Cancer and Inflammation - Genetics and Function

个体的遗传背景在他们对癌症的易感性、疾病进展和对治疗的反应中起着重要的作用。遗传变异可以作为理解复杂疾病的标记，包括癌症和免疫/炎症反应。人类和其他基因组序列的出现，微阵列的可用性，以及我们键入遗传标记和测序全基因组的能力，导致了对增强计算和信息学的需求。炎症被认为在癌症中起着重要作用。然而，人类基因中涉及炎症的遗传变异的程度和影响尚不清楚。我们正在研究癌症患者的种系基因变异、肿瘤细胞的遗传变异以及参与炎症反应的基因变异。在NCI进行的一系列全基因组关联研究（GWAS）揭示了一组与前列腺癌相关的基因座。其中一个基因座包括MSMB基因，一种编码微精原蛋白- β的基因；精液的主要成分和已知的前列腺癌生物标志物。其中一个相关的变异在基因的启动子区域发现，并影响预测的CREB转录因子结合位点。凝胶位移实验证实了这一预测。我们确定了MSMB基因启动子区域变异在前列腺癌中作用的功能基础。我们发现与前列腺癌相关的MSMB变异在该基因的调控中具有功能作用。高密度基因分型结果显示rs10993994 SNP与该病的相关性最高。该变体的C等位基因是CREB转录因子结合位点的一部分，与MSMB基因的高表达有关。而T等位基因不结合CREB，与低表达相关。这代表了癌症全基因组关联的分子基础功能表征的第一个例子之一。与纪念斯隆凯特琳癌症中心（MSKCC）的科学家合作，我们完成了一项有家族病史的德系犹太人乳腺癌全基因组关联研究。扫描确定了几个具有潜在显著关联的位点。从这些数据中，我们选择了一个基因座用于重复队列的随访研究。一个包含两个基因RNF146和ECHDC1的位点被确定与德系犹太人的乳腺癌呈正相关。我们随后在德系犹太人乳腺癌受试者的独立队列中证实了这种关联。RNF146与调节雌激素受体的蛋白具有共同的基序。在雌激素反应元件的检测中，RNF146抑制雌激素反应。为了进一步表征RNF146，我们在细菌和哺乳动物细胞中表达了该产物。我们正在与David Waugh合作，结晶蛋白质以确定其三维结构。此外，我们正在与Allan Weissman合作评估泛素E3连接酶活性的基础，并确认泛素连接酶活性。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、前列腺、胃肠道和小细胞肺肿瘤。为了进一步开发HH/PTCH抑制剂，我们设计了针对SMO蛋白的TM结构域和细胞内环的肽。SMO是g蛋白偶联受体（GPCR）超家族的成员，其他GPCR已被这种方法靶向。虽然针对几个TM结构域的肽对细胞增殖没有影响，但针对胞内环2和3的肽是有效的抑制剂。活性肽导致HH/PTCH靶基因和蛋白的表达减少，并拮抗已知途径激动剂嘌呤胺对该途径的刺激。为了将这些试剂推向临床研究，我们开发了逆转录D-肽并进行了生物分布分析。将放射性标记肽注射到小鼠体内，发现该肽分布在全身。目前正在计划进行疗效研究。我们还描述了SMO基因启动子区域的甲基化。在SMO高表达的细胞系中，启动子区域未甲基化；而启动子在低表达细胞中是甲基化的。我们与危地马拉主要的儿科肿瘤医院——危地马拉城的国家儿科医院（UNOP）建立了合作。项目厅是该国最大的儿童癌症医院。与St. Judes医院的同事一起，我们获得了向Francisco Maroquin医学院提交IRB方案的许可，并协助他们接受FWA注册，并获得了患者群体样本收集和药物遗传学研究的批准，以确定土著和混合人群中TPMT（参与硫唑嘌呤代谢和免疫抑制）和其他药物代谢基因的变异频率。已开始收集样本，已获得200多个样本。流行病学研究表明，首都的视网膜母细胞瘤发病率升高，农村病例的确定不足，特别是在几个土著玛雅人群中。这表明某些疾病的潜在环境原因。急性淋巴细胞白血病（ALL）的分析也显示，儿童ALL在农村和土著地区的代表性明显不足。为了进一步了解肾细胞癌中发生的基因组事件，我们对肾癌细胞系和肿瘤进行了全外显子组和全转录组测序。使用Nimblegen外显子捕获芯片，我们捕获了786-0和SN12C肾癌细胞系的所有外显子和miRNA位点，并对得到的DNA进行了454高通量测序。这导致了基因组中大多数外显子的高覆盖率和大量基因变异的鉴定。为了验证这些NextGen测序结果，我们从相同的细胞系中提取RNA，并在相同的技术平台上进行全转录组测序。许多癌症相关基因在基因组和cDNA中都显示出高置信度的变异，是对更大组肿瘤dna进行后续分析的候选基因。一些潜在的杂交基因转录本也被鉴定出来，将在后续分析中得到验证。我们还对两个原发性肾细胞癌肿瘤的cDNA文库进行了Solexa高通量测序。这项分析已经确定了一组独立的癌症相关基因变异，这些变异正在大量的肿瘤样本中得到验证。与Richard Lempicki博士和david Huang博士合作，我们成功建立了RT-PCR条件来分析选择性剪接转录物的表达。我们通过RT-PCR实验验证了外显子阵列数据，并证明了人类CD45， ABLIM1, PRDM1， C1orf21和BRCA1基因[摘要截断为7800个字符]

Summary An individual's genetic background plays an important role in their susceptibility to cancer, disease progression, and response to therapy. Genetic variations can be used as markers to understand complex diseases including cancer, and the immune/inflammatory response. The advent of the sequence of the human and other genomes, the availability of microarrays, and our ability to type genetic markers and sequence whole genomes has led to the need for enhanced computing and informatics. Inflammation is recognized as playing an important role in cancer. However the extent and implications of genetic variation in human genes involved in inflammation is poorly characterized. We are interrogating the germline gene variation in cancer patients, the genetic variation of tumor cells and the variation of genes involved in the inflammatory response. Prostate Cancer Analysis of the MSMB protein A series genome-wide association studies (GWAS) performed at the NCI revealed a set of loci genetically associated with prostate cancer. One of these loci include the gene for MSMB, a gene that encodes microseminoprotein-beta; a major component of seminal fluid and a known prostate cancer biomarker. One of the associated variants is found in the promoter region of the gene, and affects a predicted CREB transcription factor binding site. Gel shift experiments confirm this prediction. We identified the functional basis of the role of variants in the promoter region of the MSMB gene in prostate cancer. We showed that variants in MSMB associated with prostate cancer have a function role in the regulation of this gene. High density genotyping of this locus revealed that the rs10993994 SNP is the most highly associated. The C allele of this variant is part of a functional CREB transcription factor binding site and is associated with high expression of the MSMB gene. Whereas the T allele does not bind CREB and is associated with low expression. This represents one of the first examples of functional characterization of the molecular basis for a genome wide association in cancer. A genome wide association study for breast cancer In collaboration with scientists at the Memorial Sloan Kettering Cancer Center (MSKCC) we completed a whole genome association study of breast cancer in Ashkenazi Jewish subjects with a family history of disease. The scan identified several loci with potentially significant associations. From this data we selected loci for follow up studies in a replication cohort. A locus containing two genes, RNF146 and ECHDC1, was identified as positively associated with breast cancer in Ashkenazi subjects. We subsequently confirmed this association in an independent cohort of Ashkenazi breast cancer subjects. RNF146 has motifs in common with proteins that regulate estrogen receptor. In an assay for estrogen response elements, RNF146 inhibited estrogen response. To further characterize RNF146, we expressed the product in bacterial and mammalian cells. We are collaborating with David Waugh to crystallize the protein to determine its 3-dimensional structure. In addition we are collaborating with Allan Weissman to assess the basis for ubiquitin E3 ligase activity and confirmed the ubiquitin ligase activity. Inhibitors of the Hedgehog/Patched Pathway in 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. To further the development of HH/PTCH inhibitors we designed peptides against TM domains and intracellular loops of the SMO protein. SMO is a member of the G-protein-coupled receptor (GPCR) superfamily, and other GPCRs have been targeted by this approach. While peptides against several TM domains failed to have an effect on cell proliferation, peptides against intracellular loops 2 and 3 were potent inhibitors. The active peptides result in a decrease in expression of HH/PTCH target genes and proteins and antagonize the stimulation of the pathway by purmorphamine, a known pathway agonist. To move these reagents forward into clinical studies we have developed retro inverso D- peptides and performed biodistribution analysis. Radiolabelled peptide was injected into mice and the peptide was found to distribute throughout the body. Efficacy studies are now planned. We have also characterized the methylation of the promoter region of the SMO gene. In cell lines with high expression of SMO, the promoter region is unmethylated; whereas the promoter is methylated in low expressing cells. Retinoblastoma and Pediatric Cancer in Guatemala We have established a collaboration with the principal pediatric onclology hospital in Guatemala the Unidad Nacional de Pediatria (UNOP) in Guatemala City. UNOP is the largest childrens cancer hospital in the country. With colleagues from St. Judes hospital we received permission to submit IRB protocols to the Francisco Maroquin Medical School and assisted them in receiving FWA registration, and received approval for a patient population sample collection and pharmacogenetic study to determine the frequency of variants in the indigenous and admixed populations of such genes as TPMT (involved in azathiopurine metabolism and immunosuppression) and other drug metabolism genes. Sample collection has begun and over 200 samples have been obtained. Epidemiology studies have revealed that the incidence of retinoblastoma is elevated in the capital and that there is an under-ascertainment of rural cases, especially in the several indigenous Mayan populations. This suggests a potential environmental cause for some of the disease. Analysis of acute lymphocytic leukemia (ALL) also reveals significant under-representation of childhood ALL in rural and indigenous regions. Genomics of Renal Cell Carcinoma To further understand the genomic events occurring in renal cell cancers we have undertaken whole exome and whole transcriptome sequencing of renal cancer cell lines and tumors. Using Nimblegen exon capture chips, we have captured all exons and miRNA loci from the 786-0 and SN12C renal cancer cell lines and subjected the resulting DNA to 454 high throughput sequencing. This has resulted in high coverage of most of the exons in the genome and in the identification of variants in a large number of genes. On order to validate these NextGen sequencing results we have taken RNA from the same cell lines and performed whole transcriptome sequencing on the same technology platform. A number of cancer-related genes display high confidence variants in both the genome and cDNA and are candidates for followup analysis on larger sets of tumor DNAs. A number of potential hybrid gene transcripts were also identified, that will be validated in followup analysis. We have also performed Solexa high throughput sequencing on a cDNA library from two primary renal cell carcinoma tumors. This analysis has identified an independent set of variants in cancer-related genes that are being validated in larger numbers of tumor samples. Alternative Splicing and T-cell activation In collaboration with Dr. Richard Lempicki, and Dr. Dawei Huang we have successfully established RT-PCR conditions for analyzing the expression of alternatively spliced transcripts. We have experimentally validated the exon array data by RT-PCR and demonstrated that human CD45, ABLIM1, PRDM1, C1orf21, and BRCA1 genes [summary truncated at 7800 characters]