Human ATP-Binding Cassette Transporter ABCG2 Confers

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Human ATP-Binding Cassette transporter ABCG2 confers resistance to CUDC-907, a dual inhibitor of histone deacetylase and phosphatidylinositol 3-kinase Chung-Pu Wu, Ya-Ju Hsieh, Sung-Han Hsiao, Ching-Ya Su, Yan-Qing Li, YangHui Huang, Chiun-Wei Huang, Chia-Hung Hsieh, Jau-Song Yu, and Yu-Shan Wu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00687 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on January 30, 2016

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Molecular Pharmaceutics

Human ATP-Binding Cassette transporter ABCG2 confers resistance to CUDC-907, a dual inhibitor of histone deacetylase and phosphatidylinositol 3-kinase

Chung-Pu Wu a,b,c,*, Ya-Ju Hsieh c, Sung-Han Hsiao a, Ching-Ya Su a, Yan-Qing Li b, Yang-Hui Huang c, Chiun-Wei Huang d, Chia-Hung Hsieh e,f , Jau-Song Yu a, c and Yu-Shan Wu g, **

Authors' Affiliations: a

Graduate Institute of Biomedical Sciences, b Department of Physiology and

Pharmacology, and c Molecular Medicine Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan. d Center for Advanced Molecular Imaging and Translation, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan. e

Graduate Institute of Basic Medical Science, and f Department of Medical Research,

China Medical University Hospital, Taichung, Taiwan. g

Department of Chemistry, Tunghai University, Taichung, Taiwan.

* Corresponding author at: 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan. Phone: +886-3-2118800, ext. 3754. Fax: +886-3-2118700. E-mail address: [email protected] **Corresponding author at: 181 Taichung Harbor Road Section 3, Taichung, Taiwan. Phone: +886-4-2359-0121, ext 32248. Fax: +886-4-2359-0426. E-mail address: [email protected]

Keywords: Multidrug resistance; ABCG2; HDAC; PI3K; CUDC-907.

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Running Title: ABCG2 confers resistance to CUDC-907.

Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; HDAC, histone deacetylase; PI3K, phosphatidylinositol 3-kinase.

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ABSTRACT

CUDC-907 is a novel, dual-acting small molecule compound designed to simultaneously inhibit the activity of histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K). Treatment with CUDC-907 led to sustained inhibition of HDAC and PI3K activity, inhibition of RAF-MEK-MAPK signaling pathway and cancer cell growth. CUDC-907 is currently under evaluation in phase I clinical trials in patients with lymphoma or multiple myeloma, and in patients with advanced solid tumors. However, the risk of developing acquired resistance to CUDC-907 can present a significant therapeutic challenge to clinicians in the future, and should be investigated. The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1, ABCC1 or ABCG2 is one of the most common mechanisms of developing multidrug resistance (MDR) in cancers and a major obstacle in chemotherapy. In this study, we reveal that ABCG2 reduces the intracellular accumulation of CUDC-907 and confers significant resistance to CUDC-907, which lead to reduced activity of CUDC-907 to inhibit HDAC and PI3K in human cancer cells. Moreover, although CUDC-907 affects the transport function of ABCG2, it was not potent enough to reverse drug resistance mediated by ABCG2 or affect the expression level of ABCG2 in human cancer cells. Taken together, our findings indicate that ABCG2-mediated CUDC-907 resistance can have serious clinical implications and should be further investigated. More importantly, we demonstrate that the activity of CUDC-907 in ABCG2-overexpressing cancer cells can be restored by inhibiting the function of ABCG2, provides support for the rationale of combining CUDC-907 with modulators of ABCG2 to improve the pharmacokinetics and efficacy of CUDC-907 in future treatment trials.

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1. Introduction

The phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases that have critical roles in regulating a wide range of cellular processes, including cell proliferation and differentiation 1. Collectively, they transduce signals from growth factors and cytokines into intracellular messages, and subsequently activate the serine-threonine kinase AKT and other downstream effectors 1. The PI3K pathway is crucial in cancer proliferation. Through mutations of receptor tyrosine kinases, it is one of the most commonly activated signaling pathway in human cancer, making this pathway one of the most attractive targets for cancer therapy 1-3. Over the past decade, researchers have made significant progress in developing inhibitors that target PI3K and other key components in the pathway. For instance, PI3K pathway inhibitors GDC0941, GSK1059615 and XL147 showed promising activity against various types of cancers and are currently under clinical investigation in patients with solid tumors 4. Unfortunately, numerous studies reported that the efficacy of these PI3K inhibitors can be reduced by concurrent activation of other cellular pathways 5, 6. The overexpression of histone deacetylases (HDACs) has been reported in a wide range of cancers 7, and the inhibition of HDAC activity by agents such as SAHA (vorinostat) has been shown to induce tumor cell apoptosis 8, suppress DNA damage repair 9 and angiogenesis 10. Recent studies demonstrated synergistic anti-cancer activity by the combination of HDAC and PI3K inhibitors in human lymphoma, endometrial and prostate cancer cells 11-13. Moreover, inhibition of HDAC activity can overcome concurrent activation of alternative survival related pathways that reduces the efficacy of PI3K inhibitors 14-16. CUDC-907 is a novel, dual-acting small molecule drug developed based on the experimental evidence demonstrating a synergistic anticancer effect of PI3K and HDAC inhibitors. It was 4


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rationally designed and synthesized by integrating key components of several HDAC inhibitors and PI3K inhibitors into a single scaffold 17. Remarkably, CUDC-907 inhibited class I and class II enzymes in HDAC complexes, as well as PI3K activity at nanomolar concentrations. Treatment with CUDC-907 led to sustained AKT inhibition, inhibition of RAF-MEK-MAPK signaling pathway, induction of G2/M phase cell cycle arrest and inhibition of human cancer cell growth 17. CUDC-907 is currently under evaluation in phase I clinical trials in patients with lymphoma or multiple myeloma (ClinicalTrials.gov number NCT01742988), and in patients with advanced solid tumors (ClinicalTrials.gov number NCT02307240).

The development of multidrug resistance (MDR) to chemotherapeutic agents is a major obstacle to the effective treatment of cancer. Although several factors are known to contribute to MDR phenotype of cancer, the overexpression of ATP-binding cassette (ABC) drug transporter protein ABCB1, ABCC1 or ABCG2 is still one of the most common mechanisms for the development of MDR 18, 19. Human ABCB1 (also known as P-glycoprotein, Pgp) 20 and ABCC1 (also known as multidrug resistance protein 1, MRP1) 21 are members of the mammalian ABC protein family known to transport drug substrates across cell membranes by utilizing energy derived from ATP hydrolysis. Collectively, they are able to actively efflux a majority of conventional anticancer drugs such as Vinca alkaloids, anthracyclines, etoposide, taxanes, methotrexate and colchicines out of cancer cells, causing MDR and cancer relapse 19. Human ABCG2, also known as breast cancer resistance protein (BCRP), is another member of the mammalian ABC protein family that is linked to the development of MDR phenotype in cancer cells 22. ABCG2 functions as a homodimer 22, capable of transporting a wide range of conventional anticancer 5



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agents such as mitoxantrone, methotrexate, topotecan and SN-38 out of cancer cells 19

. Moreover, studies indicated that ABCG2 can also transport many

molecular-targeted anticancer agents, including gefitinib, imatinib, sunitinib, sorafenib and vemurafenib 23-31. ABCG2 confers resistance to these anticancer agents and is associated with clinical drug resistance, such as in patients with acute lymphocytic leukemia (ALL) or acute myelogenous leukemia (AML) 32-34. The fact that ABCG2 is also highly expressed at barrier sites in normal tissues, including the blood-brain barrier (BBB), kidney and the gastrointestinal wall, can negatively affect drug absorption, distribution, metabolism and excretion of chemotherapeutic drugs 35-38. Therefore, it is important to evaluate whether ABC drug transporter activity can lead to reduced efficacy of CUDC-907 in cancer cells.

Here, we discovered that ABCG2-overexpressing cells were significantly less sensitive to CUDC-907 treatment, and that G2/M cell cycle arrest induction, HDAC and PI3K inhibition by CUDC-907 were significantly reduced by ABCG2 in human cancer cells. More importantly, we demonstrated that the reduced sensitivity of ABCG2-overexpressing cells to CUDC-907 was caused by reduced accumulation of CUDC-907 mediated by ABCG2, which can be fully restored by inhibiting the function of ABCG2 transporter.

2. Materials and methods

2.1. Chemicals DMEM, fetal calf serum (FCS), trypsin-EDTA, penicillin, streptomycin and PBS were purchased from Gibco, Invitrogen (CA, USA). Cell Counting Kit-8 (CCK-8), MTT dye, Ko 143, SAHA, pheophorbide A, mitoxantrone and other 6


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chemicals were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise. Cell Cycle Kit was purchased from BD Pharmingen (San Diego, CA, USA). CUDC-907, GDC-0980 and lapatinib (99% purity by HPLC) were purchased from Selleckchem (Houston, TX, USA).

2.2. Cell lines and culture conditions The human epidermal tumor line KB-3-1 and its drug-selected ABCB1-overexpressing variants KB-C-1 and KB-V-1, human ovarian tumor line OVCAR-8 and its ABCB1-overexpressing variant NCI-ADR-RES, human breast MCF7 cell line and its ABCG2-overexpressing MCF7-FLV1000 and MCF7-AdVp3000 sublines, as well as mouse NIH3T3 and NIH3T3-G185 fibroblast cells were cultured in DMEM, supplemented with 10 % fetal calf serum (FCS), 2 mM L-glutamine and 100 units of penicillin/streptomycin/mL. KB-C-1 cells were maintained in media containing 1 µg/mL of colchicine, whereas KB-V-1 cells were maintained in media containing 1 mg/mL vinblastine 39. MCF7-FLV1000 cells were cultured in the presence of 1 µg/mL flavopiridol, and MCF7-AdVP3000 cells were maintained in the presence of 3 µg/mL doxorubicin and 5 µg/mL verapamil 40, 41. NIH3T3-G185 cells were maintained in the presence of 60 ng/mL colchicine 42. The human large cell lung tumor line COR-L23/P and its doxorubicin-selected ABCC1-overexpressing variant COR-L23/R, as well as the human colon carcinoma cell line S1 and its ABCG2-overexpressing subline S1-M1-80 were cultured in RPMI 1640 medium (Gibco, Invitrogen), supplemented with 10 % FCS and 100 units of penicillin/streptomycin/mL (Invitrogen, Carlsbad, CA). Doxorubicin was added to the COR-L23/R cell culture medium 43, whereas S1-M1-80 was cultured in 80 µM of mitoxantrone as described previously 44. pcDNA3.1-HEK293, MDR19-HEK293 (HEK293 cells transfected with human ABCB1), R482-HEK293 7



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(HEK293 cells transfected with human ABCG2) and MRP1-HEK293 (HEK293 cells transfected with human ABCC1) cells were cultured in DMEM, supplemented with 10 % FCS, 2 mM L-glutamine, 100 units of penicillin/streptomycin/mL and 2 mg/mL G418 30. Cell lines were generous gifts from Dr. Suresh V. Ambudkar (National Cancer Institute, NIH, Bethesda, MD, USA), all maintained at 37 °C in 5 % CO2 humidified air. Cells were placed in drug-free medium 7 days prior to assay.

2.3. Cytotoxicity assay MTT and CCK-8 assays were used to determine the sensitivity of cells to the tested compounds according to the method described by Ishiyama et al 45. MTT assays were performed to determine the cytotoxicity of drugs in cancer cell lines, whereas CCK-8 assays were used to determine the cytotoxicity of drugs in HEK293 and HEK293 cells transfected with a particular ABC transporter. Briefly, cells were plated into 96-well plates at a density of 5,000 cells per well in 100 µL of culture medium at 37 °C for 24 h before adding drugs to make a final volume of 200 µL. Cells were incubated for an additional 72 h with various concentrations of drugs before developed with CCK-8 reagent or MTT as described previously 46. For the reversal of cytotoxicity assays, a nontoxic concentration of CUDC-907 or Ko 143 was added to the cytotoxicity assay, and the extent of reversal was then calculated based on the relative resistance values.

2.4. Cell cycle analysis Cell cycle experiments were performed using a standard propidium iodide (PI) staining method and cells were analyzed using a FACSort flow cytometer equipped with the Cell Quest software (Becton-Dickinson). Briefly, cells were treated with the indicated regimens for 24 h before being harvested in PBS and fixed in ethanol 8


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overnight. Cells were washed once with PBS, then treated with 0.5 % TritonX-100 and 0.05 % RNase in PBS at 37 °C for 1 h. Cells were washed, propidium iodide (50 µg/mL) added, then incubated the cells at 4 °C for at least 20 min before analysis.

2.5. Immunoblotting Antibodies anti-acetyl-Histone H3, anti-acetylated tubulin, anti-Akt, antiphospho-Akt (Thr308), antiphospho-Akt (Ser473), BXP-21 and anti-α-tubulin were used to detect class I HDAC, class II HDAC, total Akt, phosphorylated Akt and ABCG2, respectively, and tubulin as positive control for Western blotting. The secondary antibodies used were the Horseradish peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG. Signals were detected as described previously. 30, 47, 48

.

2.6. CUDC-907 accumulation assay and HPLC-MS/MS analysis CUDC-907 was quantified by HPLC-MS/MS based assay according to the method described by Cihalova et al 49 and Shen et al 50 with slight modification. Briefly, 2 x 106 cells were treated with the indicated regimens at 37 °C for 60 min, washed twice with cold PBS, harvested and resuspended in 3 x volume of methanol and stored at -80 °C. After thawed, lysates with methanol extraction were spin down by 10,000 rpm for 30 min at 4 °C. Supernatants were dried with speed vacuum, redissolved in 50 % methanol/H2O and 0.1 % formic acid, and analyzed using Selected Reaction Monitoring (SRM). Cell contents were analyzed using multiplexed HPLC-SRM-MS on a Waters nanoACQUITY ultra-performance liquid chromatography (UPLC) system coupled with HCT ultra (Bruker Daltonik GmbH, Bremen, Germany). Mobile system A: 0.1 % formic acid in water; B: acetonitrile with 0.1 % formic acid. A flow rate of 60 µL/min with a linear gradient was set as 9



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follows: 0 min, 5 % B; 2 min, 5 % B; 3 min, 15 % B; 13 min, 35 % B; 25 min, 70 % B; 28 min, 99 % B; 32 min, 99 % B; 32.1 min 5 % B. CUDC-907 data was acquired by SRM mode; isolation 509.2 with peak width 4 amu, followed by smart fragmentation ramping from 0.3 to 2 volt. MS2 fragment 491.2 was selected, performed isolation and smart fragmentation to acquire MS3, 342.1. One blank run was inserted between every sample injections. The peak area of MS3 fragment 342.1 was detected and integrated using software package DataAnalysis 4.2. (Bruker Corporation). The standard response curves were generated with cell lysate extracts as background, combined with six different concentrations of CUDC-907. The response curves were 1 : 5 serial dilutions, and CUDC-907 ranging from 10 pmol to 3.2 fmol. The precursor ion m/z of CUDC-907 used for SRM analysis is 509.2, MS2 fragment is 491.2, and MS3 fragment is 342.1. For higher specificity, peak area of MS3 fragment (342.1) was selected and calculated as response curves.

2.7. Fluorescent drug accumulation assay ABCG2-mediated efflux assays were carried out using a FACSort flow cytometer equipped with CellQuest software. Briefly, cells were harvested after trypsinization by centrifugation at 500 x g and then resuspended in Iscove's modified Dulbecco's medium (IMDM) supplemented with 5 % FCS. Mitoxantrone (MX) or pheophorbide A (PhA) was added to 3 x 105 cells in 4 mL of IMDM in the presence or absence of tested drugs. The effect of CUDC-907 or Ko 143 on ABCG2-mediated efflux was measured and analyzed according to the method described by Gribar et al 51.

2.8. Statistical analysis 10


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GraphPad Prism software (La Jolla, CA, USA ) was used to plot the curves and statistical analysis. Data are presented as mean ± S.E.M, whereas IC50 values were calculated as mean ± SD from at least three independent experiments. Differences between any mean values were analyzed by two-sided Student’s t-test and results were considered statistically significant at P < 0.05.

3. Results

3.1. ABCG2-overexpressing cells are resistant to CUDC-907 In order to evaluate the potential interactions between CUDC-907 and three major ABC drug transporters, we examined the cytotoxicity of CUDC-907 in multiple human cancer cell lines, including cells overexpressing ABCB1, ABCG2 or ABCC1. The calculated IC50 values for CUDC-907 are summarized in Table 1. The degree of cellular resistance to CUDC-907 caused by the overexpression of a particular ABC transporter was represented by the resistance factor (RF) value, which was calculated by dividing the IC50 value of MDR subline by the IC50 value of the respective parental line. Our data revealed that although CUDC-907 was highly effective against most cancer cell lines, ABCG2-overexpressing human colon S1-M1-80, breast MCF7-FLV1000 and MCF7-AdVp3000 cancer cells were significantly resistant to CUDC-907 as compared with the ABCG2-negative parental cells, with calculated RF values of approximately 34, 174 and 131, respectively. To further confirm our findings, we determined the cytotoxicity of CUDC-907 in cells transfected with either human ABCB1, ABCG2 or ABCC1. Similar to what we observed in ABCG2-overexpressing human cancer cells, human embryonic kidney HEK293 cells transfected with human ABCG2 (R482-HEK293) were significantly resistant to CUDC-907 treatment than the parental HEK293 cells, with calculated 11



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RF value of 8.56.

3.2. CUDC-907 is less effective in inhibiting the PI3K pathway and HDAC activity in ABCG2-overexpressing MDR cancer cells Next, we examined the effect of CUDC-907 on cell cycle phase distribution in drug sensitive human S1 colon cancer cell line and ABCG2-overexpressing S1-M1-80 subline since CUDC-907 was shown to induce significant cell cycle arrest in human cancer cells 17. We found that at 1 µM, CUDC-907 induced significant G2/M cell cycle arrest in S1 cell, from 14.2 ± 1.2 % basal to 44.9 ± 3.0 % (Fig. 1A). In contrast, the effect of G2/M arrest induced by CUDC-907 in S1-M1-80 cells was considerably less, from 18.1 ± 1.3 % basal to 24.9 ± 4.2 % (Fig. 1B). Given that CUDC-907 is a dual-acting inhibitor designed to inhibit PI3K signaling pathway and HDAC activity 17, and that CUDC-907 appeared to be less cytotoxic in ABCG2-overexpressing cells (Table 1), we decided to compare the effect of CUDC-907 on AKT phosphorylation and acetylation of histone H3 and H4 in S1 and S1-M1-80 cells. As expected, CUDC-907 (10 µM) strongly reduced the level of AKT phosphorylation on threonine 308 and serine 473 in parental S1 cancer cells (left panels, Fig. 2A). However, the inhibitory effect of CUDC-907 on AKT phosphorylation was significantly less in drug resistant S1-M1-80 cells (right panels, Fig 2A). In contrast, we found that GDC-0980, a known inhibitor of PI3K/mTOR kinase 52, was equally effective in inhibiting AKT phosphorylation on threonine 308 and serine 473 in both drug sensitive S1 and drug resistant S1-M1-80 cells. Interestingly, we found that we were able to restore the activity of CUDC-907 to inhibit AKT phosphorylation with 1 µM of Ko 143, a reference inhibitor of ABCG2. Of note, treatment with CUDC-907 alone or in combination with Ko 143 for 1 h had no significant effect on ABCG2 protein expression in these cell lines (Fig. 2A and 12


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2B). Next, we examined the effect of CUDC-907 on histone acetylation of histone H3 and H4 in drug sensitive and drug resistant cancer cell lines (Fig. 2C). The human colon S1 and S1-M1-80 cancer cells were maintained in the absence or presence of 10 µM of CUDC-907 or 20 µM of SAHA (a reference inhibitor of HDAC) alone, or in combination with 1 µM of Ko 143 for 1 h as indicated in Materials and methods. We discovered that treatment with CUDC-907 led to significantly more histone acetylation in S1 cells than in S1-M1-80 cells, while SAHA was equally effective in both cell lines (Fig. 2D). Again, we discovered that in the presence of Ko 143, the ability of CUDC-907 to increase acetylation of histone H3 and H4 in drug resistant S1-M1-80 cells was restored to the same extent as in drug sensitive S1 cells (Fig. 2C and D). Collectively, our data indicate that the function of ABCG2 can reduce the capability of CUDC-907 to inhibit AKT phosphorylation and HDAC activity in cancer cells.

To further determine whether increased level of CUDC-907 resistance corresponds to reduced CUDC-907 accumulation in ABCG2-overexpressing cells, we directly measured the intracellular accumulation of CUDC-907 (Fig. 3A) in S1, S1-M1-80, HEK293 and R482-HEK293 cells after exposing cells to 10 µM of CUDC-907 for 1 h as described in Materials and methods. We found that the level of CUDC-907 accumulation in ABCG2-expressing S1-M1-80 and R482 HEK293 cells was significantly lower than in ABCG2-negative S1 and HEK293 cells. Moreover, results showed that ABCG2 reduces the accumulation of CUDC-907 by approximately 63 % in S1-M1-80 cells, and approximately 43 % in R482-HEK293 cells (Fig. 3B), which can be restored by ABCG2 inhibitor Ko 143 (1 µM). We cannot rule out that a portion of CUDC-907 measured in the experiment can be contributed by non-specific binding of CUDC-907 to membranes, in which case the 13



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percentage of CUDC-907 accumulated in ABCG2-overexpressing cells, as compared to cells in the presence of Ko 143, would be even lower. Of note, the level of CUDC-907 detected in S1-M1-80 cells appeared to be lower than the level in R482-HEK293 cells. We suspect that this may due to the higher ABCG2 protein expression in S1-M1-80 cells than in R482-HEK293 cells (Fig. 3B, inset).

3.3. Inhibition of ABCG2 function can re-sensitize ABCG2-overexpressing cancer cells to CUDC-907 Since we have discovered earlier that the activity of CUDC-907 in ABCG2-overexpressing cancer cells can be restored by directly inhibiting the transport function of ABCG2 (Fig. 2), we thus evaluated whether we can resensitize ABCG2-overexpressing cells to CUDC-907 in the same way. First, we examined the effect of Ko 143 (1 µM) and lapatinib (0.3 µM) on ABCG2-mediated resistance to CUDC-907 in human S1 colon cells. Many studies have shown that by competing with the transport of another substrate, inhibitors and some drug substrates of ABCG2 can restore drug sensitivity in ABCG2-overexpressing MDR cancer cells 30, 53, 54

. In agreement with previous findings, our results showed that Ko 143

completely reversed ABCG2-mediated resistance to CUDC-907, whereas lapatinib, a known drug substrate of ABCG2 54, partially but significantly reversed CUDC-907 resistance from approximately 34-fold to 4-fold resistance in resistant S1-M1-80 cancer cells. In order to further confirm our findings, we also examined the effect of Ko 143 and lapatinib on ABCG2-mediated resistance to CUDC-907 in ABCG2-transfected HEK293 cells. As expected, both Ko 143 and lapatinib significantly restored drug sensitivity to CUDC-907 in R482-HEK293 cells (Table 2). Of note, Ko 143 or lapatinib alone did not produce significant cytotoxic effect in S1 or HEK293 cell lines. 14


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3.4. CUDC-907 inhibits transport mediated by ABCG2 Knowing that ABCG2 overexpression confers acquired resistance to CUDC-907 in cancer cells, we decided to evaluate the drug-drug interaction of CUDC-907 with ABCG2 by examining the effect of CUDC-907 on ABCG2-mediated drug efflux and drug resistance in ABCG2-overexpressing S1-M1-80 cancer cells and ABCG2-transfected R482-HEK293 cells. Moreover, we also examined the effect of 72 h CUDC-907 exposure on ABCG2 protein expression in S1 and S1-M1-80 cancer cells. First, we examine the effect of CUDC-907 on ABCG2-mediated efflux of two known fluorescent substrates of ABCG2, mitoxantrone and pheophorbide A (PhA) 46, 55. We found that without affecting parental cells (left panels, Fig. 4), 10 µM of CUDC-907 was required to inhibit ABCG2-mediated efflux of mitoxantrone (right panels, Fig. 4A and B) and PhA (right panels, Fig. 4C and D) in S1-M1-80 and R482-HEK293 cells to the same extent as the benchmark inhibitor Ko 143 (5 µM). Next, we examined the effect of CUDC-907 on ABCG2-mediated resistance to mitoxantrone in S1-M1-80 and R482-HEK293 cells. Non-toxic concentrations of 1 nM and 2 nM were used in the experiment based on the cytotoxicity of CUDC-907 determined in S1 and HEK293 cell lines. The reversal effect of CUDC-907 in these MDR cell lines is summarized in Table 3. The relative resistance (RR) value here represents the relative resistance of ABCG2-overexpressing cells to mitoxantrone in the presence or absence of CUDC-907 or Ko 143. In contrast to Ko 143, CUDC-907 was unable to reverse ABCG2-mediated resistance to mitoxantrone in S1-M1-80 or R482-HEK293 cells at the concentrations tested (Table 3). Lastly, we tested the effect of CUDC-907 on the ABCG2 protein expression in S1 and S1-M1-80 cancer cells. Cells were maintained in medium in the absence or presence of increasing concentrations of CUDC-907 (1 15



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- 5 µM) for 72 h and processed for immunoblotting. Our results indicate that short-term exposure of S1 and S1-M1-80 cancer cells to CUDC-907 had no significant effect on the protein expression level of ABCG2 (Fig. 5).

4. Discussion

We have seen rapid progress in the development of new molecular targeted agents in recent years, especially in the introduction of oncogenic kinase inhibitors, which has resulted in improved response rates and survival in cancer patients. CUDC-907 is a novel dual-acting inhibitor of PI3K and HDAC, rationally developed based on experimental evidence that combined treatment of PI3K and HDAC inhibitors exhibited a synergistic anticancer effect 14, 16. It is believed that a single-dual acting inhibitor has therapeutic advantages over combination therapy, such as reduced toxicity, improved pharmacokinetics characteristics and patient compliance. It was suggested that due to its ability to simultaneously block PI3K activity and regulate signaling proteins through HDAC inhibition, CUDC-907 could potentially prevent the development of drug resistance 17. Unfortunately, in addition to activation of alternative or downstream signaling pathways, the overexpression of ABC drug transporters is also a known cause of acquired resistance to kinase inhibitors 23-25, 56, 57. Therefore, we sought to evaluate if overexpression of ABCB1, ABCC1 or ABCG2 could mediate resistance to CUDC-907.

In the present study, we first determined the cytotoxicity of CUDC-907 in several drug sensitive cancer cell lines and MDR cancer cell lines overexpressing human ABCB1, ABCG2 or ABCC1. CUDC-907 has been shown to be highly effective against a broad range of human cancer cell lines, including colon, lung and 16


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breast cancer cell lines, with IC50 values lower than 100 nM 17. In our study, the IC50 values of CUDC-907 for human epidermal, ovary, colon, breast and lung cancer cells (ranging from 5 to 35 nM) were comparable to previously reported values. However, we discovered that all three ABCG2-overexpressing cancer cell lines (S1-M1-80, MCF7-FLV1000 and MCF7-AdVp3000) were significantly less sensitive to CUDC-907 compared to other cancer cell lines. The fact that HEK293 cells transfected with human ABCG2 (R482-HEK293) were also resistant to CUDC-907, implicating ABCG2 as a mechanism of resistance for CUDC-907 (Table 1). This result was not surprising to us since ABCG2 overexpression is one of the most common mechanisms for the development of MDR in cancer cells 18, 19, and is responsible for reduced bioavailability and efficacy of many kinase inhibitors 23, 25, 27, 30, 46, 55, 58, 59

.

A key characteristic of CUDC-907 is to inhibit cancer cell proliferation by targeting PI3K signaling and HDAC activity in cancer cells 17. Therefore, we examined the activity of CUDC-907 in drug sensitive human colon cancer cell line S1 and resistant subline S1-M1-80. Of note, S1-M1-80 was the ABCG2-overexpressing cancer cell line we have tested with the lowest RF value (Table 1), thus was used specifically to avoid overstating the impact of ABCG2 function on the efficacy of CUDC-907. We discovered that CUDC-907 was highly effective in S1 cells, inhibited the phosphorylation of AKT at residues S473 and Thr308 to the same extent as GDC-0980. Similarly, CUDC-907 inhibited the activity of HDACs to the same extent as SAHA in S1 cells. However, CUDC-907 was significantly less effective in S1-M1- cells, suggesting the involvement of ABCG2 in reducing the activity of CUDC-907 in cancer cells. In contrast, GDC-0980 and SAHA were unaffected by the function of ABCG2. Our suspicion 17



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was supported by the fact that ABCG2-overexpressing S1-M1-80 cells accumulated significantly less CUDC-907 than ABCG2-negative S1 cells (Fig. 3), and that ABCG2 inhibitor Ko 143 was able to restore the intracellular concentration and thus activity of CUDC-907 in S1-M1-80 cells (Fig. 2 and 3). Of note, we were expecting considerably more reduction of CUDC-907 accumulation in ABCG2-expressing cells considering the high expression of ABCG2 (Fig. 3B, inset). We suspected that a portion of CUDC-907 measured in the experiment was contributed by non-specific binding of CUDC-907 to membranes, and the true effect of ABCG2 on CUDC-907 accumulation should be even greater. Nevertheless, we were able to restore the chemosensitivity of ABCG2-overexpressing S1-M1-80 cells and ABCG2 transfected R482-HEK293 to CUDC-907 by inhibiting the function of ABCG2 with Ko 143 or a competitive drug substrate lapatinib (Table 2).

The search for potent inhibitor of ABCG2 has been ongoing for years without success, but some ABCG2 interacting kinase inhibitors have recently been shown to reverse ABCG2-mediated drug resistance 30, 54, 60-62, either by competitive inhibition 63

, or by transiently reducing the expression of ABCG2 in cancer cells 64, 65.

Therefore, we examined the drug-drug interactions of CUDC-907 with mitoxantrone and ABCG2 protein expression in S1-M1-80 cancer cells. Unfortunately, due to the highly toxic nature of CUDC-907, we were unable to reverse ABCG2-mediated mitoxantrone resistance (Table 3) or downregulation ABCG2 expression over a period of 72 h (Fig. 5). Considering that 10 µM of CUDC-907 was required to inhibit ABCG2-mediated transport of mitoxantrone (Fig. 4), it is not surprising that CUDC-907 at non-toxic concentrations (1nM and 2nM) failed to have any significant impact on mitoxantrone resistance mediated by ABCG2 (Table 3). Nevertheless, the effect of prolonged treatment with CUDC-907 remains to be 18


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determined.

In summary, although the clinical relevance of cell line-based model in the study of mechanisms of MDR in cancer remains controversial 66, our results indicated that reduced sensitivity of CUDC-907 in ABCG2-overexpressing cells could have clinical implications, such as reduced therapeutic efficacy, pharmacokinetics and pharmacodynamics in some patients, and should be further investigated. Moreover, the fact that we were able to restore the chemosensitivity of ABCG2-overexpressing MDR cells to CUDC-907 by inhibiting the function of ABCG2, suggests the need for developing a more effective drug combination strategy to overcome multidrug resistance in cancer.

Conflict of Interest None.

Acknowledgments The authors thank Dr. Suresh V. Ambudkar (National Cancer Institute, NIH) for generously providing cell lines. This work was supported by funds from the Chang Gung Medical Research Program (CMRPD1D0152), the Ministry of Science and Technology of Taiwan (MOST-103-2320-B-182-011) and a grant to Chang Gung University from the Ministry of Education (EMRPD1E1571).

19



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ABCC10-multidrug resistance xenograft models. Cancer Lett 2013, 328, (2), 307-17. 61. Sodani, K.; Tiwari, A. K.; Singh, S.; Patel, A.; Xiao, Z. J.; Chen, J. J.; Sun, Y. L.; Talele, T. T.; Chen, Z. S. GW583340 and GW2974, human EGFR and HER-2 inhibitors, reverse ABCG2- and ABCB1-mediated drug resistance. Biochem Pharmacol 2012, 83, (12), 1613-22. 62. Shi, Z.; Tiwari, A. K.; Shukla, S.; Robey, R. W.; Singh, S.; Kim, I. W.; Bates, S. E.; Peng, X.; Abraham, I.; Ambudkar, S. V.; Talele, T. T.; Fu, L. W.; Chen, Z. S. Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance. Cancer Res 2011, 71, (8), 3029-41. 63. Robey, R. W.; Obrzut, T.; Shukla, S.; Polgar, O.; Macalou, S.; Bahr, J. C.; Di Pietro, A.; Ambudkar, S. V.; Bates, S. E. Becatecarin (rebeccamycin analog, NSC 655649) is a transport substrate and induces expression of the ATP-binding cassette transporter, ABCG2, in lung carcinoma cells. Cancer Chemother Pharmacol 2009, 64, (3), 575-83. 64. Cuestas, M. L.; Castillo, A. I.; Sosnik, A.; Mathet, V. L. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles. Bioorg Med Chem Lett 2012, 22, (21), 6577-9. 65. Natarajan, K.; Bhullar, J.; Shukla, S.; Burcu, M.; Chen, Z. S.; Ambudkar, S. V.; Baer, M. R. The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms. Biochem Pharmacol 2013, 85, (4), 514-24. 66. Gillet, J. P.; Calcagno, A. M.; Varma, S.; Marino, M.; Green, L. J.; Vora, M. I.; Patel, C.; Orina, J. N.; Eliseeva, T. A.; Singal, V.; Padmanabhan, R.; Davidson, B.; Ganapathi, R.; Sood, A. K.; Rueda, B. R.; Ambudkar, S. V.; Gottesman, M. M. Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance. Proc Natl Acad Sci U S A 2011, 108, (46), 18708-13.

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Figure Legends

Fig. 1. Effect of CUDC-907 on G2/M cell cycle arrest in (A) drug sensitive S1 and (B) resistant ABCG2-overexpressing S1-M1-80 cells. Cells were plated and maintained in the absence (left panels) or presence of 1 µM CUDC-907 (right panels, 1 µM) for 24 h before harvest for cell cycle analysis. Representative histograms of five independent experiments are shown.

Fig. 2. CUDC-907 inhibits PI3K signaling and HDAC activity in drug sensitive S1 cells. (A) Human colon carcinoma S1 cells and the MDR subline S1-M1-80 cells were treated with DMSO (control), 10 µM of CUDC-907 (CUDC), 10 µM of GDC-0980 (GDC), 1 µM of Ko 143 as indicated for 1 h before processed for immunoblotting. GDC-0980 is a dual PI3K/mTOR inhibitor, used here as a control. Human EGF (50 ng/mL) was added to the culture medium for 15 min to stimulate phosphorylation. (B) Quantifications of Western blots showing the relative level of AKT phosphorylation in S1 (open bars) and S1-M1-80 (closed bars) cells. (C) S1 and S1-M1-80 cells were treated with DMSO (control), 10 µM of CUDC-907, 20 µM of SAHA, 1 µM of Ko 143 as indicated for 1 h before processed for immunoblotting. SAHA is a known inhibitor of HDAC, used here as a control. (D) Quantifications of Western blots showing the relative level of histone acetylation in S1 (open bars) and S1-M1-80 (closed bars) cells. Representative Western blots are shown and values are presented as mean ± S.D. calculated from more than three independent experiments.*P < 0.05; **P < 0.01, versus the same treatment in parental cells.

Fig. 3. ABCG2 reduces CUDC-907 accumulation in ABCG2-overexpressing 27



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Page 28 of 41

S1-M1-80 and R482-HEK293 cells. (A) The structure and product ion mass spectra of CUDC-907. (B) Quantification of CUDC-907 accumulated in human S1 colon cancer cells and ABCG2-overexpressing S1-M1-80 cells, as well as in HEK293 and ABCG2-transfected R482-HEK293 cells. S1 cells (open bars), S1-M1-80 (closed bars), HEK293 (open bars) and R482-HEK293 (closed bars) were treated with 10 µM of CUDC-907 in the presence or absence of 1 µM of Ko 143 as indicated for 1 h before the intracellular concentration of CUDC-907 was quantified as described in Materials and methods. Representative Western blots (B, inset) showing the relative level of ABCG2 protein in S1, S1-M1-80, HEK293 and R482-HEK293 cells. Values are presented as mean ± S.D. calculated from at least three independent experiments. ***

P < 0.001

Fig. 4. Effect of CUDC-907 on ABCG2-mediated transport of fluorescent substrates. The accumulation of fluorescent mitoxantrone in (A) drug sensitive S1 cells (left panel) and ABCG2-overexpressing drug resistant S1-M1-80 cells (right panel), and (B) parental pcDNA-HEK293 (left panel) and ABCG2-overexpressing R482-HEK293 cells (right panel); or fluorescent pheophorbide A in (C) S1 cells (left panel) and S1-M1-80 cells (right panel), and (D) pcDNA-HEK293 (left panel) and R482-HEK293 cells (right panel), was measured in the absence or presence of 10 µM of CUDC-907 or 5 µM of Ko 143, a reference inhibitor of ABCG2, and analyzed immediately by flow cytometry as described in Materials and methods. Representative histograms of three independent experiments are shown.

Fig. 5. Effect of short-term treatment with CUDC-907 on ABCG2 protein expression. Immunoblot detection and quantification of ABCG2 protein in total cell 28


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lysate (10 µg) in S1 and S1-M1-80 cells treated with increasing concentrations of CUDC-907 for 72 h as indicated. α-Tubulin was used as an internal control for equal loading. Values are presented as mean ± S.D. calculated from three independent experiments.

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Page 30 of 41

Table 1 Sensitivity of various parental and respective MDR cell lines to CUDC-907. IC50 (nM) †

R.F‡

-

5.58 ± 0.78

1.00

epidermal

ABCB1

4.82 ± 0.78

0.86

epidermal

ABCB1

8.56 ± 1.70

1.53

OVCAR-8

ovarian

-

34.98 ± 6.82

1.00

NCI-ADR-RES

ovarian

ABCB1

36.50 ± 13.02

1.04

S1

colon

-

5.35 ± 0.96

1.00

S1-M1-80

colon

ABCG2

180.59 ± 35.51**

33.76

MCF7

breast

-

32.74 ± 5.93

1.00

MCF7-FLV1000

breast

ABCG2

5702.22 ± 959.55***

174.17

MCF7-AdVp3000

breast

ABCG2

4273.40 ± 1165.41**

130.53

COR-L23/P

lung

ABCC1

7.08 ± 1.04

1.00

COR-L23/R

lung

ABCC1

9.87 ± 0.57

1.39

NIH3T3

-

-

21.58 ± 2.39

1.00

NIH3T3-G185

-

ABCB1

31.23 ± 4.93

1.45

pcDNA-HEK293

-

-

16.34 ± 1.35

1.00

MDR19-HEK293

-

ABCB1

24.54 ± 3.75

1.68

R482-HEK293

-

ABCG2

139.82 ± 18.98***

8.56

ABCC1

16.67 ± 2.62

1.02

Cell line

Cancer

Transporter

origin

expressed

KB-3-1

epidermal

KB-C-1 KB-V-1

MRP1-HEK293

Abbreviation: RF, resistance factor. † IC50 values are mean ± SD calculated from dose-response curves obtained from three independent experiments using cytotoxicity assay as described in Materials and methods. ‡ RF values were calculated by dividing 30


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IC50 values of ABC transporter overexpressing cells by IC50 values of respective parental cells. *P < 0.05; **P < 0.01 ; ***P < 0.001

31



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Page 32 of 41

Table 2 Reference inhibitor of ABCG2 restores drug sensitivity of CUDC-907 in ABCG2-overexpressing cell lines. IC50 (µM) †

Cell line CUDC-907

S1 S1-M1-80 pcDNA-HEK293 R482-HEK293

CUDC-907 +

CUDC-907 +

Ko 143 (1 µM)

lapatinib (0.3 µM)

5.35 ± 0.96

3.87 ± 0.88

4.07 ± 0.72

180.59 ± 35.51

4.53 ± 1.47**

14.41 ± 3.09**

16.34 ± 1.35

14.62 ± 1.89

13.70 ± 2.21

139.82 ± 18.98

14.26 ± 2.81***

60.63 ± 11.30**

Abbreviation: N.D, not determined. †IC50 values are mean ± SD in the presence and absence of CUDC-907 or other tested compounds. The IC50 values were calculated from dose-response curves obtained from three independent experiments. *P < 0.05; **P < 0.01 ; ***P < 0.001

32


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Table 3 Effect of CUDC-907 on ABCG2-mediated mitoxantrone resistance in ABCG2-overexpressing S1-M1-80 cells and ABCB1-transfected R482-HEK293 cells. Drug

IC50 †

Concentration

RR‡

(nM) S1

S1-M1-80

(nM)

(µM)

Mitoxantrone

-

4.36 ± 0.61

11.95 ± 2.14

2741

+ CUDC-907

1

2.93 ± 0.55*

12.25 ± 2.35

4181

+ CUDC-907

2

1.94 ± 0.50**

12.74 ± 2.45

6567

1000

3.37 ± 0.49

0.31 ± 0.03***

92

pcDNA-HEK293

R482-HEK293

(nM)

(nM)

+ Ko 143

Mitoxantrone

-

4.19 ± 1.13

63.79 ± 8.03

15

+ CUDC-907

1

5.39 ± 1.48

59.02 ± 8.37

11

+ CUDC-907

2

4.19 ± 1.00

68.89 ± 11.22

16

1000

3.72 ± 0.68

6.53 ± 1.12***

2

+ Ko 143

Abbreviation: RR, relative resistance. † IC50 values are mean ± SD calculated from dose-response curves obtained from three independent experiments using cytotoxicity assay as described in Materials and Methods. ‡ RR values were obtained by dividing IC50 values of ABCG2-overexpressing cells by IC50 values of respective sensitive cells. *P < 0.05; **P < 0.01 ; ***P < 0.001

33



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Page 34 of 41

For Table of Contents Only

34


Wu al.,41 Figure 1 Page et 35 of

Control

+ CUDC-907

Control

+ CUDC-907

S1

A

B

S1-M1-80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58




Page 36 of 41

Wu et al., Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A S1 CUDC-907 －＋ GDC-0980 －－－－ Ko143

＋－－－－＋＋－＋－＋＋

S1-M1-80 －＋＋－－－－－－＋＋－－－＋－＋＋

p-Akt (T308) p-Akt (S473) Total Akt ABCG2 Tubulin

B


Page 37 of 41


Wu et al., Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

C S1 CUDC-907 －－＋＋－－－－－－＋＋ SAHA －＋－＋－＋ Ko 143

S1-M1-80 －－＋＋－－－－－－＋＋－＋－＋－＋

Ac-H3 Ac-Tub Tubulin

D



Wu et al., Figure 3 A CUDC-907

ABCG2

B R482

HEK293

S1

Tubulin S1-M1-80

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58


Page 38 of 41

Wu al.,41 Figure 4 Page et 39 of A

S1 MX

Cell Number

MX

S1-M1-80

Control + CUDC-907 + Ko143

Fluorescent Intensity

B


pcDNA-HEK293 MX

Cell Number

MX

R482-HEK293




S1

S1-M1-80

C PhA

PhA

Cell Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58



ACS Paragon Plus EnvironmentIntensity Fluorescent Intensity Fluorescent

Wu et al., Figure 4 D

pcDNA-HEK293 PhA

Page 40 of 41

R482-HEK293 PhA PhA

Cell Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58






Page 41 of 41


Wu et al., Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

S1 0

1

S1-M1-80 2

5

0

1

2

5

CUDC-907 [nM] ABCG2 Tubulin


Human ATP-Binding Cassette Transporter ABCG2 Confers

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