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

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死性癌症。这主要归因于称为干细胞(CSCs)的一小部分癌细胞的存活，这些细胞具有自我更新、抵抗化疗杀伤和转移的能力(广义上讲CSC表型)。我们已经产生了对5-氟尿嘧啶(5-FU)和奥沙利铂(FUOX)的联合耐药细胞，奥沙利铂是结直肠癌化疗药物的主干。这些耐FUOX的细胞富含CSCs，表现出总的和激活的IGF-1R及其配体IGF-2的表达增加。酪氨酸激酶抑制剂GSK1904529A抑制IGF-1R导致结肠球形成(衡量CSCs生长的指标)呈剂量依赖性减少，这在P53野生型细胞中尤为明显。此外，FUOX与GSK1904529A联合使用不仅能协同抑制原发结肠球的形成，还能抑制次级结肠球的形成，提示IGF-1R可能在CSCs的自我更新中发挥作用。MicroRNAs(MiRs)已被确定为重要的阴性 胚胎和癌症干细胞生长中的基因表达调控。我们假设抑制IGF-1R会导致特异性miRs表达的变化。事实上，我们发现在IGF-1R抑制后，miR-363和miR-215高度过表达(分别是28倍和6倍)。miR-363是非p53调节的miR，而miR-215是p53调节的miR。更重要的是， 我们发现，在富含CSCs的FUOX耐药细胞中，miR-363和-215的表达降低了数倍。同时，miR-215的靶点胸苷酸合酶(TS)、整合素α-V(CD51)和髓系细胞白血病-1(MCL-1)在FUOX耐药细胞中高表达。此外，抑制IGF-1R可以很好地调节miRs和它们各自的靶点。基于上述观察，我们假设，抑制IGF-1R可以改变肿瘤干细胞的表型，从而提高耐FUOX的结直肠癌细胞的化疗效果。我们进一步假设，这种增强的疗效部分是由于诱导了依赖于p53的(miR-215)和非依赖的(miR-363)microRNA。为了验证这一假设，我们将检测在原代人类结肠癌细胞(以结肠球形式繁殖)中抑制IGF-1R对CSC表型的影响，以及miR-215和miR-363在这一过程中的作用(目标1)。我们还将在体内测试IGF-1R耗尽/抑制对肿瘤形成和异种移植瘤生长(使用和不使用FUOX)的影响(目标2)。最后，我们将描述特定的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)。拟议中的实验结果 将扩大我们对结肠CSCs生长和化疗机制的理解 耐药性导致结直肠癌治疗模式的转变，改善了这一致命疾病的预后。

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.