Abstract
Thymidine phosphorylase (TP) catalyzes the conversion of thymidine to thymine and 2-deoxyribose-1-phosphate. The latter plays an important role in induction of angiogenesis. As such, many human malignancies exhibit TP overexpression that correlates with increased microvessel density, formation of aggressive tumors, and dismal prognosis. Because TP is frequently overexpressed in cancer, pro-drugs were developed that utilize TP activity for their bioactivation to cytotoxic drugs. In this respect, TP is indispensable for the pharmacologic activity of the chemotherapeutic drug capecitabine, as it converts its intermediary metabolite 5'-deoxyfluorouridine to 5-fluorouracil. Thus, loss of TP function confers resistance to the prodrug capecitabine, currently used for the treatment of metastatic colorectal cancer and breast cancer. However, drug resistance phenomena may frequently emerge that compromise the pharmacologic activity of capecitabine. Deciphering the molecular mechanisms underlying resistance to TP-activated prodrugs is an important goal toward the overcoming of such drug resistance phenomena. Here, we discovered that lack of TP protein in drug-resistant tumor cells is due to unsplicing of its pre-mRNA. Advanced bioinformatics identified the family of heterogeneous nuclear ribonucleoproteins (hnRNP) H/F as candidate splicing factors potentially responsible for impaired TP splicing. Indeed, whereas parental cells lacked nuclear localization of hnRNPs H1/H2 and F, drug-resistant cells harbored marked levels of these splicing factors. Nuclear RNA immunoprecipitation experiments established a strong binding of hnRNP H1/H2 to TP pre-mRNA, hence implicating them in TP splicing. Moreover, introduction of hnRNP H2 into drug-sensitive parental cells recapitulated aberrant TP splicing and 5'-deoxyfluorouridine resistance. Thus, this is the first study identifying altered function of hnRNP H1/H2 in tumor cells as a novel determinant of aberrant TP splicing thereby resulting in acquired chemoresistance to TP-activated fluoropyrimidine anticancer drugs.
Highlights
Introduction of heterogeneous nuclear ribonucleoproteins (hnRNP) F and hnRNPH2 Inflicts Impaired Thymidine phosphorylase (TP) Splicing upon Parental Cells— hnRNPs H2 and hnRNP F were previously shown to bind similar sequences with counter-effects on pre-mRNA cleavage and polyadenylation (29 – 31), whereas hnRNP F and hnRNP H2 were involved in the splicing process of various genes [26, 32–34]. hnRNP H1/H2 were currently shown to physically bind to TP pre-mRNA, and hnRNP F does not, both were absent from parental cells
Sulfasalazine-resistant Cells Exhibit a Unique Splicing Defect of the TP Pre-mRNA—Recently, we have identified a novel mechanism of drug resistance that is based upon the complete loss of TP expression in the human acute monocyticmacrophage leukemia cell lines THP-1 and U937 subjected to a stepwise selection to the anti-rheumatic drug SSZ [13]
Because TP is up-regulated in various malignant tumors and is a target/mediator of anticancer therapy (20 –22), we undertook this study to decipher the molecular mechanism underlying the loss of TP expression in these SSZ-resistant tumor cell lines
Summary
TP has recently gained a great deal of interest as a novel enzymatic mediator of the activity of fluoropyrimidine (pro)drugs that are bioconverted by TP to their active chemotherapeutic drug within malignant tumors. In this respect, antitumor and antiangiogenic activity of 6-(2-aminoethyl)amino-5-chlorouracil, a novel small molecule inhibitor of TP in combination with the vascular endothelial growth factor Trap, has been recently demonstrated in human small cell lung cancer xenografts [12]. We have shown that exposure of the human monocytic/macrophage leukemia cell lines THP-1 and U937 to the anti-rheumatic drug sulfasalazine (SSZ) resulted in a complete down-regulation of TP both at the mRNA and protein levels [13]. We further demonstrate that this impaired splicing confers drug resistance to the fluoropyrimidine 5Ј-DFUR
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
