Overexpression Of ABCG2 Research Articles

Brain accumulation of the EML4-ALK inhibitor ceritinib is restricted by P-glycoprotein (P-GP/ABCB1) and breast cancer resistance protein (BCRP/ABCG2)

We aimed to clarify the roles of the multidrug transporters ABCB1 and ABCG2 in oral availability and brain accumulation of ceritinib, an oral anaplastic lymphoma kinase (ALK) inhibitor used to treat metastatic non-small cell lung cancer (NSCLC) after progression on crizotinib. Importantly, NSCLC is prone to form brain metastases. Transport of ceritinib by human (h) ABCB1 or hABCG2 or mouse (m) Abcg2 was assessed in vitro. To study the single and combined roles of Abcb1a/1b and Abcg2 in ceritinib disposition in vivo, we used appropriate knockout mouse strains. Ceritinib was very efficiently transported by hABCB1, and efficiently by hABCG2 and mAbcg2 in vitro, and transport was specifically inhibited by the ABCB1 inhibitor zosuquidar and ABCG2 inhibitor Ko143, respectively. Absorption and 24-h oral availability were not significantly affected by the absence of Abcb1 and/or Abcg2, but the brain concentrations were greatly increased (>38-fold) in Abcb1a/1b−/− mice at 3 and 24h after oral administration of 20mg/kg ceritinib. The brain concentrations increased another ∼3-fold (to >90-fold) in Abcb1a/1b;Abcg2−/− mice, indicating that there was a significant additional effect of Abcg2-mediated transport of ceritinib as well in vivo. Overall, brain accumulation, but not the 24-h oral availability of ceritinib were profoundly restricted by Abcb1a/1b and Abcg2, with Abcb1a/1b being the dominant efflux protein. Our data suggest that coadministration of ceritinib with a dual ABCB1 and ABCG2 inhibitor may improve treatment of brain (micro) metastases positioned behind a functionally intact blood-brain barrier, and possibly also of tumors resistant to ceritinib due to ABCB1 or ABCG2 overexpression.

Dinaciclib, a cyclin-dependent kinase inhibitor, is a substrate of human ABCB1 and ABCG2 and an inhibitor of human ABCC1 in vitro

Dinaciclib is a novel cyclin-dependent kinase inhibitor (CDKI) with significant activity against various cancers in vitro and in vivo. ABC efflux transporters play an important role in drug disposition and are responsible for multidrug resistance in cancer cells. Inhibitors and substrates of these transporters may participate in pharmacokinetic drug–drug interactions (DDIs) that alter drug disposition during pharmacotherapy. To assess such risks associated with dinaciclib we evaluated its possible effects on efflux activities of ABCB1, ABCC1 and ABCG2 transporters in vitro. Monolayer transport, XTT cell proliferation, ATPase and intracellular accumulation assays were employed. Here, we show that the transport ratio of dinaciclib was far higher across monolayers of MDCKII-ABCB1 and MDCKII-ABCG2 cells than across MDCKII parental cell layers, demonstrating that dinaciclib is a substrate of ABCB1 and ABCG2. In addition, overexpression of ABCB1, ABCG2 and ABCC1 conferred resistance to dinaciclib in MDCKII cells. In ATPase assays, dinaciclib decreased stimulated ATPase activity of ABCB1, ABCG2 and ABCC1, confirming it has interactive potential toward all three transporters. Moreover, dinaciclib significantly inhibited ABCC1-mediated efflux of daunorubicin (EC50=18μM). The inhibition of ABCC1 further led to a synergistic effect of dinaciclib in both MDCKII-ABCC1 and human cancer T47D cells, when applied in combination with anticancer drugs. Taken together, our results suggest that ABC transporters can substantially affect dinaciclib transport across cellular membranes, leading to DDIs. The DDIs of dinaciclib with ABCC1 substrate chemotherapeutics might be exploited in novel cancer therapies.

Tetronic® 904-containing polymeric micelles overcome the overexpression of ABCG2 in the blood-brain barrier of rats and boost the penetration of the antiretroviral efavirenz into the CNS.

To assess the involvement of ABCG2 in the pharmacokinetics of efavirenz in the blood-brain barrier (BBB) and investigate a nanotechnology strategy to overcome its overexpression under a model of chronic oral administration. Materials & methods A model of chronic efavirenz (EFV) administration was established in male Sprague-Dawley rats treated with a daily oral dose over 5 days. Then, different treatments were conducted and drug concentrations in plasma and brain measured. Chronic treatment with oral EFV led to the overexpression of ABCG2 in the BBB that was reverted after a brief washout period. Moreover, gefitinib and the polymeric amphiphile Tetronic(®) 904 significantly inhibited the activity of the pump and potentiated the accumulation of EFV in CNS. The same effect was observed when the drug was administered within mixed micelles containing TetronicT904 as the main component. Tetronic 904-containing polymeric micelles overcame the overexpression of ABCG2 in the BBB caused by chronic administration of EFV then boosting its penetration into the CNS.

Abstract 4432: A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo

Abstract ABCG2 is a member of the ABC transporter superfamily, which has been implicated in the development of multidrug resistance (MDR) in cancer. Its diverse range of substrates includes many antineoplastic agents such as topotecan, doxorubicin and mitoxantrone. ABCG2 expression has been significantly increased in some solid tumors and hematologic malignancies, which is correlated to poorer clinical outcomes. In addition, ABCG2 expression is a distinguishing feature of cancer stem cells, whereby this membrane transporter impacts 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 ability of a tetrodotoxin-resistant sodium channel blocker, A-803467 to reverse ABCG2-mediated MDR. We found that A-803467 at non-toxic concentration could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies indicated that A-803467 (7.5 μM) significantly increased the intracellular accumulation of mitoxantrone by inhibiting the transport activity of ABCG2, without altering its expression level. In addition, A-803467 stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that A-803467 might be a substrate of ABCG2. Binding interactions of A-803467 were found to be in transmembrane region of homology modeled human ABCG2. Interactions of A-803467 with ABCG2 were relatively stronger when compared to the interactions of topotecan with ABCG2. Furthermore, A-803467 (30 mg/kg) with topotecan (3 mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. Our findings indicate that A-803467 has the potential to be used in combination with ABCG2 chemotherapeutic substrates to improve the response in drug resistant cancers. Citation Format: Nagaraju Anreddy, Atish Patel, Yun-Kai Zhang, Yi-Jun Wang, Suneet Shukla, Rishil J. Kathawala, Priyank Kumar, Pranav Gupta, Suresh V. Ambudkar, John ND Wurpel, Zhe-Sheng Chen. A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4432. doi:10.1158/1538-7445.AM2015-4432

Abstract 194: Deregulation of miR-222-ABCG2 regulatory module in tongue squamous cell carcinoma contributes to chemoresistance and enhanced metastatic potential

Abstract Chemoresistance often associated with other clinical characteristics, such as enhanced metastatic potential. However, the underlying molecular mechanism remains unclear. Our previous studies had revealed that microRNA-222 (miR-222) regulates the cell invasion [Cancer Genomics Proteomics 2009 6:131-138], and plays a role in cell proliferation in tongue squamous cell carcinoma (TSCC) [FEBS Lett. 2010 584(18):4115-20]. The aim of this study is to elucidate the role of miR-222-ABCG2 pathway in the association of cisplatin (cDDP) resistance with enhanced cell migration and invasion ability in TSCC. First, we confirmed the association between cDDP resistance (measured by IC50) and metastatic potential (assessed by migration and invasion assays) using TSCC cell lines and primary cultures from TSCC cases. The siRNA-mediated ABCG2 knockdown led to enhanced cDDP responsiveness, and reduced metastatic potential in TSCC cells. On the contrary, ABCG2 overexpression induced cDDP resistance, and enhanced cell migration and invasion in TSCC cells. Bioinformatics analysis revealed that ABCG2 is a target gene of miR-222, and the direct interaction of miR-222 with ABCG2 mRNA was confirmed by luciferase reporter gene assay. Functional analyses indicated that miR-222 downregulated the expression of ABCG2 gene and enhanced cDDP responsiveness, as well as reduced cell migration and invasion in TSCC cells. Thus, our results confirmed the association between cDDP resistance and enhanced metastatic potential in TSCC. ABCG2 is a direct target gene of miR-222, and deregulation of miR-222-ABCG2 regulatory module in TSCC contributes to both cDDP resistance and enhanced metastatic potential. Citation Format: Anxun Wang, Luodan Zhao, Qianting He, Tingting Zhao, Wei Wang, Xiaofeng Zhou. Deregulation of miR-222-ABCG2 regulatory module in tongue squamous cell carcinoma contributes to chemoresistance and enhanced metastatic potential. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 194. doi:10.1158/1538-7445.AM2015-194

ABCG2 overexpression in patients with acute myeloid leukemia: Impact on stem cell transplantation outcome.

ABGG2 protein overexpression in acute myeloid leukemia (AML) has been associated with poor response to conventional chemotherapy and increased relapse risk. No data are available on the role of allogeneic stem cell transplantation (SCT) in reversing its negative prognostic role. We have reviewed the outcome of 142 patients with high risk AML who underwent allogeneic SCT in complete remission (n = 94) or with active disease (n = 48). Patients with ABCG2 overexpression at AML diagnosis have lower leukemia free survival (LFS) and increased cumulative incidence of relapse (CIR) compared with ABCG2- patients (5-year LFS 50% vs. 65%, P = 0.01; 5-year CIR 46% vs. 27%, P = 0.003). Five-year overall survival was not significantly different between ABCG2+ and ABCG2- patients (39% vs. 51%, P = 0.1). However, if we consider only disease-related deaths, ABCG2 maintains its negative role (64% vs. 78%, P = 0.018). The negative impact of ABCG2 overexpression was higher in patients undergoing SCT in CR compared with patients receiving transplant with active disease. Conditioning regimen did not abrogate the effect of ABCG2 overexpression, as CIR was higher in ABCG2+ patients receiving both myeloablative (44% vs. 22%, P = 0.018) or reduced intensity conditioning (50% vs. 32%, P = 0.03). In conclusion, ABCG2 overexpression at AML diagnosis identifies a subset of patients with poor outcome also after allogeneic SCT, mainly in terms of higher relapse rates. Prospective studies employing conditioning drugs or post-transplant strategies able to target ABCG2 are needed to maximize the curative potential of stem cell transplantation.

Inhibiting ABCG2 by Ko143 May Enhance the Efficacy of Irinotecan Treatment in Colon Cancer

ABCG2 has been supposed as a promising target conferring multidrug resistance on cancer cells. However, ABCG2 studies in cancer had conflicting results. The aim of this study was to evaluate the effect of Ko143, an ABCG2 specific inhibitor, in the treatment of colon cancer with irinotecan (and its active metabolite SN38), a basic chemotherapeutic agent in colon cancer. The correlations between the ABCG2 protein expression with the effect of SN38 were confirmed in human colon cancer (HCT116) and liver metastatic (KOSA2) cell lines established from HCT116. Inhibiting ABCG2 function by Ko143 was increase SN38 sensitivity in both of cell lines, additionally, this effect was proportional to the concentration and exposure time of Ko143. Using xenografted tumor models with HCT116 cells, efficacy of combination Ko143 and irinotecan in tumor killing were shown significant increase compare with irinotecan alone treatment (P < .05). Moreover, coordination treatment with irinotecan and prolonged Ko143 extended time of tumor size doubled by arresting the cell cycle at the G0 phase which has been verified by immunostaning using an Ki-67 and p21 antibody. Finally, immunohistochemistry staining showed ABCG2 over-expression on membrane of colon cancer cells after irinotecan treatment, especially combined with Ko143. Altogether, our results suggested that combination therapy involving Ko143, an ABCG2 inhibitor, and irinotecan may improve the efficacy of colon cancer treatment. These promising results warrant further investigation in clinical trials.

Abnormal Wnt signaling and overexpression of ABCG2 contributes to drug efflux properties of side population cells in nasopharyngeal carcinoma.

The presence of cancer stem cells (CSCs) has major implications in the choice of cancer treatment strategy and is responsible for tumor relapse. CSCs have been isolated and characterized in several types of cancer; however, studies concerning the CSCs from nasopharyngeal carcinoma (NPC) are limited. Thus, the present study was designed to isolate and characterize the cancer stem-like side population (SP) cells from NPC samples. The fluorescence-activated cell sorting (FACS)-based Hoechst 33342 dye exclusion technique identified that 3.9% of cells from NPC samples were cancer stem-like SP cells. Upon treatment with verapamil (ABC transporter inhibitor), the percentage of SP cells was significantly reduced to 0.7%, which confirms that the ABC transporter protein exhibits a significant role in drug exclusion. Fluorescence microscopy analysis revealed that the FACS purified SP cells showed increased expression of ABCG2 (ATP transporter protein), Oct-4 and CD44 (stem cell surface protein). Furthermore, these SP cells exhibited increased mRNA expression of ABCG2 and anti-apoptotic factor Bmi-1, which contribute to multi-drug resistance and increased cell survival rate. Notably, the Wnt/β-catenin signaling pathways are altered in SP cells. In addition, using reverse transcription‑quantitative polymerase chain reaction analysis it was observed that the cells exhibited increased expression of DKK1 and AXIN2. In conclusion, data from the present study clearly demonstrated that the presence of cancer stem-like SP cells from NPC may be responsible for chemotherapeutic drug resistance, tumor recurrence and invasion.

FL118, a novel camptothecin derivative, is insensitive to ABCG2 expression and shows improved efficacy in comparison with irinotecan in colon and lung cancer models with ABCG2-induced resistance.

BackgroundIrinotecan is a camptothecin analogue currently used in clinical practice to treat advanced colorectal cancer. However, acquired resistance mediated by the drug efflux pump ABCG2 is a recognized problem. We reported on a novel camptothecin analogue, FL118, which shows anticancer activity superior to irinotecan. In this study, we sought to investigate the potency of FL118 versus irinotecan or its active metabolite, SN-38, in both in vitro and in vivo models of human cancer with high ABCG2 activity. We also sought to assess the potency and ABCG2 affinity of several FL118 analogues with B-ring substitutions.MethodsColon and lung cancer cells with and without ABCG2 overexpression were treated with FL118 in the presence and absence of Ko143, an ABCG2-selective inhibitor, or alternatively by genetically modulating ABCG2 expression. Using two distinct in vivo human tumor animal models, we further assessed whether FL118 could extend time to progression in comparison with irinotecan. Lastly, we investigated a series of FL118 analogues with B-ring substitutions for ABCG2 sensitivity.ResultsBoth pharmacological inhibition and genetic modulation of ABCG2 demonstrated that, in contrast to SN-38, FL118 was able to bypass ABCG2-mediated drug resistance. FL118 also extended time to progression in both in vivo models by more than 50% compared with irinotecan. Lastly, we observed that FL118 analogues with polar substitutions had higher affinity for ABCG2, suggesting that the nonpolar nature of FL118 plays a role in bypassing ABCG2-mediated resistance.ConclusionsOur results suggest that in contrast to SN-38 and topotecan, FL118 is a poor substrate for ABCG2 and can effectively overcome ABCG2-mediated drug resistance. Our findings expand the uniqueness of FL118 and support continued development of FL118 as an attractive therapeutic option for patients with drug-refractory cancers resulting from high expression of ABCG2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0362-9) contains supplementary material, which is available to authorized users.

GAIP interacting protein C-terminus regulates autophagy and exosome biogenesis of pancreatic cancer through metabolic pathways.

GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.

Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo.

Multidrug resistance (MDR) to chemotherapeutic drugs is a formidable barrier to the success of cancer chemotherapy. Expressions of ATP-binding cassette (ABC) transporters contribute to clinical MDR phenotype. In this study, we found that afatinib, a small molecule tyrosine kinase inhibitor (TKI) targeting EGFR, HER-2 and HER-4, reversed the chemoresistance mediated by ABCG2 in vitro, but had no effect on that mediated by multidrug resistance protein ABCB1 and ABCC1. In addition, afatinib, in combination with topotecan, significantly inhibited the growth of ABCG2- overexpressing cell xenograft tumors in vivo. Mechanistic investigations exhibited that afatinib significantly inhibited ATPase activity of ABCG2 and downregulated expression level of ABCG2, which resulted in the suppression of efflux activity of ABCG2 in parallel to the increase of intracellular accumulation of ABCG2 substrate anticancer agents. Taken together, our findings may provide a new and useful combinational therapeutic strategy of afatinib with chemotherapeutical drug for the patients with ABCG2 overexpressing cancer cells.

Human ATP-Binding Cassette transporters ABCB1 and ABCG2 confer resistance to CUDC-101, a multi-acting inhibitor of histone deacetylase, epidermal growth factor receptor and human epidermal growth factor receptor 2

CUDC-101 is the first small-molecule inhibitor designed to simultaneously inhibit epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and histone deacetylase (HDAC) in cancer cells. Recently, in its first in human phase I study, CUDC-101 showed promising single agent activity against advanced solid tumors and favorable pharmacodynamic profile. However, the risk of developing drug resistance to CUDC-101 can still present a significant therapeutic challenge to clinicians in the future. One of the most common mechanisms of developing multidrug resistance (MDR) in cancer is associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1 and ABCG2. Together, they are able to reduce the efficacy and modify the pharmacological properties of anti-cancer agents, including many small molecule tyrosine kinase inhibitors (TKIs). Here, we have investigated the impact of ABCB1 and ABCG2 on the efficacy of CUDC-101 in human cancer cells. We revealed that although CUDC-101 has potent antiproliferative and proapoptotic activities against most cancer cell lines, the overexpression of ABCB1 or ABCG2 in cancer cells significantly reduced the activity of CUDC-101 against HDAC, EGFR and HER2, as well as its cytotoxicity and proapoptotic activity. Moreover, we showed that CUDC-101 modulated the function of both transporters without affecting the protein expression of either ABCB1 or ABCG2. More importantly, our study provides support for the rationale of combining CUDC-101 with modulators of ABC drug transporters to improve drug efficacy and overcome multidrug resistance associated with the overexpression of ABCB1 and ABCG2.

Abstract 766: Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364

Abstract The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) has been detected in various types of cancer, and thus has fast become an attractive therapeutic target for cancer therapy. Plk1 inhibitors BI 2536, volasertib and GSK461364, were designed to selectively inhibit cancer cell proliferation by promoting G2/M cell cycle arrest at nanomolar concentrations. Unfortunately, alike most chemotherapeutic agents, the development of acquired resistance to Plk1 inhibitors is prone to present a significant therapeutic challenge. One of the most common mechanisms for acquired resistance in cancer chemotherapy is associated with the overexpression of ATP-binding cassette (ABC) transporters ABCB1, ABCC1 and ABCG2. Here, we discovered that in human cancer cells, the overexpression of ABCB1 and/or ABCG2 can lead to acquired resistance to three selective Plk1 inhibitors, BI 2536, volasertib and GSK461364. Moreover, these Plk1 inhibitors stimulate the ATPase activity of ABCB1 and ABCG2, as well as competitively inhibit the drug substrate transport mediated by ABCB1 and ABCG2. More significantly, the reduced chemosensitivity and Plk1 inhibitors-mediated G2/M cell cycle arrest in cancer cells overexpressing either ABCB1 or ABCG2 can be significantly restored in the presence of selective inhibitor of ABCB1 and ABCG2. Taken together, our findings indicate that in order to circumvent ABCB1 or ABCG2-mediated acquired resistance to Plk1 inhibitors, a combined regimen of Plk1 inhibitors and modulators or clinically active drugs that potently inhibit the function of ABC drug transporters, should be considered as a potential treatment strategy in the clinic. Citation Format: Chung-Pu Wu, Hong-May Sim, Suresh V. Ambudkar. Human ABCB1 and ABCG2 confer acquired resistance to Polo-like kinase 1 inhibitors, BI 2536, volasertib and GSK641364. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2014-766

Abstract 5002: Modulation of E-cadherin transcription mediated by nuclear localization of ABCG2

Abstract Breast cancer resistance protein (BCRP/ABCG2) is a member of ATP binding cassette (ABC) transporter family. Functional overexpression of ABCG2 at plasma membrane in variety of human cancers contributes to multidrug resistance during chemotherapy. Furthermore, previous studies also demonstrate ABCG2 can localize to the intracellular compartments for efflux of its substrates. In addition, ABCG2 is responsible for Hoechst dye efflux pattern and confers the SP cell phenotype in various organs, and to play a role in protecting hematopoietic stem cells. In this study, confocal immunofluorescence microscopy and cellular fractionation showed endogenous ABCG2 localized at nucleus. Thus, we hypothesis the nuclear localization of ABCG2 may play a novel role in stemness-related gene regulation. We established ABCG2 overexpression and knockdown stable cell line by lentivirus infections and performed RNA microarray analysis in A549 cells to elucidate the effect of ABCG2 expression. We reported that ABCG2 can shuttle into the nucleus and bind to E-cadherin promoter, and then regulate E-cadherin expression in lung cancer cells. In addition, ABCG2 was able to affect cell migration and colony formation. These results demonstrated the ABCG2 adds further complexity to play a pivotal role in E-cadherin transcriptional regulation and subsequence decrease cell motility. Citation Format: Shu-Ching Liang, Chih-Yung Yang, Chi-Hung Lin. Modulation of E-cadherin transcription mediated by nuclear localization of ABCG2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5002. doi:10.1158/1538-7445.AM2014-5002

Abstract 829: The novel camptothecin derivative and IAP inhibitor FL118 is an effective treatment for irinotecan-refractory colorectal cancer

Abstract The current standard care for metastatic colorectal cancer is combination of leucovorin, 5-fluorouracil, and irinotecan. Irinotecan is a semisynthetic analogue of the alkaloid camptothecin and functions by inducing DNA double-strand breaks through inhibition of topoisomerase I (Top1) activity. The camptothecin-derived Top1 inhibitors irinotecan and topotecan are effective anti-cancer agents that are currently utilized as first-line therapies in several human cancers. Unfortunately, resistance to these agents usually develops during treatment, often through upregulation of the drug efflux pump ABCG2, an ATP-binding cassette transporter. By increasing the rate of drug export, ABCG2 decreases the amount of irinotecan or topotecan that accumulates intracellularly, thereby protecting cancer cells from the cytotoxic effects of these drugs. Therefore, a camptothecin derivative that is also a poor substrate for ABCG2 could potentially overcome ABCG2-induced irinotecan resistance and restore therapeutic efficacy. Our lab recently reported on a novel camptothecin derivative, FL118, which shows strong anti-cancer activity both in vitro and in vivo. Here we report the ability of FL118 to overcome irinotecan resistance. Several irinotecan-resistant colon cancer cell lines derived from HCT116 were treated with either FL118 or SN38, the active metabolite of irinotecan. We observed a decrease in the potency of SN38 in irinotecan-resistant cells that overexpressed ABCG2 compared to cells that did not overexpress ABCG2. In contrast, this loss of potency was not observed for FL118. To confirm that the decrease in potency was ABCG2-dependent, HCT116-A2, an ABCG2 overexpressing cell line, was treated with SN38 or FL118 in the presence or absence of Ko143, an ABCG2 inhibitor. We observed that Ko143 could restore potency to SN38 in HCT116-A2 cells, confirming that irinotecan resistance in HCT116-A2 cells is mediated by ABCG2. However, Ko143 could not modulate the potency of FL118, further suggesting that FL118 is not affected by ABCG2 activity. Similar results were observed in studies with anti-ABCG2 shRNA. Based on these observations, we conclude that FL118 is not a substrate for ABCG2, and that further testing is warranted to determine whether FL118 would be an effective anti-cancer agent for patients with irinotecan-resistant colorectal cancer overexpressing ABCG2. Citation Format: David Westover, Xiang Ling, Xiaojun Liu, Hong Lam, Celine Gongora, Maguy Del Rio, Fengzhi Li. The novel camptothecin derivative and IAP inhibitor FL118 is an effective treatment for irinotecan-refractory colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2014-829

ABCG2 regulates the pattern of self-renewing divisions in cisplatin-resistant non-small cell lung cancer cell lines.

Overexpression of ABCG2 is considered a major mechanism of cancer drug resistance. Recent studies have shown that ABCG2 can regulate the switch between symmetric and asymmetric cell division in adult stem cells; however, the relationship between ABCG2 and cell division in drug-resistant cancer cells remains to be determined. In the present study, we demonstrated that ABCG2 is involved in the cell division of drug-resistant cancer cells. We first established drug-resistant H460 and A549 cell lines by repeated exposure to cisplatin and found that the expression of ABCG2 in these cell lines was significantly increased. As evidenced by PKH-26 staining, these drug-resistant cell lines favored symmetric division, which differed from the asymmetric division of the parental cells. Furthermore, we established stable ABCG2‑overexpressing and stable shRNA-ABCG2‑knockdown cell lines to evaluate the potential role of ABCG2 in cancer cell division. The results showed that overexpression of ABCG2 in A549 parental cells significantly increased the proportion of symmetric division, whereas knockdown of ABCG2 in drug-resistant A549 cells significantly increased the proportion of asymmetric division. Taken together, our findings suggest that ABCG2 is involved in the modulation of cancer drug resistance by regulating the pattern of cell division. The present study provides novel insight into the role of ABCG2 in cancer treatment resistance.

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.

Collateral sensitivity: ABCG2-overexpressing cells are more vulnerable to oxidative stress

Multidrug resistance (MDR), which is the main obstacle to cancer chemotherapy, is mainly due to overexpression of ATP-binding cassette (ABC) transporters, especially ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP). A novel idea to overcome MDR is that of collateral sensitivity, i.e., finding a treatment to which cells overexpressing ABC transporters are more sensitive than cells that do not overexpress them. In this study we demonstrate for the first time that MDCKII-BCRP cells, overexpressing ABCG2, are more vulnerable to exogenous oxidative stress induced by several oxidants, viz. paraquat, menadione, hydrogen peroxide, tert-butylperoxide, and 2,2-azobis(2-methylpropionamidine) dihydrochloride. MDCKII-BCRP cells have significantly decreased glutathione level and decreased activities of glutathione S-transferase and glutathione reductase, which may underlie their augmented vulnerability to oxidative stress. These results suggest the possibility of using agents that induce oxidative stress to selectively kill cells overexpressing BCRP.

The Overexpression of ABCG2 Reduces the Efficacy of Volasertib (BI 6727) and GSK641364 in Human S1-M1-80 Colon Carcinoma Cells

The polo-like kinase 1 (Plk1) is one of the key regulators in cell cycle progression. Plk1 is overexpressed in many types of cancer and promotes the proliferation of cancer cells. Inhibition of Plk1 activity induces G2/M cell cycle arrest and reduces cancer cell viability. Volasertib and GSK461364 are selective inhibitors of Plk1, active against a wide range of tumor cells at nanomolar concentrations. In this study, while examining the effectiveness of Plk1 inhibitors against multiple human colon cancer cell lines, we discovered that the overexpression of ATP-binding cassette (ABC) drug transporter ABCG2 in human S1-M1-80 colon cancer cells confers resistance to volasertib and GSK461364. Moreover, we found that ABCG2-transfected HEK293 cells were also resistant to both Plk1 inhibitors. We revealed that volasertib and GSK461364 inhibited the function of ABCG2 in a concentration dependent manner, and had no significant effect on the protein expression of ABCG2. More importantly, we showed that the G2/M cell cycle arrest induced by volasertib or GSK461364 was significantly reduced in S1-M1-80 cells, and that ABCG2-mediated drug resistance to Plk1 inhibitors can be restored by inhibition of ABCG2 function. Therefore, the development of ABCG2-mediated drug resistance to volasertib and GSK461364 in cancer clearly present a significant therapeutic challenge, and a better treatment strategy should be further investigated.

Telatinib reverses chemotherapeutic multidrug resistance mediated by ABCG2 efflux transporter in vitro and in vivo

Multidrug resistance (MDR) is a phenomenon where cancer cells become simultaneously resistant to anticancer drugs with different structures and mechanisms of action. MDR has been shown to be associated with overexpression of ATP-binding cassette (ABC) transporters. Here, we report that telatinib, a small molecule tyrosine kinase inhibitor, enhances the anticancer activity of ABCG2 substrate anticancer drugs by inhibiting ABCG2 efflux transporter activity. Co-incubation of ABCG2-overexpressing drug resistant cell lines with telatinib and ABCG2 substrate anticancer drugs significantly reduced cellular viability, whereas telatinib alone did not significantly affect drug sensitive and drug resistant cell lines. Telatinib at 1μM did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines. Telatinib at 1μM significantly enhanced the intracellular accumulation of [3H]-mitoxantrone (MX) in ABCG2-overexpressing cell lines. In addition, telatinib at 1μM significantly reduced the rate of [3H]-MX efflux from ABCG2-overexpressing cells. Furthermore, telatinib significantly inhibited ABCG2-mediated transport of [3H]-E217βG in ABCG2 overexpressing membrane vesicles. Telatinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that telatinib might be a substrate of ABCG2. Binding interactions of telatinib were found to be in transmembrane region of homology modeled human ABCG2. In addition, telatinib (15mg/kg) with doxorubicin (1.8mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. These results, provided that they can be translated to humans, suggesting that telatinib, in combination with specific ABCG2 substrate drugs may be useful in treating tumors that overexpress ABCG2.