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Article

Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes

Cancer Research Center, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2022, 23(19), 11322; https://doi.org/10.3390/ijms231911322
Submission received: 14 August 2022 / Revised: 12 September 2022 / Accepted: 17 September 2022 / Published: 26 September 2022

Abstract

The five lysyl-oxidase genes share similar enzymatic activities and contribute to tumor progression. We have knocked out the five lysyl-oxidase genes in MDA-MB-231 breast cancer cells using CRISPR/Cas9 in order to identify genes that are regulated by LOX but not by other lysyl-oxidases and in order to study such genes in more mechanistic detail in the future. Re-expression of the full-length cDNA encoding LOX identified four genes whose expression was downregulated in the knock-out cells and rescued following LOX re-expression but not re-expression of other lysyl-oxidases. These were the AGR2, STOX2, DNAJB11 and DNAJC3 genes. AGR2 and STOX2 were previously identified as promoters of tumor progression. In addition, we identified several genes that were not downregulated in the knock-out cells but were strongly upregulated following LOX or LOXL3 re-expression. Some of these, such as the DERL3 gene, also promote tumor progression. There was very little proteolytic processing of the re-expressed LOX pro-enzyme in the MDA-MB-231 cells, while in the HEK293 cells, the LOX pro-enzyme was efficiently cleaved. We introduced point mutations into the known BMP-1 and ADAMTS2/14 cleavage sites of LOX. The BMP-1 mutant was secreted but not cleaved, while the LOX double mutant dmutLOX was not cleaved or secreted. However, even in the presence of the irreversible LOX inhibitor β-aminoproprionitrile (BAPN), these point-mutated LOX variants induced the expression of these genes, suggesting that the LOX pro-enzyme has hitherto unrecognized biological functions.
Keywords: lysyl-oxidase; breast cancer lysyl-oxidase; breast cancer

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MDPI and ACS Style

Liburkin-Dan, T.; Nir-Zvi, I.; Razon, H.; Kessler, O.; Neufeld, G. Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes. Int. J. Mol. Sci. 2022, 23, 11322. https://doi.org/10.3390/ijms231911322

AMA Style

Liburkin-Dan T, Nir-Zvi I, Razon H, Kessler O, Neufeld G. Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes. International Journal of Molecular Sciences. 2022; 23(19):11322. https://doi.org/10.3390/ijms231911322

Chicago/Turabian Style

Liburkin-Dan, Tatyana, Inbal Nir-Zvi, Hila Razon, Ofra Kessler, and Gera Neufeld. 2022. "Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes" International Journal of Molecular Sciences 23, no. 19: 11322. https://doi.org/10.3390/ijms231911322

APA Style

Liburkin-Dan, T., Nir-Zvi, I., Razon, H., Kessler, O., & Neufeld, G. (2022). Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes. International Journal of Molecular Sciences, 23(19), 11322. https://doi.org/10.3390/ijms231911322

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