The Cancer Preventive Potential of Tea Polyphenol EGCG in HER2


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Chapter 28

The Cancer Preventive Potential of Tea Polyphenol E G C G in HER2-Positive Breast Cancer 1

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Min-Hsiung Pan and Wei-Jen Chen 1

Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan Department of Biomedical Sciences, Chung Shan Medical University, Taichung 402, Taiwan 2

Breast cancer is recognized as one of the leading causes of death among women in many countries and its incidence is closely linked to HER2 (human epidermal growth factor receptor 2) gene amplification. HER2, a second member of receptor tyrosine kinase of erbB family, is overexpressed in about 25% of human breast cancers with poor prognosis and chemoresistance, and considered as a target for breast cancer therapy. (-)-Epigallocatechin 3-gallate (EGCG), the most bioactive and abundant green tea catechin, has been thought as a chemopreventive agent by inhibition of growth and induction of apoptosis in various cancers including breast cancer. However, the molecular mechanisms responsible for cancer preventive effects of EGCG on breast cancer development are not fully elucidated. EGCG was found to suppress heregulinβ1-stimulated HER2/HER3 hetero-dimerization in breast cancer cells which initiates mitogenic signal transduction required for cancer progression. Also, the aggressive downstream phenotypes controlled by HER2 were inhibited by treatment of EGCG in breast cancer cells. These findings suggest that blockade of HER2/HER3 co-receptor formation by EGCG may be one of the possible cancer preventive mechanisms of EGCG in HER2-positive breast cancer.

© 2008 American Chemical Society

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336 Breast cancer is the most common and frequent cause of death among women in the industrialized world and its incidence closely correlates with genetic abnormalities. Several genes (such as p53, BRCA1 and BRCA2), frequently identified in primary human breast tumor, have been characterized in pathogenesis of breast cancer (7, 2). One of them is HER2 (human epidermal growth factor receptor 2) oncogene, which is amplified in about 25 % of human breast cancers with poor prognosis and chemoresistance (3, 4). HER2-positive breast cancer is highly proliferative and invasive with metastatic potential, as demonstrated by ectopic overexpression of HER2 in mouse embryo fibroblast 3T3 cells (5). Although amplification of HER2 is originally identified in breast cancer, aberrant HER2 expression has been found in a variety of other human cancers such as ovarian, gastric, and salivary cancers (6), implicating a critical role for HER2 in the development of human cancers. The HER2 gene encodes a transmembrane receptor tyrosine kinase (RTK) and belongs to the second member of erbB receptor family. ErbB receptor family, also known as epidermal growth factor (EGF) receptor family, comprises four homologous transmembrane receptor tyrosine kinases (erbB 1-4 or Her 1-4) which organizes a complex growth factor-mediated cellular signaling and appears to be important regulators of cell proliferation and differentiation (7). Ligand binding initiates signaling via erbB receptor homo- or heterodimerization, which in turn recruits specialized adaptors and kinases, thereby triggers and activates a network of signaling pathways (8). Increased expression and activation of erbB are tightly associated with development and progression in various types of cancer (9), suggesting erbB family receptors as targets for cancer therapy. Despite many intrinsic erbB family ligands have been recognized (70), HER2 remains an orphan receptor different from the other members and none of erbB family ligands can bind to HER2 with high affinity. In fact, activation of HER2 bases on its own overexpression to form the active homodimer or trans-activation by heregulins (HRGs) (77). HRGs are natural EGF-like ligands for HER3 or HER4 and highly expressed in breast cancer biopsies (72). Under HRG stimulation, HER2 prefers to heterodimerize to HER3 and proceeds signaling through activating intracellular signal pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways (75). Increasing evidences have indicated that suppression of HRGs expression down-regulates HER2 activation and results in the inhibition of downstream mitogenic responses, suggesting a pivotal role of HRGs in the development and the pathogenesis of breast cancer (14). Tea (Camellia sinensis) is the most worldwide consumed beverage and its constituents have been extensively investigated. Epidemiological studies suggest that tea consumption may have a protective effect against human cancer development (75). Among a number of bioactive compounds of tea, (-)epigallocatechin gallate (EGCG) (Figure 1) is thought to be the most powerful

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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337 cancer chemopreventive agent. Studies in animal models have demonstrated that EGCG can block all stages of carcinogenesis (16) and inhibit tumor angiogenesis, metastasis and invasion (17, 18). One possible mechanism responsible for the cancer preventive effects of EGCG is known to inhibit growth factor-related proliferation (19). EGCG has been shown to block the activation of EGF receptor tyrosine kinase and lead to the inhibition of cell growth in A431 epidermoid carcinoma cells (20). EGFR-dependent kinases, including ERK 1/2 and Akt, have been demonstrated as molecular targets for EGCG (21). In human head and neck squamous cell carcinoma (HNSCC) cells, EGCG inhibits TGF-a-mediated cyclin DI and c-fos promoter activities responsible for cell cycle progression, accompanied by cell cycle arrest and apoptosis (22). In addition, several investigations have demonstrated that EGCG suppresses activation of HER2, HER3 and the corresponding downstream signaling pathways in HNSCC, breast and human colon cancer cells (23-25). These findings implicate that EGCG may inhibits the downstream phenotype controlled by growth factor receptor, including ErbB receptor family.

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Figure 1. Chemical structure of (-)-epigallocatechin-3 catechin (EGCG)

In spite of many reports regarding the anti-cancer properties of EGCG, the molecular mechanisms responsible for cancer preventive effects of EGCG on breast cancer carcinogenesis is not fully known. In this brief contribution, we selected several human breast cancer cell lines expressing basal or over level of HER2 protein to evaluate the effects of EGCG on the interaction and activation of HER2 and HER3 under heregulin-pi (HRG-pl, a specific HER3 ligand) stimulation. The inhibition of HER2/HER3 heterodimer function by EGCG may be an effective strategy for suppression of HER2-mediated carcinogenesis in breast cancer cells.

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Materials and Methods Materials

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Recombinant human heregulin-pi was purchased from R&D Systems (Minneapolis, MN). EGCG was obtained from Sigma (St. Louis, MO). Antibody against FAS was obtained from BD Biosciences (Los Angeles, CA). The antibodies to HER2, HER3, MMP-2, VEGF-A and P-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture Monolayer cultures of MCF-7 and AU565 cells were grown in Dulbecco's minimal essential medium (DMEM), and MDA-MB-453 cells were maintained in DMEM/F12 (Invitrogen). All cells were supplemented with 10% fetal calf serum (Gibco BRL, Grand Island, NY), 100 units/mL of penicillin, 100 pg/mL of streptomycin, and kept at 37°C in a humidified atmosphere of 5% C 0 in air. 2

Immunoprecipitation Five hundred micrograms of total cellular proteins in MCF-7 cell lysates were first pre-cleared by incubating with protein A-agarose (10 pL, 50% slurry; Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min. The clarified supernatants were collected by microfiigation, and then incubated with HER2 antibody for 2 h at 4°C. The reaction mixtures were added with 20 ^ L of protein A-agarose to absorb the immunocomplexes at 4°C overnight. Immunoprecipitated proteins were subjected to 8% SDS-PAGE, and then transferred onto PVDF membrane (Millipore). The HER3 proteins were visualized by Western blotting.

Western Blotting Cell extracts were prepared in a lysis buffer (50 mM Tris-HCl, pH 8.0; 5 mM EDTA; 150 mM NaCl; 0.5% NP-40; 0.5 mM phenylmethylsulfonyl fluoride; and 0.5 mM dithiothreitol) for 30 min at 4°C. Equal amounts of total cellular proteins (50 pg) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (6% for FAS; 10% for MMP-2 and p-actin; 12% for VEGF-A), transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA), and then probed with primary antibody

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

339 followed by secondary antibody conjugated with horseradish peroxidase. The immunocomplexes were visualized with enhanced chemiluminescence kits (Amersham, UK).

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Results and Discussion Tyrosine phosphorylation of HER3 can be observed under HRG-pl stimulation; however, HER3 defects the intrinsic tyrosine kinase activity (26) and it means that receptor heterodimerization is needed for HER3 activation. In fact, HER2 is the preferred partner of co-receptor for HER3 and HER2/HER3 heterodimer appears to be the most transforming and mitogenic receptor complex (27). To determine the effect of EGCG on the interaction between HER2 and HER3, MCF-7 breast cancer cells expressing basal level of HER2 were treated with different concentrations of EGCG and then stimulated by HRG-pi. HER2/HER3 interaction was detected by co-immunoprecipitation using a HER2 antibody. After HRG-pi stimulation, HER3 protein was apparently observed compared with that in unstimulated cells as demonstrated by Western blotting with a HER3 antibody, and the co-immunoprecipitated HER3 with HER2 by HRG-pi stimulation were gradually disappeared under EGCG pre-treatment (Figure 2).

HRG (20 ng/mL) EGCG(jiM)

+ -

+ 10

+ 20

IP: HER2 WB: HER3 Figure 2. EGCG down-regulates HER2/HER3 heterodimerization due to HRGpi stimulation. Serum-starved MCF-7 cells werepre-incubated with various doses of EGCG for 30 min, and then stimulated with 20 ng/mL ofHRG-/31 for 10 min. At the end of incubation, cell lysates were harvested and immunoprecipitated with HER2 antibody, and then the co-precipitated HER3 was determined by Western blotting using a HER3 antibody.

Moreover, the tyrosine phosphorylation of HER2 and HER3 was positively correlated with the HER2/HER3 heterodimer formation (unpublished data). These findings indicate that EGCG could inhibit HER2/HER3 signaling by preventing the coordination of HER2 and HER3 by HRG-pl in MCF-7 breast cancer cells.

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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340 To farther investigate the effects of EGCG on the downstream phenotype controlled by HER2/HER3 signaling, the protein levels of fatty acid synthase (FAS), matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor A (VEGF-A) were determined by Western blotting in several breast cancer cell lines. All of these proteins have been considered as the downstream effectors up-regulated by HER2 or HER3 (28-30). As shown in Figure 3, EGCG markedly inhibited HRG-pi -induced FAS expression in breast cancer cell lines, MDA-MB-453 and AU565. The increased expression of MMP-2 and VEGF-A by HRG-pl were also inhibited by EGCG treatment in MDA-MD-453 and MCF-7 breast cancer cells. These data suggest that EGCG down-regulates the induction of FAS, MMP-2 and VEGF-A by HRG-pl via the suppression of HER2/HER3 coordination. Interestingly, serum-starved MDA-MB-453 breast cancer cells expressed high level of FAS protein; even there was no HRG-pi stimulus. The reason may be that MDA-MB-453 cells are more malignant than AU565 cells, and HER2 in MDA-MD-453 cells are overexpressed and highly active through HER2 homodimer formation despite serum deprivation. Therefore, the expression of fully level of FAS due to HER2 overexpression is predictable in serum-starved MDA-MD-453 cells. Based on these preliminary data, we propose a molecular model for cancer preventive action of EGCG in HER2- or/and HER3-overexpresing breast cancer (Figure 4). EGCG inhibits the formation of HER2/HER3 co-receptor by HRGpi and finally leads to down-regulation of FAS, MMP-9 and VEGF-A levels in breast cancer cells. Inhibition of these HER2- or HER3-related proteins by treatment of EGCG (Figure 3) may contribute to cancer prevention before breast cancer onset, because FAS, MMP-9 and VEGF-A have been shown as important mediators for malignant progression of tumor cells. FAS has been implicated in tumorigenesis through its role in cell proliferation and membrane lipid incorporation of neoplastic cells, and suppression of FAS function in cancer cells leads to growth inhibition and the induction of apoptosis (31). Also, MMP-2 activity is involved in tumor invasion and metastasis by its capacity for degradation of extracellular matrix (ECM) of basement membrane. Statistics indicates that MMP-2 is associated with a poor prognosis in breast carcinoma patients (32) and thought to be a target of developed MMP inhibitors. Additionally, VEGF-A secreted by tumor cells has been demonstrated as a key regulator of angiogenesis (33). Accumulating studies indicate that either HER2 gene amplification or HRG-pl stimulation regulates elevated VEGF-A expression in breast cancer cells (30\ implicating that VEGF-A may be a crucial mediator for the aggressive phenotype of HER2 or/and HER3-overexpressing breast cancer. Based on these observations, we suggest that anti-proliferation, metastasis and angiogenesis will be accompanied by suppression of FAS, MMP2 and VEGF-A, respectively, by EGCG via inhibition of HER2/HER3 signaling. This hypothesis may explain why drinking green tea can lower a recurrence rate among breast cancer patients.

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Figure 3. EGCG inhibits the expression of FAS, MMP-2 and VEGF-A proteins by HRG-fil in breast cancer cell lines. Serum-starved breast cancer cells as indicated were pre-incubated with various doses of EGCG for 30 min, and then stimulated with HRG-fil (20 or 50 ng/mL) for 9 h. Western blotting analysis was performed using specific antibodies to FAS, MMP-2, VEGF-A or p-actin. P-actin represented as a loading control

In Functional Food and Health; Shibamoto, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

342 Q

HRG-p1

EGCG

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FAS |, MMP-2 f , VEGF-A |

Proliferation f

Metastasis f

Angiogenesis f

Figure 4. Proposed model for cancer preventive action of EGCG in HER2 or/and HER3 -over expressing breast cancer. EGF-like growth factor HRG-pl, a HER3 ligand, binds to HER3, initiates signaling via HER2/HER3 heterodimerization, and consequently increases the expression ofFAS, MMP-2 and VEGF-A which are responsible for the aggressive phenotype such as proliferation, metastasis and angiogenesis, respectively, in breast cancer cells. EGCG may achieve its cancer preventive effects on breast cancer by inhibiting the formation ofHER2/HER3 co-receptor by HRG-pi and finally down-regulating the downstream effectors that contribute to malignancy of breast cancer.

Although our current study suggests that EGCG may be a useful chemopreventive agent for breast cancer carcinogenesis, how EGCG interrupts HER2/HER3 interaction essential for tumor progression still remains elusive. Recent evidence showed that EGCG can block EGFR-mediated proliferation in A431 epidermoid carcinoma by competition with EGF binding to EGFR (20). It raises the possibility that EGCG may prevent HRG-pl binding to HER3 receptor by similar pathway; however, this issue requires further study. To sum up, our findings point out a new insight on the molecular mechanism by which EGCG blocks breast cancer development or progression and suggest that EGCG may by an effective agent in the prevention of cases of breast carcinoma where HER2 or/and HER3 overexpression.

Acknowledgement This study was supported by the National Science Council NSC 94-2312B-040-001 and NSC 95-2320-B-040-037.

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