Abstract
Integrin receptors for the extracellular matrix play critical roles at all stages of carcinogenesis, including tumor growth, tumor progression and metastasis. The laminin-binding integrin α3β1 is expressed in all epithelial tissues where it has important roles in cell survival, migration, proliferation, and gene expression programs during normal and pathological tissue remodeling. α3β1 signaling and adhesion functions promote tumor growth and metastasis in a number of different types of cancer cells. Previously, we used RNA interference (RNAi) technology to suppress the expression of the ITGA3 gene (encoding the α3 subunit) in the triple-negative breast cancer cell line, MDA-MB-231, thereby generating variants of this line with reduced expression of integrin α3β1. This approach revealed that α3β1 promotes pro-tumorigenic functions such as cell invasion, lung metastasis, and gene regulation. In the current study, we used CRISPR technology to knock out the ITGA3 gene in MDA-MB-231 cells, thereby ablating expression of integrin α3β1 entirely. RNA-seq analysis revealed that while the global transcriptome was altered substantially by RNAi-mediated suppression of α3β1, it was largely unaffected following CRISPR-mediated ablation of α3β1. Moreover, restoring α3β1 to the latter cells through inducible expression of α3 cDNA failed to alter gene expression substantially, suggesting that use of CRISPR to abolish α3β1 led to a decoupling of the integrin from its ability to regulate the transcriptome. Interestingly, both cell invasion in vitro and metastatic colonization in vivo were reduced when α3β1 was abolished using CRISPR, as we observed previously using RNAi to suppress α3β1. Taken together, our results show that pro-invasive/pro-metastatic roles for α3β1 are not dependent on its ability to regulate the transcriptome. Moreover, our finding that use of RNAi versus CRISPR to target α3β1 produced distinct effects on gene expression underlines the importance of using multiple approaches to obtain a complete picture of an integrin’s functions in cancer cells.
Highlights
Integrins are heterodimeric, transmembrane proteins consisting of an α and a β subunit that function as the major cell surface receptors for cell adhesion to the extracellular matrix (ECM) [1]
MDA-MB-231 cells were transfected with purified CRISPR-associated protein 9 (Cas9) protein complexed with a CRISPR RNA (crRNA) that targets exon 1 of the ITGA3 gene
While RNA interference (RNAi) and CRISPR have each been applied to the investigation of integrins, studies are lacking that directly compare resulting phenotypes when these two approaches are used to manipulate expression of the same integrin
Summary
Transmembrane proteins consisting of an α and a β subunit that function as the major cell surface receptors for cell adhesion to the extracellular matrix (ECM) [1]. There are 24 distinct integrins with different ligand binding specificities and signaling functions [1, 2]. Genetic studies using mouse knockout models have revealed distinct phenotypes caused by the deletion of the genes that encode individual α or β subunits, indicating that different integrins have non-redundant, sometimes overlapping functions [2,3,4]. One of the most common genetic approaches to identify functions of a specific integrin is use of RNA interference (RNAi) to suppress the expression of either the α or β subunit, resulting in reduced cell surface expression of the αβ heterodimer [8,9,10,11,12,13,14]. Since some siRNAs/shRNAs can produce off-target effects that may obfuscate the target gene’s function, inclusion of a non-targeting siRNA/shRNA as an experimental control is essential [15, 16]
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