Antifolate-resistant Cells Research Articles

Folylpolyglutamate synthetase splicing alterations in acute lymphoblastic leukemia are provoked by methotrexate and other chemotherapeutics and mediate chemoresistance

Methotrexate (MTX), a folate antagonist which blocks de novo nucleotide biosynthesis and DNA replication, is an anchor drug in acute lymphoblastic leukemia (ALL) treatment. However, drug resistance is a primary hindrance to curative chemotherapy in leukemia and its molecular mechanisms remain poorly understood. We have recently shown that impaired folylpolyglutamate synthetase (FPGS) splicing possibly contributes to the loss of FPGS activity in MTX-resistant leukemia cell line models and adult leukemia patients. However, no information is available on the possible splicing alterations in FPGS in pediatric ALL. Here, using a comprehensive PCR-based screen we discovered and characterized a spectrum of FPGS splicing alterations including exon skipping and intron retention, all of which proved to frequently emerge in both pediatric and adult leukemia patient specimens. Furthermore, an FPGS activity assay revealed that these splicing alterations resulted in loss of FPGS function. Strikingly, pulse-exposure of leukemia cells to antifolates and other chemotherapeutics markedly enhanced the prevalence of several FPGS splicing alterations in antifolate-resistant cells, but not in their parental antifolate-sensitive counterparts. These novel findings suggest that an assortment of deleterious FPGS splicing alterations may constitute a mechanism of antifolate resistance in childhood ALL. Our findings have important implications for the rational overcoming of drug resistance in individual leukemia patients.

Abstract 4335: Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed

Abstract Background: Approximately 40% of malignant pleural mesothelioma (MPM) patients respond to the combination treatment with the antifolate pemetrexed (PMX) and platinum compounds. Several studies highlighted the predictive role of thymidylate synthase (TS), but additional resistance mechanisms are poorly understood. This study aimed to evaluate the role of the reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) as potential biological markers of intrinsic PMX-resistance in MPM. Materials and methods: PCFT promoter methylation was evaluated by combined bisulfite restriction analysis as well as MethyLight PCR in 14 cell lines and 10 MPM tissues, while PCFT and RFC mRNA levels were studied by Real-Time-PCR in MPM specimens from a test and a validation cohort of 73 and 51 PMX-treated patients, respectively. PCFT protein expression was evaluated by immunohistochemistry in a tissue microarray with representative MPM core biopsies from 35 patients. Data were analyzed by t-test, Fisher/log-rank test and Cox proportional hazards models. Results: PCFT expression inversely correlated with promoter methylation and treatment with 5-Aza-2′-deoxycytidine (5-Aza-dC) induced its marked restoration in MPM cells. Patients with low PCFT mRNA levels had significantly shorter overall survival (OS), in both the test (median OS, 11.3 vs. 17.6 months, P = 0.01) and in the validation cohort (OS, 12.6 vs. 30.3 months, P = 0.02). Multivariate analysis, including age, sex, EORTC prognostic score and histology, confirmed PCFT prognostic relevance (HR, 2.1 and 2.4 in the test and validation cohorts, respectively). Low PCFT protein expression was also associated with significantly shorter OS (11.8 vs. 30.3 months, P = 0.02). Remarkably, the subgroup of patients with both low PCFT and high TS mRNA levels (30% of the patients in the test cohort) had the worse prognosis (median OS, 5.5 months, P<0.01), while no correlation was found in a series of 40 MPM patients not PMX-treated. No significant associations were found between RFC and clinical outcome. Conclusions: These results define PCFT as a novel prognostic factor for MPM patients treated with PMX. Prospective trials for the validation of the prognostic/predictive role of PCFT in MPM patients treated with PMX-based regimens are warranted. Furthermore preclinical data suggest that exposure to 5-Aza-dC might restore PCFT expression and result in efficient eradication of antifolate-resistant cells. Citation Format: Elisa Giovannetti, Paolo A. Zucali, Yehuda G. Assaraf, Niccola Funel, Maria Gemelli, Michal Stark, Leticia G. Leon, Zhanjun Hou, Matteo Perrino, Larry H. Matherly, Godefridus J. Peters. Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2015-4335

Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss

AbstractThe reduced folate carrier (RFC) is the dominant route for the uptake of various antifolates including PT523, a potent dihydrofolate reductase inhibitor (Ki = 0.35 pM) and an excellent transport substrate of the RFC (Kt = 0.7 μM). Here, we describe the multiple mechanisms of RFC inactivation in human leukemia PT523-resistant cells originally harboring 3 RFC alleles. Cellular exposure to gradually increasing PT523 concentrations resulted in sublines displaying up to 3500-fold resistance to various hydrophilic antifolates that rely on RFC for their cellular uptake. Antifolate-resistant cells lost RFC gene expression (65%-99% loss) due to impaired promoter binding of various transcription factors that regulate RFC gene expression. Additionally, DNA sequencing revealed that PT523-resistant cells contained a cluster of 4 nearly consecutive mutations residing on a single RFC allele including L143P, A147V, R148G, and Q150Stop. Southern blot analysis established the loss of an RFC allele in PT523-resistant cells. These alterations resulted in markedly decreased RFC protein levels (∼80%-99% loss) and consequently impaired [3H]methotrexate transport (87%-99% loss). This study provides the first evidence that acquisition of PT523 resistance in human leukemia cells harboring 3 RFC alleles is due to multiple coexisting alterations including transcriptional silencing, inactivating mutations, and RFC allele loss.

Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier

AbstractThe reduced folate carrier (RFC) is the dominant influx transporter for antifolates. A major mechanism of antifolate resistance is loss of RFC (SLC19A1) gene expression due to decreased GC-box-dependent transcription. However, despite the poor GC-box binding in multiple antifolate-resistant cell lines, normal Sp1 levels were retained. Here we explored the post-translational modifications that may disrupt Sp1 function. Phospho-affinity purification of nuclear proteins revealed that resistant cells contained approximately 8-fold more phosphorylated Sp1 than parental cells; treatment of nuclear proteins from these cells with alkaline phosphatase restored GC-box binding. As protein kinase A phosphorylates Sp1, resistant cells were treated with various cAMP-reactive agents, revealing no apparent effect on GC-box binding except for the general phosphodiesterase inhibitor IBMX. As cGMP levels also may be affected by IBMX, resistant cells were treated with 8-pCPT-cGMP, resulting in the complete restoration of GC-box binding, luciferase reporter activity, and RFC mRNA levels. This restoration was abolished in the presence of the protein phosphatase 2A inhibitor (PP2A) okadaic acid. Importantly, whereas resistant cells showed multiple phosphorylated Sp1 forms barely detectable in parental cells, treatment with 8-pCPT-cGMP resulted in their elimination; this disappearance, however, was prevented by the copresence of okadaic acid. These findings provide the first evidence that loss of RFC gene expression in antifolate-resistant cells is associated with an inhibitory Sp1 phosphorylation that can be eliminated by a cGMP-dependent activation of PP2A. (Blood. 2006;107:708-715)

Impaired CREB-1 phosphorylation in antifolate-resistant cell lines with down-regulation of the reduced folate carrier gene.

The human reduced folate carrier (hRFC) is the dominant transporter for the uptake of antifolates used in cancer chemotherapy. We have shown recently that decreased cAMP-responsive element (CRE)-dependent transcription contributes to the loss of hRFC gene expression in multiple antifolate-resistant cell lines. This was associated with markedly decreased levels of phosphorylated cAMP response element-binding protein 1 (pCREB-1) and CRE-binding. Consistent with the autoregulation of CREB-1 gene expression by pCREB-1, prominently decreased CREB-1 mRNA levels were observed in antifolate-resistant cells. We therefore explored the possibility that these cells were defective in CREB-1 phosphorylation, thereby resulting in down-regulation of some cAMP-responsive genes. Two-dimensional gel electrophoresis revealed that CREB-1 and its phosphoisoforms were markedly decreased in these cells. Treatment with forskolin, an activator of adenylyl cyclase, restored both CREB-1 and pCREB-1 levels; this resulted in the restoration of CRE-binding, CRE-reporter activity, and CREB-1 and RFC mRNA levels. Hence, the protein kinase A pathway was examined using various agents that augment intracellular cAMP levels, including cholera toxin, an upstream agonist that renders stimulatory G-proteins (Galphas) constitutively active. Treatment of antifolate-resistant cells with these agents resulted in the restoration of pCREB-1 levels and CRE-reporter activity. Furthermore, transient transfection with a constitutively transcriptionally active VP16-CREB-1 that does not require phosphorylation for its activity resulted in restoration of CREB mRNA levels but not pCREB-1 levels. This is the first demonstration that resistance to various antifolates may potentially be associated with impaired activity of Galphas or their coupled receptors, resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP-responsive genes.

Reduced Folate Carrier Gene Silencing in Multiple Antifolate-resistant Tumor Cell Lines Is Due to a Simultaneous Loss of Function of Multiple Transcription Factors but Not Promoter Methylation

The human reduced folate carrier (hRFC) is the major uptake route for antifolates used in cancer chemotherapy. Here we explored the molecular basis for the decrease or loss of hRFC gene expression in seventeen tumor cell lines with resistance to multiple antifolates due to impaired antifolate transport. We studied the role of various cis-acting elements including CRE/AP-1-like element and GC-box in hRFC promoters A and B, respectively, as well as AP-2, Mzf-1 and E-box that are contained within or near four tandemly repeated sequences upstream of promoter A. Decreased or abolished binding either to [32P]GC-box, Mzf-1, AP-1, E-box, or CRE oligonucleotides was detected in approximately 50-80% of antifolate-resistant cell lines. Strikingly, approximately 80% of the cell lines displayed a simultaneously decreased binding to three or more of these hRFC promoter elements, whereas normal AP-2 binding was retained. The possible contribution of promoter methylation to hRFC gene silencing was also explored. None of the antifolate-resistant cell lines, except for MDA-MB-231 cells, showed hRFC promoter methylation; consistently, MDA-MB-231 was the only cell line that retained binding to all six cis-acting elements. Western blot analysis demonstrated decreased expression of transcriptional activators (pCREB-1, pATF-1, USF-1, c-Fos, c-Jun, Sp1, and Sp3) and/or increased expression of repressors (short Sp3 isoforms), whereas normal AP2alpha levels were retained. Transient expression of the relevant transcription factors restored, at least partially, both promoter binding and hRFC gene expression. This is the first report that transcriptional silencing of the hRFC gene in multiple tumor cell lines with resistance to various novel antifolates is a result of a simultaneous loss of function of multiple transcription factors but not promoter methylation.

Alterations in the Expression of Transcription Factors and the Reduced Folate Carrier as a Novel Mechanism of Antifolate Resistance in Human Leukemia Cells

The human reduced folate carrier (hRFC) is the dominant transporter mediating the uptake of reduced folate cofactors and antifolate anticancer drugs. Defective antifolate uptake due to inactivating mutations in the hRFC gene is an established mechanism of drug resistance in various tumor cells. However, while antifolate transport is frequently impaired, either no or only a single hRFC allele is inactivated, suggesting that additional mechanism(s) of resistance are operative. Here we studied the relationship between the expression and function of transcription factors and antifolate resistance in transport-defective leukemia cells that poorly express or completely lack RFC mRNA. Stable transfection with a hRFC expression construct resulted in restoration of normal RFC mRNA expression and nearly wild type drug sensitivity in these antifolate-resistant cells. The loss of RFC gene expression prompted us to explore transcription factor binding to the hRFC promoter. The hRFC promoter contains an upstream GC-box and a downstream cAMP-response element (CRE)/AP-1-like element. Electrophoretic mobility shift assays and oligonucleotide competition revealed a substantial loss of nuclear factor binding to CRE and GC-box in these drug-resistant cell lines. Consistently, antibody-mediated supershift analysis showed a marked decrease in the binding of CRE-binding protein 1 (CREB-1) and specificity protein 1 (Sp1) to CRE and GC-box, respectively. Western blot analysis revealed undetectable expression of CREB-1, decreased ATF-1 levels, parental Sp1 levels, and increased levels of the short Sp3 isoforms, recently shown to repress hRFC gene expression. Transient transfections into these antifolate-resistant cells demonstrated a marked loss of GC-box-dependent, and CRE-driven reporter gene activities and introduction of CREB-1 or Sp1 expression constructs resulted in restoration of hRFC mRNA expression. These results establish a novel mechanism of antifolate resistance that is based on altered expression and function of transcription factors resulting in transcriptional silencing of the hRFC promoter.

Characterization by flow cytometry and fluorescein-methotrexate labeling of hydrophilic and lipophilic antifolate resistance in cultured mammalian cells.

The aim of this review is to summarize currently available information on the rapid screening and initial characterization of the different mechanisms of resistance to hydrophilic [e.g. methotrexate (MTX)] and lipophilic antifolates [e.g. trimetrexate (TMTX)] in cultured mammalian cells using fluorescein-methotrexate (F-MTX) and flow cytometry. Toward this end an integrative F-MTX labeling and flow cytometry-based protocol is proposed here to facilitate the rapid identification of modes of antifolate resistance in a heterogenous drug-resistant cell population or in clonal derivatives. Following antifolate selection, drug-resistant cells are first labeled with F-MTX in order to saturate intracellular dihydrofolate reductase (DHFR). F-MTX-labeled cells are then subjected to flow cytometric analysis and mean fluorescence/cell is determined. Thus, increased F-MTX staining is an indication of overproduction of the target enzyme for antifolates, DHFR, as a result of DHFR gene amplification. In contrast, significantly reduced cellular F-MTX labeling could be an indication of the existence of a structurally altered DHFR displaying a decreased affinity for antifolates. Alternatively, antifolate-resistant cells frequently display wild-type F-MTX labeling; these cells are subjected to competition with hydrophilic and lipophilic antifolates in order to examine whether the process of antifolate accumulation is deficient. Cells that lose F-MTX labeling upon competition with lipophilic antifolates yet still retain it with hydrophilic antifolates, are likely to possess transport alteration(s) that impair or abolish the accumulation of hydrophilic but not of lipophilic antifolates. In contrast, cells that lose their F-MTX labeling after competition with hydrophilic antifolates but retain it with lipophilic antifolates, possess a deficient accumulation of lipophilic antifolates.(ABSTRACT TRUNCATED AT 250 WORDS)