Functional Characterization of the RNF146 Gene Associated with Breast Cancer We identified the RNF146 gene as a candidate for the 6q22.33 locus associated with breast cancer. Since this locus demonstrates a higher association with estrogen receptor positive versus estrogen receptor negative tumors, we investigated the functional consequences of RNF146. We found that overexpression of RNF146 under control of a dynein light chain 1 (DLC1) promoter with a luciferase reporter in MCF-7 cells resulted in remarkably decreased luciferase activity. The DLC1 promoter contains half of an estrogen receptorbinding site. This implies that RNF146 plays a role in estrogen-mediated cellular activities. To verify this possibility, we transfected RNF146 and pCMV-3XERE-luciferase-luciferase constructs into MCF-7 cells. Similarly, in this experiment the luciferase activity was significantly inhibited. In summary, these experiments demonstrate diminished luciferase activity from the ERE reporter vector in the presence of RNF146 overexpression, suggesting that RNF146 plays a role in down-regulation of estrogen response elements. 2. Determined that the rs10993994 SNP Controls Expression of MSMB and a Hybrid Product with NCOA4, an Androgen Receptor Regulator We described the association in prostate cancer subjects of the rs10993994 SNP in the promoter of the MSMB gene. To further characterize the promoter, a 1-kb region was sequenced in 64 cancer cell lines. Only the two previously identified common SNPs in the proximal promoter region were observed, rs12770171 at -242 and rs10993994 at -57, and no new variants were observed. To elucidate whether these variants influence promoter activity, the four possible DNA fragments were cloned and promoter activities determined in 293T, PC-3, and MCF-7 cells. The transcriptional activities of the MSMB promoter fragments with C at rs10993994 are higher than those of fragments with T at rs10993994. The presence of a C residue at the SNP rs10993994 is associated with a putative CREB-binding site downstream of a GATA site and close to the TATA box. To investigate the effect of SNP rs10993994 within the proximal MSMB promoter region on CREB binding to the MSMB promoter, we performed an electrophoretic mobility shift assay (EMSA) analysis with oligonucleotide probes containing the polymorphisms. The allele C of rs10993994 increased promoter activity (PSP94-C) and thus stronger CREB binding, whereas the allele, already shown to have weak promoter activity (PSP94-T), had undetectable CREB binding. To confirm the predicted effect of the observed changes in promoter activity associated with SNP rs10993994 in the MSMB promoter, the mRNA expression levels of the MSMB gene were measured. Nineteen cancer cell lines had detectable MSMB mRNA expression, whereas the mean of MSMB mRNA expression level with rs10993994-C was significantly higher than that of rs10993994-T. By examination of the EST clones in the MSMB gene region, we identified several that represent apparent trans-splicing events between the first exons of MSMB and the adjacent gene, NCOA4. Because NCOA4 encodes a protein known to interact with and regulate the androgen receptor, these transcripts could be relevant to prostate cancer. We validated that these hybrid transcripts are present in cancer cell lines and prostate cancer tissue, and demonstrated that their abundance is elevated in cells from individuals that contain the rs10993994-C allele. We cloned and expressed several of these transcripts, and using MSMB and NCOA4 sera demonstrated that they produce a stable hybrid protein. 3. Development of Agents to Target SMO and Cancer Stem Cells The identification of a population of self-renewing cells in several solid tumor types extends the previous work in leukemias and suggests that many, or all, tumors contain a small population of cancer stem cells. The HH/PTCH pathway has been demonstrated to be mutated in virtually all basal cell carcinomas and a portion of medulloblastomas. In addition, many tumors display ligand-dependent activation of the HH/PTCH pathway including pancreas, prostate, gastrointestinal tract, and small cell lung tumors. Small molecule inhibitors of SMO, the downstream regulator of the HH/PTCH pathway, have been validated preclinically, and several agents are in clinical trials. To further the development of HH/PTCH inhibitors, we previously designed dominant negative inhibitors derived from transmembrane (TM) domains and intracellular loops of the SMO protein that are highly potent and selective. To understand the biodistribution of these peptides, we radiolabeled one of the most active derivatives and delivered it to mice by several routes. Intravenous injection of the peptide results in rapid distribution to nearly all organ sites, with the highest accumulation in the lungs. With subcutaneous and intraperitoneal injection, more than 99% of the peptide stays at the injection site. Topical application results in nearly complete retention at the application site, suggesting that the peptides could be used in topical formulations. 4. Function and Targeting of ABC Transporters Involved in Multidrug Resistance The ABCG2 gene encodes an ABC transporter protein with high normal tissue expression in the brain endothelium, gastrointestinal tract, and placenta, ABCG2 is believed to be important in the protection from xenobiotics, regulating oral bioavailability, and forming part of the blood-brain barrier, the blood-testis barrier, and the maternal-fetal barrier. ABCG2 is highly expressed in early embryonic stem cells and functions in part to protect these cells from toxins. As the inhibition of ABCG2 may have applications in both drug pharmacology and in the elimination of cancer stem cells, we previously described a screen for new ABCG2 inhibitors. Based on the activity profiles and the availability of materials, five inhibitors were examined for their ability to compete with [125I]-iodoarylazidoprazosin labeling of ABCG2, increase binding of the anti-ABCG2 antibody 5D3, and prevent P-glycoprotein or multidrug resistance protein 1-mediated transport. At a concentration of 20 micromol/L, all of the compounds reduced iodoarylazidoprazosin labeling by 50% to 80% compared with controls. All five compounds also increased 5D3 labeling of ABCG2, indicating that these compounds are inhibitors but not substrates of ABCG2. None of the compounds affected P-glycoprotein-mediated rhodamine 123 transport. However, three affected multidrug resistance protein-1-mediated calcein transport at 25 mumol/L, suggesting that the compounds mediated calcein transport at 25 mumol/L, and are therefore relatively specific for ABCG2. These five novel inhibitors of ABCG2 activity may provide a basis for further investigation of ABCG2 function and its relevance in multidrug resistance. ABCB5 is a member of the ABC protein superfamily, which includes the transporters ABCB1, ABCC1, and ABCG2, which are responsible for causing drug resistance in cancer patients. This superfamily also includes a number of other transporters that have been linked to human disease. The function of ABCB5 is presently unknown, although it is known to be expressed in melanoma stem cells, normal melanocytes, and other pigment cells. The ABCB5 full transporter (ABCB5.ts) has been found to express only in the human testis, and its functional significance is currently unknown. The ABCB5 protein has important clinical implications, as it may be involved with causing multidrug resistance in melanoma stem cells, allowing these stem cells to survive chemotherapeutic treatments.
Showing the most recent 10 out of 30 publications
