Comparative use of CRISPR and RNAi to modulate integrin α3β1 in triple negative breast cancer cells reveals that some pro-invasive/pro-metastatic α3β1 functions are independent of global regulation of the transcriptome.

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.