ANTICANCER RESEARCH 29: 4879-4886 (2009)
`
`Review
`
`Efficacy of Ligand-based Targeting
`for the EGF System in Cancer
`FUSANORI YOTSUMOTO1, AYAKO SANUI2, TATSUYA FUKAMI2, KYOKO SHIROTA2, SHINJI HORIUCHI2,
`HIROSHI TSUJIOKA2, TOSHIYUKI YOSHIZATO2, MASAHIDE KUROKI1 and SHINGO MIYAMOTO1,2
`Departments of 1Biochemistry, and 2Obstetrics and Gynecology, School of Medicine,
`Fukuoka University, 45-1, 7-chome, Nanakuma, Jonan-ku, Fukuoka, 814-0180, Japan
`
`Abstract. Although drugs inhibiting ErbB receptors such
`as epidermal growth factor receptor (EGFR) and HER2 have
`been developed as anticancer agents targeting the EGF
`family, they are not effective for all types of cancer and
`instead target only certain types. We propose the following
`four main reasons for these observations: (i) although seven
`EGFR ligands exist, effective inhibition of specific EGFR
`ligands may occur because their expression levels differ in
`different malignancies; (ii) suppressing EGFR ligands
`inhibits aggregation of EGFR and other ErbB receptors and
`activation of ERK and Akt signals; (iii) EGFR ligands may
`have various combinations for signal transduction through
`the EGFR pathway and other receptor signals; and (iv) the
`intracellular C-terminals of EGFR ligands move into the
`nucleus and strongly regulate cell proliferation. In this
`review, we describe important implications for targeted
`cancer therapy against EGFR ligands and describe the
`current situation in the development of ligand-based
`therapies for cancer.
`Epidermal growth factor (EGF) ligands, comprising EGF-
`related peptides, activate EGF receptor (EGFR, also known as
`ErbB1/HER1) and other ErbB receptors (ErbB3/HER3 and
`ErbB4/HER4). They are broadly classified into EGFR family
`ligands that bind to EGFR (comprising EGF, transforming
`growth factor (TGF)-α, heparin-binding EGF-like growth
`factor (HB-EGF), amphiregulin (AR), epiregulin, betacellulin
`(BTC) and epigen) and neuregulin (NRG) family ligands that
`
`Correspondence to: Shingo Miyamoto, Department of Biochemistry,
`School of Medicine, Fukuoka University, 45-1, 7-chome, Nanakuma,
`Jonan-ku, Fukuoka, 814-0180, Japan. Tel: +81 928011011, Fax: +81
`928013600, e-mail: smiya@cis.fukuoka-u.ac.jp
`Key Words: Cancer, HB-EGF, amphiregulin, targeted therapy,
`review.
`
`bind to ErbB3 or ErbB4 (comprising NRG1, NRG2, NRG3
`and NRG4) (1). Binding of ligands to the extracellular
`domains of ErbB receptors initiates their homodimerization or
`heterodimerization with other ErbB
`receptors
`and
`phosphorylation of tyrosine residues within their cytoplasmic
`domains, which in turn activates downstream growth and
`survival signals such as the mitogen-activated protein kinase
`(MAPK) and phosphoinositol 3-kinase/v-akt murine thymoma
`viral oncogene homolog (PI3K/AKT) pathways (2-4). No
`ligands that bind ErbB2 have been identified, and the kinase
`activity of ErbB3 is defective. These receptors are capable of
`generating intracellular signals by forming heterodimers with
`other ErbB receptors (5, 6). The EGF family members play
`important roles in normal tissue processes including ontogeny,
`morphogenesis, migration, differentiation and proliferation.
`Dysregulation of EGF family members and related signaling
`molecules can contribute to carcinogenesis and is associated
`with tumorigenesis, invasion and metastasis (2).
`EGFR and ErbB receptors have been especially focused
`upon as target molecules for cancer treatments because
`overexpression and mutations of these receptors are
`frequently observed in human malignancies. A variety of
`small molecule kinase inhibitors targeting EGFR (e.g.
`erlotinib: Tarceva™) and monoclonal antibodies targeting
`EGFR
`(e.g.
`cetuximab: Erbitux) and HER2
`(e.g.
`trastuzumab: Herceptin™) have been developed and some of
`them have already been used for treatment of lung cancer
`and breast cancer (7). However, these medicines have not
`exhibited the expected levels of clinical efficacy thus far,
`despite numerous cases of administration to patients with
`malignant tumors targeting EGFR and HER2 (8). One of the
`reasons for these observations is that EGFR and HER2 form
`complexes with HER3 and other signal receptors. The
`proliferation of cancer cells is subsequently accelerated by
`these complexes, whose formation cannot be inhibited by
`targeted therapies against EGFR and HER2. Another reason
`is that the anti-EGFR drugs can suppress the proliferation of
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`ANTICANCER RESEARCH 29: 4879-4886 (2009)
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`extracellular signal-related kinase (ERK) signals located
`downstream of EGFR but cannot suppress protein kinase B
`(AKT) survival signals. This background has given rise to
`an increasing demand for further development of targeted
`medicines against the EGF family. The belief that cancer
`agents targeting EGFR ligands are less effective than those
`targeting receptors has delayed the development of such
`medicines until now, but our research has revealed that this
`notion is not necessarily true.
`In this review, we will discuss the significance of targeting
`EGFR
`ligands for cancer
`therapy and describe
`the
`characteristics and present state of the development of
`anticancer agents targeting EGFR ligands.
`Significance of Targeting EGFR
`Ligands for Cancer Therapy
`Predominant expression of HB-EGF or AR in cancer (Figure
`1a). Enhancement of EGFR ligand expression, an autocrine
`loop mediated by an EGFR ligand itself, is the main
`mechanism
`implicated
`in cancer development and
`progression (9-11). First, in order to identify the EGFR
`ligands forming autocrine loops in cancer, we examined the
`expression levels of EGFR ligands in a variety of cancer cell
`lines (12). HB-EGF expression was dominantly elevated in
`ovarian, gastric and breast cancer, melanoma and
`glioblastoma. In pancreatic, colon and prostate cancer, renal
`cell carcinoma and cholangiocarcinoma, the expression of
`AR was primarily enhanced. Next, in order to examine
`whether inhibition of EGFR ligands exerts antitumor effects,
`we transfected individual small interfering RNAs (siRNAs)
`for these EGFR ligands into the cancer cell lines. In the cell
`lines with dominant expression of HB-EGF, a siRNA for
`HB-EGF increased the number of apoptotic cells and
`suppressed the activation of EGFR and ERK, whereas
`transfection of siRNAs for other EGFR ligands had no
`effects. Similarly, in the cell lines with abundant expression
`of AR, apoptosis and attenuation of EGFR and ERK signals
`were significantly mediated by inhibition of AR, while
`inhibition of the other EGFR ligands had no effects. Taken
`together, these findings suggest that HB-EGF and AR play
`pivotal roles in cancer cell proliferation and should be
`considered as promising target molecules for cancer therapy.
`Increases in the levels of HB-EGF or AR can also
`contribute to oncogenic transformation. In the absence of
`growth factors, Ha-ras-transformed human mammary
`epithelial cells do not form colonies in soft agar, and
`exogenous recombinant human HB-EGF is able to promote
`their anchorage-independent growth (13). AR is not
`expressed in the healthy liver, but is induced in the
`regenerating liver after partial hepatectomy and behaves as
`a pivotal mitogenic and antiapoptotic factor for normal
`hepatocytes (14, 15). Conversely, suppression of AR
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`production dramatically reduces the aggressiveness of
`hepatocellular carcinoma cancer cells
`in anchorage-
`independent growth (16). Furthermore, EGFR ligands play
`important roles in inflammatory and neoplastic lesions in
`human tissues. Secretion of HB-EGF or AR, a heparin-
`binding EGFR ligand with undetectable expression in normal
`gastric tissues, is stimulated by the hormone gastrin, which
`induces gastric mucosa proliferation, and the inflammatory
`cytokine interleukin-1β. Inflammation of the gastric mucosa
`is itself associated with the proliferation of parietal cells in
`the gastric gland and
`the development of gastric
`malignancies (17). These pieces of evidence indicate that
`among the EGFR ligands, HB-EGF and AR are the main
`contributing growth factors for human carcinomas.
`
`Activation of signals-mediated ErbB receptor heterodime-
`rization resistance to anti-EGFR or anti-HER2 therapy
`(Figure 1b). Recently, there have been a large number of
`studies confirming the notion that dimerization of ErbB
`receptors is associated with resistance to anti-EGFR or anti-
`HER2 therapy (18-20). Dimerization is necessary for the
`signaling activity of ErbB receptors. The ligand-induced
`formation of a receptor complex stimulates the intrinsic
`tyrosine kinase activities of the receptors and induces
`autophosphorylation of specific tyrosine residues within their
`cytoplasmic domains. These phosphorylated residues serve
`as docking sites for various adaptor proteins and enzymes
`involved in potent signaling cascades, such as the raf proto-
`oncogene serine/threonine protein kinase/mitogen-activated
`protein kinase kinase/mitogen-activated protein kinase
`(Raf/MEK/MAPK) and phosphoinositide 3 kinase/v-akt
`murine thymoma viral oncogene homolog (PI3K/AKT)
`pathways (21, 22). When one receptor is functionally
`inactivated, its function as a receptor tyrosine kinase can be
`replaced by another receptor among the HER receptors.
`Gefinitib down-regulates the signaling pathway via EGFR,
`but does not block dimer formation between EGFR and other
`HER receptors. EGFR/HER2 (and EGFR/HER3) complex
`formation is increased in PC-9/ZD cells, a non-small cell
`lung cancer cell line with acquired gefinitib resistance (23).
`In addition, we tested the abovementioned hypothesis using
`MDA-MB-468 cells, a breast cancer cell line that secretes
`abundant soluble HB-EGF (12). In serum-free medium
`supplemented with HB-EGF, MDA-MB-468 cells formed
`EGFR/HER2 complexes, and this complex formation was
`enhanced by trastuzumab but reduced by CRM197, a
`specific HB-EGF
`inhibitor
`(unpublished
`data).
`Correspondingly, CRM197 attenuated the phosphorylation of
`ERK as well as AKT and led to significant apoptotic cell
`death compared with trastuzumab (unpublished data). Taken
`together, it is assumed that ligand-induced dimerization of
`ErbB receptors plays important roles in retrieving the
`intracellular signaling for cell survival against targeted
`
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`Yotsumoto et al: EGFR Ligands in Targeted Cancer Therapy (Review)
`
`Figure 1. Multidirectional functions of ligands in the EGF family. (a) Expression of a specific EGFR ligand (HB-EGF or AR) is enhanced in fourteen
`types of carcinomas and associated with cancer progression. (b) Binding of a ligand induces heterodimerization of ErbB receptors and subsequently
`activates resistance signals to targeted therapies for ErbB receptors. (c) Ligand-bound EGFR interacts with other growth factor receptors and
`SGLT1. (d) The shed extracellular domain and the CTF of HB-EGF transmit cell proliferation signals.
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`ANTICANCER RESEARCH 29: 4879-4886 (2009)
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`therapy against EGFR or HER2 alone, and that targeting of
`a dominantly expressed EGFR ligand is an available strategy
`for the development of cancer therapies.
`
`Combinations of EGFR and other signaling pathways
`(Figure 1c). EGFR signaling can be modulated by several
`mechanisms that include transactivation by or crosstalk with
`other growth factor receptors such as insulin-like growth
`factor-I receptor (IGF-IR) (24) or steroid hormone receptor
`(25). Enhancement of IGF-IR expression was reported to be
`associated with
`trastuzumab
`resistance
`in HER2-
`overexpressing breast cancer cells. Inhibition of IGF-IR
`signaling rescued trastuzumab sensitivity (26). In recent
`studies, IGF-I was identified as another ligand that uses
`EGFR for MAPK and AKT activation (27, 28). IGF-I
`binding to IGF-IR activates matrix metalloproteinase
`(MMP)-2 and MMP-9, which cleave HB-EGF and release it
`to bind to EGFR, thereby leading to stimulation of MAPK.
`In MCF-7 breast cancer cells, IGF-IR activation induced by
`17β-estradiol/estrogen receptor α complexes on the cell
`membrane can also trigger a downstream signaling cascade
`through MMP-2, MMP-9, HB-EGF and EGFR and finally
`active MAPK (29). MDA-MB-468 cells are also a model for
`gefinitib resistance via constitutive activation of the
`intracellular signaling downstream of EGFR (i.e. the PI3K-
`AKT and MEK-MAPK pathways) (30-32). Under gefinitib
`treatment, MDA-MB-468 cells exhibit significant up-
`regulation (by up to 21-fold) of the EGFR ligands EGF, AR
`and BTC (33).
`Although the significance of EGFR, an oncogenic protein,
`has been sufficiently proven, as described above, this review
`begins to clarify a new role of EGFR in malignancy. The
`Authors have demonstrated that the extracellular domain of
`EGFR associates with and stabilizes the sodium/glucose co-
`transporter SGLT1 to promote glucose uptake into cancer
`cells (34). In PC-3MM2 prostate cancer cells, a siRNA for
`EGFR induced autophagic cell death with a decrease in the
`intracellular glucose level, whereas inhibition of EGFR
`kinase did not exert these effects. Furthermore, EGFR
`increased its complex formation with SGLT1 and glucose
`uptake following stimulation of EGFR by exposure to EGF
`in serum-free medium. These novel insights into EGFR
`functions could widen its potential in the development of
`anticancer agents for EGF family members.
`Transfer of HB-EGF-CTF to the nucleus (Figure 1d). Recent
`studies have reported that the intracellular HB-EGF
`carboxyl-terminal fragment (CTF) translocates from the
`plasma membrane to the nucleus and regulates the cell cycle
`when membrane-anchored HB-EGF
`is proteolytically
`cleaved by a disintegrin and metalloprotease (35, 36). BAG-
`1, promyelocytic leukemia zinc finger (PLZF) and B-cell
`leukemia 6 (Bcl6) have been identified as binding proteins
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`for HB-EGF-CTF by yeast two-hybrid screening with the
`cytoplasmic region of HB-EGF (amino acids 185-208).
`Interactions between BAG-1, a prosurvival co-chaperone,
`and HB-EGF-CTF lead to attenuation of cell adhesion,
`resistance to apoptosis and enhancement of soluble HB-EGF
`expression (37). PLZF and Bcl6 are
`transcriptional
`repressors, and function as negative regulators within the cell
`cycle. Internalized HB-EGF-CTF co-localizes with PLZF or
`Bcl6 at the nuclear periphery, which releases suppression of
`the cell cycle. In addition, inhibition of HB-EGF-CTF
`nuclear translocation has been shown to reduce gastric
`cancer cell growth (38). Treatment using KB-R7785, an
`inhibitor of HB-EGF shedding, with or without EGFR
`activation by cetuximab interferes with the transfer of HB-
`EGF-CTF from the plasma membrane to the nucleus. As a
`result, KB-R7785 induces cell cycle arrest and increases the
`subG1 DNA content because PLZF remains in the nucleus
`and suppresses the cell cycle. Investigation of these functions
`of HB-EGF-CTF will lead to further understanding of the
`actions of EGFR ligands as growth factors and also provide
`new aspects for targeted therapies against EGFR ligands.
`Present State of the Development of Anticancer
`Agents Targeting EGFR Ligands
`Development of targeted therapeutic agents using CRM197.
`As discussed earlier, HB-EGF may be a promising target for
`ovarian cancer (39-41). Diphtheria toxin secreted by
`Corynebacterium diphtheriae binds to the EGF domain of
`HB-EGF and inhibits cell proliferation activity. Diphtheria
`toxin cannot be used as an HB-EGF inhibitor owing to its
`strong
`toxicity. However, cross-reacting material 197
`(CRM197), a mutated diphtheria toxin, can be used because it
`is a non-toxic protein with a variation in the active site and
`binds to HB-EGF more strongly than, or at least as strongly
`as, diphtheria toxin. We investigated the anticancer effects of
`CRM197 on ovarian cancer by evaluating the proliferation of
`human ovarian cancer cell lines (namely SKOV3, RMG1 and
`OVMG1) subcutaneously
`implanted
`into nude mice.
`CRM197 significantly suppressed peritoneal dissemination in
`the nude mice peritoneally injected with RMG1 or high HB-
`EGF-expressing SKOV3 cells (42). Furthermore, concomitant
`administration of CRM197 and paclitaxel induced complete
`disappearance of tumors at concentrations that showed no
`satisfactory antitumor effects in single treatments with either
`agent (43). The above findings suggest that CRM197 should
`be considered as a promising antineoplastic agent against
`ovarian cancer because it shows synergistic antitumor effects
`with a conventional chemotherapeutic agent and exerts effects
`on peritoneal dissemination. We have already started phase 1
`of a clinical trial of CRM197 administration for intractable
`advanced or recurrent ovarian cancer patients under the
`approval of an ethical committee.
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`Yotsumoto et al: EGFR Ligands in Targeted Cancer Therapy (Review)
`
`Pan-ligand trapping as a targeted therapy for cancer. In
`2008, a new strategy was reported that targets multiple
`ligands in the EGF family using a bispecific ligand trap,
`RB200 (44). RB200 was designed as a chimeric molecule
`composed of the full-length extracellular domains (ECDs) of
`EGFR and HER3 fused with the Fc domain of human
`immunoglobulin G1. cDNAs for EGFR and HER3 linked to
`the Fc domain (EGFR/Fc and HER3/Fc, respectively) were
`transiently co-transfected into HEK293T cells. RB200 was
`obtained by purification of conditioned medium harvested
`from the co-transfected cells. The purified RB200 bound
`EGFR ligands including EGF, TGFα and HB-EGF as well
`as HER3 ligands including NRG1-α, NRG1-β1 and NRG1-
`β3. Experiments were carried out to elucidate whether
`RB200 could inhibit ligand-stimulated phosphorylation of
`ErbB receptors and cell proliferation in a variety of cancer
`cells. Compared with C225 (the murine parent of cetuximab)
`and trastuzumab, RB200 successfully competed with the
`receptors for binding to ligands and suppressed both EGF-
`and NRG1-β1-induced tyrosine phosphorylation of EGFR,
`HER2 and HER3. Furthermore, RB200 exhibited inhibitory
`effects on cell growth in monolayer cultures of nine tumor
`cell lines and in vivo antitumor efficacy in two xenograft
`models (A431 human epidermoid carcinoma cells and H1437
`non-small cell lung cancer cells). Furthermore, mutants with
`amino acid substitutions in the EGFR and HER3 ECDs
`showed enhanced ligand-binding affinities and were more
`powerful than RB200 for inhibition of cell proliferation (45).
`In conclusion, EGFR and HER3 ligand traps may be potent
`tools for cancer therapy.
`Future Directions
`treatments with conventional
`Limitations of cancer
`anticancer agents, such as nucleic acid analogs and cell
`division inhibitors, developed so far have already been
`proven, making it all the more crucial to develop molecular
`targeted medicines on the basis of cancer characteristics.
`The targeted therapies that have achieved certain treatment
`outcomes to date have involved either plasma membrane
`molecules (receptors: EGFR, HER2; ligands: vascular
`endothelial growth factor, HB-EGF) or nuclear receptors
`(estrogen
`receptor,
`androgen
`receptor, peroxisome
`proliferator-activated receptor γ). We have demonstrated
`through non-clinical basic science investigations that
`CRM197 targets HB-EGF, blocks the autocrine loop created
`by HB-EGF and shows efficacy exceeding that of targeted
`therapies for EGFR. As a consequence, CRM197 is
`currently undergoing a clinical trial for administration to
`cancer patients.
`In future studies, we aim to elucidate the transcriptional
`mechanism involved in the acceleration of the autocrine loop
`mechanism by HB-EGF and identify novel target molecules
`
`that can inhibit nuclear signal transduction. The targeted
`medicines developed based on the results of these studies are
`expected to possess synergistic antitumor effects when used
`concomitantly with CRM197. Since these studies aim to
`identify transcription factors that induce the expression of
`HB-EGF, they appear to represent research into creating a
`novel concept targeting the molecules controlling the EGF
`system. According to our understanding, introduction of the
`HER2 gene or a mutated K-ras gene accelerates HB-EGF
`expression in breast cancer. These observations suggest that
`the transcription factor controlling the expression of HB-
`EGF is likely to be a target molecule not only for breast
`cancer but also for stomach and pancreatic cancer with
`abnormalities in the K-ras gene. Consequently, we presume
`that these findings will help to improve the prognosis of
`cancer patients as well as clarify how EGF family members
`promote cancer progression.
`Acknowledgements
`The Authors would like to thank Professor Eisuke Mekada
`(Department of Cell Biology, Research Institute for Microbial
`Diseases, Osaka University) for helpful discussions. This work was
`supported in part by a Grant-in-Aid for Scientific Research on
`Priority Areas (No. 19591947) and Research Promotion for
`Innovative Therapies against Cancers from the Ministry of
`Education, Culture, Sports, Science and Technology to E.M.
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`Received April 28, 2009
`Revised July 28, 2009
`Accepted September 16, 2009
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