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Open AccessArticle

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

1
Department of Biochemistry and Molecular & Cellular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA
2
Amelia Technologies, Rockville, MD 20850, USA
3
Department of Oncology, Georgetown University School of Medicine, Washington, DC 20007, USA
4
Department of Pathology, Georgetown University School of Medicine, Washington, DC 20007, USA
*
Author to whom correspondence should be addressed.
Cancers 2019, 11(10), 1490; https://doi.org/10.3390/cancers11101490
Received: 8 September 2019 / Revised: 26 September 2019 / Accepted: 1 October 2019 / Published: 3 October 2019
(This article belongs to the Special Issue Cancer Invasion and Metastasis)
CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) ‘melanoma-initiating cells’ are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(−) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma. View Full-Text
Keywords: melanoma initiating cells; invasion; metastasis; matrix metalloproteinases; CRISPR-Cas9 knockdown melanoma initiating cells; invasion; metastasis; matrix metalloproteinases; CRISPR-Cas9 knockdown
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Simbulan-Rosenthal, C.M.; Dougherty, R.; Vakili, S.; Ferraro, A.M.; Kuo, L.-W.; Alobaidi, R.; Aljehane, L.; Gaur, A.; Sykora, P.; Glasgow, E.; Agarwal, S.; Rosenthal, D.S. CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis. Cancers 2019, 11, 1490.

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