ErbB4/HER4: Role in Mammary Gland Development, Differentiation and Growth Inhibition

The ErbB receptor tyrosine kinase family has often been associated with increased growth of breast epithelial cells, as well as malignant transformation and progression. In contrast, ErbB4/HER4 exhibits unique attributes from a two step proteolytic cleavage which releases an 80 kilodalton, nuclear localizing, tyrosine kinase to a signal transduction mechanism that slows growth and stimulates differentiation of breast cells. This review provides an overview of ErbB4/HER4 in growth and differentiation of the mammary epithelium, including its physiologic role in development, the contrasting growth inhibition/tumor suppression and growth acceleration of distinct ErbB4/HER4 isoforms and a description of the unique cell cycle regulated pattern of nuclear HER4 ubiquitination and destruction

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

Ack1 Mediated AKT/PKB Tyrosine 176 Phosphorylation Regulates Its Activation

The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery

The HER4 Cytoplasmic Domain, But Not Its C Terminus, Inhibits Mammary Cell Proliferation

Unlike the proliferative action of other EGF receptor family members, HER4/ErbB4 is often associated with growth inhibitory and differentiation signaling. These actions may involve HER4 two-step proteolytic processing by intra-membraneous γ-secretase, releasing the soluble, intracellular 80kDa HER4 cytoplasmic domain, s80HER4. We demonstrate that pharmacologic inhibition of either γ-secretase activity or HER4 tyrosine kinase activity blocked heregulin-dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80HER4 (GFP fused to the N-terminus of the HER4 cytoplasmic domain, residues 676–1308), GFP-CTHER4 (GFP fused to N-terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989–1308) or GFP alone. Both GFP-s80HER4 and GFP-CTHER4 were found in the nucleus, but GFP-s80HER4 accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated and treatment of cells with a specific HER family tyrosine kinase inhibitor i) blocked tyrosine phosphorylation; ii) markedly diminished GFP-s80HER4 nuclear localization, and iii) reduced STAT5A tyrosine phosphorylation and nuclear localization as well as GFP-s80HER4:STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80HER4 (SUM44, MDA-MB-453, MCF10A, SUM102, and HC11) were growth inhibited compared to the same cell line expressing GFP-CTHER4, or GFP alone. The s80HER4-induced cell number reduction was due to slower growth, as rates of apoptosis were equivalent in GFP, GFP-CTHER4, and GFP-s80HER4 expressing cells. Lastly, GFP-s80HER4 enhanced differentiation signaling as indicated by increased basal and prolactin-dependent β–casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand-dependent release of s80HER4 are necessary, and s80HER4 signaling is sufficient for HER4-dependent growth inhibition

HER4 D-Box Sequences Regulate Mitotic Progression and Degradation of the Nuclear HER4 Cleavage Product s80HER4

Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80HER4, containing nuclear localization sequences) and results in G2/M delay by unknown signaling mechanisms. We report herein that s80HER4 contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by APC/C, a multisubunit ubiquitin ligase. s80HER4 ubiquitination and ptoteosomal degradation occurred during mitosis but not during S-phase. Inhibition of an APC subunit (APC2) using siRNA knock-down impaired s80HER4 degradation. Mutation of the s80HER4 D-box sequence stabilized s80HER4 during mitosis, and s80HER4-dependent growth inhibition via G2/M delay was significantly greater with the D-box mutant. Polyomvirus middle-T antigen-transformed HC11 cells expressing s80HER4 resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80HER4 or the empty vector. Cells expressing s80HER4 with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80HER4 limits its growth inhibitory action, and stabilization of s80HER4 enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control

Cell Death or Survival Promoted by Alternative Isoforms of ErbB4

The report demonstrates that two distinct isoforms of the ErbB4 receptor tyrosine kinase stimulate either proliferation or apoptosis by mechanisms involving differential transcriptional regulation of the PDGFRA gene. These data have implications for developing approaches to target ErbB4 signaling in cancer

Frequent Gene Amplification Predicts Poor Prognosis in Gastric Cancer

Gastric cancer is one of the most common malignancies worldwide. However, genetic alterations leading to this disease are largely unknown. Gene amplification is one of the most frequent genetic alterations, which is believed to play a major role in the development and progression of gastric cancer. In the present study, we identified three frequently amplified genes from 30 candidate genes using real-time quantitative PCR method, including ERBB4, C-MET and CD44, and further explored their association with clinicopathological characteristics and poor survival in a cohort of gastric cancers. Our data showed amplification of these genes was significantly associated with certain clinicopathological characteristics, particularly tumor differentiation and cancer-related death. More importantly, amplification of these genes was significantly related to worse survival, suggesting that these amplified genes may be significant predictors of poor prognosis and potential therapeutic targets in gastric cancer. Targeting these genes may thus provide new possibilities in the treatment of gastric cancer

SFRP1 reduction results in an increased sensitivity to TGF-β signaling

Background Transforming growth factor (TGF)-β plays a dual role during mammary gland development and tumorigenesis and has been shown to stimulate epithelial-mesenchymal transition (EMT) as well as cellular migration. The Wnt/β-catenin pathway is also implicated in EMT and inappropriate activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway and loss of SFRP1 expression is frequently observed in breast tumors and breast cancer cell lines. We previously showed that when SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells (TERT-siSFRP1) acquire characteristics associated with breast tumor initiating cells. The phenotypic and genotypic changes that occur in response to SFRP1 loss are consistent with EMT, including a substantial increase in the expression of ZEB2. Considering that ZEB2 has been shown to interact with mediators of TGF-β signaling, we sought to determine whether TGF-β signaling is altered in TERT-siSFRP1 cells. Methods Luciferase reporter assays and real-time PCR analysis were employed to measure TGF-β transcriptional targets. Western blot analysis was used to evaluate TGF-β-mediated ERK1/2 phosphorylation. Migration chamber assays were utilized to quantify cellular migration. TERT-siSFRP1 cells were transfected with Stealth RNAi™ siRNA in order to knock-down the expression of ZEB2. Results TERT-siSFRP1 cells exhibit a significant increase in both TGF-β-mediated luciferase activity as well as TGF-β transcriptional targets, including Integrin β3 and PAI-1. Phosphorylation of ERK1/2 is increased in TERT-siSFRP1 cells in response to enhanced TGF-β signaling. Furthermore, when the TGF-β pathway is blocked with a TGF-βR antagonist (LY364947), cellular migration is significantly hindered. Finally, we found that when ZEB2 is knocked-down, there is a significant reduction in the expression of exogeneous and endogenous TGF-β transcriptional targets and cellular migration is impeded. Conclusions We demonstrate that down-regulation of SFRP1 renders mammary epithelial cells more sensitive to TGF-β signaling which can be partially ameliorated by blocking the expression of ZEB2