R e vie w

Author for correspondence: Department of Medical Biochemistry and Genetics, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland n Tel.: +358 2333 7240 n Fax: +358 2333 7300 n [email protected] †

Antibodies targeting the extracellular domains of ErbB receptors have been extensively studied for cancer drug development. This work has led to clinical approval of monoclonal antibodies against the well-known oncogenes EGFR and ErbB2. Here we discuss the biological activities of ErbB4, a less-studied member of the EGFR/ErbB growth factor receptor family and speculate on the potential clinical relevance of antibodies targeting ErbB4. In addition to their significance as therapeutics, the role of ErbB4 antibodies in prognostic and predictive applications is surveyed. Introduction to the relevance of ErbB receptors in cancer biology

The ErbB/HER subfamily of receptor tyrosine kinases includes four members: EGFR (also known as ErbB1 or HER1), ErbB2 (c-Neu, HER2), ErbB3 (HER3), and ErbB4 (HER4) [1,2] . Since the initial discovery that EGFR and ErbB2 share structural similarities with the potent animal oncogenes v-ErbB and c-Neu, a considerable number of studies have demonstrated the significance of altered ErbB signaling in malignant transformation. As a result, ErbB overactivity is commonly implicated in the pathogenesis of various epithelial and central nervous system malignancies, and ErbB expression is typically associated with poor clinical outcome [3] . The identification of specific abnormalities in gene expression and mutational status provides both prognostic and predictive information [4] , and the alterations form the basis for therapeutics aiming to specifically inhibit ErbB activity in cancer [5] . By contrast to EGFR and ErbB2, the role of ErbB4 as a tumor-driving oncogene has been controversial [6] . Association of ErbB4 with normal mammary gland differentiation [7,8] as well as with estrogen receptor (ER) signaling in breast cancer [9,10] , has supported a view of ErbB4 as a marker of well-differentiated phenotype. However, ErbB4 is frequently present in various cancer tissues [11–15] , and experimental downregulation of ErbB4 in different tumor cells suppresses growth [16–19] . Moreover, recent findings about somatic mutations activating ErbB4 in metastatic melanoma [17] , have started to shift the paradigm of ErbB4 in carcinogenesis to include a causal role and to support development of tools, such as ErbB4 antibodies, to target ErbB4 in cancer [20] . 10.2217/FON.09.132 © 2009 Future Medicine Ltd

Review

Maija Hollmén & Klaus Elenius†

Future Oncology

Potential of ErbB4 antibodies for cancer therapy

In this review, we will first describe the general structure and signaling mechanisms of ErbB receptors and present clinically relevant EGFR and ErbB2 antibodies as examples of rational anti-ErbB development. We will then focus on ErbB4 by reviewing the prognostic and predictive significance of ErbB4 expression in different cancers. Finally, current preclinical evidence about blocking ErbB4 function in different cancer models and experimentation with ErbB4 antibodies will be discussed. Structure & signaling mechanisms of the ErbB receptors

The four ErbB receptors are selectively activated by a number of EGF-like growth factors leading to cellular responses, such as proliferation, differentiation, migration, or survival. ErbB receptors of approximately 180 kD consist of a glycosylated extracellular domain divided into four subdomains (I, I, III and IV), a single transmembrane domain and an intracellular domain (ICD) including a tyrosine kinase enzyme (Figure 1) . Ligand binding to the receptor extracellular domain triggers receptor dimerization, subsequent activation of the kinase domain, receptor autophosphorylation and multiple downstream signaling cascades [21] . Upon ligand interaction, the extracellular subdomains I and II rotate away from subdomains III and IV, extending the receptor conformation and exposing important structures to form an optimal dimer interface [22] . The structure of the extracellular domain of ErbB2 is strikingly different from the other ErbBs and it constitutively resembles the conformation of a ligand-bound active receptor [23] (Figure 1) . Thus, its activity is not dependent on ligand interactions and ErbB2 seems to be the preferred and most mitogenic Future Oncol. (2010) 6(1), xxx–xxx

Keywords alternative splicing n biomarkers n EGFR n ELISA n HER4 n shedding n therapeutic antibodies n tumor growth

part of

ISSN 1479-6694

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II III

IV

I

I Extracellular domain

III

II

IV Transmembrane domain Tyrosine kinase Intracellular domain

EGFR unoccupied and tethered

EGFR occupied and extended

ErbB2 ligandless and extended

Activated EGFR/ErbB2 dimer Ligand Phosphorylation

Figure 1. Structure and activation of ErbB receptors. ErbB receptors consist of a glycosylated extracellular domain divided into four subdomains (I–IV), a single transmembrane domain and an intracellular domain, including a tyrosine kinase enzyme. When unoccupied (as shown for EGFR in the figure), the receptors are in a tethered conformation and remain inactive. Upon ligand binding, the receptor conformation extends revealing important elements for receptor dimerization leading to asymmetric intermolecular interaction and allosteric activation of the kinase domains and receptor autophosphorylation. In contrast to the other ErbBs, there is no known ligand for ErbB2 and its conformation is constantly extended and ready to form active dimers with the other ErbB family members.

dimerization partner among the ErbB family members [24] . ErbB3, in turn, is unique within the ErbB family as its intrinsic kinase activity is weak and it only mediates signaling as a heterodimeric partner [25] . Therapeutic ErbB antibodies

Several structural and functional properties of the ErbB family members make them potential targets for antibody-based drug development. The extracellular cell surface location of the ErbB ectodomain is a prerequisite for targeting by immunotherapy, as the relatively large (150 kD) immunoglobulins cannot freely pass through the lipid-composed plasma membranes. Overexpression of ErbBs in malignant tissues facilitates preferential targeting of diseased tissues, a phenomenon that is particularly significant for cytotoxic mechanisms, such as antibody-dependent cell cytotoxicity (ADCC), which recruits the immune cells regardless of the functional characteristics of the cell surface antigen. Importantly, the extracellular domains also regulate the activation of ErbB receptors through ligand binding and dimerization. Thus 2

Future Oncol. (2010) 6(1)

antibodies binding to the ectodomains may interfere with receptor function. To elucidate how therapeutic antibodies may regulate ErbB activity by interacting with specific structural motifs in ErbB ectodomains, five antibodies targeting EGFR and two targeting ErbB2 are discussed as illustrative examples (Table 1) . The EGFR targeting monoclonal antibodies cetuximab, panitumumab, matuzumab, zalutumumab and nimotuzumab, all bind residues on the ligand-binding subdomain III [26–30] . Cetuximab and panitumumab have US FDA approval for the treatment of metastasized colorectal carcinoma (CRC) and cetuximab also for advanced squamous cell carcinoma of the head and neck. Matuzumab, zalutumumab and nimotuzumab are in clinical trials. All these subdomain III-binding antibodies, except matuzumab, compete for ligand binding and consequent EGFR activation [26–28,30] . Matuzumab, as well as cetuximab, have been reported to inhibit EGFR activity by sterically preventing conformational changes required for the formation of receptor dimers [28,29] . The properties of zalutumumab include bivalent binding that limits future science group


Review

Table 1. Therapeutic EGFR and ErbB2 antibodies. Antibody

Epitope

Mechanism of action

Approval

Ref.

EGFR

IMC-C225/cetuximab/Erbitux® (Bristol-Myers Squibb, NY, USA) Chimeric

Subdomain III ligand binding region residues Q384, Q408, S418, S440, K443, K465, S468 and N473

Blocks ligand binding and prevents conformational changes required for dimerization. ADCC activity.

Approved 2004 for CRC, 2006 for HNSCC

[28]

ABX-EGF/panitumumab/Vectibix® (Amgen, CA, USA) Fully human

Subdomain III ligand binding region

Blocks ligand binding and receptor activation. Subtype IgG2. Weak ADCC activity.

Approved 2006 for CRC

[30] [33] [36]

EMD 72000/matuzumab (Merck KGaA, Darmstadt, Germany) Humanized

Subdomain III residues 454–464, D434 and N449

Prevents conformational changes required for dimerization. ADCC activity.

Trials for CRC, ovarian, lung and gastric cancer

[29]

HuMax-EGFrTM / zalutumumab (Genmab, Copenhagen, Denmark) Fully human

Subdomain III ligand binding region residues K465 and M467

Blocks ligand binding and restricts intra- and intermolecular flexibility. ADCC activity.

US FDA fast track status 2006 for HNSCC. Trials for NSCLC and CRC.

[26] [31] [32]

h-R3/ nimotuzumab/TheraCIM (Innogene, Singapore) Humanized

[37]

Subdomain III linear epitope in Blocks ligand binding. ADCC region 400–410 activity.

Limited national approval for HNSCC and glioma. Several ongoing Phase III trials.

ErbB2

Trastuzumab/Herceptin® (Genentech, CA, USA) Humanized

Subdomain IV (juxtamembrane Blocks ectodomain shedding. Approved 1998 for ErbB2-positive metastatic ADCC activity. Disrupts region) residues 557–561, breast cancer and in 2006 ErbB2/ErbB3 activation. 570–573 and 593–603 for adjuvant therapy

Pertuzumab/OmnitargTM (Genentech, CA, USA) Humanized

Subdomain II (dimerization arm) residues Y252, F257, K311 and H296

Prevents heterodimerization preferably with ErbB3.

Phase III trials in combination with trastuzumab and chemotherapy

[27] [34]

[38] [39] [41]

[42]

ADCC: Antibody-dependent cell cytotoxity; CRC: Colorectal cancer; HNSCC: Head and neck squamous cell carcinoma; NSCLC: Non-small cell lung cancer.

both intra- and intermolecular flexibility of the EGFR extracellular domain [26,31] . While the antibodies have overlapping binding sites, and are thus expected similarly to regulate EGFR function, they differ in their affinities for EGFR, which may affect dosing as well as adverse effects [32–34] . In addition, the IgG1 subtype activates ADCC efficiently enhancing the cytotoxic activity of the antibody [35] . Panitumumab, unlike the other four antibodies, belongs to the IgG2 subtype of immunoglobulins and is thus less effective in mediating ADCC [36] . However, it has a relatively long half-life compared with the antibodies of the IgG1 subtype and can be administered less frequently than the typical weekly dose used for most protocols [30] . Panitumumab and zalutumumab are both fully human antibodies and are thus not expected to evoke human antimouse future science group

antibody (HAMA) production similarly to the chimeric or humanized antibodies that include short mouse-specific sequences [36,37] . The unique structure of ErbB2 partially explains why the therapeutic ErbB2 antibodies trastuzumab and pertuzumab recognize different structural motifs when compared with the EGFR antibodies (Figure 1 & Table 1) . In the ErbB2 structure, the subdomain III makes contact with the subdomain I and leaves the dimerization surfaces in subdomains II and IV exposed. Trastuzumab binds to subdomain IV [38] and disrupts the formation of active ErbB2/ErbB3 heterodimers [39] . In addition, the binding site of trastuzumab is located in a region that is susceptible to proteolytic cleavage and trastuzumab has been reported to inhibit ErbB2 ectodomain shedding by ADAM10, as well as subsequent ligand-independent activation of the truncated www.futuremedicine.com

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TACE

JM-a (exon 16, 23 aa) JM-b (exon 15, 13 aa) ErbB4 I

III

II

IV

Kinase

C-terminus

TM

PI3-k CYT-1 (exon 26, 16 aa) CYT-2 (no exon 26)

Itch

Isoforms JM-a CYT-1

JM-a CYT-2

JM-b CYT-1

JM-b CYT-2

TACE

Cleavage, nuclear signaling

PI3-k

PI3-k/Akt survival

Itch

Ubiquitination, degradation

Figure 2. Structures and distinct biological activities of the ErbB4 isoforms. The ErbB4 gene is transcribed into four different isoforms by alternative splicing. The juxtamembrane isoforms are formed either from exon 16 encoding 23 amino acids (JM-a) or exon 15 encoding 13 amino acids (JM-b). The longer JM-a includes a TACE cleavage site that results in proteolytic processing by TACE, and subsequently by g-secretase activity, and nuclear signaling by the soluble intracellular receptor fragment. The C-terminal isoforms either include sequence from exon 26 encoding 16 amino acids (CYT-1) or not (CYT-2). The 16 amino acid stretch includes binding motifs for PI3-K and the ubiquitin ligase Itch. aa: Amino acid; TACE: TNFa-converting enzyme; TM: Transmembrane domain.

membrane-anchored receptor fragment [40,41] . Pertuzumab, in turn, binds epitopes on the exposed dimerization motif on subdomain II and inhibits ErbB2 dimerization with other ErbB family members [42] . A recent review on the interaction of antibodies with ErbB extracellular regions by Schmitz and Ferguson broadly covers the area of antibodies targeting ErbB receptors [43] . ErbB4 isoforms have distinct biological activities

The biology of ErbB4 is complicated when compared with EGFR or ErbB2 as ErbB4 is expressed as four alternatively spliced and functionally distinct isoforms (Figure 2) . Two of the isoforms differ in the intracellular cytoplasmic domain (isoforms CYT-1 and CYT-2) and two in the extracellular juxtamembrane region (isoforms JM-a and JM-b). The CYT-1 isoform contains a 16 amino acid sequence transcribed from exon 26 that is capable of coupling to phosphoinositide 3-kinase (PI3-K) via a YTPM sequence 4


[44] . The CYT-2 isoform lacks this insertion and cannot directly couple to the PI3-K pathway. The stretch of unique amino acids within the CYT-1 isoform-specific sequence also includes an interaction motif (PPAY ) for the Itch E3 ubiquitin ligase that regulates ErbB4 monoubiquitination, endocytosis and degradation in an isoform-specific manner [45,46] . Additionally, E3 ubiquitin ligases WWP1 and Nedd4 have been reported to preferably target ErbB4 CYT-1 isoforms for degradation [47,48] . These properties differentiate ErbB4 isoforms in their stability and signal transduction and result in dissimilar cellular responses. The CYT-1 isoforms, but not the CYT-2 isoforms, may, for example, directly mediate PI3-K-regulated survival and migration in vitro [49] . Also, the ICD of CYT-1 isoform has been demonstrated to promote differentiation of mammary epithelial cells in vitro and in vivo, whereas CYT-2 ICD supports proliferation [50] . The extracellular juxtamembrane isoforms are alternatively spliced to include either sequences from the 23 amino acid long exon 16 (JM-a)

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or from the 13 amino acid long exon 15 (JMb). The longer JM-a isoform can be cleaved by TNF-a-converting enzyme (TACE) [51] , whereas the JM-b isoform is proteinase-resistant [52] . The cleavage site is located between His651 and Ser652 within the JM-a-specific extracellular juxtamembrane region [53] . Thus, TACE activity generates a shed soluble extracellular domain of approximately 120 kD, as well as a membrane-bound truncated receptor fragment of 80 kD (called the m80). Extracellular cleavage by TACE triggers a second cleavage of ErbB4 in the transmembrane region by g-secretase activity [54,55] . This process of two consecutive proteolytic events is also known as regulated intramembrane proteolysis (RIP) [56] . The g-secretase activity releases a soluble ICD (or s80), which can translocate into the nucleus and function as a transcriptional coactivator or corepressor. Transcription factors that have been reported to associate with the ICD of ErbB4 include signal transducer and activator of transcription 5 (STAT5) [57] , yes-associated protein (YAP) [58] , ETO2 [59] , ER [60] and the TAB2-NCoR complex [61] . This signaling via the released ICD is only possible for the cleavable JM-a isoform thus, enhancing the functional diversity between the ErbB4 isoforms. For example, the cleavable JM-a CYT-2, but not noncleavable JM-b CYT-2, promotes growth of 32D myeloid cell and MCF-7 breast cancer cell transfectants [62] , and induces tubulogenesis of Madin-Darby canine kidney (MDCK) kidney epithelial cells [63] . Moreover, released ErbB4 ICD may promote apoptosis either by utilizing a BH3-like proapoptotic domain within ErbB4 itself [64] or by associating with the p53 regulator Hdm2 [65] , whereas a full-length ErbB4 JM-b CYT-1 capable of coupling to PI3-K/Akt pathway promotes survival [49] . While most of this experimentation addressing functional roles of the ErbB4 ICD has been carried out in vitro in variable engineered cellular backgrounds, interaction of nuclear ICD with TAB2-CoR has been demonstrated to regulate astrogenesis in the developing mouse brain in vivo and represents a biologically relevant example of transcriptional regulation by endogenous soluble ErbB4 ICD [61] . Expression & prognostic significance of ErbB4 in cancer

ErbB4 is widely expressed in normal adult and fetal tissues including the gastrointestinal, urinary, reproductive and respiratory tracts, as well as the skin, skeletal muscle, circulatory, endocrine and central nervous systems [66] . The future science group

Review

different ErbB4 isoforms are represented in a tissue-specific manner. For example, JM-a is the only JM isoform expressed in kidney, salivary gland, trachea, thyroid gland, prostate and mammary gland [11] . Brain, in turn, has been shown to express both juxtamembrane isoforms and the heart expresses predominantly the JM-b isoform [11] . The CYT isoforms are both present when ErbB4 is expressed in normal tissues. To elucidate the presence and prognostic significance of ErbB4 in human cancers, we collected data from published clinical studies fulfilling the following two criteria: they were carried out using either IHC or reverse transcription (RT)-PCR-based techniques, and the series included at least 20 tumor samples. Data collected by this approach are shown in Table 2 and include information about ErbB4 expression in malignancies of breast, central nervous system, colon, head and neck, ovary, endometrium, bladder, lung, thyroid, prostate and connective tissues. If information about the expression of different ErbB4 isoforms in cancer tissues is available, it is discussed below. Breast cancer is the most studied cancer type for ErbB4 expression and activity, and the role of ErbB4 in breast cancer has been specifically covered in recent reviews [9,10] . A view shared in the majority of reports seems to be that ErbB4 is expressed in a subpopulation (estimates vary between 12 and 92%) of breast cancer patients and that its expression associates with ER- and progesterone receptor (PgR)-positivity, ErbB2negativity, well-differentiated phenotype and favorable outcome in both primary and invasive tumors [67–77] . Also, loss of ErbB4 expression has been associated with resistance to the ER antagonists [78–80] . Interestingly, Biéche et al. report both under- as well as overexpression of ErbB4 mRNA in breast cancer and demonstrate that overexpression of ErbB4 mRNA is associated with ER-positivity but also with shorter relapsefree survival [81] . Lodge et al. have also showed that ErbB4 overexpression correlates with decreased survival in tumors with lymph node involvement [82] . Significantly, in both of these studies [81,82] , ErbB4 expression was associated with ErbB3 expression. ErbB3 is a major mediator of survival signals downstream of ErbB heterodimers and has recently been demonstrated to contribute to resistance for both hormonal and tyrosine kinase-targeted therapies [83,84] . It is plausible that the different ErbB4 isoforms with different growth-, migration-, survival- and differentiation-promoting activities [7,49,62] , have different roles also when present in breast cancer www.futuremedicine.com

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Table 2. ErbB4 in cancer. Cancer

n

Method Ab

ErbB4 expression

Association with ErbB4 expression

Ref.

Breast

ErbB2-negative

171

IHC

268

RT-PCR

278

FISH/ IHC 83B10

37% overexpression

ER+

89

IHC

RB-9045

[78]

402

IHC

DCIS

129

IHC

HFR-1, HA.77.16 sc-283

458/62

HFR-1

Invasive

1513

IHC/ RT-PCR IHC

Overexpression in 17% Loss of ErbB4 independent marker tamoxifene -sensitive, 9% in -resistant of tamoxifene resistance Decreased survival when ErbB4 nuclear 63% in nonrecurred, 36% in recurred Nonrecurrence and ER+ DCIS IHC: strong 46%, weak 34%; RT-PCR: Decreased survival when ErbB4 19% JM-a overexpression nuclear vs membrane-associated 45% strong, 34% weak Increased DFS

130

RT-PCR

-

ER+ and decreased RFS

[81]

220

IHC

HA.77.16

30% overexpression, 25% downregulation 12% overexpression

ER+ and increased survival

[77]

LN+

66

IHC

HFR-1

92% cytoplasmic, 85% nuclear

[82]

100

sc-283

82%

365

IHC/ RT-PCR RT-PCR

Decreased survival when ErbB4 in cytoplasm Increased DFS in ErbB2+ tumors Increased RFS and OS

[138]

Invasive

178

IHC

18% high, 7% low, 49% nuclear

Low grade when ErbB4 nuclear

[75]

127

IHC

HFR-1/ sc-283 HFR-1

58% cytoplasmic, 41% membranous, 25% nuclear

Differentiated phenotype

[71]

47

RT-PCR

-

ER+ and EGFR-

[72]

94

IHC

sc-283

50%

ER+, PgR+, ErbB2-

[68]

Brain

Meningioma

RT-PCR

-

Downregulation

[92]

MB/PA

RT-PCR

-

JM-a isoform upregulated in MB and PA 100%

[12]

[70]

19% moderate or strong -

HFR-1

-

Glioblastoma

21

IHC

MB

26

sc-283

Ependymoma

121

MB

65

IHC RT-PCR IHC/ RT-PCR IHC

Colorectal

Primary and metastatic Stage II/III

Increased DFS

[73]

Tendency for decreased OS

[74]

[137] [69] [11] [67]

[76]

[91]

CYT-1:CYT-2 ratio associates with more aggressive disease Coexpression with ErbB2 associates with enhanced proliferation Decreased survival

[93]

sc-283

IHC: 42% overexpression, nuclear RT-PCR: upregulation of CYT-1 IHC: 100% coexpressed with ErbB2 RT-PCR: overexpression of JM-a. 54% coexpression with ErbB2

64

IHC

RB-9045

109

IHC

RB-9045

81% in both primary and metastatic tumors 11% membranous, 19% cytoplasmic

sc-283

[90] [89]

[13]

Membranous ErbB4 staining associates with recurrence

[96]

Ab: Antibody; DCIS: Ductal carcinoma in situ; DFS: Disease-free survival; ER: Estrogen receptor; FISH: Fluorescence in situ hybridization; HNSCC: Head and neck squamous cell carcinoma; IHC: Immunohistochemistry; LN: Lymph node; MB: Medulloblastoma; NSCLC: Non-small cell lung cancer; OS: Overall survival; PgR: Progesterone receptor; PA: Pilocytic astrocytoma; n: Number of patients; RFS: Relapse-free survival; RT: Reverse transcription.

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Review


n

Method Ab

ErbB4 expression

Colon/metastatic LN

127/47

IHC

sc-283

37%/27% overexpression

106

IHC

HFR-1

19% membranous, 30% cytoplasmic

LN+

[95]

125

IHC

sc-283

18% +, 4% ++ immunoreactivity

Coexpression with ErbB2 in late stage associates with decreased OS

[94]

SCC

Esophageal

61

IHC

sc-283

Increased survival; reduced survival when ErbB4 nuclear

[15]

Dysplastic/ HNSCC (oral) HNSCC

23/26

IHC

HFR-1

84% cytoplasmic and membranous, 61% nuclear 30%/26% nuclear

38

IHC

sc-283

26% strong, 50% weak

HNSCC (oral)

111

IHC

sc-283

14% high, 36% intermediate

Nuclear ErbB4 immunoreactivity independent of overexpression and differentiation Metastasis and decreased survival

Cervix

56

IHC

sc-283

68% overexpression, coexpression with EGFR

Ovarian

Benign/ malignant 200

53

Endometrial

ELISA/ RT-PCR IHC/ RT-PCR

HFR-1/ HA.77.16

Overexpression in malignant disease, coexpression with ErbB2 and ErbB3 93%/89% with the two antibodies RT-PCR: both JM-a and JM-b


Ref. [139]

[140] [97]

[98] [141]

[105] [107]

No associations with grade or stage [106]

106

IHC

45

41

IHC/ RT-PCR IHC

HFR-1

Overexpression of both protein and mRNA 15% overexpression

Bladder

248

IHC

RB-9045

Downregulation

Nonpapillary histology, high grade, invasiveness and decreased survival

[99]

73

RT-PCR

-

Downregulation

88

RT-PCR

-

63% high expression

245

IHC

sc-283

HFR-1

[142] [108]

[143] [100]

30%

Increased survival in tumors coexpressing EGFR or ErbB2 No significant associations

Lung

Carcinoid

31

IHC

sc-283

100% moderate-to-intense staining

NSCLC

40

IHC

NSCLC

20

IHC

Thyroid

205

[144]

[14]

50%

No significant associations

[145]

sc-283

25%

Resistance to chemotherapy

[102]

IHC

HFR-1

73%, decreased compared with benign

Small tumor size

[101]

Prostate

50

IHC

Overexpression 60% of which cytoplasmic 38%, nuclear 34%

No significant associations

[109]



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n

Method Ab

ErbB4 expression


Ref.

Bone

21

IHC

sc-283

48%

Soft-tissue

29

IHC

sc-283

Overexpression

Poor histologic response and short [103] DFS [104] Upregulation of ErbB4 associates with response to chemotherapy and short DFS

Sarcoma


tissues in vivo. Unfortunately, no IHC analyses of cancer tissue series have so far been reported with antibodies that would differentiate between the ErbB4 isoforms or their cleavage status. However, RT-PCR analyses have demonstrated that breast cancer tissues exclusively express the cleavable JM-a isoform [11] . Interestingly, the immunohistochemically detected ErbB4 staining pattern either in the nucleus or in other cellular compartments excluding the nucleus may reflect the extent to which this cleavable ErbB4 isoform is indeed cleaved. When the prognostic significance of different ErbB4 subcellular staining patterns are compared within the subset of ErbB4-positive breast cancer, nuclear ErbB4 immunoreactivity associates with worse survival when compared with ErbB4 staining on the cell membrane [11] . Also, membranous ErbB4 staining associates with favorable survival when compared with tumors without such ErbB4 expression [85] . Interestingly, the tumor suppressor WW domain-containing oxidoreductase (Wwox) directly binds ErbB4, prevents nuclear translocation of ErbB4 ICD, and promotes accumulation of full-length ErbB4 at the membrane of breast cancer cells [85,86] . Taken together, these findings are consistent with a conclusion that although ErbB4 expression in general associates with favorable outcome in breast cancer, this may not be the case for the subpopulation of ErbB4-positive cancers that show ErbB4 in their nuclei [11] , or that coexpress ErbB4 with ErbB3 [81,82] . ErbB4 expression and activity is required for normal development of the central nervous system [61] and its abnormal activity is implicated in several pathologies of the brain including malignancies [87,88] . ErbB4 overexpression has been reported in medulloblastomas, ependymomas, pilocytic astrocytomas and glioblastomas [12,89–91] . By contrast, ErbB4 is underexpressed in meningiomas that are usually benign [92] . Although significantly overexpressed, ErbB4 8


alone is not a prognostic marker in medulloblastoma. However, when coexpressed with ErbB2 it significantly associates with poor prognosis, as compared with tumors solely expressing ErbB4 or ErbB2 [89] . Ependymomas also show an increased proliferation index (Ki-67) when ErbB4 is expressed together with ErbB2 [90] . Medulloblastoma, ependymoma and pilocytic astrocytomas all seem to demonstrate selectively elevated levels of mRNA encoding the cleavable JM-a isoform [12,90] . A PCR-based analysis has also indicated a shift in the CYT-1:CYT-2 ratio to favor the CYT-1 isoform in the more aggressive histological subtypes of medulloblastoma [93] . In conclusion, most data about central nervous system malignancies are consistent with a tumor-promoting role of ErbB4. The ErbB4 status in primary or metastatic CRC has been evaluated in five independent IHC studies all demonstrating overexpression of ErbB4. Lee et al. reported an increase of ErbB4 expression in late-stage tumors and an association of ErbB4/ErbB2 coexpression with short overall survival (OS) when compared with expression of any ErbB receptor alone [94] . Another study showed a significant increase of ErbB4/ErbB3 coexpression in metastatic tumors [13] . In addition, membranous ErbB4 expression, but not cytoplasmic expression, associates with lymph node involvement [95] , and is an independent prognostic factor of recurrence [96] . Nuclear ErbB4 immunoreactivity has not been evaluated in any of the CRC studies. Taken together, ErbB4 is overexpressed in a subset of CRCs and its expression seems to associate with unfavorable survival. The squamous cell carcinomas (SCC) of the esophagus, head and neck and cervix all show overexpression of ErbB4. Consistent with breast cancer data [11] , cytoplasmic or membranous ErbB4 staining in esophageal SCC associates with favorable survival, and nuclear immunoreactivity is an independent marker of future science group


Review

Cancer-associated somatic mutations of ErbB4 I

II

III

IV TM

Kinase

Ref. [17] (melanoma) *functional analysis L 39F Y 111H M313I *E 317K S 341L R 393W P 409L *E 452K R 491K *E 542K *R 544W *E 563K D609N P 700S *E 836K *E 872K G 936R P 1033S R 1174Q S 1246N Ref. [114] (NSCLC) S 303Y

C-terminus

Ref. [112] (NSCLC) N181S T 244R Y 285C R 306S V 348L D595V H618P D931Y K 935I Ref. [113] V721I A773S R782Q *G802dup E810K P854Q *D861Y E872K T926M

CRC Gastric NSCLC NSCLC NSCLC CRC CRC Breast NSCLC

*functional analysis Ref. [116] Ref. [115] (CRC) I1030M

Figure 3. Cancer-associated somatic mutations of ErbB4. Somatic mutations affecting the coding sequence of ErbB4 detected in melanoma, non-small cell lung cancer (NSCLC), colorectal carcinoma (CRC), breast, and gastric cancer are indicated by dots next to the schematic figure on the left. The amino acid changes, cancer tissues harbouring the mutations, and original references are listed on the right. The mutations are mostly located in regions important for ErbB4 activation: subdomain I or III (ligand-binding), subdomain II or IV (dimerization), or kinase domain (phosphorylation). TM indicates the transmembrane domain.

poor survival [15] . Bei et al. reported that 18% of patients with head and neck SCC demonstrate nuclear ErbB4 immunoreactivity that is independent of total ErbB4 overexpression and differentation stage of the tumor cells [97] . Xia et al. demonstrated that overexpression of ErbB4 significantly associates with distant metastasis and decreased overall survival in oral SCCs [98] . The presence of ErbB4 has also been demonstrated in several other cancer types from which less data are available. ErbB4 expression in bladder cancer seems to associate with favorable prognosis: low ErbB4 expression associates with low grade of differentiation, invasiveness and short survival [99] , whereas high ErbB4 expression associates with favorable prognosis [100] . The majority of thyroid cancer samples are positive for ErbB4, and ErbB4 expression associates with small tumor size [101] . In one study, expression of ErbB4 in 25% of patients with non-small cell lung cancer (NSCLC) did not correlate future science group

with survival but negative staining for ErbB4 significantly associated with response to chemotherapy [102] . Another report demonstrated ErbB4 expression in 48% of patients with bone sarcoma, and association of ErbB4 expression with poor response to chemotherapy as well as short disease-free survival [103] . Similarly, in limb soft-tissue sarcoma, increased ErbB4 expression correlated with resistance to chemotherapy and short disease-free survival [104] . ErbB4 is also expressed in ovarian, endometrial and prostate cancers but only limited data about clinical associations are currently available [105–109] . Cancer-associated somatic mutations of ErbB4

EGFR and ErbB2 genes are known to harbor mutations in cancer tissues [4] . The tyrosine kinase domain mutations of EGFR (most commonly DE746-A750 or L858R) in NSCLC cause a gainof-function phenotype that results from enhanced www.futuremedicine.com

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kinase activity of the receptor [110] . The mutations also confer sensitivity to EGFR tyrosine kinase inhibitors gefitinib and erlotinib providing a predictive marker. Another clinically relevant mutation of EGFR is T790M that promotes resistance to the tyrosine kinase inhibitors [111] . A recent report by Prickett et al. described somatic ErbB4 mutations in 19% of patients with metastatic melanoma [17] . These mutations were scattered throughout the ErbB4 gene (Figure 3) and all the seven missense mutations that were functionally analyzed induced kinase activity and promoted anchorage-independent growth [17] , supporting a driving role for ErbB4 in tumor progression [20] . In addition to melanoma, somatic ErbB4 mutations affecting different functional domains have been described in 2.3–4.8% of NSCLC, 0.68–2.9% of CRC, 1.7% of gastric cancer and 1.1% of breast cancer patients (Figure 3) [112–115] . Two of the mutations (D861Y and G802dup; found from CRC and NSCLC, respectively) cause a significant loss of ErbB4 tyrosine kinase activity indicating a loss-of-function phenotype for ErbB4 in cancer [116] . However, the attenuated kinase activity only kills the intrinsic kinase activity of ErbB4 necessary for signaling via ErbB4 homodimers and the ability to activate STAT5 but does not affect the ability of ErbB4 to heterodimerize with ErbB2 and to promote signaling via Erk and Akt pathways [116] . Thus, these two kinase-dead somatic mutants of ErbB4 may actually shift the balance of signaling outcome to favor Erk- and Akt-mediated growth and survival, as opposed to STAT5-dependent signaling that, in the context of ErbB4, has been associated with differentiation [8,9,116] . These findings, together with the functional observations about the melanoma-derived mutations indicate that at least part of the reported cancer-associated ErbB4 mutations have oncogenic properties. What have we learned from nonimmunological approaches to target ErbB4-positive cancer cells?

Whether ErbB4 signaling contributes to tumorigenesis has been addressed by analyzing both the effects of enhanced as well as downregulated ErbB4 activity. ErbB4 activity has been experimentally promoted by overexpressing wild type or constitutively active receptor constructs or using activating ligands, and downregulated using ErbB4-specific ribozymes, RNA interference, or antibodies. Several reports about the effects of the ErbB4 10


ligand neuregulin (NRG)-1 have indicated that NRG-1 suppresses breast cancer cell growth by delaying mitotic progression and inducing differentiation and apoptosis [7,55,64,117–119] , implying a tumor suppressor role for ErbB4. However, NRG-1 is not specific only for ErbB4 as it can also directly bind ErbB3 and promote different cellular responses depending on ErbB receptor dimerization [120,121] . Approaches to overexpress wild type ErbB4 in cellular backgrounds of no or endogenous ErbB4 expression have demonstrated either growth and survival advantage [18,62] , or enhanced apoptosis for cells expressing greater amounts of ErbB4 [64] . One explanation for the variable observations may be that cDNAs encoding different ErbB4 isoforms seem to promote different cellular responses [50,62] . Overexpression of the cleavable JM-a CYT-2 isoform or the sole CYT-2-type of soluble ICD promotes growth of breast cancer cells in vitro [11] and of normal mammary epithelium in vivo [50] . By contrast, the CYT-1 ICD promotes mammary epithelial cell differentiation [50] or apoptosis [64] . The Riese laboratory has studied the effects of a constitutively active ErbB4 (JM-a CYT-1) mutant Q646C and concluded that retroviral overexpression of this engineered construct suppresses, rather than promotes, colony formation of breast and prostate cancer cell lines [122,123] . In accordance, ectopic expression of another constitutively active ErbB4 (JM-a CYT-1) mutant, I658E, has been demonstrated to promote breast, prostate and ovarian cancer cell apoptosis [124] . However, cre-lox-mediated deletion of ErbB4 has no effect on the occurrence of mammary tumors induced by the neu transgene in mice arguing against a tumor-suppressor role for ErbB4 in vivo [125] . Given the difficulties in drawing conclusions about ErbB4 function from approaches utilizing nonspecific ligands or engineered ErbB4 mutants, analyses involving direct downregulation of ErbB4 expression in cells with natural ErbB4 expression may be more informative and relevant for possible clinical applications. Interestingly, to our knowledge most studies that have used specific reagents to downregulate endogenous ErbB4 in tumor cells have indicated a tumor growth-supporting role for ErbB4. Inhibition of ErbB4 activity with specific ribozymes decreases formation of breast tumors both in vitro and in vivo [19,126] . Proliferation of breast cancer cells is also suppressed by siRNAs targeting ErbB4 [60,62,127] . RNA interference approaches have further demonstrated that ErbB4 is necessary for survival of Ewing sarcoma future science group


ErbB4 JM-a isoform signaling

Review

Inhibition of signaling by mAb 1479

ErbB4 extracellular domain

1° shedding TACE

TACE

2° phosphorylation

ErbB4 intracellular domain

Ub

Ub

3° degradation

Endosome

Transcriptional regulation

Proteosome

Figure 4. Blocking ErbB4 signaling by the anti-ErbB4 mAb 1479. The ErbB4 JM-a isoform undergoes regulated intramembrane proteolysis (RIP) initiated by TNFa-converting enzyme cleavage that results in the release and translocation of the ErbB4 intracellular domain to the nucleus where it regulates transcription. mAb 1479 specifically interacts with the subdomain IV of the cleavable JM-a isoform of ErbB4. mAb 1479 inhibits ErbB4 signaling by decreasing shedding of ErbB4 which reduces the release of ErbB4 intracellular domain. Additionally, binding of mAb 1479 decreases ErbB4 phosphorylation possibly by preventing allosteric activation of the kinase domain, and promotes ubiquitination, endocytosis and finally receptor degradation. The outcome is decreased signaling via ErbB4 JM-a, and suppressed proliferation and tumor formation of breast cancer cells.

cells [128] , as well as of both normal colon epithelial [129] and CRC [130] cells. Finally, ErbB4specific siRNAs as well as the ErbB inhibitor lapatinib have been reported to suppress growth of melanoma cell lines with activating ErbB4 mutations [17] . Thus, although an ErbB4targeted siRNA has also been reported to suppress NRG-stimulated breast cancer cell apoptosis [64] , the outcome of ErbB4 downregulation in the great majority of the in vitro models seems to be reduced tumor cell growth and survival. Immunological approaches to target ErbB4-positive cancer cells

Although ribozymes and siRNAs are illustrative tools for experimental models, ErbB antibodies have already been successfully applied into clinical practice. The inhibition of ErbB4 with specific antibodies has been demonstrated to block tumor formation of lung, prostate and future science group

breast cancer cells in vitro and in vivo [16,18,131] . A monoclonal ErbB4 antibody, MAb-3 (clone H.72.8), suppresses the growth of lung [18] and prostate cancer [131] cells both in vitro and in mouse xenograft models, and inhibits ErbB4 activity by targeting the ligand-binding site in the receptor extracellular domain. Treatment with this antibody has been demonstrated to stimulate lung cancer cell apoptosis [18] and to sensitize prostate cancer cells to radiation therapy [131] . An antibody specific for the cleavable, and putatively more oncogenic, JM-a isoform of ErbB4 has been described to block breast cancer cell growth [16] . This IgG2 monoclonal antibody, mAb 1479, blocks ErbB4 ectodomain shedding and induces ErbB4 downregulation by promoting ubiquitination, endocytosis and degradation (Figure 4) [16] . The epitope of mAb 1479 has been mapped to the subdomain IV of ErbB4 ectodomain [Hollmen et al., University of Turku, www.futuremedicine.com

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Turku, Finland, Unpublished data] similar to trastuzumab that also blocks shedding of its target [38] . As mAb 1479 does not bind the alternative JM-b isoform of ErbB4, exclusively expressed in tissues such as heart, treatment with mAb 1479 could be expected to cause fewer side effects than nondiscriminating targeting of all ErbB4 isoforms. Interestingly, mAb 1479 is capable of inhibiting ErbB4 shedding and endocytosis independent of ErbB4 tyrosine kinase activity [16] , suggesting that it could also be used to block the naturally-occurring kinase-dead somatic mutants of ErbB4 [116] .

Potential prognostic & predictive use of ErbB4 antibodies

ErbB4 antibodies have also been used to immunohistochemically analyze ErbB4 overexpression and prognostic significance in different cancer tissues. As described above, ErbB4 is overexpressed in subpopulations of patients with either favorable or poor prognosis. In addition, IHC data about the subcellular localization of ErbB4 may provide prognostically relevant information. One of the major challenges in the development of modern targeted cancer drugs is to identify the subset of patients sensitive to the drug. Potential ErbB4 inhibiting therapy could optimally be targeted to patients who overexpress ErbB4, have activating ErbB4 mutations or, as discussed above, demonstrate ErbB4 cleavage in their cancer tissues. In addition to being indirectly assessed by IHC, ErbB4 cleavage could be more directly measured by estimating the amount of released ErbB4 ectodomain from serum samples using ELISA-based techniques. A similar approach has been used to quantify ErbB2 ectodomain in the sera of breast cancer patients. High levels of the shed ErbB2 ectodomain in patient serum has been proposed to decrease sensitivity to trastuzumab by neutralizing and preventing it from reaching the target tissues [132] . Thus, ELISAs measuring ErbB2 ectodomain level have been evaluated as possible predictive tools for trastuzumab response [133] . Direct evidence about ErbB4 cleavage in breast cancer tissues in vivo has been provided by Western analyses [16] . Consistent with IHC analyses demonstrating ErbB4 ICD in the nuclei of breast cancer tissues [11,75] , more ErbB4 ectodomain is present by Western analysis in breast cancer tissue when compared with histologically normal breast tissue from the same patients [16] . In accordance, significant quantities of soluble ErbB4 ectodomain are present in serum samples derived from breast cancer patients when 12


analyzed by ELISA using two specific ErbB4 monoclonal antibodies [Hollmén et al., University of Turku, Turku, Finland, Unpublished data] . In addition, expression and activity of TACE, the primary enzyme responsible of ErbB4 shedding [51] , is increased in breast, ovarian and prostate cancers and associates with ErbB4 expression [62,134] . These observations indicate that ErbB4 shedding is induced in tumors, such as breast cancer, and imply that techniques utilizing ErbB4 antibodies specifically detecting ErbB4 or the ErbB4 cleavage products could be of predictive value for future anti-ErbB4 therapies. As different ErbB heterodimers have different activities in promoting tumorigenesis [121] , measuring ErbB4 from clinical samples may also provide predictive information for existing therapies primarily targeting EGFR or ErbB2. Conclusion

ErbB4 protein is frequently present in several different types of cancer tissues, such as breast and colorectal cancer, SSCs, melanoma and various central nervous system malignancies. Somatic ErbB4 mutations have also been observed in melanoma, NSCLC, CRC, and in breast and gastric cancers. Some of the cell biological and clinical data about the role of ErbB4 in tumorigenesis and tumor progression have produced conflicting results. Most of the controversial data have been reported from analyses of breast cancer in which the biology of ErbB4 seems to be closely linked to the biology of ER in the prognostically favorable subgroup of patients. However, approaches to downregulate endogenous ErbB4 expression by antibodies, ribozymes or RNA interference have consistently resulted in growth suppression of ER-positive breast as well as other cancer types. Unlike RNA-interference and ribozymes, monoclonal antibodies targeting ErbB receptors (trastuzumab, cetuximab and panitumumab) have already been introduced into the clinical practice, and have demonstrated both efficacy as well as a favorable safety profile. An ErbB4 antibody, mAb 1479, was recently described to specifically target the cleavable tumor-associated ErbB4 JM-a isoform and to suppress breast cancer cell growth by a mechanism involving inhibition of receptor cleavage and stimulation of degradation. Antibodies against ErbB4 may also have prognostic or predictive value. Future perspective

To further elucidate the potential of targeting ErbB4 in cancer, more preclinical future science group


Review

Executive summary Targeting EGFR or ErbB2 with monoclonal antibodies has demonstrated clinical effect in the treatment of breast, colorectal and head and neck cancer. n While the role of ErbB4 as a driver of tumor progression is not clear, ErbB4 is overexpressed and/or mutated in a subset of cancer patients and experimental downregulation of ErbB4 in cancer cells suppresses growth. n ErbB4 is expressed as four functionally different alternatively spliced isoforms which may explain some of the controversial data and has implications for the outcome of targeting ErbB4 in cancer. n A monoclonal ErbB4 antibody, mAb1479, specifically recognizes a tumor-associated cleavable ErbB4 isoform, blocks signaling via ErbB4, and suppresses breast cancer cell growth. Monoclonal antibodies against ErbB4 have also demonstrated effect on lung and prostate cancer cells in vitro and in vivo. n In addition to potential therapeutic value, ErbB4 antibodies may have prognostic and predictive relevance for cancer therapy. n The cancer types that could be included in future preclinical and clinical tests with ErbB4 antibodies include breast, colorectal, and lung cancer, melanoma, squamous cell carcinomas, and malignancies of the central nervous system. n

experimentation is needed. For example, data from transgenic mice expressing different ErbB4 isoforms and cleavage products under specific promoters are expected to be informative. Functional data about recently described SNPs harboring the ErbB4 gene in CRC and breast cancer [135,136] and possibly also affecting ErbB4 splicing [87] , may provide interesting new insights into the cancer biology of ErbB4, similar to further functional analysis of the somatic ErbB4 mutations [17,112,113,116] . To specifically address the role of ErbB4 antibodies, such as mAb 1479, analyses of different in vivo cancer models need to be carried out. If justified based on the preclinical models, the cancer types to be assessed in possible future clinical trials with ErbB4 antibodies could include breast cancer, CRC, melanoma and SCCs of various tissues. While in breast cancer ErbB4 seems to be predominantly expressed in the ER-positive and ErbB2-negative subtype with relatively favorable survival and wellestablished current therapy, ErbB4 expression in CRC, SCCs and different neural malignancies is typically associated with poor clinical outcome [10] . Thus, in particular CRC and SCCs, the targeting of which does not need to comply


with the problems of penetrating the blood– brain barrier, may represent future targets for trials with humanized ErbB4 antibodies. Other potentially clinically relevant cancer types for future analyses are NSCLC and metastatic melanoma with somatic ErbB4 mutations and preclinical data supporting the concept of inhibiting tumor growth by targeting ErbB4. Selection of patients for these trials may include bioassays, such as anti-ErbB4 ELISAs from serum samples, to demonstrate the presence and tumor-promoting activity of the ErbB4 target. Finally, ErbB4 antibodies may be used as prognostic markers for different tumor types and for prediction of responsiveness to therapies targeted to other ErbB family members. Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.


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Maija Hollmén, MSc Department of Medical Biochemistry and Genetics, and Medicity Research Laboratory, University of Turku, and Turku Graduate School of Biomedical Sciences, Turku, Finland Klaus Elenius, MD Department of Medical Biochemistry and Genetics, and Medicity Research Laboratory, University of Turku, and Department of Oncology, Turku University Central Hospital, Kiinamyllynkatu 10, FIN-20520 Turku, Finland Tel.: +358 233 37 240 Fax: +358 233 37 300 [email protected]

145. Sasaki H, Okuda K, Kawano O et al.: ErbB4

expression and mutation in Japanese patients

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