CANCER AND INFLAMMATION: FUNCTION AND THERAPY

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1. Analysis of MSMB transcripts and Hybrid Products 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. 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. We are currently further exploring the promoter regions of the MSMB gene, and expressing the hybrid protein to determine its role in androgen regulation. 2. 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. 3. 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. The second leading cause of cancer death for women in the U.S. is breast cancer, however, nearly 50% of patients with breast tumors acquire resistance to drugs during therapy. To develop targeted therapeutic strategies to combat drug resistance it is essential to understand the basic molecular mechanisms through which cancer cells control sensitivity to chemotherapeutics. To identify new candidate genes and facilitate the discovery of novel drug resistance pathways, we have generated a resistance profile or a resistome of MCF7 breast cancer cells resistant to etoposide. Differential expression of over 5000 genes (fold change > 2, P value < 0.05) indicate that several drug resistance mechanisms may be operating in etoposide resistant breast cancer cells, including the up-regulation of ABC transporter genes, down-regulation of the drug target gene and down-regulation of apoptotic genes. We also found evidence that genes involved in another novel mechanism of resistance called Extra Cellular Matrix (ECM) mediated drug resistance were up-regulated. Several transcription factors such as RUNX2, SOX9, ETS1 and SMAD3 were up-regulated and may be potential therapeutic targets/biomarkers of etoposide resistance. Differential miRNA (microRNA) expression was observed among the drug resistant and sensitive cells suggesting that miRNA may also play a role in regulation of drug resistance. Hsa-miR-218 was down-regulated in the drug resistant cell line. Transfection of a miR-218 mimic could down-regulate the expression of the efflux pump ABCC6 by almost 65% in drug resistant cells suggesting that miRNA mimics may be explored as a regulatory mechanism in drug resistance.