Impact of Complex Apoptotic Signaling Pathways on Cancer Cell Sensitivity to Therapy.

Anticancer drugs induce apoptotic and non-apoptotic cell death in various cancer types. The signaling pathways for anticancer drug-induced apoptotic cell death have been shown to differ between drug-sensitive and drug-resistant cells. In atypical multidrug-resistant leukemia cells, the c-Jun/activator protein 1 (AP-1)/p53 signaling pathway leading to apoptotic death is altered. Cancer cells treated with anticancer drugs undergo c-Jun/AP-1-mediated apoptotic death and are involved in c-Jun N-terminal kinase activation and growth arrest- and DNA damage-inducible gene 153 (Gadd153)/CCAAT/enhancer-binding protein homologous protein pathway induction, regardless of the p53 genotype. Gadd153 induction is associated with mitochondrial membrane permeabilization after anticancer drug treatment and involves a coupled endoplasmic reticulum stress response. The induction of apoptosis by anticancer drugs is mediated by the intrinsic pathway (cytochrome c, Cyt c) and subsequent activation of the caspase cascade via proapoptotic genes (e.g., Bax and Bcl-xS) and their interactions. Anticancer drug-induced apoptosis involves caspase-dependent and caspase-independent pathways and occurs via intrinsic and extrinsic pathways. The targeting of antiapoptotic genes such as Bcl-2 enhances anticancer drug efficacy. The modulation of apoptotic signaling by Bcl-xS transduction increases the sensitivity of multidrug resistance-related protein-overexpressing epidermoid carcinoma cells to anticancer drugs. The significance of autophagy in cancer therapy remains to be elucidated. In this review, we summarize current knowledge of cancer cell death-related signaling pathways and their alterations during anticancer drug treatment and discuss potential strategies to enhance treatment efficacy.

Transient resistance to DNA damaging agents is associated with expression of microRNAs-135b and -196b in human leukemia cell lines.

The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance.

Concordance of deregulated mechanisms unveiled in underpowered experiments: PTBP1 knockdown case study.

BACKGROUND: Genome-wide transcriptome profiling generated by microarray and RNA-Seq often provides deregulated genes or pathways applicable only to larger cohort. On the other hand, individualized interpretation of transcriptomes is increasely pursued to improve diagnosis, prognosis, and patient treatment processes. Yet, robust and accurate methods based on a single paired-sample remain an unmet challenge. METHODS: "N-of-1-pathways" translates gene expression data profiles into mechanism-level profiles on single pairs of samples (one p-value per geneset). It relies on three principles: i) statistical universe is a single paired sample, which serves as its own control; ii) statistics can be derived from multiple gene expression measures that share common biological mechanisms assimilated to genesets; iii) semantic similarity metric takes into account inter-mechanisms' relationships to better assess commonality and differences, within and cross study-samples (e.g. patients, cell-lines, tissues, etc.), which helps the interpretation of the underpinning biology. RESULTS: In the context of underpowered experiments, N-of-1-pathways predictions perform better or comparable to those of GSEA and Differentially Expressed Genes enrichment (DEG enrichment), within-and cross-datasets. N-of-1-pathways uncovered concordant PTBP1-dependent mechanisms across datasets (Odds-Ratios≥13, p-values≤1 × 10-5), such as RNA splicing and cell cycle. In addition, it unveils tissue-specific mechanisms of alternatively transcribed PTBP1-dependent genesets. Furthermore, we demonstrate that GSEA and DEG Enrichment preclude accurate analysis on single paired samples. CONCLUSIONS: N-of-1-pathways enables robust and biologically relevant mechanism-level classifiers with small cohorts and one single paired samples that surpasses conventional methods. Further, it identifies unique sample/ patient mechanisms, a requirement for precision medicine.

Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1.

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5' untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model.

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.

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 (N1(IC)), 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 N1(IC) and presenilin 1 (PSEN1), the catalytic subunit of γ-secretase required for Notch activation. We observed higher levels of N1(IC) and PSEN1 proteins as well as higher activity of N1(IC) in ABCC1-expressing MDR MCF7/VP cells compared with parental MCF7/WT cells. Reducing N1(IC) levels in MCF7/VP cells with either a γ-secretase inhibitor or shRNA led to reduction of ABCC1. By contrast, ectopic expression of N1(IC) 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 N1(IC)-overexpressing stable cells. Using an ABCC1 promoter construct, we observed both its reduced transcriptional activity after blocking the generation of N1(IC) and its increased transcriptional activity in stable cells overexpressing N1(IC). ChIP and gel-shift assays revealed an interaction between a specific promoter region of ABCC1 and the N1(IC)-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.

Identification and characterization of the major alternative promoter regulating Bcrp1/Abcg2 expression in the mouse intestine.

Mouse models are often used to predict drug absorption in humans. Mouse Bcrp1 protein exhibits sequence and functional homology with human BCRP protein. Additionally, BCRP/Bcrp1 expression is regulated by alternative promoter usage in humans and mice; however, the precise intestine-specific alternative promoter utilized in either species is yet to be determined. Therefore we sought to identify and characterize the mouse intestinal Bcrp1 promoter. Using real-time quantitative RT-PCR and 5' RACE PCR we first established the predominance of a single Bcrp1 first exon (E1b) in the Bcrp1 mRNA isolated throughout the mouse intestine. Simultaneously using 5' RACE PCR we identified E1C as the predominant BCRP 5' UTR expressed in the human intestine. Next we established functional activity for the murine promoter upstream of E1b using reporter assays. Subsequently using deletion-construct analysis we found the core promoter region to span -231 to -42bps from the transcriptional start site of E1b. We then predicted a cAMP response element (CRE) as a transcription factor binding site unique only to the E1b promoter region, using in silico methods. We finally established functional interaction of phospho-CREB (p-CREB) protein with the CRE on the E1b promoter using both functional assays and chromatin immunoprecipitation assays. In conclusion, mouse intestinal Bcrp1 expression is regulated by a single alternative promoter upstream of E1b, the predominant Bcrp1 mRNA isoform expressed in the mouse intestine. Furthermore, Bcrp1 E1b mRNA expression is regulated by binding of p-CREB to its cis site on the mouse E1b promoter region.

Structure-activity relationship and molecular mechanisms of ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) and its analogues.

Multidrug resistance (MDR) in cancer is a phenomenon in which administration of a single chemotherapeutic agent causes cross-resistance of cancer cells to a variety of therapies even with different mechanisms of action. Development of MDR against standard therapies is a major challenge in the treatment of cancer. Previously we have demonstrated a unique ability of CXL017 (5) to selectively target MDR cancer cells and synergize with mitoxantrone (MX) in HL60/MX2 MDR cells. Here we expand its scope and demonstrate that 5 can synergize with both vincristine and paclitaxel in three different MDR cell lines (HL60/DNR, K562/HHT300, and CCRF-CEM/VLB100). We also demonstrate that 5 has potent cytotoxicity in the NCI-60 panel of cell lines with an average IC(50) of 1.04 µM. In addition, 5 has a unique mechanism of action in comparison with standard agents in the NCI database based on COMPARE analysis. Further structure-activity relationship study led to the development of a more potent analogue, compound 7d, with an IC(50) of 640 nM in HL60/MX2. Additionally, one enantiomer of 5 is 13-fold more active than the less active enantiomer. Taken together, our study has led to the discovery of a series of analogues that selectively target drug-resistant cancer cells with the potential for the treatment of drug-resistant cancers.

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.

Dynamic assessment of mitoxantrone resistance and modulation of multidrug resistance by valspodar (PSC833) in multidrug resistance human cancer cells.

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, is one of the major causes for multidrug resistance (MDR). We report using confocal microscopy to study the roles of Pgp in mediating the efflux of the anticancer agent mitoxantrone and the reversal of MDR by the specific Pgp inhibitor valspodar (PSC833). The net uptake and efflux of mitoxantrone and the effect of PSC833 were quantified and compared in Pgp-expressing human cancer MDA-MB-435 (MDR) cells and in parental wild-type cells. The MDR cells, transduced with the human Pgp-encoding gene MDR1 construct, were approximately 8-fold more resistant to mitoxantrone than the wild-type cells. Mitoxantrone accumulation in the MDR cells was 3-fold lower than that in the wild-type cells. The net uptake of mitoxantrone in the nuclei and cytoplasm of MDR cells was only 58 and 67% of that in the same intracellular compartment of the wild-type cells. Pretreatment with PSC833 increased the accumulation of mitoxantrone in the MDR cells to 85% of that in the wild-type cells. In living animals, the accumulation of mitoxantrone in MDA-MB-435mdr xenograft tumors was 61% of that in the wild-type tumors. Administration of PSC833 to animals before mitoxantrone treatment increased the accumulation of mitoxantrone in the MDR tumors to 94% of that in the wild-type tumors. These studies have added direct in vitro and in vivo visual information on how Pgp processes anticancer compounds and how Pgp inhibitors modulate MDR in resistant cancer cells.

MicroRNA-mediated regulation of Ubc9 expression in cancer cells.

PURPOSE: As an E2-conjugating enzyme for sumoylation, Ubc9 plays a critical role in sumoylation-mediated cellular pathways, ultimately impacting cell growth and cancer development. The aim of this study was to investigate the regulation of Ubc9 in cancer cells. EXPERIMENTAL DESIGN: Immunohistochemistry and Western blot were used to determine Ubc9 expression in paraffin-embedded tumor tissue and frozen specimens of the matched tumors from the same patient, respectively. To establish the causal relationship between miR-30e and Ubc9 expression, we overexpressed miR-30e and then determined the resultant effects on Ubc9 expression. To determine whether miR-30e directly targets Ubc9, we did luciferase assays using luciferase reporters carrying the 3'-untranslated region (3'-UTR) of the Ubc9 gene. RESULTS: We found that Ubc9 is up-regulated in breast, head and neck, and lung cancer specimens. In addition, an examination of eight pairs of matched breast tumor specimens by Western blot analysis revealed that, on average, the level of Ubc9 is 5.7-fold higher in tumor than in the matched normal breast tissue. Of interest, we present evidence that Ubc9 is subjected to posttranscriptional regulation by microRNA, and the miR-30 family, such as miR-30e, negatively regulates Ubc9 expression. In contrast to Ubc9, miR-30e is underexpressed in tumors. Moreover, ectopic expression of miR-30e suppresses cell growth, which can be partially reversed by Ubc9. Finally, using luciferase-Ubc9-3'-UTR reporters, we show that Ubc9 is a direct target for miR-30e by interactions with the putative miR-30e binding sites. CONCLUSION: These results provide new insight into regulation of Ubc9 in cancer cells.

Quantitation of doxorubicin uptake, efflux, and modulation of multidrug resistance (MDR) in MDR human cancer cells.

P-glycoprotein (Pgp), a membrane transporter encoded by the MDR1 gene in human cells, mediates drug efflux from cells, and it plays a major role in causing multidrug resistance (MDR). Confocal microscopy was used to study in vitro and in vivo drug accumulation, net uptake and efflux, and MDR modulation by P-glycoprotein inhibitors in MDR1-transduced human MDA-MB-435mdr (MDR) cancer cells. The MDR cells were approximately 9-fold more resistant to the anticancer drug doxorubicin than their parental wild-type MDA-MB-435wt (WT) cells. Doxorubicin accumulation in the MDR cells was only 19% of that in the WT cells. The net uptake of doxorubicin in the nuclei of the MDR cells was 2-fold lower than that in the nuclei of the WT cells. Pgp inhibitors verapamil, cyclosporine A, or PSC833 increased doxorubicin accumulation in the MDR cells up to 79%, and it reversed drug resistance in these cells. In living animals, doxorubicin accumulation in MDA-MB-435mdr xenograft tumors was 68% of that in the wild-type tumors. Administration of verapamil, cyclosporine A, or PSC833 before doxorubicin treatment of the animals increased doxorubicin accumulation in the MDR tumors up to 94%. These studies have added direct in vitro and in vivo information on the capacity of the transporter protein Pgp to efflux doxorubicin and on the reversal of MDR by Pgp inhibitors in resistant cancer cells.

Gam1-associated alterations of drug responsiveness through activation of apoptosis.

An early gene product, Gam1, encoded by the avian adenovirus CELO, is an inhibitory protein for the sumoylation machinery, which has been implicated in regulating a variety of cellular pathways. In this study, we found that Gam1 effectively suppressed both constitutive and inducible sumoylation and caused significant cell growth inhibition. This Gam1-mediated cell growth inhibition was associated with induction of apoptosis. In particular, Gam1 induced caspase-3 activity as detected by immunostaining and Western blot. Of interest, like the Ubc9 dominant-negative mutant, Gam1 also sensitized cells to DNA-damaging agents such as topotecan and doxorubicin and non-DNA-damaging agents such as paclitaxel and vincristine. Taken together, our findings suggest that activation of the caspase pathways is at least in part responsible for the increased apoptosis in Gam1-expressing cells and, thus, contributes to the growth inhibition and enhanced chemosensitivity.

Topoisomerase II binds importin alpha isoforms and exportin/CRM1 but does not shuttle between the nucleus and cytoplasm in proliferating cells.

Resistance to anticancer drugs that target DNA topoisomerase II (topo II) isoforms alpha and/or beta is associated with decreased nuclear and increased cytoplasmic topo IIalpha. Earlier studies have confirmed that functional nuclear localization and export signal sequences (NLS and NES) are present in both isoforms. In this study, we show that topo II alpha and beta bind and are imported into the nucleus by importin alpha1, alpha3, and alpha5 in conjunction with importin beta. Topo IIalpha also binds exportin/CRM1 in vitro. However, wild-type topo IIalpha has only been observed in the cytoplasm of cells that are entering plateau phase growth. This suggests that topo IIalpha may shuttle between the nucleus and the cytoplasm with the equilibrium towards the nucleus in proliferating cells but towards the cytoplasm in plateau phase cells. The CRM1 inhibitor Leptomycin B increases the nuclear localization of GFP-tagged topo IIalpha with a mutant NLS, suggesting that its export is being inhibited. However, homokaryon shuttling experiments indicate that fluorescence-tagged wild-type topo II alpha and beta proteins do not shuttle in proliferating Cos-1 or HeLa cells. We conclude that topo II alpha and beta nuclear export is inhibited in proliferating cells so that these proteins do not shuttle.

Identification of estrogen-responsive genes using a genome-wide analysis of promoter elements for transcription factor binding sites.

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

A role for Ubc9 in tumorigenesis.

The post-translational modifications ubiquitination and sumoylation have been implicated in regulating many critical cellular pathways. Like ubiquitination, sumoylation is a multistep process involving maturation, activation, conjugation and deconjugation. Ubc9 is a sole E2-conjugating enzyme essential for sumoylation. We have previously shown that alterations of Ubc9 expression affect tumor drug responsiveness. However, it is not clear whether there is any link between sumoylation and tumorigenesis, even though alterations of the ubiquitination pathway can lead to the development of cancer. In this study, we found that Ubc9 expression levels were elevated in ovarian tumors compared to the matched normal ovarian specimens, suggesting that Ubc9 may play a role in tumorigenesis. To test this, we overexpressed a dominant-negative mutant of Ubc9 (Ubc9-DN) and wild-type Ubc9 (Ubc9-WT) in the MCF-7 human breast tumor cells. Inoculating these cells as xenografts in mice revealed that tumors expressing Ubc9-WT grew better than the vector control, while tumors expressing Ubc9-DN exhibited reduced growth. This pattern was also seen in these cells when grown in culture. To better understand the mechanism behind this observation, we profiled gene expressions in these cells by microarray analysis and found alterations in expression of the pro-oncogene bcl-2 in these Ubc9-DN- and Ubc9-WT-expressing cells. Consistent with the bcl-2 results, subsequent studies revealed a higher rate of apoptosis and poor survival for the MCF-7 cells expressing Ubc9-DN, which are associated with downregulation of bcl-2. Together, these results suggest a role for Ubc9 in tumorigenesis at least partially through regulation of bcl-2 expression.

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.

Overexpression of a dominant-negative mutant Ubc9 is associated with increased sensitivity to anticancer drugs.

Ubc9 is an E2-conjugating enzyme required for sumoylation and has been implicated in regulating several critical cellular pathways. We have shown previously that Ubc9 is important for sumoylation and nucleolar delocalization of topoisomerase (topo) I in response to topo I inhibitors such as topotecan. However, the role for Ubc9 in tumor drug responsiveness is not clear. In this study, we found that although MCF7 cells expressing a Ubc9 dominant-negative mutant (Ubc9-DN) display decreased activity of topo I, these cells are more sensitive to the topo I inhibitor topotecan and other anticancer agents such as VM-26 and cisplatin. In addition, we found that alteration of Ubc9 expression correlates with drug responsiveness in tumor cell lines. To understand possible mechanisms of Ubc9-associated drug responsiveness, we examined several proteins that have been shown to interact with Ubc9 and that may be involved in drug responsiveness. One such protein is Daxx, which is a Fas-associated protein that plays a role in Fas-mediated apoptosis by participating in a caspase-independent pathway through activation of apoptosis signal-regulating kinase 1 and c-Jun NH(2)-terminal kinase. We found that cells expressing Ubc9-DN accumulate more cytoplasmic Daxx than the control cells. Because cytoplasmic Daxx is believed to participate in cellular apoptosis, we suggest that the interaction of Ubc9 with Daxx and subsequent alteration in the subcellular localization of Daxx may contribute to the increased sensitivity to anticancer drugs in the cells expressing Ubc9-DN. Finally, we found that overexpression of Daxx sensitizes cells to anticancer drugs possibly in part through alterations of the ratio of cytoplasmic and nuclear Daxx. Together, our results suggest a role for Ubc9 in tumor drug responsiveness.

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 ERalpha. 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 ERalpha to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.