ErbB2-driven downregulation of the transcription factor Irf6 in breast epithelial cells is required for their 3D growth

Abstract

BackgroundThe ability of solid tumor cells to resist anoikis, apoptosis triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. ErbB2/Her2 oncoprotein is often overproduced by breast tumor cells and blocks their anoikis by partially understood mechanisms. In our effort to understand them better, we observed that detachment of nonmalignant human breast epithelial cells from the ECM upregulates the transcription factor Irf6. Irf6 is thought to play an important role in mammary gland homeostasis and causes apoptosis by unknown mechanisms. We noticed that ErbB2, when overproduced by detached breast epithelial cells, downregulates Irf6.MethodsTo test whether ErbB2 downregulates Irf6 in human ErbB2-positive breast cancer cells, we examined the effect of ErbB2 inhibitors, such as the anti-ErbB2 antibody trastuzumab or the ErbB2/epidermal growth factor receptor small-molecule inhibitor lapatinib, on Irf6 in these cells. Moreover, we performed Irf6 IHC analysis of tumor samples derived from the locally advanced ErbB2-positive breast cancers before and after neoadjuvant trastuzumab-based therapies. To examine the role of Irf6 in anoikis of nonmalignant and ErbB2-overproducing breast epithelial cells, we studied anoikis after knocking down Irf6 in the former cells by RNA interference and after overproducing Irf6 in the latter cells. To examine the mechanisms by which cell detachment and ErbB2 control Irf6 expression in breast epithelial cells, we tested the effects of genetic and pharmacological inhibitors of the known ErbB2-dependent signaling pathways on Irf6 in these cells.ResultsWe observed that trastuzumab and lapatinib upregulate Irf6 in ErbB2-positive human breast tumor cells and that neoadjuvant trastuzumab-based therapies tend to upregulate Irf6 in human breast tumors. We found that detachment-induced Irf6 upregulation in nonmalignant breast epithelial cells requires the presence of the transcription factor ∆Np63α and that Irf6 mediates their anoikis. We showed that ErbB2 blocks Irf6 upregulation in ErbB2-overproducing cells by activating the mitogen-activated protein kinases that inhibit ∆Np63α-dependent signals required for Irf6 upregulation. Finally, we demonstrated that ErbB2-driven Irf6 downregulation in ErbB2-overproducing breast epithelial cells blocks their anoikis and promotes their anchorage-independent growth.ConclusionsWe have demonstrated that ErbB2 blocks anoikis of breast epithelial cells by downregulating Irf6.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1080-1) contains supplementary material, which is available to authorized users.

The foggy world(s) of p63 isoform regulation in normal cells and cancer

“…Additional support for a negative role of PI3K/Akt signalling comes from perturbation of the epigenetic reader protein BRD4, where p63 levels increased after Akt inhibition or removal of EGF and IGF, which was relieved by inhibiting nuclear FOXO1 activity [128]. MCF10A cells overexpressing either HER2 or an activating Mek mutant also showed reduced ΔNp63 levels in 3D suspension culture, and Mek inhibition increased ΔNp63 in HER2‐overexpressing cells [129]. EGFR/HER2 activation also decreased p63 in non‐neoplastic squamous oesophageal cells [130].…”

“…These discrepant results are difficult to reconcile, but one possibility is the duration/intensity of the signal; stable cell lines with hyperactive Ras/PI3K/Akt associate with reduced p63 [59,69,129], whereas short‐term growth factor exposure associates with increased p63 [121,122,132], as does short‐term inducible expression of K‐RasG12D [94]. This notion is supported by the lack of ΔNp63 downregulation by H‐RasV12 within 2 days of induced expression [59].…”

“…Certain cells showed reduced levels of p63 at low density [54,172] (and see supplementary material, Figure S4), implying cell–cell contact regulation, but this is not universal [195,196]. ΔNp63 is reduced after detachment [129,160,197], suggesting cell–matrix regulation. Indeed, inhibitors of Src/FAK focal adhesion signalling reduced ΔNp63 [197] and Src/FAK signalling from integrins increased ΔNp63, with YAP activation implicated in this pathway [198,199].…”

Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity

“…To search for proteins whose EV levels correlate with breast tumor trastuzumab response, we generated a trastuzumab-resistant variant of ErbB2-positive breast cancer cells BT-474 [18] (BT-474TR) by exposing the cells in three-dimensional (3D) culture (when cells were detached from the extracellular matrix (ECM) [19]) to trastuzumab and expanding the surviving cells in the presence of the drug [19]. We used 3D culture in these and further studies since breast tumors and metastases grow in vivo as 3D masses [20].…”

Screening circulating proteins to identify biomarkers of fetal macrosomia

“…Many of these proteins have been barely studied in placenta (such as XAGE-3, and COBLL1), while others have well reported roles in placental function. For example, CSH1 (or placental lactogen) has an important role in growth control [16], whilst ErbB2 encodes an epidermal growth factor receptor that abundantly localises to the placental surface and reportedly plays important roles in placental function [24]. Similarly, PAPPA1 is involved with insulin-like growth factor binding protein cleavage resulting in the insulin-like growth factor pathway activation [25].…”