Guoqing Du

Mrp4 Confers Resistance to Topotecan and Protects the Brain from Chemotherapy

ABSTRACT The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries. Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF. The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors.

Identification of a mammalian mitochondrial porphyrin transporter

The movement of anionic porphyrins (for example, haem) across intracellular membranes is crucial to many biological processes, but their mitochondrial translocation and coordination with haem biosynthesis is not understood. Transport of porphyrins into isolated mitochondria is energy-dependent, as expected for the movement of anions into a negatively charged environment. ATP-binding cassette transporters actively facilitate the transmembrane movement of substances. We found that the mitochondrial ATP-binding cassette transporter ABCB6 is upregulated (messenger RNA and protein in human and mouse cells) by elevation of cellular porphyrins and postulated that ABCB6 has a function in porphyrin transport. We also predicted that ABCB6 is functionally linked to haem biosynthesis, because its mRNA is found in both human bone marrow and CD71+ early erythroid cells (by database searching), and because our results show that ABCB6 is highly expressed in human fetal liver, and Abcb6 in mouse embryonic liver. Here we demonstrate that ABCB6 is uniquely located in the outer mitochondrial membrane and is required for mitochondrial porphyrin uptake. After ABCB6 is upregulated in response to increased intracellular porphyrin, mitochondrial porphyrin uptake activates de novo porphyrin biosynthesis. This process is blocked when the Abcb6 gene is silenced. Our results challenge previous assumptions about the intracellular movement of porphyrins and the factors controlling haem biosynthesis.

Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2

Oxaliplatin is an integral component of colorectal cancer therapy, but its clinical use is associated with a dose-limiting peripheral neurotoxicity. We found that the organic cation transporter 2 (OCT2) is expressed on dorsal root ganglia cells within the nervous system where oxaliplatin is known to accumulate. Cellular uptake of oxaliplatin was increased by 16- to 35-fold in cells overexpressing mouse Oct2 or human OCT2, and this process was associated with increased DNA platination and oxaliplatin-induced cytotoxicity. Furthermore, genetic or pharmacologic knockout of Oct2 protected mice from hypersensitivity to cold or mechanical-induced allodynia, which are established tests to assess acute oxaliplatin-induced neurotoxicity. These findings provide a rationale for the development of targeted approaches to mitigate this debilitating toxicity.

Influence of Polymorphic OATP1B-Type Carriers on the Disposition of Docetaxel

Purpose: Docetaxel is extensively metabolized by CYP3A4 in the liver but mechanisms by which the drug is taken up into hepatocytes remain poorly understood. We hypothesized that (i) liver uptake of docetaxel is mediated by the polymorphic solute carriers OATP1B1 and OATP1B3 and (ii) inherited genetic defects in this process may impair systemic drug elimination. Experimental Design: Transport of docetaxel was studied in vitro using various cell lines stably transfected with OATP1B1*1A (wild-type), OATP1B1*5 [c.521T>C (V174A); rs4149056], OATP1B3, or the mouse transporter Oatp1b2. Docetaxel clearance was evaluated in wild-type and Oatp1b2-knockout mice as well as in two cohorts of patients with multiple variant transporter genotypes (n = 213). Results: Docetaxel was found to be a substrate for OATP1B1, OATP1B3, and Oatp1b2 but was not transported by OATP1B1*5. Deficiency of Oatp1b2 in mice was associated with an 18-fold decrease in docetaxel clearance (P = 0.0099), which was unrelated to changes in intrinsic metabolic capacity in mouse liver microsomes. In patients, however, none of the studied common reduced function variants in OATP1B1 or OATP1B3 were associated with docetaxel clearance (P > 0.05). Conclusions: The existence of at least two potentially redundant uptake transporters in the human liver with similar affinity for docetaxel supports the possibility that functional defects in both of these proteins may be required to confer substantially altered disposition phenotypes. In view of the established exposure–toxicity relationships for docetaxel, we suggest that caution is warranted if docetaxel has to be administered together with agents that potently inhibit both OATP1B1 and OATP1B3. Clin Cancer Res; 18(16); 4433–40. ©2012 AACR.

ATP-dependent Mitochondrial Porphyrin Importer ABCB6 Protects against Phenylhydrazine Toxicity

Background: The role of the mitochondrial ABC transporter, Abcb6, in vivo is unknown. Results: Abcb6-null mice are incapable of ATP-dependent import of mitochondrial porphyrins. Despite compensatory changes in the porphyrin pathway, Abcb6-null mice are less viable after a porphyrin-inducing stress. Conclusion: Abcb6 absence abolished ATP-dependent mitochondrial porphyrin uptake and deregulated porphyrin pathway genes. Significance: Disrupted Abcb6 function may produce porphyria after certain stresses. Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6+/−) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6−/− mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6−/− mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6−/− mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival.

Cell survival under stress is enhanced by a mitochondrial ATP-binding cassette transporter that regulates hemoproteins

The ATP-binding cassette (ABC) transporter ABCB6 localizes to the mitochondria, where it imports porphyrins and up-regulates de novo porphyrin synthesis. If ABCB6 also increases the intracellular heme concentration, it may broadly affect the regulation and physiology of cellular hemoproteins. We tested whether the ability of ABCB6 to accelerate de novo porphyrin biosynthesis alters mitochondrial and extramitochondrial heme levels. ABCB6 overexpression increased the quantity of cytosolic heme but did not affect mitochondrial heme levels. We then tested whether the increased extramitochondrial heme would increase the concentration and/or activity of cellular hemoproteins (hemoglobin, catalase, and cytochrome c oxidase). ABCB6 overexpression increased the activity and quantity of hemoproteins found in several subcellular compartments, and reduction of ABCB6 function (by small interfering RNA or knockout) reversed these findings. In complementary studies, suppression of ABCB6 expression sensitized cells to stress induced by peroxide and cyanide, whereas overexpression of ABCB6 protected against both stressors. Our findings show that the ability of ABCB6 to increase cytosolic heme levels produces phenotypic changes in hemoproteins that protect cells from certain stresses. Collectively, these findings have implications for the health and survival of both normal and abnormal cells, which rely on heme for multiple cellular processes.

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Significance Acute kidney injury (AKI) is a common clinical condition caused by loss of kidney function. Lack of therapeutic options has contributed to high mortality rates in AKI patients. Drug-induced AKI, as observed during cisplatin-based anticancer therapy, is responsible for about 20% of renal failure cases. The initial injury triggers a proliferative response in renal tubular cells, which in the presence of cellular damage can further accelerate renal injury. Our study provides evidence that the small-molecule cell-cycle inhibitors palbociclib and LEE011 can prevent cisplatin-induced AKI by inhibiting two relevant targets: renal cell-cycle progression and organic cation transporter 2, a renal uptake transporter of cisplatin. The future development of cyclin-dependent kinase 4/6 inhibitors as renal protective agents could have significant clinical benefits. Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.

Multikinase Inhibitors Induce Cutaneous Toxicity through OAT6-Mediated Uptake and MAP3K7-Driven Cell Death.

The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand-foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo and that this effect could be reversed by cotreatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR.

Influence of OATP1B1 Function on the Disposition of Sorafenib‐β‐D‐Glucuronide

The oral multikinase inhibitor sorafenib undergoes extensive UGT1A9‐mediated formation of sorafenib‐β‐D‐glucuronide (SG). Using transporter‐deficient mouse models, it was previously established that SG can be extruded into bile by ABCC2 or follow a liver‐to‐blood shuttling loop via ABCC3‐mediated efflux into the systemic circulation, and subsequent uptake in neighboring hepatocytes by OATP1B‐type transporters. Here we evaluated the possibility that this unusual process, called hepatocyte hopping, is also operational in humans and can be modulated through pharmacological inhibition. We found that SG transport by OATP1B1 or murine Oatp1b2 was effectively inhibited by rifampin, and that this agent can significantly increase plasma levels of SG in wildtype mice, but not in Oatp1b2‐deficient animals. In human subjects receiving sorafenib, rifampin acutely increased the systemic exposure to SG. Our study emphasizes the need to consider hepatic handling of xenobiotic glucuronides in the design of drug–drug interaction studies of agents that undergo extensive phase II conjugation.