Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

Abstract

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.

Introduction

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTK), a family that also includes the Epidermal Growth Factor (EGF) Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These family members share organizational homology; they all contain an extracellular ligand-binding domain, a hydrophobic single-pass transmembrane domain, and an intracellular tyrosine kinase domain. The agonists for these receptors are members of the EGF family of peptide hormones, which consists of more than 20 different members [1], [2], [3]. The signaling network composed of these RTKs and their complementary peptide hormones regulates many cellular functions, including proliferation, survival, differentiation, motility, growth arrest, and apoptosis [4], [5], [6]. Moreover, deregulation of this network, typically due to inappropriate receptor or ligand expression, often plays a significant role in tumorigenesis [7], [8], [9], [10], [11].

EGFR, ErbB2, and ErbB3 overexpression is observed in many tumor types and this overexpression is associated with the progression and malignancy of these tumors [2], [7], [12], [13], [14], [15]. The roles that ErbB4 plays in tumorigenesis remain topics of some controversy. Some studies indicate that ErbB4 expression is an adverse prognostic factor in breast cancer [16], [17], [18], [19], [20]. Moreover, ErbB4 ligands stimulate the proliferation of some human tumor cell lines [17], [21], [22]. Thus, these studies support the hypothesis that ErbB4 is an oncogene. However, other studies indicate that ErbB4 expression correlates with a favorable outcome for breast cancer patients [23], [24], [25], [26], [27], [28]. Furthermore, the ErbB4 ligand neuregulin (NRG) induces differentiation of the mammary epithelium into secretory lobuloalveoli in vivo [22] and the ErbB4 ligands NRG and heparin-binding EGF-like growth factor (HB-EGF) induce growth arrest and differentiation in some human breast cancer cell lines in vitro [29], [30], [31], [32]. Moreover, betacellulin, an ErbB4 ligand endogenous to the pancreas, induces differentiation of intra islet precursor cells to β-cells in vivo [33] and together with activin-A causes differentiation of exocrine AR42J rat pancreatic tumor cells into insulin-secreting cells [34], [35]. These data indicate that ErbB4 signaling may couple to terminal differentiation and growth arrest and that ErbB4 may be a tumor suppressor. Consistent with this model, published and unpublished data from our laboratory indicate that the constitutively-active ErbB4 Q646C mutant inhibits clonogenic proliferation by human breast and prostate tumor cell lines [25], [26], [36]. Introduction of a glutamate residue into the transmembrane domain of ErbB4 results in constitutive ErbB4 dimerization, tyrosine phosphorylation, and coupling to apoptosis in a variety of cancer cell lines [37]. The s80 ICD, formed when the ErbB4 intracellular domain is released from the membrane by α and γ-secretase following ligand stimulation, forms tyrosine phosphorylated homodimers that inhibit cellular proliferation [38], [39].

Pancreatic cancer is one of the predominant cancers in developed countries. It is the fourth leading cause of cancer death in the United States and the sixth leading cause of cancer death in Europe [40]. Indeed, it has been estimated that approximately 43,000 people in the United States will be diagnosed with pancreatic cancer in 2010 and that approximately 37,000 Americans will die from this disease [41]. The median survival time of pancreatic cancer patients usually does not exceed 6 months [42]. Late diagnosis, chemoresistance, and radioresistance of these tumors are the main reasons for poor patient outcome [43], [44].

The deregulation of several signaling networks has been associated with the malignant growth transformation of pancreatic tumor cells. Examples include a gain-of-function mutation of the c-K-ras oncogene [45], a dominant negative mutation of the p53 tumor suppressor gene [46], [47], a loss-of-function mutation of the p16 tumor suppressor gene, deletion of the DPC4 tumor suppressor gene [47] and overexpression of growth factors [48], [49], [50] and their receptors, including EGFR [50], ErbB2 [51], and ErbB3 [52].

The roles that ErbB4 plays in pancreatic cancer have not been determined. However, ErbB4 transcription is decreased in the early stages of pancreatic cancer, indicating that loss of ErbB4 expression may be a prerequisite for tumorigenesis [53]. Indeed, ErbB4 expression in pancreatic tumor cells correlates with favorable staging [54]. However, an alternative explanation for the expression data is that ErbB4 expression may merely be a marker for the proliferative or differentiation state of these cells. To address this issue, we have determined the level of ErbB4 expression and basal ErbB4 signaling (basal ErbB4 tyrosine phosphorylation) in four human pancreatic tumor cell lines. We have also assessed the effect of a constitutively-dimerized and constitutively-active ErbB4 mutant on clonogenic proliferation of these cell lines. Finally, we have evaluated the effect of wild-type ErbB4 expression on the stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1β (NRG1β). The data presented indicate that ErbB4 has multiple functions and suggest that ErbB4 functions as a context-sensitive tumor suppressor and oncogene.