A new model system identifies epidermal growth factor receptor-human epidermal growth factor receptor 2 (HER2) and HER2-human epidermal growth factor receptor 3 heterodimers as potent inducers of oesophageal epithelial cell invasion

Abstract

Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas display distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homo- or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion of early carcinogenesis remains unknown. Here, a new cellular model system for controlled activation of EGFR or HER2 and EGFR/HER2 or HER2/HER3 homo- and heterodimers was studied in non-neoplastic squamous oesophageal epithelial Het-1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA / DmrA and DmrC), and transduced into Het-1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR/HER2, HER2/HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct down-stream signalling pathways, such as PLCγ1, Akt, STAT and Src family kinases. Phenotypically, ErbB homo- and heterodimers caused cell rounding and non-apoptotic blebbing in EGFR/HER2 and HER2/HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2-dimer as compared to empty vector cells. In addition, HER2-dimer cells showed in increased cell invasion, reaching significance for induced HER2/HER3 heterodimers (p=0.015). Importantly, in three-dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2-dimer cells (HER2 homodimers) were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of CK7 (when HER2 homodimers were modelled) and p63 (when EGFR/HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homo- and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three dimensional microenvironment, thereby functionally identifying ErbB homo- and heterodimers as important drivers of oesophageal carcinogenesis.

∆Np63/p40 correlates with the location and phenotype of basal/mesenchymal cancer stem‐like cells in human ER+ and HER2+ breast cancers

“…Of note, these patient samples pre-date the clinical use of trastuzumab. Other studies have shown positive and negative associations between ΔNp63/p40 and HER2 (or other ERBB family members) [18,23,[40][41][42] and it is therefore likely that HER2-ΔNp63/p40 crosstalk accounts for our observations. Investigation of ΔNp63/p40 as a marker of improved prognosis deserves further study, potentially in the setting of HER2 directed clinical trials.…”

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

“…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]. To add further confusion, mutant RasG12V increased and a dominant negative Ras mutant decreased ΔNp63 activity in HaCaT keratinocytes [131], whilst EGF treatment of MCF10A cells [132] and mutant K‐RasG12D in prostate basal cells increased ΔNp63 levels [94].…”

“…Activating the laminin receptor increased ΔNp63 levels in keratinocytes [203] and integrin‐linked kinase (ILK) inhibition reduced ΔNp63 [204]. The ECM component TGFBIp increased ΔNp63 in limbal epithelial cells, also linked to FAK activation [205], whilst decreased FAK was associated with ΔNp63 downregulation in oesophageal squamous cells with EGFR/HER2 activation [130]. These cell–cell and cell–matrix interactions may account for some of the discrepancies on p63 regulation, where growth factors, TGFβ, and other stimuli may cause EMT‐induced loss of cell–cell and/or cell–ECM signalling.…”

The promise of stem cell markers in the diagnosis and therapy of epithelial dysplasia and oral squamous cell carcinoma

“…In a study performed by Chiou et al (2008), the expression of EGFR1 was increased in nearly 90% of the OSCC cases. Aggressive tumor behavior, radiation resistance, and poor prognosis are associated with the enhanced expression of EGFR1 (Fichter et al, 2017;Olayioye, Neve, Lane, & Hynes, 2000). EGFR activates several downstream pathways through binding to growth factors and causing conformational changes in the extracellular domain (Sandfeld-Paulsen, Folkersen, Rasmussen, Meldgaard, & Sorensen, 2016).…”

“…Moreover, such mutations predispose to numerous malignancies including breast, lung, colorectal, stomach, pancreatic, oral and head and neck cancers (Hynes, 2016). Fichter et al (2017) demonstrated that the activation of EGFR, HER2, and HER3 homo-and hetero-dimers resulted in the migration and invasion of epithelial cells. They concluded that ErbB homo-and hetero-dimers played a key role in most cancers.…”