The Yin and Yang of ERBB4: Tumor Suppressor and Oncoprotein.

ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are oncoproteins and validated targets for therapeutic intervention in a variety of solid tumors. In contrast, the role that ERBB4 plays in human malignancies is ambiguous. Thus, here we review the literature regarding ERBB4 function in human malignancies. We review the mechanisms of ERBB4 signaling with an emphasis on mechanisms of signaling specificity. In the context of this signaling specificity, we discuss the hypothesis that ERBB4 appears to function as a tumor suppressor protein and as an oncoprotein. Next, we review the literature that describes the role of ERBB4 in tumors of the bladder, liver, prostate, brain, colon, stomach, lung, bone, ovary, thyroid, hematopoietic tissues, pancreas, breast, skin, head, and neck. Whenever possible, we discuss the possibility that ERBB4 mutants function as biomarkers in these tumors. Finally, we discuss the potential roles of ERBB4 mutants in the staging of human tumors and how ERBB4 function may dictate the treatment of human tumors. SIGNIFICANCE STATEMENT: This articles reviews ERBB4 function in the context of the mechanistic model that ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR or ERBB4-ERBB2 heterodimers act as oncogenes. Thus, this review serves as a mechanistic framework for clinicians and scientists to consider the role of ERBB4 and ERBB4 mutants in staging and treating human tumors.

Multiple Functional Motifs Are Required for the Tumor Suppressor Activity of a Constitutively-Active ErbB4 Mutant.

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, which includes the Epidermal Growth Factor Receptor (EGFR/ErbB1), ErbB2 (HER2/Neu), and ErbB3 (HER3). Mounting evidence indicates that ErbB4, unlike EGFR or ErbB2, functions as a tumor suppressor in many human malignancies. Previous analyses of the constitutively-dimerized and -active ErbB4 Q646C mutant indicate that ErbB4 kinase activity and phosphorylation of ErbB4 Tyr1056 are both required for the tumor suppressor activity of this mutant in human breast, prostate, and pancreatic cancer cell lines. However, the cytoplasmic region of ErbB4 possesses additional putative functional motifs, and the contributions of these functional motifs to ErbB4 tumor suppressor activity have been largely underexplored. Here we demonstrate that ErbB4 BH3 and LXXLL motifs, which are thought to mediate interactions with Bcl family proteins and steroid hormone receptors, respectively, are required for the tumor suppressor activity of the ErbB4 Q646C mutant. Furthermore, abrogation of the site of ErbB4 cleavage by gamma-secretase also disrupts the tumor suppressor activity of the ErbB4 Q646C mutant. This last result suggests that ErbB4 cleavage and subcellular trafficking of the ErbB4 cytoplasmic domain may be required for the tumor suppressor activity of the ErbB4 Q646C mutant. Indeed, here we demonstrate that mutants that disrupt ErbB4 kinase activity, ErbB4 phosphorylation at Tyr1056, or ErbB4 cleavage by gamma-secretase also disrupt ErbB4 trafficking away from the plasma membrane and to the cytoplasm. This supports a model for ErbB4 function in which ErbB4 tumor suppressor activity is dependent on ErbB4 trafficking away from the plasma membrane and to the cytoplasm, mitochondria, and/or the nucleus.

Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation.

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with extraordinary sensitivity to targeted tyrosine kinase inhibitors (TKI). A single secondary EGFR mutation, T790M, arising in cis with the primary activating mutation, confers acquired resistance to these drugs. However, the T790M mutation is also detected in the absence of drug selection, suggesting that it may provide a growth advantage. We show here that although T790M alone has only a modest effect on EGFR function, when combined with the characteristic activating mutations L858R or del746-750, it results in a dramatic enhancement of EGFR activity. The double mutants show potent ligand-independent receptor autophosphorylation associated with altered cellular phenotypes, soft agar colony formation, and tumorigenesis in nude mice. The significant gain-of-function properties of these double mutants may explain their initial presence before drug selection and their rapid selection as the single drug resistance mutation during therapy with gefitinib/erlotinib, and suggests that they may contribute to the adverse clinical course of TKI-resistant NSCLC.

Phosphorylation of ErbB4 on tyrosine 1056 is critical for ErbB4 coupling to inhibition of colony formation by human mammary cell lines.

In many studies, ErbB4 expression in breast tumor samples correlates with a favorable patient prognosis. Similarly, ErbB4 signaling is coupled to cellular differentiation and growth arrest in a variety of model systems. However, in some studies, ErbB4 expression in breast tumor samples correlates with poor outcome. Likewise, studies using some human mammary tumor cell lines suggest that ErbB4 is coupled to malignant phenotypes. Thus, the roles that ErbB4 plays in human breast cancer are still poorly defined. Here we demonstrate that a constitutively active ErbB4 mutant (ErbB4-Q646C) inhibits colony formation on plastic by two human mammary tumor cell lines (SKBR3 and MCF7) and by the MCF10A immortalized human mammary cell line, but does not inhibit colony formation by the MDA-MB-453 and T47D human mammary tumor cell lines. ErbB4 kinase activity is necessary for ErbB4 function and phosphorylation of ErbB4 Tyr1056 is necessary and appears to be sufficient for ErbB4 function. The inhibition of colony formation by MCF10A cells is accompanied by growth arrest but not cell death. These data suggest that ErbB4 behaves as a mammary tumor suppressor and that loss of ErbB4 coupling to growth arrest may be an important event in mammary tumorigenesis.

Phosphorylation of ErbB4 on Tyr1056 is critical for inhibition of colony formation by prostate tumor cell lines.

We have previously demonstrated that the constitutively active Q646C mutant of the ErbB4 receptor tyrosine kinase inhibits colony formation by human prostate tumor cell lines. Here we use ErbB4 mutants to identify ErbB4 functions critical for inhibiting colony formation. A derivative of ErbB4 Q646 that lacks kinase activity fails to inhibit colony formation by prostate tumor cells. Likewise, an ErbB4 Q646C mutant in the context of the CT-b splicing isoform fails to inhibit colony formation. Mutation of tyrosine 1056 to phenylalanine abrogates inhibition of colony formation whereas an ErbB4 mutant that lacks all of the putative sites of tyrosine phosphorylation except for tyrosine 1056 still inhibits colony formation. Given that tyrosine 1056 is missing in the CT-b isoform, these results suggest that phosphorylation of tyrosine 1056 is critical for function. Indeed, an ErbB4 mutant that lacks kinase activity but has a glutamate phosphomimic residue substituted for tyrosine 1056 inhibits colony formation. Finally, 1-dimensional phosphopeptide mapping indicates that ErbB4 Q646C is phosphorylated on tyrosine 1056. These data suggest that phosphorylation of ErbB4 tyrosine 1056 is critical for coupling ErbB4 to prostate tumor suppression.

Anilinodialkoxyquinazolines: screening epidermal growth factor receptor tyrosine kinase inhibitors for potential tumor imaging probes.

The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogues, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A new EGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC50 = 0.4-51 nM) for the EGFR tyrosine kinase. All of the analogues inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 = 0.8-20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2 to 5.5. Four compounds, 4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3'-chloroanilino)- and 4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice.

A constitutively active ErbB4 mutant inhibits drug-resistant colony formation by the DU-145 and PC-3 human prostate tumor cell lines.

ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases, a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. Several groups have hypothesized that signal transduction by the ErbB4 receptor tyrosine kinase is coupled to differentiation, growth arrest, and tumor suppression in mammary and prostate epithelial cells. In this report we demonstrate that a constitutively active ErbB4 mutant inhibits the formation of drug-resistant colonies by the DU-145 and PC-3 human prostate tumor cell lines. This is consistent with our hypothesis that ErbB4 signaling is growth inhibitory and may be coupled to tumor suppression in prostate cells.

Constitutively active ErbB4 and ErbB2 mutants exhibit distinct biological activities.

ErbB4 is a member of the epidermal growth factor receptor(EGFR) family of tyrosine kinases, which includes EGFR/ErbB1, ErbB2/HER2/Neu, and ErbB3/HER3. These receptors play important roles both in normal development and in neoplasia. For example, deregulated signaling by ErbB1 and ErbB2 is observed in many human malignancies. In contrast, the roles that ErbB4 plays in tumorigenesis and normal biological processes have not been clearly defined. To identify the biological responses that are coupled to ErbB4, we have constructed three constitutively active ErbB4 mutants. Unlike a constitutively active ErbB2 mutant, the ErbB4 mutants are not coupled to increased cell proliferation, loss of contact inhibition, or anchorage independence in a rodent fibroblast cell line. This suggests that ErbB2 and ErbB4 may play distinct roles in tumorigenesis in vivo.