The role of ErbB4 in cancer

Abstract

Background

The epidermal growth factor receptor family consists of four members, ErbB1 (epidermal growth factor receptor-1), ErbB2, ErbB3, and ErbB4, which all have been found to play important roles in tumor development. ErbB4 appears to be unique among these receptors, because it is the only member with growth inhibiting properties. ErbB4 plays well-defined roles in normal tissue development, in particular the heart, the nervous system, and the mammary gland system. In recent years, information on the role of ErbB4 in a number of tumors has emerged and its general direction points towards a tumor suppressor role for ErbB4. However, there are some controversies and conflicting data, warranting a review on this topic.

Conclusions

Here, we discuss the role of ErbB4 in normal physiology and in breast, lung, colorectal, gastric, pancreatic, prostate, bladder, and brain cancers, as well as in hepatocellular carcinoma, cholangiocarcinoma, and melanoma. Understanding the role of ErbB4 in cancer is not only important for the treatment of tumors, but also for the treatment of other disorders in which ErbB4 plays a major role, e.g. cardiovascular disease.

Introduction

The ErbB family of receptor tyrosine kinases consists of four members, i.e., the epidermal growth factor receptor (EGFR/Erythroblastic leukemia viral oncogene homolog 1 or ErbB1), ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 (Fig. 1). In the last 3 decades, investigators have linked different members of the ErbB family to the causation and/or progression of various epithelial malignancies [1–3]. Overexpression and activation of ErbB receptors is often associated with poor patient outcomes and advanced tumor states [1] and has been proven therapeutically relevant in breast, colon, and other tumors [1]. Small-molecule tyrosine kinase inhibitors and monoclonal antibodies targeting ErbB1 and ErbB2 have been developed and successfully used to treat cancer patients [4, 5]. Furthermore, novel data indicate that somatic mutations in ErbB2 and ErbB3 are present in a wide variety of tumors [6]. Based on this information, the ErbB receptor family is firmly linked to cell proliferation and oncogenic events. The notable exception is ErbB4, the only member of the ErbB receptor family with growth inhibiting properties. One example is the growth inhibiting and differentiation stimulating effects of ErbB4 signaling on mammary gland epithelial cells [1]. Whereas the other members of the ErbB family are linked to aggressive forms of different cancer types, ErbB4 expression has been found to be downregulated in aggressive tumors [7]. Data from the Cancer Genome Atlas indicate that, in contrast to other ErbB receptors, in different cancer types (including lung, esophageal, cervical, and bladder carcinoma) a large number of cases shows loss of ErbB4 gene copy numbers, while only in ovarian adenocarcinoma there seems to be a gain in ErbB4 gene copy numbers (Fig. 2). These data indicate that loss of ErbB4 function may promote tumor growth in many types of cancer. A similar pattern can be deduced from mRNA expression analyses of the different ErbB receptors in tumor-derived cell lines. The Cancer Cell Line Encyclopedia (CCLE) provides RNA sequencing data from over 1100 tumor cell lines. These data indicate that ErbB4 mRNA expression is only present in a small fraction of tumor cell lines, whereas the other ErbB receptors are expressed at high levels in the majority of tumor cell lines (Fig. 3). The controversies around the anti- or pro-oncogenic role of ErbB4 can in part be explained by the multiple ligands that can activate ErbB4, its numerous intracellular phosphorylation sites, the presence of alternative splice variants, the different intracellular signaling pathways affected, and the different downstream responses in different cell types and in different disease stages.

Fig. 1.

Different ErbB receptors and their ligands. ErbB2 has a defective ligand binding domain and ErbB3 does not have a tyrosine kinase domain. Phosphorylation of ErbB3 occurs through transphosphorylation by the tyrosine kinase domain on the dimerization partner

Fig. 2.

Data from the Cancer Genome Atlas (TCGA) on ErbB receptor copy number variation in different forms of cancer. (A) Number of cases per cancer type within the TCGA with gain of copy number of different receptors. (B) Number of cases per cancer type within the TCGA with loss of copy number of different receptors. Threshold is set at deletion of at least one copy of a particular ErbB gene

Fig. 3.

mRNA expression levels in different tumor cell lines. Data from the Cancer Cell Line Encyclopedia (CCLE). (A) EGFR (ErbB1) expression in the X-axis and ErbB4 expression in the Y-axis, showing a high EGFR expression in most tumor cell lines compared to a low ErbB4 expression in most tumor cell lines. (B) ErbB2 expression in the X-axis and ErbB4 expression in the Y-axis, showing a high ErbB2 expression in most tumor cell lines (even more pronounced than EGFR in panel A) compared to a low ErbB4 expression in most tumor cell lines. (C) ErbB3 expression in the X-axis and ErbB4 expression in the Y-axis, showing a high ErbB3 expression in most tumor cell lines (in a pattern similar to EGFR) compared to a low ErbB4 expression in most tumor cell lines

Role of ErbB4 in embryonic development and adult physiology

ErbB4 plays important roles during the embryonic development of many different organs and has been found to be indispensable for normal cardiac development, as ErbB4 null mice die by embryonic day 11 due to defective heart development [8, 9]. When ErbB4 expression in the heart is rescued by expressing ErbB4 under a cardiac-specific myosin promoter in ErbB4-null mice, the mutant mice reach adulthood and are fertile. During pregnancy, however, the mammary glands of these mutant mice fail to differentiate correctly, resulting in defective lactation [8]. ErbB4-null mice with rescued cardiac ErbB4 expression also display aberrant cranial nerve and cerebellar architectures [8]. Accordingly, the ErbB4 signaling pathway has been found to regulate many biological processes in neurodevelopment, including neuronal migration, dendritic and glial cell development, axon myelination and guidance, and neurotransmitter signaling [10].

Data derived from the Genotype-Tissue Expression (GTEx) project, which contains deep phenotyping data from adult human samples from a wide variety of tissues, show that ErbB4 mRNA expression is particularly high in samples from different regions of the brain, mammary tissue, large arteries, heart, kidneys, testis, and thyroid (Fig. 4). Expression of ErbB2 in the cardiovascular system matches ErbB4 expression, whereas ErbB3 expression is low (Fig. 4). Both ErbB4 and its ligand neuregulin-1 (NRG-1) are highly expressed in different parts of the nervous system (Fig. 4). The neuropsychiatric disease entity most commonly linked to ErbB4 signaling is schizophrenia [10]. Both the NRG1 and ERBB4 genes have been linked to schizophrenia by multiple association studies [10]. Accordingly, it has been found that impaired ErbB4 signaling in transgenic mouse models induces behavioral abnormalities relevant to schizophrenia and that these abnormalities can be alleviated by antipsychotic treatment [10].

Fig. 4.

ErbB4 expression levels in different human tissues. ErbB4 expression is particularly high in arteries, different regions of the brain, mammary tissue, heart, kidneys, testis, and thyroid. Data derived from The Genotype-Tissue Expression (GTEx) project. TPM: transcripts per kilobase million

ErbB1, ErbB2, and ErbB3 all stimulate the proliferation of mammary epithelial cells during puberty and/or pregnancy. ErbB4 signaling is unique among the ErbB receptors in the mammary epithelium, i.e., it is not required for driving epithelial cell proliferation but, instead, induces the differentiation of epithelial cells during pregnancy and in vitro in cell culture [1]. Studies on primary mammary tissues of humans, mice, and rats indicate that ErbB4 expression is lowest during epithelial cell proliferation (puberty and early pregnancy) and highest during differentiation (late pregnancy and early lactation) [11]. Moreover, mammary glands from multiple transgenic mice with a mammary-specific ErbB4 deletion show lactation defects, as measured by a decrease in the expression of milk proteins, due to impaired cellular differentiation [8, 12, 13]. Overall, these data provide strong evidence that ErbB4 directs the differentiation, but not proliferation, of the mammary epithelium.

In the heart, NRG-1 that is secreted by endothelial cells [14] has been found to activate ErbB4 receptors on cardiomyocytes and fibroblasts in a paracrine manner. Thus, the NRG-1/ErbB4 axis serves as an important regulator of cardiac homeostasis [14–17]. Moreover, it has become clear in the last 15 years that NRG-1 exhibits protective properties in the cardiovascular system, and that most of these protective properties are mediated by activation of ErbB4 receptors [5, 16, 18–20]. Based on a large preclinical dataset [21, 22], NRG-1 is currently tested in phase 3 clinical trials for the treatment of heart failure (NCT03388593).

Recently, also the anti-inflammatory and anti-fibrotic properties of ErbB4 activation have become apparent. Activation of ErbB4 by NRG-1 has been found to elicit anti-fibrotic effects in multiples tissues, including the heart, lung, skin, and kidney [23–25]. These anti-fibrotic effects have been linked to anti-inflammatory effects of ErbB4 activation [24]. It has also been shown that activation of ErbB4 on macrophages inhibits the release of inflammatory cytokines [24]. The anti-inflammatory effects of ErbB4 may potentially contribute to the inhibition of cancer development, since chronic tissue inflammation is a risk factor for the development of different types of cancer.

Ligands of the ErbB4 receptor

The signaling of the ErbB receptor system is complex, because at least 10 different ligands can activate one or more ErbB receptors, which can form homodimers (ErbB1 and ErbB4) or heterodimers (mostly ErbB1/ErbB2, ErbB3/ErbB2, and ErbB4/ErbB2) that activate different intracellular signaling pathways [5, 26]. Spontaneous dimerization of ErbB4 has been described for certain mutants of the receptor. As such, the constitutively dimerized ErbB4 Q646C mutant has been found to inhibit the proliferation of several pancreatic tumor cell lines [27], as also breast and prostate cancer cell lines [28, 29], suggesting that ErbB4 acts as a tumor suppressor.

Most ErbB ligands share an EGF-like domain and 7 of them have been shown to activate ErbB4 [18, 19]. Betacellulin, heparin binding-EGF, and epiregulin activate both ErbB4 and ErbB1 receptors, NRG-1, and NRG-2 activate both ErbB4 and ErbB3, and NRG-3 and NRG-4 activate only ErbB4 [1]. Besides these 7 classic ligands, ErbB4 can also be regulated by non-conventional agonists. For instance, crypto-1 has been found to stimulate the phosphorylation of ErbB4 without directly binding to it [30], and also tomoregulin has been found to act as a weak activator of ErbB4 [31]. However, the exact roles of these non-conventional ErbB ligands in ErbB4 biology and/or cancer biology are currently unclear.

Exciting data have been published indicating that forced homodimerization of ErbB4 with an engineered bivalent NRG-1, consisting of 2 NRG-1 moieties linked by a long spacer, has growth inhibiting effects on different tumor cell types [32]. These results indicate that bivalent NRG-1 can alter ErbB4 signaling in cancer cells, resulting in their decreased migration and proliferation, whereas native NRG-1 stimulation increases the malignant potential of the same cells. This novel approach may result in new therapies for ovarian, breast, lung, and other cancers [32].

The complexity of the system is further exemplified by the fact that a single ligand can either induce growth stimulation or growth inhibition, depending on tumor cell type and disease state. NRG-1 has, for instance, been shown to stimulate the growth of breast tumor cells and to induce resistance to lapatinib (an ErbB1 and ErbB2 inhibitor) [33], but also to inhibit growth of ErbB2-overexpressing breast tumor cells [34].

Different isoforms of the ErbB4 receptor

ErbB4 is unique among the ErbB receptors by undergoing alternative splicing. Four different alternative splice variants of ErbB4 mRNA have been described based on 2 splicing sites (Fig. 5): the first alternative splicing site is located in the extracellular juxtamembrane region (JMa or JMb) [35] and the second alternative splicing site is located in the cytosolic C-terminus, distal to the tyrosine kinase domain (Cyt1 and Cyt2) [36]. Thus, ErbB4 may be present as four distinct isoforms: JMa-Cyt1, JMa-Cyt2, JMb-Cyt1, and JMb-Cyt2 [9]. The JMa isoform, but not the JMb isoform, has an extracellular proteolytic site which can be cleaved by a metalloprotease known as tumor necrosis factor-alpha converting enzyme (TACE) [37]. Cleavage by TACE occurs upon ligand-activation of ErbB4 [38]. The JMb isoform cannot be cleaved by TACE, because it lacks the cleavage site. The second alternative splicing site in ErbB4 leads to either the Cyt1 or the Cyt2 isoform. Unlike ErbB4-Cyt1, ErB4-Cyt2 lacks amino acids 1046 to 1061 from the canonical sequence, including the phosphoinositide 3-kinase (PI3-K) binding site and, thus, cannot activate the PI3-K signaling pathway [36]. Experiments in NIH3T3 cells have shown that, in contrast to ErB4-Cyt1, ErB4-Cyt2 can mediate proliferation but not chemotaxis or survival [19, 36].

Fig. 5.

Different isoforms of ErbB4. The juxtamembrane a (JMa) isoform can be cleaved by TACE in a region close to the membrane and subsequently by γ-secretase, leading to the release of the intracellular domain (ErbB4-ICD). The JMb isoform cannot be cleaved by TACE. Compared to the Cyt2 isoform, the Cyt1 isoform has an extra exon in the intracellular domain containing tyrosine 1056, which when phosphorylated functions as a binding site for PI3K

Many studies indicate that mammary tumors expressing ErbB4 generally present with a more favorable prognosis [1, 39] and, therefore, that ErbB4 may decrease tumor formation and/or progression through growth inhibition and enhanced differentiation. However, some reports have shown an unfavorable prognostic association of ErbB4 expression with breast cancer [40–42]. This discrepancy might be due to expression of different ErbB4 splice variants [43]. Supporting this hypothesis, overexpression of either the ErbB4-Cyt1 or the ErbB4-Cyt2 isoform in HC11 mammary epithelial cells has been found to result in opposite effects on the growth and formation of colonies [43]. The Cyt1 isoform differs from the Cyt2 isoform only by containing an extra 16 amino acids strand, which allows the binding of PI3K. Expression of ErbB4-Cyt1 results in growth inhibition and differentiation of ductal epithelium cells and this may explain why ErbB4 expression correlates with a more favorable prognosis in many breast cancer studies [43]. In contrast with these data, however, sustained overexpression of the Cyt1 but not of the Cyt2 isoform in transgenic mice has been found to result in the formation of neoplastic mammary lesions, suggesting a potential oncogenic function for the Cyt1 isoform [44]. Also, in vitro and in vivo data support a growth promoting role of ErbB4 [39]. It has, for instance, been shown that ErbB4 JMa-Cyt2 overexpression enhances the growth of human breast cancer cells [45]. Also, downregulation of ErbB4 with ribozymes or siRNAs has been found to reduce the growth of T47D and MCF-7 breast cancer cells, both in vitro and in immunocompromised mice [45, 46]. In patients with bladder cancer, the JMa-Cyt2 isoform of ErbB4 has been shown to have a protective role [47]. Phosphorylation of tyrosine 1056 (in the Cyt1 isoform) has been found to be crucial for the inhibition of colony formation by prostate tumor cells [48], again highlighting that different ErbB4 isoforms may exhibit differential cellular effects.

Regulation of expression by miRNAs

In recent years, it has been shown that the expression of several genes is regulated by micro-RNAs (miRNAs, miRs). Novel data from multiple studies indicate that ErbB4 mRNA levels are regulated by different miRNAs, including miR-146a [49], miR-551b [50], and miR-302b [51]. As yet, only few studies have investigated the role of ErbB4-targeting miRNAs on tumor growth; more can be expected in the near future. It has been suggested that, although ERBB4 mRNA levels are unchanged in lung cancer cells, there may exist differences in ErbB4 protein levels [52]. The ErbB4 protein levels have also been found to be negatively regulated by miR-193a-3p, and tumor growth has been found to be suppressed by miR-193a-3p in a mouse xenograft tumor model [52]. However, miR-193a-3p has many targets, thus the relative role of ErbB4 expression will have to be validated. Indirect evidence indicates that low levels of a miRNA, miR-551b, are associated with metastasis and a poor prognosis in gastric cancer patients [50], and that ErbB4 is a potential target of miR-551b [50]. Moreover, high ErbB4 expression has been found to correlate with a poor prognosis in gastric cancer patients and treatment of gastric cancer with miR-551b mimics has been found to inhibit epithelial-mesenchymal transition (EMT) [50].

Phosphorylation sites and protein interaction motifs of ErbB4

Ligand binding to ErbB4 receptors induces either homo- or heterotypic receptor dimerization and activation of the intracellular tyrosine kinase domain. Activated receptors phosphorylate each other on tyrosine residues of the intracellular domain, which serve as docking sites for downstream signaling proteins (Table 1) [54]. Recruited proteins that bind to phosphorylated ErbB4 often contain Src Homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains, which recognize these phosphotyrosine (pTyr) sites in a sequence-specific fashion. In the ErbB4 cytoplasmic domain, 19 tyrosine phosphorylation sites have been identified using tandem mass spectrometry and protein microarrays to construct signaling maps [54]. These experiments showed that ErbB4 behaves different from the other ErbB receptors, because it appears to be much more selective and to only interact with proteins that also interact with at least one of the other ErbB receptors [54]. Based on these findings, it has been speculated that one of the roles of ErbB4 in normal physiology is to furnish the missing functions of ErbB2 and ErbB3, and that ErbB4 may play a protective role in cancer by buffering the proliferative and oncogenic effects of heterodimers formed between the other ErbB receptors [54].

Table 1.

Phosphorylation sites and protein interaction motifs of ErbB4

Tyrosine location	Motif	Recruited proteins	Ref
733		Shc	[53]
875		PLCG2	[54]
950	YXXM	Src	[9, 55]
984		Crk/ PTP-2c	[56]
1022		Crk	[56]
1035	PPXY	YAP/ STAT1	[9, 54]
1056 (only in Cyt1)	YXXM/ PPXY	PI3K/ ABL2/ CBL/ YAP/ Src	[9, 54]
1066			[54]
1081		ABL2/ CRKL	[54]
1128		SRC	[54]
1150	YXXM	ABL2/ SYK/ CRK/ CRKL/ RASA1	[9, 54]
1162		ABL2/ VAV2/ Grb2	[54, 56]
1188		ABL2/ Shc/ Grb2	[53, 54, 56]
1202		SYK/ Grb2	[54, 56]
1208		Grb2	[54, 56]
1221		Grb2	[54, 56]
1242		ABL2/ Shc/ Grb2	[53, 54, 56]
1258		SHC1	[54, 56]
1268		Grb2	[54, 56]
1284	PPXY	YAP/ Shc	[9, 53, 54]
1301			[54]

ErbB4 contains a number of known protein interaction motifs. The YXXM consensus motif for the recruitment of the p85 regulatory subunit of PI3K is present at Y1056. The Cyt2 isoform lacks amino acids 1046 to 1061 and, therefore, does not recruit the p85 subunit of PI3K [57]. Additionally, ErbB4 contains two other YXXM motifs (at Y950 and Y1150), but it is currently unknown whether these are involved in PI3K binding. Protein microarray data [54] and the finding that the Cyt2 isoform does not bind PI3K argue against binding of PI3K to Y950 or Y1150. ErbB4 also contains PPXY motifs for the recruitment of class I WW domain-containing proteins, such as Yes-associated protein (YAP). These include the PPIY motifs with the Y at position 1035, with the Y at position 1056 (exists only in the Cyt1 isoform), and with the Y at position 1284 [9, 54]. The interaction between YAP and ErbB4 occurs mainly via the WW domain of YAP and the PPPY motif at position 1284 [58]. Another WW domain-containing protein that binds ErbB4 and is clinically relevant is WWOX, which is an oxidoreductase frequently deleted or mutated in cancer cells [9]. Overexpression of WWOX sequesters the ErbB4-intracellular domain (ErbB4-ICD) in the cytoplasm of cancer cells after stimulation with an ErbB4 ligand [59]. Immunohistochemical analysis of primary breast cancer samples showed absence of WWOX expression in 36% of the cases, and this absence was found to be associated with an unfavorable outcome [60]. Localization of WWOX expression has been found to be associated with membranous ErbB4, and co-expression of membranous ErbB4 and WWOX has been found to be associated with a favorable outcome [60]. Furthermore, increased expression of WWOX in vitro and in vivo has been found to correlate with increased levels of full-length membrane-associated ErbB4, whereas knockdown of endogenous WWOX in MCF-7 cells has been found to lead to reduced expression of membrane-bound ErbB4 [60]. These data are consistent with a model in which WWOX protein constitutively stabilizes ErbB4 at the cell membrane, interferes with binding of other WW domain-containing proteins and, thereby, reduces nuclear trafficking and transcriptional activity resulting from ErbB4-ICD generation [60].

In summary, ErbB4 contains 19 tyrosine phosphorylation sites on its intracellular domain, including the YXXM and PPXY consensus motifs that allow interactions with intracellular signaling proteins.

Nuclear localization of the ErbB4 receptor

The JMa isoform, once cleaved extracellularly by TACE, can undergo a second intramembrane cleavage by γ-secretase [61], releasing into the cytoplasm the soluble 80 kDa intracellular C-terminal domain of ErbB4, referred to as ErbB4-ICD [61]. Once cleaved by gamma-secretase, ErbB4-ICD translocates to the nucleus [61] and this translocation is proportional to ErbB4 activation in transfected NIH3T3 cells [61]. The intranuclear function of ErbB4-ICD is believed to be similar to that of a transcription factor [61] and, in addition, ErbB4-ICD has been found to act as a chaperone for the nuclear entry of signal transducer and activator of transcription 5A (STAT5A) [13]. Moreover, NRG-1-induced accumulation of ErbB4-ICD in the nucleus has been found to lead to increased levels of trimethylated histone H3 on lysine 9 (H3K9me3) and decreased human telomerase reverse transcriptase (hTERT) activity, an enzyme involved in cell proliferation [55]. It has also been reported that ErbB4-ICD generation is required for astrogenesis in the developing mouse [62]. ErbB4 null mice show precocious cortical astrogenesis, which can be rescued by expression of the cleavable JMa isoform of ErbB4, but not by the uncleavable JMb isoform [56]. In cardiomyocytes, ErbB4-ICD plays a role in protecting against DNA damage by inducing the tumor suppressor p53 [63].

Nuclear ErbB4 has been detected in normal human and mouse mammary tissues as well as in ErbB4-positive breast cancers and endometrial cancers [1]. It has been shown that ErbB4 can inhibit the growth of SUM44 breast cancer cells and that ErbB4-ICD is both necessary and sufficient for ErbB4-dependent growth inhibition [1]. The finding that ErbB4 can have tumor suppressor-like activities is supported by studies in other cell lines [64]. ErbB4-ICD contains a functional D-Box sequence [65], which can be ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and, subsequently, destroyed by proteosomal degradation during mitosis. Proteosomal degradation of ErbB4-ICD may be necessary for mitosis to proceed. Mutation of the ErbB4-ICD D-box sequence prolongs the ErbB4-ICD protein half-life and increases its effect on G2/M delay [1]. Expression of the D-box mutant of ErbB4-ICD with an increased stability has been found to have a more profound effect on the inhibition of mouse breast cancer tumor growth [1].

In summary, the JMa isoform of ErbB4 can be cleaved, leading to release of an intracellular domain, ErbB4-ICD, which translocates to the nucleus to regulate transcriptional activity.

Intracellular signaling pathways

The main intracellular pathways linked to ErbB4 are the Ras-MAPK-ERK and PI3K-Akt pathways (Fig. 6). ErbB4 phosphorylation induces sustained activation of the Ras-MAPK-ERK pathway, leading to cell cycle cessation and differentiation [66]. Y1056 is only present in the Cyt1 isoform of ErbB4 and its phosphorylation results in recruitment of the p85 adaptor to activate PI3K-Akt signaling [66]. A third important pathway is represented by release of ErbB4-ICD into the cytoplasm. As discussed earlier, the JMa isoform is susceptible to proteolytic cleavage by TACE and γ-secretase releasing an 80 kDa ErbB4-ICD intracellular fragment, which interacts with the transcription factor STAT5A and, subsequently, migrates into the nucleus [66].

Fig. 6.

Intracellular signaling pathways of ErbB4 receptors. ErbB4 signals mainly through the ERK and AKT signaling pathways. The JMa isoform of ErbB4 can be cleaved by TACE and γ-secretase resulting in an 80 kDa ErbB4-ICD, which dimerizes with STAT5A resulting in nuclear translocation

Evidence indicates that somatic mutations in the kinase tail of ErbB4 in cancer patients lead to cell proliferation rather than differentiation [67]. Data indicate that signaling downstream of wild type ErbB4 is enriched for the JAK-STAT pathway, whereas signaling downstream of mutant ErbB4 is enriched for the PI3K-AKT pathway. ErbB4 receptors containing these mutations can still form heterodimers with other ErbB receptors, but cannot be activated, which favors activation of the proliferative PI3K-Akt pathway instead of the JAK-STAT pathway [67]. These data support the hypothesis that ErbB4 has a pro-differentiation role in cancer.

Genetic variants of ErbB4 and the risk of cancer

Mammary gland tumors

Certain genetic variants of the ErbB4 receptor impose increased breast cancer risks in humans. For instance, SNP rs62626348 and SNP rs13393577, located in ERBB4, have been found to be associated with a higher risk of breast cancer [68, 69]. Novel data indicate that also SNP rs13423759 allele C is associated with an enhanced breast cancer risk, and in silico studies indicate that rs13423759 allele C strengthens the interaction between miR-548as (an oncomiRNA) and ERBB4 mRNA, leading to lower ErbB4 protein levels [70].

Lung cancer

Although ERBB4 mutations in lung cancer are rare and the clinical significance of these mutations is limited [71, 72], a number of somatic ERBB4 mutations has been described in non-small cell lung cancer [73]. Functional characterization of nine ERBB4 mutations revealed four activating mutations, i.e., Y285C, D595V, D931Y, and K935I, with increased basal and ligand-induced ErbB4 phosphorylation levels [74]. These four activating mutations are located at critical positions at the ErbB4 dimerization interface (Y285C and D595V) and kinase domain (D931Y and K935I), and enhance ErbB4 dimerization and phosphorylation [74]. Interestingly, these mutations enhance the proteolytic release of the ErbB4-ICD and promote the survival of 3TE cells in the absence of serum [74]. Certain ERBB4 polymorphisms (SNPs rs6747637, rs6740117 and rs6742399) have been found to be associated with a higher lung cancer risk, suggesting that germline ErbB4 variants may predispose to lung cancer development [75]. These are all novel data, and it is currently unclear whether these polymorphisms activate or deactivate the ErbB4 receptor.

Gastric cancer

Gastric cancer exhibits high morbidity and mortality rates worldwide. It is, however, a highly heterogeneous disease. A large number of studies has been published on the role of ErbB2 [76] and ErbB3 [77] in gastric cancer, but data on the role of ErbB4 in this type of cancer are just emerging. Sequencing of 294 gastric cancer samples revealed mutations in the ERBB4 gene in 20 samples (6.8%). One of these mutations, ERBB4 p.R50C, has previously been observed in melanoma [78]. 33% of the ERBB4 mutations in gastric cancers occurred in the kinase domain and 20% in the receptor domain, suggesting that these mutations may affect kinase activity or receptor ligand interactions [78].

Growth inhibiting properties of ErbB4 in cancer

Most published studies indicate that ErbB4 inhibits tumor growth or has no effect on tumor growth. Most data are derived from studies on common cancers, including breast and colorectal cancer.

Mammary gland tumors

Members of the ErbB receptor family are known to play a role in the growth of mammary epithelial cells, as well as in malignant transformation and tumor progression [1]. The exception is ErbB4, which slows down the growth and stimulates the differentiation of mammary epithelial cells [1]. In ErbB4-positive, but not ErbB4-negative breast cancer cells, NRG-1 increases the proportion of cells in the G2/M phase of the cell cycle [79] and increases the expression of the G2/M checkpoint protein BRCA1, suggesting that ErbB4 homodimers may mediate this process, as opposed to ErbB4/ErbB2 or ErbB3/ErbB2 heterodimers [79]. Therefore, activation of ErbB4 by NRG-1 delays mitosis and decreases the proliferation of breast cancer cells. Interestingly, it has been found that ErbB4 mRNA expression correlates with BRCA1 mRNA expression in human breast cancer samples and that BRCA1 activity is required for NRG-1-mediated growth inhibition of breast cells, as demonstrated by both in vitro and in vivo BRCA1 knockdown experiments [79]. These findings indicate that ErbB4 impairs the proliferation of breast cancer cells by inducing a G2/M checkpoint through an as yet unknown mechanism involving BRCA1 [79].

It has also been demonstrated in other studies that ErbB4 activity can induce apoptosis in breast cancer-derived cells through mitochondrial accumulation of ErbB4 [7] and interaction between the pro-apoptotic protein BAK and the BH3-like domain of ErbB4. In another study, a constitutively active form of ErbB4 was overexpressed in breast, prostate, and ovarian cancer cells resulting in their apoptosis [28]. Epigenetic silencing [80] of the ErbB4 promotor has also been suggested to play a role in mammary tumor development, since treatment of cells with a demethylating agent resulted in increased ErbB4 expression and increased apoptosis [81].

Colorectal cancer

ErbB4 has been found to protect colon epithelial cells from TNF-induced apoptosis, and that knockdown of ErbB4 sensitizes epithelial cells to insult, consistent with the anti-apoptotic and cell-protective effects of ErbB4 activation [82]. The relevance of ErbB4 as a cancer target in colon is, compared to ErbB1 and ErbB2, largely unexplored [83]. In all colorectal cancer (CRC) data sets from the Cancer Genome Atlas (TCGA) examined in this report, non-synonymous mutations in ERBB4 (7.5–11% of the cases) predominated, without any amplification or deletion. This suggests that ErbB4 overexpression is not due to gene duplication, but is driven by changes occurring at the transcriptional and/or protein stability level [83]. This also suggests that ErbB4 overexpression is not the primary driver of oncogenesis in CRC. Also, analysis of the Vanderbilt/MCC transcriptome dataset did not reveal any influence of ErbB4 expression on clinical treatment and/or outcome [83]. Additional analysis of genetic variants in the promotor region of ERBB4 in CRC samples revealed a -782 T allele (instead of the 782G allele), resulting in a lower promotor activity and an increased risk for CRC and breast cancer [68]. This would indicate that lower ERBB4 promotor activity may increases the risk for CRC.

Hepatocellular carcinoma

It has recently been shown that ErbB4 deletion in hepatocytes in mice increases their sensitivity for the development of hepatocellular carcinoma (HCC) in response to toxic stimuli such as diethylnitrosamine (DEN) [84]. Furthermore, it has been found that isolated ErbB4-null hepatocytes show a higher proliferation rate in vitro than control hepatocytes [84]. Also, it has been found that the expression of ErbB4 is down-regulated in tumor samples of patients with HCC, and that ErbB4 downregulation correlates with a decreased cellular differentiation and a worse prognosis [84]. Mechanistically, loss of ErbB4 leads to decreased p53 activity through inhibiting the expression of the tumor suppressor tp53inp1 [84]. Another study also examined the development of HCC in response to DEN, but used rats instead of mice and was limited to immunohistochemistry [85]. The results of this study indicated that loss of ErbB4 expression may play a role in the progression of hepatic lesions to HCC [85]. In summary, the currently published studies on ErbB4 and HCC indicate that ErbB4 suppresses the progression of HCC.

Growth stimulating properties of ErbB4 in cancer

A limited number of studies indicates that ErbB4 may have a growth stimulating effect on certain tumor cells, including CRC [83], gastric cancer [86], and melanoma cells [87].

Colorectal cancer

Some data indicate that ErbB4 may promote CRC growth. One study revealed, based on data from the Cancer Genome Atlas, elevated ErbB4 mRNA and protein levels in all stages of CRC, suggesting that ErbB4 overexpression is involved in its tumorigenesis [83]. Accordingly, knockdown of ERBB4 expression in a poorly differentiated CRC cell line impaired its anchorage-independent growth, which is associated with a more malignant phenotype [83]. Data from this latter report additionally indicate that ErbB4 overexpression may cooperate with WNT signaling to enhance the growth of mouse and human colonocytes [83]. Colorectal cancer is the most predominant example of conflicting data on the role of ErbB4 on tumor growth and progression. Currently, there are no solid data explaining these discrepancies. It could be that the role of ErbB4 in CRC differs among subtypes. It has, for example, been shown that ErbB4 attenuates inflammation in colitis and, thus, may be tumor suppressive in colitis-associated CRC [88].

Melanoma

Melanoma is a deadly skin cancer with a high metastatic potential and a rising incidence in the Western world. There is some evidence indicating that ERBB4 mutations may acts as oncogenic drivers in melanoma [87]. It has been found that ERBB4 mutations may be present in 12 to 19% of individuals with melanoma [89], and that growth can be reduced in melanoma cells expressing mutant ErbB4 through siRNA-mediated knockdown or treatment with lapatinib [87]. Later studies indicated, however, that ERBB4 mutations in melanoma may be less frequent than originally reported or even absent in specific populations [90, 91], indicating that they do not act as universal oncogenic drivers of melanoma. Therefore, more studies are warranted to delineate the controversial role of ErbB4 in melanoma.

Prognostic relevance of ErbB4 expression in tumors

In general, data on the prognostic relevance of ErbB4 expression in tumors points towards a neutral effect or a better prognosis with higher expression levels. It has, for instance, been shown that ErbB4 expression in cholangiocarcinomas is associated with a better survival [92], whereas a recent study in an Iranian population found no significant association among ERBB4 polymorphisms and the risk of prostate cancer [93].

Mammary gland tumors

Whereas ErbB1 and ErbB2 are frequently overexpressed and correlate with a poor prognosis in breast cancer, expression of ErbB4 is generally regarded as a marker for favorable prognosis [9]. In general, ErbB4 expression in breast cancer cells is much lower than that of ErbB1 and ErbB2 [39]. NRG-1, a ligand of both ErbB3 and ErbB4, has been found to inhibit the growth of a panel of ErbB4-positive human breast cancer-derived cell lines, whereas NRG-1 increased the growth of ErbB4-negative breast cancer cells [79]. Consistent with this, it has been shown that NRG-1 increases breast cancer cell motility by activating ErbB3, but not ErbB4, receptors [94]. A large number of studies has been published on the role of ErbB4 in breast cancer development and progression, with a majority of them pointing towards a growth inhibiting role. Also, a recent systematic review and meta-analysis of ErbB4 expression studies in human breast cancers showed that a higher ErbB4 expression is significantly associated with a longer relapse-free survival (HR = 0.63; CI = 0.48–0.83; p = 0.001; 8024 patients) [95].

Colorectal cancer

The number of studies on the prognostic relevance of ErbB4 expression in CRC patients is limited [96–100]. Three studies failed to find a significant relationship between ErbB4 expression and patient prognosis [98–100], while one study found that positive ErbB4 expression may serve as an independent prognostic factor for recurrence [97], and another study found that positive ErbB4 expression may predict a worse prognosis [96]. Only a minority of CRCs expresses ErbB4 receptors. Immunohistological examination of human CRC tissue samples revealed that ErbB1, ErbB2, ErbB3, and ErbB4 expression was positive in 51%, 26%, 36% and 21% of the cases, respectively, and that phosphorylated pErbB1, pErbB2, pErbB3, and pErbB4 expression was positive in 32%, 21%, 18% and 16% of the cases, respectively [96]. Importantly, positivity for pErbB3 and pErbB4 was found to serve as a negative predictor of survival in CRC patients [96].

Pancreatic cancer

In the USA, pancreatic cancer is the fourth cause of cancer-related death and, in general, it has a poor outcome [101]. Expression of ErbB4 in human pancreatic cancers appears to be low [101]. Moreover, ERBB4 transcription has been found to decrease in the early stages of pancreatic cancer development, indicating that loss of ErbB4 expression is required for tumorigenesis [101]. Also, higher ErbB4 expression in pancreatic cancers has been found to correlate with favorable staging [27]. Together, current data on the role of ErbB4 in pancreatic cancer point towards a role in tumor suppression.

Bladder cancer

Some reports have shown increased ErbB4 mRNA and protein expression levels in bladder cancer [102, 103], whereas others have shown absence of ErbB4 expression in bladder cancer [104]. It has also been found that low ErbB4 expression is associated with high grade and invasive bladder tumors as well as with significantly shorter survival times [105]. Increased expression of ErbB4 and one of its ligands, NRG-4, has been found to lead to a better prognosis in patients with bladder cancer. Furthermore, a higher combined expression of ErbB3 and ErbB4 has been found to be associated with an improved survival in patients [106]. Also, tumors with a high ErbB1/ErbB2 expression turned out to be less aggressive when they exhibited at the same time a higher ErbB4 expression [107]. Overall, the role of ErbB4 in bladder cancer seems to be limited, and the available evidence largely points towards a better outcome in cases with a higher ErbB4 expression level.

Brain cancer

Glioblastoma is a common form of brain cancer in adults. ErbB4 mRNA levels have been found to be lower in glioblastomas than in normal brain samples, and ErbB4 protein has been found to be ubiquitously expressed in glioblastomas, but not to be associated with survival [108]. Publicly available data from the CCLE indicates copy number loss of the ERBB4 gene across numerous glioblastoma cell lines, suggesting a role as tumor suppressor [109], but it has been found that the same variants occur at approximately the same frequencies in the general population [109]. Although overall low ErbB4 expression levels are found in glioblastoma, a high pErbB4 expression has been found to be present in 11% of the cases and to be associated with a shorter survival than no pErbB4 expression [108]. Thus, despite low ErbB4 mRNA levels in glioblastoma, increased ErbB4 activity may have prognostic and/or therapeutic potential. In summary, although ErbB4 plays a crucial role in brain development and adult brain physiology, remarkably little solid evidence exists on its potential role in glioblastoma.

Role of ErbB4 in the immune system

Tumor growth is accompanied by suppression of the immune system, which allows tumor cells to evade destruction by immune cells. Thus, pathways that activate or suppress the immune system may also affect tumor growth. Research on the role of ErbB4 in immune cells is relatively new. The first study investigating the expression of ErbB4 in immune cells showed that a decreased expression of this protein in peripheral blood mononuclear cells, T cells, monocytes, and B cells is associated with multiple sclerosis [110]. A functional role of ErbB4 in immune cells has been demonstrated more recently, and activation of ErbB4 receptors has been shown to induce macrophage apoptosis in a mouse model of colitis [88]. Using a mouse model of cardiac and skin fibrosis, we found that activation of ErbB4 receptors on macrophages attenuates inflammation and fibrosis [17, 24]. Another interesting study demonstrated that NRG-1 therapy enhanced interleukin-10 (IL-10) production by immune cells, which was mediated through ErbB2 and ErbB4 receptor activation [111]. It has also been shown that IL-10 can inhibit tumor growth [112] and, as such, may mediate some of the growth-inhibiting properties of ErbB4. As the role of ErbB4 in the immune system is just emerging, more studies are needed to gain further insight in this exciting topic.

Conclusions and perspectives

The ErbB4 signaling system is complex due to the involvement of different natural ligands, different dimerization partners, different alternative splicing events, different phosphorylation sites, and different downstream signaling pathways. ErbB4 has been found to be expressed in several tumors and tumor cell lines, but evidence for its potential as an oncogenic driver is limited, in contrast to the other ErbB family members. Some tyrosine kinase inhibitors are able to inhibit ErbB4 (Table 2) but, as yet, ErbB4-specific inhibitors do not exist. In order to gain a better understanding of the role of ErbB4 in cancer, small molecules or antibodies that specifically inhibit ErbB4 would be very useful. In vitro studies with cancer cell lines expressing ErbB4 have indicated that its inhibition can slow down their growth but, conversely, when ErbB4 was exogenously overexpressed the growth of most cell lines was also found to be inhibited (Table 3). Current evidence that ErbB4 may act as a proto-oncogene is primarily based on its association with other ErbB receptors. As yet, there is no definitive prove that either ERBB4 mutation or overexpression can induce cancer development and/or progression. Therefore, more pre-clinical studies are needed to assess the impact of overexpression of native or mutated ErbB4 on the development and/or progression of different types of cancer.

Table 2.

Therapies targeting ErbB receptors in cancer

Name	Reversibility	ErbB1	ErbB2	ErbB3	ErbB4	Other
Tyrosine kinase inhibitors
Afatinib	No	X	X		X
Dacomitinib	No	X	X		X
Erlotinib	Yes	X
Gefitinib	Yes	X
Ibrutinib	No		X		X	BTK
Neratinib	No	X	X		X
Lapatinib	Yes	X	X
Vandetanib		X
Antibodies
Ado-trastuzumab emtansine			X
Cetuximab		X
Panitumumab		X
Patritumab				X
Pertuzumab			X
Trastuzumab			X

BTK: Bruton’s tyrosine kinase.

Table 3.

Growth inhibiting and stimulating effects of ErbB4 activation

Cell type	Cell line	Growth inhibition	Growth stimulation	Ref
Breast cancer	MCF7, SKBr3	ErbB4 overexpression		[28]
Ovarian cancer	SKOv3	ErbB4 overexpression		[28]
HCC	Huh-7, QGY-7703	p53 dependent		[84]
Pancreatic cancer	CaPan-1, HPAC, MIA PaCa-2, PANC-1	ErbB4 overexpression inhibits proliferation		[101]
Prostate cancer	LNCAP, PC3	ErbB4 overexpression		[28]
Mesenchymal cell		ErbB4-ICD dependent		[113]
Breast cancer	MCF7, T47D		Cell growth	[114, 115]
Ovarian carcinoma	OVCAR-3		Cyt1 dependent	[116]
Endometrial carcinoma	KLE		Wnt5A dependent	[117]
CRC	H716E		Wnt5A dependent	[117]
Gastric cancer	SGC-7901, MNK-45		PI3K/Akt depdent	[86]
Lung cancer	H661, H522		Wnt5A dependent	[117]

Based on our present knowledge, growth of mammary gland tumors and hepatocellular carcinoma could potentially be inhibited by ErbB4 agonists, such as e.g. bivalent NRG-1. The tumors that would be most likely to be inhibited by ErbB4 antagonists are melanoma and colorectal cancer. At the moment, however, data on the response of different tumor cell lines to ErbB4 activation or inhibition are lacking. One reason for this lack of data is that ErbB4 has been a neglected target in most studies of tumor growth, and another reason is the limited availability of specific ErbB4 agonists and antagonists. In the coming years, the field of ErbB4 research in cancer would benefit tremendously from studies in which the effects of ErbB4 overexpression or downregulation on tumor growth are evaluated.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.