CANCER AND INFLAMMATION: FUNCTION AND THERAPY

癌症和炎症：功能和治疗

• 批准号: 8553090
• 负责人: MICHAEL DEAN
• 金额: $ 87.94万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553090
• 关键词: ABCG2 gene; ATP-Binding Cassette Transporters; Alleles; Androgen Receptor; Androgens; Apoptotic; Area; Basal cell carcinoma; Binding; Binding Sites; Biodistribution; Biological Availability; Biological Markers; Blood - brain barrier anatomy; Blood-Testis Barrier; Brain; Breast Cancer Cell; Breast Cancer Genetics; CREB1 gene; Cancer Etiology; Cancer cell line; Candidate Disease Gene; Cell Line; Cell physiology; Cells; Cessation of life; Childhood Acute Lymphocytic Leukemia; Clear Cell; Clinical Trials; Cytoskeleton; Development; Disease; Dominant-Negative Mutation; Drug Delivery Systems; Drug Formulations; Drug resistance; Drug resistance pathway; Drug-sensitive; ETS1 gene; Electrophoretic Mobility Shift Assay; Endothelium; Etoposide; Event; Exons; Expressed Sequence Tags; Future; Gastrointestinal Neoplasms; Gastrointestinal tract structure; Gene Proteins; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Haplotypes; Hybrids; Individual; Inflammation; Injection of therapeutic agent; Intraperitoneal Injections; Kidney Neoplasms; Lead; Ligands; Lung; Lung Neoplasms; MADH3 gene; MCF7 cell; MSMB gene; Malignant Neoplasms; Malignant neoplasm of prostate; Mammary Neoplasms; Measures; Mediating; Methods; MicroRNAs; Molecular; Multi-Drug Resistance; Mus; Mutate; Mutation; Mutation Spectra; NCOA4 gene; Normal tissue morphology; Nuclear Receptor Coactivator 4; Oligonucleotide Probes; Oral; Organ; PTCH gene; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Placenta; Play; Population; Promoter Regions; Prostate; Prostatic Neoplasms; Protein Binding; Proteins; Pseudoxanthoma Elasticum; Radiolabeled; Regulation; Renal carcinoma; Research; Resistance; Resistance profile; Role; Route; Serum; Site; Solid Neoplasm; Stem Cell Research; Subcutaneous Injections; TATA Box; Therapeutic; Therapeutic Uses; Tissues; Topical application; Toxin; Trans-Splicing; Transcript; Transfection; Transmembrane Domain; Up-Regulation; Urologic Diseases; Validation; Variant; Woman; Work; Xenobiotics; base; cancer cell; cancer site; cancer stem cell; clinical application; combat; design; efflux pump; embryonic stem cell; fetal; gene repression; genetic association; genetic variant; hybrid protein; inhibitor/antagonist; interest; intravenous injection; leukemia; mRNA Expression; malignant breast neoplasm; medulloblastoma; next generation; novel; pancreatic neoplasm; pre-clinical; promoter; radiotracer; resistance mechanism; self renewing cell; small molecule; stem; success; therapeutic target; therapy development; transcription factor; tumor

1. Analysis of MSMB transcripts and Hybrid Products with NCOA4, an Androgen Receptor RegulatorWe 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 CellsThe 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 ResistanceThe 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.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.

1。对MSMB转录本和NCOA4的混合产物的分析，Androgen受体调节仪描述了MSMB基因启动子中RS10993994 SNP的前列腺癌受试者的关联。 SNP RS10993994的C残基存在与GATA站点下游的假定Creb结合位点有关，并靠近TATA盒子。为了研究SNP RS10993994在近端MSMB启动子区域中对CREB与MSMB启动子结合的影响，我们对含有多态性的寡核苷酸探针进行了电泳迁移率转移测定（EMSA）分析。 RS10993994的等位基因C增加了启动子活性（PSP94-C），因此CREB结合更强，而等位基因已经显示出具有较弱的启动子活性（PSP94-T），具有无法检测到的CREB结合。为了确认与SNP RS10993994在MSMB启动子中观察到的启动子活性变化的预测作用，测量了MSMB基因的mRNA表达水平。 十九个癌细胞系具有可检测到的MSMB mRNA表达，而MSMB mRNA表达水平的平均值显着高于对MSMB基因区域中EST克隆的rs10993994-T.By rs10993994-T.的平均值，我们确定了MSMB和MSMB之间的明显移植事件，我们确定了明显的移植事件。由于NCOA4编码已知与雄激素受体相互作用并调节的蛋白质，因此这些转录本可能与前列腺癌有关。我们验证了这些杂种转录本存在于癌细胞系和前列腺癌组织中，并证明它们的丰度在包含RS10993994-C等位基因的个体的细胞中升高。我们克隆并表达了其中几个转录本，并使用MSMB和NCOA4 Sera表明它们产生了稳定的混合蛋白。我们目前正在进一步探索MSMB基因的启动子区域，并表达混合蛋白来确定其在雄激素调节中的作用。2。靶向SMO和癌症细胞的药物的开发鉴定几种实体瘤类型中的自我更新细胞种群扩展了白血病的先前工作，并表明许多或全部肿瘤含有少量癌症干细胞。 已经证明HH/PTCH途径在几乎所有基底细胞癌和一部分髓母细胞瘤中被突变。 此外，许多肿瘤都显示出HH/PTCH途径的配体依赖性激活，包括胰腺肿瘤，前列腺肿瘤，胃肠道肿瘤和小细胞肺肿瘤。 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.为了了解这些肽的生物分布，我们放射标记了最活跃的衍生物之一，并通过多个路线将其传递给了小鼠。静脉注射肽会导致几乎所有器官部位的快速分布，肺部积聚最高。皮下注射和腹膜内注射，超过99％的肽停留在注射部位。局部应用几乎可以在应用地点完全保留，这表明该肽可以用于局部配方中。 3. Function and Targeting of ABC Transporters Involved in Multidrug ResistanceThe 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,以及母亲障碍。 ABCG2在早期的胚胎干细胞中高度表达，部分作用是保护这些细胞免受毒素的影响。 美国妇女癌症死亡的第二大原因是乳腺癌，但是，乳腺肿瘤患者中有近50％在治疗期间对药物具有抵抗力。为了开发针对抗药性抗药性的有针对性的治疗策略，必须了解癌细胞控制化学疗法敏感性的基本分子机制。为了鉴定新的候选基因并促进了新型耐药性途径的发现，我们产生了耐药性或MCF7乳腺癌细胞抵抗依托泊苷的抗性。超过5000个基因的差异表达（倍数变化> 2，p值<0.05）表明，几种耐药性机制可能在依托泊苷耐药性乳腺癌细胞中起作用，包括ABC转运蛋白基因的上调，药物靶标基因的下调和凋亡基因的下调。我们还发现证据表明，涉及另一种新型抗性机制的基因称为额外的细胞基质（ECM）介导的耐药性。诸如Runx2，Sox9，Ets1和Smad3之类的几种转录因子被上调，可能是依托泊苷耐药性的潜在治疗靶标/生物标志物。在耐药性和敏感细胞中观察到差异miRNA（microRNA）表达，这表明miRNA也可能在调节耐药性的调节中起作用。 HSA-MIR-218在耐药细胞系中下调。 miR-218模拟物的转染可能会在耐药细胞中下调几乎65％的外排泵ABCC6的表达，这表明可以将miRNA模拟物作为耐药性的调节机制探索。4。我们已经使用了下一代测序方法来鉴定PXE患者ABCC6基因中的突变，并测序了其他几个潜在的基因座的基因。5。 TET2基因在一些前列腺肿瘤中突变，并且该基因启动子的变异与前列腺癌有关。我们已经开始鉴定与特定TET2的特定启动子单倍型结合的蛋白质。 PBRM1基因经常在肾癌中突变，我们已经确定了与PBRM1结合的新蛋白质。 ARID5B基因与儿童期有关，我们已经定位了感兴趣的区域，并开始识别调节蛋白。6。我们使用100个透明细胞肾脏肿瘤的Affymetrix外显子芯片研究了表达，并鉴定出将这些肿瘤分为两种亚型的基因。我们正在设计一个进一步的面板，以准确量化这些基因，以迈向基因面板的临床应用。