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Enhancement of paclitaxel-induced apoptosis in HER2-overexpressing human breast cancer cells by a pertuzumab mimetic peptide, HRAP
Journal of Bioscience and Bioengineering VOL. 110 No. 2, 250 – 253, 2010 www.elsevier.com/locate/jbiosc
Enhancement of paclitaxel-induced apoptosis in HER2-overexpressing human breast cancer cells by a pertuzumab mimetic peptide, HRAP Hiroo Nakajima,1 Naruhiko Mizuta,2 Koichi Sakaguchi,2 Ikuya Fujiwara,2 Atsushi Yoshimori,3 Junji Magae,4,⁎ and Sei-ichi Tanuma3 Tsujigiwa Daigo Clinic, 1-19 Daigo Shinmachi-uramachi, Fushimi-ku, Kyoto 601-1326, Japan 1 Department of Endocrine and Breast Surgery, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan 2 Department of Biochemistry, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan 3 and Radiation Safety Research Center, Central Institute of Electric Power Industry, 2-11-1 Iwado-Kita, Komae-shi, Tokyo 201-8511, Japan 4 Received 5 October 2009; accepted 9 February 2010 Available online 11 March 2010
HRAP, a pentapeptide designed to bind with the pertuzumab interacting site in an extracellular domain of the HER2 molecule, enhanced the cytotoxicity of paclitaxel in HER2-overexpressing human breast cancer cell lines, BT474 and SKBR-3, but not in MDA-231 cells, which express lower levels of HER2. HRAP enhanced mitochondria-dependent apoptosis induced by paclitaxel in SKBR-3 and BT-474, but not in MDA-231. HRAP enhanced the inhibition of phosphorylation of serine 473 in Akt and Ser380/Thy382/The383 in PTEN. These results suggest that HRAP enhances paclitaxel-induced apoptosis in a manner dependent on the PTEN/Akt signal transduction pathway. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: HRAP; HER2; Paclitaxel; Pertuzumab; Breast cancer; Apoptosis]
HER2, a member of the ErbB family of tyrosine kinase receptors, is overexpressed in 20–30% of human breast cancer cells. Overexpression of HER2 correlates to the malignant characteristics of these cells, including invasiveness, metastatic ability and resistance to chemotherapy (1). HER2 transduces tyrosine kinase signaling via phosphatidylinositide 3′-kinase (PI3K)/Akt pathways, leading to phosphorylation and activation of Akt kinase, a major survival kinase affecting cellular proliferation and apoptosis (2). HER2, unlike other family members, has no specific ligand for activation. Rather, it is activated through homodimerization or through heterodimerization with another family member that has been activated by its specific ligand (2). Dimerization results in the multiple phosphorylation of tyrosine residues in the intracellular tail, an essential event for evoking intracellular signaling. Thus, blocking of signal transduction pathways initiated by HER2 is believed to be an effective therapeutic approach in the treatment of HER2-positive breast cancer. Antibody therapy is one of the most powerful strategies used to suppress HER2-mediated malignant signals (3). Trastuzumab, a humanized monoclonal antibody, binds domain IV of the HER2 extracellular domain. Trastuzumab inhibits Akt phosphorylation and suppresses proliferation of HER2 overexpressing breast cancer cells in vivo and in vitro (4). Moreover, trastuzumab overcomes the drug resistance of HER2positive breast cancer cells and increases their sensitivity to anticancer drugs and radiation (5-7). Trastuzumab was approved as a chemotherapeutic agent for HER2-overexpressing breast cancers by the US Food and ⁎ Corresponding author. Tel.: +81 3 3480 2111; fax: +81 3 3480 3113. E-mail address: [email protected] (J. Magae).
Drug Administration (FDA) in 1998 and has since dramatically improved the prognosis for HER2-positive breast cancer patients. Because Akt phosphorylation induced by HER2 overexpression is dependent on the inactivation of phosphatase and tensin homolog (PTEN), a protein encoded by the PTEN gene on chromosome 10 that dephosphorylates polyphosphoinositides, HER2-positive breast cancer cells lacking PTEN are resistant to trastuzumab treatment in vivo and in vitro (8). Trastuzumab binding rapidly decouples Src from HER2, and the resulting loss of Src activity reduces PTEN C2 domain phosphorylation. This event allows PTEN to translocate to the plasma membrane and inhibit the PI3K/ Akt pathway. Pertuzumab is another humanized antibody that binds to the extracellular domain of HER2. Pertuzumab, unlike trastuzumab, binds to domain II of the extracellular domain of HER2, which directly contributes to dimerization. 2C4, the original mouse monoclonal antibody for pertuzumab, inhibits heterodimerization of HER2 and HER3, tyrosine phosphorylation of the intracellular domain of HER3, proliferation of the ligand-activated breast cancer cells and tumor growth in nude mouse in vivo (9). The effects of 2C4 are evident in trastuzumabresistant breast cancer cells expressing lower levels of HER2. Breast cancers without HER2 overexpression tend to metastasize to bone, whereas HER2-overexpressing breast cancers are more likely to spread to visceral organs such as lung, liver and brain (10). When patients with metastatic breast cancer are treated with trastuzumab, the breast cancer frequently progresses in the central nervous system (CNS) (11). CNS disease progression during trastuzumab therapy does not result from a loss of HER2 overexpression in the brain, but from poor penetration of trastuzumab into the brain. Intracerebral administration of trastuzumab through microinfusion significantly
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improved the survival of rat with intracerebrally transplanted HER2overexpressing human breast cancer cells, while systemic i.p. administration of trastuzumab failed to deliver the drug to the brain, and did not significantly improve survival (12). Another approach to preventing the metastasis of HER2-positive breast cancer into the CNS is to develop molecules small enough to penetrate into the brain, and experimental therapies employing trastuzumabmimetic peptides or tyrosine kinase inhibitors have been reported (11). Small molecule therapy has the added benefit for patients of a significantly lower cost than antibody therapy. We recently developed the pertuzumab-mimetic pentapeptide HRAP (HER2 reactive antagonistic peptide: acetyl-Pro-His-Ala-HisPhe-NH2), through computational methods (13). An in silico docking study demonstrates specific interaction of HRAP at the HER2/ pertuzumab interacting surface present in domain II of the HER2 extracellular domain. HRAP induces growth arrest but not apoptosis of HER2-overexpressing breast cancer cells, and suppresses phosphorylation of PTEN and Akt, which suggests a synergistic effect between HRAP and chemotherapeutic agents. In this paper, we report that HRAP enhances the apoptosis-inducing activity of paclitaxel, a microtubule toxin used for breast cancer chemotherapy. MATERIALS AND METHODS Reagents HRAP was purchased from the Peptide Institute (Tokyo, Japan). Paclitaxel was kindly provided Kyowa Hakko Inc. (Tokyo, Japan). Cell culture Human breast cancer cell lines, MDA-231, BT-474 and SKBR-3, were purchased from the American Type Culture Collection. Cells were maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (BioWhittaker, Maryland, USA) at 37 °C under a humidified atmosphere containing 5% carbon dioxide. MTT dye reduction assay Cell viability was determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay, which measures mitochondrial respiratory function (14, 15). Breast cancer cells were exponentially grown (5 × 104 cells/well) and plated at 100 μl volume in 96-well flatbottom plates (Nunc, Roskilde, Denmark) and allowed to adhere for 24 h. After incubation for 48 h with the drug, cells were incubated with MTT dye (1 mg/ml) for 2 h, medium was removed and 100 μl of 0.04 M HCl in isopropanol was added to extract the reduced formazan deposit. The optical densities of the samples were measured at 590 nm. Western blotting Western blotting was performed as described previously (13). Cell pellets were lysed in 2 × SDS sample buffer containing 100 mM dithiothreitol and then boiled for 5 min. A uniform amount of each sample (10 μg of protein) was resolved by SDS-PAGE (12% acrylamide). Proteins in each gel were transferred to Immobilon-P membrane (Milipore, Bedford, MA). After the blots were blocked for 1 h in 5% nonfat dry milk in 20 mM Tris, 0.8% NaCl, 0.1% Tween 20, pH 7.5, the blots were incubated with the primary antibody for 1 h and then with the appropriate horseradish peroxidase-conjugated secondary antibody (MBL, Nagoya, Japan). Signals were visualized by fluorescence emission using commercial detection kits (Roche Diagnosis, Mannheim, Germany), according to the manufacturer's instructions. Antibodies for Akt, phospho-Ser473-Akt, PTEN, and phopho-Ser380/Thr382/Thr383-PTEN were purchased from Cell Signaling Technology (Danvers, MA, USA); those for PARP and β-actin from Santa Cruz (Santa Cruz, CA, USA); and those for caspase-3, caspase-8 and caspase-9 from MBL (Nagoya, Japan). Relative protein expression was quantified by densitometry. Statistics Differences between two experimental groups were statistically evaluated by two-sided t-test. Differences with p values less than 0.05 were considered statistically significant.
RESULTS Enhancement of the cytotoxic effect of paclitaxel on HER2 overexpressing human breast cancer cells by HRAP Human breast cancer cells were cultured with paclitaxel in the presence of trastuzumab or HRAP for 48 h, and cytotoxic activity was evaluated by MTT assay (Fig. 1). Although trastuzumab and HRAP inhibit proliferation of HER2-overexpressing breast cancer cells as judged by 5-bromo-2′-deoxyuridine uptake, they had no significant effect on cell viability as judged by MTT assay, suggesting that their growth arrest is cytostatic and not due to apoptosis (13). In contrast, paclitaxel treatment significantly reduced cell viability as judged by MTT assay, and trastuzumab and HRAP significantly enhanced the
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FIG. 1. Enhancement of the cytotoxic effect of paclitaxel by HRAP in HER2overexpressing breast cancer cells. Cells in triplicate culture were incubated with drugs for 48 h, and cell viability was determined by MTT assay. ⁎Statistically significant compared to paclitaxel-treated control (p b 0.05, two-sided t-test).
cytotoxic activity induced by paclitaxel in HER2-overexpressing breast cancer cells, BT-474 and SKBR-3. The enhancement was not observed with MDA-231, a breast cancer cell line expressing low levels of HER2 (13). The enhancement by HRAP required 1 mM concentration, and lower doses did not have significant effect on the paclitaxel-induced cytotoxic effect. Enhancement of paclitaxel-induced mitochondria-dependent apoptosis in HER2-overexpressing human breast cancer cells by HRAP The reduced cell viability shown by MTT assay suggests that cells die through induction of apoptosis. To confirm the induction of apoptosis, cleavage of caspase was determined by Western blotting (Fig. 2A), and the expression was quantified by densitometry (Fig. 2B). Paclitaxel induced cleavage of caspase-3 after 48 h incubation, and the cleavage was enhanced by HRAP in BT-474 and SKBR-3. The activation of caspase-3 was confirmed by the cleavage of poly(ADPribose) polymerase (PARP), a major substrate of caspase-3 (16), which was induced by paclitaxel and enhanced by HRAP. Cleavages of caspase-3 and PARP were also detected in paclitaxel-treated MDA231 cells. The cleavage of caspase-3 was further enhanced by HRAP, while that of PARP was not as evident as in HER2-overexpressing breast cancer cells. Cleavage of caspase-9, an initiator caspase activated by mitochondria-dependent apoptotic signals (17), but not caspase-8, an initiator caspase activated by the death-inducing signaling complex (DISC) (18), was detected in HER2-overexpressing cells. HRAP had only negligible effect on the cleavage of caspase-9 in BT-474 cells, while showed slight enhancement in SKBR-3 cells. These results suggest that HRAP mainly enhances the apoptosis-executing mechanism in the mitochondria-dependent apoptotic pathway induced by paclitaxel. Blockade of phosphorylation of survival factors by HRAP To verify the pertuzumab mimetic effect of HRAP, we examined the phosphorylation status of Akt and PTEN, two major targets of pertuzumab present downstream in the HER2-mediated signaling pathway (Fig. 2A and B). Paclitaxel itself reduced phosphorylation of Ser380/Thr382/Thr383 in PTEN, a phosphorylation site at the Cterminal that reduces the activity but increases the stability of the phosphatase (19). The reduction was enhanced by HRAP in BT-474 and SKBR-3 cells. In contrast, this phosphorylation was neither induced by paclitaxel treatment nor affected by HRAP in MDA-231 cells. Phosphorylation of Akt at Ser473, required for activation of the kinase (20), was also reduced by treatment with paclitaxel in HER2-overexpressing breast cancer cells. HRAP enhanced the reduction in BT-474 cells, but not in SKBR-3 cells. Paclitaxel and HRAP again had no marked effect on the phosphorylation of Ser473 at Akt in MDA231 cells. Taken together, our results suggest that HRAP-enhances
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FIG. 2. Activation profile of caspases and phosphorylation of survival factors downstream of HER2/3 signaling in HER2-overexpressing breast cancer cells treated with HRAP and/or paclitaxel. Cells were incubated with drugs for 48 h. Protein expression in the resultant cell lysate was determined by Western blotting (A). The relative protein expression normalized by β-actin expression was quantified by densitometry (B).
paclitaxel-induced apoptosis through the suppression of HER2mediated phosphorylation events involving PTEN phosphorylation. DISCUSSION Because trastuzumab blocks the drug resistance of HER2-overexpressing tumor cells by suppressing survival factors including PTEN and Akt (4), combined therapy with other chemotherapeutic agents greatly improves its efficacy (4), while trastuzumab monotherapy does not improve the prognosis for breast cancer patients. Trastuzumab enhances the therapeutic effects of radiation and chemotherapeutic agents including anthracyclines and taxanes in vitro and in vivo (5-7). We observed similar enhancement of the therapeutic efficacy of paclitaxel by HRAP, a pertuzumab-mimetic pentapeptide, demonstrating its clinical potential in breast cancer therapy. In HER2 overexpressing human breast cancer cells, HRAP enhanced the inhibitory effect of paclitaxel on Akt phosphorylation and/or PTEN phosphorylation, and potentiated the paclitaxel-induced cleavage of caspase-3 and PARP. These effects were not observed in the HER2-dull cell line MDA-231. These results suggest that HRAP, like trastuzumab, improves therapeutic efficacy of paclitaxel, through the blockade of HER2-mediating activation signals and the potentiation of paclitaxelinducing apoptosis. It is likely that this apoptosis is mediated by mitochondria activation because cleavage of caspase-9, but not of caspase-8, was observed in paclitaxel-treated HER2 overexpressing cells. Because trastuzumab potentiates effect of radiation and other chemotherapeutic agents including doxorubicin and etoposide, HRAP could enhance the chemotherapeutic effect of agents other than paclitaxel. A trastuzumab-mimetic cyclic dodecapeptide, anti-HER2/neu peptide (AHNP), has been reported (21, 22). AHNP was designed based on the complementarity determining regions (CDR)-3 of antibodies against HER2 domain IV, including trastuzumab, and
contains the five amino acids in this region. AHNP specifically binds to HER2 with high affinity, inhibits proliferation of HER2-overexpressing tumor cells and suppresses tumor growth in athymic mice. AHNP, like HRAP, enhances the suppressive effect of doxorubicin on cell proliferation and apoptosis induced by radiation in HER2-overexpressing cells. Compared to trastuzumab, pertuzumab more directly inhibits the dimerization of HER2 and more profoundly inhibits HER2/HER3 phosphorylation induced by neuregulin. Pertuzumab inhibits ligand-induced proliferation of MCF-7 in vitro and the tumor growth in nude mice, a breast cancer expressing a lower level of HER2 (9). Thus, it is expected that pertuzumab-mimicry peptides including HRAP have some advantage, compared to trastuzumabmimicry peptides. Although AHNP has high affinity for HER2 (Kd = 300 nM), more than 67 μM is required to attain proliferation inhibition similar to trastuzumab (22). In the case of HRAP, a dose as high as 1 mM suppressed proliferation only partially, and a synergistic effect with paclitaxel was not observed at lower doses. Doses higher than 10 mM kill fraction of cells nonspecifically. IC50 for SKBR-3 is 6.8 mM in a 72h MTT assay (unpublished result). Clearly improvements of affinity and specificity are necessary before HRAP could be used clinically. Based on the three-dimensional structure of HRAP, we are now attempting to create a compound with a different, nonpeptide structure through computational design. Another strategy is to create molecular targeting compounds by conjugating the toxic molecules on HRAP. Fantin et al. (23) have engineered a bifunctional, HER-2blocking and apoptosis-inducing peptide (BHAP) which consists of a HER2 targeting/neutralizing domain, AHNP, and a mitochondria-toxic domain. In this report, BHAP effectively suppresses proliferation, induces apoptosis and prevents tumor growth in athymic mice in a manner selective for HER2 overexpressing breast cancer, in contrast to the moderate effects of AHNP. The present results demonstrate that HRAP could be a lead compound for creating such molecular targeting
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agents small enough to penetrate into the brain for treating malignant breast cancers. References 1. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A, and McGuire, W. L.: Human breast cancer: correlation of relapse and survival with amplification on the HER-2/neu oncogene, Science, 235, 177–182 (1987). 2. Crowder, R. J., Lombardi, D. P., and Ellis, M. J.: Successful targeting of ErbB2 receptors-is PTEN the key, Cancer Cell, 6, 103–104 (2004). 3. Zhang, H., Richter, M., and Greene, M. I.: Therapeutic monoclonal antibodies for the ErbB family of receptor tyrosine kinases, Cancer Biol. Ther., 2, S122–S126 (2003). 4. Hudis, C. A.: Trastuzumab—mechanism of action and use in clinical practice, N. Engl. J. Med., 357, 39–51 (2007). 5. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and Mendelsohn, J.: Recombinant humanized anti-HER2 antibody (Herceptin™) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenograft, Cancer Res., 58, 2825–2831 (1998). 6. Lee, S., Yang, W., Lan, K. H., Sellappan, S., Klos, K., Hortobagyi, G., Hung, M. C., and Yu, D.: Enhanced sensitization to Taxol-induced apoptosis by Herceptin pretreatment in ErbB2-overexpressing breast cancer cells, Cancer Res., 62, 5703–5710 (2002). 7. Liang, K., Lu, Y., Jin, W., Ang, K. K., Milas, L., and Fan, Z.: Sensitization of breast cancer cells to radiation by trastuzumab, Mol. Cancer Ther., 2, 1113–1120 (2003). 8. Nagata, Y., Lan, K. H., Zhou, X., Tan, M., Esteva, F. J., Sahin, A. A., Klos, K. S., Li, P., Monia, B. P., Nguyen, N. T., and other 3 authors: PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients, Cancer Cell, 6, 117–127 (2004). 9. Agus, D. A., Akita, R. W., Fox, W. D., Lewis, G. D., Higgins, B., Pisacane, P. I., Lofgren, J. A., Tindell, C., Evans, D. P., Maiese, K., Scher, H. I., and Sliwkowski, M. X.: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth, Cancer Cell, 2, 127–137 (2002). 10. Kallioniemi, O. P., Holli, K., Visakorpi, T., Koivula, T., Helin, H. H., and Isola, J. J.: Association of c-ErbB-2 protein over-expression with high rate of cell proliferation,
11. 12.
13.
14. 15. 16.
17. 18. 19.
20. 21.
22.
23.
253
increased risk of visceral metastasis and poor long-term survival in breast cancer, Int. J. Cancer, 49, 650–655 (1991). Kirsch, D. G. and Hochberg, F. H.: Targeting HER2 in brain metastasis from breast cancer, Clin. Cancer Res., 9, 5435–5439 (2003). Grossi, M., Ochiai, H., Archer, G. E., McLendon, R. E., Zalutsky, M. R., Friedman, A. H., Friedman, H. S., Bigner, D. D., and Sampson, J. H.: Efficacy of intracerebral microinfusion of trastuzumab in an athymic rat model of intracerebral metastatic breast cancer, Clin. Cancer Res., 9, 5514–5520 (2003). Nakajima, H., Mizuta, N., Sakaguchi, K., Fujiwara, I., Yoshimori, A., Takahashi, S., Takasawa, R., and Tanuma, S.: Development of HER2-antagonistic peptides as novel anti-breast cancer drugs by in silico methods, Breast Cancer, 15, 65–72 (2008). Mosmann, T.: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65, 55–63 (1983). Gerlier, D. and Thomasset, N.: Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods, 94, 57–63 (1986). Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M.: Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose)polymerase, Cell, 81, 801–809 (1995). Hengartner, M. O.: The biochemistry of apoptosis, Nature, 407, 770–776 (2000). Nagata, S.: Apoptosis by death factor, Cell, 88, 355–365 (1997). Vazquez, F., Ramaswamy, S., Nakamura, N., and Sellers, W. R.: Phosphorylation of the PTEN tail regulates protein stability and function, Mol. Cell. Biol., 20, 5010–5018 (2000). Datta, S. R., Brunet, A., and Greenberg, M. E.: Cellular survival: a play in three Akts, Genes Dev., 13, 2905–2927 (1999). Berezov, A., Zhang, H. T., Greene, M. I., and Murali, R.: Disabling ErbB receptors with rationally designed exocyclic mimetics of antibodies: structure-function analysis, J. Med. Chem., 44, 2565–2574 (2001). Park, B. W., Zhang, H. T., Wu, C., Berezov, A., Zhang, X., Dua, R., Wang, Q., Kao, G., O'Rourke, D. M., Greene, M. I., and Murali, R.: Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo, Nat. Biotechnol., 18, 194–198 (2000). Fantin, V. R., Berardi, M. J., Babbe, H., Michelman, M. V., Manning, C. M., and Leder, P.: A bifunctional targeted peptide that blocks HER-2 tyrosine kinase and disables mitochondrial function in HER-2-positive carcinoma cells, Cancer Res., 65, 6891–6900 (2005).
Enhancement of paclitaxel-induced apoptosis in HER2-overexpressing human breast cancer cells by a pertuzumab mimetic peptide, HRAP Hiroo Nakajima,1 Naruhiko Mizuta,2 Koichi Sakaguchi,2 Ikuya Fujiwara,2 Atsushi Yoshimori,3 Junji Magae,4,⁎ and Sei-ichi Tanuma3 Tsujigiwa Daigo Clinic, 1-19 Daigo Shinmachi-uramachi, Fushimi-ku, Kyoto 601-1326, Japan 1 Department of Endocrine and Breast Surgery, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan 2 Department of Biochemistry, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan 3 and Radiation Safety Research Center, Central Institute of Electric Power Industry, 2-11-1 Iwado-Kita, Komae-shi, Tokyo 201-8511, Japan 4 Received 5 October 2009; accepted 9 February 2010 Available online 11 March 2010
HRAP, a pentapeptide designed to bind with the pertuzumab interacting site in an extracellular domain of the HER2 molecule, enhanced the cytotoxicity of paclitaxel in HER2-overexpressing human breast cancer cell lines, BT474 and SKBR-3, but not in MDA-231 cells, which express lower levels of HER2. HRAP enhanced mitochondria-dependent apoptosis induced by paclitaxel in SKBR-3 and BT-474, but not in MDA-231. HRAP enhanced the inhibition of phosphorylation of serine 473 in Akt and Ser380/Thy382/The383 in PTEN. These results suggest that HRAP enhances paclitaxel-induced apoptosis in a manner dependent on the PTEN/Akt signal transduction pathway. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: HRAP; HER2; Paclitaxel; Pertuzumab; Breast cancer; Apoptosis]
HER2, a member of the ErbB family of tyrosine kinase receptors, is overexpressed in 20–30% of human breast cancer cells. Overexpression of HER2 correlates to the malignant characteristics of these cells, including invasiveness, metastatic ability and resistance to chemotherapy (1). HER2 transduces tyrosine kinase signaling via phosphatidylinositide 3′-kinase (PI3K)/Akt pathways, leading to phosphorylation and activation of Akt kinase, a major survival kinase affecting cellular proliferation and apoptosis (2). HER2, unlike other family members, has no specific ligand for activation. Rather, it is activated through homodimerization or through heterodimerization with another family member that has been activated by its specific ligand (2). Dimerization results in the multiple phosphorylation of tyrosine residues in the intracellular tail, an essential event for evoking intracellular signaling. Thus, blocking of signal transduction pathways initiated by HER2 is believed to be an effective therapeutic approach in the treatment of HER2-positive breast cancer. Antibody therapy is one of the most powerful strategies used to suppress HER2-mediated malignant signals (3). Trastuzumab, a humanized monoclonal antibody, binds domain IV of the HER2 extracellular domain. Trastuzumab inhibits Akt phosphorylation and suppresses proliferation of HER2 overexpressing breast cancer cells in vivo and in vitro (4). Moreover, trastuzumab overcomes the drug resistance of HER2positive breast cancer cells and increases their sensitivity to anticancer drugs and radiation (5-7). Trastuzumab was approved as a chemotherapeutic agent for HER2-overexpressing breast cancers by the US Food and ⁎ Corresponding author. Tel.: +81 3 3480 2111; fax: +81 3 3480 3113. E-mail address: [email protected] (J. Magae).
Drug Administration (FDA) in 1998 and has since dramatically improved the prognosis for HER2-positive breast cancer patients. Because Akt phosphorylation induced by HER2 overexpression is dependent on the inactivation of phosphatase and tensin homolog (PTEN), a protein encoded by the PTEN gene on chromosome 10 that dephosphorylates polyphosphoinositides, HER2-positive breast cancer cells lacking PTEN are resistant to trastuzumab treatment in vivo and in vitro (8). Trastuzumab binding rapidly decouples Src from HER2, and the resulting loss of Src activity reduces PTEN C2 domain phosphorylation. This event allows PTEN to translocate to the plasma membrane and inhibit the PI3K/ Akt pathway. Pertuzumab is another humanized antibody that binds to the extracellular domain of HER2. Pertuzumab, unlike trastuzumab, binds to domain II of the extracellular domain of HER2, which directly contributes to dimerization. 2C4, the original mouse monoclonal antibody for pertuzumab, inhibits heterodimerization of HER2 and HER3, tyrosine phosphorylation of the intracellular domain of HER3, proliferation of the ligand-activated breast cancer cells and tumor growth in nude mouse in vivo (9). The effects of 2C4 are evident in trastuzumabresistant breast cancer cells expressing lower levels of HER2. Breast cancers without HER2 overexpression tend to metastasize to bone, whereas HER2-overexpressing breast cancers are more likely to spread to visceral organs such as lung, liver and brain (10). When patients with metastatic breast cancer are treated with trastuzumab, the breast cancer frequently progresses in the central nervous system (CNS) (11). CNS disease progression during trastuzumab therapy does not result from a loss of HER2 overexpression in the brain, but from poor penetration of trastuzumab into the brain. Intracerebral administration of trastuzumab through microinfusion significantly
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improved the survival of rat with intracerebrally transplanted HER2overexpressing human breast cancer cells, while systemic i.p. administration of trastuzumab failed to deliver the drug to the brain, and did not significantly improve survival (12). Another approach to preventing the metastasis of HER2-positive breast cancer into the CNS is to develop molecules small enough to penetrate into the brain, and experimental therapies employing trastuzumabmimetic peptides or tyrosine kinase inhibitors have been reported (11). Small molecule therapy has the added benefit for patients of a significantly lower cost than antibody therapy. We recently developed the pertuzumab-mimetic pentapeptide HRAP (HER2 reactive antagonistic peptide: acetyl-Pro-His-Ala-HisPhe-NH2), through computational methods (13). An in silico docking study demonstrates specific interaction of HRAP at the HER2/ pertuzumab interacting surface present in domain II of the HER2 extracellular domain. HRAP induces growth arrest but not apoptosis of HER2-overexpressing breast cancer cells, and suppresses phosphorylation of PTEN and Akt, which suggests a synergistic effect between HRAP and chemotherapeutic agents. In this paper, we report that HRAP enhances the apoptosis-inducing activity of paclitaxel, a microtubule toxin used for breast cancer chemotherapy. MATERIALS AND METHODS Reagents HRAP was purchased from the Peptide Institute (Tokyo, Japan). Paclitaxel was kindly provided Kyowa Hakko Inc. (Tokyo, Japan). Cell culture Human breast cancer cell lines, MDA-231, BT-474 and SKBR-3, were purchased from the American Type Culture Collection. Cells were maintained in DMEM (Nissui, Tokyo, Japan) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (BioWhittaker, Maryland, USA) at 37 °C under a humidified atmosphere containing 5% carbon dioxide. MTT dye reduction assay Cell viability was determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay, which measures mitochondrial respiratory function (14, 15). Breast cancer cells were exponentially grown (5 × 104 cells/well) and plated at 100 μl volume in 96-well flatbottom plates (Nunc, Roskilde, Denmark) and allowed to adhere for 24 h. After incubation for 48 h with the drug, cells were incubated with MTT dye (1 mg/ml) for 2 h, medium was removed and 100 μl of 0.04 M HCl in isopropanol was added to extract the reduced formazan deposit. The optical densities of the samples were measured at 590 nm. Western blotting Western blotting was performed as described previously (13). Cell pellets were lysed in 2 × SDS sample buffer containing 100 mM dithiothreitol and then boiled for 5 min. A uniform amount of each sample (10 μg of protein) was resolved by SDS-PAGE (12% acrylamide). Proteins in each gel were transferred to Immobilon-P membrane (Milipore, Bedford, MA). After the blots were blocked for 1 h in 5% nonfat dry milk in 20 mM Tris, 0.8% NaCl, 0.1% Tween 20, pH 7.5, the blots were incubated with the primary antibody for 1 h and then with the appropriate horseradish peroxidase-conjugated secondary antibody (MBL, Nagoya, Japan). Signals were visualized by fluorescence emission using commercial detection kits (Roche Diagnosis, Mannheim, Germany), according to the manufacturer's instructions. Antibodies for Akt, phospho-Ser473-Akt, PTEN, and phopho-Ser380/Thr382/Thr383-PTEN were purchased from Cell Signaling Technology (Danvers, MA, USA); those for PARP and β-actin from Santa Cruz (Santa Cruz, CA, USA); and those for caspase-3, caspase-8 and caspase-9 from MBL (Nagoya, Japan). Relative protein expression was quantified by densitometry. Statistics Differences between two experimental groups were statistically evaluated by two-sided t-test. Differences with p values less than 0.05 were considered statistically significant.
RESULTS Enhancement of the cytotoxic effect of paclitaxel on HER2 overexpressing human breast cancer cells by HRAP Human breast cancer cells were cultured with paclitaxel in the presence of trastuzumab or HRAP for 48 h, and cytotoxic activity was evaluated by MTT assay (Fig. 1). Although trastuzumab and HRAP inhibit proliferation of HER2-overexpressing breast cancer cells as judged by 5-bromo-2′-deoxyuridine uptake, they had no significant effect on cell viability as judged by MTT assay, suggesting that their growth arrest is cytostatic and not due to apoptosis (13). In contrast, paclitaxel treatment significantly reduced cell viability as judged by MTT assay, and trastuzumab and HRAP significantly enhanced the
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FIG. 1. Enhancement of the cytotoxic effect of paclitaxel by HRAP in HER2overexpressing breast cancer cells. Cells in triplicate culture were incubated with drugs for 48 h, and cell viability was determined by MTT assay. ⁎Statistically significant compared to paclitaxel-treated control (p b 0.05, two-sided t-test).
cytotoxic activity induced by paclitaxel in HER2-overexpressing breast cancer cells, BT-474 and SKBR-3. The enhancement was not observed with MDA-231, a breast cancer cell line expressing low levels of HER2 (13). The enhancement by HRAP required 1 mM concentration, and lower doses did not have significant effect on the paclitaxel-induced cytotoxic effect. Enhancement of paclitaxel-induced mitochondria-dependent apoptosis in HER2-overexpressing human breast cancer cells by HRAP The reduced cell viability shown by MTT assay suggests that cells die through induction of apoptosis. To confirm the induction of apoptosis, cleavage of caspase was determined by Western blotting (Fig. 2A), and the expression was quantified by densitometry (Fig. 2B). Paclitaxel induced cleavage of caspase-3 after 48 h incubation, and the cleavage was enhanced by HRAP in BT-474 and SKBR-3. The activation of caspase-3 was confirmed by the cleavage of poly(ADPribose) polymerase (PARP), a major substrate of caspase-3 (16), which was induced by paclitaxel and enhanced by HRAP. Cleavages of caspase-3 and PARP were also detected in paclitaxel-treated MDA231 cells. The cleavage of caspase-3 was further enhanced by HRAP, while that of PARP was not as evident as in HER2-overexpressing breast cancer cells. Cleavage of caspase-9, an initiator caspase activated by mitochondria-dependent apoptotic signals (17), but not caspase-8, an initiator caspase activated by the death-inducing signaling complex (DISC) (18), was detected in HER2-overexpressing cells. HRAP had only negligible effect on the cleavage of caspase-9 in BT-474 cells, while showed slight enhancement in SKBR-3 cells. These results suggest that HRAP mainly enhances the apoptosis-executing mechanism in the mitochondria-dependent apoptotic pathway induced by paclitaxel. Blockade of phosphorylation of survival factors by HRAP To verify the pertuzumab mimetic effect of HRAP, we examined the phosphorylation status of Akt and PTEN, two major targets of pertuzumab present downstream in the HER2-mediated signaling pathway (Fig. 2A and B). Paclitaxel itself reduced phosphorylation of Ser380/Thr382/Thr383 in PTEN, a phosphorylation site at the Cterminal that reduces the activity but increases the stability of the phosphatase (19). The reduction was enhanced by HRAP in BT-474 and SKBR-3 cells. In contrast, this phosphorylation was neither induced by paclitaxel treatment nor affected by HRAP in MDA-231 cells. Phosphorylation of Akt at Ser473, required for activation of the kinase (20), was also reduced by treatment with paclitaxel in HER2-overexpressing breast cancer cells. HRAP enhanced the reduction in BT-474 cells, but not in SKBR-3 cells. Paclitaxel and HRAP again had no marked effect on the phosphorylation of Ser473 at Akt in MDA231 cells. Taken together, our results suggest that HRAP-enhances
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FIG. 2. Activation profile of caspases and phosphorylation of survival factors downstream of HER2/3 signaling in HER2-overexpressing breast cancer cells treated with HRAP and/or paclitaxel. Cells were incubated with drugs for 48 h. Protein expression in the resultant cell lysate was determined by Western blotting (A). The relative protein expression normalized by β-actin expression was quantified by densitometry (B).
paclitaxel-induced apoptosis through the suppression of HER2mediated phosphorylation events involving PTEN phosphorylation. DISCUSSION Because trastuzumab blocks the drug resistance of HER2-overexpressing tumor cells by suppressing survival factors including PTEN and Akt (4), combined therapy with other chemotherapeutic agents greatly improves its efficacy (4), while trastuzumab monotherapy does not improve the prognosis for breast cancer patients. Trastuzumab enhances the therapeutic effects of radiation and chemotherapeutic agents including anthracyclines and taxanes in vitro and in vivo (5-7). We observed similar enhancement of the therapeutic efficacy of paclitaxel by HRAP, a pertuzumab-mimetic pentapeptide, demonstrating its clinical potential in breast cancer therapy. In HER2 overexpressing human breast cancer cells, HRAP enhanced the inhibitory effect of paclitaxel on Akt phosphorylation and/or PTEN phosphorylation, and potentiated the paclitaxel-induced cleavage of caspase-3 and PARP. These effects were not observed in the HER2-dull cell line MDA-231. These results suggest that HRAP, like trastuzumab, improves therapeutic efficacy of paclitaxel, through the blockade of HER2-mediating activation signals and the potentiation of paclitaxelinducing apoptosis. It is likely that this apoptosis is mediated by mitochondria activation because cleavage of caspase-9, but not of caspase-8, was observed in paclitaxel-treated HER2 overexpressing cells. Because trastuzumab potentiates effect of radiation and other chemotherapeutic agents including doxorubicin and etoposide, HRAP could enhance the chemotherapeutic effect of agents other than paclitaxel. A trastuzumab-mimetic cyclic dodecapeptide, anti-HER2/neu peptide (AHNP), has been reported (21, 22). AHNP was designed based on the complementarity determining regions (CDR)-3 of antibodies against HER2 domain IV, including trastuzumab, and
contains the five amino acids in this region. AHNP specifically binds to HER2 with high affinity, inhibits proliferation of HER2-overexpressing tumor cells and suppresses tumor growth in athymic mice. AHNP, like HRAP, enhances the suppressive effect of doxorubicin on cell proliferation and apoptosis induced by radiation in HER2-overexpressing cells. Compared to trastuzumab, pertuzumab more directly inhibits the dimerization of HER2 and more profoundly inhibits HER2/HER3 phosphorylation induced by neuregulin. Pertuzumab inhibits ligand-induced proliferation of MCF-7 in vitro and the tumor growth in nude mice, a breast cancer expressing a lower level of HER2 (9). Thus, it is expected that pertuzumab-mimicry peptides including HRAP have some advantage, compared to trastuzumabmimicry peptides. Although AHNP has high affinity for HER2 (Kd = 300 nM), more than 67 μM is required to attain proliferation inhibition similar to trastuzumab (22). In the case of HRAP, a dose as high as 1 mM suppressed proliferation only partially, and a synergistic effect with paclitaxel was not observed at lower doses. Doses higher than 10 mM kill fraction of cells nonspecifically. IC50 for SKBR-3 is 6.8 mM in a 72h MTT assay (unpublished result). Clearly improvements of affinity and specificity are necessary before HRAP could be used clinically. Based on the three-dimensional structure of HRAP, we are now attempting to create a compound with a different, nonpeptide structure through computational design. Another strategy is to create molecular targeting compounds by conjugating the toxic molecules on HRAP. Fantin et al. (23) have engineered a bifunctional, HER-2blocking and apoptosis-inducing peptide (BHAP) which consists of a HER2 targeting/neutralizing domain, AHNP, and a mitochondria-toxic domain. In this report, BHAP effectively suppresses proliferation, induces apoptosis and prevents tumor growth in athymic mice in a manner selective for HER2 overexpressing breast cancer, in contrast to the moderate effects of AHNP. The present results demonstrate that HRAP could be a lead compound for creating such molecular targeting
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agents small enough to penetrate into the brain for treating malignant breast cancers. References 1. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A, and McGuire, W. L.: Human breast cancer: correlation of relapse and survival with amplification on the HER-2/neu oncogene, Science, 235, 177–182 (1987). 2. Crowder, R. J., Lombardi, D. P., and Ellis, M. J.: Successful targeting of ErbB2 receptors-is PTEN the key, Cancer Cell, 6, 103–104 (2004). 3. Zhang, H., Richter, M., and Greene, M. I.: Therapeutic monoclonal antibodies for the ErbB family of receptor tyrosine kinases, Cancer Biol. Ther., 2, S122–S126 (2003). 4. Hudis, C. A.: Trastuzumab—mechanism of action and use in clinical practice, N. Engl. J. Med., 357, 39–51 (2007). 5. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and Mendelsohn, J.: Recombinant humanized anti-HER2 antibody (Herceptin™) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenograft, Cancer Res., 58, 2825–2831 (1998). 6. Lee, S., Yang, W., Lan, K. H., Sellappan, S., Klos, K., Hortobagyi, G., Hung, M. C., and Yu, D.: Enhanced sensitization to Taxol-induced apoptosis by Herceptin pretreatment in ErbB2-overexpressing breast cancer cells, Cancer Res., 62, 5703–5710 (2002). 7. Liang, K., Lu, Y., Jin, W., Ang, K. K., Milas, L., and Fan, Z.: Sensitization of breast cancer cells to radiation by trastuzumab, Mol. Cancer Ther., 2, 1113–1120 (2003). 8. Nagata, Y., Lan, K. H., Zhou, X., Tan, M., Esteva, F. J., Sahin, A. A., Klos, K. S., Li, P., Monia, B. P., Nguyen, N. T., and other 3 authors: PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients, Cancer Cell, 6, 117–127 (2004). 9. Agus, D. A., Akita, R. W., Fox, W. D., Lewis, G. D., Higgins, B., Pisacane, P. I., Lofgren, J. A., Tindell, C., Evans, D. P., Maiese, K., Scher, H. I., and Sliwkowski, M. X.: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth, Cancer Cell, 2, 127–137 (2002). 10. Kallioniemi, O. P., Holli, K., Visakorpi, T., Koivula, T., Helin, H. H., and Isola, J. J.: Association of c-ErbB-2 protein over-expression with high rate of cell proliferation,
11. 12.
13.
14. 15. 16.
17. 18. 19.
20. 21.
22.
23.
253
increased risk of visceral metastasis and poor long-term survival in breast cancer, Int. J. Cancer, 49, 650–655 (1991). Kirsch, D. G. and Hochberg, F. H.: Targeting HER2 in brain metastasis from breast cancer, Clin. Cancer Res., 9, 5435–5439 (2003). Grossi, M., Ochiai, H., Archer, G. E., McLendon, R. E., Zalutsky, M. R., Friedman, A. H., Friedman, H. S., Bigner, D. D., and Sampson, J. H.: Efficacy of intracerebral microinfusion of trastuzumab in an athymic rat model of intracerebral metastatic breast cancer, Clin. Cancer Res., 9, 5514–5520 (2003). Nakajima, H., Mizuta, N., Sakaguchi, K., Fujiwara, I., Yoshimori, A., Takahashi, S., Takasawa, R., and Tanuma, S.: Development of HER2-antagonistic peptides as novel anti-breast cancer drugs by in silico methods, Breast Cancer, 15, 65–72 (2008). Mosmann, T.: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65, 55–63 (1983). Gerlier, D. and Thomasset, N.: Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods, 94, 57–63 (1986). Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M.: Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose)polymerase, Cell, 81, 801–809 (1995). Hengartner, M. O.: The biochemistry of apoptosis, Nature, 407, 770–776 (2000). Nagata, S.: Apoptosis by death factor, Cell, 88, 355–365 (1997). Vazquez, F., Ramaswamy, S., Nakamura, N., and Sellers, W. R.: Phosphorylation of the PTEN tail regulates protein stability and function, Mol. Cell. Biol., 20, 5010–5018 (2000). Datta, S. R., Brunet, A., and Greenberg, M. E.: Cellular survival: a play in three Akts, Genes Dev., 13, 2905–2927 (1999). Berezov, A., Zhang, H. T., Greene, M. I., and Murali, R.: Disabling ErbB receptors with rationally designed exocyclic mimetics of antibodies: structure-function analysis, J. Med. Chem., 44, 2565–2574 (2001). Park, B. W., Zhang, H. T., Wu, C., Berezov, A., Zhang, X., Dua, R., Wang, Q., Kao, G., O'Rourke, D. M., Greene, M. I., and Murali, R.: Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo, Nat. Biotechnol., 18, 194–198 (2000). Fantin, V. R., Berardi, M. J., Babbe, H., Michelman, M. V., Manning, C. M., and Leder, P.: A bifunctional targeted peptide that blocks HER-2 tyrosine kinase and disables mitochondrial function in HER-2-positive carcinoma cells, Cancer Res., 65, 6891–6900 (2005).