Imidazoles as Potential Anticancer Agents: An Update on Recent Studies.

Nitrogen-containing heterocyclic rings are common structural components of marketed drugs. Among these heterocycles, imidazole/fused imidazole rings are present in a wide range of bioactive compounds. The unique properties of such structures, including high polarity and the ability to participate in hydrogen bonding and coordination chemistry, allow them to interact with a wide range of biomolecules, and imidazole-/fused imidazole-containing compounds are reported to have a broad spectrum of biological activities. This review summarizes recent reports of imidazole/fused imidazole derivatives as anticancer agents appearing in the peer-reviewed literature from 2018 through 2020. Such molecules have been shown to modulate various targets, including microtubules, tyrosine and serine-threonine kinases, histone deacetylases, p53-Murine Double Minute 2 (MDM2) protein, poly (ADP-ribose) polymerase (PARP), G-quadraplexes, and other targets. Imidazole-containing compounds that display anticancer activity by unknown/undefined mechanisms are also described, as well as key features of structure-activity relationships. This review is intended to provide an overview of recent advances in imidazole-based anticancer drug discovery and development, as well as inspire the design and synthesis of new anticancer molecules.

Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3.

Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.

Identification of Structural and Molecular Features Involved in the Transport of 3'-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3.

Combination antiretroviral drug treatments depend on 3'-deoxy-nucleoside analogs such as 3'-azido-3'-deoxythymidine (AZT) and 2'3'-dideoxyinosine (DDI). Despite being effective in inhibiting human immunodeficiency virus replication, these drugs produce a range of toxicities, including myopathy, pancreatitis, neuropathy, and lactic acidosis, that are generally considered as sequelae to mitochondrial damage. Although cell surface-localized nucleoside transporters, such as human equilibrative nucleoside transporter 2 (hENT2) and human concentrative nucleoside transporter 1 (hCNT1), are known to increase the carrier-mediated uptake of 3'-deoxy-nucleoside analogs into cells, another ubiquitously expressed intracellular nucleoside transporter (namely, hENT3) has been implicated in the mitochondrial transport of 3'-deoxy-nucleoside analogs. Using site-directed mutagenesis, generation of chimeric hENTs, and 3H-permeant flux measurements in mutant/chimeric RNA-injected Xenopus oocytes, here we identified the molecular determinants of hENT3 that dictate membrane translocation of 3'-deoxy-nucleoside analogs. Our findings demonstrated that whereas hENT1 had no significant transport activity toward 3'-deoxy-nucleoside analogs, hENT3 was capable of transporting 3'-deoxy-nucleoside analogs similar to hENT2. Transport analyses of hENT3-hENT1 chimeric constructs demonstrated that the N-terminal half of hENT3 is primarily responsible for the hENT3-3'-deoxy-nucleoside analog interaction. In addition, mutagenic studies identified that 225D and 231L in the N-terminal half of hENT3 partially contribute to the ability of hENT3 to transport AZT and DDI. The identification of the transporter segment and amino acid residues that are important in hENT3 transport of 3'-deoxy-nucleoside analogs may present a possible mechanism for overcoming the adverse toxicities associated with 3'-deoxy-nucleoside analog treatment and may guide rational development of novel nucleoside analogs.

Chemokine-Like Receptor 1 Is a Novel Wnt Target Gene that Regulates Mesenchymal Stem Cell Differentiation.

Bone remodeling is a dynamic process requiring the coordinated action of formative (osteoblast) and resorptive (osteoclast) cell populations. An imbalance of the development and function of these cell types underlies several chronic bone loss disorders such as osteoporosis. Increased bone marrow adipocyte numbers commonly occur with bone loss disorders and numerous studies have documented an inverse relationship between bone marrow fat and bone formation. Osteoblasts and adipocytes derive in a competitive fashion from a common mesenchymal stem cell (MSC) precursor. Generally, factors that promote MSC adipogenesis inhibit osteoblastogenesis and thereby, reduce bone formation. Previously we established that the secreted protein chemerin regulates adipogenic and osteoblastogenic differentiation of MSCs by signaling through chemokine-like receptor 1 (CMKLR1). However, the fundamental mechanisms by which chemerin/CMKLR1 influences lineage determination remain largely uncharacterized. Herein, we provide experimental evidence that chemerin/CMKLR1 regulates canonical Wnt signaling in MSCs by influencing the expression, subcellular location, and transcriptional activity of the central Wnt transducer, ß-catenin. Moreover, we provide evidence that CMKLR1 is a novel Wnt responsive gene that functions in a negative feedback loop to limit osteoblastogenic Wnt signaling. Mechanistically, this entails Notch-dependent changes in the expression and function of key adipogenic and osteoblastogenic transcription factors, cell cycle proteins and chromatin remodeling enzymes. Consistent with this, MSCs from CMKLR1 knockout (-/-) mice exhibited similar dependency on Notch signaling to maintain osteoblastogenic differentiation. Taken together, our findings support a fundamental biological function for chemerin/CMKLR1 to balance osteoblastogenic and adipogenic signaling and thereby contribute to the maintenance of pluripotency in MSCs. Stem Cells 2017;35:711-724.

Bone Marrow Adipose Tissue and Skeletal Health.

PURPOSE OF REVIEW: To summarize and discuss recent progress and novel signaling mechanisms relevant to bone marrow adipocyte formation and its physiological/pathophysiological implications for bone remodeling. RECENT FINDINGS: Skeletal remodeling is a coordinated process entailing removal of old bone and formation of new bone. Several bone loss disorders such as osteoporosis are commonly associated with increased bone marrow adipose tissue. Experimental and clinical evidence supports that a reduction in osteoblastogenesis from mesenchymal stem cells at the expense of adipogenesis, as well as the deleterious effects of adipocyte-derived signaling, contributes to the etiology of osteoporosis as well as bone loss associated with aging, diabetes mellitus, post-menopause, and chronic drug therapy. However, this view is challenged by findings indicating that, in some contexts, bone marrow adipose tissue may have a beneficial impact on skeletal health. Further research is needed to better define the role of marrow adipocytes in bone physiology/pathophysiology and to determine the therapeutic potential of manipulating mesenchymal stem cell differentiation.

The role of the equilibrative nucleoside transporter 1 on tissue and fetal distribution of ribavirin in the mouse.

Ribavirin is used for the treatment of hepatitis C virus (HCV) infection. The equilibrative nucleoside transporter 1 (ENT1) expressed in hepatocytes transports ribavirin into the liver, the site of efficacy of the drug. However, it is still unclear whether ENT1 plays a dominant role in the hepatic distribution of the drug in vivo. In addition, due to fetal toxicity, administration of ribavirin to pregnant women with HCV infection is contraindicated. ENT1 might play a role in the fetal distribution and therefore the fetal toxicity of ribavirin. The aim of the present study was to investigate the in vivo contribution of ENT1 to the tissue distribution of ribavirin. When compared with that in Ent1(+/+) mice, the ribavirin tissue to plasma concentration ratio (including phosphorylated metabolites) in Ent1(-/-) mice at 15 min and 6 h after intravenous [(3) H]-ribavirin (3 mg/kg) administration was consistently and significantly decreased in the liver and the pancreas. Likewise, when compared with the Ent1(+/+) mice, the fetal distribution of ribavirin at 15 min after administration was significantly reduced in Ent1(-/-) fetuses and placenta. In contrast, there was no significant difference between Ent1(+/+), Ent1(+/-) and Ent1(-/-) mice in the fetal or placental to maternal plasma ribavirin concentration ratio at 2 h after ribavirin administration. The findings in the present study suggest that ENT1 plays a pivotal role in the distribution of ribavirin into tissues including the liver and pancreas, but affects only the rate, but not the extent, of ribavirin distribution into the fetus. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetic assessment of low dose decitabine in combination therapies: Development and validation of a sensitive UHPLC-MS/MS method for murine plasma analysis.

Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine.

2'-Fluoro-6'-methylene-carbocyclic adenosine phosphoramidate (FMCAP) prodrug: in vitro anti-HBV activity against the lamivudine-entecavir resistant triple mutant and its mechanism of action.

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (FMCA) monophosphate prodrug (FMCAP) was synthesized and evaluated for its in vitro anti-HBV potency against a lamivudine-entecavir resistant clone (L180M+M204V+S202G). FMCA demonstrated significant antiviral activity against wild-type as well as lamivudine-entecavir resistant triple mutant (L180M+M204V+S202G). The monophosphate prodrug (FMCAP) demonstrated greater than 12-fold (12×) increase in anti-HBV activity without increased cellular toxicity. Mitochondrial and cellular toxicity studies of FMCA indicated that there is no significant toxicity up to 100 µM. Mode of action studies by molecular modeling indicate that the 2'-fluoro moiety by hydrogen bond as well as the Van der Waals interaction of the carbocyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the drug-resistant mutants.

Combination of Tissue Microarray Profiling and Multiplexed IHC Approaches to Investigate Transport Mechanism of Nucleoside Analog Drug Resistance.

Nucleoside analogs (NAs) are an established class of anticancer agents being used clinically for the treatment of diverse cancers, either as monotherapy or in combination with other established anticancer or pharmacological agents. To date, nearly a dozen anticancer NAs are approved by the FDA, and several novel NAs are being tested in preclinical and clinical trials for future applications. However, improper delivery of NAs into tumor cells because of alterations in expression of one or more drug carrier proteins (e.g., solute carrier (SLC) transporters) within tumor cells or cells surrounding the tumor microenvironment stands as one of the primary reasons for therapeutic drug resistance. The combination of tissue microarray (TMA) and multiplexed immunohistochemistry (IHC) is an advanced, high-throughput approach over conventional IHC that enables researchers to effectively investigate alterations to numerous such chemosensitivity determinants simultaneously in hundreds of tumor tissues derived from patients. In this chapter, taking an example of a TMA from pancreatic cancer patients treated with gemcitabine (a NA chemotherapeutic agent), we describe the step-by-step procedure of performing multiplexed IHC, imaging of TMA slides, and quantification of expression of some relevant markers in these tissue sections as optimized in our laboratory and discuss considerations while designing and carrying out this experiment.

Increased renal elimination of endogenous and synthetic pyrimidine nucleosides in concentrative nucleoside transporter 1 deficient mice.

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.

Adriamycin-Induced Nephropathy is Robust in N and Modest in J Substrain of C57BL/6.

Adriamycin (ADR)-induced nephropathy remains the leading model to study human primary focal segmental glomerulosclerosis (FSGS), a common pathway for podocyte damage and glomerular loss of function that leads to chronic kidney disease. However, the use of this model for reverse genetics is limited by historical categorization of C57BL/6 mice as an ADR-resistant strain, which is also the most common genetically modified strain. Additionally, conflicting reports exist utilizing C57BL/6 for ADR-nephrosis due to lack of understanding of substrain differences (J/N) and variability in ADR dosage, timing, and frequency to induce damage. We have undertaken a systematic approach to elucidate the specifics of ADR-nephrosis in C57BL/6 N and J substrains. We induced nephropathy with 2 doses of ADR, and measured albuminuria for 6 weeks and performed histological evaluations. Our findings revealed induction of robust and modest proteinuria in N and J substrains, respectively. The serum creatinine levels were elevated in N, but not J substrain. Both the substrains showed reduction in body weight with N greater than J, although mortality remained at 0% in both substrains. Histological analysis showed worse renal lesions in the N than the J substrain. Podocyte markers synaptopodin, nephrin, podocin, and WT1 were reduced to a greater extent in the N than the J substrain. In summary, we provide the nephrology community with a reproducible mouse model for FSGS, in a strain otherwise assumed to be ADR-resistant and highlight the differences between J and N substrains. This enables future studies, especially concerning genetically manipulated animal models in C57BL/6.

Nucleoside transporters and immunosuppressive adenosine signaling in the tumor microenvironment: Potential therapeutic opportunities.

Adenosine compartmentalization has a profound impact on immune cell function by regulating adenosine localization and, therefore, extracellular signaling capabilities, which suppresses immune cell function in the tumor microenvironment. Nucleoside transporters, responsible for the translocation and cellular compartmentalization of hydrophilic adenosine, represent an understudied yet crucial component of adenosine disposition in the tumor microenvironment. In this review article, we will summarize what is known regarding nucleoside transporter's function within the purinome in relation to currently devised points of intervention (i.e., ectonucleotidases, adenosine receptors) for cancer immunotherapy, alterations in nucleoside transporter expression reported in cancer, and potential avenues for targeting of nucleoside transporters for the desired modulation of adenosine compartmentalization and action. Further, we put forward that nucleoside transporters are an unexplored therapeutic opportunity, and modulation of nucleoside transport processes could attenuate the pathogenic buildup of immunosuppressive adenosine in solid tumors, particularly those enriched with nucleoside transport proteins.

Alternatively Spliced Landscape of PPARγ mRNA in Podocytes Is Distinct from Adipose Tissue.

Podocytes are highly differentiated epithelial cells, and their structural and functional integrity is compromised in a majority of glomerular and renal diseases, leading to proteinuria, chronic kidney disease, and kidney failure. Traditional agonists (e.g., pioglitazone) and selective modulators (e.g., GQ-16) of peroxisome-proliferator-activated-receptor-γ (PPARγ) reduce proteinuria in animal models of glomerular disease and protect podocytes from injury via PPARγ activation. This indicates a pivotal role for PPARγ in maintaining glomerular function through preservation of podocytes distinct from its well-understood role in driving insulin sensitivity and adipogenesis. While its transcriptional role in activating adipokines and adipogenic genes is well-established in adipose tissue, liver and muscle, understanding of podocyte PPARγ signaling remains limited. We performed a comprehensive analysis of PPARγ mRNA variants due to alternative splicing, in human podocytes and compared with adipose tissue. We found that podocytes express the ubiquitous PPARγ Var 1 (encoding γ1) and not Var2 (encoding γ2), which is mostly restricted to adipose tissue and liver. Additionally, we detected expression at very low level of Var4, and barely detectable levels of other variants, Var3, Var11, VartORF4 and Var9, in podocytes. Furthermore, a distinct podocyte vs. adipocyte PPAR-promoter-response-element containing gene expression, enrichment and pathway signature was observed, suggesting differential regulation by podocyte specific PPARγ1 variant, distinct from the adipocyte-specific γ2 variant. In summary, podocytes and glomeruli express several PPARγ variants, including Var1 (γ1) and excluding adipocyte-specific Var2 (γ2), which may have implications in podocyte specific signaling and pathophysiology. This suggests that that new selective PPARγ modulators can be potentially developed that will be able to distinguish between the two forms, γ1 and γ2, thus forming a basis of novel targeted therapeutic avenues.

EMT alterations in the solute carrier landscape uncover SLC22A10/A15 imposed vulnerabilities in pancreatic cancer.

The involvement of membrane-bound solute carriers (SLCs) in neoplastic transdifferentiation processes is poorly defined. Here, we examined changes in the SLC landscape during epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. We show that two SLCs from the organic anion/cation transporter family, SLC22A10 and SLC22A15, favor EMT via interferon (IFN) α and γ signaling activation of receptor tyrosine kinase-like orphan receptor 1 (ROR1) expression. In addition, SLC22A10 and SLC22A15 allow tumor cell accumulation of glutathione to support EMT via the IFNα/γ-ROR1 axis. Moreover, a pan-SLC22A inhibitor lesinurad reduces EMT-induced metastasis and gemcitabine chemoresistance to prolong survival in mouse models of pancreatic cancer, thus identifying new vulnerabilities for human PDAC.

Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective.

Anticancer nucleoside analogs produce adverse, and at times, dose-limiting hematological toxicities that can compromise treatment efficacy, yet the mechanisms of such toxicities are poorly understood. Recently, cellular nucleoside transport has been implicated in normal blood cell formation with studies from nucleoside transporter-deficient mice providing additional insights into the regulation of mammalian hematopoiesis. Furthermore, several idiopathic human genetic disorders have revealed nucleoside transport as an important component of mammalian hematopoiesis because mutations in individual nucleoside transporter genes are linked to various hematological abnormalities, including anemia. Here, we review recent developments in nucleoside transporters, including their transport characteristics, their role in the regulation of hematopoiesis, and their potential involvement in the occurrence of adverse hematological side effects due to nucleoside drug treatment. Furthermore, we discuss the putative mechanisms by which aberrant nucleoside transport may contribute to hematological abnormalities and identify the knowledge gaps where future research may positively impact treatment outcomes for patients undergoing various nucleoside analog therapies.

Selective modulator of nuclear receptor PPARγ with reduced adipogenic potential ameliorates experimental nephrotic syndrome.

Glomerular disease manifests as nephrotic syndrome (NS) with high proteinuria and comorbidities, and is frequently refractory to standard treatments. We hypothesized that a selective modulator of PPARγ, GQ-16, will provide therapeutic advantage over traditional PPARγ agonists for NS treatment. We demonstrate in a pre-clinical NS model that proteinuria is reduced with pioglitazone to 64%, and robustly with GQ-16 to 81% of nephrosis, comparable to controls. Although both GQ-16 and pioglitazone restore glomerular-Nphs1, hepatic-Pcsk9 and serum-cholesterol, only GQ-16 restores glomerular-Nrf2, and reduces hypoalbuminemia and hypercoagulopathy. GQ-16 and pioglitazone restore common and distinct glomerular gene expression analyzed by RNA-seq and induce insulin sensitizing adipokines to various degrees. Pioglitazone but not GQ-16 induces more lipid accumulation and aP2 in adipocytes and white adipose tissue. We conclude that selective modulation of PPARγ by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria, NS associated comorbidities, and adipogenic side effects of full PPARγ agonists.

Human equilibrative nucleoside transporter-3 (hENT3) spectrum disorder mutations impair nucleoside transport, protein localization, and stability.

Accumulating evidence reveals that sole mutations in hENT3 cause a spectrum of human genetic disorders. Among these include H syndrome, characterized by scleroderma, hyperpigmentation, hypertrichosis, hepatomegaly, cardiac abnormalities and musculoskeletal deformities, pigmented hypertrichotic dermatosis with insulin-dependent diabetes syndrome, characterized by autoantibody-negative diabetes mellitus and skin deformities, familial Rosai-Dorfman disease, characterized by short stature, familial histiocytosis and sinus histiocytosis with massive lymphadenopathy (SHML), characterized by severe tissue infiltration of immune cells and swollen lymph nodes. hENT3 spectrum disorders share a common mutation and share overlapping clinical manifestations that display many intriguing resemblances to mitochondrial and lysosomal disorders. Although earlier studies identify hENT3 as a mitochondrial and a lysosomal nucleoside transporter, the precise connections between hENT3 and the pathophysiology of these disorders remain unresolved. In this study, we performed functional and biochemical characterization of these mutations in hENT3. We report severe reductions/losses of hENT3 nucleoside transport functions of hENT3 syndrome mutants. In addition to transport alterations, we provide evidence for possible loss of hENT3 functions in all H and pigmented hypertrichotic dermatosis with insulin-dependent diabetes syndromes due to either mistrafficking or altered stability of mutant hENT3 proteins.

Antiviral activity of novel 2'-fluoro-6'-methylene-carbocyclic adenosine against wild-type and drug-resistant hepatitis B virus mutants.

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (9) was synthesized and evaluated its anti-HBV activity. The titled compound demonstrated significant antiviral activity against wild-type as well as lamivudine, adefovir and double lamivudine/entecavir resistant mutants. Molecular modeling study indicate that the 2'-fluoro moiety by a hydrogen bond, as well as the van der Waals interaction of the carbocyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the drug resistant mutants.

Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors.

Mutations in human equilibrative nucleoside transporter 3 (ENT3) encoded by SLC29A3 results in anemia and erythroid hypoplasia, suggesting that ENT3 may regulate erythropoiesis. Here, we demonstrate that lysosomal ENT3 transport of taurine-conjugated bile acids (TBA) facilitates TBA chemical chaperone function and alleviates endoplasmic reticulum (ER) stress in expanding mouse hematopoietic stem and progenitor cells (HSPCs). Slc29a3-/- HSPCs accumulate less TBA despite elevated levels of TBA in Slc29a3-/- mouse plasma and have elevated basal ER stress, reactive oxygen species (ROS), and radiation-induced apoptosis. Reintroduction of ENT3 allows for increased accumulation of TBA into HSPCs, which results in TBA-mediated alleviation of ER stress and erythroid apoptosis. Transplanting TBA-preconditioned HSPCs expressing ENT3 into Slc29a3-/- mice increase bone marrow repopulation capacity and erythroid pool size and prevent early mortalities. Together, these findings suggest a putative role for a facilitative lysosomal transporter in the bile acid regulation of ER stress in mouse HSPCs which may have implications in erythroid biology, the treatment of anemia observed in ENT3-mutated human genetic disorders, and nucleoside analog drug therapy.

EMT-Induced Gemcitabine Resistance in Pancreatic Cancer Involves the Functional Loss of Equilibrative Nucleoside Transporter 1.

Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.