Fusanori Yotsumoto

Heparin-binding epidermal growth factor-like growth factor as a novel targeting molecule for cancer therapy

HB‐EGF, a member of the EGF family of growth factors, exerts its biological activity through activation of the EGFR and other ErbB receptors. HB‐EGF participates in diverse biological processes, including heart development and maintenance, skin wound healing, eyelid formation, blastocyst implantation, progression of atherosclerosis and tumor formation, through the activation of signaling molecules downstream of ErbB receptors and interactions with molecules associated with HB‐EGF. Recent studies have indicated that HB‐EGF gene expression is significantly elevated in many human cancers and its expression level in a number of cancer‐derived cell lines is much higher than those of other EGFR ligands. Several lines of evidence have indicated that HB‐EGF plays a key role in the acquisition of malignant phenotypes, such as tumorigenicity, invasion, metastasis and resistance to chemotherapy. Studies in vitro and in vivo have indicated that HB‐EGF expression is essential for tumor formation of cancer‐derived cell lines. CRM197, a specific inhibitor of HB‐EGF, and an antibody against HB‐EGF are both able to inhibit tumor growth in nude mice. These results indicate that HB‐EGF is a promising target for cancer therapy, and that the development of targeting tools against HB‐EGF could represent a novel type of therapeutic strategy, as an alternative to targeting ErbB receptors. (Cancer Sci 2006; 97: 341–347)

Heparin-binding epidermal growth factor–like growth factor promotes transcoelomic metastasis in ovarian cancer through epithelial-mesenchymal transition

Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is involved in several biological processes including cell adhesion, invasion, and angiogenesis. HB-EGF also plays a pivotal role in the progression of ovarian cancer. To investigate the significance of HB-EGF in peritoneal dissemination, we examined the roles of HB-EGF in cell adhesion, invasion, and angiogenesis in ovarian cancer. Through the suppression of focal adhesion kinase and EGF receptor activation, cell adhesive properties mediated by integrin β1 were diminished by the inhibition of HB-EGF expression. The reduction of HB-EGF expression attenuated the chemotactic invasive ability and the expression of matrix metalloprotease (MMP)-2 and vascular endothelial growth factor (VEGF), leading to the inhibition of cell invasion and angiogenesis. Suppression of the Snail family, which regulates the epithelial-mesenchymal transition, blocked the cell adhesion properties on extracellular matrices, the chemotactic invasive ability, and the expression of MMP9 and VEGF through the reduction of HB-EGF expression. The volume of tumor burden in the peritoneal cavity was dependent on the expression of HB-EGF. According to these results, HB-EGF contributes to cell adhesion, invasion, and angiogenesis, which are integral to transcoelomic metastasis in ovarian cancer. CRM197, an inhibitor of HB-EGF, resulted in a significant decrease of tumor burden in peritoneal dissemination, accompanied with a reduction in both cellular spreading, when assayed on an extracellular matrix, and invasive ability, when assayed in a chemotaxis chamber, as well as decreased expression of MMP9 and VEGF. Thus, HB-EGF is a mutual validating target in the peritoneal dissemination of ovarian cancer, and CRM197 may be useful as a anticancer agent for advanced ovarian cancer. [Mol Cancer Ther 2008;7(9):3441–51]

Synergistic anti‐tumor effect of paclitaxel with CRM197, an inhibitor of HB‐EGF, in ovarian cancer

Heparin‐binding EGF‐like growth factor (HB‐EGF) plays a pivotal role in tumor growth and clinical outcomes in patients with ovarian cancer, leading to the validation of HB‐EGF as a target for ovarian cancer therapy. In this study, we investigated the anti‐tumor effects of paclitaxel, as an anti‐cancer agent, and CRM197, as a specific inhibitor off HB‐EGF, in ovarian cancer. Paclitaxel induced transient ERK activation and sustained activation of JNK and p38 MAPK through the ectodomain shedding of HB‐EGF in SKOV3 cells. In addition, the overexpression of HB‐EGF in paclitaxel‐treated SKOV3 cells resulted in modulation of paclitaxel‐evoked MAPK signaling, including marked activation of ERK and Akt, and minimized activation of JNK and p38 MAPK, indicating that HB‐EGF is involved in drug sensitivity through the balance of anti‐apoptotic and pro‐apoptotic signals induced by paclitaxel. The combination of paclitaxel with CRM197 had an inhibitory effect on cell proliferation and enhanced apoptosis via the inhibition of ERK and Akt activation and the stimulation of p38 and JNK activation. More prominently, the administration of paclitaxel with CRM197 resulted in synergistic anti‐tumor effects in SKOV3 cells and in SKOV3 cells overexpressing HB‐EGF in xenografted mice. Accordingly, inhibitory agents against HB‐EGF, such as CRM197, represent possible chemotherapeutic and chemosensitizing agents for ovarian cancer.

Amphiregulin regulates the activation of ERK and Akt through epidermal growth factor receptor and HER3 signals involved in the progression of pancreatic cancer

Pancreatic cancer is one of the most lethal malignancies. Epidermal growth factor receptor (EGFR), HER3, Akt, and amphiregulin have been recognized as targets for pancreatic cancer therapy. Although gemcitabine + erlotinib has been the recommended chemotherapy for pancreatic cancer, the prognosis is extremely poor. The development of molecularly targeted therapies has been required for patients with pancreatic cancer. To assess the validation of amphiregulin as a target for pancreatic cancer therapy, we examined its expression in pancreatic cancer using real‐time PCR analyses and ELISA. We also measured the apoptotic cell rate using TUNEL assays. In addition, alterations in signaling pathways were detected by immunoblotting analyses. Treatment with gemcitabine, which reduced the cell viability and augmented the cell apoptotic rate, activated and subsequently attenuated ERK and EGFR signals. However, gemcitabine, paclitaxel, or cisplatin treatment enhanced the Akt activation, heterodimer formation of EGFR with HER3, and secretion of amphiregulin, indicating that the presence of gemcitabine promoted the activity of targeted molecules including amphiregulin, Akt, and HER3 for pancreatic cancer therapy. Combined treatment with an inhibitor for amphiregulin and gemcitabine, paclitaxel, or cisplatin induced synergistic antitumor effects, accompanied by the suppression of Akt and ERK activation. Blockade of amphiregulin suppressed the activities of EGFR, HER3, and Akt and the expression of amphiregulin itself. According to this evidence, combination chemotherapy of conventional anticancer drugs plus an inhibitor for amphiregulin would allow us to provide more favorable clinical outcomes for patients with pancreatic cancer. (Cancer Sci 2010; 101: 2351–2360)

HB-EGF orchestrates the complex signals involved in triple-negative and trastuzumab-resistant breast cancer.

A number of therapeutic strategies targeting epidermal growth factor receptor (EGFR) have not always led to success in the present state of breast cancer therapy. Notably, there is currently no way to treat trastuzumab‐resistant and triple‐negative breast cancer (TNBC). Here, we found that heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a member of the EGFR ligands, was predominantly expressed in breast cancer and that treatment with crossreacting material 197 (CRM197), a specific inhibitor of HB‐EGF, blocked ERK as well as AKT activation via complexes of EGFR and unknown growth factor receptors in TNBC or through complexes of EGFR and human epidermal growth factor receptor‐2 in trastuzumab‐resistant breast cancer, caused significant cell apoptosis and inhibited tumor growth. Accordingly, we can provide a novel concept that a certain EGFR ligand is recognized as a rational target against breast cancer. In addition, it is plausible that CRM197 could be an effective anticancer agent for molecularly targeted therapies.

Targeting Bcl-2 family proteins in adult T-cell leukemia/lymphoma: in vitro and in vivo effects of the novel Bcl-2 family inhibitor ABT-737.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-1). ABT-737, a small molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, significantly induced apoptosis in HTLV-1 infected T-cell lines as well as in fresh ATLL cells, and synergistically enhanced the cytotoxicity and apoptosis induced by conventional cytotoxic drugs. Moreover, ABT-737 significantly inhibited the in vivo tumor growth of an ATLL mouse model. These results suggest that the use of an agent targeting anti-apoptotic bcl-2 family proteins, either alone or in combination with other conventional drugs, represents a novel promising approach for ATLL.

Amphiregulin is much more abundantly expressed than transforming growth factor-alpha and epidermal growth factor in human follicular fluid obtained from patients undergoing in vitro fertilization–embryo transfer

OBJECTIVE To identify the most important epidermal growth factor (EGF) receptor ligand in the LH or hCG signal pathway in human ovary. DESIGN A retrospective clinical study. SETTING Tertiary university hospital. PATIENT(S) Ninety-eight infertile patients who underwent IVF-embryo transfer. INTERVENTION(S) Sera and follicular fluid were collected at the time of oocyte retrieval. The levels of EGF, transforming growth factor-alpha (TGFalpha), and amphiregulin (AR) were measured in follicular fluid and sera by using ELISA. MAIN OUTCOME MEASURE(S) The relationships between the level of AR and level of hCG, fertilization rate, and embryo quality. RESULT(S) Amphiregulin was abundantly expressed in follicular fluid after hCG stimulation. Although large differences were found between AR and both EGF and TGFalpha in follicular fluid, no significant difference was detected in the levels of the three EGF receptor ligands in sera. The level of AR was inversely correlated with the fertilization rate and hCG level, whereas little significant association was observed between the level of AR and embryo quality. CONCLUSION(S) Amphiregulin was expressed most dominantly among EGF receptor ligands tested and may mediate the hCG signal in human oocyte maturation. Elaborate interaction between AR and hCG may be required for an optimal oocyte maturation.

Proteolytic activation of heparin‐binding EGF‐like growth factor by membrane‐type matrix metalloproteinase‐1 in ovarian carcinoma cells

Increased expression of heparin‐binding EGF‐like growth factor (HB‐EGF) and membrane‐type matrix metalloproteinase‐1 (MT1‐MMP) is frequently associated with various types of malignant tumor. HB EGF‐like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB‐EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1‐MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co‐expression of MT1‐MMP and HB‐EGF in gastric carcinoma cells leads to cleavage of HB‐EGF within its N‐terminal heparin‐binding region, converting it into a potent heparin‐independent growth factor. In this study, we evaluated the importance of regulation of HB‐EGF by MT1‐MMP in clinical samples of ovarian carcinoma. We detected co‐expression of HB‐EGF and MT1‐MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB‐EGF alone was expressed in non‐invasive borderline ovarian tumor tissue. Furthermore, a soluble HB‐EGF fragment that corresponds to that processed by MT1‐MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1‐MMP and HB‐EGF exhibited enhanced cell growth in a 3D‐collagen matrix and anchorage‐independent growth in suspension. These results indicate that MT1‐MMP co‐expressed with HB‐EGF in ovarian carcinoma cells potentiates the activity of HB‐EGF to promote invasive tumor growth and spreading in vivo. (Cancer Sci 2011; 102: 111–116)

A novel anti-human HB-EGF monoclonal antibody with multiple antitumor mechanisms against ovarian cancer cells

Purpose: Heparin-binding epidermal growth factor (EGF)–like growth factor (HB-EGF) is a member of the EGF family and plays a pivotal role in tumor progression in ovarian cancer. We developed an anti-HB-EGF monoclonal antibody (mAb) and investigated its antitumor activities in vitro and in vivo to evaluate its potential as a therapeutic antibody against ovarian cancer. Experimental Design: We prepared mAbs from HB-EGF null mice immunized with recombinant human soluble HB-EGF and evaluated their binding and neutralizing activity against HB-EGF. Next, we generated a mouse–human chimeric antibody and examined its in vitro and in vivo antitumor activities. Results: Two murine anti-HB-EGF mAbs were developed, and one of them, KM3566, was revealed to have a high binding reactivity for membrane-anchored HB-EGF (pro-HB-EGF) expressed on the cell surface, as well as neutralizing activity against growth promoting activity of soluble HB-EGF. The mouse–human chimeric counterpart for KM3566 (cKM3566) induced dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against cancer cells expressing HB-EGF in vitro, and significantly inhibited tumor growth in severe combined immunodeficient mice inoculated with MCAS or ES-2 human ovarian cancer cells. Conclusions: A novel anti-HB-EGF chimeric antibody, cKM3566, with two antitumor mechanisms, neutralization and ADCC, exhibits potent in vivo antitumor activity. These results indicate that cKM3566 is a promising antiovarian cancer therapeutic antibody. Clin Cancer Res; 17(21); 6733–41. ©2011 AACR.

Molecular Hierarchy of Heparin-Binding EGF-like Growth Factor–Regulated Angiogenesis in Triple-Negative Breast Cancer

Heparin-binding EGF-like growth factor (HB-EGF) is one of several proangiogenic factors and represents a possible therapeutic target for patients with triple-negative breast cancer (TNBC). However, the role of HB-EGF in promoting tumor aggressiveness in TNBC remains unclear. To investigate specific genes and pathways involved in TNBC tumorigenesis, we profiled gene expression changes in two TNBC cell lines under two-dimensional culture (2DC) and three-dimensional culture (3DC) and in a tumor xenograft model. We identified simultaneous upregulation of HB-EGF, VEGFA, and angiopoietin-like 4 (ANGPTL4) in 3DC and tumor xenografts, compared with 2DC. We show that HB-EGF regulates the expression of VEGFA or ANGPTL4 via transcriptional regulation of hypoxia-inducible factor-1α and NF-κB. Furthermore, suppression of VEGFA or ANGPTL4 expression enhanced HB-EGF expression, highlighting a unique regulatory loop underlying this angiogenesis network. Targeted knockdown of HB-EGF significantly suppressed tumor formation in a TNBC xenograft model, compared with individual knockdown of either VEGFA or ANGPTL4, by reducing the expression of both VEGFA and ANGPTL4. In patients with TNBC, VEGFA or ANGPTL4 expression was also significantly correlated with HB-EGF expression. Low concentrations of exogenously added HB-EGF strongly activated the proliferation of endothelial cells, tube formation, and vascular permeability in blood vessels, in a similar fashion to high doses of VEGFA and ANGPTL4. Taken together, these results suggest that HB-EGF plays a pivotal role in the acquisition of tumor aggressiveness in TNBC by orchestrating a molecular hierarchy regulating tumor angiogenesis. Mol Cancer Res; 11(5); 506–17. ©2013 AACR.