Reactivation of epigenetically silenced HER4/ERBB4 results in apoptosis of breast tumor cells

Experimental and clinical data support a growth inhibitory role for HER4 in breast cancer. Clinically HER4 expression is extinguished during breast tumorigenesis supporting a tumor suppressor function for HER4, however, a molecular mechanism to explain the selective loss of HER4 expression has remained elusive. Epigenetic mechanisms, for example, aberrant gene promoter hypermethylation, have been shown to ablate tumor suppressor gene expression in breast carcinomas. We identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite-treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Treatment of the HER4-negative BT20 cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC)-enhanced HER4 expression, confirming a role for DNA methylation in suppressed HER4 expression. DAC treatment to reactive HER4 expression in combination with the HER4 ligand heregulin-β1 (HRG) resulted in apoptosis of BT20 cells providing a novel therapeutic strategy for triple-negative tumors. The BT20 cells were rescued from apoptosis when preincubated with HER4 small interfering RNA, thereby confirming a role for HER4 in DAC/HRG-induced apoptosis. We verified HER4 promoter methylation in primary breast carcinomas and detected a significant increase in HER4 promoter methylation in HER4-negative breast tumors (P<0.001). Furthermore, increased levels of HER4 promoter methylation were significantly associated with worse patient prognosis (P=0.0234). Taken together, our data support a tumor suppressor function for HER4, which is epigenetically suppressed in breast tumors through promoter hypermethylation.