A specific vascular endothelial growth factor receptor tyrosine kinase inhibitor enhances the antiproliferative effect of trastuzumab in human epidermal growth factor receptor 2 overexpressing breast cancer cell lines

A specific vascular endothelial growth factor receptor tyrosine kinase inhibitor enhances the antiproliferative effect of trastuzumab in human epidermal growth factor receptor 2 overexpressing breast cancer cell lines

The American Journal of Surgery (2009) 197, 331–336 The Midwest Surgical Association A specific vascular endothelial growth factor receptor tyrosine...

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The American Journal of Surgery (2009) 197, 331–336

The Midwest Surgical Association

A specific vascular endothelial growth factor receptor tyrosine kinase inhibitor enhances the antiproliferative effect of trastuzumab in human epidermal growth factor receptor 2 overexpressing breast cancer cell lines Elizabeth M.H. Kim, M.D.*, Catherine Lobocki, M.S., Linda Dubay, M.D., Vijay K. Mittal, M.D. Department of Surgery, Providence Hospital and Medical Centers, 16001 W. Nine Mile Rd., Southfield, MI 48075, USA KEYWORDS: Trastuzumab; HER-2; VEGFR; Breast cancer

Abstract BACKGROUND: Trastuzumab has been found to have potent antiproliferative effects in human epidermal growth factor receptor 2 (HER-2)– overexpressing human breast tumors. Inhibition of vascular endothelial growth factor receptor (VEGFR), a protein often overexpressed in breast carcinoma, has been shown to induce apoptosis. METHODS: Breast carcinoma cell lines were cultured with increasing doses of trastuzumab and/or a VEGFR tyrosine kinase inhibitor (TKI). Growth inhibition and apoptosis were assessed after 5 days and 48 hours of treatment, respectively. Combination index values were calculated to determine the effectiveness of this drug combination. RESULTS: A dose-dependent growth inhibition was shown in all cell lines tested with the VEGFR TKI, whereas trastuzumab was effective only in the HER-2–positive cells. A synergistic interaction was shown in the HER-2– overexpressing cell lines, accompanied by an increase in apoptosis. CONCLUSIONS: The combination of trastuzumab and a VEGFR TKI may be of therapeutic value in select breast cancer patients. © 2009 Elsevier Inc. All rights reserved.

Breast cancer is a devastating disease. The recent clinical success of therapies that target specific proteins expressed by tumor cells has shown that growth factor receptors and tyrosine kinase inhibitors (TKIs) have important clinical benefits.1,2 Two such targets, vascular endothelial growth The manuscript has been seen and approved by all authors and the material is previously unpublished. * Corresponding author. Tel.: ⫹1-248-849-5546; fax: ⫹1-248-8493304. E-mail address: [email protected] Manuscript received August 1, 2008; revised manuscript October 24, 2008

0002-9610/$ - see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.amjsurg.2008.10.009

factor receptor (VEGFR) and human epidermal growth factor receptor 2 (HER-2) have been shown to be mediators of breast cancer pathogenesis. Also, overexpression of these receptors correlates strongly with aggressive behavior in breast carcinomas.3–5 HER-2, a receptor tyrosine kinase, is activated constitutively and amplified in up to 30% of all breast cancers.3 Multiple signaling pathways related to cellular proliferation are activated downstream of HER-2 (Fig. 1). Therapy targeting the HER-2 oncoprotein using monoclonal antibodies is a promising treatment strategy for breast cancer patients with HER-2 positive tumors. Trastuzumab (Herceptin) is a

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Figure 1 Mechanism of HER-2 activation in the presence of trastuzumab.

recombinant DNA-derived humanized monoclonal antibody that selectively binds with high affinity to the extracellular domain of the HER-2 protein (Fig. 1). It has been shown in vitro and in vivo to inhibit the proliferation of human tumor cells that overexpress HER-2.6 Although trastuzumab has been heralded as a newfound hope for many breast cancer patients with HER-2 overexpression, it is not without limitations. Studies have shown that less than 35% of patients with HER-2– overexpressing metastatic breast cancer will respond to trastuzumab as a single agent.7 Furthermore, a significant number of patients who achieve an initial response acquire resistance within 1 year.1,8,9 Clinically, the cardiotoxic side effects of trastuzumab can be grave and require strict evaluation.10 For adjuvant treatment in patients with HER-2–positive breast cancer, trastuzumab currently is approved as part of a regimen consisting of doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel.8,11–13 VEGFR is a receptor tyrosine kinase, which plays an important role in promoting tumorigenesis by affecting not only the tumor vasculature, but the cancer cells as well.14 Recent data have indicated a translational up-regulation of the VEGF protein in HER-2– overexpressing breast cancer cells,2,15–17 which suggested that this up-regulation may contribute to the aggressive phenotype observed in these tumors. VEGF produced by breast cancer cells promotes angiogenesis through a paracrine effect on tumor vascular endothelial cells,18,19 which is essential for breast cancer progression.17 In addition, expression of VEGFR-1 in breast carcinoma has been found to be a prognostic indicator of poor outcome in patients with node-negative tumors.20 Coexpression of VEGFR-1 and its ligand (VEGF-A) suggests the presence of an autocrine-signaling loop that enables these tumor cells to promote their own growth.21 This autocrine loop represents an attractive therapeutic target, especially because in vivo blockade of VEGF and VEGFR signaling can inhibit angiogenesis, as well as tumor cell proliferation and migration.2 The presence of both VEGFR and HER-2 might suggest a cooperation between these 2 receptors in sustaining

The American Journal of Surgery, Vol 197, No 3, March 2009 the autonomous proliferation of breast cancer cells.22 Furthermore, it has been shown that the overexpression of both HER-2 and VEGFR is associated with poorer prognosis than overexpression of either protein alone in breast carcinoma patients.23 The results of these studies suggest that targeting both HER-2 and VEGFR may be an effective therapeutic strategy. The recent development of directed therapy against receptor tyrosine kinases and their ligands includes either monoclonal antibodies such as trastuzumab (HER-2) and bevacizumab (VEGF), or TKIs such as imatinib mesylate (stem cell factor receptor), erlotinib (epidermal growth factor receptor), and sunitinib (multiple targets including platelet-derived growth factor receptor and VEGFR). In this study, the VEGFR TKI we examined is a potent, selective inhibitor of VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR).24 We chose to examine the antiproliferative and apoptotic effects of this novel reagent in combination with trastuzumab in human breast carcinoma cells in vitro.

Materials and Methods Three human breast carcinoma cell lines from the American Type Culture Collection (Manassas, VA) were studied: 2 HER-2– overexpressing cell lines (BT474 and SKBR3) and 1 cell line that expresses low levels of the receptor (MCF-7). BT474 and SKBR3 show a 25- and 30-fold increase, respectively, in HER-2 expression. Trastuzumab was obtained from Genentech, Inc. (South San Francisco, CA), and the VEGFR TKI (4-[(4=-chloro-2=-fluoro) phenylamino]⫺6,7-dimethoxyquinazoline) was from EMD Biosciences. (San Diego, CA). Growth inhibition was assessed after 5 days of drug treatment using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric (MTT) colorimetric assay (Sigma Chemical Co., St. Louis, MO). Results (% of control) represent the relative absorbance of drug-treated cells compared with untreated control cells. Caspase 3 activity, as an indicator of apoptosis, was determined using a caspase 3 fluorometric assay kit (Assay Designs, Ann Arbor, MI). Crude lysates, prepared from cells treated for 48 hours with drugs, were used in the assay following the manufacturer’s instructions. Caspase 3 levels were tested in duplicate and normalized per mg of cytosol protein.

Data analysis For the MTT assays, cells were plated in triplicate for each treatment group, and the average of 3 separate growth inhibition experiments was determined. Results for the 4 treatment groups (control, trastuzumab alone, VEGFR TKI alone, and the combination of these agents) were analyzed for statistical significance by one-way analysis of variance (P ⬍ .05 was considered significant). Also, drug interaction was examined by the combination index (CI) method of

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Trastuzumab plus VEGFR inhibition

333 line (Fig. 2C). However, as expected, little effect was seen with trastuzumab in the low HER-2– expressing MCF-7 breast cancer cell line, and no increase in growth inhibition was seen with the combination of drugs compared with VEGFR TKI inhibition alone. At a selected dosage, trastuzumab (.5 nmol/L) inhibited cell growth by 35.6% and 31.9%, and the VEGFR TKI (5 ␮mol/L) by 37.4% and 54.3% in BT474 and SKBR3 cells, respectively; however, at these doses, the combination of trastuzumab and VEGFR TKI significantly inhibited growth in BT474 and SKBR3 cells by 60.4% and 75.8% (P ⬍ .001, 1-way analysis of variance). The CI values for the combination of trastuzumab and VEGF TKI indicated a synergistic interaction. At the 50% inhibition level the CI was .62 for the BT474 cells and .87 for the SKBR3 cells. Caspase 3 activation (Fig. 3) after 48 hours of treatment was used as a marker of apoptosis. Trastuzumab, at the highest dose tested (2 nmol/L) did not induce apoptosis in any of the cell lines examined. BT474 cells showed the highest level of caspase 3 activity (15 U/mg) after treatment with VEGFR TKI (20 ␮mol/L), but no difference compared with untreated controls was observed for SKBR3 or MCF-7 at this time point. However, the combination of drugs resulted in a 10.9- and 1.9-fold increase in caspase 3 activity in BT474 and SKBR3, respectively.

Comments Figure 2 Growth inhibition after 5 days of treatment using increasing doses of trastuzumab, a VEGFR TKI, or the combination in three breast cancer cell lines: (A) BT474, (B) SKBR3, and (C) MCF7. Trastuzumab (0.125-2.0 nmol/L), VEGFR TKI (1.25-20 ␮mol/L). *P ⬍ 0.001 compared with single agents. , .125, 1.25; □, .25, 2.5; , .5, 5; , 1.0, 10; , 2.0, 20.

median dose-effect analysis. The CI values were calculated at the 50% inhibition level for the BT474 and SKBR3 cell lines treated with trastuzumab and the VEGFR TKI at a fixed molar ratio. A CI of less than 1 indicates synergy, a CI of 1 indicates additivity, and a CI of greater than 1 indicates antagonism.25

This study shows that the combination of trastuzumab and a specific VEGFR TKI can significantly decrease proliferation and increase apoptosis in HER-2– overexpressing breast cancer cell lines. These findings strongly suggest that simultaneous blockade of different growth factor– driven signal transduction pathways might result in a more significant antitumor effect. The significance of this combined therapy approach with a VEGFR TKI and trastuzumab may prove effective in high-risk patients who overexpress both receptors. These findings are consistent with a number of other studies that showed that trastuzumab exerts only a modest effect on growth inhibition when used as a single agent, and it has no effect on apoptosis.1,7,8 However, significant

Results To assess the potential inhibitory effects of trastuzumab and/or the VEGFR TKI, the growth inhibition of cell lines differing in HER-2 expression was tested. Both HER-2– overexpressing cell lines, BT474 (Fig. 2A) and SKBR3 (Fig. 2B) showed a dose-dependent inhibition with trastuzumab or the VEGFR TKI after 5 days of treatment. Furthermore, the combination of trastuzumab and the VEGFR TKI significantly enhanced growth inhibition at all doses compared with either agent alone (P ⬍ .001). The VEGFR TKI also reduced cellular proliferation in the MCF-7 cell

Figure 3 Caspase 3 activation as an indicator of apoptosis in the 3 breast carcinoma cell lines after 48 hours of drug treatment. , Control; □, trastuzumab; , VEGF-R TKI; , trastuzumab ⫹ VEGF-R TKI.

334 growth inhibition was shown in all breast cancer cell lines using this specific VEGFR TKI. These findings are consistent with the body of literature that showed VEGFR and VEGF expression in SKBR3, BT474, and MCF-7 breast carcinoma cell lines.2,21 Although the intracellular mechanism is not completely clear, by blocking VEGFR tyrosine kinase activity, the growth inhibition and apoptosis caused by trastuzumab were enhanced in HER-2– overexpressing cell lines. The role of VEGF in breast cancer progression is evident from clinical studies showing increased serum VEGF levels in invasive breast cancers.17,26 Interestingly, VEGF levels in serum were found to be increased in 75% of patients during disease progression compared with 15% of breast cancer patients who responded to therapy.26 In addition, tumors from 95% of patients (n ⫽ 260) with node-negative breast cancer had detectable or increased levels of VEGF.13 In vivo, targeting VEGF and VEGFR signaling has been shown to inhibit angiogenesis, tumor cell proliferation, and migration.27 Current research also suggests an up-regulation of VEGF in HER-2– overexpressing breast cancer cells, which may contribute to the aggressive phenotype observed in this subset of patients. Clinical data thus suggest the significance of targeting this growth factor and its receptor in the treatment of breast cancer. Prior research studies have suggested that a network consisting of multiple ligands and receptor molecules are sustaining the autonomous proliferation and the survival of human breast carcinoma cells. Neutralizing antibodies against HER-2 and epidermal growth factor receptors resulted in down-regulation of VEGF, and inhibition of tumor growth.28 It is suggested that the activity derived from autocrine and paracrine pathways communicates through a downstream intracellular pathway leading to amplification of their response. We believe that the signaling pathways of HER-2 and VEGFR may communicate in such a fashion because a synergistic interaction was shown with the combination of trastuzumab and the VEGFR TKI. Future research endeavors should be directed at analyzing the specific molecular targets and downstream signaling involved after treatment with this drug combination. The effect of using a VEGF angiogenic inhibitor has successfully achieved clinical benefit in other cancer models. For instance, bevacizumab (Avastin, San Francisco, CA, USA), a VEGF-specific monoclonal antibody, is used as a standard therapeutic agent in treating colorectal and lung cancer patients.29 Currently, regional clinical trials are evaluating the efficacy of bevacizumab in metastatic and locally invasive breast cancer patients. The following is currently unknown: (1) if HER-2–positive breast cancer patients specifically will benefit from this combination therapy clinically, or (2) if there is a potential adverse interaction when using 2 monoclonal antibodies. The attractiveness of our study is the fact that this VEGFR TKI is not a monoclonal antibody, but a biochemical inhibitor that selectively inhibits the tyrosine kinase activity of VEGFR. We

The American Journal of Surgery, Vol 197, No 3, March 2009 can possibly avoid drug interactions by using this innovative biochemical inhibitor. Targeting VEGFR potentially could disrupt the following: (1) the autocrine pathway, and thus induce tumor growth arrest and apoptosis; and (2) the paracrine effect on the vascular endothelium, and thus reduce angiogenesis and tumor invasion. This would be especially beneficial in HER-2–positive breast tumors, which are known to be especially aggressive. Targeted therapies that focus on molecular mechanisms have gained much attention in recent years in an effort to improve the cytotoxic specificity directed toward malignant cells. The rationale for the use of targeted combination therapy for breast carcinoma is convincing. This work provides an example of a treatment regimen that can serve as an innovative approach against primary or metastatic breast cancers that overexpress HER-2. This novel combination can have important clinical therapeutic implications in treating breast cancer patients. Our next step will be to investigate the efficacy of this combination in an animal model.

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13. Gasparini G, Toi M, Gion M, et al. Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma. J Natl Cancer Inst 1997;89:139 – 47. 14. Camp ER, Summy J, Bauer TW, et al. Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor. Clin Cancer Res 2005;11:397– 405. 15. Konecny GE, Meng YG, Untch M, et al. Association between HER2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients. Clin Cancer Res 2004; 10:1706 –16. 16. Viloria Petit AM, Rak J, Hung M-C, et al. Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol 1997;151:1523–30. 17. Pegram MD, Reese DM. Combined biological therapy of breast cancer using monoclonal antibodies directed against HER2/neu protein and vascular endothelial growth factor. Semin Oncol 2002;29(Suppl 11): 29 –37. 18. Guo P, Fang Q, Tao H-Q, et al. Overexpression of vascular endothelial growth factor by MCF-7 breast cancer cells promotes estrogen-independent tumor growth in vivo. Cancer Res 2003;63:4684 –91. 19. Ferrara N. Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol 1999;237:1–30. 20. Meunier-Carpentier S, Dales JP, Djemli A, et al. Comparison of the prognosis indication of VEGFR-1 and VEGFR-2 and Tie2 receptor expression in breast carcinoma. Int J Oncol 2005;26:977– 84. 21. Weigand M, Hantel P, Kreienberg R, et al. Autocrine vascular endothelial growth factor signalling in breast cancer. Evidence from cell lines and primary breast cancer cultures in vitro. Angiogenesis 2005; 8:197–204. 22. Nakopoulou L, Stefanaki K, Panayotopoulou E, et al. Expression of the vascular endothelial growth factor receptor-2/Flk-1 in breast carcinomas: correlation with proliferation. Hum Pathol 2002;33:863–70. 23. Dales JP, Garcia S, Bonnier P, et al. Prognostic significance of VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) in breast carcinoma. Ann Pathol 2003;23:297–305. 24. Hennequin LF, Thomas AP, Johnstone C, et al. Design and structureactivity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J Med Chem 1999;42:5369 – 89. 25. Chou T, Talalay P. Median effect/combination-index isobologram method of multiple drug effect analysis. Adv Enzym Regul 1984;22: 27–55. 26. Dirix LY, Vermeulen PB, Pawinski A, et al. Elevated levels of the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in sera of cancer patients. Br J Cancer 1997; 76:238 – 43. 27. Sledge GW Jr. Vascular endothelial growth factor in breast cancer: biologic and therapeutic aspects. Semin Oncol 2002;29(Suppl 11): 104 –10. 28. Yen L, You X-L, Al Moustafa A-E, et al. Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis. Oncogene 2000;19:3460 –9. 29. Wang Y, Fei D, Vanderlaan M, et al. Biological activity of bevacizumab, a humanized anti-VEGF antibody in vitro. Angiogenesis 2004; 7:335– 45.

Discussion Dr. Arthur M. Carlin (Detroit, MI): Your group has addressed a very important issue affecting many breast cancer patients: HER-2 overexpression, which correlates with aggressive behavior. Your study nicely demonstrates a synergistic inhibition of growth and apoptosis when trastu-

335 zumab is used in combination with the VEGFR tyrosine kinase inhibitor. I have a few questions. First, could you please discuss why you chose to assess growth inhibition after 5 days and apoptosis after 2 days? Were there any other time points or drug concentrations evaluated, and, if so, what were your results? Since trastuzumab and vascular endothelial growth factor tyrosine kinase inhibitors both affect apoptosis, how do you explain your lack of effect on apoptosis when they were used alone in most of your assays? What future studies are you planning to help you identify the specific molecular mechanism involved in the synergistic antiproliferative effect that you have identified with this combination of agents? What potential deleterious effects might you anticipate that this biochemical inhibitor would have in animals or humans? Dr. Kim: In regards to your first question, from our pilot studies we have measured the IC50 levels (concentration of drug required to exert a 50% growth inhibitory effect) for both the trastuzumab and VEGFR tyrosine kinase inhibitors for all of these cell lines, and we selected treatment concentration that was close to our measured IC50. Our previous studies also revealed that it takes at least 5 days for trastuzumab to show a measurable growth inhibition. And it takes at least 24 hours of treatment to have an effect. And, hence, we chose these time points for that reason. Also, we assess for the growth inhibition with MTT assay at time points such as day 3, day 5, and day 7. In addition, we looked at various different dosing of our agents, trastuzumab from .01 to as high as 25 ␮mol/L, and VEGF tyrosine kinase inhibitor from 1 to as high as 50 ␮mol/L. What was so interesting was that we still have both the dose- and time-dependent growth inhibition with individual treatments, as well as in combination. In regards to your second question, trastuzumab does not induce apoptosis, an observation supported by the literature. As shown in Fig. 3, the apoptotic activity for trastuzumab was no different from controls. And that was true for both the SKBR3 and BT474 cell lines. As for VEGF, it did induce apoptosis in SKBR3 and BT474 cells. It was interesting that apoptotic activity was enhanced when we added trastuzumab. In regards to your third question, the literature suggests that HER-2 and VEGF tyrosine kinase receptors function via the autocrine and paracrine pathways and that the activity creates intracellular and amplified communication. Thus, when we combined trastuzumab and VEGF, there was a synergistic response. This observation could have interesting implications for our breast cancer patients. In regards to your fourth question, the VEGF tyrosine kinase inhibitor, which we use in our study, belongs to a family of quinazolines. And these derivatives are known to cause gastrointestinal disturbances, such as nausea, vomiting, and diarrhea, in human subjects. Our specific tyrosine kinase inhibitor is under study using an in vivo model, so we will let you know about side effects as soon as we find out.

336 Dr. Herbert Chen (Madison, WI): I have 2 quick questions. One, can you tell us the name of the VEGF inhibitor? And, second, you showed very nice MTT growth curves, but do you have any data to show that your drug is working through the specific mechanism you say it is? Have you done Western blots to show that you are indeed blocking the downstream pathway of the VEGF receptor?

The American Journal of Surgery, Vol 197, No 3, March 2009 Dr. Kim: The name of the drug is VEGF receptor tyrosine kinase inhibitor (4-[(4’-chloro-2’-fluoro) phenylamino]-6,7dimethoxyquinazoline) from EMD Biosciences. We measured growth inhibition and apoptosis in our controls and also in a MCF7 control cell line. We were looking at the inhibition of proliferation and have not as yet examined the specific mechanism involved.