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 remain 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-kB 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 Notch1, IKKa, RELA and RELB modulated NF-kB transactivation, the CD133+ population, and cellular and stemness phenotypes. Immunofluorescent staining confirmed co-localization of CD133+ and IKKa expression in SCC tumor specimens. Our functional, genetic and pharmacologic studies uncovered a novel linkage between Notch1, IKKα and NF-B pathway activation in maintaining the CD133+ stem SCC phenotypes. Studies investigating markers of activation and modulators of NOTCH, IKK/NF-kB and other pathways regulating these cancer stem gene signatures could further accelerate the development of effective therapeutic strategies to treat cSCC recurrence and metastasis.
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