CANCER AND INFLAMMATION: FUNCTION AND THERAPY

1. MSMB转录本及其与雄激素受体调控因子NCOA4杂交产物的分析我们描述了前列腺癌患者中MSMB基因启动子rs10993994 SNP的相关性。SNP rs10993994上的C残基与GATA位点下游的creb结合位点有关，并且靠近TATA盒。为了研究近端MSMB启动子区域内SNP rs10993994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量分析（EMSA）。rs10993994的等位基因C增加了启动子活性（PSP94-C），因此更强的CREB结合，而已经显示弱启动子活性的等位基因（PSP94-T）没有检测到CREB结合。为了证实观察到的MSMB启动子中与SNP rs10993994相关的启动子活性变化的预测效果，我们测量了MSMB基因的mRNA表达水平。19株肿瘤细胞株检测到MSMB mRNA的表达，而rs10993994-C的MSMB mRNA平均表达量显著高于rs10993994-T。通过检查MSMB基因区域的EST克隆，我们确定了几个在MSMB的第一外显子与相邻基因NCOA4之间表现出明显的反式剪接事件。由于NCOA4编码一种已知与雄激素受体相互作用和调节的蛋白质，这些转录本可能与前列腺癌有关。我们证实了这些杂交转录本存在于癌细胞系和前列腺癌组织中，并证明了它们的丰度在含有rs10993994-C等位基因的个体细胞中升高。我们克隆并表达了这些转录本，并使用MSMB和NCOA4血清证明它们产生了稳定的杂交蛋白。我们目前正在进一步探索MSMB基因的启动子区域，并表达杂交蛋白以确定其在雄激素调节中的作用。2. 在几种实体肿瘤类型中发现的自我更新细胞群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的癌症干细胞群。HH/PTCH通路已被证明在几乎所有基底细胞癌和部分髓母细胞瘤中发生突变。此外，许多肿瘤显示HH/PTCH通路的配体依赖性激活，包括胰腺肿瘤、前列腺肿瘤、胃肠道肿瘤和小细胞肺肿瘤。SMO的小分子抑制剂（HH/PTCH通路的下游调节因子）已经在临床前得到验证，并且有几种药物正在临床试验中。为了进一步开发HH/PTCH抑制剂，我们先前设计了来自SMO蛋白的跨膜（TM）结构域和细胞内环的显性阴性抑制剂，这些抑制剂具有高度的有效性和选择性。为了了解这些肽的生物分布，我们对其中最活跃的衍生物进行了放射性标记，并通过几种途径将其传递给小鼠。静脉注射肽可迅速分布到几乎所有器官部位，在肺部积聚最多。通过皮下和腹腔注射，99%以上的肽停留在注射部位。局部应用结果在应用部位几乎完全保留，表明肽可用于局部配方。3. ABCG2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，ABCG2被认为在保护机体免受外源药物侵害、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具有重要作用。abcg2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。在美国，导致女性癌症死亡的第二大原因是乳腺癌，然而，近50%的乳腺癌患者在治疗期间对药物产生耐药性。为了制定有针对性的治疗策略来对抗耐药性，了解癌细胞控制化疗敏感性的基本分子机制是至关重要的。为了确定新的候选基因并促进发现新的耐药途径，我们已经生成了对依托泊苷耐药的MCF7乳腺癌细胞的耐药谱或抗性组。超过5000个基因的差异表达（fold change > 2， P值&lt； 0.05）表明，依托泊苷耐药乳腺癌细胞可能存在多种耐药机制，包括ABC转运蛋白基因上调、药物靶基因下调和凋亡基因下调。我们还发现了另一种新的耐药机制——细胞外基质（ECM）介导的耐药基因被上调的证据。RUNX2、SOX9、ETS1和SMAD3等转录因子上调，可能是依托opo苷耐药的潜在治疗靶点/生物标志物。在耐药细胞和敏感细胞中观察到miRNA （microRNA）表达的差异，提示miRNA也可能在耐药调控中发挥作用。Hsa-miR-218在耐药细胞系中下调。转染miR-218模拟物可使耐药细胞中外排泵ABCC6的表达下调近65%，这表明miRNA模拟物可能是耐药的一种调节机制。4. 我们使用新一代测序方法鉴定了PXE患者的ABCC6基因突变，并对其他几个可能修饰位点的基因进行了测序。5. TET2基因在某些前列腺肿瘤中发生突变，该基因启动子的变异与前列腺癌有关。我们已经开始识别与TET2特定启动子单倍型结合的蛋白质。PBRM1基因在肾癌中经常发生突变，我们已经发现了与PBRM1结合的新蛋白。ARID5B基因与儿童ALL相关，我们已经定位了感兴趣的区域，并开始鉴定调节蛋白。6. 我们使用Affymetrix外显子芯片研究了100个透明细胞肾肿瘤的表达，并确定了一组将这些肿瘤分为两个亚型的基因。我们正在设计一个进一步的面板，以准确地定量这些基因，作为基因面板临床应用的进一步步骤。

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 leukemia 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 pancreatic tumors, prostate tumors, gastrointestinal tract tumors, 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. ABCG2is 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. 4. We have used a Next-generation sequencing method to identify mutations in The ABCC6 gene in PXE patients and have sequenced several other genes that are potential modifying loci. 5. The TET2 gene is mutated in some prostate tumors and variation in the promoter of the gene are associated with prostate cancer. we have begun identifying proteins that bind to specific promoter haplotypes of TET2. The PBRM1 gene is frequently mutated in kidney cancer and we have identified new proteins that bind to PBRM1. The ARID5B gene is associated with childhood ALL, and we have localized the region of interest and begun identifying regulatory proteins. 6. We have studied the expression using Affymetrix Exon chips of 100 clear cell kidney tumors and identified a panel of genes that divide these tumors into two subtypes. We are designing a further panel to accurately quantitate these genes as a further step towards clinical application of the gene panel.