Molecular Targets - Colon Cancer

我们采用了一种新的策略来确定治疗和预防常见癌症的分子靶点，最初的重点是结肠癌。化疗药物的主要局限性之一是相对较窄的治疗：毒性比，这是由于癌症和正常组织中治疗靶点的共性造成的。因此，我们试图通过使用公共数据库中可用的基因表达信息结合高通量基因表达阵列分析来识别相对癌症特异性的潜在治疗靶点。使用数字基因表达显示器挖掘通过NCIS癌症基因组解剖项目获得的表达文库，我们鉴定了665个在结肠癌文库中表达但在必要的正常组织中不表达的序列。在这些基因中，有356个表达在Affymetrix表达阵列上，这使得可以比较这些基因在结肠癌组织和细胞系以及基本正常组织中的表达。这项研究再加上随后使用定量RT-PCR进行的确认，导致鉴定出两个以前未描述的基因，它们的表达对相当大比例的结肠癌样本具有相对特异性。我们进行了实验，以确定siRNA介导的候选基因的抑制是否抑制了HCT15结肠癌细胞系的细胞增殖；使用Cyquant试剂盒进行了细胞增殖检测，与siRNA阴性对照相比，细胞增殖抑制了10%。SiRNA介导的这些基因的沉默导致了HCT15细胞增殖的轻微下降，表明这些基因可能在结肠癌中起到促进细胞增殖的作用。进一步的实验表明，IGFL2在MAP激酶途径的调节中起着假定的作用。随后的研究将涉及IGFL2保守区的定点突变，以确定这些区域在MAP激酶激活中的结构作用。我们采用了一种新的策略来确定治疗和预防常见癌症的分子靶点，最初的重点是结肠癌。化疗药物的主要局限性之一是相对较窄的治疗：毒性比，这是由于癌症和正常组织中治疗靶点的共性造成的。因此，我们试图通过使用公共数据库中可用的基因表达信息结合高通量基因表达阵列分析来识别相对癌症特异性的潜在治疗靶点。使用数字基因表达显示器挖掘通过NCIS癌症基因组解剖项目获得的表达文库，我们鉴定了665个在结肠癌文库中表达但在必要的正常组织中不表达的序列。在这些基因中，有356个表达在Affymetrix表达阵列上，这使得可以比较这些基因在结肠癌组织和细胞系以及基本正常组织中的表达。这项研究再加上随后使用定量RT-PCR进行的确认，导致鉴定出两个以前未描述的基因，它们的表达对相当大比例的结肠癌样本具有相对特异性。我们进行了实验，以确定siRNA介导的候选基因的抑制是否抑制了HCT15结肠癌细胞系的细胞增殖；使用Cyquant试剂盒进行了细胞增殖检测，与siRNA阴性对照相比，细胞增殖抑制了10%。SiRNA介导的这些基因的沉默导致了HCT15细胞增殖的轻微下降，表明这些基因可能在结肠癌中起到促进细胞增殖的作用。进一步的实验表明，IGFL2在MAP激酶途径的调节中起着假定的作用。随后将进行涉及IGFL2保守区的定点突变的研究，以确定这些区域在MAP激酶激活中的结构作用。

We have employed a novel strategy to identify molecular targets for the therapy and prevention of common cancers, with an initial focus on colon cancer. One of the major limitations of chemotherapeutic agents is the relatively narrow therapeutic: toxic ratio that results from the commonality of the therapeutic target in both the cancerous and normal tissues. Therefore, we sought to identify potential therapeutic targets that were relatively cancer specific by using gene expression information available in public databases coupled with the use of high-throughput gene expression array analysis. Using the Digital Gene expression Displayer to mine expression libraries available through the NCIs Cancer Genome Anatomy Project, we identified 665 sequences that were expressed in colon cancer libraries but not in essential normal tissues. Of these, 356 were represented on Affymetrix expression arrays and this allowed for a comparison of the expression of these genes in colon cancer tissues and cell lines, and essential normal tissues. This investigation coupled with subsequent confirmation using quantitative RT-PCR resulted in the identification of two previously uncharacterized genes whose expression was relatively specific to a substantial proportion of colon cancer samples tested. We carried out experiments in order to determine whether the siRNA mediated suppression of our candidate genes inhibits cell proliferation in the HCT15 colon cancer cell line; cell proliferation assays were carried out using a Cyquant kit inhibit cell proliferation by 10% as compared to the siRNA negative control. siRNA mediated silencing of these gene resulted in a small reduction of cell proliferation in HCT15 cell lines indicating that these genes may play a role in promoting cell proliferation in colon cancer. Additional experiments revealed that IGFL2 plays a putative role in the regulation of the MAP Kinase pathway. Subsequent studies involving site directed mutagenesis in the conserved regions of the IGFL2 will be carried out in order to determine the structural role of these regions in the activation of MAP Kinases.We have employed a novel strategy to identify molecular targets for the therapy and prevention of common cancers, with an initial focus on colon cancer. One of the major limitations of chemotherapeutic agents is the relatively narrow therapeutic: toxic ratio that results from the commonality of the therapeutic target in both the cancerous and normal tissues. Therefore, we sought to identify potential therapeutic targets that were relatively cancer specific by using gene expression information available in public databases coupled with the use of high-throughput gene expression array analysis. Using the Digital Gene expression Displayer to mine expression libraries available through the NCIs Cancer Genome Anatomy Project, we identified 665 sequences that were expressed in colon cancer libraries but not in essential normal tissues. Of these, 356 were represented on Affymetrix expression arrays and this allowed for a comparison of the expression of these genes in colon cancer tissues and cell lines, and essential normal tissues. This investigation coupled with subsequent confirmation using quantitative RT-PCR resulted in the identification of two previously uncharacterized genes whose expression was relatively specific to a substantial proportion of colon cancer samples tested. We carried out experiments in order to determine whether the siRNA mediated suppression of our candidate genes inhibits cell proliferation in the HCT15 colon cancer cell line; cell proliferation assays were carried out using a Cyquant kit inhibit cell proliferation by 10% as compared to the siRNA negative control. siRNA mediated silencing of these gene resulted in a small reduction of cell proliferation in HCT15 cell lines indicating that these genes may play a role in promoting cell proliferation in colon cancer. Additional experiments revealed that IGFL2 plays a putative role in the regulation of the MAP Kinase pathway. Subsequent studies involving site directed mutagenesis in the conserved regions of the IGFL2 will be carried out in order to determine the structural role of these regions in the activation of MAP Kinases.