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1.  Histone deacetylase inhibitors induce CXCR4 mRNA but antagonize CXCR4 migration 
Cancer Biology & Therapy  2013;14(2):175-183.
The stromal cell-derived factor-1α SDF-1α (CXCL12)/CXCR4 axis has been linked to poor prognosis in some cancers. As histone deacetylase inhibitors (HDIs) exert antitumor effects by targeting proteins affecting cell migration, we sought to evaluate the effects of the HDIs apicidin, vorinostat, entinostat (MS-275) and romidepsin on the expression and function of CXCR4 in human cancer cell lines. After treatment with romidepsin, CXCR4 mRNA expression increased 12-fold in UOK121 renal cancer cells, 16-fold in H460 non-small cell cancer cells and 4-fold in SF295 glioma cells; treatment with other HDIs yielded similar effects. CXCR4 induction was not observed in MCF7 breast cancer cells or SW620 colon cancer cells. To evaluate the corresponding functional increase, the effect of CXCR4 ligand, CXCL12, on ERK1/2, STAT3 and c-SRC activation and cell migration was examined in UOK121, SF295 and H460 cells. Alone, the HDIs increased pERK1/2, while reducing pSTAT-3 and pSRC. Following CXCL12 exposure, pERK1/2 induction was maintained, but STAT3 and SRC phosphorylation was impaired. These findings resulted in reduced basal and CXCL12-mediated cell migration. In conclusion, HDIs upregulated CXCR4 mRNA expression but impaired CXCL12-dependent signaling cascades through STAT3 and c-SRC, suggesting a potential role for HDIs in delaying or preventing metastatic processes in solid tumors.
doi:10.4161/cbt.22957
PMCID: PMC3571999  PMID: 23192271
CXCR4; CXCL12; histone deacetylase inhibitor; romidepsin; migration
2.  Flavonoids from Eight Tropical Plant Species That Inhibit the Multidrug Resistance Transporter ABCG2 
Journal of natural products  2011;74(2):262-266.
Overexpression of ABCG2, a membrane-bound multidrug transporter, can make tumor cells resistant to treatment with conventional chemotherapeutic agents. A high-throughput screening effort with the NCI repository of natural product extracts revealed that eight tropical plant extracts significantly inhibited the function of ABCG2. This activity was tracked throughout the extract fractionation process to a series of ABCG2 inhibitory flavonoids (1–13). Their structures were identified by a combination of NMR, mass spectrometry, and circular dichroism studies, and this resulted in the elucidation of (2S)-5,7,3′-trihydroxy-4′-methoxy-8-(3″-methylbut-2″-enyl)-flavonone (1), (2S)-5,7,3′,5′-tetrahydroxy-8-[3″,8″ -dimethylocta-2″(E),7″-dienyl]flavonone (3), and 5,7,3′-trihydroxy-3,5′-dimethoxy-2′-(3′-methylbut-2-enyl)flavone (12) as new compounds.
doi:10.1021/np100797y
PMCID: PMC3805045  PMID: 21275386
3.  The Challenge of Exploiting ABCG2 in the Clinic 
ABCG2, or breast cancer resistance protein (BCRP), is an ATP-binding cassette half transporter that has been shown to transport a wide range of substrates including chemotherapeutics, antivirals, antibiotics and flavonoids. Given its wide range of substrates, much work has been dedicated to developing ABCG2 as a clinical target. But where can we intervene clinically and how can we avoid the mistakes made in past clinical trials targeting P-glycoprotein? This review will summarize the normal tissue distribution, cancer tissue expression, substrates and inhibitors of ABCG2, and highlight the challenges presented in exploiting ABCG2 in the clinic. We discuss the possibility of inhibiting ABCG2, so as to increase oral bioavailability or increase drug penetration into sanctuary sites, especially the central nervous system; and at the other end of the spectrum, the possibility of improving ABCG2 function, in the case of gout caused by a single nucleotide polymphism. Together, these aspects of ABCG2/BCRP make the protein a target of continuing interest for oncologists, biologists, and pharmacologists.
PMCID: PMC3091815  PMID: 21118093
ABCG2/BCRP; Blood Brain Barrier; CNS Penetration; Drug Resistance; Gout; Oral Bioavailability; Q141K; Single nucleotide polymorphism
4.  ABC Transporters: Unvalidated Therapeutic Targets in Cancer and the CNS 
The discovery of the multidrug transporter P-glycoprotein (Pgp) over 35 years ago in drug resistant cells prompted several decades of work attempting to overcome drug resistance by inhibition of drug efflux. Despite convincing laboratory data showing that drug transport can be inhibited in vitro, efforts to translate this discovery to the clinic have not succeeded. Since overexpression of Pgp and related transporters including ABCG2 and members of the ABCC family have been linked with poor outcome, it remains a reasonable hypothesis that this poor outcome is linked to reduction of drug exposure by efflux, and thus to drug resistance. In this review, we will discuss the question of whether ABC transporters mediate drug resistance in cancer through a reduction in drug accumulation in tumors, and whether the “Pgp inhibition hypothesis” might be wrong. The hypothesis, which holds that increased chemotherapy effectiveness can be achieved by inhibiting Pgp-mediated drug efflux has only been validated in model systems. Possible explanations for the failure to validate this clinically include the existence of other modulators of drug accumulation and uptake in tumors. Despite these difficulties, a potential role has emerged for drug transporters as therapeutic targets in the central nervous system (CNS). Both lines of investigation point to the need for imaging agents to facilitate the study of drug accumulation in human cancer. This is a critical need for targeted therapies where an important dose-response relationship is likely to exist, and where drug resistance renders many of the novel targeted agents ineffective in a subset of patients.
PMCID: PMC3119022  PMID: 21189132
ABC transporters; drug resistance; P-glycoprotein
5.  Escape from hsa-miR-519c enables drug resistant cells to maintain high expression of ABCG2 
Molecular cancer therapeutics  2009;8(10):2959-2968.
Overexpression of ABCG2 has been reported in cell lines selected for drug resistance and it is widely believed to be important in the clinical pharmacology of anticancer drugs. We and others have previously identified and validated two microRNAs (hsa-miR-519c and hsa-miR-520h) targeting ABCG2. In this study, the shortening of the ABCG2 3′ untranslated region (3′UTR) was found to be a common phenomenon in a number of ABCG2-overexpressing resistant cell lines, which as a result removes the hsa-miR-519c binding site and its repressive effects on mRNA stability and translation blockade, thereby contributing to drug resistance. On the other hand, reduced expression of hsa-miR-520h, previously thought to have allowed ABCG2 overexpression, was found to be caused by the sequestering of the microRNA by the highly expressed ABCG2. In drug sensitive cells, inhibitors against hsa-miR-519c and hsa-miR-520h could augment the cytotoxic effect of mitoxantrone, suggesting a substantial role for both microRNAs in controlling ABCG2 level and thereby anticancer drug response. However, in drug resistant cells, altering the levels of the two microRNAs did not have any effect on sensitivity to mitoxantrone. Taken together, these studies suggest that in ABCG2 overexpressing drug resistant cells, hsa-miR-519c, is unable to impact ABCG2 expression because the mRNA lacks its binding site; whereas hsa-miR-520h is sequestered and unable to limit ABCG2 expression. Given the recent observation that a truncated 3′UTR is also observed in ABCG2-overexpressing human embryonic stem cells, our results in drug resistant cell lines suggest that 3′UTR truncation is a relatively common mechanism of ABCG2 regulation.
doi:10.1158/1535-7163.MCT-09-0292
PMCID: PMC3680354  PMID: 19825807
ABCG2; microRNA; multidrug resistance; gene translocation; mRNA stability
6.  Targeting MDR in Breast and Lung Cancer: Discriminating its Potential Importance from the Failure of Drug Resistance Reversal Studies 
This special issue of Drug Resistance Updates is dedicated to multidrug resistance protein 1 (MDR-1), 35 years after its discovery. While enormous progress has been made and our understanding of drug resistance has become more sophisticated and nuanced, after 35 years the role of MDR-1 in clinical oncology remains a work in progress. Despite clear in vitro evidence that P-glycoprotein (Pgp), encoded by MDR-1, is able to dramatically reduce drug concentrations in cultured cells, and that drug accumulation can be increased by small molecule inhibitors, clinical trials testing this paradigm have mostly failed. Some have argued that it is no longer worthy of study. However, repeated analyses have demonstrated MDR-1 expression in a tumor is a poor prognostic indicator leading some to conclude MDR-1 is a marker of a more aggressive phenotype, rather than a mechanism of drug resistance. In this review we will re-evaluate the MDR-1 story in light of our new understanding of molecular targeted therapy, using breast and lung cancer as examples. In the end we will reconcile the data available and the knowledge gained in support of a thesis that we understand far more than we realize, and that we can use this knowledge to improve future therapies.
doi:10.1016/j.drup.2012.02.002
PMCID: PMC3680361  PMID: 22464282
MDR-1/Pgp; 99mTc-sestamibi; 18F fluoropaclitaxel; breast cancer; lung cancer; drug penetration
7.  ABCG2: A Perspective 
ABCG2, or Breast Cancer Resistance Protein (BCRP), is an ABC transporter that has been the subject of intense study since its discovery a decade ago. With high normal tissue expression in the brain endothelium, gastrointestinal tract, and placenta, ABCG2 is believed to be important in protection from xenobiotics, regulating oral bioavailability, forming part of the blood-brain barrier, the blood-testis barrier, and the maternal-fetal barrier. Notably, ABCG2 is often expressed in stem cell populations, where it likely plays a role in xenobiotic protection. However, clues to its epigenetic regulation in various cell populations are only beginning to emerge. While ABCG2 overexpression has been demonstrated in cancer cells after in vitro drug treatment, endogenous ABCG2 expression in certain cancers is likely a reflection of the differentiated phenotype of the cell of origin and likely contributes to intrinsic drug resistance. Notably, research into the transporter’s role in cancer drug resistance and its development as a therapeutic target in cancer has lagged. Substrates and inhibitors of the transporter have been described, among them chemotherapy drugs, tyrosine kinase inhibitors, antivirals, HMG-CoA reductase inhibitors, carcinogens, and flavonoids. This broad range of substrates complements the efficiency of ABCG2 as a transporter in laboratory studies and suggests that, while there are redundant mechanisms of xenobiotic protection, the protein is important in normal physiology. Indeed, emerging studies in pharmacology and toxicology assessing polymorphic variants in man, in combination with murine knockout models have confirmed its dynamic role. Work in pharmacology may eventually lead us to a greater understanding of the physiologic role of ABCG2.
doi:10.1016/j.addr.2008.11.003
PMCID: PMC3105088  PMID: 19135109
ABCG2; BCRP; drug-resistance; ABC transporter; chemotherapy; pharmacology
8.  Other Paradigms: Growth Rate Constants and Tumor Burden Determined Using Computed Tomography Data Correlate Strongly With the Overall Survival of Patients With Renal Cell Carcinoma 
Cancer journal (Sudbury, Mass.)  2009;15(5):441-447.
In solid tumors, where curative therapies still elude oncologists, novel paradigms are needed to assess the efficacy of new therapies and those already approved. We used radiologic measurements obtained in patients with metastatic renal cell carcinoma enrolled in a phase II study of the epothilone B analog, ixabepilone (Ixempra), to address this issue. Using a novel 2-phase mathematical equation, we used the radiologic measurements to estimate the concomitant rates of tumor regression and growth (regression and growth rate constants). Eighty-one patients were enrolled on the ixabepilone trial at the time of this analysis. Growth rate constants were determined using computed tomography measurements obtained exclusively while a patient was enrolled on study. The growth rate constants of renal cell carcinomas treated with ixabepilone were significantly reduced compared with those of tumors in patients who received placebo in a previous trial. Furthermore, a correlation with overall survival was found for both the growth rate constant and the initial tumor burden; and this correlation was even stronger when both the growth rate constant and the initial tumor burden were combined. The readily amenable mathematical model described herein has potential applications to many tumor types that can be assessed with imaging modalities. Because the growth rate constant seems to be a surrogate for survival, assessment could aid in the evaluation of relative efficacies of different therapies and perhaps in assessing the potential individual benefit of an experimental therapy.
doi:10.1097/PPO.0b013e3181be1b90
PMCID: PMC3667552  PMID: 19826366
RECIST; chemotherapy efficacy; cancer clinical trials; phase II studies; chemotherapy assessment; chemotherapy evaluation
9.  Analyzing the pivotal trial that compared sunitinib and interferon alfa in renal cell carcinoma, using a method that assesses tumor regression and growth 
Clinical Cancer Research  2012;18(8):2374-2381.
Purpose
We applied a method that analyzes tumor response, quantifying the rates of tumor growth (g) and regression (d), using tumor measurements obtained while patients receive therapy. We used data from the phase III trial comparing sunitinib and interferon-alfa (IFN-α) in metastatic renal cell carcinoma (mRCC) patients.
Methods
The analysis used an equation that extracts d and g.
Results
For sunitinib, overall survival (OS) was strongly correlated with log g (Rsq=0.44, p<0.0001); much less with log d (Rsq=0.04; p=0.0002). The median g of tumors in these patients (0.00082 per days; log g=−3.09) was about half that (p<0.001) of tumors in patients receiving IFN-α (0.0015 per day; log g=−2.81). With IFN-α, the OS/log g correlation (Rsq=0.14) was weaker. Values of g from measurements obtained by study investigators or central review were highly correlated (Rsq=0.80). No advantage resulted in including data from central review in regressions. Further, g can be estimated accurately four months before treatment discontinuation. Extrapolating g in a model that incorporates survival generates the hypothesis that g increased after discontinuation of sunitinib but did not accelerate.
Conclusions
In patients with mRCC, sunitinib reduced tumor growth rate, g, more than did IFN-α. Correlating g with OS confirms earlier analyses suggesting g may be an important clinical trial endpoint, to be explored prospectively and in individual patients.
doi:10.1158/1078-0432.CCR-11-2275
PMCID: PMC3328595  PMID: 22344231
Renal cell carcinoma; Tumor growth rates; Tumor regression rates; Sunitinib; Interferon
10.  A Pharmacodynamic Study of the P-glycoprotein Antagonist CBT-1® in Combination With Paclitaxel in Solid Tumors 
The Oncologist  2012;17(4):512.
Background:
This pharmacodynamic trial evaluated the effect of CBT-1® on efflux by the ATP binding cassette (ABC) multidrug transporter P-glycoprotein (Pgp/MDR1/ABCB1) in normal human cells and tissues. CBT-1® is an orally administered bisbenzylisoquinoline Pgp inhibitor being evaluated clinically. Laboratory studies showed potent and durable inhibition of Pgp, and in phase I studies CBT-1® did not alter the pharmacokinetics of paclitaxel or doxorubicin.
Methods:
CBT-1® was dosed at 500 mg/m2 for 7 days; a 3-hour infusion of paclitaxel at 135 mg/m2 was administered on day 6. Peripheral blood mononuclear cells (PBMCs) were obtained prior to CBT-1® administration and on day 6 prior to the paclitaxel infusion. 99mTc-sestamibi imaging was performed on the same schedule. The area under the concentration–time curve from 0–3 hours (AUC0–3) was determined for 99mTc-sestamibi.
Results:
Twelve patients were planned and enrolled. Toxicities were minimal and related to paclitaxel (grade 3 or 4 neutropenia in 18% of cycles). Rhodamine efflux from CD56+ PBMCs was a statistically significant 51%–100% lower (p < .0001) with CBT-1®. Among 10 patients who completed imaging, the 99mTc-sestamibi AUC0–3 for liver (normalized to the AUC0–3 of the heart) increased from 34.7% to 100.8% (median, 71.9%; p < .0001) after CBT-1® administration. Lung uptake was not changed.
Conclusion:
CBT-1® is able to inhibit Pgp-mediated efflux from PBMCs and normal liver to a degree observed with Pgp inhibitors studied in earlier clinical trials. Combined with its ease of administration and lack of toxicity, the data showing inhibition of normal tissue Pgp support further studies with CBT-1® to evaluate its ability to modulate drug uptake in tumor tissue.
Discussion:
Although overexpression of ABCB1 and other ABC transporters has been linked with poor outcome following chemotherapy efforts to negate that through pharmacologic inhibition have generally failed. This is thought to be a result of several factors, including (a) failure to select patients with tumors in which ABCB1 is a dominant resistance mechanism; (b) inhibitors that were not potent, or that impaired drug clearance; and (c) the existence of other mechanisms of drug resistance, including other ABC transporters. Although an animal model for Pgp has been lacking, recent studies have exploited a Brca1−/−; p53−/− mouse model of hereditary breast cancer that develops sporadic tumors similar to cancers in women harboring BRCA1 mutations. Treatment with doxorubicin, docetaxel, or the poly(ADP-ribose) polymerase inhibitor olaparib brings about shrinkage, but resistance eventually emerges. Overexpression of the Abcb1a gene, the mouse ortholog of human ABCB1, has been shown to be a mechanism of resistance in a subset of these tumors. Treating mice with resistant tumors with olaparib plus the Pgp inhibitor tariquidar resensitized the tumors to olaparib. Although results in this animal model support a new look at Pgp as a target, in this era of “targeted therapies,” trial designs that directly assess modulation of drug uptake, including quantitative nuclear imaging, should be pursued before clinical efficacy assessments are undertaken. Such assessment should be performed with compounds that inhibit tissue Pgp without altering the pharmacokinetics of chemotherapeutic agents. This pharmacodynamic study demonstrated that CBT-1®, inhibits Pgp-mediated efflux from PBMCs and normal liver.
doi:10.1634/theoncologist.2012-0080
PMCID: PMC3336838  PMID: 22416063
11.  Drug resistance: Still a daunting challenge to the successful treatment of AML 
Drug Resistance Updates  2012;15(1-2):62-69.
Resistance to chemotherapy remains a challenging issue for patients and their physicians. P-glycoprotein (Pgp, MDR1, ABCB1), as well as a family of structurally and functionally related proteins, are plasma membrane transporters able to efflux a variety of substrates from the cell cytoplasm, including chemotherapeutic agents. The discovery of ABCB1 made available a potential target for pharmacologic down-regulation of efflux-mediated chemotherapy resistance. In patients with acute myeloid leukemia (AML), a neoplasm characterized by proliferation of poorly differentiated myeloid progenitor cells, leukemic cells often express ABCB1 at high levels, which may lead to the development of resistance to chemotherapy. Thus, AML seemed to be a likely cancer for which the addition of drug efflux inhibitors to the chemotherapeutic regimen would improve outcomes in patients. Despite this rational hypothesis, the majority of clinical trials evaluating this strategy have failed to reach a positive endpoint, most recently the Eastern Cooperative Oncology Group E3999 trial. Here we review data suggesting the importance of ABCB1 in AML, address the failure of clinical trials to support a therapeutic strategy aimed at modulating ABCB1-mediated resistance, and consider the type of research that should be conducted in this field going forward.
doi:10.1016/j.drup.2012.02.001
PMCID: PMC3348380  PMID: 22409994
acute myeloid leukemia; multidrug resistance; ABC transporters; cancer stem cells; gene signatures
12.  EZH2 mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3 and NGFR 
Cancer research  2011;72(1):315-324.
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb complex histone methytransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNAi-mediated knockdown or pharmacological inhibiton with 3-deazaneplanocin A (DZNep) increased CASZ1 expression, inhibited NB cell growth and induced neurite extension. Similarly, EZH2−/− mouse embryonic fibroblasts (MEFs) displayed 3-fold higher levels of CASZ1 mRNA compared to EZH2+/+ MEFs. In cells with increased expression of CASZ1, treatment with HDAC inhibitors decreased expression of EZH2 and the Polycomb complex component SUZ12. Under steady-state conditions H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors.
doi:10.1158/0008-5472.CAN-11-0961
PMCID: PMC3487161  PMID: 22068036
CASZ1; neuroblastoma; EZH2; NGFR; CLU; RUNX3
13.  Targeting the Epigenome in Lung Cancer: Expanding Approaches to Epigenetic Therapy 
Frontiers in Oncology  2013;3:261.
Epigenetic aberrations offer dynamic and reversible targets for cancer therapy; increasingly, alteration via overexpression, mutation, or rearrangement is found in genes that control the epigenome. Such alterations suggest a fundamental role in carcinogenesis. Here, we consider three epigenetic mechanisms: DNA methylation, histone tail modification and non-coding, microRNA regulation. Evidence for each of these in lung cancer origin or progression has been gathered, along with evidence that epigenetic alterations might be useful in early detection. DNA hypermethylation of tumor suppressor promoters has been observed, along with global hypomethylation and hypoacetylation, suggesting an important role for tumor suppressor gene silencing. These features have been linked as prognostic markers with poor outcome in lung cancer. Several lines of evidence have also suggested a role for miRNA in carcinogenesis and in outcome. Cigarette smoke downregulates miR-487b, which targets both RAS and MYC; RAS is also a target of miR-let-7, again downregulated in lung cancer. Together the evidence implicates epigenetic aberration in lung cancer and suggests that targeting these aberrations should be carefully explored. To date, DNA methyltransferase and histone deacetylase inhibitors have had minimal clinical activity. Explanations include the possibility that the agents are not sufficiently potent to invoke epigenetic reversion to a more normal state; that insufficient time elapses in most clinical trials to observe true epigenetic reversion; and that doses often used may provoke off-target effects such as DNA damage that prevent epigenetic reversion. Combinations of epigenetic therapies may address those problems. When epigenetic agents are used in combination with chemotherapy or targeted therapy it is hoped that downstream biological effects will provoke synergistic cytotoxicity. This review evaluates the challenges of exploiting the epigenome in the treatment of lung cancer.
doi:10.3389/fonc.2013.00261
PMCID: PMC3793201  PMID: 24130964
epigenetics; non-small cell lung cancer; small-cell lung cancer; DNA methylation; histone modification; microRNA
14.  Potential Pitfalls of Crossover and Thoughts on Iniparib in Triple-Negative Breast Cancer 
As recruitment for oncology clinical trials has become more difficult, there appears to have been an increase in the number of studies that allow patients in the control arm to “crossover” and receive the experimental therapy after disease progression. Although some researchers worry that allowing such crossover may abolish gains in progression-free survival in the experimental arm, the possibility that crossover might inadvertently benefit the experimental arm has not been addressed. In clinical trials in which the experimental agent has little or no intrinsic activity and is used to modulate an active combination, such crossover might negatively affect the overall survival of the control arm. Because resistance to the active combination—manifested as disease progression—has occurred, the likelihood of benefit from adding the experimental drug is reduced. Consequently, patients who were randomly assigned to the control arm continue to receive the now inactive combination after crossover, whereas patients in the experimental arm who discontinue study participation may seek out potentially effective salvage regimens. This difference in subsequent therapies may confer an advantage to the experimental arm that is manifested as gains beyond those achieved in progression-free survival, gains that occur not because the experimental therapy induced a change in tumor biology that persists beyond treatment discontinuation but because the control arm suffers by continuing to receive a therapy on which their tumor is progressing. Such an outcome may explain the recently reported trial results for iniparib in triple-negative breast cancer. Given that allowing patients in the control arm to receive the experimental agent may confound interpretation of overall survival, such crossover should not be used indiscriminately, especially if the experimental agent has little or no intrinsic activity.
doi:10.1093/jnci/djr386
PMCID: PMC3247787  PMID: 22045362
15.  Histone deacetylase inhibitors: emerging mechanisms of resistance 
Molecular pharmaceutics  2011;8(6):2021-2031.
The histone deacetylase inhibitors (HDIs) have shown promise in the treatment of a number of hematologic malignancies, leading to the approval of vorinostat and romidepsin for the treatment of cutaneous T-cell lymphoma and romidepsin for the treatment of peripheral T-cell lymphoma by the U. S. Food and Drug Administration. Despite these promising results, clinical trials with the HDIs in solid tumors have not met with success. Examining mechanisms of resistance to HDIs may lead to strategies that increase their therapeutic potential in solid tumors. However, relatively few examples of drug-selected cell lines exist, and mechanisms of resistance have not been studied in depth. Very few clinical translational studies have evaluated resistance mechanisms. In the current review, we summarize many of the purported mechanisms of action of the HDIs in clinical trials and examine some of the emerging resistance mechanisms.
doi:10.1021/mp200329f
PMCID: PMC3230675  PMID: 21899343
histone deacetylase inhibitor; resistance; romidepsin; vorinostat; panobinostat
16.  Schedule-dependent synergy of histone deacetylase inhibitors with DNA damaging agents in small cell lung cancer 
Cell Cycle  2011;10(18):3119-3128.
Small cell lung cancer (SCLC) is an aggressive lung cancer subtype in need of better therapies. Histone deacetylase inhibitors (HDIs) promote increased lysine acetylation in nucleosomal histones and are thought to relax chromatin, thereby allowing increased access of transcription factors and DNA damaging agents alike to DNA. We studied whether two HDIs, belinostat and romidepsin, could be effectively combined with cisplatin or etoposide (VP-16) for SCLC cells. Analysis of cell survival and synergy was performed using CalcuSyn mathematical modeling to calculate a combination index. Immunostaining of γH2AX was performed to evaluate persistence of DNA damage following simultaneous or sequential exposure. Based on CalcuSyn modeling, HDIs synergized with DNA damaging agents only when added simultaneously. An additive-to-antagonistic effect was seen with HDI pretreatment for 24 h or with addition after cisplatin or etoposide. Furthermore, pretreatment with HDIs resulted in normalization of cell cycle and reduced PARP degradation as compared with simultaneous treatment. The increase in γH2AX phosphorylation confirmed that simultaneous but not sequential treatment enhanced double-stranded DNA breaks. These results suggest that DNA relaxation is not required for synergy of HDIs with DNA damaging agents, and that scheduling of drug administration will be critical for rational development of clinical protocols.
doi:10.4161/cc.10.18.17190
PMCID: PMC3218622  PMID: 21900747
combination index; drug interaction; γH2AX phosphorylation; dsDNA break; synergy; PARP degradation
17.  Rapid detection of ABC transporter interaction: potential utility in pharmacology 
Introduction
The ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are known to transport a wide range of structurally diverse compounds. Their high level of expression at the blood-brain, maternal-fetal, and blood-testis barriers as well as their purported roles in oral absorption suggests that ABC transporters play important pharmacologic roles.
Methods
We have developed a method to characterize the function and inhibition of ABC transporters using an automated cell counter with fluorescence detection capability. The assay was performed using stably-transfected HEK293 cells expressing P-gp, MRP1, or ABCG2 and examining transport of fluorescent substrates in the presence or absence of known inhibitors and compared to results obtained with a flow cytometer. Fold-increase in intracellular fluorescence was then calculated for cells incubated with fluorescent substrate in the absence of inhibitor versus in the presence of inhibitor.
Results
Fold-increase values obtained either with the cell counter or flow cytometer were comparable for cells expressing either MRP1 or ABCG2; slightly higher fold-increase values were observed when cells expressing P-gp were read on a flow cytometer compared to the cell counter.
Discussion
The assay described provides an inexpensive detection method to aid in the development of novel ABC transporter inhibitors or to characterize potential drug-drug interactions.
doi:10.1016/j.vascn.2010.11.003
PMCID: PMC3086650  PMID: 21112407
ABCG2; methods; MRP1; fluorescence; P-glycoprotein; ABC transporter
18.  Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance 
Cancer research  2011;71(8):3029-3041.
Sildenafil is a potent and selective inhibitor of the type 5 cGMP-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1 and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, non-toxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs, such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E217βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [125I]-IAAP, whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [125I]-IAAP. In contrast, Sildenafil did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anti-cancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions.
doi:10.1158/0008-5472.CAN-10-3820
PMCID: PMC3078184  PMID: 21402712
Sildenafil; multidrug resistance; ABCB1; ABCG2; chemosensitivity
19.  Tumor Growth Rates Derived from Data for Patients in a Clinical Trial Correlate Strongly with Patient Survival: A Novel Strategy for Evaluation of Clinical Trial Data 
The Oncologist  2008;13(10):1046-1054.
Purpose
The slow progress in developing new cancer therapies can be attributed in part to the long time spent in clinical development. To hasten development, new paradigms especially applicable to patients with metastatic disease are needed.
Patients and Methods
We present a new method to predict survival using tumor measurement data gathered while a patient with cancer is receiving therapy in a clinical trial. We developed a two-phase equation to estimate the concomitant rates of tumor regression (regression rate constant d) and tumor growth (growth rate constant g).
Results
We evaluated the model against serial levels of prostate-specific antigen (PSA) in 112 patients undergoing treatment for prostate cancer. Survival was strongly correlated with the log of the growth rate constant, log(g) (Pearson r=−0.72) but not with the log of the regression rate constants, log(d)(r=−0.218). Values of log(g) exhibited a bimodal distribution. Patients with log(g) values above the median had a mortality hazard of 5.14 (95% confidence interval, 3.10–8.52) when compared with those with log(g) values below the median. Mathematically, the minimum PSA value (nadir) and the time to this minimum are determined by the kinetic parameters d and g, and can be viewed as surrogates.
Conclusions
This mathematical model has applications to many tumor types and may aid in evaluating patient outcomes. Modeling tumor progression using data gathered while patients are on study, may help evaluate the ability of therapies to prolong survival and assist in drug development.
doi:10.1634/theoncologist.2008-0075
PMCID: PMC3313464  PMID: 18838440
20.  Bevacizumab Reduces the Growth Rate Constants of Renal Carcinomas: A Novel Algorithm Suggests Early Discontinuation of Bevacizumab Resulted in a Lack of Survival Advantage 
The Oncologist  2008;13(10):1055-1062.
Background
To hasten cancer drug development, new paradigms are needed to assess therapeutic efficacy. In a randomized phase II study in patients with renal cell carcinoma, 10 μg/kg bevacizumab (Avastin®; Genentech, Inc., South San Francisco, CA) administered every 2 weeks resulted in a longer time to progression but a statistically significant difference in overall survival could not be demonstrated.
Methods
We developed a novel two-phase equation to estimate concomitant rates of tumor regression (regression rate constant) and tumor growth (growth rate constant). This method allows us to assess therapeutic efficacy using tumor measurements gathered while a patient receives therapy in a clinical trial.
Results
The growth rate constants of renal cell carcinomas were significantly lower during therapy with 10 μg/kg bevacizumab than those of tumors in patients receiving placebo. In all cohorts the tumor growth rate constants were correlated with survival. That a survival advantage was not demonstrated with bevacizumab appears to have been a result of early discontinuation of bevacizumab.
Conclusions
Single-agent bevacizumab significantly affects the growth rate constants of renal cell carcinoma. Extrapolating from the growth rate constants, we conclude that the failure to demonstrate a survival advantage in the original study was a result of premature discontinuation of bevacizumab. The mathematical model described herein has applications to many tumor types and should aid in evaluating the relative efficacies of different therapies. Quantitating tumor growth rate constants using data gathered while patients are enrolled in a clinical trial, as in the present study, may streamline and assist in drug development.
doi:10.1634/theoncologist.2008-0016
PMCID: PMC3306833  PMID: 18827177
Bevacizumab; Chemotherapy efficacy; Clear-cell carcinoma; Drug efficacy; Growth rate constant; Premature discontinuation; RECIST; Renal cell carcinoma; Tumor assessment; Tumor measurements
21.  Histone Modifications at the ABCG2 Promoter following Treatment with Histone Deacetylase Inhibitor Mirror Those in Multidrug-Resistant Cells 
Molecular Cancer Research  2008;6(1):151-164.
ABCG2 is a ubiquitous ATP-binding cassette transmembrane protein that is important in pharmacology and may play a role in stem cell biology and clinical drug resistance. To study the mechanism(s) regulating ABCG2 expression, we used ChIP to investigate the levels of acetylated histone H3, histone deacetylases (HDAC), histone acetyltransferases, and other transcription regulatory proteins associated with the ABCG2 promoter. Following selection for drug resistance and the subsequent overexpression of ABCG2, an increase in acetylated histone H3 but a decrease in class I HDACs associated with the ABCG2 promoter was observed. Permissive histone modifications, including an increase in histone H3 lysine 4 trimethylation (Me3-K4 H3) and histone H3 serine 10 phosphorylation (P-S10 H3), were observed accompanying development of the resistance phenotype. These changes mirrored those in some cell lines treated with a HDAC inhibitor, romidepsin. A repressive histone mark, trimethylated histone H3 lysine 9 (Me3-K9 H3), was found in untreated parental cells and cells that did not respond to HDAC inhibition with ABCG2 up-regulation. Interestingly, although all five studied cell lines showed global histone acetylation and MDR1 up-regulation upon HDAC inhibition, only those cells with removal of the repressive mark, and recruitment of RNA polymerase II and a chromatin remodeling factor Brg-1 from the ABCG2 promoter, showed increased ABCG2 expression. In the remaining cell lines, HDAC1 binding in association with the repressive Me3-K9 H3 mark apparently constrains the effect of HDAC inhibition on ABCG2 expression. These studies begin to address the differential effect of HDAC inhibitors widely observed in gene expression studies.
doi:10.1158/1541-7786.MCR-07-0175
PMCID: PMC3306834  PMID: 18234970
22.  Impact of ABCB1 allelic variants on QTc interval prolongation 
Purpose
While the ABCB1 (P-glycoprotein) drug transporter is a constituent of several blood-tissue barriers (i.e. blood-brain and blood-nerve), its participation in a putative blood-heart barrier has been poorly explored. ABCB1 could decrease the intracardiac concentrations of drugs that cause QT-prolongation and cardiotoxicity.
Experimental design
ABCB1-related romidepsin transport kinetics were explored in LLC-PK1 cells transfected with different ABCB1 genetic variants. ABCB1 plasma and intracardiac concentrations were determined in Abcb1a/1b (−/−) mice and wild-type FVB controls. These same mice were used to evaluate romidepsin-induced QTc prolongation over time. Finally, a cohort of 83 individuals with available QTcB and ABCB1 genotyping data were used to compare allelic variation in ABCB1 versus QTc-prolongation phenotype.
Results
Here, we demonstrate that mice lacking the ABCB1-type P-glycoprotein have higher intracardiac concentrations of a model ABCB1 substrate, romidepsin, that correspond to changes in QT-prolongation from baseline (ΔQTc) over time. Consistent with this observation, we also demonstrate that patients carrying genetic variants that could raise ABCB1 expression in the cardiac endothelium have lower ΔQTc following a single dose of romidepsin.
Conclusions
To our knowledge, this is the first evidence that Abcb1-type P-glycoprotein can limit intracardiac exposure to a drug that mediates QT-prolongation and suggests that certain commonly inherited polymorphisms in ABCB1 may serve as markers for QT-prolongation following the administration of ABCB1-substrate drugs.
doi:10.1158/1078-0432.CCR-10-0925
PMCID: PMC3074531  PMID: 21106724
Depsipeptide; FK228; polymorphisms; ABCB1; QTc
23.  A Pharmacodynamic Study of Docetaxel in Combination with the P-glycoprotein Antagonist, Tariquidar (XR9576) in Patients with Lung, Ovarian, and Cervical Cancer 
Purpose
P-glycoprotein (Pgp) antagonists have been difficult to develop because of complex pharmacokinetic interactions and a failure to demonstrate meaningful results. Here we report the results of a pharmacokinetic and pharmacodynamic trial using a third generation, potent, non-competitive inhibitor of Pgp, tariquidar (XR9576), in combination with docetaxel.
Experimental Design
In the first treatment cycle, the pharmacokinetics of docetaxel (40 mg/m2) were evaluated after day 1 and day 8 doses, which were administered with or without tariquidar (150 mg). 99mTc-sestamibi scanning and CD56+ mononuclear cell rhodamine efflux assays were performed to assess Pgp inhibition. In subsequent cycles, 75 mg/m2 docetaxel was administered with 150 mg tariquidar every three weeks.
Results
Forty-eight patients were enrolled onto the trial. Non-hematologic grade 3/4 toxicities in 235 cycles were minimal. Tariquidar inhibited Pgp-mediated rhodamine efflux from CD56+ cells and reduced 99mTc-sestamibi clearance from the liver. A 12 to 24% increase in sestamibi uptake in visible lesions was noted in 8 of 10 patients with lung cancer. No significant difference in docetaxel disposition was observed in pairwise comparison with and without tariquidar. Four PRs were seen (4/48); three in the non-small cell lung cancer (NSCLC) cohort, measuring 40%, 57% and 67% by RECIST and one PR in a patient with ovarian cancer.
Conclusions
Tariquidar is well-tolerated with less observed systemic pharmacokinetic interaction than previous Pgp antagonists. Variable effects of tariquidar on retention of sestamibi in imageable lung cancers suggest that follow-up studies assessing tumor drug uptake in this patient population would be worthwhile.
doi:10.1158/1078-0432.CCR-10-1725
PMCID: PMC3071989  PMID: 21081657
P-glycoprotein; ABC transporter; drug resistance; sestamibi imaging; lung cancer
24.  New Strategies in Peripheral T-cell lymphoma: Understanding Tumor Biology and Developing Novel Therapies 
Peripheral T-cell lymphomas (PTCLs) constitute a group of heterogeneous diseases that are uncommon, representing, in Western countries, only approximately 10% of all non-Hodgkin lymphomas. They are typically associated with a poor prognosis compared to their B-cell counterparts and are much less well understood with respect to tumor biology, due to their rarity and biologic heterogeneity, and to the fact that characteristic cytogenetic abnormalities are few compared to B-cell lymphomas. While the outcome for patients with anaplastic large cell lymphoma (ALCL), particularly ALK-positive ALCL, is good, other types of peripheral T-cell lymphomas are associated with a poor prognosis even with aggressive anthracycline-based chemotherapy. In this respect, there is a need for new approaches in these diseases and this review focuses on and explores recent experience with novel therapies in PTCL.
doi:10.1158/1078-0432.CCR-09-1995
PMCID: PMC3058794  PMID: 21138864
25.  Gout, genetics and ABC transporters 
Gout is a chronic arthritic disease associated with high levels of urate in blood. Recent advances in research have permitted the identification of several new and common genetic factors underlying the disease. Among them, a polymorphism in the ABC transporter gene ATP-binding cassette transporter isoform G2 has been highlighted. ATP-binding cassette transporter isoform G2 was found to be involved in renal urate elimination, and the presence of the Q141K polymorphism to induce a 2-fold decrease in urate efflux. The Q141K variant has been shown to have impaired trafficking, leading to its intracellular retention, whereas the wild type protein is expressed on the cell surface. Several agents are being studied for the purpose of improving folding, trafficking and function of various ABC transporters, including ATP-binding cassette transporter isoform G2. If successful, this strategy opens doors to potential new therapies for gout.
doi:10.3410/B3-23
PMCID: PMC3206739  PMID: 22065982

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