Carrillo Jaime, García-Aragoncillo Eva, Azorín Daniel, Agra Noelia, Sastre Ana, González-Mediero Imelda, García-Miguel Purificación, Pestaña Angel, Gallego Soledad, Segura Dolores, Alonso Javier
Laboratorio de Patología Molecular de Tumores Sólidos Infantiles, Departamento de Biología Molecular y Celular del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC-UAM), Spain.
Clin Cancer Res. 2007 Apr 15;13(8):2429-40. doi: 10.1158/1078-0432.CCR-06-1762.
Tumors of the Ewing family are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype. In the present study, we show that cholecystokinin (CCK) is a new target of the EWS/FLI1 oncoprotein and assess its functional role in Ewing tumor pathogenesis.
Relevant EWS/FLI1 targets were identified using a combination of cell systems with inducible EWS/FLI1 expression, Ewing tumors and cell lines, microarrays, and RNA interference with doxycycline-inducible small hairpin RNA (shRNA) vectors. A doxycycline-inducible CCK-shRNA vector was stably transfected in A673 and SK-PN-DW Ewing cell lines to assess the role of CCK in cell proliferation and tumor growth.
Microarray analysis revealed that CCK was up-regulated by EWS/FLI1 in HeLa cells. CCK was overexpressed in Ewing tumors as compared with other pediatric malignancies such as rhabdomyosarcoma and neuroblastoma, with levels close to those detected in normal tissues expressing the highest levels of CCK. Furthermore, EWS/FLI1 knockdown in A673 and SK-PN-DW Ewing cells using two different doxycycline-inducible EWS/FLI1-specific shRNA vectors down-regulated CCK mRNA expression and diminished the levels of secreted CCK, showing that CCK is a EWS/FLI1 specific target gene in Ewing cells. A doxycycline-inducible CCK-specific shRNA vector successfully down-regulated CCK expression, reduced the levels of secreted CCK in Ewing cell lines, and inhibited cell growth and proliferation in vitro and in vivo. Finally, we show that Ewing cell lines and tumors express CCK receptors and that the growth inhibition produced by CCK silencing can be rescued by culturing the cells with medium containing CCK.
Our data support the hypothesis that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be promising in the treatment of Ewing tumors.
尤因家族肿瘤的特征是染色体易位产生嵌合转录因子，如EWS/FLI1，其调节有助于恶性表型的特定基因的表达。在本研究中，我们表明胆囊收缩素（CCK）是EWS/FLI1癌蛋白的新靶点，并评估其在尤因肿瘤发病机制中的功能作用。
使用具有可诱导EWS/FLI1表达的细胞系统、尤因肿瘤和细胞系、微阵列以及用强力霉素诱导的小发夹RNA（shRNA）载体进行RNA干扰的组合来鉴定相关的EWS/FLI1靶点。将强力霉素诱导的CCK-shRNA载体稳定转染到A673和SK-PN-DW尤因细胞系中，以评估CCK在细胞增殖和肿瘤生长中的作用。
微阵列分析显示，在HeLa细胞中CCK被EWS/FLI1上调。与其他小儿恶性肿瘤如横纹肌肉瘤和神经母细胞瘤相比，CCK在尤因肿瘤中过表达，其水平接近在表达最高水平CCK的正常组织中检测到的水平。此外，使用两种不同的强力霉素诱导的EWS/FLI1特异性shRNA载体在A673和SK-PN-DW尤因细胞中敲低EWS/FLI1可下调CCK mRNA表达并降低分泌的CCK水平，表明CCK是尤因细胞中EWS/FLI1的特异性靶基因。强力霉素诱导的CCK特异性shRNA载体成功下调了CCK表达，降低了尤因细胞系中分泌的CCK水平，并在体外和体内抑制了细胞生长和增殖。最后，我们表明尤因细胞系和肿瘤表达CCK受体，并且通过用含有CCK的培养基培养细胞可以挽救由CCK沉默产生的生长抑制。
我们的数据支持CCK作为自分泌生长因子刺激尤因细胞增殖的假说，并表明靶向CCK的疗法在尤因肿瘤治疗中可能很有前景。
