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: 8764674
• 负责人: Bhaumik B Patel
• 金额: --
• 依托单位: VA VETERANS ADMINISTRATION HOSPITAL
• 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/8764674
• 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.

尽管最近在治疗方面取得了进展，但结直肠癌仍然是美国第三大致命癌症。这主要归因于称为干细胞（CSC）的小群体癌细胞的存活，其具有自我更新、抵抗化疗杀伤和转移的能力（广义地说CSC表型）。我们已经产生了对5-氟尿嘧啶（5-FU）和奥沙利铂（FUOX）组合（结直肠癌化疗药物的支柱）具有抗性的细胞。这些FUOX抗性细胞富含CSC，并且显示出IGF-1 R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GSK 1904529 A对IGF-1 R的抑制导致结肠球形成（CSC生长的量度）的剂量依赖性降低，这在p53野生型细胞中尤其明显。此外，FUOX与GSK 1904529 A的组合不仅导致对初级结肠球形成的协同抑制，而且还导致次级球形成，这表明IGF-1 R可能在CSC自我更新中起作用。microRNAs（miRs）被认为是重要的负 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-1 R抑制会导致特定miR表达的变化。事实上，我们鉴定了miR-363（一种非p53调节的miR）和miR-215（一种p53调节的miR）在IGF-1 R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miR-363和-215的表达在富含CSC的FUOX抗性细胞中降低了数倍。同时，胸苷酸合成酶（TS）（miR-215的假定靶点）以及整合素α-V（CD 51）和髓样细胞白血病-1（MCL-1）（两者均为miR-363的靶点和CSC表型的调节因子）的表达在FUOX耐药细胞中高度过表达。此外，miR和它们各自的靶标都可以通过IGF-1 R抑制有利地调节。基于上述观察，我们假设IGF-1 R抑制改变了癌症干细胞表型，从而增强了化疗在FUOX耐药结直肠细胞中的有效性。我们进一步假设，这种增强的功效部分是由于p53依赖性（miR-215）和非依赖性（miR-363）microRNA的诱导。为了验证这一假设，我们将检查在原代人结肠癌细胞（作为结肠球繁殖）中IGF-1 R抑制对体外CSC表型的影响以及miR-215和miR-363在该过程中的作用（目的1）。我们还将在体内检测IGF-1 R消耗/抑制对肿瘤形成和异种移植物生长（有和无FUOX）的影响（目的2）。最后，我们将描述特定的miR-215（TS和ABCG 2）以及miR-363（CD 51和MCL-1）靶基因在调节CSC表型中的作用（目的3）。从拟议的实验结果 将扩大我们对结肠CSCs生长和化疗机制的理解 耐药性导致结肠直肠癌治疗的范式转变，改善了这种致命疾病的结果。