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.

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.

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.

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.

Crocetin promotes clearance of amyloid-ß by inducing autophagy via the STK11/LKB1-mediated AMPK pathway.

Alzheimer disease (AD) is usually accompanied by two prominent pathological features, cerebral accumulation of amyloid-ß (Aß) plaques and presence of MAPT/tau neurofibrillary tangles. Dysregulated clearance of Aß largely contributes to its accumulation and plaque formation in the brain. Macroautophagy/autophagy is a lysosomal degradative process, which plays an important role in the clearance of Aß. Failure of autophagic clearance of Aß is currently acknowledged as a contributing factor to increased accumulation of Aß in AD brains. In this study, we have identified crocetin, a pharmacologically active constituent from the flower stigmas of Crocus sativus, as a potential inducer of autophagy in AD. In the cellular model, crocetin induced autophagy in N9 microglial and primary neuron cells through STK11/LKB1 (serine/threonine kinase 11)-mediated AMP-activated protein kinase (AMPK) pathway activation. Autophagy induction by crocetin significantly increased Aß clearance in N9 cells. Moreover, crocetin crossed the blood-brain barrier and induced autophagy in the brains' hippocampi of wild-type male C57BL/6 mice. Further studies in transgenic male 5XFAD mice, as a model of AD, revealed that one-month treatment with crocetin significantly reduced Aß levels and neuroinflammation in the mice brains and improved memory function by inducing autophagy that was mediated by AMPK pathway activation. Our findings support further development of crocetin as a pharmacological inducer of autophagy to prevent, slow down progression, and/or treat AD.Abbreviations: Aß: amyloid-ß; ABCB1/P-gp/P-glycoprotein: ATP-binding cassette, subfamily B (MDR/TAP), member 1; AD: Alzheimer disease; AMPK/PRKAA: AMP-activated protein kinase; APP: amyloid beta (A4) precursor protein; ATG: autophagy related; BBB: blood-brain barrier; BECN1: beclin 1, autophagy related; CAMKK2/CaMKKß: calcium/calmodulin-dependent protein kinase kinase 2, beta; CSE: Crocus sativus extract; CTSB: cathepsin B; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GFAP: glial fibrillary acidic protein; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; Kp: brain partition coefficient; LRP1: low density lipoprotein receptor-related protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK/ERK: mitogen-activated protein kinase; MAPT/tau: microtubule-associated protein tau; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTOR: mechanistic target of rapamycin kinase; MWM: Morris water maze; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NMDA: N-methyl-d-aspartic acid; RPTOR: regulatory associated protein of MTOR; RPS6KB1/p70S6K: ribosomal protein S6 kinase 1; SQSTM1: sequestosome 1; SRB: sulforhodamine B; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; TSC2: TSC complex subunit 2; ULK1: unc-51 like kinase 1.