CANCER AND INFLAMMATION: FUNCTION AND THERAPY

1. MSMB转录物和具有雄激素受体调节剂NCOA 4的杂合产物的分析我们描述了前列腺癌受试者中MSMB基因启动子中rs 10993994 SNP的相关性。SNP rs 10993994处C残基的存在与加塔位点下游且靠近TATA盒的推定CREB结合位点相关。为了研究近端MSMB启动子区域内SNP rs 10993994对CREB与MSMB启动子结合的影响，我们使用含有多态性的寡核苷酸探针进行了电泳迁移率变动分析（EMSA）。rs 10993994的等位基因C增加了启动子活性（PSP 94-C），从而增强了CREB结合，而已经显示具有弱启动子活性（PSP 94-T）的等位基因具有不可检测的CREB结合。为了证实所观察到的与MSMB启动子中SNP rs 10993994相关的启动子活性变化的预测效果，测量了MSMB基因的mRNA表达水平。19个癌细胞系具有可检测的MSMB mRNA表达，而rs 10993994-C的MSMB mRNA表达水平的平均值显著高于rs 10993994-T。通过检查MSMB基因区域的EST克隆，我们确定了几个代表MSMB第一外显子和相邻基因NCOA 4之间明显的反式剪接事件。由于NCOA 4编码一种已知与雄激素受体相互作用并调节雄激素受体的蛋白质，因此这些转录物可能与前列腺癌相关。我们验证了这些杂合转录本存在于癌细胞系和前列腺癌组织中，并证明它们在含有rs 10993994-C等位基因的个体的细胞中丰度升高。我们克隆并表达了这些转录本中的几种，并使用MSMB和NCOA 4血清证明它们产生稳定的杂合蛋白。我们目前正在进一步探索MSMB基因的启动子区域，并表达杂合蛋白以确定其在雄激素调节中的作用。2.靶向SMO和癌症干细胞的药物开发在几种实体瘤类型中鉴定出一群自我更新细胞扩展了先前在白血病中的工作，并表明许多或所有肿瘤都含有少量癌症干细胞。已证明HH/PTCH途径在几乎所有基底细胞癌和部分髓母细胞瘤中均发生突变。此外，许多肿瘤显示HH/PTCH通路的配体依赖性激活，包括胰腺、前列腺、胃肠道和小细胞肺肿瘤。SMO（HH/PTCH通路的下游调节剂）的小分子抑制剂已在临床前得到验证，几种药物正在进行临床试验。为了进一步开发HH/PTCH抑制剂，我们先前设计了源自SMO蛋白的跨膜（TM）结构域和细胞内环的显性负性抑制剂，其是高度有效和选择性的。为了了解这些肽的生物分布，我们放射性标记了最活跃的衍生物之一，并通过几种途径将其递送给小鼠。静脉注射该肽导致快速分布到几乎所有器官部位，在肺中积累最多。通过皮下和腹膜内注射，超过99%的肽停留在注射部位。局部施用导致在施用部位几乎完全保留，表明肽可用于局部制剂。 3. ABCG 2基因编码一种ABC转运蛋白，在脑内皮、胃肠道和胎盘中具有高正常组织表达，ABCG 2被认为在保护免受外源性物质、调节口服生物利用度以及形成血脑屏障、血睾丸屏障和母胎屏障的一部分方面是重要的。ABCG 2在早期胚胎干细胞中高度表达，部分功能是保护这些细胞免受毒素的侵害。 美国女性癌症死亡的第二大原因是乳腺癌，然而，近50%的乳腺肿瘤患者在治疗期间对药物产生耐药性。为了开发靶向治疗策略以对抗耐药性，必须了解癌细胞控制对化疗药物敏感性的基本分子机制。为了鉴定新的候选基因并促进新的耐药途径的发现，我们已经产生了对依托泊苷耐药的MCF 7乳腺癌细胞的耐药谱或耐药基因组。超过5000个基因的差异表达（倍数变化2，P值0.05）表明，在依托泊苷耐药的乳腺癌细胞中可能存在几种耐药机制，包括ABC转运蛋白基因的上调，药物靶基因的下调和凋亡基因的下调。我们还发现证据表明，参与另一种新的耐药机制（称为细胞外基质（ECM）介导的耐药性）的基因被上调。RUNX 2、SOX 9、ETS 1和SMAD 3等转录因子表达上调，可能是足叶乙甙耐药的潜在治疗靶点/生物标志物。在耐药和敏感细胞中观察到差异miRNA（microRNA）表达，表明miRNA也可能在耐药调节中起作用。Hsa-miR-218在耐药细胞系中下调。miR-218模拟物的转染可以在耐药细胞中将外排泵ABCC 6的表达下调近65%，这表明miRNA模拟物可以被探索为耐药的调节机制。

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.