Xiaolong He

Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene.

The breast cancer resistance protein (BCRP) is an ATP-binding cassette half transporter that confers resistance to anticancer drugs such as mitoxantrone, anthracyclines, topotecan, and SN-38. Initial characterization of the BCRP promoter revealed that it is TATA-less with 5 putative Sp1 sites downstream from a putative CpG island and several AP1 sites (K. J. Bailey-Dell et al., Biochim. Biophys. Acta, 1520: 234–241, 2001). Here, we examined the sequence of the 5′-flanking region of the BCRP gene and found a putative estrogen response element (ERE). We showed that estrogen enhanced the expression of BCRP mRNA in the estrogen receptor (ER)-positive T47D:A18 cells and PA-1 cells stably expressing ERα. In BCRP promoter-luciferase assays, sequential deletions of the BCRP promoter showed that the region between −243 and −115 is essential for the ER effect. Mutation of the ERE found within this region attenuated the estrogen response, whereas deletion of the site completely abrogated the estrogen effect. Furthermore, electrophoretic mobility shift assays revealed specific binding of ERα to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.

Knockdown of polypyrimidine tract-binding protein suppresses ovarian tumor cell growth and invasiveness in vitro.

Polypyrimidine tract-binding protein (PTB) is an RNA-binding protein with multiple functions in the regulation of RNA processing and IRES-mediated translation. We report here overexpression of PTB in a majority of epithelial ovarian tumors revealed by immunoblotting and tissue microarray (TMA) staining. By western blotting, we found that PTB was overexpressed in 17 out of 19 ovarian tumor specimens compared to their matched-normal tissues. By TMA staining, we found PTB expression in 38 out of 44 ovarian cancer cases but only in two out of nine normal adjacent tissues. PTB is also overexpressed in SV40 large T-antigen immortalized ovarian epithelial cells compared to normal human ovarian epithelial cells. Using doxycycline-inducible small interfering RNA technology, we found that knockdown of PTB expression in the ovarian tumor cell line A2780 substantially impaired tumor cell proliferation, anchorage-independent growth and in vitro invasiveness. These results suggest that overexpression of PTB is an important component of the multistep process of tumorigenesis, and might be required for the development and maintenance of epithelial ovarian tumors. Moreover, because of its novel role in tumor cell growth and invasiveness, shown here for the first time, PTB may be a novel therapeutic target in the treatment of ovarian cancer.

Alternative Splicing of the Multidrug Resistance Protein 1/ATP Binding Cassette Transporter Subfamily Gene in Ovarian Cancer Creates Functional Splice Variants and Is Associated with Increased Expression of the Splicing Factors PTB and SRp20

Purpose: Overexpression of multidrug resistance protein 1 (MRP1) confers resistance to a range of chemotherapeutic agents in cell lines and could be involved in clinical drug resistance of some tumor types also. We examined MRP1 expression in a small series of untreated human ovarian tumors and matched normal tissues. Experimental Design: We analyzed ten pairs of snap-frozen ovarian tumor and matched normal total ovarian tissues from the same patients for expression of MRP1 by reverse transcription-PCR. Amplified PCR products were sequenced to reveal splicing events of MRP1. MRP1 splice variants were expressed as enhanced green fluorescent fusion proteins in HEK293T cells to demonstrate their localization in the cell and their activity in conferring resistance to doxorubicin. The expression of splicing factors PTB and SRp20 was examined by Western blot. Results: MRP1 was expressed in all 10 of the pairs of specimens. Multiple MRP1 cDNA fragments of various sizes were amplified between exons 10 and 19. Of interest, more MRP1 cDNA fragments were detected in ovarian tumors than in matched normal tissues in 9 of 10 pairs. We identified 10 splicing forms between exons 10 and 19 of the MRP1 gene with exon skipping ranging from 1 to 7. Amplification of the entire coding region of MRP1 from 1 ovarian tumor revealed >20 splice variants. We found whole and partial exon skipping and partial intron inclusion in these splice variants. We expressed 3 of these MRP1 splice variants in HEK293T cells and found that they appeared to localize to the plasma membrane and were functional in conferring resistance to doxorubicin. In addition, we identified a few nucleotide variations in this gene. To understand the basis for increased splice variants in the tumors, we examined splicing factor expression in these tissues. Western blot analysis revealed that two splicing factors, PTB and SRp20, were overexpressed in most ovarian tumors compared with their matched normal ovarian tissues. Importantly, overexpression of both of these splicing factors was associated with the increased number of MRP1 splicing forms in the ovarian tissues. Conclusion: The MRP1 gene undergoes alternative splicing at a higher frequency in ovarian tumors than in matched normal tissues. Some of these splice variants confer resistance to doxorubicin. Expression of splicing factors PTB and SRp20 is strongly associated with the alternative splicing of the MRP1 gene.

Knockdown of splicing factor SRp20 causes apoptosis in ovarian cancer cells and its expression is associated with malignancy of epithelial ovarian cancer

Our previous study revealed that two splicing factors, polypyrimidine tract-binding protein (PTB) and SRp20, were upregulated in epithelial ovarian cancer (EOC) and knockdown of PTB expression inhibited ovarian tumor cell growth and transformation properties. In this report, we show that knockdown of SRp20 expression in ovarian cancer cells also causes substantial inhibition of tumor cell growth and colony formation in soft agar and the extent of such inhibition appeared to correlate with the extent of suppression of SRp20. Massive knockdown of SRp20 expression triggered remarkable apoptosis in these cells. These results suggest that overexpression of SRp20 is required for ovarian tumor cell growth and survival. Immunohistochemical staining for PTB and SRp20 of two specialized tissue microarrays, one containing benign ovarian tumors, borderline/low malignant potential (LMP) ovarian tumors as well as invasive EOC and the other containing invasive EOC ranging from stage I to stage IV disease, reveals that PTB and SRp20 are both expressed differentially between benign tumors and invasive EOC, and between borderline/LMP tumors and invasive EOC. There were more all-negative or mixed staining cases (at least two evaluable section cores per case) in benign tumors than in invasive EOC, whereas there were more all-positive staining cases in invasive EOC than in the other two disease classifications. Among invasive EOC, the majority of cases were stained all positive for both PTB and SRp20, and there were no significant differences in average staining or frequency of positive cancer cells between any of the tumor stages. Therefore, the expression of PTB and SRp20 is associated with malignancy of ovarian tumors but not with stage of invasive EOC.

Notch1 regulates the expression of the multidrug resistance gene ABCC1/MRP1 in cultured cancer cells

Multidrug resistance (MDR) is a barrier to successful cancer chemotherapy. Although MDR is associated with overexpression of ATP-binding cassette (ABC) membrane transporters, mechanisms behind their up-regulation are not entirely understood. The cleaved form of the Notch1 protein, intracellular Notch1 (N1IC), is involved in transcriptional regulation of genes. To test whether Notch1 is involved in the expression of multidrug resistance-associated protein 1 (ABCC1/MRP1; herein referred to as ABCC1), we measured N1IC and presenilin 1 (PSEN1), the catalytic subunit of γ-secretase required for Notch activation. We observed higher levels of N1IC and PSEN1 proteins as well as higher activity of N1IC in ABCC1-expressing MDR MCF7/VP cells compared with parental MCF7/WT cells. Reducing N1IC levels in MCF7/VP cells with either a γ-secretase inhibitor or shRNA led to reduction of ABCC1. By contrast, ectopic expression of N1IC in MCF7/WT cells led to increased expression of ABCC1 and associated drug resistance, consistent with expression of this transporter. Inhibition of ABCC1 reversed drug resistance of N1IC-overexpressing stable cells. Using an ABCC1 promoter construct, we observed both its reduced transcriptional activity after blocking the generation of N1IC and its increased transcriptional activity in stable cells overexpressing N1IC. ChIP and gel-shift assays revealed an interaction between a specific promoter region of ABCC1 and the N1IC-activated transcription factor CBF1, suggesting that the regulation of ABCC1 expression by Notch1 is mediated by CBF1. Indeed, deletion or site-directed mutagenesis of these CBF1 binding sites within the ABCC1 promoter region attenuated promoter-reporter activity. Overall, our results reveal a unique regulatory mechanism of ABCC1 expression.

Novel regulation of nuclear factor-YB by miR-485-3p affects the expression of DNA topoisomerase IIα and drug responsiveness.

Nuclear factor (NF)-YB, a subunit of the transcription factor nuclear factor Y (NF-Y) complex, binds and activates CCAAT-containing promoters. Our previous work suggested that NF-YB may be a mediator of topoisomerase IIα (Top2α), working through the Top2α promoter. DNA topoisomerase II (Top2) is an essential nuclear enzyme and the primary target for several clinically important anticancer drugs. Our teniposide-resistant human lymphoblastic leukemia CEM cells (CEM/VM-1-5) express reduced Top2α protein compared with parental CEM cells. To study the regulation of Top2α during the development of drug resistance, we found that NF-YB protein expression is increased in CEM/VM-1-5 cells compared with parental CEM cells. This further suggests that increased NF-YB may be a negative regulator of Top2α in CEM/VM-1-5 cells. We asked what causes the up-regulation of NF-YB in CEM/VM-1-5 cells. We found by microRNA profiling that hsa-miR-485-3p is lower in CEM/VM-1-5 cells compared with CEM cells. MicroRNA target prediction programs revealed that the 3′-untranslated region (3′-UTR) of NF-YB harbors a putative hsa-miR-485-3p binding site. We thus hypothesized that hsa-miR-485-3p mediates drug responsiveness by decreasing NF-YB expression, which in turn negatively regulates Top2α expression. To test this, we overexpressed miR-485-3p in CEM/VM-1-5 cells and found that this led to reduced expression of NF-YB, a corresponding up-regulation of Top2α, and increased sensitivity to the Top2 inhibitors. Results in CEM cells were replicated in drug-sensitive and -resistant human rhabdomyosarcoma Rh30 cells, suggesting that our findings represent a general phenomenon. Ours is the first study to show that miR-485-3p mediates Top2α down-regulation in part by altered regulation of NF-YB.

A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing

Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre–messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.

Abstract 3201: PTBP1 stability is increased in ovarian and breast cancer cell lines compared to matched controls.

PTBP1 (polypyrimidine tract-binding) protein is a splicing factor that is frequently upregulated in ovarian and breast tumors. Knockdown (KD) of PTBP1 expression by shRNA impairs ovarian tumor cell growth in vivo and in vitro, as well as colony formation and invasiveness in vitro (He et al, Oncogene 26:4961-8, 2007). We have also found that PTBP1 is progressively upregulated in immortalized human mammary epithelial cells (HMECs) and human ovarian surface epithelial (HOSE) cells, correlating with their transformation state. These results prompted us to determine the factors that play a role in the upregulation of PTBP1 protein expression. We first asked whether the increased PTBP1 protein in human ovarian and breast cell lines is accompanied by proportional increases of PTBP1 mRNA. We found that there was a discordance between PTBP1 mRNA and protein levels, as measured by RT-PCR and western blot, in these cancer cell lines in relation to the control cells (finite lifespan HMEC or HOSE, respectively). Accordingly, we attempted to understand the basis for this discrepancy, and measured PTBP1 protein half-life. We treated A2780 (ovarian cancer), MCF7 (breast cancer) cells, and “finite lifespan” HMEC (184A1) with cycloheximide to terminate protein synthesis and followed the fate of PTBP1 protein over time by western blot. We found that the half-life of PTBP1 protein is ∼3-5 h in the HMECs with finite lifespan, whereas it ranges from ∼9-12h in human epithelial ovarian and breast cancer cells. These results demonstrate that PTBP1 expression is regulated in part by events that control the stability of the protein and suggest that the normal regulation of PTBP1 protein turnover is altered in cancer cell lines. Bioinformatics analysis identified one possible SUMO-binding motif site within the PTBP1 protein (SUMOsp 2.0 - SUMOylation Site Prediction, http://sumosp.biocuckoo.org/), suggesting that SUMOylation may play a role in PTBP1 stability; we are currently examining this possibility. Findings presented herein will enhance our understanding of the regulation of PTBP1 expression and the mechanisms underlying its variation in protein and mRNA levels in ovarian and breast tumors. (Support in part by NCI grants R01 CA40570 and R01 CA138762 [to WTB], by OCRF [to XH], and by UIC.) Citation Format: Ahmet Dirim Arslan, Szilard Asztalos, Martha Stampfer, Debra Tonetti, Xiaolong He, William T. Beck. PTBP1 stability is increased in ovarian and breast cancer cell lines compared to matched controls. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3201. doi:10.1158/1538-7445.AM2013-3201

Abstract 796: Novel mechanisms of drug resistance in leukemia cells associated with changes in HoxA9 and microRNA expression

The acquisition of resistance to anticancer drugs is a key obstacle to successful cancer therapy. An increasing number of studies have investigated the roles of microRNAs in drug resistance. We previously reported that the upregulation of miR-135b and miR-196b correlated positively with acquired drug resistance in the human T-cell leukemia cell line, CCRF-CEM (CEM), and that the elevation of expression of these two microRNAs in response to the DNA damaging agent etoposide appears to be histiotype-specific (T-T Ho et al, Proceedings of the 102nd Annual Meeting of the AACR 2011). To develop a mechanistic understanding of why these microRNAs are differentially expressed, we have studied miR-196b, which maps between the HoxA9 and HoxA10 genes on chromosome 7p15.2, suggesting that miR-196b and HoxA genes might be co-activated. Indeed, we found upregulation of HoxA9 mRNA after short-term (48 h) exposure of CEM cells to the topoisomerase II inhibitors etoposide and doxorubicin. Of note, we also found that HoxA9 is constitutively overexpressed in multidrug-resistant CEM/VM-1-5 cells. Of interest, we found that HoxA9 is clearly associated with drug resistance, as knockdown of HoxA9 using esiRNA (a heterogeneous mixture of siRNAs that all target the same mRNA) partially sensitized multidrug-resistant CEM/VM-1-5 cells to etoposide. This indicates that the expression of miR-196b is linked to HoxA gene transcription and to drug responsiveness. We subsequently confirmed that the expression of HoxA9 mRNA follows the same pattern in other leukemia cells. For example, both miR-196b and HoxA9 are upregulated in response to short-term etoposide exposure in HL-60 (acute promyelocytic leukemia), but not in solid tumor-derived MCF7 (breast cancer cell line) and its etoposide-resistant subline MCF7/VP, or Rh30 (rhabdomyosarcoma cell line) and its etoposide-resistant subline, Rh30/V1. We are presently assessing the role of HoxA9 in drug resistance in other leukemia cell lines and investigating the mechanistic basis for this effect. In summary, we report here a novel mechanism of anticancer drug resistance in leukemia cells that is associated with HoxA9 and specific changes in microRNA expression. This is the first report that HoxA9 may impact on anticancer drug resistance. (Supported in part by R01 CA40570 from NCI to WTB, and in part by UIC.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 796. doi:1538-7445.AM2012-796

Abstract A180: Development of a high-throughput screening (HTS) assay to detect splicing inhibitors.

Background and Hypothesis: Alternative splicing (AS) of RNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract binding protein (PTB) is overexpressed in ovarian tumors (He et al, Clin Cancer Res, 10:4652–60, 2004), compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness (He et al, Oncogene 26:4961–8, 2007). PTB is a widely expressed RNA binding protein whose main molecular function is regulating alternative splicing. Because KD of PTB with shRNA impairs ovarian cancer cell growth and other malignant properties, we wished to identify small molecule inhibitors of PTB that will have the same effect as the shRNA. We hypothesized that PTB is a drugable therapeutic target and that its activity in live cells can be monitored by measuring the splicing of a PTB target gene. Accordingly, we have developed a cell-based fluorescent reporter assay for monitoring RNA splicing by using a GFP minigene. This assay allows HTS of small molecule libraries to identify splicing modulator agents that inhibit PTB. Materials and Methods: In this pilot study, we screened 89 NCI-approved oncology drugs (http://.goo.gl/Lf1Jl) in engineered A2780 epithelial ovarian tumor sublines that carried the splicing reporter minigene construct and/or an inducible shRNA against PTB. Three different sublines were used for this assay: (i) a positive control that carries the splicing reporter construct and an inducible shRNA against PTB; (ii) a negative control that carries the splicing reporter construct only; and (iii) a blank control that consist of parental cells only. For the assay, all cells were seeded in 96-well plates (black, clear bottom) at 1.5×106 cells/mL. After 24 h, compounds were added using a PerkinElmer Janus workstation (http://goo.gl/zbK71), and plates were incubated with compounds (final concentration, 1μM) for 48 h at 37C, 5% CO2. Plates were washed inside the sterile workstation with 1× PBS solution before measuring activity. Fluorescence measurements were carried out on a PerkinElmer EnVision plate reader (http://goo.gl/ZxDN8), using filters at 485-nm for excitation and 520-nm for emission. The activation percent was assessed by quantifying fluorescence intensity changes in the compound-treated cells in relation to the positive and negative control cells. Results: We found that our cell-based splicing reporter assay can be use to detect PTB activity in live cells. Moreover, because compounds in this library are intrinsically cytotoxic, 18% (16 of 89) showed toxicity in the A2780 cells. Of importance, this assay was able to detect cytotoxic compounds. These compounds were separated for further investigation to test them at lower concentrations, and to determine whether any of them affect splicing in the reporter assay. Conclusions: This study describes the preliminary assessment of a novel HTS approach to identify small molecule inhibitors of the splicing factor PTB. This cell-based HTS allows large numbers of agents to be tested against engineered cell lines to identify small molecule inhibitors of PTB with the goal of translating positive hits to the clinic. Supported in part by grants RO1 CA40570 and RO1 CA138762 (to WTB), by OCRF (to XH), and by UIC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A180.