ATP-binding Cassette Sub-family G Member 2 Expression Research Articles

Abstract P1-05-05: Hyperthermia regulates transporter expression via ROS production and enhances the cytotoxicity of doxorubicin

Abstract Introduction: Hyperthermia (HT) is a non-invasive cancer therapy. Treatment temperature between 41°C to 44°C has no cytotoxic damage in normal cells, however shows cytotoxicity in cancer cells because of the underdeveloped vascular system. HT often used with other cancer therapy such as radiation therapy and chemotherapy. However mechanism of synergistic effect using these therapies remains unclear. Compared to 37°C, 42°C is mild heat stress for cells, thus superoxide anion is released from tissue. Superoxide anion is produced by mitochondrial electron transport chain. Reactive oxygen species (ROS), produced by mild heat stress, can be released from mitochondria. We have previously reported that ATP-binding cassette sub-family G member 2 (ABCG2) expression was suppressed by increasing mitochondrial ROS, and induction of the cancer specific porphyrin accumulation. ABCG2 is a transporter of doxorubicin (DOX), therefore we hypothesized that synergistic effect of HT and chemotherapy would be induced by down-regulation of ABCG2 expression via intracellular ROS increase. In this study, we investigated if cytotoxic effect of breast cancer cell using DOX can be enhance by HT via intracellular ROS increase. Materials and methods: The murine breast cancer cell line, 4T1E was incubated at 37°C or 42°C for 1h. Intracellular ROS generation after HT treatment was detected by electron spin resonance (ESR). Twenty four hours after HT treatment, cells were incubated in medium containing 0, 0.1 and 1 μM DOX for 24 h. Cell viability was measured using the Cell Counting Kit 8, a water-soluble tetrazolium-8 based colorimetric assay. ABCG2 expression in whole cells was analyzed by Western blotting. Results and discussion: ESR signal peak with HT treatment became high as compared to without HT treatment, indicating intracellular ROS level was increased by HT treatment. Cell viability and ABCG2 expression were decreased by DOX exposure and by HT treatment. The enhancement of HT treatment effect by DOX is considered to be result of down-regulation of ABCG2 expression by ROS. When cells were exposed to DOX with 5-aminolevulinic acid (ALA), cell viability reduced further. Since it is known that porphyrin is introduced by ALA and is transported by ABCG2, we speculate that ALA worked as a competitive inhibitor of DOX excretion transporter to enhance cell death. ESR signal peak in ALA treatment cells was higher than that in non-ALA treatment cells. Significant increase in cellular damage by HT treatment was shown by adding ALA, but not without ALA. Moreover, cell death induced by HT and ALA treatment was suppressed by adding N-acetylcysteine (NAC), which is an antioxidant. These results suggest that cellular damage of HT treatment is due to ROS production induced by ALA. Conclusion: HT treatment involved intracellular ROS production and down-regulated the expression of ABCG2 protein. HT treatment also enhanced the cell damage by DOX. Cell death by DOX was enhanced by combination with HT and ALA treatment, possibly via intracellular ROS generation, and was suppressed by additing antioxidant. Citation Format: Terasaki A, Kurokawa H, Terasaki M, Ito H, Matsui H, Ichioka E, Tsushima Y, Manaka-Iguchi A, Bando H, Hara H. Hyperthermia regulates transporter expression via ROS production and enhances the cytotoxicity of doxorubicin [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-05.

Rab23 promotes the cisplatin resistance of ovarian cancer via the Shh-Gli-ABCG2 signaling pathway.

As a novel member of the Rab GTPase family, the role of Rab23 has been reported in multiple types of tumor. However, to the best of our knowledge, the role of Rab23 in ovarian cancer (OC) has not yet been reported. In the present study, immunohistochemistry analysis demonstrated that Rab23 was upregulated in OC tissue; survival analysis indicated that Rab23 expression was associated with a reduced overall survival (OS) rate and disease-free survival (DFS) time. In vitro experiments also demonstrated the increased expression of Rab23 in the OC cells lines, A2780 and SKOV-3, compared with in the normal ovarian cell line, IOSE80. Following the silencing of ABCG2 in SKOV-3 cells, ATP-binding cassette sub-family G member 2 (ABCG2) expression was significantly downregulated both at the RNA and protein levels. The cisplatin (DDP) IC50 declined from 43.09±7.12 µmol/l in control cells to 26.46±5.38 µmol/l in SKOV-3 cells with silenced Rab23. In contrast, in A2780 cells overexpressing Rab23 (A2780-Rab23), ABCG2 expression was significantly upregulated and the DDP IC50 increased from 27.42±6.54 µmol/l in control cells to 45.92±5.23 µmol/l in A2780-Rab23. Investigation into the potential molecular mechanisms for this revealed that the expression of sonic hedgehog (Shh) and Gli family zinc finger 1 (Gli1) was increased in A2780-Rab23 cells, whereas silencing Rab23 in SKOV-3 cells significantly inhibited the expression of Shh and Gli1. The Gli1 inhibitor GANT-61 significantly abrogated the increased ABCG2 expression in A2780-Rab23 cells. Furthermore, the DDP IC50 in A2780-Rab23 cells decreased significantly following the silencing of ABCG2 expression; the IC50 declined from 51.66±8.32 µmol/l in A2780-Rab23 cells to 25.61±6.17 µmol/l in A2780-Rab23 cells with silenced ABCG2. Collectively, the results indicate that Rab23 promotes the DDP resistance of OC cells via the Shh-Gli1-ABCG2 pathway, providing the proof of principle for the further investigation of drug resistance therapy targeting Rab23.

Identification of side population cells in human lung adenocarcinoma A549 cell line and elucidation of the underlying roles in lung cancer.

The present study aimed to isolate and characterize side population (SP) cells in the human lung cancer A549 cell line, and elucidate the molecular mechanism of SP cells underlying lung cancer. The SP and non-SP (NSP) cells in A549 cells were isolated and their differentiation was analyzed by fluorescence-activated cell sorting. An in vitro plate clone assay, Matrigel® Transwell assay and chemoresistance analysis of the sorted SP and NSP cells were performed. In addition, the sorted SP and NSP cells were injected into BALB/c nude mice to detect their tumorigenic potential in vivo. The expression of ATP-binding cassette sub-family G member 2 (ABCG2) in transplanted tumors was detected by immunohistochemistry. The SP and NSP cells were successfully isolated. The results demonstrated that SP cells accounted for 1.09% of live A549 cells. SP cells produced SP and NSP cells, while NSP cells only produced NSP cells. In addition, SP cells formed more colonies, exhibited improved invasive ability and increased levels of chemoresistance compared with NSP cells in vitro. SP cells demonstrated a higher tumorigenic potential in BALB/c nude mice, and the number of ABCG2-positive cells in the SP xenograft tumors were significantly increased compared with that in the NSP xenograft tumors. The present study indicated that SP cells isolated from the human lung cancer A549 cell line demonstrated increased tumorigenicity, and improved invasive ability and chemoresistance compared with NSP cells. In addition, detection of ABCG2 expression may assist in predicting the chemotherapeutic outcome of patients, and serve as a target for treating lung cancer.

ABCG2 (BCRP) m RNA expression level by using real- time PCR and immunohistochemistry associated with clinicopathological features in Iraqi women with stage II-III breast cancer.

Background: Breast cancer is the most frequent cancer and cause of death among women worldwide. ABCG2 (ATP–binding cassette sub-family G member 2) is an ABC transporter superfamily and endogenous expression of ABCG2 in different certain cancer reflect intrinsic drug resistance. It is also a molecular determinant of pharmacokinetic properties of many drugs in humans. In this study, we determined the expression levels of ABCG2 in breast tissues of Iraqi women with stage II and III breast cancer. The correlation between the expression levels of ABCG2 and clinicopathological features was analyzed. Methods: The expression levels of ABCG2 in the breast was determined by using real- time PCR and immunohistochemistry in 64 patients with stage II and III samples and in 21 benign tumors from Iraqi women. Results: We found that the expression level of ABCG2 mRNA were significantly increased in breast cancer stage II-III tissues than those in benign tumor tissues. There was a significant variation between the mRNA levels of ABCG2 in stage II and stage III at P< 0.05. Immunohistochemistry revealed that the protein expression levels of ABCG2, was also increased in 83% of patients with stage II and III breast cancer as compared to 17% in benign tumor. The increased expression levels of ABCG2, in stage II-III breast cancer were correlated to tumor stages (P=0.03), tumor grades (P=0.01), tumor types (P=0.01) and lymph node metastasis (p=0.0001), respectively. Conclusion: ABCG2 expression level in Iraqi women with stage II and III breast cancer were highly correlated with tumor stages, grades, types and metastasis and they could be used a potential markers which can prediction tumor behavior, progression and prognosis. Over expression of ABCG2 protein lead to treatment failure, tumor relapse and tumor metastasis by induction cancer cells against cytotoxic drugs.

Upregulation of Breast Cancer Resistance Protein Expression was Decreased in Plasma Membrane of Colon Cancer with Metastasis of Lymphatic Node.

ATP-binding cassette sub-family G member 2 (ABCG2), an ABC transport protein involved in the efflux of anticancer compounds, has been reported to have altered expression levels in several cancers including breast, colon, and gastric cancer, etc. However, its expression change (up- or down-regulated in cancer) is still contradictory. We performed immunohistochemistry to examine ABCG2 expression in the microarray with 90 pairs of colon cancer and their adjacent normal tissues. To find the expression of ABCG2 in lymphatic node metastasis (N1) and N0, we performed immunofluorescence, analyzed by Confocal technology. Compared to adjacent normal tissues, the percentage and density of positive cells expressing ABCG2 in colon cancer were significantly increased. In addition, ABCG2 expression in plasma membrane was related to lymph node metastasis in colon cancer, which was further verified to be downregulated by 2.7-fold in N1 to N0 through immunofluorescent analysis. ABCG2 expression in colon cancer was up-regulated, and its expression was decreased in the plasma membrane of colon cancer with lymphatic node metastasis (N metastasis).

125 - ROS Increase Chemo-reagent Concentration by the Down Regulation of BCRP During Both Hyperthermia and Chemotherapy

Hyperthermia (HT) is a non-invasive cancer therapy. Treatment temperature between 41 to 44°C has no cytotoxic damage in normal cells, while the temperature does in cancer cells because of the underdeveloped vascular system. HT often used with other cancer therapy such as radiation-therapy and chemotherapy. However mechanisms of synergistic effects among these therapies remains unclear. The 42°C environment is a cellular mild heat stress generating O2‒ from mitochondrial electron transport chain. We have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), which is known as breast cancer resistant protein (BCRP), was suppressed by increasing mitochondrial reactive oxygen species (ROS) to induce cancer specific porphyrin accumulations. Since ABCG2 is a transporter of doxorubicin (DOX), we hypothesized that synergistic effect of HT and chemotherapy may be induced by down-regulation of ABCG2 expression via intracellular ROS increase. In this study, we elucidated whether HT with intracellular ROS increase by HT can enhance the cytotoxic effect of DOX for breast cancer cells. The murine breast cancer cell line, 4T1E was incubated at 37 or 42°C for 1h. Intracellular ROS generation was detected by electron spin resonance (ESR). Cytotoxicity of DOX was measured using the Cell Counting Kit 8. ABCG2 expression was analyzed by Western blotting. ESR signal peak with HT treatment became high as compared to without HT treatment, indicating intracellular ROS level was increased by HT treatment. Cell viability and ABCG2 expression were decreased by DOX exposure and by HT treatment. Thus we conclude that the enhancement of HT treatment effect by DOX is considered to be result of down-regulation of BCRP expression by ROS.

The developmental transcription factor IRF6 attenuates ABCG2 gene expression and distinctively reverses stemness phenotype in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), which originates from the nasopharynx, is highly prevalent in Southern China and Southeast Asia, and more than 90% of all NPCs are non-keratinizing undifferentiated cells or poorly differentiated squamous cells. Cancer stem cells (CSCs) are capable of self-renewal and have differentiation potential. These properties form the basis of cancer initiation, development, and radiochemoresistance. However, the molecular mechanisms underlying NPC CSC maintenance remain poorly understood. Here, genomic expression profiling using our previously established monoclonal cellular and animal models revealed that interferon regulatory factor 6 (IRF6) was downregulated in highly metastatic NPC cells, cancer stem-like NPC cells and animal models. Functional assays revealed that elevated IRF6 expression suppressed cell proliferation, growth, CSCs properties and enhanced cell chemotherapeutic sensitivity. However, silencing IRF6 resulted in opposing effects. Moreover, we determined that as a tumor suppressor gene and transcription factor, IRF6 directly bound the upstream region of the ATP-binding cassette sub-family G member 2 (ABCG2) DNA element and suppressed target ABCG2 expression in NPC cells. Consistently, an inverse correlation was observed between the mRNA levels of IRF6 and ABCG2 in clinical NPC samples. With these results, we provide the first evidence that IRF6 directly targets the ABCG2 gene and selectively kills CSCs in NPC and that IRF6 may be a valuable tool for developing new CSC-targeted treatment strategies for undifferentiated NPC patients.

Vasohibin2 promotes adriamycin resistance of breast cancer cells through regulating ABCG2 via AKT signaling pathway.

As a well‑known angiogenic factor in different histology and pathological conditions, the pro‑progressive role of vasohibin2 (VASH2) has been reported in various types of tumors. However, its role in drug resistance of breast cancer has not been reported so far. The present study demonstrated that MCF‑7 cells with increased expression of VASH2 demonstrate stronger adriamycin (ADM) resistance compared with MDA‑MB‑231 cells with decreased expression of VASH2. Overexpression of VASH2 in MDA‑MB‑231 cells increased ADM resistance and silencing VASH2 in MCF‑7 cells inhibited ADM resistance. Furthermore, in newly established ADM resistant cell lines, VASH2 was significantly upregulated. These results revealed the promotive role of VASH2 in the ADM resistance of breast cancer cells. In addition, overexpression of VASH2 in MDA‑MB‑231 cells significantly upregulated ATP‑binding cassette sub‑familyG member 2 (ABCG2), however silencing VASH2 in MCF‑7 cells inhibited ABCG2 significantly. Silencing ABCG2 abrogated increase of ADM resistance induced by VASH2 overexpression in MDA‑MB‑231 cells. This proved that VASH2 induced ADM resistance through promoting expression of ABCG2, at least in part. Further study regarding the underlying molecular mechanism demonstrated that VASH2 promoted ABCG2 via the protein kinaseB (AKT) signaling pathway. Overall, VASH2 may promote drug resistance of breast cancer cells through regulating ABCG2 via the AKT signaling pathway. This suggests a novel therapeutic target to inhibit drug resistance in breast cancer, for a more efficient therapeutic outcome.

LncRNA CCAT1/miR-130a-3p axis increases cisplatin resistance in non-small-cell lung cancer cell line by targeting SOX4

ABSTRACTBackground: Colon cancer-associated transcript-1 (CCAT1) has been demonstrated to act as an oncogene and promote chemoresistance in several cancers. However, little is known about the underlying mechanism of CCAT1 in cisplatin (DDP) resistance of non-small-cell lung cancer (NSCLC) cells.Methods: qRT-PCR was performed to detect the expression levels of CCAT, miR-130a-3p, or sex-determining region Y-box 4 (SOX4) mRNA. Luciferase reporter assay, RNA immunoprecipitation (RIP), and qRT-PCR analysis were carried out to explore the potential targets of CCAT1 or miR-130a-3p. Effect of CCAT1, miR-130a-3p, or SOX4 on IC50 value of DDP and ATP binding cassette subfamily G member 2 (ABCG2) level in NSCLC cells were determined by cell counting kits-8 (CCK-8) assay and western blot, respectively.Results: CCAT1 and SOX4 were up-regulated, and miR-130a-3p was down-regulated in DDP-resistant NSCLC cells compared with their parental NSCLC cells. CCAT1 directly interacted with miR-130a-3p and negatively regulated miR-130a-3p expression. CCAT1 contributed to DDP resistance of A549/DDP cells by down-regulating miR-130a-3p. miR-130a-3p was found to directly target SOX4 to suppress its expression. SOX4 knockdown reversed miR-130a-3p-inhibition-induced increase of DDP resistance and ABCG2 expression in NSCLC cells. Exogenous expression of SOX4 abrogated CCAT1-knockdown-mediated decrease of DDP resistance and ABCG2 expression in DDP-resistant NSCLC cells.Conclusion: CCAT1/miR-130a-3p axis enhanced DDP resistance of NSCLC cells by targeting SOX4, providing potential targets to overcome DDP resistance and improve efficacy of chemotherapy for patients with NSCLC.

Photodynamic Therapy Using Photosensitizer-Encapsulated Polymeric Nanoparticle to Overcome ATP-Binding Cassette Transporter Subfamily G2 Function in Pancreatic Cancer.

Chlorin-based photosensitizers are commonly used in photodynamic therapy (PDT). These drugs are effluxed by cell membrane transporters, such as the ATP-binding cassette subfamily G member 2 (ABCG2). PDT efficacy is limited in tumor cells expressing high levels of these proteins. Pancreatic cancer cell lines AsPC-1 and MIA PaCa-2, which have high and low ABCG2 expression, respectively, were used, and ABCG2-overexpressing MIA PaCa-2 cells were generated. We compared PDT efficacy between chlorin e6 (Ce6) and cationic photosensitizer-encapsulated polymeric nanoparticle (PS-pNP), which is comprised with Ce6, polyethylene glycol, and polyethylenimine. The intracellular concentration of Ce6 was significantly higher in MIA PaCa-2 cells than in AsPC-1 or ABCG2-overexpressing MIA PaCa-2 cells. PS-pNP increased intracellular levels of the photosensitizer in all cell lines. The cell viability experiments indicated increased Ce6 resistance in ABCG2-overexpressing cells. In contrast, PS-pNP produced similar levels of cytotoxicity in each of the cancer cell lines tested. Singlet oxygen production was higher in cells treated with PS-pNP than in those treated with Ce6. Furthermore, in heterotopic and orthotopic AsPC-1 xenograft mouse models, PDT using PS-pNP significantly reduced tumor volume in comparison with that of Ce6 treatment. PS-pNP could increase intracellular Ce6 concentration, which was related with reduced ABCG2-mediated efflux of Ce6, thereby enhancing the effects of PDT in pancreatic cancer cells. Mol Cancer Ther; 16(8); 1487-96. ©2017 AACR.

Abstract 4757: Radioresistance is linked with stem-like properties via activation of aryl hydrocarbon receptor

Abstract Most cancer patients receive radiotherapy in the course of their disease and the occurrence of radioresistance is associated with a poor prognosis for cancer patients. The molecular pathways that drive the enhanced tumorigenic potential during the development of radioresistance are poorly understood. Here, we demonstrate that the aryl hydrocarbon receptor (AhR) plays a vital and previously unrecognized role in the maintenance of stem-like cells that are isolated from radioresistant epithelial lung cancer and nasopharyngeal carcinoma. AhR promotes the stem-like phenotype and drives metastasis by directly targeting the promoters of ‘stemness’ genes, such as the ATP-binding cassette sub-family G member 2 (ABCG2) gene. Cancer cell populations enriched for ABCG2 expression (CD338+) show an elevated ‘stemness’ signature and are more prone to cancer progression in vitro and in vivo. Ubiquitin C-terminal hydrolase-L3 (UCHL3), a deubiquitinating enzyme, may counteract AhR, while IKKα, a bone fide chromatin modifier, may directly activate stemness related genes. The radioresistant cancer cell sublines are associated with a hybrid state in epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), and elevated tumor associated macrophages and proinflammatory factors. Moreover, the radioresistant sublines display high levels of oncometabolites, including α-ketoglutarate, and treatment of cancer cells with α-ketoglutarate enhances their stem-like properties through activation of the AhR. Thus AhR is functionally linked with radioresistance in lung cancer and nasopharyngeal carcinoma, and it drives tumorigenesis by an epithelial and mesenchymal composition, tumor associated macrophages and oncometabolites. Note: This abstract was not presented at the meeting. Citation Format: SHuang Liu, Bin Yan, Yongguang Tao. Radioresistance is linked with stem-like properties via activation of aryl hydrocarbon receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2017-4757

Expression of the cancer stem cell markers ABCG2 and OCT-4 in right-sided colon cancer predicts recurrence and poor outcomes.

Right-sided colon cancer (RCC) has a poorer prognosis and a higher relapse rate than left-sided colon cancer (LCC). Like cancer stem cells (CSCs), RCC cells cannot be fully eradicated and are often involved in relapse or metastasis. Because CSCs may be linked with poor outcomes, CSC markers may have prognostic value in RCC. ATP-binding cassette sub-family G member 2 (ABCG2) and OCT-4 (also known as POU5F1) are among the most useful markers for CSC identification. We therefore examined the malignant behavior of ABCG2 and OCT-4 in vitro and in vivo, and their expression was assessed in pathology tissues obtained from clinicopathologically recurrent and non-recurrent cases. Our survey suggested associations between ABCG2 and OCT-4 expression and RCC clinicopathological variables. No correlations were detected between ABCG2 or OCT-4 expression and age, gender, tumor size, or tumor shape, but ABCG2 expression correlated with TNM stage, tumor differentiation, and lymphovascular invasion. Additionally, expression of both ABCG2 and OCT-4 correlated with RCC recurrence and poor outcomes.

Estrogen Enhances the Expression of the Multidrug Transporter Gene ABCG2-Increasing Drug Resistance of Breast Cancer Cells through Estrogen Receptors.

Background: Multidrug resistance is a major obstacle in the successful therapy of breast cancer. Studies have proved that this kind of drug resistance happens in both human cancers and cultured cancer cell lines. Understanding the molecular mechanisms of drug resistance is important for the reasonable design and use of new treatment strategies to effectively confront cancers. Results: In our study, ATP-binding cassette sub-family G member 2 (ABCG2), adenosine triphosphate (ATP) synthase and cytochrome c oxidase subunit VIc (COX6C) were over-expressed more in the MCF-7/MX cell line than in the normal MCF7 cell line. Therefore, we believe that these three genes increase the tolerance of MCF7 to mitoxantrone (MX). The data showed that the high expression of COX6C made MCF-7/MX have more stable on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) expression than normal MCF7 cells under hypoxic conditions. The accumulation of MX was greater in the ATP-depleted treatment MCF7/MX cells than in normal MCF7/MX cells. Furthermore, E2 increased the tolerance of MCF7 cells to MX through inducing the expression of ABCG2. However, E2 could not increase the expression of ABCG2 after the inhibition of estrogen receptor α (ERα) in MCF7 cells. According to the above data, under the E2 treatment, MDA-MB231, which lacks ER, had a higher sensitivity to MX than MCF7 cells. Conclusions: E2 induced the expression of ABCG2 through ERα and the over-expressed ABCG2 made MCF7 more tolerant to MX. Moreover, the over-expressed ATP synthase and COX6c affected mitochondrial genes and function causing the over-expressed ABCG2 cells pumped out MX in a concentration gradient from the cell matrix. Finally lead to chemoresistance.

Dexamethasone reduces side population fraction through downregulation of ABCG2 transporter in MCF-7 breast cancer cells.

Side population (SP) cells represent a rare population among breast cancer cells. SP cells have been reported to act as cancer stem‑like cells, and to participate in the development of multidrug resistance via modulating the expression of ATP-binding cassette subfamily G member 2 (ABCG2). Dexamethasone is a corticosteroid drug that has been used as an adjuvant treatment to enhance the efficacy of chemotherapeutic agents; however, its effects in breast cancer have yet to be thoroughly investigated. In the present study, the effects of dexamethasone were investigated using the human MCF‑7 breast cancer cell line, and SPs were examined in detail. Cellular proliferation, SP fractions and ABCG2 expression were examined following treatment of MCF‑7 cells with dexamethasone. Dexamethasone was revealed to cause a dose‑ and time‑dependent decrease in cancer cell proliferation, and it also decreased the size of the SP fraction of MCF‑7 cells and the expression of the ABCG2 transporter. The effects of dexamethasone on cellular proliferation, SP fraction and ABCG2 expression were abolished following the administration of the glucocorticoid antagonist RU486. These results suggested that dexamethasone may target breast cancer cell SPs and thus increase the sensitivity of tumor cells to chemotherapy. Therefore, it may be hypothesized that dexamethasone can be used as a chemosensitizer in the adjuvant treatment of patients with breast cancer.

YAP1 regulates ABCG2 and cancer cell side population in human lung cancer cells.

A small population of cancer cells called cancer-initiating cells or cancer stem cells (CSCs) are involved in drug resistance, metastasis, and cancer relapse. Finding pathways that regulate CSC is very important for clinical therapy. ATP-binding cassette sub-family G member 2 (ABCG2) plays a role in side population (SP) cell formation and contributes to chemotherapy resistance in common forms of cancer. Yes-associated protein 1 (YAP1) is a major transcriptional effector of the Hippo pathway, which plays important roles in organ size control and tumorigenesis. In this study, we found ABCG2 and YAP1 were both overexpressed in lung cancer SP cells. Disruption of YAP1 expression by siRNA attenuated the expression of ABCG2 transcript and significantly reduced the percentage of SP cells and sphere formation in lung cancer cells. Overexpression of YAP1 in lung cancers led to an increase in ABCG2 expression and increased the percentage of SP cells. However, overexpression of YAP1 in purified non-SP cells did not increase ABCG2 expression and the percentage of SP cells, which may be due to the inhibition of YAP activity through phosphorylation. YAP1 directly transcriptionally regulated ABCG2 by binding to the promoter of ABCG2. Moreover, the YAP1 inhibitor verteporfin and YAP1 siRNA downregulated ABCG2 level through inhibition of YAP1 in lung cancer cells and sensitized them to the chemotherapy drug doxorubicin. Our study adds a new function for YAP1 that may be relevant to drug resistance and cancer therapy through regulation of ABCG2 and side population cell formation in lung cancer.

Elevated expression of Nrf2 mediates multidrug resistance in CD133+ head and neck squamous cell carcinoma stem cells

Enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC sub-family G member 2 (ABCG2) in cancer stem cells (CSCs) plays a major role in chemotherapeutic drug efflux, which results in therapy failure and tumor relapse. In addition to downregulating apoptosis in CSCs, it has been reported that the transcriptional upregulation of the redox sensing factor Nrf2 is involved in the upregulation of ABCG2 expression and consequent chemoresistance. The current study investigated the presence of cancer stem-like side population (SP) cells from head and neck squamous cell carcinoma (HNSCC) samples, and evaluated the Nrf2 expression profile and multidrug resistance properties of HNSCC stem cells. Fluorescence-activated cell sorting was used for SP cells detection, while reverse transcription-polymerase chain reaction was used for the analysis of Nrf2 expression. The present study identified ~2.1% SP cells present in HNSCC specimens, which were positive for cluster of differentiation (CD)133 expression and displayed significantly elevated messenger RNA expression of Nrf2, compared with non-SP cells. These data suggest that the ABC transporter ABCG2 is highly upregulated in SP cells, and this results in multidrug resistance. In addition, these CD133+ cells underwent rapid proliferation and exhibited high self-renewal and tumorigenic properties. Taken together, the present findings suggest that elevated expression of Nrf2 mediated drug resistance in HNSCC CSCs, which may be one of the causative factors for cancer treatment failure. Therefore, novel anti-cancer drugs that downregulate the Nrf2 signaling pathway could effectively improve the treatment and survival rate of patients with HNSCC.

ABCG2 is required for self-renewal and chemoresistance of CD133-positive human colorectal cancer cells.

There is increasing evidence supporting the cancer stem cell (CSC) hypothesis, which suggests thata population of tumor cells with stem cell characteristics is responsible for tumor growth, resistance, and recurrence as well as drug resistance. In colorectal cancer, the CD133 antigen definesdistinct cell subpopulations that are rich in tumor-initiating cells; however, the drug resistance properties of these CD133-positive cells have not been well defined. The breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) ispresent on the plasma membrane of many types of human cancer cells and contributes to multidrug resistance during chemotherapy. Theresults of the present study showed that ABCG2 is expressed in CD133-positive CSCs from human colorectal tumors. Furthermore, the downregulation of ABCG2 expression inhibited the self-renewal capacity of these cells, and significantly enhanced the efficacy of chemotherapy-induced apoptosis in LS174T colon adenocarcinoma cells and CD133-positive colorectal carcinoma cells. Together, these data show that ABCG2 expression correlates with the presence of CD133-positive cancer cells, and thus is a possible therapeutic target for colorectal cancer.

Upregulation of ABCG2 via the PI3K-Akt pathway contributes to acidic microenvironment-induced cisplatin resistance in A549 and LTEP-a-2 lung cancer cells.

Hypoxia always exists in the processes involved in the development of lung cancer and contributes to an acidic microenvironment. Despite that hypoxia in the tumor microenvironment is associated with the formation of chemotherapeutic resistance, the exact role of an acidic microenvironment in the development of hypoxia-induced lung cancer multidrug resistance is still unknown. In the present study, we acidized the medium with 2-(N-morpholino)-ethanesulfonic acid (MES monohydrate) to mimic the acidic tumor microenvironment and observed the effects of acidification on lung cancer cell viability, the expression of ATP-binding cassette sub-familyG member2 (ABCG2) and myeloid cell leukemia‑1 (Mcl-1), and activation of the PI3K-Akt pathway. Thereafter, we investigated the mechanisms involved in the acidification-induced expression of ABCG2 and Mcl-1, and the potential therapeutic strategy to overcome acidification-associated multidrug resistance formation. We demonstrated that acidification obviously increased the expression of ABCG2 and Mcl-1 via PI3K‑Akt‑mTOR-S6 pathway activation and contributed to multidrug resistance. Inhibition of PI3K-Akt activity efficiently abolished the effect of acidification on cell viability, indicating that the PI3K-Akt pathway may include potential therapeutic target molecules in acidized microenvironment-associated lung cancer chemotherapeutic resistance.

A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo.

ATP-binding cassette subfamily G member 2 (ABCG2) is a member of the ABC transporter superfamily proteins, which has been implicated in the development of multidrug resistance (MDR) in cancer, apart from its physiological role to remove toxic substances out of the cells. The diverse range of substrates of ABCG2 includes many antineoplastic agents such as topotecan, doxorubicin and mitoxantrone. ABCG2 expression has been reported to be significantly increased in some solid tumors and hematologic malignancies, correlated to poor clinical outcomes. In addition, ABCG2 expression is a distinguishing feature of cancer stem cells, whereby this membrane transporter facilitates resistance to the chemotherapeutic drugs. To enhance the chemosensitivity of cancer cells, attention has been focused on MDR modulators. In this study, we investigated the effect of a tetrodotoxin-resistant sodium channel blocker, A-803467 on ABCG2-overexpressing drug selected and transfected cell lines. We found that at non-toxic concentrations, A-803467 could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies demonstrated that A-803467 (7.5 μM) significantly increased the intracellular accumulation of [(3)H]-mitoxantrone by inhibiting the transport activity of ABCG2, without altering its expression levels. In addition, A-803467 stimulated the ATPase activity in membranes overexpressed with ABCG2. In a murine model system, combination treatment of A-803467 (35 mg/kg) and topotecan (3 mg/kg) significantly inhibited the tumor growth in mice xenografted with ABCG2-overexpressing cancer cells. Our findings indicate that a combination of A-803467 and ABCG2 substrates may potentially be a novel therapeutic treatment in ABCG2-positive drug resistant cancers.

Correlation between the promoter methylation status of ATP-binding cassette sub-family G member 2 and drug sensitivity in colorectal cancer cell lines

Resistance to chemotherapeutic agents has been considered as a major reason for the high incidence rate of recurrence and metastasis suffered by colorectal cancer (CRC) patients. ATP-binding cassette sub-family G member 2 (ABCG2) is involved in drug resistance. DNA methylation of the ABCG2 promoter site has a significant influence on the regulation of epigenetic gene expression. In the present study, we investigated whether the methylation status of the ABCG2 promoter is related to drug sensitivity in CRC cell lines. In order to examine the ABCG2 expression level and identify the methylation status, RT-PCR, qRT-PCR analysis, MS-PCR and bisulfite sequencing were conducted on 32 CRC cell lines. SNU-C4, LS174T and NCI-H716 were selected as low ABCG2-expressing and high promoter methylated cell lines. The cell proliferation assay for 5-fluorouracil, oxaliplatin and irinotecan was performed after 5-aza-2'-deoxycytidine (5-aza) treatment in these cell lines. In the 32 CRC cell lines, 25% of the cell lines expressed low or no ABCG2 expression. Of these cell lines, SNU-C4, LS174T and NCI-H716 were hypermethylated at the promoter region, ~20%. Demethylation of ABCG2 was induced by 5-aza, which enhanced the ABCG2 expression level and influenced the cell proliferation similar to treatment with the anticancer agents. Our data suggest that the ABCG2 expression level regulated by methylation is related to anticancer drug sensitivity. Based on these results, it can be applied to predict the anticancer drug response.