NRG1/ERBB3/ERBB2 Axis Triggers Anchorage-Independent Growth of Basal-like/Triple-Negative Breast Cancer Cells

Abstract

ERBB3, also known as HER3, is a tyrosine kinase transmembrane receptor of the ERBB family. Upon binding to neuregulin 1 (NRG1), ERBB3 preferentially dimerizes with HER2 (ERBB2), in turn inducing aggressive features in several cancer types. The analysis of a dataset of breast cancer patients unveiled that higher ERBB3 mRNA expression correlates with shorter relapse-free survival in basal-like breast cancers, despite low ERBB3 expression in this breast cancer subtype. Administration of neuregulin 1 beta (NRG1β) significantly affected neither cellular proliferation nor the basal migratory ability of basal-like/triple-negative quasi-normal MCF10A breast cells, cultured in mono-layer conditions. Furthermore, no significant regulation in cell morphology or in the expression of basal/myoepithelial and luminal markers was observed upon stimulation with NRG1β. In non-adherent conditions, NRG1β administration to MCF10A cells did not significantly influence cell survival; however, it robustly induced cell growth as spheroids (3D growth). Intriguingly, a remarkable upregulation of ERBB3 and ERBB2 protein abundance was observed in 3D compared to 2D cell cultures, and NRG1β-induced 3D cell growth was efficiently prevented by the anti-HER2 monoclonal antibody pertuzumab. Similar results were obtained by the analysis of basal-like/triple-negative breast cancer cellular models, MDA-MB-468 and MDA-MB-231 cells, in which NRG1β induced anchorage-independent cell growth that in turn was prevented or reduced by the simultaneous administration of anti-HER2 neutralizing antibodies. Finally, the ability of pertuzumab in suppressing NRG1β-induced 3D growth was also evaluated and confirmed in MCF10A engineered with HER2-overexpression. We suggest that the NRG1/ERBB3/ERBB2 pathway promotes the anchorage-independent growth of basal-like breast cancer cells. Importantly, we provide evidence that ERBB2 neutralization, in particular by pertuzumab, robustly inhibits this process. Our results pave the way towards the development of novel anticancer strategies for basal-like breast cancer patients based on the interception of the NRG1/ERBB3/ERBB2 signaling axis.

Neuregulin 4 Boosts the Efficacy of Anti-ERBB2 Neutralizing Antibodies

“…Clinical data supported a link between ERBB4 and increased survival in luminal and HER2+ breast cancer patients. Of note, ERBB4 activation, induced by stimulation with neuregulin 1 (NRG1), has been shown to promote the differentiation and impair the growth of SUM44 breast cancer cells, which belong to the luminal subtype ( 32 ) and express very high levels of ERBB4 ( Supplementary Figure S2B ) and low levels of ERBB2 ( 10 ). Thus, we decided to test whether the activation of ERBB4 signaling by neuregulins may restrain the aggressiveness of HER2+ breast cancers, if ERBB2 is inhibited by anti-HER2 agents.…”

“…Our observation of a positive correlation between ERBB4 mRNA levels and increased relapse-free patients’ survival in the luminal A subtype is in line with a previous study showing that the activation of ERBB4 signaling by NRG1 restrains the growth of breast cancer luminal cells ( 32 ). Importantly, the luminal breast cancer subtype expresses high levels of ERBB4 (please consult Figure 1E ) and ERBB3 ( 10 ) and quite low ERBB2 and EGFR levels ( 50 ) [reviewed in ( 51 )]; by consequence, the activation of ERBB4 by ligands in this breast tumor subtype is expected to preferentially activate ERBB4-ERBB4 homodimers or ERBB4-ERBB3 heterodimers. Apart from a modest reduction in ERBB4 (please consult Figure 1E ), the abundance of ERBB receptors in the luminal B subgroup does not differ that much as compared to luminal A ( 10 ).…”

“…Importantly, the luminal breast cancer subtype expresses high levels of ERBB4 (please consult Figure 1E ) and ERBB3 ( 10 ) and quite low ERBB2 and EGFR levels ( 50 ) [reviewed in ( 51 )]; by consequence, the activation of ERBB4 by ligands in this breast tumor subtype is expected to preferentially activate ERBB4-ERBB4 homodimers or ERBB4-ERBB3 heterodimers. Apart from a modest reduction in ERBB4 (please consult Figure 1E ), the abundance of ERBB receptors in the luminal B subgroup does not differ that much as compared to luminal A ( 10 ). Why patients’ stratification based on ERBB4 abundance showed no difference in survival of breast cancer patients of the luminal B subtype deserves further investigation.…”

“…After 30 minutes of treatments, cells were washed and scrapped in cold RIPA buffer supplemented with a protease inhibitor cocktail (P8340, Sigma-Aldrich, Saint Louis, MO, USA, 1:100) and Na 3 VO 4 (1 mM). Protein extracts were then analyzed by western blotting, as previously described ( 10 ). Briefly, protein lysates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (AmershamTM ProtranTM Premium 0.45 μm 300 mm × 4 m).…”

“…ERBB3-ERBB2 heterodimers are considered the most powerful oncogenic driver units among ERBB potential combinations ( 8 ) [reviewed in ( 1 , 9 )]. Intriguingly, higher ERBB3 expression levels correlate with lower relapse-free survival in basal-like/triple-negative (HER2-/ER-/PR-) breast cancer patients, and NRG1/ERBB3/ERBB2 axis was shown to promote anchorage-independent cell growth in basal-like/triple-negative breast cancer cells ( 10 ). EGFR (also known as ERBB1) is associated with cancer progression in several cancer types, including lung, head and neck, colorectal and pancreatic cancers [reviewed in ( 1 – 3 )].…”