Article in press - uncorrected proof Horm Mol Biol Clin Invest 2011;5(2):97–103
2011 by Walter de Gruyter • Berlin • New York. DOI 10.1515/HMBCI.2011.010

Extending aromatase inhibitor sensitivity in hormone resistant breast cancer

Angela M.H. Brodie1–3,*, Saranya Chumsri2,3, Sara Sukumar4 and Gauri J. Sabnis1 1

Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD, USA 2 Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA 3 Department of Oncology, University of Maryland Greenebaum Cancer Center, Baltimore, MD, USA 4 Johns Hopkins University School of Medicine, Baltimore, MD, USA

Abstract Aromatase inhibitors (AIs) are first-line treatment for ERq breast cancer. However, despite responses initially, some patients can eventually acquire resistance. Moreover, 25% of all breast cancer patients do not express the estrogen receptor (ERa) and are innately resistance. In tumors of mouse models with acquired AI letrozole resistance, expression of ERa was reduced whereas HER2/growth factor signaling was enhanced. Treatment of mice with trastuzumab (HER2 antibody) reduced HER2/p-MAPK but restored ERa expression. The addition of trastuzumab to letrozole treatment when tumors progressed resulted in significantly longer tumor suppression than these drugs alone. Thus, inhibition of both HER2 and ERa signaling pathways was necessary to overcome resistance. In ERa-negative tumors, the receptor has been shown to be silenced by epigenetic modifications. Treatment of MDA-MB-231 ER-negative tumors with a histone deacetylase inhibitor, entinostat (ENT) increased expression of ERa and also aromatase. When ENT was combined with letrozole, tumor growth rate was markedly reduced compared with control tumors. ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with significant reduction in visible and microscopic foci. These novel strategies could improve treatment for patients with acquired and innate resistance to AIs. Keywords: aromatase inhibitors; breast cancer; letrozole; trastuzumab. *Corresponding author: Angela M.H. Brodie, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Health Science Facility-I, Room 580G, 685 W. Baltimore St, Baltimore, MD 21201, USA Phone: q1-410-706-3137, Fax: q1-410-706-0032, E-mail: [email protected] Received January 24, 2011; accepted February 1, 2011

Introduction Aromatase inhibitors (AIs) have become the first-line choice for treatment of ERq breast cancer, because they are proving to have greater efficiency than tamoxifen w1x. Nevertheless, although most patients might respond to treatment initially, some can eventually relapse and become unresponsive to continued treatment. Furthermore, approximately 25% of all breast cancer patients do not express either the estrogen receptor alpha (ERa) or the progesterone receptor (PR), both of which are essential for response to hormone therapy, including AIs. As 210,000 new cases of breast cancer are expected in 2010 w2x in the United States alone, the number of patients who are unresponsive to antihormone therapy is significant. Therefore, a greater understanding of how tumors become resistant to AIs is required to improve our efforts to manage and treat breast cancer. Recently, we have investigated the underlying mechanisms of acquired resistance to AIs that allow tumors to adapt and survive the pressure of estrogen deprivation therapy. In addition, we have investigated mechanisms involved in hormone receptor-negative breast cancer and whether they can be reprogrammed to be sensitive to AIs. In this article, our findings concerning mechanisms associated with resistance to AIs and novel strategies to reverse both the acquired and innate resistance to AIs are reviewed.

Materials and methods Cell culture ERa-positive MCF-7Ca aromatase expressing cells were provided by Dr. S. Chen, City of Hope. ERa-negative MDA-MB-231, Hs578T, and SKBR3 cells were obtained from ATCC. These cell lines were authenticated by ATCC using short tandem repeat profiling, karyotyping, and by monitoring cell morphology. In vitro assay conditions and data analysis are described previously.

Inhibitors Letrozole was provided by Dr. Dean Evans, Novartis, Basel, Switzerland. Entinostat (ENT, MS-275) was supplied by Dr. Peter Odentlich, Syndax, Watham, MA, USA. PD98059 and UO126 were purchased from Sigma Inc., St. Louis, MO, USA.

Tumor growth rate analysis All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the University of Maryland, Baltimore, MD, USA. Tumor xenografts of MCF-7Ca cells or MDA-MB-231 cells were inoculated into each flank of the female ovariectomized athymic nude mouse as previously described w3–8x.

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Tumors were measured weekly for MCF7Ca xenografts and twice weekly for MDA-M-231 xenografts. Volumes were calculated from 4 /3pr12r2 where r1-r2.

Lung colonization assay Mice received injections of 3=106 of MDA-MB-231 cells via the tail vein. Groups of mice began treatment 3 weeks later with vehicle (control), ENT, letrozole, or ENT plus letrozole. Mice were treated for 6 weeks, and then euthanized.

Western blotting Cell lysates were prepared as described previously w3–7, 9x and 50 mg of protein from each sample was analyzed by SDS-PAGE. The densitometric values were corrected using b-actin as a loading control.

Aromatase activity Aromatase activity in cells was determined using a radiometric assay by measuring 3H2O formed on conversion of w1-b3Hxandrostenedione (aromatase substrate) to estrone w3, 10x.

RNA extraction and reverse transcription (RT) and PCR RNA was extracted and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) as per manufacturer’s protocol. Analysis of ERa, CYP-19 and pS2 mRNA expression was carried out by conventional RT-PCR as described previously w4, 11x.

Statistics The tumor volumes were analyzed with S-PLUS (7.0, Insightful Corp., Seattle, Washington, USA) to estimate and compare an exponential parameter (bi) controlling the growth rate for each treatment group. The original values for tumor volumes were log transformed. All p-values -0.05 were considered statistically significant. The graphs are presented as mean"SEM.

Results Acquired resistance to aromatase inhibitors in ER-positive tumors

To investigate the mechanisms of acquired resistance, we utilized a xenograft mouse model that mimics postmenopausal women with hormone receptor-positive breast cancer. MCF-7 human ER-positive breast cancer cell line stably transfected with aromatase gene (MCF-7Ca) is inoculated into oophorectomized, immune suppressed mice. Growth of MCF-7Ca xenograft tumors is stimulated by estrogens produced by aromatization of androstenedione within the tumor, thus modeling the postmenopausal patient. The mice were administered AI letrozole for approximately 56 weeks and tumor growth was suppressed for 16 weeks. Tumors subsequently became resistant to continued treatment with letrozole w8, 10x. Tumors were removed at intervals up to 56 weeks and cells were isolated. This cell line is designated long-term letrozole treated (LTLT) cells w8, 10x. Western blot

analysis of tumors collected at 4 weeks revealed that ERa expression initially increased while tumors were still responding to letrozole. However, the total ERa protein level was progressively reduced as tumors became resistant at 28 and 56 weeks. Nevertheless, levels of phosphorylated form of ERa (p-ERa) were significantly higher than p-ERa level at baseline. Furthermore, PR, an estrogen response gene, remained unchanged from baseline. This indicates that the ER signaling pathway continues to be activated in AI resistant tumors. The finding that the ER signaling pathway can be activated despite the lack of its ligand can be explained by crosstalk between ERa and other signaling pathways such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases (PI3K)/Akt pathways w12–15x. Thus, AI resistant tumors appeared to adapt and become dependent on growth factor receptor pathways, especially the human epidermal growth factor receptor 2 (HER2/MAPK) pathway. Thus, at 28 and 56 weeks of letrozole treatment, the expression of HER2 and its downstream effectors (p-Shc, Grb-2, p-Raf, p-Mek1/2, and p-MAPK) were all significantly increased compared with baseline. Moreover, the downstream targets of MAPK such as p90 ribosomal S6 kinase (p90RSK) and the ETS-domain containing protein (Elk) were also found to be activated w8x. To demonstrate that growth of AI resistant cells is dependent on the MAPK pathway, LTLT cells were treated with the MEK inhibitor (U0126) and the MAPK inhibitor (PD98059). Both inhibitors significantly reduced proliferation of LTLT in a dose-dependent manner. By contrast, there was no effect of these inhibitors on the parental MCF-7Ca cells w8x. The plasticity of the cells was evident when they were treated with MAPK inhibitor (PD98059) and ER restored, suggesting that the sensitivity to endocrine therapy can be re-established by disruption of the MAPK signaling cascade. This can also be achieved by disruption of the HER2 pathway. Trastuzumab is a monoclonal antibody against the extracellular domain of HER2 and is currently used in the clinic for patients with HER2-positive breast cancer pathway w16x. In letrozole-resistant cells, trastuzumab treatment dramatically inhibits the proliferation of LTLT cells. Trastuzumab by inhibiting p-HER2 and p-MAPK expression restored ERa expression and E2 sensitivity to LTLT cells w4x. LTLT cells are insensitive to growth stimulation by E2 w8x. However, when pretreated with trastuzumab (100 mg/mL) for 72 h, LTLT cells exhibited a marked stimulation in proliferation with relatively low concentration of E2. Transcriptional activation of ERa was induced in LTLT cells to a similar extent as in parental MCF-7Ca cells stimulated with E2. To investigate whether inhibition of HER2 could be utilized as a strategy to reverse resistance to AI therapy, we used our xenograft model. Following long-term treatment with letrozole, trastuzumab was added at week 16 when tumors started to double in size w4x. The addition of trastuzumab to letrozole at the time of tumor progression resulted in significantly prolonged tumor suppression when compared with either trastuzumab or letrozole alone (Figure 1). The addition of trastuzumab resulted in significant downregulation of HER2 and p-MAPK, as well as restoration of ERa signaling path-

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ways are required to reverse resistance to estrogen signaling and for prolonged tumor suppression. Consistent with this finding, trastuzumab alone did not have antitumor activity in the parental endocrine responsive MCF-7Ca as ER expression was increased and unchallenged. The combination of

trastuzumab and letrozole from the start of the experiment where tumors were not resistant could not prolong or avert development of the resistance w4x. Recent clinical data support our preclinical findings. Lipton et al. found that approximately 26% of patients treated

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.

Figure 1 (A) The effect of trastuzumab alone or in combination with letrozole on the growth of MCF-7Ca xenografts. Trastuzumab (5 mg/kg/week) did not inhibit the growth of MCF-7Ca tumors, whereas the combination was highly effective. The difference in the exponential variable governing growth rate of control vs. trastuzumab treatment was 0.02"0.14 (ps0.86); trastuzumab vs. trastuzumab plus letrozole was 0.49 (ps0.0001); trastuzumab vs. letrozole was 0.32 (ps0.0009); letrozole vs. letrozole switched to letrozole plus trastuzumab was 0.21"0.08 (ps0.008); letrozole plus trastuzumab vs. letrozole switched to letrozole plus trastuzumab was 0.39"0.09 (p-0.0001); letrozole switched to trastuzumab vs. letrozole switched to letrozole plus trastuzumab was 0.2"0.08 (ps0.011) over weeks 15–28. When compared with week 29, the difference in the exponential variable governing growth rate of letrozole vs. letrozole switched to trastuzumab was 0.005"0.08 (ps0.97). (B) Effect of trastuzumab on the tumor weight of the mice bearing MCF-7Ca xenografts. The mean tumor weight of trastuzumab-treated mice was 2.68"0.57 g, which was not significantly different from those of the D4A-treated mice (1.37"0.57 g; ps0.14). The tumor weights of other groups are not compared owing to differences in the time of termination. (C) Effect of trastuzumab on the uterine wet weight of mice bearing MCF-7Ca xenografts. The average weight of the atrophic uterus in ovariectomized mice is -10 mg; the greater uterine weight of mice receiving D4A (22.42"0.92 mg) indicates that aromatase in the tumors is producing enough estrogens to maintain the uterine weight similar to intact mice in diestrus. When mice were treated with trastuzumab, the uteri weighed significantly more (74.8"0.92 mg) than D4A-treated mice (Wilcoxon rank sum test, two-sided exact ps0.008). The uterus weights of other groups are not compared owing to differences in the time of termination. (D) Effect of trastuzumab and letrozole alone or in combination on protein expression of ERa, Her-2, MAPK, and CYP-19 in MCF-7Ca xenografts. Expression of proteins was examined using Western immunoblotting as described in the materials and methods section. Blot shows ERa at 66 kDa, Her-2 at 185 kDa, p-MAPK and MAPK at 42–44 kDa, CYP-19 at 55 kDa, and b-actin at 45 kDa. The blots show a single representative of three independent experiments. The blots were stripped and reprobed for b-actin to verify equal loading w5x.

with letrozole converted from serum HER2-negative to positive at the time of disease progression w17x. The extracellular domain of HER2 protein can be detected in the peripheral blood and has been demonstrated to correlate with the overexpression of HER2 protein in tumor cells w18x. In a neoadjuvant study, HER2 levels increased during AI treatment in 18 out of 26 patients and in 15 out of 21 respondents w19x. Several clinical trials have confirmed the benefit of targeting both ER and HER2 pathways. A Phase II trial of letrozole and trastuzumab in ER-positive/HER2-negative metastatic breast cancer patients demonstrated that the combination was well tolerated with a clinical benefit rate of 50% w20x. A subsequent Phase III trial (TANDEM trial) of anastrozole in combination with trastuzumab demonstrated significant improvement in progression free survival (PFS) with addition of trastuzumab (3.8 vs. 5.6 months; ps0.0059) w21x. Moreover, a recent randomized Phase III trial of letrozole in combination with lapatinib, an oral dual tyrosine kinase inhibitor of HER2 and EGFR, also demonstrated a significant benefit of adding lapatinib to letrozole with a PFS of 8.2 vs. 3.0 months wHazard ratio (HR) of 0.71; ps0.019x w22x. However, the benefit only appears to be in the group of patients with HER2 overexpression. Nonetheless, the preplanned Cox regression analysis of patients with HER2-negative tumors who relapse less than 6 months after tamoxifen discontinuation demonstrated a non-significant trend toward improvement in PFS for the combination group (HR 0.78; ps0.117) w22x. This result supports our preclinical finding that the upfront combination of AI with anti-HER2 therapy does not prolong or avert the resistance but the combination is more beneficial where HER2 is expressed and at the time of the resistance. Innate resistance to aromatase inhibitors in ER-negative tumors

It is well known that breast cancers that lack expression of ER or PR do not respond to endocrine therapy. Recently, the

American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) recommended that ER and PR tests should be considered as positive if there are at least 1% positive tumor nuclei in the breast cancer w23x. Nevertheless, one-third of breast cancers are intrinsically resistant to endocrine therapy including AIs. Previous studies have demonstrated that expression of the ERa occurs via epigenetic mechanisms. It has been demonstrated that ERa is downregulated in ERa-negative breast cancer. Unlike healthy normal breast tissue, the CpG island of the ERa promoter has been found to be hypermethylated in all of the ER-negative breast cancer cell lines and tumors w24, 25x. However, the expression levels of functional ERa can be restored in ERa-negative breast cancer cells with a DNA methyltransferase (DNMT) inhibitor, 5-azacytidine w26x. Furthermore, the expression of ERa transcription is increased by 300- to 400-fold when combining a histone deacetylase inhibitor (HDACi), Trichostatin A, with 5-azactidine w27x. However, cytotoxicity limits its clinical use. Nevertheless the same study showed that ERa expression can also be restored via HDAC inhibitors and that the ERa is functional. More recently, we have shown that HDACis can be used to induce not only ER but also aromatase and that ER/PR-negative cancer cells can be reprogrammed by epigenetic modulators to render them sensitive to aromatase inhibitors. Expression of ERa protein is undetectable in MDA-MB-231 cells by Western blotting and no significant binding of E2 occurs without ENT treatment. In addition, growth of MDA-MB-231 cells was not inhibited by AEs or AIs nor stimulated by estrogen. After treatment with 10 nM of ENT, expression of ERa protein was upregulated 8-fold and expression of aromatase was upregulated 2.6-fold with ENT. Treatment with ENT resulted in upregulation of ERa and aromatase mRNA in a time-dependent manner. To test whether these findings can be recapitulated in vivo, we used a mouse xenograft model with ER-negative tumors. As shown in Figure 2, the combined treatment of ENT and letro-

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Figure 2 Effect of ENT alone or in combination with letrozole on the growth of ER-negative MDA-MB-231 xenografts. The mice bearing MDA-MB-231 xenografts were treated with ENT alone or in combination with letrozole. Mice receiving the combined treatment were pretreated for 3 days before starting letrozole. Tumor volumes were measured twice a week. Tumor growth rate of the mice in the combination group was significantly lower than control (ps0.004), single agent ENT (ps0.009) or letrozole (ps0.049) w4x.

zole resulted in almost complete inhibition of tumor growth. The ENT plus letrozole treatment significantly inhibited tumor growth compared with the control, ENT or letrozole treatment alone. At necropsy, the mean tumor weight of mice treated with ENT plus letrozole was also significantly lower than the control (p-0.001), ENT or letrozole groups (p0.01). These results suggest that aromatase activity induced by ENT was blocked by letrozole, resulting in little or no estrogen production. Reduction in uterine weight in the ENT plus letrozole treated mice supports this conclusion. Western blot analysis of the MDA-MB-231 tumors confirmed that in ENT-treated tumors, protein expression of ERa, PR and aromatase was upregulated. The tumors of mice treated with ENT plus letrozole had increased levels of aromatase protein compared with controls and ERa/PR levels were higher than with letrozole alone. Furthermore, ENT treatment significantly increased intratumoral aromatase activity in comparison to controls (p-0.0001), which was markedly inhibited by treatment with ENT plus letrozole compared with ENT alone. Similar responses were also seen in tumors of a second ER-negative cell line, Hs578T w28x. ERa is also known to function in pathways not related to transcriptional regulation. ERa was recently shown to interact with HDAC6 to deacetylate tubulin, allowing breast cancer cells to have greater mobility w29x. This suggests that it might be possible for HDACi and AI combinational therapy to prevent the motility of cellular migration and subsequent metastases. We observed that mice receiving both entinostat and letrozole exhibited a markedly lower frequency of metastasis to the lungs w30x. The mice were injected with MDA-MB-231 cells via the tail vein. Colonization of cells

into the lungs resulted in visible or microscopic tumor foci in six out of six control mice with an average of 60 visible metastases and 15 micrometastases per animal. Five out of six mice in the ENT group had visible metastases (;25 on average) and micrometastases (;13). Three out of 6 mice in the letrozole group had visible metastases (;20) and micrometastases (;7). However, in the group treated with ENTqletrozole, only one out of six mice showed one visible and two micrometastases in the lungs. These results provide strong evidence that the combination of ENT plus letrozole not only inhibits tumor proliferation but markedly inhibits growth of tumor foci in the lungs. This suggests that metastases could also be inhibited by the combined treatment.

Conclusions Our preclinical data as well as those of others demonstrate that crosstalk between ER and other signaling pathways particularly MAPK and PI3k/Akt are key resistant mechanisms of ER-positive tumors contributing to acquired resistance to hormone therapy. Inhibiting both estrogen and growth factor signaling appears to be an effective strategy to overcome resistance to AI therapy in breast cancer. Several studies suggest that HER2-negative tumors can convert to HER2-positive in patients treated with AIs w17, 30x. Clinical trials are now in progress to evaluate therapies that target the mechanisms of resistance to AIs. These agents include HER2 inhibitors, MEK inhibitors, Raf inhibitors, PI3K inhibitors, mTOR inhibitors and Akt inhibitors. If effective, this strategy could extend the benefits of AIs in these patients.

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ERa-negative breast cancers remain difficult to manage and treatment options are limited to chemotherapy because the tumors are more aggressive and resistant to endocrine therapy. Genetic alternations such as mutations, loss of heterozygosity or homozygous deletions are rare. Our studies in ER-negative tumors demonstrate that the HDACi, ENT, induces expression of functional ERa and aromatase activity, thereby converting ER-negative tumors to ERa-positive tumors. These tumors are sensitive to the growth stimulatory effects of estrogens produced locally by the aromatization of androstenedione in the tumor. When the HDACi ENT was combined with AI letrozole, the tumors were deprived of estrogen, preventing ER regulated gene transcription that support tumor growth and tumor cell colonization in the lungs. The detailed molecular mechanism of the conversion of ER-negative breast cancer cells from hormone-independent to hormone-dependent cells expressing ER and aromatase is unknown at this time. However, it suggests phenotypic plasticity of the tumor cells that enables them to adapt to changes in their microenvironment. Our studies show that methods that restore the ER and aromatase in the tumor can reverse resistance to AIs. These novel strategies could improve treatment and delay the need for cytotoxic chemotherapy not only for patients with acquired resistance but also for those with innate resistance.

References 1. Burstein HJ, Prestrud AA, Seidenfeld J, Anderson H, Buchholz TA, Davidson NE, Gelmon KE, Giordano SH, Hudis CA, Malin J, Mamounas EP, Rowden D, Solky AJ, Sowers MR, Stearns V, Winer EP, Somerfield MR, Griggs JJ. American Society of Clinical Oncology Clinical Practice Guideline: update on adjuvant endocrine therapy for women with hormone receptor-positive breast cancer. J Clin Oncol 2010;28:3784–96. 2. The American Cancer Society A, Georgia. Breast cancer facts & figures 2009–2010. 3. Jelovac D, Sabnis G, Long BJ, Macedo L, Goloubeva OG, Brodie AM. Activation of mitogen-activated protein kinase in xenografts and cells during prolonged treatment with aromatase inhibitor letrozole. Cancer Res 2005;65:5380–9. 4. Sabnis G, Goloubeva O, Jelovac D, Schayowitz A, Brodie A. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway improves response of long-term estrogen-deprived breast cancer xenografts to antiestrogens. Clin Cancer Res 2007;13:2751–7. 5. Sabnis G, Schayowitz A, Goloubeva O, Macedo L, Brodie A. Trastuzumab reverses letrozole resistance and amplifies the sensitivity of breast cancer cells to estrogen. Cancer Res 2009; 69:1416–28. 6. Sabnis GJ, Macedo LF, Goloubeva O, Schayowitz A, Brodie AM. Stopping treatment can reverse acquired resistance to letrozole. Cancer Res 2008;68:4518–24. 7. Sabnis G, Goloubeva O, Gilani R, Macedo L, Brodie A. Sensitivity to the aromatase inhibitor letrozole is prolonged after a ‘‘break’’ in treatment. Mol Cancer Therap 2010;9:46–56. 8. Sabnis GJ, Macedo L, Goloubeva O, Schayowitz A, Zhu Y, Brodie A. Toremifene-atamestane; alone or in combination: predictions from the preclinical intratumoral aromatase model. J Steroid Biochem Mol Biol 2008;108:1–7.

9. Sabnis GJ, Jelovac D, Long B, Brodie A. The role of growth factor receptor pathways in human breast cancer cells adapted to long-term estrogen deprivation. Cancer Res 2005;65:3903– 10. 10. Long BJ, Jelovac D, Handratta V, Thiantanawat A, MacPherson N, Ragaz J, Goloubeva O, Brodie AM. Therapeutic strategies using the aromatase inhibitor letrozole and tamoxifen in a breast cancer model. J Natl Cancer Inst 2004;96:456–65. 11. Kazi AA, Jones JM, Koos RD. Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogeninduced recruitment of both estrogen receptor alpha and hypoxia-inducible factor 1 to the vascular endothelial growth factor promoter. Mol Endocrinol 2005;19:2006–19. 12. Jelovac D, Macedo L, Goloubeva OG, Handratta V, Brodie AM. Additive antitumor effect of aromatase inhibitor letrozole and antiestrogen fulvestrant in a postmenopausal breast cancer model. Cancer Res 2005;65:5439–44. 13. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995;270:1491–4. 14. Joel PB, Traish AM, Lannigan DA. Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase. J Biol Chem 1998;273:13317–23. 15. Chen D, Washbrook E, Sarwar N, Bates GJ, Pace PE, Thirunuvakkarasu V, Taylor J, Epstein RJ, Fuller-Pace FV, Egly JM, Coombes RC, Ali S. Phosphorylation of human estrogen receptor alpha at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene 2002;21:4921–31. 16. Hudis CA. Trastuzumab – mechanism of action and use in clinical practice. N Engl J Med 2007;357:39–51. 17. Lipton A, Leitzel K, Ali SM, Demers L, Harvey HA, ChaudriRoss HA, Evans D, Lang R, Hackl W, Hamer P, Carney W. Serum HER-2/neu conversion to positive at the time of disease progression in patients with breast carcinoma on hormone therapy. Cancer 2005;104:257–63. 18. Lipton A, Ali S, Leitzel K. HER-2 protein expression predicts response to trastuzumab in FISH-positive patients with metastatic breast cancer. In: 31st AACR-CTRC San Antonio Breast Cancer Symposium 2008, San Antonio, TX, USA, 2008. 19. Fla˚geng MH, Moi LL, Dixon JM, Geisler J, Lien EA, Miller WR, Lonning PE, Mellgren G. Nuclear receptor co-activators and HER-2/neu are upregulated in breast cancer patients during neo-adjuvant treatment with aromatase inhibitors. Br J Cancer 2009;10:1253–60. 20. Marcom PK, Isaacs C, Harris L, Wong ZW, Kommarreddy A, Novielli N, Mann G, Tao Y, Ellis MJ. The combination of letrozole and trastuzumab as first or second-line biological therapy produces durable responses in a subset of HER2 positive and ER positive advanced breast cancers. Breast Cancer Res Treat 2007;102:43–9. 21. Mackey J, Kaufman B, Clemens M. Trastuzumab prolongs progression-free survival in hormone-dependent and HER2-positive metastatic breast cancer. Breast Cancer Res Treat 2006; 100(Suppl 1):abstract 3. 22. Johnston S, Pippen J Jr, Pivot X, Lichinitser M, Sadeghi S, Dieras V, Gomez HL, Romieu G, Manikhas A, Kennedy MJ, Press MF, Maltzman J, Florance A, O’Rourke L, Oliva C, Stein S, Pegram M. Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hor-

Article in press - uncorrected proof Brodie et al.: Reversing aromatase inhibitor resistance 103

23.

24.

25.

26.

mone receptor-positive metastatic breast cancer. J Clin Oncol 2009;27:5538–46. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol 2010;28:2784–95. Ottaviano YL, Issa JP, Parl FF, Smith HS, Baylin SB, Davidson NE. Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res 1994;54:2552–5. Lapidus RG, Nass SJ, Butash KA, Parl FF, Weitzman SA, Graff JG, Herman JG, Davidson NE. Mapping of ER gene CpG island methylation-specific polymerase chain reaction. Cancer Res 1998;58:2515–9. Ferguson AT, Lapidus RG, Baylin SB, Davidson NE. Deme-

27.

28.

29.

30.

thylation of the estrogen receptor gene in estrogen receptornegative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res 1995;55:2279–83. Yang X, Phillips DL, Ferguson AT, Nelson WG, Herman JG, Davidson NE. Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells. Cancer Res 2001;61:7025–9. Sabnis G, Goloubeva O, Chumsri S, Nguyen N, Sukumar S, Brodie A. Functional activation of the estrogen receptor-a and aromatase by the HDAC inhibitor, entinostat, sensitizes of ERnegative tumors to letrozole. Cancer Res 2011;71:1893–903. Azuma K, Urano T, Horie-Inoue K, Hayashi S, Sakai R, Ouchi Y, Inoue S. Association of estrogen receptor alpha and histone deacetylase 6 causes rapid deacetylation of tubulin in breast cancer cells. Cancer Res 2009;69:2935–40. Munzone E, Curigliano G, Rocca A, Bonizzi G, Renne G, Goldhirsch A, Nole F. Reverting estrogen-receptor-negative phenotype in HER-2-overexpressing advanced breast cancer patients exposed to trastuzumab plus chemotherapy. Breast Cancer Res 2006;8:R4.

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Copyright of Hormone Molecular Biology & Clinical Investigation is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.