Role of proton-coupled folate transporter in pemetrexed resistance of mesothelioma: clinical evidence and new pharmacological tools

Abstract

Background: Thymidylate synthase (TS) has a predictive role in pemetrexed treatment of mesothelioma; however, additional chemoresistance mechanisms are poorly understood. Here, we explored the role of the reduced-folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) in antifolate resistance in mesothelioma.

Patients and methods: PCFT, RFC and TS RNA and PCFT protein levels were determined by quantitative RT-PCR of frozen tissues and immunohistochemistry of tissue-microarrays, respectively, in two cohorts of pemetrexed-treated patients. Data were analyzed by t-test, Fisher's/log-rank test and Cox proportional models. The contribution of PCFT expression and PCFT-promoter methylation to pemetrexed activity were evaluated in mesothelioma cells and spheroids, through 5-aza-2'-deoxycytidine-mediated demethylation and siRNA-knockdown.

Results: Pemetrexed-treated patients with low PCFT had significantly lower rates of disease control, and shorter overall survival (OS), in both the test (N = 73, 11.3 versus 20.1 months, P = 0.01) and validation (N = 51, 12.6 versus 30.3 months, P = 0.02) cohorts. Multivariate analysis confirmed PCFT-independent prognostic role. Low-PCFT protein levels were also associated with shorter OS. Patients with both low-PCFT and high-TS levels had the worst prognosis (OS, 5.5 months), whereas associations were neither found for RFC nor in pemetrexed-untreated patients. PCFT silencing reduced pemetrexed sensitivity, whereas 5-aza-2'-deoxycytidine overcame resistance.

Conclusions: These findings identify for the first time PCFT as a novel mesothelioma prognostic biomarker, prompting prospective trials for its validation. Moreover, preclinical data suggest that targeting PCFT-promoter methylation might eradicate pemetrexed-resistant cells characterized by low-PCFT expression.

Keywords: PCFT; expression; malignant pleural mesothelioma; methylation; outcome; pemetrexed.

Figure 1.

Correlation of PCFT mRNA expression with clinical outcome. (A and B) Overall survival (OS) and progression-free survival (PFS) curves segregated according to PCFT mRNA expression in the MPM patients from the test cohort. High/low PCFT levels are relative to the median values acquired by RT-PCR data available from 69 patients; (C and D) OS and PFS curves according to RT-PCR data of PCFT mRNA expression in the MPM patients from the validation cohort (mRNA expression data were available from 51 patients); (E) OS curves subgrouped according TS mRNA expression levels in the MPM patients from the validation cohort. High/low levels are relative to the median values acquired by RT-PCR data available from 51 patients; (F) OS curves according to RT-PCR data of both TS and PCFT mRNA levels in the MPM patients from both the test and the validation cohorts (mRNA expression data were available for 120 patients). The curves were compared using the log-rank test.

Figure 2.

Correlation of PCFT protein expression with clinical outcome. (A) Representative pictures of the tissue-microrrays (TMAs) including cores obtained from paraffin-embedded tumor material (from four different tumor areas from each of the 35 MPM patients of the validation cohort) incubated with an anti-PCFT specific antibody, as described in the methods; (upper panels) TMA slide (original magnification 1× magnification) and single spots of negative and positive controls (original magnification 20×); (Lower panels) Example of weak, intermediate and strong PCFT staining (original magnification 20×). Staining results were evaluated using a four-tier system (supplementary Figure S1A, available at Annals of Oncology online). (B and C) Overall survival (OS) and progression-free survival (PFS) curves segregated according to PCFT protein expression levels in the MPM patients from the validation cohort.

Figure 3.

PCFT promoter methylation and PCFT expression affect pemetrexed cytotoxicity. (A) DNA methylation of the PCFT promoter involves a 414-bp-long fragment (termed fragment-1) spanning nucleotide positions −309/+104. Fragment-1 was subdivided into fragment-1A and fragment-1B for bisulfite-based DNA sequencing in H28 and MSTO-211H MPM cells. Each column represents a different clone, whereas each row represents a different CpG site. (B) Quantitative RT-PCR analysis of PCFT mRNA expression in MPM cell lines. Results are presented relative to the expression levels of PCFT in CCRF-CEM cells, assigned a value of 1. Columns, mean values obtained from three independent experiments; bars, SEM. (C) PCFT silencing successfully reduced PCFT mRNA levels in the three MPM cell lines. Conversely, exposure to the demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR, 1 μM) increased the mRNA expression of PCFT. *Significantly different (P < 0.05) from untreated cells (cells treated with the siRNA negative control had similar results, data not shown). (D) Representative growth inhibition curves of MPM cells treated with pemetrexed (PMX, 0.001–50 μM, 72-h exposure), with and without transfection with a specific anti-PCFT siRNA; Points, mean values obtained from three independent experiments; bars, SEM. (E) (Upper panels) Representative images of trypan blue-stained H2452 cells following treatment with PMX after exposure to 5-aza-CdR; (Lower panel). Cell growth of cells treated with PMX (at the cell line specific IC50) after exposure to 5-aza-CdR (1 μM). Cell growth of treated cells was compared to growth of untreated control cells set at 100%. *Significantly different (P < 0.05) from cells treated with PMX alone.