Polypyrimidine Tract-binding Protein (PTB) Differentially Affects Malignancy in a Cell Line-dependent Manner

Abstract

RNA processing is altered during malignant transformation, and expression of the polypyrimidine tract-binding protein (PTB) is often increased in cancer cells. Although some data support that PTB promotes cancer, the functional contribution of PTB to the malignant phenotype remains to be clarified. Here we report that although PTB levels are generally increased in cancer cell lines from multiple origins and in endometrial adenocarcinoma tumors, there appears to be no correlation between PTB levels and disease severity or metastatic capacity. The three isoforms of PTB increase heterogeneously among different tumor cells. PTB knockdown in transformed cells by small interfering RNA decreases cellular growth in monolayer culture and to a greater extent in semi-solid media without inducing apoptosis. Down-regulation of PTB expression in a normal cell line reduces proliferation even more significantly. Reduction of PTB inhibits the invasive behavior of two cancer cell lines in Matrigel invasion assays but enhances the invasive behavior of another. At the molecular level, PTB in various cell lines differentially affects the alternative splicing pattern of the same substrates, such as caspase 2. Furthermore, overexpression of PTB does not enhance proliferation, anchorage-independent growth, or invasion in immortalized or normal cells. These data demonstrate that PTB is not oncogenic and can either promote or antagonize a malignant trait dependent upon the specific intra-cellular environment.The polypyrimidine tract-binding protein (PTB), 3 also termed heterogeneous nuclear ribonucleoprotein I, is a 57-kDa RNA-binding protein that binds preferentially to pyrimidinerich sequences (1-3). PTB contains four RNA recognition motifs (RRMs). RRM 1 and 2 at the N terminus of the protein are involved in the dimerization of PTB, whereas RRM 3 and 4 are responsible for high affinity interactions with RNA (4, 5). PTB has been shown to be involved in many aspects of pre-mRNA and mRNA metabolism. PTB participates in pre-mRNA splicing (6) and acts as a splicing repressor in alternative splicing of pre-mRNA (5, 7-12). PTB is also involved in 3Ј end polyadenylation of pre-mRNA (13-15) and is important for translational regulation of certain RNA transcripts through internal ribosome entry sites (16 -20). In addition, PTB shuttles between the nucleus and the cytoplasm (21), which is regulated through phosphorylation by 3Ј,5Ј-cAMP-dependent protein kinase (22).Alternative splicing is a process that allows multiple different proteins to be made from the same pre-mRNA by either including or excluding particular exons during pre-mRNA splicing. PTB plays a key role in alternative site selection for many gene products by acting as a splicing repressor that prevents the inclusion of target exons (11,(23)(24)(25). Changes in alternative splicing sites have been previously correlated with malignant transformation (26 -29), and the expression level of PTB has been found elevated in transformed cells. Such an elevation is responsible for the increases...

Upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased interactions with and expression of PTB-1 and YB-1

“…Similarly, PTB-1 overexpression has been implicated in tumourigenesis; it is overexpressed in certain cancers (He et al, 2007;Wang et al, 2008) and a reduction in PTB-1 expression in ovarian cancers reduces cell proliferation (He et al, 2007). The data form our study show that a reduction in PTB-1 expression alone in cell lines derived from patients with MM had a dramatic effect on cell proliferation although, interestingly, there was little effect on the control B cells used.…”

Splicing-factor alterations in cancers

“…Furthermore, PTB KD decreases breast and ovarian tumor cell proliferation, anchorage-independent growth, and invasiveness in vitro (He et al 2007(He et al , 2014, and is accompanied by an increased ratio of PKM1 versus PKM2 isoform and increased oxygen consumption (He et al 2014). PTB overexpression also enhances anchorage-dependent growth of immortalized human mammary epithelial cells (HMECs) (He et al 2014); however, overexpression of PTB alone is not sufficient to transform murine fibroblasts (Wang et al 2008). Together, these results suggest that PTB plays a role in breast tumorigenesis.…”

“…PTB controls alternative splicing of target genes, such as pyruvate kinase PKM2 (Clower et al 2010), fibroblast growth factor receptor-1α-exon FGFR-1 (Jin et al 2000), and multidrug resistance protein 1 (MRP1), which contributes to the drug-resistance phenotype associated with many cancers (He et al 2004). PTB levels are elevated in cancer cell lines, as well as endometrial tumor tissues, compared to normal tissues, although no correlation was observed between PTB expression and tumor grading (Wang et al 2008). PTB is also overexpressed in most ovarian tumors, compared with matched normal tissue, and its overexpression is associated with an increased number of expressed MRP1 spliced isoforms (He et al 2004(He et al , 2007.…”

Identification of Cardiac Glycoside Molecules as Inhibitors of c-Myc IRES-Mediated Translation

“…However, one cannot exclude this might be a specific cell type effect. 24 Cardiac glycosides have been shown previously to be potent inhibitors, at the level of translation, of the expression of the tumor suppressor p53 as well as the hypoxiainducible transcription factor 1 (HIF-1) in hypoxic tumor cells. 25,26 It was shown that digoxin very potently inhibits HIF-1 mRNA translation and affects mouse xenograft tumor growth associated with modest effects on global protein synthesis.…”