Cancer stem–like cells are hypothesized to be the major tumor-initiating cell population of human cutaneous squamous cell carcinoma (cSCC), but the landscape of molecular alterations underpinning their signaling and cellular phenotypes as drug targets remains undefined. In this study, we developed an experimental pipeline to isolate a highly enriched CD133+CD31−CD45−CD61−CD24− (CD133+) cell population from primary cSCC specimens by flow cytometry. The CD133+ cells show enhanced stem–like phenotypes, which were verified by spheroid and colony formation in vitro and tumor generation in vivo. Gene expression profiling of CD133+/− cells was compared and validated, and differentially expressed gene signatures and top pathways were identified. CD133+ cells expressed a repertoire of stemness and cancer-related genes, including NOTCH and NOTCH1-mediated NF-κB pathway signaling. Other cancer-related genes from WNT, growth factor receptors, PI3K/mTOR, STAT pathways, and chromatin modifiers were also identified. Pharmacologic and genetic targeting of NOTCH1, IKKα, RELA, and RELB modulated NF-κB transactivation, the CD133+ population, and cellular and stemness phenotypes. Immunofluorescent staining confirmed colocalization of CD133+ and IKKα expression in SCC tumor specimens. Our functional, genetic, and pharmacologic studies uncovered a novel linkage between NOTCH1, IKKα, and NF-κB pathway activation in maintaining the CD133+ stem SCC phenotypes. Studies investigating markers of activation and modulators of NOTCH, IKK/NF-κB, and other pathways regulating these cancer stem gene signatures could further accelerate the development of effective therapeutic strategies to treat cSCC recurrence and metastasis. Mol Cancer Ther; 17(9); 2034–48. ©2018 AACR.

中文翻译：

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靶向 Notch1 和 IKKa 增强 CD133+皮肤癌干细胞中的 NF-κB 活化

癌症干细胞样细胞被假设为人类皮肤鳞状细胞癌 (cSCC) 的主要肿瘤起始细胞群，但支持其信号传导和细胞表型作为药物靶点的分子改变景观仍未确定。在这项研究中，我们开发了一个实验管道，通过流式细胞术从原代 cSCC 标本中分离出高度富集的 CD133+CD31-CD45-CD61-CD24-(CD133+) 细胞群。CD133+ 细胞显示出增强的干细胞样表型，这通过体外球体和集落形成和体内肿瘤生成得到证实。比较和验证了 CD133+/- 细胞的基因表达谱，并确定了差异表达的基因特征和主要途径。CD133+ 细胞表达了一系列干细胞和癌症相关基因，包括 NOTCH 和 NOTCH1 介导的 NF-κB 通路信号。还鉴定了来自 WNT、生长因子受体、PI3K/mTOR、STAT 通路和染色质修饰剂的其他癌症相关基因。NOTCH1、IKKα、RELA 和 RELB 的药理学和基因靶向调节 NF-κB 反式激活、CD133+ 群体以及细胞和干细胞表型。免疫荧光染色证实了 SCC 肿瘤标本中 CD133+ 和 IKKα 表达的共定位。我们的功能、遗传和药理学研究揭示了 NOTCH1、IKKα 和 NF-κB 通路激活在维持 CD133+ 干 SCC 表型方面的新联系。研究 NOTCH、IKK/NF-κB、和其他调节这些癌症干基因特征的途径可以进一步加速开发治疗 cSCC 复发和转移的有效治疗策略。摩尔癌症治疗; 17(9); 2034-48。©2018 AACR。