HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport.

Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported [3H] or [14C] pyrimidine and purine nucleosides once inside cells. With the exception of adenosine, transported nucleosides were generally subject to minimal intracellular metabolism. We also used radiochemical HPLC analysis to study the mechanism by which adenosine functions as a low Km, low Vmax permeant of hCNT1. hCNT1-producing oocytes were pre-loaded with [3H] uridine, after which efflux of accumulated radioactivity was measured in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. hCNT1-mediated [3H]-efflux was stimulated by extracellular uridine, but inhibited by extracellular adenosine, with >95% of the radioactivity exiting cells being unmetabolized uridine, consistent with a low transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC 29) family, hENT1-4. Of these, hENT1 and hENT2 transport both nucleosides and nucleobases into and out of cells, but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. We therefore used HPLC to determine plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice, and to compare the findings with knockout of mCNT3. Results demonstrated that ENT1 was more important than ENT2 or CNT3 in determining plasma adenosine concentrations, indicated modest roles of ENT1 in the homeostasis of other nucleosides, and suggested that none of the transporters is a major participant in handling of nucleobases.

Fine-mapping of a novel premenopausal breast cancer susceptibility locus at Chr4q31.22 in Caucasian women and validation in African and Chinese women.

We previously identified a novel breast cancer susceptibility variant on chromosome 4q31.22 locus (rs1429142) conferring risk among women of European ancestry. Here, we report replication of findings, validation of the variant in diverse populations and fine-mapping of the associated locus in Caucasian population. The SNP rs1429142 (C/T, minor allele frequency 18%) showed association for the overall breast cancer risk in Stages 1-4 (n = 4,331 cases/4271 controls; p = 4.35 × 10-8 ; odds ratio, ORC-allele ,1.25), and an elevated risk among premenopausal women (n = 1,503 cases/4271 controls; p = 5.81 × 10-10 ; ORC-allele 1.40) in European populations. SNP rs1429142 was associated with premenopausal breast cancer risk in women of African (T/C; p-value 1.45 × 10-02 ; ORC-allele 1.2) but not from Chinese ancestry. Fine-mapping of the locus revealed several potential causal variants which are present within a single association signal, revealed from the conditional regression analysis. Functional annotation of the potential causal variants revealed three putative SNPs rs1366691, rs1429139 and rs7667633 with active enhancer functions inferred based on histone marks, DNase hypersensitive sites in breast cell line data. These putative variants were bound by transcription factors (C-FOS, STAT1/3 and POL2/3) with known roles in inflammatory pathways. Furthermore, Hi-C data revealed several short-range interactions in the fine-mapped locus harboring the putative variants. The fine mapped locus was predicted to be within a single topologically associated domain, potentially facilitating enhancer-promoter interactions possibly leading to the regulation of nearby genes.

The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.

2-Nitroimidazole-Furanoside Derivatives for Hypoxia Imaging-Investigation of Nucleoside Transporter Interaction, 18F-Labeling and Preclinical PET Imaging.

The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives-mimicking nucleosides-have proven their potential with [18F]FAZA ([18F]fluoro-azomycin-α-arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios. We recently evaluated [18F]fluoro-azomycin-ß-deoxyriboside (ß-[18F]FAZDR), with a structure more similar to nucleosides than [18F]FAZA and possible interaction with nucleoside transporters. For a deeper insight, we comparatively studied the interaction of FAZA, ß-FAZA, α-FAZDR and ß-FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions of the compounds. The highest interactions being for ß-FAZDR (IC50 124 ± 33 µM for SLC28A3), but also for FAZA with the non-nucleosidic α-configuration, the interactions were remarkable (290 ± 44 µM {SLC28A1}; 640 ± 10 µM {SLC28A2}). An improved synthesis was developed for ß-FAZA. For a PET study in tumor-bearing mice, α-[18F]FAZDR was synthesized (radiochemical yield: 15.9 ± 9.0% (n = 3), max. 10.3 GBq, molar activity > 50 GBq/µmol) and compared to ß-[18F]FAZDR and [18F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios (TMR) for ß-[18F]FAZDR already at 1 h p.i. (2.52 ± 0.94, n = 4) in comparison to [18F]FMISO (1.37 ± 0.11, n = 5) and α-[18F]FAZDR (1.93 ± 0.39, n = 4), with possible mediation by the involvement of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers (2.5⁻3.0). Highest clearance from tumor tissue was observed for ß-[18F]FAZDR (56.6 ± 6.8%, 2 h p.i.), followed by α-[18F]FAZDR (34.2 ± 7.5%) and [18F]FMISO (11.8 ± 6.5%). In conclusion, both isomers of [18F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

Multitargeted kinase inhibitors imatinib, sorafenib and sunitinib perturb energy metabolism and cause cytotoxicity to cultured C2C12 skeletal muscle derived myotubes.

Tyrosine kinase inhibitors (TKIs) have advanced cancer treatment and prognosis but have also resulted in adverse effects such as fatigue, diarrhea, hypothyroidism, and other toxicities. We investigated TKI effects on skeletal muscle as a possible explanation of TKI induced fatigue. Changes in mitochondrial function due to inhibition of oxidative phosphorylation complexes, generation of superoxides, and inhibition of key transporters involved in uptake of glucose and/or nucleosides may result in alteration of energy metabolism and/or mitochondrial function. We investigated effects of imatinib, sorafenib and sunitinib on these processes in cultured C2C12 murine skeletal muscle cells. Imatinib, sorafenib and sunitinib were cytotoxic to C2C12 cells with IC50 values of 20, 8 and 8 µM, respectively. Imatinib stimulated glucose uptake and inhibited complex V activity by 35% at 50 µM. Sorafenib inhibited complex II/III and V with IC50 values of 32 and 28 µM, respectively. Sorafenib caused activation of caspase 3/7 and depolarization of mitochondrial membranes occurred very rapidly with complete loss at 5-10 µM. Sunitinib inhibited Complex I with an IC50 value of 38 µM and caused ATP depletion, caspase 3/7 activation, an increase in reactive oxygen species (ROS), and decreased nucleoside and glucose uptake. In conclusion, imatinib, sunitinib and sorafenib caused changes in mitochondrial complex activities, glucose and nucleoside uptake leading to decreased energy production and mitochondrial function in a skeletal muscle cell model, suggesting that these changes may play a role in fatigue, one of the most common adverse effects of TKIs.

Breast cancer associated germline structural variants harboring small noncoding RNAs impact post-transcriptional gene regulation.

Copy Number Variants (CNVs) are a class of structural variations of DNA. Germline CNVs are known to confer disease susceptibility, but their role in breast cancer warrants further investigations. We hypothesized that breast cancer associated germline CNVs contribute to disease risk through gene dosage or other post-transcriptional regulatory mechanisms, possibly through tissue specific expression of CNV-embedded small-noncoding RNAs (CNV-sncRNAs). Our objectives are to identify breast cancer associated CNVs using a genome wide association study (GWAS), identify sncRNA genes embedded within CNVs, confirm breast tissue (tumor and normal) expression of the sncRNAs, correlate their expression with germline copy status and identify pathways influenced by the genes regulated by sncRNAs. We used an association study design and accessed germline CNV data generated on Affymetrix Human SNP 6.0 array in 686 (in-house data) and 495 (TCGA data) subjects served as discovery and validation cohorts. We identified 1812 breast cancer associated CNVs harboring miRNAs (n = 38), piRNAs (n = 9865), snoRNAs (n = 71) and tRNAs (n = 12) genes. A subset of CNV-sncRNAs expressed in breast tissue, also showed correlation with germline copy status. We identified targets potentially regulated by miRNAs and snoRNAs. In summary, we demonstrate the potential impact of embedded CNV-sncRNAs on expression and regulation of down-stream targets.

Germline copy number variations are associated with breast cancer risk and prognosis.

Breast cancer is one of the most common cancers among women, and susceptibility is explained by genetic, lifestyle and environmental components. Copy Number Variants (CNVs) are structural DNA variations that contribute to diverse phenotypes via gene-dosage effects or cis-regulation. In this study, we aimed to identify germline CNVs associated with breast cancer susceptibility and their relevance to prognosis. We performed whole genome CNV genotyping in 422 cases and 348 controls using Human Affymetrix SNP 6 array. Principal component analysis for population stratification revealed 84 outliers leaving 366 cases and 320 controls of Caucasian ancestry for association analysis; CNVs with frequency > 10% and overlapping with protein coding genes were considered for breast cancer risk and prognostic relevance. Coding genes within the CNVs identified were interrogated for gene- dosage effects by correlating copy number status with gene expression profiles in breast tumor tissue. We identified 200 CNVs associated with breast cancer (q-value < 0.05). Of these, 21 CNV regions (overlapping with 22 genes) also showed association with prognosis. We validated representative CNVs overlapping with APOBEC3B and GSTM1 genes using the TaqMan assay. Germline CNVs conferred dosage effects on gene expression in breast tissue. The candidate CNVs identified in this study warrant independent replication.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance.

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C-), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C-) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C-) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.

Synthesis and preclinical characterization of 1-(6'-deoxy-6'-[18F]fluoro-ß-d-allofuranosyl)-2-nitroimidazole (ß-6'-[18F]FAZAL) as a positron emission tomography radiotracer to assess tumor hypoxia.

Positron emission tomography (PET) using fluorine-18 (18F)-labeled 2-nitroimidazole radiotracers has proven useful for assessment of tumor oxygenation. However, the passive diffusion-driven cellular uptake of currently available radiotracers results in slow kinetics and low tumor-to-background ratios. With the aim to develop a compound that is actively transported into cells, 1-(6'-deoxy-6'-[18F]fluoro-ß-d-allofuranosyl)-2-nitroimidazole (ß-[18F]1), a putative nucleoside transporter substrate, was synthetized by nucleophilic [18F]fluoride substitution of an acetyl protected labeling precursor with a tosylate leaving group (ß-6) in a final radiochemical yield of 12±8% (n=10, based on [18F]fluoride starting activity) in a total synthesis time of 60min with a specific activity at end of synthesis of 218±58GBq/µmol (n=10). Both radiolabeling precursor ß-6 and unlabeled reference compound ß-1 were prepared in multistep syntheses starting from 1,2:5,6-di-O-isopropylidene-α-d-allofuranose. In vitro experiments demonstrated an interaction of ß-1 with SLC29A1 and SLC28A1/2/3 nucleoside transporter as well as hypoxia specific retention of ß-[18F]1 in tumor cell lines. In biodistribution studies in healthy mice ß-[18F]1 showed homogenous tissue distribution and excellent metabolic stability, which was unaffected by tissue oxygenation. PET studies in tumor bearing mice showed tumor-to-muscle ratios of 2.13±0.22 (n=4) at 2h after administration of ß-[18F]1. In ex vivo autoradiography experiments ß-[18F]1 distribution closely matched staining with the hypoxia marker pimonidazole. In conclusion, ß-[18F]1 shows potential as PET hypoxia radiotracer which merits further investigation.

Selective Inhibition of Human Equilibrative and Concentrative Nucleoside Transporters by BCR-ABL Kinase Inhibitors: IDENTIFICATION OF KEY hENT1 AMINO ACID RESIDUES FOR INTERACTION WITH BCR-ABL KINASE INHIBITORS.

Human nucleoside transporters (hNTs) mediate cellular influx of anticancer nucleoside drugs, including cytarabine, cladribine, and fludarabine. BCR-ABL tyrosine kinase inhibitors (TKIs) imatinib and dasatinib inhibit fludarabine and cytarabine uptake. We assessed interactions of bosutinib, dasatinib, imatinib, nilotinib, and ponatinib with recombinant hNTs (hENT1, 2; hCNT1, -2, and -3) produced individually in yeast Saccharomyces cerevisiae Nilotinib inhibited hENT1-mediated uridine transport most potently (IC50 value, 0.7 µm) followed by ponatinib > bosutinib > dasatinib > imatinib. Imatinib inhibited hCNT2 with an IC50 value of 2.3 µm Ponatinib inhibited all five hNTs with the greatest effect seen for hENT1 (IC50 value, 9 µm). TKIs inhibited [(3)H]uridine uptake in a competitive manner. Studies in yeast with mutants at two amino acid residues of hENT1 (L442I, L442T, M33A, M33A/L442I) previously shown to be involved in uridine and dipyridamole binding, suggested that BCR-ABL TKIs interacted with Met(33) (TM1) and Leu(442) (TM11) residues of hENT1. In cultured human CEM lymphoblastoid cells, which possess a single hNT type (hENT1), accumulation of [(3)H]cytarabine, [(3)H]cladribine, or [(3)H]fludarabine was reduced by each of the five TKIs, and also caused a reduction in cell surface expression of hENT1 protein. In conclusion, BCR-ABL TKIs variously inhibit five different hNTs, cause a decrease in cell surface hENT1 expression, and decrease uridine accumulation when presented together with uridine or when given before uridine. In experiments with mutant hENT1, we showed for the first time interaction of Met(33) (involved in dipyridamole binding) with BCR-ABL inhibitors and reduced interaction with M33A mutant hENT1.

A Uridine Glucuronosyltransferase 2B7 Polymorphism Predicts Epirubicin Clearance and Outcomes in Early-Stage Breast Cancer.

BACKGROUND: Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7), an enzyme rich in single nucleotide polymorphisms (SNPs). We studied whether the -161 C > T germline SNP in UGT2B7 was related to epirubicin metabolism and whether differences exist in the toxicity and efficacy of epirubicin-based chemotherapy among patients who were TT homozygotes, CT heterozygotes, and CC homozygotes. PATIENTS AND METHODS: A total of 132 women with non-metastatic breast cancer receiving FEC (5-fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2), cyclophosphamide 500 mg/m(2)) were prospectively enrolled. Toxicity was assessed in cycle 1 using the National Cancer Institute Common Toxicity Criteria, version 2.0. RESULTS: The sequence at -161 was studied in 132 subjects; 37 were TT homozygotes, 63 were CT heterozygotes, 26 were CC homozygotes, and 6 could not be genotyped. The CC genotype patients had decreased epirubicin clearance (median, 103.3 L/hr) compared with the CT/TT genotype patients (median, 134.0 L/hr; P = .002). The CC homozygous patients had an increased risk of grade 3 to 4 leukopenia compared with the TT homozygotes or heterozygotes (P = .038 and P = .032, respectively). TT homozygotes or heterozygotes had an increased risk of early recurrence (P = .039; χ(2) test). CONCLUSION: The results of the present prospective pharmacogenetic study suggest that the UGT2B7 -161 C > T SNP correlate with drug metabolism, toxicity, and efficacy in patients receiving epirubicin chemotherapy. Further studies of this UGT2B7 SNP as a predictor of epirubicin toxicity and efficacy are warranted.

Inhibition of sodium-independent and sodium-dependent nucleobase transport activities by tyrosine kinase inhibitors.

PURPOSE: Effects of tyrosine kinase inhibitors (TKIs) on equilibrative nucleobase transport (ENBT) and sodium-dependent nucleobase transport (SNBT) activities were investigated in normal human renal proximal tubule epithelial cells (hRPTECs) and in pig kidney cell line (LLC-PK1). METHODS: Uptake assays were performed by assessing accumulation of radiolabeled nucleobases over time into hRPTECs or LLC-PK1 cell lines which express ENBT and SNBT activities, respectively. Dose-response curves for inhibition of 1 µM [(3)H]adenine or 1 µM [(3)H]hypoxanthine were examined in hRPTECs and in LLC-PK1 cells with varying TKI concentrations (0-100 µM) to calculate the IC50 values (mean ± S.E) for inhibition. RESULTS: Gefitinib inhibited ENBT activity with an IC50 value of 0.7 µM, thus indicating strong interactions of ENBT with gefitinib in hRPTECs. Erlotinib > sorafenib > imatinib > sunitinib inhibited ENBT with IC50 values of 15, 40, 60, 78 µM, respectively, whereas dasatinib, lapatinib, and vandetanib were not inhibitory at concentrations >100 µM. Similar studies in LLC-PK1 cells which exhibit SNBT activity showed that vandetanib was the most potent inhibitor followed by sorafenib > erlotinib > gefitinib > sunitinib > imatinib with IC50 values of 14, 25, 28, 40, 47, 94 µM, respectively, whereas dasatinib and lapatinib were not inhibitory at concentrations >100 µM. CONCLUSIONS: These results suggest for the first time inhibition of both ENBT and SNBT transport activities by TKIs. These results suggest that it is important to consider potential effects on combination regimens using TKIs with nucleobase drugs such as 5-FU in cancer treatment.

Designing and Testing of Novel Taxanes to Probe the Highly Complex Mechanisms by Which Taxanes Bind to Microtubules and Cause Cytotoxicity to Cancer Cells.

Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of ß-tubulin. Since ß-tubulin isotypes have tissue-dependent expression--specifically, the ßIII isotype is very abundant in aggressive tumors and much less common in normal tissues--this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects. Seven derivatives of paclitaxel were designed and four of these were amenable for synthesis in sufficient purity and yield for further testing in breast cancer model cell lines. None of the derivatives studied were superior to currently used taxanes, however computer simulations provided insights into the activity of the derivatives. Our results suggest that neither binding to the intermediate binding site nor the final binding site is sufficient to explain the activities of the derivative taxanes studied. These findings highlight the need to iteratively improve on the design of taxanes based on their activity in model systems. Knowledge gained on the ability of the engineered drugs to bind to targets and bring about activity in a predictable manner is a step towards personalizing therapies.

Interactions of multitargeted kinase inhibitors and nucleoside drugs: Achilles heel of combination therapy?

Multitargeted tyrosine kinase inhibitors (TKI) axitinib, pazopanib, and sunitinib are used to treat many solid tumors. Combination trials of TKIs with gemcitabine, a nucleoside anticancer drug, in pancreas, renal, lung, ovarian, and other malignancies resulted in little benefit to patients. TKI interactions with human nucleoside transporters (hNT) were studied by assessing inhibition of [(3)H]uridine uptake in yeast producing recombinant hNTs individually and in cultured human cancer cell lines. Axitinib, pazopanib, and sunitinib inhibited hENT1 at low micromolar concentrations. In A549, AsPC-1, and Caki-1 cells, [(3)H]uridine, [(3)H]thymidine, [(3)H]gemcitabine, and [(3)H]fluorothymidine (FLT) accumulation was blocked by all three TKIs. Pazopanib > axitinib ≥ sunitinib inhibited hENT1 with IC50 values of 2, 7, and 29 µmol/L, respectively, leading to reduced intracellular gemcitabine and FLT accumulation. Pretreatment or cotreatment of Caki-1 cells with TKIs reduced cellular accumulation of [(3)H]nucleosides, suggesting that TKI scheduling with nucleoside drugs would influence cytotoxicity. In combination cytotoxicity experiments that compared sequential versus simultaneous addition of drugs in Caki-1 cells, cytotoxicity was greatest when gemcitabine was added before TKIs. In clinical settings, TKI inhibitor concentrations in tumor tissues are sufficient to inhibit hENT1 activity, thereby reducing nucleoside chemotherapy drug levels in cancer cells and reducing efficacy in combination schedules. An additional unwanted interaction may be reduced FLT uptake in tumor tissues that could lead to aberrant conclusions regarding tumor response.

Assessing SNP-SNP interactions among DNA repair, modification and metabolism related pathway genes in breast cancer susceptibility.

Genome-wide association studies (GWASs) have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs) associated with breast cancer susceptibility. Although GWASs are primarily focused on single-locus effects, gene-gene interactions (i.e., epistasis) are also assumed to contribute to the genetic risks for complex diseases including breast cancer. While it has been hypothesized that moderately ranked (P value based) weak single-locus effects in GWASs could potentially harbor valuable information for evaluating epistasis, we lack systematic efforts to investigate SNPs showing consistent associations with weak statistical significance across independent discovery and replication stages. The objectives of this study were i) to select SNPs showing single-locus effects with weak statistical significance for breast cancer in a GWAS and/or candidate-gene studies; ii) to replicate these SNPs in an independent set of breast cancer cases and controls; and iii) to explore their potential SNP-SNP interactions contributing to breast cancer susceptibility. A total of 17 SNPs related to DNA repair, modification and metabolism pathway genes were selected since these pathways offer a priori knowledge for potential epistatic interactions and an overall role in breast carcinogenesis. The study design included predominantly Caucasian women (2,795 cases and 4,505 controls) from Alberta, Canada. We observed two two-way SNP-SNP interactions (APEX1-rs1130409 and RPAP1-rs2297381; MLH1-rs1799977 and MDM2-rs769412) in logistic regression that conferred elevated risks for breast cancer (P(interaction)<7.3 × 10(-3)). Logic regression identified an interaction involving four SNPs (MBD2-rs4041245, MLH1-rs1799977, MDM2-rs769412, BRCA2-rs1799943) (P(permutation) = 2.4 × 10(-3)). SNPs involved in SNP-SNP interactions also showed single-locus effects with weak statistical significance, while BRCA2-rs1799943 showed stronger statistical significance (P(correlation/trend) = 3.2 × 10(-4)) than the others. These single-locus effects were independent of body mass index. Our results provide a framework for evaluating SNPs showing statistically weak but reproducible single-locus effects for epistatic effects contributing to disease susceptibility.

Expression of nucleoside transporters and deoxycytidine kinase proteins in muscle invasive urothelial carcinoma of the bladder: correlation with pathological response to neoadjuvant platinum/gemcitabine combination chemotherapy.

PURPOSE: In pancreatic cancer, deoxycytidine kinase and the human equilibrative nucleoside transporter 1 have been validated as predictive markers for benefit from gemcitabine therapy. Gemcitabine is used with cisplatin or carboplatin as neoadjuvant chemotherapy for muscle invasive urothelial cancer of the bladder before radical cystectomy and patients rendered disease-free at surgery tend to have better outcomes. In this trial we examined if nucleoside transporter or deoxycytidine kinase protein abundance in biopsy specimens before chemotherapy is related to the response to neoadjuvant chemotherapy. MATERIALS AND METHODS: A total of 62 consecutive patients undergoing neoadjuvant chemotherapy with platinum/gemcitabine at a single institution were accrued. Initial transurethral resection of bladder tumor specimens and cystectomy specimens were collected, and scored for nucleoside transporter and deoxycytidine kinase expression. Pathological response rates and survival data were collected. RESULTS: Of the 62 patients 17 (27%) achieved a complete pathological response (pT0) to neoadjuvant chemotherapy. Nucleoside transporter and deoxycytidine kinase protein expression in the transurethral resection of bladder tumor specimens did not predict for pT0 status to neoadjuvant chemotherapy. Median overall survival was not reached for the group achieving pT0 status and was 46 months for those with persistent cancer at definitive surgery (p = 0.07). Median followup for the cohort was 30 months. CONCLUSIONS: Nucleoside transporter and deoxycytidine kinase expression in transurethral resection of bladder tumor samples do not predict for response to gemcitabine and platinum neoadjuvant chemotherapy. Patients should continue to be offered neoadjuvant chemotherapy before radical cystectomy based on clinical and pathological staging.

Erlotinib, gefitinib, and vandetanib inhibit human nucleoside transporters and protect cancer cells from gemcitabine cytotoxicity.

PURPOSE: Combinations of tyrosine kinase inhibitors (TKI) with gemcitabine have been attempted with little added benefit to patients. We hypothesized that TKIs designed to bind to ATP-binding pockets of growth factor receptors also bind to transporter proteins that recognize nucleosides. EXPERIMENTAL DESIGN: TKI inhibition of uridine transport was studied with recombinant human (h) equilibrative (E) and concentrative (C) nucleoside transporters (hENT, hCNT) produced individually in yeast. TKIs effects on uridine transport, gemcitabine accumulation, regulation of hENT1 activity, and cell viability in the presence or absence of gemcitabine were evaluated in human pancreatic and lung cancer cell lines. RESULTS: Erlotinib, gefitinib and vandetanib inhibited [(3)H]uridine transport in yeast and [(3)H]uridine and [(3)H]gemcitabine uptake in the four cell lines. Treatment of cell lines with erlotinib, gefitinib, or vandetanib for 24 hours reduced hENT1 activity which was reversed by subsequent incubation in drug-free media for 24 hours. Greater cytotoxicity was observed when gemcitabine was administered before erlotinib, gefitinib, or vandetanib than when administered together and synergy, evaluated using the CalcuSyn Software, was observed in three cell lines resulting in combination indices under 0.6 at 50% reduction of cell growth. CONCLUSIONS: Vandetanib inhibited hENT1, hENT2, hCNT1, hCNT2, and hCNT3, whereas erlotinib inhibited hENT1 and hCNT3 and gefitinib inhibited hENT1 and hCNT1. The potential for reduced accumulation of nucleoside chemotherapy drugs in tumor tissues due to inhibition of hENTs and/or hCNTs by TKIs indicates that pharmacokinetic properties of these agents must be considered when scheduling TKIs and nucleoside chemotherapy in combination.

Comparative in vitro evaluation of transportability and toxicity of capecitabine and its metabolites in cells derived from normal human kidney and renal cancers.

The goal of this study was to understand roles of nucleoside and nucleobase transport processes in capecitabine pharmacology in cells derived from human renal proximal tubule cells (hRPTCs) and three human renal cell carcinoma (RCC) cell lines, A498, A704, and Caki-1. Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) mediated activities and a sodium-independent nucleobase activity were present in hRPTCs. In hRPTCs, uptake of 5'-deoxy-5-fluorouridine (DFUR), a nucleoside metabolite of capecitabine, was pH dependent with highest uptake seen at pH 6.0. In RCC cell lines, hENT1 was the major nucleoside transporter. Nucleobase transport activity was variable among the three RCC cell lines, with Caki-1 showing the highest and A498 showing the lowest activities. Treatment of RCC cell lines with interferon alpha (IFN-α) increased thymidine phosphorylase levels and prior treatment of RCC cell lines with IFN-α followed by 5-FU or DFUR resulted in enhanced sensitivity of all cell lines to 5-FU and two of three cell lines to DFUR. We report for the first time a nucleobase transport activity in hRPTCs and RCC cell lines. In addition, our in vitro cytotoxicity results showed that RCC cell lines differed in their response to 5-FU and DFUR and prior treatment with IFN-α potentiated cytotoxic response to metabolites of capecitabine.

Breast cancer prediction using genome wide single nucleotide polymorphism data.

BACKGROUND: This paper introduces and applies a genome wide predictive study to learn a model that predicts whether a new subject will develop breast cancer or not, based on her SNP profile. RESULTS: We first genotyped 696 female subjects (348 breast cancer cases and 348 apparently healthy controls), predominantly of Caucasian origin from Alberta, Canada using Affymetrix Human SNP 6.0 arrays. Then, we applied EIGENSTRAT population stratification correction method to remove 73 subjects not belonging to the Caucasian population. Then, we filtered any SNP that had any missing calls, whose genotype frequency was deviated from Hardy-Weinberg equilibrium, or whose minor allele frequency was less than 5%. Finally, we applied a combination of MeanDiff feature selection method and KNN learning method to this filtered dataset to produce a breast cancer prediction model. LOOCV accuracy of this classifier is 59.55%. Random permutation tests show that this result is significantly better than the baseline accuracy of 51.52%. Sensitivity analysis shows that the classifier is fairly robust to the number of MeanDiff-selected SNPs. External validation on the CGEMS breast cancer dataset, the only other publicly available breast cancer dataset, shows that this combination of MeanDiff and KNN leads to a LOOCV accuracy of 60.25%, which is significantly better than its baseline of 50.06%. We then considered a dozen different combinations of feature selection and learning method, but found that none of these combinations produces a better predictive model than our model. We also considered various biological feature selection methods like selecting SNPs reported in recent genome wide association studies to be associated with breast cancer, selecting SNPs in genes associated with KEGG cancer pathways, or selecting SNPs associated with breast cancer in the F-SNP database to produce predictive models, but again found that none of these models achieved accuracy better than baseline. CONCLUSIONS: We anticipate producing more accurate breast cancer prediction models by recruiting more study subjects, providing more accurate labelling of phenotypes (to accommodate the heterogeneity of breast cancer), measuring other genomic alterations such as point mutations and copy number variations, and incorporating non-genetic information about subjects such as environmental and lifestyle factors.