CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis

Abstract

CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably acancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+)‘melanoma-initiating cells’ are associated with chemoresistance, contributing to poor patientoutcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magneticactivatedcell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealedthat CD133(+) cells are significantly more invasive than CD133(−) cells. Conditional reprogrammingof BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stemcell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cellsshowed upregulated CD133 expression, and consequently were more invasive and metastatic thanBAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAKR-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAKR-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA orCRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels,doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9concentrations. CD133 may therefore play an essential role in invasion and metastasis viaupregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target forintervention in melanoma.