Inhibition of IGF-1R: A Therapeutic Strategy for Colon Cancer Stem-like Cells

抑制 IGF-1R：结肠癌干细胞样细胞的治疗策略

• 批准号: 8142628
• 负责人: Bhaumik B Patel
• 金额: --
• 依托单位: JOHN D DINGELL VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142628
• 关键词: ABCG2 gene; Age-Years; Aging; Cancer Diagnostics; Cells; Clinic; Colon; Colon Carcinoma; Colorectal; Colorectal Cancer; Disease; Dose; Effectiveness; Embryo; Fluorouracil; Gene Expression; Gene Targeting; Genes; Goals; Growth; Health; Human; In Vitro; Incidence; Insulin-Like Growth Factor II; Integrin alphaV; Investigation; Lead; Ligands; Malignant Neoplasms; Measures; MicroRNAs; Myeloid Cells; Neoplasm Metastasis; Outcome; Pathway interactions; Phenotype; Play; Population; Process; Recurrent disease; Regulator Genes; Resistance; Role; Stem cells; Testing; Therapeutic; Thymidylate Synthase; Time; Treatment outcome; Tyrosine Kinase Inhibitor; Vertebral column; Veterans; Xenograft procedure; base; cancer cell; cancer stem cell; cell growth; cell type; chemotherapy; improved; in vivo; killings; leukemia; mortality; overexpression; oxaliplatin; patient population; preclinical study; research study; self-renewal; stem; tumor

Despite recent advances in therapeutics, colorectal cancer remains the third deadliest cancer in the USA. This is mainly attributable to survival of a small population of cancer cells called stem cells (CSCs) with the ability to self-renew, resist chemotherapy killing and metastasize (broadly speaking CSC phenotype). We have generated cells resistant to a combination of 5-flurouracil (5-FU) and oxaliplatin (FUOX), the backbone of colorectal cancer chemotherapeutics. These FUOX-resistant cells are enriched in CSCs and show increased expression of total and activated form of IGF-1R as well as its ligand IGF-2. Inhibition of IGF-1R by a tyrosine kinase inhibitor, GSK1904529A, results in a dose dependant decrease in colonosphere formation (a measure of CSCs growth) which is especially pronounced in p53 wild type cells. Moreover, combination of FUOX with GSK1904529A results in not only synergistic inhibition of primary colonosphere formation but also secondary sphere formation suggesting IGF-1R may play a role in CSCs self-renewal. MicroRNAs (miRs) have been identified as important negative regulators of gene expression in embryonic and cancer stem cell growth. We hypothesized that IGF-1R inhibition would lead to a change in expression of specific miRs. Indeed, we identified miR-363, a non p53 regulated miR, and miR-215, a p53 regulated miR, to be highly overexpressed (28-fold and 6-fold respectively) following IGF-1R inhibition. More importantly, we found that expression of miR-363 and -215 is decreased several fold in FUOX-resistant cells that are enriched in CSCs. At the same time, expression of thymidylate synthase (TS), the putative target of miR-215, as well as integrin alpha-V (CD51) and myeloid cell leukemia-1 (MCL-1), both targets of miR-363 and regulator of CSC phenotype, were highly overexpressed in FUOX-resistant cells. Moreover, both miRs and their respective targets can be favorably modulated by IGF-1R inhibition. Based on the above observation we hypothesize, that IGF-1R inhibition alters the cancer stem cell phenotype thus enhancing effectiveness of chemotherapy in FUOX-resistant colorectal cells. We further hypothesize that this enhanced efficacy is due, in part, to induction of p53 dependent (miR-215) and independent (miR-363) microRNAs. To test this hypothesis, we will examine the effect of IGF-1R inhibition in primary human colon cancer cells (propagated as colonospheres) on CSC phenotype in vitro and the role of miR-215 and miR-363 in the process (Aim 1). We will also test the effect of IGF-1R depletion/inhibition on tumor formation and xenograft growth (with and without FUOX) in vivo (Aim 2). Lastly, we will delineate the role of specific miR-215 (TS and ABCG2) as well as miR-363 (CD51 and MCL-1) target genes in modulating CSC phenotype (Aim 3). The results from the proposed experiments would expand our understanding of mechanisms of colon CSCs growth and chemotherapy resistance resulting in a paradigm shift in treatment of colorectal cancer improving outcome for this deadly disease.

尽管治疗学最近取得了进展，结直肠癌仍然是美国第三大致命癌症。这主要归因于一小群称为干细胞 (CSC) 的癌细胞的存活，它们具有自我更新、抵抗化疗杀伤和转移的能力（广义上的 CSC 表型）。我们已经培育出对 5-氟尿嘧啶 (5-FU) 和奥沙利铂 (FUOX) 组合具有耐药性的细胞，这是结直肠癌化疗药物的支柱。这些 FUOX 抗性细胞在 CSC 中富集，并显示 IGF-1R 及其配体 IGF-2 的总形式和活化形式的表达增加。酪氨酸激酶抑制剂 GSK1904529A 对 IGF-1R 的抑制会导致结肠球形成（CSC 生长的指标）呈剂量依赖性减少，这在 p53 野生型细胞中尤其明显。此外，FUOX 与 GSK1904529A 的组合不仅可以协同抑制初级菌落球的形成，还可以协同抑制次级球的形成，表明 IGF-1R 可能在 CSC 的自我更新中发挥作用。 MicroRNA (miR) 已被确定为重要的阴性 胚胎和癌症干细胞生长中基因表达的调节因子。我们假设 IGF-1R 抑制会导致特定 miR 表达的变化。事实上，我们发现 miR-363（一种非 p53 调节的 miR）和 miR-215（一种 p53 调节的 miR）在 IGF-1R 抑制后高度过表达（分别为 28 倍和 6 倍）。更重要的是， 我们发现，在 CSC 中富集的 FUOX 抗性细胞中，miR-363 和 -215 的表达降低了几倍。同时，胸苷酸合酶 (TS)（miR-215 的假定靶标）以及整合素 α-V (CD51) 和骨髓细胞白血病-1 (MCL-1)（miR-363 和 miR-363 的靶标）的表达CSC 表型的调节因子在 FUOX 耐药细胞中高度过表达。此外，IGF-1R 抑制可以有利地调节 miR 及其各自的靶标。基于上述观察，我们假设 IGF-1R 抑制改变了癌症干细胞表型，从而增强了 FUOX 耐药结直肠细胞化疗的有效性。我们进一步假设这种增强的功效部分归因于 p53 依赖性 (miR-215) 和独立 (miR-363) microRNA 的诱导。为了检验这一假设，我们将在体外研究原代人结肠癌细胞（作为结肠球繁殖）中 IGF-1R 抑制对 CSC 表型的影响以及 miR-215 和 miR-363 在此过程中的作用（目标 1）。我们还将测试 IGF-1R 消耗/抑制对体内肿瘤形成和异种移植物生长（使用和不使用 FUOX）的影响（目标 2）。最后，我们将描述特定 miR-215（TS 和 ABCG2）以及 miR-363（CD51 和 MCL-1）靶基因在调节 CSC 表型中的作用（目标 3）。拟议实验的结果 将扩大我们对结肠 CSC 生长和化疗机制的理解 耐药性导致结直肠癌治疗模式的转变，改善了这种致命疾病的治疗结果。