ABCB1 Expression Research Articles

Abstract 1685: Overcoming acquired resistance to PROTAC degraders

Abstract Background: Proteolysis-targeting chimera (PROTAC) technology has been widely investigated for cancer treatment and there have been several PROTAC degrader-based drugs used for clinical trials in the past few years. PROTAC degraders are able to target crucial proteins traditionally thought to be “undruggable”, such as transcriptional factors. Hence, there is increasing interest in these drugs due to their highly efficacy and low off-target toxicity. However, upon further investigation, it has been found that after a period of treatment with PROTAC degraders, cancer cells can acquire resistance to these compounds. Thus, it is of increasing importance to figure out the mechanisms of the resistance and to overcome the acquired resistance to these PROTAC degraders. In our study, we classified resistance mechanisms of prostate cancer cells to PROTAC degraders into two classes and identified factors that can induce them to preferentially develop specific resistance mechanisms. Methods and Results: AU-15330 is a PROTAC that specifically degrades the subunits (BRG1, PBRM1, BRM) of SWI/SNF complex. We treated 22rv1, a prostate cancer cell line, with different AU-15330 concentrations over a month to establish AU-15330-resistant cell lines. Whole exome sequencing (WES) analysis of these resistant cell lines found that the resistant cell lines that were developed through high-dose (1uM) AU-15330 treatment acquired multiple point mutations within or adjacent to the AU-15330 targeting bromodomain of BRG1. However, BRG1 point mutations were absent in the resistant cell lines that developed through lower concentration of AU-15330 treatment (100nM). Instead, RNA-seq analysis revealed that ABCB1 (also known as Multidrug Resistance Protein 1) expression was upregulated in cells that developed resistance to AU-15330 at 100nM. This was further confirmed by Western blot and qPCR. Next, we overexpressed ABCB1 in LNCaP and 22RV1, and found that overexpression of ABCB1 indeed made prostate cancer cells resistant to AU15330. In addition, we tested with several different potent PROTAC degraders (ARD616, ZBC260, etc) in ABCB1-expressing AU-15530 resistant cells, and found the ABCB1 expression drove the cells to become resistant to all tested PROATC degraders compared to the parental cells. Lastly, we demonstrated that Zosuquidar, an ABCB1 inhibitor, can overcome the ABCB1-mediated resistance to AU-15330 and other PROTAC degraders, suggesting that inhibiting ABCB1 might be a direct and effective strategy to restore PROTAC degrader’s potency when ABCB1-mediated drug resistance is developed. Conclusion: Our study found that the mechanisms of resistance of PROTAC degraders can vary and may be dependent on drug concentrations. Such concepts may inform future clinical decision-making regarding drug dosing. Citation Format: Tongchen He, Caleb Cheng, Abhijit Parolia, Alex Hopkins, Yuanyuan Qiao, Lanbo Xiao, Arul Chinnaiyan. Overcoming acquired resistance to PROTAC degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1685.

Abstract 418: Elacridar potentiates sunitinib efficacy in colorectal cancer models

Abstract For unresectable metastatic colorectal cancer (mCRC), the main treatment strategy is combination chemotherapy, targeted therapy, or immunotherapy. Long-term treatment benefit of these therapies is limited to a small subgroup of patients. To improve treatment outcome, many tyrosine kinase inhibitors (TKIs) have been clinically tested in patients with mCRC. Most failed already in early-phase clinical trials due to intrinsic resistance. ATP Binding Cassette Transporter (ABC)B1- and ABCG2-transporters are known contributors to treatment resistance in mCRC. Therefore, we evaluated whether inhibition of these transporters could potentiate the efficacy of TKIs in colorectal cancer (CRC) models. CRC cell lines, HT29, COLO320 and CACO2 and newly established patient-derived tumor organoids (PDTOs) from mCRC biopsies were first analyzed for the presence of ABCB1 and ABCG2 expression. Models that expressed ABCB1 or ABCG2 were pre-incubated with a non-toxic dose of elacridar, a potent transporter inhibitor, and exposed to a concertation range of three TKIs; sunitinib, cediranib and osimertinib. Next, we measured intracellular drug concentrations using LC/MS-MS one hour after TKI exposure (20 µM) with or without pre-incubation of elacridar. Using the fluorescent properties of sunitinib, we performed live-cell imaging to compare intracellular sunitinib distribution in cells that were or were not pre-incubated with elacridar. ABCB1 and ABCG2 expression was observed in all CRC models except for HT29 cells, which did not express ABCB1. Pre-incubation with elacridar significantly improved sunitinib efficacy in CRC cells. The IC50 decreased from 2.15 µM to 1.17 µM (p = 0.04), from 1.32 µM to 0.44 µM (p = 0.03), and from 4.94 µM to 3.43 µM (p = 0.03) for HT29, COLO320 and CACO2 cells respectively. This effect was not observed for cediranib and osimertinib. Although not significant, for both PDTO models tested, the IC50 for sunitinib decreased from 8.04 µM to 3.29 µM and from 6.93 µM to 4.16 µM. Intracellular sunitinib concentrations in the tested CRC cell models after sunitinib exposure ranged from 2.46 mM to 4.13 mM without elacridar and from 0.38 mM µM to 1.31 mM when pre-incubated with elacridar (p < 0.01). Live-cell imaging of CRC cells revealed a different pattern of intracellular sunitinib accumulation with and without pre-incubation with elacridar. Inhibition of multi-drug resistance proteins ABCB1 and ABCG2 with the clinically tested selective transporter inhibitor elacridar potentiates sunitinib efficacy in CRC models. Interestingly, this was accompanied with reduced intracellular sunitinib accumulation. Further research in mCRC PDTOs is warranted to confirm whether this mechanism is involved in intrinsic TKI resistance and therefore contribute to the early-phase failure of clinical trials testing TKIs for mCRC. Citation Format: Dennis Poel, Kirti K. Iyer, Bob van Gasteren, Beau Dagniaux, Erik van den Hombergh, Nielka P. van Erp, Daniele V. Tauriello, Henk M. Verheul. Elacridar potentiates sunitinib efficacy in colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 418.

Abstract 3660: Bone mechanics influence cancer stem cell formation in osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary tumor of bone and is characterized by rapid bone remodeling accompanied by structural and mechanical heterogeneity across the tumor. Chemoresistance and recurrence of OS can be attributed to a subpopulation of OS cells known as cancer stem-like cells (OS-CSCs). It can be hypothesized that tumor niches possessing distinct structural and mechanical properties could be harboring different OS cell populations, and certain niches might be more conducive for OS-CSCs formation and maintenance. To evaluate the presence of OS-CSCs, mineralized collagen scaffolds mimicking heterogenous bone niches with distinct structural and mechanical properties were fabricated by changing collagen content and pore alignment and were cultured with the highly metastatic human OS cell line 143B. Scaffolds with 47% collagen by weight but different pore alignment were chosen for further analysis because they possessed different stiffness and topographical properties. Cytoskeletal changes, epigenetic changes, chemoresistance and chemoresistance-related pathways, and the levels of stemness genes were assessed in the 3D scaffold cultures. A consistent trend of OS-CSC phenotype was observed in the cell population grown in the softer bone niches (12 kPa) with aligned pores as compared to stiffer niche (31 kPa) with non-aligned pores. The cell population growing in softer, aligned scaffolds had a significantly higher expression of stemness genes (OCT4, NANOG, SOX2, ALDH1A1) and drug efflux gene ABCB1 and increased chemoresistance to doxorubicin and cisplatin. Epigenetic changes associated with cisplatin resistance in OS such as a decrease in methyl transferase EZH2 accompanied by the upregulation of LIF/Notch and PRKCA pathways was observed in cells grown in softer, aligned niches. Fluorescent imaging of actin organization showed rounder cells in the aligned niche indicative of the CSC phenotype, and stretched, elongated cells in the non-aligned niche indicative of a differentiated phenotype. Our study showed that the mechanical and structural properties of bone niches influence the formation of distinct OS cell populations. A softer niche with an aligned structure is more conducive for maintaining OS-CSCs. Citation Format: Zunaira Shoaib, Aleczandria S. Tiffany, Brendan Harley, Joseph Irudayaraj, Timothy M. Fan. Bone mechanics influence cancer stem cell formation in osteosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3660.

Abstract 3709: Hyperglycemia induces metabolic reprogramming and promotes epithelial-mesenchymal transitions in pancreatic cancer: an in vitro and in vivo experiment

Abstract Background: Pancreatic cancer is a serious life-threatening disease due to its highly invasive and metastatic potential. Hyperglycemia is a well-known initial symptom in patients with pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming in cancer, described as the Warburg effect, can induce epithelial-mesenchymal transition (EMT). In this study, we elucidate the biological and clinical impact of hyperglycemia in PDAC progression. Methods: Biological impact of hyperglycemia on malignant behavior in PDAC was examined by in vitro and in vivoexperiment. Results: Hyperglycemia promoted EMT by inducing metabolic reprogramming into a glycolytic phenotype via yes-associated protein (YAP)/PDZ-binding motif (TAZ) overexpression, accompanied by GLUT1 overexpression and enhanced phosphorylation Akt in PDAC. In addition, hyperglycemia enhanced chemoresistance by upregulating ABCB1 expression, and triggered PDAC switch into pure-basal-like subtype with activated Hedgehog pathway (GLI1 high, GATA6 low expression) through YAP/TAZ overexpression. PDAC is characterized by abundant stroma that harbors tumor-promoting properties and chemoresistance. Hyperglycemia promotes production of collagen fiber-related proteins (fibronectin, fibroblast activation protein, COL1A1, and COL11A1) by stimulating YAP/TAZ expression in cancer-associated fibroblasts (CAFs). Knockdown of YAP and/or TAZ or treatment with YAP/TAZ inhibitor (K975) abolished EMT, chemoresistance, and a favorable tumor microenvironment even under hyperglycemic conditions in vitro and in vivo. Indeed, YAP/TAZ overexpression was significantly associated with worse prognostic outcomes in human PDAC patients. Conclusion: Hyperglycemia induce metabolic reprogramming into a glycolytic phenotype and promote EMT via the YAP/TAZ-Hedgehog signaling axis in PDAC. Hyperglycemia as an initial symptom may be the cause of highly invasive and metastatic potential by inducing YAP/TAZ overexpression, while YAP/TAZ could be a novel therapeutic target in PDAC patients. Citation Format: Zhao Liu, Hiromitsu Hayashi, Kazuki Matsumura, Yoko Ogata, Hiroki Sato, Yuta Shiraishi, Norio Uemura, Tatsunori Miyata, Takaaki Higashi, Shigeki Nakagawa, Kosuke Mima, Katsunori Imai, Hideo Baba. Hyperglycemia induces metabolic reprogramming and promotes epithelial-mesenchymal transitions in pancreatic cancer: an in vitro and in vivo experiment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3709.

ABCB1 expression is increased in human first trimester placenta from pregnant women classified as overweight or obese

Obesity has become a global health challenge also affecting reproductive health. In pregnant women, obesity increases the risk of complications such as preterm birth, macrosomia, gestational diabetes, and preeclampsia. Moreover, obesity is associated with long-term adverse effects for the offspring, including increased risk of cardiovascular and metabolic diseases and neurodevelopmental difficulties. The underlying mechanisms are far from understood, but placental function is essential for pregnancy outcome. Transporter proteins P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are important for trans-placental transport of endogenous substances like lipids and cortisol, a key hormone in tissue maturation. They also hold a protective function protecting the fetus from xenobiotics (e.g. pharmaceuticals). Animal studies suggest that maternal nutritional status can affect expression of placental transporters, but little is known about the effect on the human placenta, especially in early pregnancy. Here, we investigated if overweight and obesity in pregnant women altered mRNA expression of ABCB1 encoding P-gp or ABCG2 encoding BCRP in first trimester human placenta. With informed consent, 75 first trimester placental samples were obtained from women voluntarily seeking surgical abortion (< gestational week 12) (approval no.: 20060063). Villous samples (average gestational age 9.35 weeks) were used for qPCR analysis. For a subset (n = 38), additional villi were snap-frozen for protein analysis. Maternal BMI was defined at the time of termination of pregnancy. Compared to women with BMI 18.5–24.9 kg/m2 (n = 34), ABCB1 mRNA expression was significantly increased in placenta samples from women classified as overweight (BMI 25–29.9 kg/m2, n = 18) (p = 0.040) and women classified as obese (BMI ≥ 30 kg/m2, n = 23) (p = 0.003). Albeit P-gp expression did not show statistically significant difference between groups, the effect of increasing BMI was the same in male and female pregnancies. To investigate if the P-gp increase was compensated, we determined the expression of ABCG2 which was unaffected by maternal obesity (p = 0.291). Maternal BMI affects ABCB1 but not ABCG2 mRNA expression in first trimester human placenta. Further studies of early placental function are needed to understand how the expression of placental transport proteins is regulated by maternal factors such as nutritional status and determine the potential consequences for placental–fetal interaction.

ABCB1 and ABCC1 Function during TGF-β-Induced Epithelial-Mesenchymal Transition: Relationship between Multidrug Resistance and Tumor Progression.

Multidrug resistance (MDR) and induction of metastasis are some of the puzzles encountered during cancer chemotherapy. The MDR phenotype is associated with overexpression of ABC transporters, involved in drug efflux. Metastasis originates from the epithelial-mesenchymal transition (EMT), in which cells acquire a migratory phenotype, invading new tissues. ABC transporters' role during EMT is still elusive, though cells undergoing EMT exhibit enhanced ABCB1 expression. We demonstrated increased ABCB1 expression but no change in activity after TGF-β-induced EMT in A549 cells. Moreover, ABCB1 inhibition by verapamil increased snail and fibronectin expression, an event associated with upregulation of ABCB1, evidencing coincident cell signaling pathways leading to ABCB1 and EMT-related markers transcription, rather than a direct effect of transport. Additionally, for the first time, increased ABCC1 expression and activity was observed after EMT, and use of ABCC1 inhibitors partially inhibited EMT-marker snail, although increased ABCC1 function translated into collateral sensibility to daunorubicin. More investigations must be done to evaluate the real benefits that the gain of ABC transporters might have on the process of metastasis. Considering ABCC1 is involved in the stress response, affecting intracellular GSH content and drug detoxification, this transporter could be used as a therapeutic target in cancer cells undergoing EMT.

TNFα Activates the Liver X Receptor Signaling Pathway and Promotes Cholesterol Efflux from Human Brain Pericytes Independently of ABCA1.

Neuroinflammation and brain lipid imbalances are observed in Alzheimer's disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (LXR) signaling pathways are involved in both processes. However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the LXR pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (ABCA1), while ABCG1 is not expressed. Apolipoprotein E (APOE) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when ABCA1 or LXR are blocked. Moreover, as for TNFα, direct LXR activation by the agonist (T0901317) increases ABCA1 expression and the associated cholesterol efflux. However, this process is abolished when LXR/ABCA1 are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases ABCB1 expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an LXR/ABCA1 independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders.

Identification and Empiric Evaluation of New Inhibitors of the Multidrug Transporter P-Glycoprotein (ABCB1).

The expression of the drug efflux pump ABCB1 correlates negatively with cancer survival, making the transporter an attractive target for therapeutic inhibition. In order to identify new inhibitors of ABCB1, we have exploited the cryo-EM structure of the protein to develop a pharmacophore model derived from the best docked conformations of a structurally diverse range of known inhibitors. The pharmacophore model was used to screen the Chembridge compound library. We identified six new potential inhibitors with distinct chemistry compared to the third-generation inhibitor tariquidar and with favourable lipophilic efficiency (LipE) and lipophilicity (CLogP) characteristics, suggesting oral bioavailability. These were evaluated experimentally for efficacy and potency using a fluorescent drug transport assay in live cells. The half-maximal inhibitory concentrations (IC50) of four of the compounds were in the low nanomolar range (1.35 to 26.4 nM). The two most promising compounds were also able to resensitise ABCB1-expressing cells to taxol. This study demonstrates the utility of cryo-electron microscopy structure determination for drug identification and design.

The Molecular Mechanisms of Oleanane Aldehyde-β-enone Cytotoxicity against Doxorubicin-Resistant Cancer Cells.

Oleanane aldehyde-β-enone (OA), being the semi-synthetic derivative of the triterpenoid betulin, effectively inhibits the proliferation of HBL-100 and K562 cancer cells (IC50 0.47-0.53 µM), as well as the proliferation of their resistant subclones with high P-gp expression HBL-100/Dox, K562/i-S9 and K562/i-S9_Dox (IC50 0.45-1.24 µM). A molecular docking study, rhodamine efflux test, synergistic test with Dox, and ABC transporter gene expression were used to investigate the ability of OA to act as a P-gp substrate or inhibitor against Dox-resistant cells. We noted a trend toward a decrease in ABCB1, ABCC1 and ABCG2 expression in HBL-100 cells treated with OA. The in silico and in vitro methods suggested that OA is neither a direct inhibitor nor a competitive substrate of P-gp in overexpressing P-gp cancer cells. Thus, OA is able to overcome cellular resistance and can accumulate in Dox-resistant cells to realize toxic effects. The set of experiments suggested that OA toxic action can be attributed to activating intrinsic/extrinsic or only intrinsic apoptosis pathways in Dox-sensitive and Dox-resistant cancer cells, respectively. The cytotoxicity of OA in resistant cells is likely mediated by a mitochondrial cell death pathway, as demonstrated by positive staining with Annexin V-FITC, an increasing number of cells in the subG0/G1 phase, reactive oxygen species generation, mitochondrial dysfunction, cytochrome c migration and caspases-9,-6 activation.

Effect of Polyunsaturated Fatty Acids on Temozolomide Drug-Sensitive and Drug-Resistant Glioblastoma Cells.

Glioblastomas (GBMs) are notoriously difficult to treat, and the development of multiple drug resistance (MDR) is common during the course of the disease. The polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have been reported to improve MDR in several tumors including breast, bladder, and leukaemia. However, the effects of PUFAs on GBM cell MDR are poorly understood. The present study investigated the effects of PUFAs on cellular responses to temozolomide (TMZ) in U87MG cells and the TMZ-resistant (TMZR) cells derived from U87MG. Cells were treated with PUFAs in the absence or presence of TMZ and dose-response, viable cell counting, gene expression, Western blotting, flow cytometry, gas chromatography-mass spectrometry (GCMS), and drug efflux studies were performed. The development of TMZ resistance caused an increase in ABC transporter ABCB1 and ABCC1 expression. GLA-, EPA-, and DHA-treated cells had altered fatty acid composition and accumulated lipid droplets in the cytoplasm. The most significant reduction in cell growth was seen for the U87MG and TMZR cells in the presence of EPA. GLA and EPA caused more significant effects on ABC transporter expression than DHA. GLA and EPA in combination with TMZ caused significant reductions in rhodamine 123 efflux from U87MG cells but not from TMZR cells. Overall, these findings support the notion that PUFAs can modulate ABC transporters in GBM cells.

Imidazo[1,2-a]Pyridine Derivatives as Novel Dual-Target Inhibitors of ABCB1 and ABCG2 for Reversing Multidrug Resistance.

ABCB1 and ABCG2 are the important ATP-binding cassette (ABC) transporters associated with multidrug resistance (MDR). Herein, we designed a series of imidazo[1,2-a]pyridine derivatives as dual-target inhibitors of ABCB1 and ABCG2 through the scaffold hopping strategy. Compound Y22 displayed potential efflux function inhibitory toward both ABCB1 and ABCG2 (reversal fold: ABCB1 = 8.35 and ABCG2 = 2.71) without obvious cytotoxicity. Y22 also enhanced the potency of antiproliferative drugs in vitro. Mechanistic studies demonstrated that Y22 slightly suppressed ATPase activity but did not affect the protein expression of ABCB1 or ABCG2. Notably, Y22 exhibited negligible CYP3A4 inhibition and enhanced the antiproliferative activity of adriamycin in vivo by restoring the sensitivity of resistant cells. Thus, Y22 may be effective clinically in combination with common chemotherapy agents. In summary, Y22 is a potential dual-target inhibitor that reverses MDR by blocking the efflux function of ABCB1 and ABCG2.

Molecular aspects of ABCB1 and ABCG2 in Gallbladder cancer and its clinical relevance.

The function of ABC transporters in the body is manifold; such as maintenance of homeostasis, effect on multi-drug resistance and their role in tumor initiation & progression. Evidence pointing towards the direct or indirect role of ABC transporter genes in particular; ABCB1 and ABCG2 in cancer genesis is increasing. However, their role in gallbladder cancer is unexplored. Therefore, we investigated the methylation status and expression pattern of ABCB1 and ABCG2in gallbladder carcinogenesis. The methylation and expression study of ABCB1/MDR1 and ABCG2/BCRP was performed in tumour and normal fresh tissue samples collected from 61 histopathologically diagnosed gallbladder cancer patients. The methylation status was analysed by Methylation-Specific PCR and expression was determined by Real-Time PCR and Immunohistochemistry. Hypomethylation of ABCB1 and ABCG2 was found in 44 (72.13%) and 48 (78.6%) cases, respectively. ABCB1 hypomethylation pattern showed association with female patients (p = 0.040) and GradeII tumors (p = 0.036) while, ABCG2hypomethylation was more frequent in early tumors (T1-T2). The mRNA expression ofABCB1 and ABCG2 was up-regulated in 33 (54.10%) and 41 (67.21%) patients with fold change of 4.7 and 5.5, respectively. The mRNA expression of both genes showed association with Grade II tumours and the increased fold change of ABCG2 was higher in (T1-T2) depth of invasion (p = 0.02) and Stage I-II disease (p = 0.08). The protein expression on IHC was strongly positive for ABCB1/MDR1and ABCG2/BCRP in 32 (52.46%) and 45 (73.77%) patients, respectively. The protein expression in ABCG2 showed association with patients age > 50years (p = 0.04) and GradeII differentiation (p = 0.07). Interestingly, the hypomethylation of both the genes showed significant correlation with increased expression. ABCB1/MDR1 and ABCG2/BCRP hypomethylation and overexpression could have a potential role in gallbladder cancer tumorigenesis especially in early stages. The epigenetic change might be a plausible factor for altered gene expression of ABCB1 and ABCG2 in gallbladder cancer.

RETRACTED ARTICLE: LncRNA BCAR4 promotes migration, invasion, and chemo-resistance by inhibiting miR-644a in breast cancer

BackgroundMetastasis and drug resistance of breast cancer have become a barrier to treating patients successfully. Long noncoding RNAs (lncRNAs) are known as vital players in cancer development and progression. MethodsThe RT-qPCR were used to detect the gene expression. Colony formation assay, would healing assay, and transwell assay were performed to investigate oncogenic functions of cells. CCK8 assay was used to detect the cell viability. Western blot was applied to detect the protein level. Dual-luciferase reporter assay was used to determine the relationship between molecules. Mouse orthotopic xenograft tumor models were established to evaluate the effects of BCAR4 on tumor growth and metastasis in vivo. ResultsLncRNA BCAR4 was significantly increased in breast cancer patients’ tissues and plasma and upregulated in breast cancer cell lines. BCAR4 upregulation was correlated with the TNM stages and decreased after surgical removal of breast tumors. Silencing of BCAR4 suppressed breast cancer cell colony formation, migration, invasion, and xenograft tumor growth and promoted chemo-sensitivity. Mechanistically, BCAR4 facilitates breast cancer migration and invasion via the miR-644a-CCR7 axis of the MAPK pathway. BCAR4 promotes ABCB1 expression indirectly by binding to and down-regulating miR-644a to induce chemo-resistance in breast cancer.ConclusionsOur findings provide insights into the oncogenic role of BCAR4 and implicate BCAR4 as a potential diagnostic biomarker and a promising therapeutic agent to suppress metastasis and inhibit chemo-resistance of breast cancer.

Protoporphyrin IX (PpIX) Fluorescence during Meningioma Surgery: Correlations with Histological Findings and Expression of Heme Pathway Molecules.

Background: The usefulness of 5-ALA-mediated fluorescence-guided resection (FGR) in meningiomas is controversial, and information on the molecular background of fluorescence is sparse. Methods: Specimens obtained during 44 FGRs of intracranial meningiomas were analyzed for the presence of tumor tissue and fluorescence. Protein/mRNA expression of key transmembrane transporters/enzymes involved in PpIX metabolism (ABCB6, ABCG2, FECH, CPOX) were investigated using immunohistochemistry/qPCR. Results: Intraoperative fluorescence was observed in 70 of 111 specimens (63%). No correlation was found between fluorescence and the WHO grade (p = 0.403). FGR enabled the identification of neoplastic tissue (sensitivity 84%, specificity 67%, positive and negative predictive value of 86% and 63%, respectively, AUC: 0.75, p < 0.001), and was improved in subgroup analyses excluding dura specimens (86%, 88%, 96%, 63% and 0.87, respectively; p < 0.001). No correlation was found between cortical fluorescence and tumor invasion (p = 0.351). Protein expression of ABCB6, ABCG2, FECH and CPOX was found in meningioma tissue and was correlated with fluorescence (p < 0.05, each), whereas this was not confirmed for mRNA expression. Aberrant expression was observed in the CNS. Conclusion: FGR enables the intraoperative identification of meningioma tissue with limitations concerning dura invasion and due to ectopic expression in the CNS. ABCB6, ABCG2, FECH and CPOX are expressed in meningioma tissue and are related to fluorescence.

Hyperglycaemia induces metabolic reprogramming into a glycolytic phenotype and promotes epithelial-mesenchymal transitions via YAP/TAZ-Hedgehog signalling axis in pancreatic cancer.

Hyperglycaemia is a well-known initial symptom in patients with pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming in cancer, described as the Warburg effect, can induce epithelial-mesenchymal transition (EMT). The biological impact of hyperglycaemia on malignant behaviour in PDAC was examined by in vitro and in vivo experiments. Hyperglycaemia promoted EMT by inducing metabolic reprogramming into a glycolytic phenotype via yes-associated protein (YAP)/PDZ-binding motif (TAZ) overexpression, accompanied by GLUT1 overexpression and enhanced phosphorylation Akt in PDAC. In addition, hyperglycaemia enhanced chemoresistance by upregulating ABCB1 expression and triggered PDAC switch into pure basal-like subtype with activated Hedgehog pathway (GLI1 high, GATA6 low expression) through YAP/TAZ overexpression. PDAC is characterised by abundant stroma that harbours tumour-promoting properties and chemoresistance. Hyperglycaemia promotes the production of collagen fibre-related proteins (fibronectin, fibroblast activation protein, COL1A1 and COL11A1) by stimulating YAP/TAZ expression in cancer-associated fibroblasts (CAFs). Knockdown of YAP and/or TAZ or treatment with YAP/TAZ inhibitor (K975) abolished EMT, chemoresistance and a favourable tumour microenvironment even under hyperglycemic conditions in vitro and in vivo. Hyperglycaemia induces metabolic reprogramming into glycolytic phenotype and promotes EMT via YAP/TAZ-Hedgehog signalling axis, and YAP/TAZ could be a novel therapeutic target in PDAC.

Prognostic and Predictive Significance of mRNA Expression of ABC-transporter Genes in Patients with Non-small Cell Lung Cancer

Introduction. To date, one of the reasons for the ineffectiveness of chemotherapy in various malignant neoplasms, including lung cancer, is the formation of the multidrug resistance (MDR) phenotype in tumor cells, which is caused by the expression of ABC transporter genes.Aim. The aim of this work was to assess the expression of ABC-transporter genes during chemotherapy and to analyze the relationship with the effectiveness of chemotherapy and prognosis of the disease.Materials and methods. We used biopsy and surgical material from 91 patients with stage IIB–IIIA non-small cell lung cancer (NSCLC). The treatment regimen included: 2 courses of neoadjuvant chemotherapy (NAC), surgery and 3 courses of adjuvant chemotherapy (ACT) with platinum doublets. RNA was isolated from the samples, followed by quantitative PCR to assess the expression of genes ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2.Results and discussion. It was shown that the level of expression of the studied genes was not associated with the effect of NAC in patients with lung cancer, except for the ABCC5 gene, which showed a relationship at the level of a pronounced trend (p = 0.07). It was also shown that in the group of patients with an objective response to chemotherapy, the frequency of decreased expression of the ABCC1 (p = 0.01) and ABCC5 (p = 0.004) genes was statistically significantly higher than in the group of patients with stabilization. Further, using the Kaplan – Meier method, it was found that a decrease in expression is associated with high rates of metastatic-free survival (MFS). The highest rates of 5-year MFS (more than 85 %) are observed in patients with a decrease in the expression of the ABCB1 and ABCG2 genes, log-rank test p = 0.0007 and p = 0.002, respectively.Conclusion. Thus, it has been shown that changes in the expression of ABC transporter genes are associated with the effectiveness of chemotherapy and the prognosis of the disease. The data obtained can be used as a basis for the detection of potential drug targets.

Expression and clinical significance of TYRP1, ABCB5, and MMP17 in sinonasal mucosal melanoma.

Sinonasal mucosal melanoma (SNMM) is a lethal malignancy with poor prognosis. Treatment outcomes of SNMM are poor. Novel prognostic or progression markers are needed to help adjust therapy. RNA-seq was used to analyze the mRNA expression of tumor tissues and normal nasal mucosa from primary SNMM patients (n= 3). Real-time fluorescent quantitative PCR (qRT-PCR) was used to validate the results of RNA-seq (n= 3), while protein expression was analyzed by immunohistochemistry (IHC, n= 31) and western blotting (n= 3). Retrospective studies were designed to determine the clinical parameters and the total survival rate, and correlation between the protein expression levels of the most significant key genes and prognosis was analyzed. In total, 668 genes were upregulated and 869 genes were downregulated in SNMM (fold change ⩾ 2, adjusted p value < 0.01). Both mRNA and protein expression levels of the key genes in SNMM tumor tissues were higher than those in the normal control nasal mucosal tissues. The expression rates of TYRP1, ABCB5, and MMP17 in 31 primary SNMM cases were 90.32%, 80.65%, and 64.52%, respectively. In addition, age, typical symptoms, and AJCC stage were related to overall survival rate of patients with SNMM (p< 0.05). Furthermore, the expression of ABCB5 was age-related (p= 0.002). Compared with individuals with negative ABCB5 expression, those with positive expression exhibited significantly poor overall survival (p= 0.02). The expression levels of TYRP1, ABCB5, and MMP17 were significantly upregulated in SNMM tissues, and the expression of ABCB5 was related to poor prognosis in SNMM. Thus, ABCB5 may serve as a progression marker and can predict unfavorable prognosis in patients with SNMM.

Natural Hemp-Ginger Extract and Its Biological and Therapeutic Efficacy.

The prevention and treatment of skin diseases remains a major challenge in medicine. The search for natural active ingredients that can be used to prevent the development of the disease and complement treatment is on the rise. Natural extracts of ginger and hemp offer a wide range of bioactive compounds with potential health benefits. This study evaluates the effectiveness of hemp and ginger extract as a supportive treatment for skin diseases. It reports a synergistic effect of hemp and ginger extract. The contents of cannabinoids and components of ginger are determined, with the highest being CBD (587.17 ± 8.32 µg/g) and 6-gingerol (60.07 ± 0.40 µg/g). The minimum inhibitory concentration for Staphylococcus aureus (156.5 µg/mL), Escherichia coli (625.2 µg/mL) and Candida albicans (78.3 µg/mL) was also analyzed. Analysis of WM-266-4 cells revealed the greatest decrease in metabolic activity in cells exposed to the extract at a concentration of 1.00 µg/mL. Regarding the expression of genes associated with cellular processes, melanoma aggressiveness, resistance and cell survival, a significant difference was found in the expression of ABCB5, CAV1 and S100A9 compared with the control (cells not exposed to the extract).

Expression of ABCB1, ABCC1 and 3 and ABCG2 in glioblastoma and their relevance in relation to clinical survival surrogates.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumors in adults. Patients invariably relapse during or after first-line therapy and the median overall survival is 14.6 months. Such poor clinical response is partly ascribed to the activity of ATP-binding cassette (ABC) transporters. The activity of these proteins, severely reduces the amount of therapeutics that penetrates the tumor cells. We hypothesized that ABC transporter expression could correlate with survival surrogates. In this study, we assessed the expression of four commonly expressed ABC transporters in GBM samples and investigated if mRNA levels could serve as a prognostic biomarker. Human specimens were analyzed by qPCR to assess ABCB1, ABCC1/3 and ABCG2 expression. Kaplan-Meier and multivariate analyses were then used to evaluate the correlation with overall survival (OS) and progression-free survival (PFS). Our cohort included 22 non-tumoral samples as well as 159 GBM tumor specimens. ABC transporters were significantly more expressed in GBM samples compared to non-tumoral tissue. Moreover ABCC1 and 3 mRNA expression were significantly increased at recurrence. Statistical analyses revealed that increased expression of either ABCC1 or ABCC3 did not confer a poorer prognosis. However, increased ABCC1 mRNA levels did correlate with a significantly shorter PFS. In this manuscript, the analyses we conducted suggest that the expression of the four ABC transporters evaluated would not be suitable prognostic biomarkers. We believe that, when estimating prognosis, the plethora of mechanisms implicated in chemoresistance should be analyzed as a multi-facetted entity rather than isolated units.

Importance of selected ABCB1 SNPs for the level of severity of depressive symptoms and effectiveness of recurrent depressive disorder therapy

The expression level of mRNA and also the function of P-gp are strictly connected with the polymorphic nature of the ABCB1 gene. In this study, we evaluated the association between promoter SNP, i.e. T-129C, three other SNPs investigated earlier and ABCB1 expression in the depression group. To assess the additive significance of these SNPs on clinicopathological features a mathematical model was also built.102 patients suffering from recurrent depressive disorder (rDD) and 94 healthy individuals from a local blood bank were enrolled in this study. ABCB1 gene polymorphism was identified by the RFLP method. The relative level of ABCB1 expression was measured by real-time PCR.For SNP T-129C no statistically significant differences in allele and genotype frequencies between depression and control groups were found (p = 0.3176). There was no statistically significant association between the expression value and 4 studied SNPs in ABCB1 (T-129C, C1236T, G2677T/A and C3435T) or the investigated clinicopathological features. Furthermore, a correlation between the initial HDRS score (lower than 23) and presence of at least 1236 T allele was observed, in particular in combination with 3435 T or 2677 T/A. Mutated allele of each SNP was also significantly associated with declined response to antidepressant therapy, both individually and in combination with others.Results of this study suggest that T-129C does not play an important role in the rDD development. The influence of the studied SNPs on ABCB1 gene expression is still unknown. However, the additive impact of 3 most frequently studied SNPs of ABCB1 on the course of depression and effectiveness of its treatment was confirmed.