Induction of p 53 Up-Regulated Modulator of Apoptosis Messenger RNA by Chemotherapeutic Treatment of Locally Advanced Breast Cancer

Purpose: In biopsies of patients with locally advanced breast cancer, we investigated the in vivo changes of the gene expression pattern induced by chemotherapy to find genes that are potentially responsible for the efficacy of the drug. Experimental Design: Early cellular responses to chemotherapy-induced damage, both in vivo and in vitro , were investigated by analyzing chemotherapy-induced changes in gene expression profiles. Core biopsies were taken from nine patients with locally advanced breast cancer, before and at 6 hours after initiation of doxorubicinbased chemotherapy. Both samples were cohybridized on the same microarray containing 18,000 cDNA spots. Results: The analysis revealed marked differences in gene expression profile between treated and untreated samples. The gene which was most frequently found to be differentially expressed was p53 up-regulated modulator of apoptosis (PUMA). This gene was up-regulated in eight of nine patients with an average factor of 1.80 (range, 1.36-2.73). In vitro MCF-7 breast cancer cells exposed to clinically achievable doxorubicin concentrations for 6 hours revealed marked induction of PUMA mRNA, as well. Conclusions: This is the first report describing PUMA mRNA to be up-regulated as a response to chemotherapy in patients. Because PUMA is a known member of the family of BH3-only proapoptotic proteins, this finding suggests PUMA’s potential importance for the response to anticancer drugs. INTRODUCTION Locally advanced breast cancer (LABC) consists of a heterogeneous group of breast tumors including stages IIB, IIIA, IIIB, and IIIC, according to the American Joint Committee on Cancer criteria (1). It has been suggested that patients with LABC often have micrometastases at presentation, which seriously reduces the effectiveness of local therapy (2–6). The use of different regimens of systemic chemotherapy, in the treatment of LABC, has therefore been investigated extensively (7–10). Recent studies in this field have investigated the use of four to six cycles of neoadjuvant chemotherapy, consisting of an intermediate-high dose of doxorubicin, cyclophosphamide and granulocyte macrophage colony-stimulating factor (11, 12). This treatment regimen has shown impressive survival rates, especially in those patients treated with six cycles of this chemotherapy. However, in spite of these advances in the treatment of LABC and the initial high rate of response to this form of chemotherapy, relapses still occur in >25% of the patients (11, 12). These clinical findings clearly indicate that, although the vast majority of tumor cells are killed during the initial treatment, some malignant cells survive and will inevitably lead to a relapse. Understanding the in vivo response of human cancer cells to the exposure to both doxorubicin and cyclophosphamide could not only help us identify those cellular mechanisms responsible for the death of most tumor cells but also those responsible for the survival of a few. From in vitro studies, it has been known for some time that doxorubicin causes DNA damage by intercalation, metal ion chelation, free radical generation, and topoisomerase II inhibition (13–17). More recently, several molecular pathways have been implied in either the repair of doxorubicin-induced damage or the induction of programmed cell death (apoptosis) in response to this damage (13, 18, 19). Cyclophosphamide is active after activation in liver microsomes to a metabolite with DNA alkylating activity (20). Therefore, it could induce similar, DNA damage–related, cellular responses. Some reports on microarray studies on long-term responses to doxorubicin treatment of breast cancer have been published (21, 22). However, these studies compared gene expression profiles in samples taken 16 weeks and 21 days, respectively, after initiation of treatment. Because we aimed to identify mechanisms involved in the early response to doxorubicin-induced cellular damage, we needed to analyze gene expression changes that are more rapidly induced. Therefore, we set out to investigate these mechanisms by identifying differences in gene expression profiles between tumor biopsies taken from LABC patients before and at only 6 hours after initiation of doxorubicin-based chemotherapeutic treatment. MCF-7 breast tumor cells were included as a model to verify that changes in gene expression as found in patients can also occur in vitro , because we would preferentially identify mechanisms Received 7/12/04; revised 11/9/04; accepted 12/10/04. Grant support: Koningin Wilhelmina Fonds Dutch Cancer Foundation project VU 2001-2508. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: H.M. Pinedo is a Spinoza Award recipient. Requests for reprints: Jan Lankelma, Department of Medical Oncology, Room BR232, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-20-444-2603; Fax: 31-20-4443844; E-mail: j.lankelma@vumc.nl. D2005 American Association for Cancer Research. Vol. 11, 1863–1869, March 1, 2005 Clinical Cancer Research 1863 Research. on May 3, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals. org Downloaded from that can be further investigated in vitro . To our knowledge, no microarray studies on the in vitro effects of cyclophosphamide and its active metabolite have been published, which is probably due to the instability of its active metabolite in culture medium (23). A microarray-based study of doxorubicin induced gene expression changes in MCF-7 breast cancer cells has already been published (24). However, the experimental conditions and microarray platform used in this study did not allow for a direct comparison of these results with the data obtained from our in vivo samples. Gene expression changes in vitro have previously been observed after only a few hours of exposure to a relatively high concentration of doxorubicin (24). However, our studies showed that slow cellular uptake and reduced penetration into certain types of tumor tissue can lead to several hours of delay time before the cellular response to doxorubicin can take effect in a clinical setting (25, 26). Therefore, we obtained our samples at 6 hours after initiation of chemotherapy, to allow for doxorubicin to induce sufficient cellular damage to provoke a detectable