Heparin-Binding Epidermal Growth Factor-Like Growth Factor as a Potent Target for Breast Cancer Therapy

Introduction

The human epidermal growth factor receptor (HER) family consists of four members: epidermal growth factor receptor (EGFR, HER1, or ErbB1), HER2 (ErbB2 or neu), HER3 (ErbB3), and HER4 (ErbB4). ¹ Upon binding ligands, these receptor tyrosine kinases, which normally exist as monomers, aggregate together through either homodimerization or heterodimerization to activate multiple downstream signaling pathways. ^2,3 Of note, HER2 does not have any known ligands but functions through heterodimerization with other members of the HER family. ⁴ Because of their ubiquitous existence in tissues and their potent effects on a variety of signaling pathways, the HER families play essential roles in physiological processes such as proliferation, differentiation, and development. More importantly, a large number of studies have shown that the functions of the HER family members are dysregulated in various types of malignancies such as lung cancer, pancreatic cancer, colon cancer, and breast cancer. ^{5 –9} A few scientists have reviewed the previous studies of cancers harboring abnormal HER-signaling from different aspects, such as the relationship between HER-signaling and different cancers, the relevant molecular mechanisms, and cancer therapy targeting HER-signaling. ^{10 –14} In this article, the authors focus on the roles of heparin-binding EGF-like growth factor (HB-EGF) in the tumorigenesis, development, and treatment of breast cancers.

As a ligand of EGFR and HER4, HB-EGF is mitogenic for fibroblasts, smooth muscle cells, hepatocytes, mesangial cells, and keratinocytes. ^{15 –18} It was initially identified as an O-glycosylated growth factor secreted by macrophage-like cells. ¹⁶ Synthesized as a type I transmembrane protein, proHB-EGF can be cleaved by a disintegrin and metalloprotease (ADAM) or a matrix metalloproteinase (MMP) through a process called “ectodomain shedding” to release soluble HB-EGF (sHB-EGF, mature form) and c-terminus of proHB-EGF (HB-EGF-C). ^19,20 HB-EGF is involved in a variety of physiological and pathological events, including wound healing, ^{21 –23} eyelid formation, ²⁴ blast implantation, ^25,26 pulmonary hypertension, ²⁷ atherosclerosis, ^{28 –30} heart development and function, ^{31 –33} adipogenesis, smooth muscle cell hyperplasia, ³⁴ brain injury, ^35,36 and tumor progression. ^{37 –40}

Breast cancer is one of the most common malignant tumors in women. Increasing reports have demonstrated that HB-EGF plays a pivotal role in mammary carcinoma progression and development, especially in metastasis, invasion, dissemination, and even drug resistance. In this review, the authors aim to elucidate the effects of HB-EGF signaling in breast cancer development and the value of HB-EGF-related pathways as targets for developing novel anticancer drugs against breast cancer.

Structure and Signaling Pathways of HB-EGF

Composed of 208 amino acids, proHB-EGF contains the signal peptide (1–24), propeptide (25–62), sHB-EGF (63–149), juxtamembrane (150–161), transmembrane (162–184), and cytoplasmic domains (185–208). ^19,41 Like other EGF family members, sHB-EGF also has an EGF domain, which has three intramolecular disulfide bonds essential for the binding of sHB-EGF to HER receptors thereby activating them. ^41,42 HB-EGF can be purified by heparin-Sepharose affinity chromatography, with 1 M NaCl for elution. ¹⁶ The membrane-anchored form, proHB-EGF, can interact with heparan-sulfate proteoglycans in adjacent cells in a juxtacrine manner, inhibiting its proteolytic cleavage, cell growth, and anoikis. ^43,44 Distinct from proHB-EGF, sHB-EGF binds EGFR in an autocrine and/or paracrine way, thus exhibiting its mitogenetic and migration promoting activities both in physiological and pathological processes. ^{45 –47} Subsequent to ectodomain shedding, HB-EGF-C translocates from the plasma into the nucleus to accelerate cell cycle by interacting with transcriptional repressors such as promyelocytic leukemia zinc finger and Bcl6. ^{48 –50} Notably, proHB-EGF can also be translocated into the nucleus, which may be correlated with cancer progression. ^{51 –53}

HB-EGF in Breast Cancer

A growing body of work supports that dysregulation of HB-EGF signaling pathway is involved in many kinds of cancers. ^{53 –55} The known functions of HB-EGF in cancer progression include supporting tumor growth, ⁵⁶ promoting angiogenesis, ⁵⁷ and increasing tumor metastasis. ^{58 –60}

More importantly, there are several studies demonstrating that HB-EGF plays a central role in mammary cancer progression, intravasation, metastasis, and dissemination. For instance, Yotsumoto et al. illustrated that HB-EGF expression was the highest among all EGFR ligands in all of 9 triple-negative breast cancer (TNBC) patients. ⁶¹ In another study of 363 breast tumors, HB-EGF was detected in 96% of the cases and its high expression was associated with larger tumors and higher histoprognostic grading. ⁶² In addition, Olsen et al. found that compared with autologous reference tissues, HB-EGF was upregulated in malignant tissues and positively associated with higher grades and poorer clinical outcome. ⁶³ Furthermore, a recent study showed that increased HB-EGF plasma levels correlated with lymph node dissemination of breast carcinomas. While the mean HB-EGF plasma concentrations of the healthy controls and lymph node dissemination-negative patients were 21.2 and 24.6 pg/mL, respectively, a significantly elevated mean HB-EGF level (97.5 pg/mL) was detected in patients with positive lymph node dissemination. ⁶⁴

Interestingly, other studies have also shown that HB-EGF is important in drug resistance of breast cancers. Fulvestrant is a selective estrogen receptor antagonist administrated in hormone receptor-positive metastatic breast cancer patients. ⁶⁵ Fulvestrant treatment was found to upregulate HB-EGF mRNA level, while mRNA levels of other EGFR ligands, such as amphiregulin and epiregulin, were decreased. Consequently, the elevated HB-EGF activated EGFR signaling and downstream extracellular signal-regulated kinase (ERK) signaling pathway, which significantly contributed to increased cell survival rates. ⁶⁶ In trastuzumab-resistant mammary carcinomas, HB-EGF also predominantly expressed among all the EGFR ligands and promoted cell survival and tumor growth through binding EGFR to activate ERK and protein kinase B (PKB) signaling pathways. ⁶¹

Mechanisms of HB-EGF Contributing to Breast Cancer Development

Through tumor cell invasion

Considerable amounts of HB-EGF were expressed in invasive breast cancer cell lines, such as MDA-MB-231, T47, and MCF7. ^{72 –74} Increased HB-EGF usually stimulates proliferation, migration, and invasion of cancer cells. ⁷⁵

In estrogen receptor-negative breast cancers, especially TNBC, the increase of autocrine HB-EGF led to elevated basal cell motility, increased invadopodium production, and enhanced invasion. ⁷⁶ Invadopodia are actin-rich protrusions capable of degrading extracellular matrix (ECM) during metastasis. ⁷⁷ Phosphorylation of invadopodia protein cortactin, which is achieved by Src, is essential for invadopodia formation and function. ⁷⁸ Both Src and MEK/MAPK activations occur as a result of EGFR activation. ⁷⁹ In addition, the MEK/MAPK pathway also contributes to the high expression of MMPs (such as MMP2 and MMP9), which can directly degrade ECM or indirectly disintegrate ECM by promoting invadopodia maturation (Fig. 1A). Degradation of ECM by MMPs and invadopodia induced by activated EGFR signaling pathways finally promotes tumor invasion and metastasis.

OPEN IN VIEWER

FIG. 1.

Autocrine and paracrine loop of HB-EGF in promoting cancer development. (A) Tumor cell-derived HB-EGF activates EGFR, then the activated EGFR upregulates the level of MMP2 and MMP9 and phosphorylates the invadopodia protein cortactin, respectively. Both pathways promote the invadopodia formation and ECM degradation, ultimately leading to tumor metastasis and invasion. (B) Cytokines, such as CCL2, produced by tumor cells, recruit monocytes and stimulate them to differentiate into macrophages, which secrete HB-EGF substantially and act on EGFR of tumor cells in a feedback way. CCL2, CC-chemokine ligand 2; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; HB-EGF, heparin-binding EGF-like growth factor; MMP, matrix metalloproteinase. Color images available online at www.liebertpub.com/cbr

Deriving from the mononuclear phagocytic lineage, ⁸⁰ tumor-associated macrophages (TAMs) ^{81 –83} are associated with tissue remodeling and tumor angiogenesis. ⁸⁴ As tumors progress, monocytes are recruited by tumor cell-secreted factors such as CC-chemokine ligand 2 (CCL2) ⁸⁵ and differentiate into macrophages, which prepare the favorable environment for the growth of tumor cells by creating a mutagenic and growth promoting inflammatory environment and suppressing antitumor immunity. In addition, it was shown that ⁶⁴ in breast carcinoma patients, TAMs substantially expressed HB-EGF, which boosts migration and invasion of tumor cells (Fig. 1B).

HB-EGF Contributes to Breast Cancer Through Its Shedding Mediated by ADAMs

The ADAMs are multidomain transmembrane proteins that have a diverse array of functions in both physiological and pathological processes. ⁸⁶ ProHB-EGF is a favorable substrate of ADAMs since many ADAMs are involved in proHB-EGF shedding, such as ADAM9, 10, 12, and 17. ^87,88

A number of ADAMs have been implicated in the development of breast cancer. ADAM9 has been suggested to be involved in the processes of breast cancer invasion and metastasis. ⁸⁹ ADAM10 and ADAM17 are reported to mediate trastuzumab resistance and correlate with survival in HER2-positive breast cancer. ⁹⁰ Besides, ADAM17 is expressed at significantly higher levels in TNBC. More importantly, Li et al. reported that there was a positive correlation between ADAM12L (a transmembrane form of ADAM12) and HB-EGF in TNBC, and increased expression of ADAM12L augmented EGFR phosphorylation, which results in a decrease of distant metastasis-free survival time. In conclusion, their results suggest that ADAM12L is responsible for EGFR activation in lymph node-negative TNBC by disintegrating HB-EGF. ⁹¹

Explorations in Breast Cancer Therapeutics Involving HB-EGF Signaling

CRM197, a nontoxic mutant of the diphtheria toxin that specifically binds to HB-EGF, blocks HB-EGF-mediated ERK (as well as PKB) activation in TNBC or trastuzumab-resistant breast cancer and inhibits tumor growth by causing significant cell apoptosis. ⁶¹ So far, clinical trials for CRM197 have been initiated.

Tyrosine142 (Y-142), an anti-HB-EGF monoclonal antibody that recognizes sHB-EGF, inhibits HB-EGF-induced proliferation of T47D breast cancer cells. Y-142 also reduces VEGF production and tube formation of human umbilical vein endothelial cells more effectively than cetuximab (an inhibitor of EGFR), CRM197, and bevacizumab (an anti-VEGF antibody). ⁷²

When the anti-HB-EGF antibody was conjugated to liposome encapsulating siRNA or anticancer drugs such as doxorubicin, the new system showed high affinity to HB-EGF and selectively interacted with cells expressing HB-EGF. This new system was found to significantly suppress tumor progression compared to the control. ^74,92

Using HB-EGF, Lois et al. constructed a cytotoxic protein delivery system, in which sHB-EGF was linked to plant ribosome-inactivating protein saporin through a 22-amino-acid flexible linker (L₂₂). The fusion protein HB-EGF-L₂₂-saporin showed both cytotoxicity to breast carcinoma cells in vitro and proliferation inhibition in vivo. ⁹³

Lysophosphatidic acid (LPA), a natural lipid mitogen and motility factor, is an extracellular signal transmitter and intracellular second messenger that acts on six G protein-coupled receptors. ^94,95 LPA signaling pathways influence the metastasis of mammary carcinoma. ^{96 –98} However, the anti-LPA therapy study is limited by the deficiency of specific biomarkers of LPA activation. LPA signaling activation can induce HB-EGF expression and stimulate proHB-EGF ectodomain shedding. ^99,100 Since the expression and maturation of HB-EGF are specifically controlled by LPA activation and sHB-EGF is easy to be detected as a secreted factor, HB-EGF would be a valuable biomarker to assist anti-LPA therapies. ¹⁰¹

Conclusions and Perspectives

The results of recent studies have shown that expression levels of HB-EGF are closely correlated with tumorigenesis, development, metastasis, and drug resistance of breast cancers, and HB-EGF is a promising target against breast cancers. Inhibition of HB-EGF through its antibody or affibody (CRM197) blocks drug-induced EGFR activation and leads to decreased tumor growth. Because of the high expression of HB-EGF in TNBC, the therapeutic strategy targeting HB-EGF is especially attractive in this cancer. In addition, as a biomarker, it can be utilized to diagnose breast cancers and provide individual antineoplastic treatment.