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Her2/Neu Induces All-Trans Retinoic Acid (ATRA) Resistance in Breast Cancer Cells

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Her2/Neu Induces All-Trans Retinoic Acid (ATRA) Resistance in Breast Cancer Cells Oncogene (2002) 21, 5224 – 5232 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Her2/neu induces all-trans retinoic acid (ATRA) resistance in breast cancer cells Ana M Tari*,1,5, Soo-Jeong Lim1,4,5, Mien-Chie Hung2, Francisco J Esteva3 and Gabriel Lopez-Berestein1 1Department of Bioimmunotherapy, Section of Immunobiology and Drug Carriers, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, TX 77030, USA; 2Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, TX 77030, USA; 3Department of Breast Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, TX 77030, USA We observed that all-trans retinoic acid (ATRA) Introduction inhibited the growth of MCF-7 breast cancer cells, but not those transfected with HER2/NEU or its transacti- All-trans retinoic acid (ATRA) inhibits the growth of vating ligand HEREGULIN. This suggests that Her2/ various cell types. ATRA mediates its growth neu causes breast cancer cells to be resistant to the inhibitory effects via the retinoic acid receptors growth inhibitory effects of ATRA. To confirm this (RARs). Upon binding to ATRA, RARs form observation, MDA-MB-453 and BT-474 cells, which heterodimers with retinoic X receptors, bind to retinoic have high levels of Her2/neu and are resistant to ATRA, acid response elements found in the promoters of were incubated with the trastuzumab (HerceptinTM) target genes, and modulate gene transcription antibody so that we could determine whether inhibition (Mangelsdorf et al., 1994). Many human breast cancer of the expression and function of Her2/neu would cell lines are sensitive to the inhibitory actions of resensitize these cells to ATRA. Indeed, we found that ATRA, but there are breast cancer cell lines that are MDA-MB-453 and BT-474 cells treated with trastuzu- resistant to ATRA (Lotan, 1979; Fraker et al., 1984; mab were growth inhibitory by ATRA. We then Fontana, 1987). ATRA-resistant breast cancer cell lines determined whether Her2/neu uses Grb2 and Akt are generally negative for the expression of estrogen proteins to induce ATRA resistance. Liposome-incorpo- receptor-a (ERa) and have low levels of RARs, rated Grb2 antisense oligonucleotides (L-Grb2) and a particularly RARa (Sheikh et al., 1994; van der Leede dominant negative (DN) AKT mutant were used to et al., 1995; Fitzgerald et al., 1997; Schneider et al., down-regulate Grb2 expression and inhibit Akt activity, 2000). respectively. When incubated with L-Grb2 or transfected Amplification of HER2/NEU (also known as with the DN AKT mutant, ATRA-resistant, Her2/neu- ERBB2) is correlated with poor prognosis and survival overexpressing cells became sensitive to ATRA. Our outcome in patients with breast cancer (Slamon et al., results indicate that Her2/neu utilizes Grb2 and Akt 1987). This is partly because Her2/neu induces breast proteins to induce ATRA resistance in breast cancer cancer cells resistance to chemotherapeutic and cells. ATRA sensitivity was also correlated with RARa biological agents, including tumor necrosis factor-a, protein levels since higher RARa protein levels were tamoxifen, cisplatin, doxorubicin, and paclitaxel observed in cells in which the Her2/neu pathway was (Hudziak et al., 1989; Benz et al., 1992; Pietras et al., inhibited. 1994; Yu et al., 1996; Baselga et al., 1998). While Oncogene (2002) 21, 5224 – 5232. doi:10.1038/sj.onc. comparing the characteristics of ATRA-sensitive and 1205660 ATRA-resistant breast cancer cell lines, we observed that many ATRA-resistant cell lines, with the excep- Keywords: HER2/neu; ATRA; Grb2; Akt tion of SKBr3 cells, have moderate to high expression levels of the Her2/neu protein (Sheikh et al., 1993; Rishi et al., 1996; De Luca et al., 1997). We hypothesized that Her2/neu induces ATRA resistance in breast cancer cells. Here we report that transfection of HER2/NEU in ATRA-sensitive breast cancer cells caused cells to become ATRA-resistant. Inhibition of TM *Correspondence: AM Tari, Department of Bioimmunotherapy, Box Her2/neu by the trastuzumab (Herceptin ) antibody 422, The University of Texas M.D. Anderson Cancer Center, 1515 sensitized Her2/neu-overexpressing cells to ATRA. Holcombe Boulevard, Houston, TX 77030, USA Inhibition of the growth factor receptor bound 4 Current address: Division of Basic Science, National Cancer Center, protein-2 (Grb2) and Akt, two well-known down- 809 Madu 1-Dong, ILsan-gu, Goyang-si, Gyeonggi-do, Korea 5The first two authors contributed equally to this study stream signaling proteins of Her2/neu, also sensitized Received 7 December 2001; revised 19 April 2002; accepted 10 Her2/neu-overexpressing cells to ATRA. Furthermore, May 2002 we could correlate inhibition of the Her2/neu signaling Her2/neu induces ATRA resistance AM Tari et al 5225 pathway with increased ATRA sensitivity and sensitivity of MCF-7 cells to ATRA by heregulin was increased RARa protein levels. observed when cells were incubated with up to 250 nM ATRA (Figure 2). However, heregulin did not affect the sensitivity of MCF-7 cells to ATRA when the cells were Results incubated with higher doses of ATRA. At 1 mM concentration, ATRA reduced the growth of MCF-7 Her2/neu and heregulin induced ATRA resistance in cells by 71% in the absence of heregulin, and 55% in the breast cancer cells presence of heregulin (P40.05). Thus, in the presence of To determine whether Her2/neu could induce ATRA heregulin, MCF-7 cells became less sensitive to low doses resistance in breast cancer cells, the sensitivity of of ATRA. Heregulin also reduced the sensitivity of parental MCF-7 cells to the growth inhibitory effects another breast cancer cell line, T-47D, to ATRA (data of ATRA was compared with those of MCF-7 cells not shown). These data indicate that overexpression or transfected with HER2/NEU or HEREGULIN, since activation of Her2/neu led to ATRA resistance in breast Her2/neu is known to be transactivated by heregulin cancer cells. (Lupu et al., 1992; Peles et al., 1992; Plowman et al., 1993; Sliwkowski et al., 1994). We chose MCF-7 cells Reduced RARa protein levels were found in MCF-7 cells because these cells are sensitive to ATRA, have very transfected with Her2/neu or heregulin low levels of Her2/neu (Figure 1a, inset), and due to their ease of transfection, have been used as a model to Since the sensitivity of breast cancer cells to ATRA has study the function of various genes in breast cancer been correlated with RARa expression (Sheikh et al., cells (Kern et al., 1994). As expected, MCF-7 cells 1994; van der Leede et al., 1995; Fitzgerald et al., 1997; stably transfected with either the HER2/NEU gene Schneider et al., 2000), we examined the expression of (MCF-7/HER2) or the HEREGULIN gene (MCF-7/ RARs in MCF-7 parental cells and its variants. HRG) express very high levels of the respective Compared with MCF-7/WT cells, MCF-7/HRG and transfected proteins (Figure 1a, inset). MCF-7/HER2 cells had lower RARa protein expres- After 5 days of treatment with ATRA, the sion (Figure 3). Densitometric data indicated that proliferation of parental MCF-7 cells (MCF-7/WT) RARa protein expression levels in the MCF-7/V, was inhibited in a dose-dependent manner (Figure 1a). MCF-7/HRG and MCF-7/HER2 cells were approxi- At 1 mM concentration, ATRA inhibited MCF-7/WT mately 90, 41 and 63% of the level for MCF-7/WT cell growth by 63%. The sensitivity of vector- cells. It is not clear why two RARa bands were transfected cells (MCF-7/V) to ATRA was similar to detected in MCF-7/HER2 cells. The two RARa bands that of MCF-7/WT cells (data not shown). However, could be phosphorylated and unphosphorylated forms ATRA did not inhibit the proliferation of MCF-7/ of RARa or two different RARa isoforms. The HER2 or MCF-7/HRG cells (Figure 1a). This is expression levels of RARb and RARg proteins were despite the fact that the expression levels of ERa are not modulated in any of the transfected MCF-7 cells similar among MCF-7/WT, MCF-7/HER2 and MCF- (Figure 3), indicating that Her2/neu, when over- 7/HRG cells (Figure 1a, inset). expressed or activated by heregulin, can decrease Since Her2/neu and EGFR are related family endogenous RARa expression. members, we determined whether EGFR could also induce ATRA resistance. The levels of EGFR in MCF- Trastuzumab increased ATRA sensitivity in Her2- 7 cells transfected with vector control (MCF-7/V) are overexpressing cells almost undetectable, whereas the levels of EGFR in MCF-7 cells stably transfected with the EGFR gene The antibody trastuzumab specifically blocks Her2/neu (MCF-7/EGFR cells) are very high (Figure 1b, inset). function and expression (Carter et al., 1992; Sliwkows- The expression levels of ERa in both cell lines are quite ki et al., 1999). Thus we determined whether similar (Figure 1b, inset). At 1 mM concentration, trastuzumab, by blocking Her2/neu signaling, could ATRA inhibited the growth of MCF-7/V cells by sensitize Her2-overexpressing breast cancer cells, such approximately 51% (Figure 1b). In the presence or as MDA-MB-453 and BT-474, to ATRA. In the absence of EGF, ATRA inhibited the growth of MCF- absence of trastuzumab, ATRA did not inhibit the 7/EGFR cells to a very similar extent, approximately growth of MDA-MB-453 (Figure 4a) or BT-474 39 – 43% (Figure 1b). (Figure 4b) cells. When 1 mM trastuzumab was added To avoid the bias of using only one clone of MCF-7/ to MDA-MB-453 cells, growth was inhibited by 56% HER2 and one clone of MCF-7/HRG cells, we decided (Figure 4a).
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