Establishing a CRISPR/Cas9 Based System Designed to Explore the Similarities and Differences between Lysyl Oxidases

Lysyl-oxidases catalyze the covalent cross-linking of collagens and contribute to the stabilization of extracellular matrixes. The LOX family contains five genes (LOX, LOXL1-4). They are characterized by a highly conserved C-terminal catalytic domain that includes a copper binding domain. Several lysyl-oxidase family members such as LOX and LOXL2 were found to promote tumor metastasis, and tumor angiogenesis. They were found to promote tumor progression by diverse mechanisms that include induction of epithelial to mesenchymal transition (EMT) and extracellular matrix remodeling that results in enhanced extracellular matrix stiffness resulting in enhanced tumor cell invasiveness and tumor metastasis. However, it is unclear if different lysyl-oxidases all promote tumor progression or if differences between individual lysyl-oxidases result in different effects of individual lysyl-oxidases on tumor progression.

We created novel MDA-MB-231 and LM2-4 cell lines in which we have knocked-out the five LOX family genes, using the CRISPR/Cas9 method. This resulted in the generation of viable LM2-4 and MDA-MB-231 cell lines that do not express any lysyl-oxidases. These cells displayed significant inhibition of invasive ability and were unable to form colonies in soft agar. To identify similarities and differences between the different lysyl-oxidases we re-expressed in these cells cDNAs encoding individual lysyl-oxidases and we then examined the effects of the re-expression on gene expression profiles using deep sequencing and mass spectrometry. This resulted in the identification of several genes whose expression was regulated by individual lysyl-oxidases.