or remain dormant. Some emerging clinical evidence supports that some tumor cells may possess metastatic properties already in the earlier stages of tumorigenesis. Because the initiation and progression of vertical growth in human melanoma is fundamental to the notion of tumor virulence and progression, we decided to immune-magnetic collect and molecularly characterize circulating melanoma cells (CMCs) from melanoma patients AJCC staged = pT1b (i.e., transition from radial to vertical phase). CMCs are phenotypically and molecularly heterogeneous, thus we performed a “home-made Liquid-Biopsy,” by targeting the melanoma-associated-antigen, MCAM/MUC18/CD146, and/or the melanoma-initiating marker, ABCB5. We assessed a biomarker qualitative expression panel, contemplating the angiogenic-potential, melanoma-initiating and melanoma-differentiation drivers, cell-cell adhesion molecules, matrix-metallo-proteinases, which was performed on three enriched subpopulations from a total of 61 blood-samples from 21 melanoma patients. At first, a significant differential expression of the specific transcripts was documented between and within the CMC fractions enriched with MCAM-, ABCB5-, and both MCAM/ABCB5-coated beads, when analyzing two distinct groups: early AJCC- (stage I–II) and advanced- staged patients (stage II–IV). Moreover, in the early-AJCC staged-group, we could distinguish “endothelial,” CD45–MCAM+ enriched-, “stem” S-CMCs, CD45–ABCB5+ enriched- and a third hybrid bi-phenotypic CD45–MCAM+/ABCB5+ enriched-fractions, due to three distinct gene-expression profiles. In particular, the endothelial-CMCs were characterized by positive expression of genes involved in migration and invasion, whilst the stem CMC-fraction only expressed stem and differentiation markers. The third subpopulation isolated based on concurrent MCAM and ABCB5 protein expression showed an invasive phenotype. All three distinct CMCs sub-populations, exhibited a primitive, “stem-mesenchymal” profile suggesting a highly aggressive and metastasizing phenotype. This study confirms the phenotypic and molecular heterogeneity observed in melanoma and highlights those putative genes involved in early melanoma spreading and disease progression.

Fish gills are a structurally and functionally complex organ at the interface between organism and the aquatic environment. Gill functions include the transfer of organic molecules, both natural ones and xenobiotic compounds. Whether the branchial exchange of organic molecules involves active transporters is currently not known. Here, we investigated the presence, diversity, and functional activity of ATP-binding cassette (ABC) transporters in gills of juvenile rainbow trout. By means of RT-qPCR, gene transcripts of members from the abcb, abcc and abcg subfamilies were identified. Comparisons with mRNA profiles from trout liver and kidney revealed that ABC transporters known for an apical localization in polarized epithelia, especially abcc2 and abcb1 were underrepresented in the gills. In contrast, ABC transporters with mainly basolateral localization showed comparable gene transcript levels in the three organs. The most prominent ABC transporter in gills was an abcb subfamily member, which was annotated as abcb5 based on the synteny and phylogeny. Functional in vivo assays pointed to a role of branchial ABC transporters in branchial solute exchange. We further assessed the utility of primary gill cell cultures to characterize transporter-mediated branchial exchange of organic molecules, we examined ABC transporter gene transcript patterns and functional activity in primary cultures. The cultures display functional transport activity, but the ABC mRNA expression patterns were different to those of the intact gills. Overall, the findings of this study provide evidence for the presence of functional ABC transporter activity in gills of fish.

Malignant pleural mesothelioma (MPM) is a tumor with high chemoresistance and poor prognosis. MPM-initiating cells (ICs) are known to be drug resistant, but it is unknown if and how stemness-related pathways determine chemoresistance. Moreover, there are no predictive markers of IC-associated chemoresistance. Aim of this work is to clarify if and by which mechanisms the chemoresistant phenotype of MPM IC was due to specific stemness-related pathways. We generated MPM IC from primary MPM samples and compared the gene expression and chemo-sensitivity profile of IC and differentiated/adherent cells (AC) of the same patient. Compared to AC, IC had upregulated the drug efflux transporter ABCB5 that determined resistance to cisplatin and pemetrexed. ABCB5-knocked-out (KO) IC clones were resensitized to the drugs in vitro and in patient-derived xenografts. ABCB5 was transcriptionally activated by the Wnt/GSK3β/β-catenin/c-myc axis that also increased IL-8 and IL-1β production. IL-8 and IL-1β-KO IC clones reduced the c-myc-driven transcription of ABCB5 and reacquired chemosensitivity. ABCB5-KO clones had lower IL-8 and IL-1β secretion, and c-myc transcriptional activity, suggesting that either Wnt/GSK3β/β-catenin and IL-8/IL-1β signaling drive c-myc-mediated transcription of ABCB5. ABCB5 correlated with lower time-to-progression and overall survival in MPM patients treated with cisplatin and pemetrexed. Our work identified multiple autocrine loops linking stemness pathways and resistance to cisplatin and pemetrexed in MPM IC. ABCB5 may represent a new target to chemosensitize MPM IC and a potential biomarker to predict the response to the first-line chemotherapy in MPM patients.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry–based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma. Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462–72. ©2018 AACR.

The continuously decreasing willingness for liver donation aggravates treatment of end-stage liver diseases requiring organ transplantation as the only curative strategy. Cell therapy approaches using human hepatocytes or stem cell-derived hepatocyte-like cells may be a therapeutic option out of this dilemma. ABCB5-positive mesenchymal stromal cells from human skin featured promising potential to treat immune-mediated diseases. Since most of chronic liver diseases involve exaggerating immune mechanisms, it was the aim to demonstrate in this study, whether ABCB5+ stem cells may serve as a resource to generate hepatocytic cells for application in liver cell transplantation. Using an established single-step protocol, which had been successfully applied to differentiate mesenchymal stromal cells into the hepatocytic lineage, ABCB5+ skin-derived stem cells did not gain significant characteristics of hepatocytes. Yet, upon culture in hepatocytic differentiation medium, ABCB5+ stem cells secreted immunomodulatory and anti-fibrotic factors as well as proteins, which may prompt hepatic morphogenesis besides others. Hepatic transplantation of ABCB5+ stem cells, which had been prior cultured in hepatocytic differentiation medium, did not cause any obvious deterioration of liver architecture suggesting their safe application. Thus, human ABCB5+ skin-derived stem cells secreted putative hepatotropic factors after culture in hepatocytic differentiation medium.

Purpose Multidrug resistance (MDR) is a major cause of treatment failure. In cancer cells, MDR is often caused by an increased efflux of therapeutic drugs mediated by an up-regulation of ATP binding cassette (ABC) transporters. It has previously been shown that oncogenic ΔNp73 plays an important role in chemo-resistance. Here we aimed at unraveling the role of ΔNp73 in regulating multidrug resistance in breast cancer and melanoma cells. Methods KEGG pathway analysis was used to identify pathways enriched in breast cancer samples with a high ΔNp73 expression. We found that the ABC transporter pathway was most enriched. The expression of selected ABC transporters was analyzed using qRT-PCR upon siRNA/shRNA-mediated knockdown or exogenous overexpression of ΔNp73 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, as well as in primary melanoma samples and in the melanoma-derived cell line SK-MEL-28. The ability to efflux doxorubicin and the concomitant effects on cell proliferation were assessed using flow cytometry and WST-1 assays. Results We found that high ΔNp73 levels correlate with a general up-regulation of ABC transporters in breast cancer samples. In addition, we found that exogenous expression of ΔNp73 led to an increase in the expression of ABCB1 and ABCB5 in the breast cancer-derived cell lines tested, while knocking down of ΔNp73 resulted in a reduction in ABCB1 and ABCB5 expression. In addition, we found that ΔNp73 reduction leads to an intracellular retention of doxorubicin in MDA-MB-231 and MCF7 cells and a concomitant decrease in cell proliferation. The effect of ΔNp73 on ABCB5 expression was further confirmed in metastases from melanoma patients and in the melanoma-derived cell line SK-MEL-28. Conclusions Our data support a role for ΔNp73 in the multidrug-resistance of breast cancer and melanoma cells. Electronic supplementary material The online version of this article (doi:10.1007/s13402-017-0340-x) contains supplementary material, which is available to authorized users.

Melanoma remains one of the most therapy-resistant forms of human cancer despite recent introductions of highly efficacious targeted therapies. The intrinsic therapy resistance of human melanoma is largely due to abundant expression of a repertoire of xenobiotic efflux pumps of the ATP-binding cassette (ABC) transporter family. Here, we report that GH action is a key mediator of chemotherapeutic resistance in human melanoma cells. We investigated multiple ABC efflux pumps (ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1, and ABCG2) reportedly associated with melanoma drug resistance in different human melanoma cells and tested the efficacy of five different anti-cancer compounds (cisplatin, doxorubicin, oridonin, paclitaxel, vemurafenib) with decreased GH action. We found that GH treatment of human melanoma cells upregulates expression of multiple ABC transporters and increases the EC50 of melanoma drug vemurafenib. Also, vemurafenib-resistant melanoma cells had upregulated levels of GH receptor (GHR) expression as well as ABC efflux pumps. GHR knockdown (KD) using siRNA in human melanoma cells treated with sub-EC50 doses of anti-tumor compounds resulted in significantly increased drug retention, decreased cell proliferation and increased drug efficacy, compared to mock-transfected controls. Our set of findings identify an unknown mechanism of GH regulation in mediating melanoma drug resistance and validates GHR as a unique therapeutic target for sensitizing highly therapy-resistant human melanoma cells to lower doses of anti-cancer drugs.

ABCB5 belongs to the ATP-binding cassette (ABC) superfamily, which is recognized for playing a role in the failure of chemotherapy. ABCB5 has also been found to be overexpressed at the transcriptional level in a number of cancer subtypes, including breast cancer. However, the exact mechanism ABCB5 uses on cancer cell metastasis is still unclear. In the present study, we demonstrate that ABCB5 expression was increased in metastatic tissues when compared with nonmetastatic tissues. ABCB5 can significantly enhance metastasis and epithelial‐mesenchymal transition (EMT), while knockdown of ABCB5 inhibited these processes. Microarray analysis indicated that ZEB1 may function as a downstream factor of ABCB5. Furthermore, the expression of ZEB1 in tissues is positively relevant to ABCB5 in breast cancer. Knocking down ZEB1 inhibits ABCB5 ectopic expression-induced migration and invasion, as well as EMT. Taken together, these results helped to realize the oncogene functions of ABCB5 in breast cancer cells and provided a new direction in treating breast cancer.

Mercury is a widespread toxicant in aquatic environment that can cause deleterious effects on fish. Although a number of mercury-regulated genes have been investigated in adult fish, the transcriptional responses of fish larvae to acute mercury exposure are not well understood. In this study, RNA sequencing was used to examine the transcriptional changes in developing zebrafish larvae under a low concentration of mercuric chloride exposure from 24 to 120 hpf. Our initial results showed that a total of 142.59 million raw reads were obtained from sequencing libraries and about 86% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 391 up- and 87 down-regulated genes. Gene ontology enrichment analysis revealed that most of the differential expressed genes are closely related to the regulation of cellular process, metabolic process, multicellular organismal process, biological regulation, pigmentation, and response to stimulus. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that antigen processing and presentation was the most significantly enriched pathway. Moreover, we characterized a novel and sensitive mercury-induced ABCB (ATP- binding cassette B subfamily) transporter gene - abcb5. This gene is localized on zebrafish chromosome 16 and contains a 4014 bp open-reading frame. The deduced polypeptide is composed of 1337 amino acids and possesses most of functional domains and critical residues defined in human and mouse ABCB5/Abcb5. Functional analysis in vitro demonstrated that overexpression of zebrafish abcb5 gene can significantly decrease the cytotoxicity of mercury in LLC-PK1 cells, implying it is a potential efflux transporter of mercury. Thus, these findings provide useful insights to help further understand the transcriptional response and detoxification ability of zebrafish larvae following acute exposure to mercury.

Defining markers of different phenotypic states in melanoma is important for understanding disease progression, determining the response to therapy, and defining the molecular mechanisms underpinning phenotype-switching driven by the changing intratumor microenvironment. The ABCB5 transporter is implicated in drug-resistance and has been identified as a marker of melanoma-initiating cells. Indeed ongoing studies are using ABCB5 to define stem cell populations. However, we show here that the ABCB5 is a direct target for the microphthalmia-associated transcription factor MITF and its expression can be induced by β-catenin, a key activator and co-factor for MITF. Consequently, ABCB5 mRNA expression is primarily associated with melanoma cells exhibiting differentiation markers. The results suggest first that ABCB5 is unlikely to represent a marker of de-differentiated melanoma stem cells, and second that ABCB5 may contribute to the non-genetic drug-resistance associated with highly differentiated melanoma cells. To reconcile the apparently conflicting observations in the field, we propose a model in which ABCB5 may mark a slow-cycling differentiated population of melanoma cells.

Background Recent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments. In this study, silymarin nanoemulsion (SN) was used along with air CAP as therapeutic agent to counter human melanoma. Methods In this study, we examined the combined treatment of CAP and SN on G-361 human melanoma cells by evaluating cellular toxicity levels, reactive oxygen and nitrogen species (RONS) levels, DNA damage, melanoma-specific markers, apoptosis, caspases and poly ADP-ribose polymerase-1 (PARP-1) levels using flow cytometer. Dual-treatment effects on the epithelial–mesenchymal transition (EMT), Hepatocyte growth factor (HGF/c-MET) pathway, sphere formation and the reversal of EMT were also assessed using western blotting and microscopy respectively. SN and plasma-activated medium (PAM) were applied on tumor growth and body weight and melanoma-specific markers and the mesenchymal markers in the tumor xenograft nude mice model were checked. Results Co-treatment of SN and air CAP increased the cellular toxicity in a time-dependent manner and shows maximum toxicity at 200 nM in 24 h. Intracellular RONS showed significant generation of ROS ( 2 times) and DNMT and showed damage in G-361 cells. Increase in Caspase 8,9,3/7 (> 1.5 times), PARP level (2.5 times) and apoptotic genes level were also observed in dual treated group and hence blocking HGF/c-MET pathway. Decrease in EMT markers (E-cadherin, YKL-40, N-cadherin, SNAI1) were seen with simultaneously decline in melanoma cells (BRAF, NAMPT) and stem cells (CD133, ABCB5) markers. In vivo results showed significant reduction in SN with PAM with reduction in tumor weight and size. Conclusions The use of air CAP using μ-DBD and the SN can minimize the malignancy effects of melanoma cells by describing HGF/c-MET molecular mechanism of acting on G-361 human melanoma cells and in mice xenografts, possibly leading to suitable targets for innovative anti-melanoma approaches in the future. Electronic supplementary material The online version of this article (10.1186/s12964-019-0360-4) contains supplementary material, which is available to authorized users.

Background Cucurbitacin E (CuE) is an oxygenated tetracyclic triterpenoid isolated from the fruits of Citrullus colocynthis (L.) Schrad. Purpose This study outlines CuE's cytotoxic activity against drug-resistant tumor cell lines. Three members of ABC transporters superfamily, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and ABCB5 were investigated, whose overexpression in tumors is tightly linked to multidrug resistance. Further factors of drug resistance studied were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). Methods Cytotoxicity assays (resazurin assays) were used to investigate the activity of Citrullus colocynthis and CuE towards multidrug resistant cancer cells. Molecular docking (In silico) has been carried out to explore the CuE's mode of binding to ABC transporters (P-gp, BCRP and ABCB5). The visualization of doxorubicin uptake was done by a Spinning Disc Confocal Microscope. The assessment of proteins expression was done by western blotting analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to cucurbitacins (CuA, CuB, CuE, CuD, CuI, and CuK). Results Multidrug-resistant cells overexpressing P-gp or BCRP were cross-resistant to CuE. By contrast, TP53 knock-out cells were sensitive to CuE. Remarkably, resistant cells transfected with oncogenic ΔEGFR or ABCB5 were hypersensitive (collateral sensitive) to CuE. In silico analyses demonstrated that CuE is a substrate for P-gp and BCRP. Immunoblot analyses highlighted that CuE targeted EGFR and silenced its downstream signaling cascades. The most striking result that emerged from the doxorubicin uptake by ABCB5 overexpressing cells is that CuE is an effective inhibitor for ABCB5 transporter when compared with verapamil. The COMPARE analyses of transcriptome-wide expression profiles of tumor cell lines of the NCI identified common genes involved in cell cycle regulation, cellular adhesion and intracellular communication for different cucurbitacins. Conclusion CuE represents a potential therapeutic candidate for the treatment of certain types of refractory tumors. To best of our knowledge, this is the first time to identify CuE and verapamil as inhibitors for ABCB5 transporter.

Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue.

Limbal stem cell deficiency has an important position among corneal blindness, it is the second most common cause of blindness in the worldwide. Hyperemia in the eye, watering of the eyes, chronic pain attacks, blepharospasm, low vision, conjunctivalization, neovascularization, and fibrovascular pannus are observed in patients and these lowering their quality of life significantly. Keratolimbal allografts, conjunctivolimbal allografts and eccentric corneal grafts were used in the treatment of these pathologies. However, as a result of the recent studies, genes and markers that belong to stem cells such as p63 gene, ABCG2, ABCB5, Vimentin, K19 and connexin were defined and thus limbal stem cells were successfully reproduced. Today, the treatment of these diseases is possible by reproducing the stem cells in a small limbal biopsy using various methods. This approach is still current and still developing. In this study we aimed to review limbal stem cells genes, markers and current treatment modalities. [Med-Science 2016; 5(1.000): 233-43]

Drug resistance remains an obstacle hindering the success of chemotherapy. Cancer stem cells (CSCs) have been recently found to confer resistance to chemotherapy. Therefore functional markers of CSCs should be discovered and specific therapies targeting these cells should be developed. In our investigation, a small population of B16F10 cells which was positive for ATP-binding cassette sub-family B member 5 (ABCB5) was isolated. This population displayed characteristics similar to those of CSCs and ABCB5 was identified to confer tumor growth and drug resistance in B16F10 cell line. Although targeting ABCB5 by small short interfering RNA delivered by VNP20009 failed to inhibit tumor growth, the combined treatment of VNP-shABCB5 and chemotherapy can act synergistically to delay tumor growth and enhance survival time in a primary B16F10 mice model. Results suggest that the combined treatment of VNP-shABCB5 and chemotherapy can improve the efficacy of chemotherapeutic drugs. Therefore, this combination therapy is of potential significance for melanoma treatment.

Melanoma is usually highly refractory to chemotherapy. This resistance to treatment is mainly due to high heterogeneity and plasticity of melanoma cells strictly connected to changes in tumor microenvironment. Hypoxia can drastically alter cancer biology. Solid tumor cells under hypoxia gain stem-like features, they are more invasive and drug-resistant than their normoxic counterparts. These effects could be mediated by changes in miRNA expression under hypoxia. MiRNAs are small non-coding RNA molecules that can negatively control gene expression. In the present study using microarray technology we evaluated the expression of miRNAs in melanoma cells derived from nodular melanoma and grown under normoxic and hypoxic conditions. Using R environment for statistical analysis we found that 70 miRNAs were differentially-expressed, and 16 of them were significantly down-regulated in melanoma cells grown in hypoxic conditions compared to cells grown in normoxia. We intended to find transcripts whose expression is increased due to down-regulation of selected miRNAs. Bioinformatics analysis revealed that increased levels of HIF-2α, ABCB5, OCT4, SOX2 and ZEB1 in different melanoma populations under hypoxia could be a result of significant down-regulation of miR-340-5p. Inhibition of miR-340-5p confirmed that this miRNA negatively influences the expression of ABCB5. This is the first study showing the relationship between miR-340-5p and expression of ABCB5, a transmembrane transporter involved in drug resistance considered as a marker of melanoma stem-like cells.

Accumulating studies have indicated that long non-coding RNAs (lncRNAs) participate in the regulation of cancer stem cells (CSCs), which are crucial in tumor initiation, metastasis, relapse, and therapy resistance. In the current study, RT-PCR analysis was employed to evaluate the expression of LINC00963 in tumor tissues and oral CSCs. Stemness phenotypes and the expression of CSCs markers in oral cancer cells transfected with sh-LINC00963 were examined. Our results showed that the expression of the lncRNA LINC00963 was up-regulated in oral cancer tissues and CSCs. We found that the downregulation of LINC00963 inhibited CSC hallmarks, such as migration, invasion and colony formation capacity. Moreover, suppression of LINC00963 reduced the activity of stemness marker ALDH1, the percentage of self-renewal, chemoresistance and the expression of multidrug-resistance transporter ABCB5. Most importantly, we demonstrated that knockdown of LINC00963 decreased self-renewal, invasion and colony formation ability via ABCB5. Analysis of TCGA (the Cancer Genome Atlas) datasets suggested that the level of LINC00963 was positively correlated with the expression of the cancer stemness markers (Sox2 and CD44) and drug resistance markers (ABCG2 and ABCB5). Altogether, our results showed that suppression of LINC00963 may be beneficial to inhibit chemoresistance and cancer relapse in oral cancer patients.

ABCB5 is an ABC transporter that was shown to confer low-level multidrug resistance in cancer. In this study, we show that ABCB5 was mutated in 13.75% of the 640 melanoma samples analyzed. Besides nonsense mutations, two mutation hotspots were found in the ABCB5 protein, in the drug-binding pocket and the nucleotide-binding domains. Four mutations, which are representative of the mutation pattern, were selected. ATPase assays showed that these mutations resulted in a decrease in basal ATP hydrolysis by ABCB5. To select informative melanoma cell lines, mutational profiles of the clinical samples were further analyzed. This study showed mutations in the tumor suppressor CDKN2A gene and the NRAS oncogene in 62.5% and 75%, respectively of the samples that had mutations in the ABCB5 gene. No mutation was found in the tumor suppressor PTEN gene, whereas the activating V600E mutation in the BRAF oncogene was found in 25% of the samples with a mutated ABCB5 gene. Studies in four melanoma cell lines with various genetic backgrounds showed an increase in the proliferation and migration capacity of mutant ABCB5-expressing cells, suggesting that ABCB5 plays a role in the development of melanoma as a tumor suppressor gene.

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+-derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5+-derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5+-derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγnull mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057–1074

P-glycoprotein (P-gp or ABCB1) and multidrug resistance associated proteins (MRPs or ABCC) belonging to the family of ATP-binding cassette (ABC) transporters protect aquatic organisms from various toxicants and pathogen exposure. The function of ABC transporters of Sinonovacula constricta in response to pathogens was explored by cloning the complete cDNA of ABCB1 and ABCC5 of S. constricta through the rapid amplification of cDNA ends. Tissue-specific expression showed that ABCB1 and ABCC5 have the highest expression in hemocytes and gills among eight examined tissues, respectively. The transport activities of ABCB1 and ABCC5 in the gills were severely inhibited after their corresponding inhibitor treatments. The expression levels and transport activities of ABCB1 and ABCB5 were also investigated by using S. constricta samples infected by Vibrio parahaemolyticus at a final concentration of 107 CFU/mL. Results showed that the mRNA expression levels of ABCB1 and ABCC5 were significantly down-regulated when exposed to V. parahaemolyticus at 6 h and 12 h (P

Background More than 50% of metastatic melanoma patients have a specific mutation in the serine/threonine kinase BRAF. This results in constitutive activation of the RAS-RAF-MEK-ERK-MAP kinase pathway, which causes uncontrolled cell growth. Vemurafenib (PLX4032) is an oral chemotherapeutic agent that targets the specific mutation V600E in the BRAF protein. Initial response rates are high in patients with BRAF mutant melanoma treated with a BRAF inhibitor such as vemurafenib, but resistance nearly always develops and disease progression ensues. There are several different mechanisms by which melanoma develops BRAF inhibitor resistance. One potential component of resistance is increased drug efflux. Overexpressed ABCB5 (ATP-binding cassette transporter, subfamily B, member 5) has been shown to efflux anti-cancer drugs from cancer cells. The purpose of this study is to determine whether ABCB5 is highly expressed in BRAF inhibitor-resistant melanoma cells and to evaluate whether ABCB5 is involved in the development of resistance to BRAF inhibitors in cutaneous melanoma. Methods We established three BRAF inhibitor-resistant melanoma cell lines with BRAF mutation. The expression level of ABCB5 in PLX-resistant cell lines was checked by real-time PCR and Western blot analysis. SK-MEL-2 melanoma cells with wild-type BRAF were used for comparison. The association of different levels of ABCB5 with the changes of ERK, p-ERK, Akt and p-Akt was also assessed by Western blotting. Re-sensitization of melanoma cells to PLX was tested by p-ERK inhibitor PD58059 and ABCB5 knockdown by ABCB5 siRNA, respectively. Results We showed that ABCB5 was overexpressed in SK-MEL-28PLXr and A2058PLXr cells but not in A375PLXr cells. ABCB5 overexpression is associated with activation of p-ERK status but not Akt. Inhibition of p-ERK re-sensitized SK-MEL-28PLXr and A2058PLXr cells to PLX treatment, but knockdown of ABCB5 did not re-sensitize A2058 PLXr and SK-MEL-28 PLXr cells to PLX treatment. Conclusion These results confirm that, even though ABCB5 was overexpressed in SK-MEL-28 and A2058 melanoma cells that develop resistance to BRAF inhibitors, ABCB5 may not be a major targetable contributor to BRAF resistance. p-ERK inhibition may play important roles in BRAF resistance in these two melanoma cell lines.

INTRODUCTION: The corneal epithelium is maintained by limbal stem cells (LSCs) that reside in the basal epithelial layer of the tissue surrounding the cornea termed the limbus. Loss of LSCs results in limbal stem cell deficiency (LSCD) that can cause severe visual impairment. Patients with partial LSCD may respond to conservative therapies designed to rehabilitate the remaining LSCs. However, if these conservative approaches fail or, if complete loss of LSCs occurs, transplantation of LSCs or their alternatives is the only option. While a number of clinical studies utilizing diverse surgical and cell culture techniques have shown favorable results, a universal cure for LSCD is still not available. Knowledge of the potential risks and benefits of current approaches, and development of new technologies, is essential for further improvement of LSCD therapies. AREAS COVERED: This review focuses on cell-based LSCD treatment approaches ranging from current available clinical therapies to preclinical studies of novel promising applications. EXPERT OPINION: Improved understanding of LSC identity and development of LSC expansion methods will influence the evolution of successful LSCD therapies. Ultimately, future controlled clinical studies enabling direct comparison of the diverse employed approaches will help to identify the most effective treatment strategies.

Background Malignant melanoma is the most malignant tumours of skin and mucous membranes mainly due to its aggressive biological behaviour and tendency to generate early metastases. Unfortunately, the mechanisms underlying the development, progression and the expression of an aggressive melanoma phenotype still remain largely unknown. Objectives The purpose of this study was to determine whether a multi-panel of molecular transcripts can be predictive for risk of recurrent disease in malignant melanoma patients. Results Peripheral blood was collected from 31 malignant melanoma patients in follow-up for melanoma and from 30 healthy volunteers randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of six markers: PAX3d, TYR, MITFm, MCAM, TGFβ2 and ABCB5. Malignant melanoma patients expressed an important number of markers, with a median value of four markers. Only PAX3d displayed a trend in terms of differences when the levels of gene expression were made in function of Breslow index. Furthermore, PAX3d showed the best diagnostic capacity among the remaining residual markers or in combination with TGFβ2 and MTIF. Conclusions We demonstrated the usefulness of multimarker qRT-PCR to detect circulating melanoma cells in blood and to potentially assessing patient disease status or progression, especially when PAX3d was used in combination with MTIFm and TGFβ2.

c-MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c-MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c-MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c-MYC increased the survival rate following 5-FU treatment in human colon cancer cells, and knockdown of endogenous c-MYC decreased it. Furthermore, c-MYC knockdown decreased the expression level of ABCB5, which is involved in 5-FU resistance. Using a chromatin immunoprecipitation assay, we found that c-MYC bound to the ABCB5 promoter region. c-MYC inhibitor (10058-F4) treatment inhibited c-MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5-FU treatment as expected, and the ABCB5 expression level was increased in 5-FU-resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5-FU and 10058-F4 treatment significantly decreased tumorigenicity in nude mice compared with 5-FU or 10058-F4 treatment alone. 10058-F4 treatment decreased the ABCB5 expression level in the presence or absence of 5-FU. In contrast, 5-FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c-MYC confers resistance to 5-FU through regulating ABCB5 expression in human colon cancer cells., Journal of Cellular and Molecular Medicine, 19(7), pp.1569-1581; 2015

According to the cancer stem-like cell (CSC) hypothesis, neoplastic clones are maintained by a small fraction of cells with stem cell properties. Also, melanoma resistance to chemo- and radiotherapy is thought to be attributed to melanoma stem-like cells (MSCs). Caffeic acid phenethyl ester (CAPE) is a bioactive molecule, whose antitumor activity is approved in different tumor types. CAPE induced both apoptosis and E2F1 expression in CD133(-), but not in CD133(+) melanoma subpopulations. The resistance of CD133(+) melanoma subpopulation is attributed to the enhanced drug efflux mediated by ATP-binding cassette sub-family B member 5 (ABCB5), since the knockdown of ABCB5 was found to sensitize CD133(+) cells to CAPE. CAPE-induced apoptosis is mediated by E2F1 as evidenced by the abrogation of apoptosis induced in response to the knockdown of E2F1. The functional analysis of E2F1 in CD133(+) melanoma subpopulation demonstrated the ability of E2F1 gene transfer to trigger apoptosis of CD133(+) cells and to enhance the activation of apoptosis signal-regulating kinase (ASK1), c-Jun N-terminal kinase and p38, and the DNA-binding activities of the transcription factors AP-1 and p53. Also, the induction of E2F1 expression was found to enhance the expression of the pro-apoptotic proteins Bax, Noxa and Puma, and to suppress the anti-apoptotic protein Mcl-1. Using specific pharmacological inhibitors we could demonstrate that E2F1 overcomes the chemo-resistance of MSCs/CD133(+) cells by a mechanism mediated by both mitochondrial dysregulation and ER-stress-dependent pathways. In conclusion, our data addresses the mechanisms of CAPE/E2F1-induced apoptosis of chemo-resistant CD133(+) melanoma subpopulation.

Apigenin is a common dietary flavonoid with considerable cytotoxic activity in vitro and in vivo. Despite many mechanistic studies, less is known about resistance factors hampering apigenin's activity. We investigated the ATP-binding cassette (ABC) transporters BCRP/ABCG2, P-glycoprotein/ABCB1 and its close relative ABCB5. Multidrug-resistant cells overexpressing these ABC transporters were not cross-resistant toward apigenin. Moreover, apigenin inhibited not only P-glycoprotein but also BCRP by increasing cellular uptake of doxorubicin and synergistic inhibition of cell viability in combination with doxorubicin or docetaxel in multidrug-resistant cells. To perform in silico molecular docking studies, we first generated homology models for human P-glycoprotein and ABCB5 based on the crystal structure of murine P-glycoprotein. Their nucleotide binding domains (NDBs) revealed the highest degrees of sequence homologies (89%-100%), indicating that ATP binding and cleavage is of crucial importance for ABC transporters. Molecular docking of apigenin bound to the NDBs of P-glycoprotein and ABCB5 in molecular docking studies. Hence, apigenin may compete with ATP for NDB-binding leading to energy depletion to fuel the transport of ABC transporter substrates. Furthermore, we performed COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression profiles of the National Cancer Institute tumor cell line panel. Microarray-based mRNA expressions of genes of diverse biological functions (signal transduction, transcriptional regulation, ubiquitination, autophagy, metabolic activity, xenobiotic detoxification and microtubule formation) significantly predicted responsiveness of tumor cells to apigenin. In conclusion, apigenin's activity is not hampered by classical mechanisms of multidrug resistance and the inhibition of ABC transporters by apigenin indicates that apigenin may overcome multidrug resistance in otherwise refractory tumors.

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self-renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor-α-Converting Enzyme (TACE) and γ-secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch-2. In addition, there was a reduction in the expression of downstream target proteins, Hes-1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ-secretase complex proteins in melanoma cells. To confirm that suppression of Notch-2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes-1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling.

For decades, natural products represented a significant source of diverse and unique bioactive lead compounds in drug discovery field. In Clinical oncology, complete tumors remission is hampered by the development of drug-resistance. Therefore, development of cytotoxic agents that may overcome drug resistance is urgently needed. Here, the natural benzophenanthridine alkaloid sanguinarine has been studied for its cytotoxic activity against multidrug resistance (MDR) cancer cells. We investigated the role of the ATP-binding cassette (ABC) transporters BCRP/ABCG2, P-glycoprotein/ABCB1 and its close relative ABCB5 in drug resistance. Further drug resistance mechanisms analyzed in this study were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). Multidrug resistant cells overexpressing BCRP, ABCB5 and mutated ΔEGFR were not cross-resistant toward sanguinarine. Interestingly, P-gp overexpressing cells were hypersensitive to sanguinarine. Doxorubicin uptake assay carried by flow cytometry revealed that sanguinarine is a potent inhibitor of the P-gp transporter. Moreover, immunoblotting analysis proved that P-gp was downregulated in a dose dependent manner after treating P-gp overexpressing cells with sanguinarine. It was surmised that The inhibition of NFκB activity might explain the collateral sensitivity in CEM/ADR5000 cells. The COMPARE and hierarchical cluster analyses of transcriptome-wide expression profiles of tumor cell lines of the National Cancer Institute identified genes involved in various cellular processes (immune response, inflammation signaling, cell migration and microtubule formation) significantly correlated with log10IC50 values for sanguinarine. In conclusion, sanguinarine may have therapeutic potential for treating multidrug resistant tumors.

Melanoma is the most aggressive type of skin cancer. Melanoma has an extremely poor prognosis because of its high potential for vascular invasion, metastasis and recurrence. The mechanism of melanoma metastasis is not well understood. ATP-binding cassette sub-family B member 5 (ABCB5) plays a key role in melanoma growth. However, it is uncertain what function ABCB5 may exert in melanoma metastasis. In this report, we for the first time demonstrate ABCB5 as a crucial factor that promotes melanoma metastasis. ABCB5 positive (ABCB5+) malignant melanoma initiating cells (MMICs) display a higher metastatic potential compared with ABCB5 negative (ABCB5-) melanoma subpopulation. Knockdown of ABCB5 expression reduces melanoma cell migration and invasion in vitro and melanoma pulmonary metastasis in tumor xenograft mice. ABCB5 and NF-κB p65 expression levels are positively correlated in both melanoma tissues and cell lines. Consequently, ABCB5 activates the NF-κB pathway by inhibiting p65 ubiquitination to enhance p65 protein stability. Our finding highlights ABCB5 as a novel pro-metastasis factor and provides a potential therapeutic target for melanoma.

Apoptosis resistance is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC).Expression of apoptosis resistance-related ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] and DNaseX (Apo10) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry.Expression of ABCB5 and Apo10 were significantly increased in the carcinogenesis of OSCC compared with normal tissue. Compared with SIN I-III, ABCB5 expression was significantly decreased in OSCC. Apo10 expression did not significantly differ from OSCC compared with SIN I-III.This study provides the first evidence of the expression of ABCB5 and Apo10 in the multi-step carcinogenesis of OSCC. Overcoming drug resistance of ABCB5+ and Apo10+ cells in precursor lesions and tumors by natural compounds may act as sensitizers for apoptosis or could be useful for chemoprevention.

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

International audience; Understanding the molecular and cellular processes underlying melanoma plasticity and heterogeneity is of paramount importance to improve the efficiency of current treatment and to overcome resistance to chemotherapy drugs. The notion of plasticity and heterogeneity implies the existence of melanoma cell populations with different phenotypic and tumorigenic properties. Using melanoma cell lines and melanoma cells freshly isolated from patient biopsies, we investigated the relationship between ABCB5+, CD271+ and low-MITF, expressing populations that were reported to display melanoma initiating cell properties. Here, we showed that ABCB5+ and CD271+ populations poorly overlap. However, we found that the CD271+ population is enriched in low-MITF cells and expresses a higher level of stemness genes, such as OCT4, NANOG and NES. These features could explain the increased tumorigenicity of the CD271+ cells. The rapid conversion of CD271+ to CD271- cells in vitro demonstrates the plasticity ability of melanoma cells. Finally, we observed that the transient slow-growing population contains only CD271+ cells that are highly tumorigenic. However, the fast growing/CD271+ population exhibits a poor tumorigenic ability. Taking together, our data show that CD271 is an imperfect marker for melanoma initiating cells, but may be useful to identify melanoma cells with an increased stemness and tumorigenic potential.

MicroRNAs (miRNAs) are small noncoding RNAs, which regulate the expression of their target genes post-transcriptionally by RNA interference. They are involved in almost all cellular processes, including proliferation, differentiation, apoptosis, cell survival and the maintenance of tissue specificity. Recent findings also suggest that efflux pumps of the ABC (ATP-binding cassette) transporter family are subject to miRNA-mediated gene regulation. Moreover, it seems that ABC transporters are embedded in a concerted and miRNA-guided network of concurrently regulated proteins that mediate altered drug transport and cell survival in changing environmental conditions. In this review, we summarize recent findings of miRNAs interacting with ABC transporters, which have been connected with drug distribution as well as with drug resistance. Additionally, we specify findings of complex miRNA–protein pathways conferring increased drug export and cell survival.

Cancer stem cells (CSCs) have been identified in various malignancies, and different properties have been examined to characterize CSCs: tumorigenicity in immunocompromised mice, stem cell surface markers, label-retaining properties, and proliferation as nonadherent spheres. This study explored the consistency and efficiency among these methods. Among the melanoma cell lines examined (A375, A875, MUM-2b, and MUM-2c), only A375 and MUM-2c grew as nonadherent spheres and continuously propagated in a defined serum-free medium in vitro. Flow cytometry and immunofluorescence analysis indicated that sphere-derived cells contained a smaller proportion of cells expressing the candidate surface markers of melanoma stem cells such as ABCB5, CD133, CD20 and CD271, and a larger proportion of cells expressing melanocytic differentiation markers such as HMB45 and S100 protein, compared with adherent cells. Surprisingly, the more highly differentiated sphere-derived melanoma cells exhibited increased tumorigenic potential in vivo, as indicated by shorter tumor incubation (A375) and smaller number of cells required to initiate tumor formation (A375 and MUM-2c) compared with those of parental cells. Despite the similarity in histopathological characteristics, the expression profile indicated that xenografts derived from sphere-derived melanoma cells exhibited a more tumorigenic phenotype with respect to the stem or the differentiation markers detected by immunohistochemical analysis. Therefore, sphere formation in nonadherent cultures may not be a preferred surrogate in-vitro method for enriching melanoma stem cells according to candidate markers but may be a favorable condition for activating potential CSCs.