Heterogeneous Nuclear Ribonucleoprotein H1/H2-dependent Unsplicing of Thymidine Phosphorylase Results in Anticancer Drug Resistance

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.

Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing

“…Altered ARVCF Expression Leads to Changes in Pre-mRNA Splicing-All three ARVCF-interacting proteins were reported to be involved in splicing of pre-mRNA (33,36,37). Therefore, we tested whether ARVCF also influences splicing.…”

“…The other genes identified were grouped into the subcategories mRNA-processing, DNA-associated, and cytoskeleton-associated (Table 1). Two independent clones corresponded to the HNRNPH2 gene that is involved in mRNA processing (33,34). Similarly, RNA helicase p68/DDX5 has been shown to contribute to mRNA processing (35,36).…”

“…Aberrant RNA processing by alternative splicing of genes related to schizophrenia had been frequently observed in the past (69,70), and this was correlated with the pathophysiology of the disorder. In future studies, it will be clarified whether ARVCF contributes to malfunctions in alternative splicing that may lead to aberrant pre-mRNA splicing events giving rise to neurological disorders, such as schizophrenia, or other human diseases, such as myotonic dystrophy or cancer (33,61,71,72).…”

Pharmacologic resistance in colorectal cancer: a review

“…This is seen in contrast to the inverse effect of TP expression on response to 5-FU. One putative mechanism of loss of function of TP is abnormal pre-mRNA splicing by increased levels heterogeneous nuclear RNP (hnRNP) H/F splicing factors which leads to loss of function of the TYMP gene [Stark et al 2011]. Further research is ongoing in regards to exact mechanisms of development of resistance specifically to capecitabine that may be distinct from other FPs.…”

High-throughput analyses of hnRNP H1 dissects its multi-functional aspect

“…16,17 Moreover, hnRNP H1 has been associated with leukemia chemo-resistance via its impact on thymidine phosphorylase (TP) splicing. 18 In addition to these targeted analyses, genome-wide studies have revealed hnRNP H1s role, in conjunction with hnRNP F, in promoting both exon inclusion and skipping. 8 Other recent studies have linked hnRNP H1 to alternative cleavage and polyadenylation, 6 and established the cooperative roles of the hnRNP proteins in the regulation of splicing.…”