Integrin α3β1 Promotes Invasive and Metastatic Properties of Breast Cancer Cells through Induction of the Brn-2 Transcription Factor

Abstract

In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

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

“…Moreover, rescue of α3β1 expression in α3-Cr cells using a doxycyclineinducible α3 model had a minimal effect on the transcriptome, suggesting that use of CRISPR to eliminate α3β1 led to a cellular adaptation such that gene regulation was no longer responsive to the integrin. Interestingly, α3-Cr cells showed reduced cell invasion in vitro and impaired metastatic colonization in vivo compared with control cells, as we reported previously for RNAi-mediated suppression of α3β1 [12,13]. Our results identify a requirement for α3β1 in invasion and metastasis that is independent of its ability to regulate gene expression on a global scale.…”

“…Previously, we showed that RNAi-mediated suppression of ITGA3 in MDA-MB-231 cells substantially reduced cell invasion in vitro and metastatic lung colonization in vivo, and we have shown that these phenotypes are due at least partly to the loss of α3β1-dependent gene regulation in these cells [11][12][13]. However, α3β1 also has important roles in cell adhesion and migration, raising the possibility that this integrin also promotes invasion and/or metastasis independently of its ability to regulate gene expression.…”

“…To build from our previous investigations of integrin α3β1 in promoting pro-tumorigenic functions of TNBC cells [11][12][13], we used CRISPR to target the ITGA3 gene (which encodes the α3 integrin subunit) within the MDA-MB-231 cell line. As α3 is known to partner exclusively with the β1 integrin subunit [1], this strategy was employed to generate cells in which expression of α3β1 is completely ablated (i.e., α3-Cr cells).…”

“…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]. RNAi is widely used to suppress or "knock down" the expression of a target gene through transient expression of a small interfering RNA (siRNA), or through stable expression of a short hairpin RNA (shRNA), that is designed to neutralize the target mRNA transcript and inhibit gene expression or translation [15].…”

“…The laminin-binding integrin α3β1 promotes tumor growth, invasion, and metastasis of breast cancer cells, although this role may be context dependent [27][28][29]. Studies using RNAi to knockdown the α3 integrin subunit in breast cancer cells have identified α3β1-dependent gene regulation that promotes tumor cell growth and invasion [11][12][13]. In the current study, we used CRISPR to target the ITGA3 gene that encodes the α3 integrin subunit in MDA-MB-231 cells, a widely used model of triple-negative breast cancer (TNBC), thereby generating a variant line in which expression of α3β1 is entirely absent (hereafter referred to as α3-Cr cells).…”