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ONCOLOGY Molecular Mechanisms of Hormone Resistance of Breast Cancer
A. M. Scherbakov, M. A. Krasil’nikov, and N. E. Kushlinskii Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 155, No. 3, pp. 363-376, March, 2013 Original article submitted January 11, 2013 More than 70% malignant mammary tumors contain steroid hormone receptors; this suggests the possibility of hormone therapy in the majority of patients with breast cancer (BC). The main cause of inefficiency of hormone therapy in BC is hormone resistance (tumor resistance to hormonal cytostatics). Here we discuss the main mechanisms of hormone resistance of BC and the mechanisms underlying the formation of hormone resistance of the tumors are analyzed at the molecular level. The data on the signal pathways of estrogen receptors (ER), the key regulators of BC cell proliferation, are presented. The most important factors of BC hormone resistance are: high activity/expression of receptor tyrosine kinases; high activity of proteins regulating cell defense mechanisms (Akt PI3K, mTOR); changes in the activities of cell cycle regulator proteins (Myc, c-Fos, Cyclin D1). Our experiments have demonstrated that estrogen-independent BC cell growth is supported by VEGF/VEGFR2 and EGF/EGFR mitogenic signal pathways. Our data indicate that NF-B transcription factor is directly involved in the regulation of hormone-resistant BC cell growth and survival, while NF-B suppression determines cell sensitivity to apoptotic activity of antitumor compounds. On the whole, the results indicate good prospects of using EGFR, HER-2/neu, mTOR, VEGFR, PI3K/Akt molecular pathways as targets for BC therapy, including therapy for BC resistant forms. Key Words: breast cancer; tamoxifen; hormone resistance; estrogen receptor ; estrogens Hormone resistance (tumor resistance to the effects of hormonal cytostatics) is the main cause of inefficiency of endocrine therapy for breast cancer (BC) [31,84,92]. Hormone resistant tumors can be divided into 2 groups: tumors with primary and acquired hormone resistance [21,91]. Tumors with the biological mechanism of hormone resistance formed de novo before therapy are referred to group 1. Group 2 includes tumors which lost sensitivity to the cytostatic effects N. N. Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, Moscow, Russia. Address for correspondence:
[email protected]. A. M. Scherbakov
of antiestrogens during endocrine therapy [21,91]. The emergence of primary and acquired hormone resistance in BC patients is caused by molecular mechanisms maintaining malignant cell growth [21,91]. Progress in understanding of BC hormone resistance mechanisms has been attained due to active research of steroid hormones and their role in the maintenance of tumor cell growth [21,92]. The effect of steroid hormones on the cell is mediated by receptors [21,43]. Two types of estrogen receptors (ER) are known by the present time: ER (ESR1 gene product) and ER (ESR2 gene product); BC cells produce both receptor forms [21,78,120]. ER and ER are char-
0007-4888/13/15530384 © 2013 Springer Science+Business Media New York
A. M. Scherbakov, M. A. Krasil’nikov, and N. E. Kushlinskii
acterized by high homology [78], but their role in BC cell growth regulation is different: the proliferative signal reaches the cell through ER, while ER seems to be involved in tumor growth inhibition [21,114]. Functional characteristics of ER are still poorly studied. We shall discuss here mainly the characteristics and functions of ER (further called estrogen receptors, or ER); the data on the presence of ER in the cells will be specified, if necessary. Estrogen receptors are nuclear transcription factors involved in the genome and other than genome interactions (Fig. 1). First, ER bind to estrogen-sensitive elements (ERE) in the target gene promoters (so-called “classical” genome ER signal pathway); second, ER regulate activities of transcription factors AP-1, SP-1, c-Fos [21,25]; this interaction is called “nonclassical” genome signal pathway of ER. In addition, ER are involved in nongenomic interactions by forming complexes with other signal proteins: PI3K, Akt, S6K, ERK1, ERK2, p38 MAPK, IGF1-R, Src [21,91]. These aspects of ER regulation have been intensely studied in recent years [76,92]. The genomic and genomic ER signal pathways form a complex multistep system of reactions. Inefficiency of tamoxifen in many cases is not associated with the absence of ER, the main target of this drug, or its loss in the course of therapy. This is confirmed by the results of clinical studies: ER is lost in just a small (17-28%) group of patients with acquired tamoxifen resistance [38,49]; about 80% hormone resistant mammary tumors (contralateral BC) retain ER after a course of tamoxifen adjuvant therapy [4]. In vitro studies have shown that tamoxifen-resistant MCF-7/LCC9 cells do not lose ER [14]. Our in vitro studies have also shown that the development of tamoxifen resistance in MCF-7 human BC cells is not associated with complete loss of ER expression: just a slight reduction of ER has been found in a resistant cell substrain [102]. Hence, the receptor-positive (ER+) phenotype of tumor cells is rather prevalent in hormone-resistant BC. Mechanisms of hormone resistance of BC. What mechanisms help the cell acquire resistance to tamoxifen and other antiestrogens? Probable changes in tumor cell metabolism, leading to complete or partial loss of estrogen dependence, have been described [7,21]. The following mechanisms of hormone resistance can be distinguished: • ligand-independent activation of ER signal pathways (ER activation by other signal proteins) [77]; • disorders in ER expression; • mutations in the ER functional activation domains [21]; • imbalance of ER activator proteins and suppressor proteins, disorders in ER interactions with activator proteins [56];
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Fig. 1. Molecular pathways of estrogen receptors. 1) ER “classical” genome signal pathway; 2) ER “nonclassical” genome signal pathway; 3) nongenomic interactions of ER with receptor tyrosine kinase signal proteins (RTKSP). TF: transcription factor.
• activation of estrogen-independent signal pathways in the tumor cell: growth factor (FGFR, EGFR, HER-2/neu) signal pathways [7,36,82,106], signal pathways of phosphatidylinositol-3-kinase (PI3K), the enzyme playing an important role in regulation of tumor cell growth, resistance to destructive factors, and survival, MAP-kinase cascades (MAPK), mTOR (mammalian target of rapamycin), the enzyme regulating protein synthesis and proliferation, some antiapoptotic proteins (Akt, NF-B [84,121]); • disorders in regulation of the epithelial-mesenchymal cell transition (Snail1, MTA3, E-cadherin) [44,100]; • cell hypersensitivity to some antiestrogens (for example, SERM group drugs) as estrogen agonists [9,21]; • emergence of BC cell hypersensitivity to estrogens [9,98]. Many signal cellular pathways are involved in the development of hormone resistance, and high level of the tumor genetic system plasticity and heterogeneity impede the detection of all regularities in the changes in tumor cell resistance to antiestrogens. The situation is aggravated by coordinated regulation of factors essential for tumor cell growth, treatment resistance, and tumor cell survival and by side effects of hormonal drugs and their estrogen agonist activity. Antiestrogens and signal molecule inhibitors in BC. The content of steroid hormone receptors (ER and PR, progesterone receptors) [45] is the main criterion for predicting the sensitivity of BC patients to antiestrogens (for example, tamoxifen). According to different authors, about 70% malignant mammary tumors contain steroid hormone receptors, this making possible hormone therapy in the majority of BC patients.
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Importantly that more than 75% tumors of patients with positive receptor status (ER+/PR+) are initially sensitive to tamoxifen therapy. As a rule, a high percentage of patients in this group objectively respond to the drug in adjuvant and nonadjuvant therapy for BC [42]. About 34% only estrogen receptor-positive tumors (ER+/PR–) and 45% tumors with the ER–/PR+ phenotype are sensitive to tamoxifen [42]. Less than 10% tumors of patients with the negative receptor status (ER–/PR–) are sensitive to tamoxifen therapy, but many scientists attribute this fact to probable falsenegative data of analysis or acquired ER–/ER+/PR– phenotype with persisting temporary sensitivity to the drug (according to Early Breast Cancer Trialists’ Collaborative Group [122]. In addition, some authors claim that the ER–/ER+/PR– phenotype is associated with the most aggressive tumor forms and unfavorable prognosis [24]. Despite the fact that about 70% mammary tumors contain steroid hormone receptors, tamoxifen therapy is highly effective only in locally disseminated BC, the 5-year survival reaching 95%. In disseminated BC the 5-year survival remains low (20-30%). About 40% patients with metastatic BC, who received tamoxifen as the first-line endocrine therapy drug, develop relapses. The signal pathways, that help the tumor cell overcome the cytostatic effects of an antiestrogen, contribute to the disease progress. Studies of the potentialities of combined therapy are now in progress: these are protocols aimed at regulation of estrogen effects on BC (SERM and SERD group drugs, aromatase inhibitors) and inhibition of signal molecules – EGFR (gefitinib/”Iressa”, erlotinib/”Tartseva”, EKB569 drugs), HER-2/neu (trastuzumab/”Herceptin”), PI3K/Akt (LY294002), mTOR (everolimus/”Afinitor”, temsirolimus/”Torisel”), IGFR-1 (AG1024), Raf (BAY43-9006), MEK 1/2, Src, CDK, NF-B [8,84,91,92]. Estrogen regulation of BC growth is an intricate mechanism of signal transmission via a ramified molecular system. It is currently accepted that numerous proteins of different signal pathways are involved in the modulation of estrogen effect on tumor cells: proteins of the classical genome estrogen pathway (ER, ER, Hsp family proteins, etc.), proteins of the nonclassical genome estrogen pathway (AP-1, SP-1, c-Fos), “nonhormonal” signal pathways (PI3K, Akt, S6K, ERK1, ERK2, p38 MAPK, IGF1-R, etc.). The result of these multistep cascades is tumor cell response to the hormone: activation or suppression of the expression of estrogen specific target genes, modification of activities of molecules regulating the cell cycle, etc. Branching and entangled estrogen signal pathways often determine inefficiency of endocrine therapy in BC patients: tumor cell has many ways to overcome the cytostatic effect of antiestrogens,
i.e. become resistant, and hence, new approaches to combined exposure of BC (aimed at various target molecules) are particularly promising. Let us analyze in detail the main signal pathways determining BC resistance to hormone therapy. The EGFR signal pathway. An appreciable part of in vitro BC models of acquired hormone resistance, obtained in various research centers, including N. N. Blokhin Oncological Center, demonstrate activation of signal pathways of the EGFR receptor tyrosine kinase family [13,21,91,92,102]. The EGFR family consists of four glycoproteins, v-ErbB viral oncogene homologs: ErbB-1 (EGFR), ErbB-2 (HER-2/neu), ErbB-3 (HER-3), and ErbB-4 (HER-4). The EGFR family proteins are membrane receptors, consisting of ligand-binding extracellular domain, transmembrane part, and intracellular tyrosine kinase [70]. Tyrosine kinase is activated by ligand interactions with ligand-binding domain and subsequent dimerization of receptors. One exclusion is HER-2/neu protein with low affinity for ligands; it is activated in heterodimerization with other EGFR family proteins [40,70]. The EGFR family receptors regulate the activities of various signal pathways, controlling cellular defense mechanisms, proliferation, migration, and differentiation [92,105] (Fig. 2). High expression of EGFR family proteins and/ or their constitutive activation promote tumor growth, neoangiogenesis, and is as a rule associated with an unfavorable prognosis of BC [70,96]. Numerous modern experimental and clinical studies have been focused on two representatives of this family: EGFR (ErbB-1) and HER-2/neu proteins [38,39,70]. The EGFR protein is involved in regulation of various molecular pathways in the cells. It is assumed that the main sublying effector molecules of EGFR signal pathway are PI3K (enzyme playing an important role in the regulation of cell growth, resistance to destructive factors, and survival), Ras (a group of
Fig. 2. EGFR signal pathways.
A. M. Scherbakov, M. A. Krasil’nikov, and N. E. Kushlinskii
minor guanosine triphosphatases, regulating cell proliferation), mTOR (enzyme regulating protein synthesis and proliferation), a group of transcription factors STAT, MAPK, etc. [40,105]. Activation of the EGFR signal pathway in tumor cells leads to changes in the activities of the cell cycle main regulator proteins, Myc, c-Fos, Cyclin D1, increases proliferation rate for cells of different origin [70,88] and, hence, reduces their sensitivity to cytostatics, specifically, to antiestrogens [75] (Fig. 2). The development of MCF-7 BC cells to Faslodex (fulvestrant) antiestrogen is paralleled by an increase of EGFR expression and MAPK activation [75]. Faslodex resistance is reversible and is observed only during cell incubation with the drugs and during the first 3 weeks after its discontinuation [75]. Recovery of Faslodex sensitivity is associated with decrease of EGFR expression [75]. The growth of Faslodex-resistant cells is effectively suppressed by specific inhibitors of EGFR (Iressa) and MAPK (PD098059) [75]. Activation of EGFR during the development of hormone resistance leads to increase of ER phosphorylation [13]. It has been shown on tamoxifenresistant MCF-7/Tam-R BC cells that ER phosphorylation by serine-118 is regulated by EGFR and MAPK [13]. The transcription activity of ER and its binding to two p68 coactivators (RNA helicase) and Src in MCF-7 in MCF-7/Tam-R cells also depend on EGFR/ MAPK signal pathway [13]. The EGFR protein family maintains hormone resistance of BC cells by the autocrine mechanism [55]. The development of resistance to tamoxifen in BC cells is associated with an increase of expression of TGF – one of the main EGFR ligands. Phosphorylation of EGFR-HER-2/neu and EGFR-ErbB-3 heterodimers and their effectors MAPK increases in tamoxifen-resistant BC cells [55]. Hence, intensive synthesis of TGF and subsequent activation of EGFR family proteins lead to the formation of the autocrine mechanism supporting BC growth and cell resistance to tamoxifen [55]. Experimental data on the important role of EGFR protein in the development of BC resistance to endocrine therapy and hence, efficiency of anti-EGFR therapy in the group of resistant patients have been confirmed in recent clinical studies [23,39,83]. The study [39] was carried out in a group of BC patients (28 patients with acquired resistance to tamoxifen with ER+ tumors and 26 ones with ER– tumors). All patients received EGFR inhibitor Iressa in a daily dose of 500 mg. Objective response to therapy or disease stabilization were observed in 33.3% patients (53.6% and 11.5% in ER+ and ER– subgroups, respectively) [39]. The therapeutic efficiency in the studied patients correlated with reduction of Ki67 proliferation marker
387 level and lesser phosphorylation of EGFR and MAPK in the tumor [39]. Phase II of clinical studies of combined protocol (Iressa+tamoxifen) efficiency was over in 2011 [83]. The study was carried out in 206 patients with receptor-positive disseminated BC. The relapse-free survival median in the combined therapy group was 23% longer (10.9 months) than in the group receiving tamoxifen monotherapy (8.8 months) [83]. High efficiency of target inhibition of EGFR signal pathway in BC patients receiving endocrine therapy is confirmed in one more report [23]. A group of 43 BC patients received anastrosole and Iressa, 50 patients received anastrosole and placebo. The relapsefree survival median in the patients receiving Iressa was 75% longer than in the placebo group and reached 14.7 months [23]. Hence, a combination of endocrine therapy for BC with EGFR inhibitors is an effective approach to overcoming the tumor hormone resistance. Proteins HER-2/neu and EGFR are highly homologous [70,92] and activate common signal pathways, which fact determines their similar roles in development of resistance to endocrine therapy for BC [84]. Experiments on MCF-7 cells transfected with HER-2/neu demonstrated that HER-2/neu inhibition by tyrphostin (AG1478) restored cell sensitivity to tamoxifen [59]. Recent clinical studies of combined use of HER2/neu signal pathway inhibitors and endocrine therapy for BC demonstrate good prospects of this approach. High efficiency of therapy in groups of patients with ER/HER-2+ tumors is proven for combinations with anastrosole+Herceptin, letrosole+Herceptin, letrosole+lapatinib [53,74,103]. Inhibition of EGFR and HER-2/neu signal pathways in cases with development of resistance to endocrine therapy for BC is sufficiently well studied. The main efforts of scientists are aimed at the development of effective protocols for combined therapy and search for new specific inhibitors of these molecular pathways. FGFR signal pathway. The role of FGFR family proteins in the development of BC hormonal resistance is far less studied. In contrast to EGFR and HER-2/neu signal pathways, whose role in resistance is realized mainly at the expense of high activity/expression of tyrosine kinases, for FGFR molecular pathway the main mechanism maintaining tumor resistance to hormonal cytostatics is gene amplification [117]. Amplification of FGFR1 gene is detected in about 9% BC cases and, according to some data [26], is a poor prognostic factor in ER+ BC status. Tamoxifen resistance of BC cells with FGFR1 amplification has been demonstrated [117]; tamoxifen sensitivity is restored by suppressing FGFR1 expression by specific siRNA (small interfering RNA).
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Clinical studies of the efficiency of combined therapy with endocrine drugs and FGFR signal pathway inhibitors in BC have started in 2010 (# NCT01202591 in the National Institutes of Health database). The study is carried out in postmenopausal patients with ER+ BC; the efficiency of FGFR specific inhibitor AZD4547 in combination with aromazine in comparison with aromazine+placebo protocol is going to be analyzed. The main results will be published in 2014. VEGF signal pathway. The synthesis of VEGF (vascular endothelial growth factor, VEGF-A), the key factor of neoangiogenesis – is regulated by several factors, the central of which is hypoxia [29,52]. Transcription of VEGF gene, induced by hypoxia, is mediated by HIF-1 transcription factor, triggering activation cascades of the main proangiogenic factors in tumors [57,81]. Estrogens and antiestrogens also play an important role in VEGF synthesis regulation, which fact is particularly interesting for studies of the mechanisms of BC hormone resistance development. The MCF-7 BC cells culturing with estradiol causes an increase of VEGF mRNA synthesis and accumulation of VEGF in culture medium [93]. Addition of Faslodex (antiestrogen) to culture medium inhibits estrogen-induced synthesis of VEGF, this indicating the involvement of estrogen receptors in regulation of VEGF expression. On the other hand, antiestrogens (tamoxifen and toremifene) do not inhibit estrogeninduced synthesis of VEGF in MCF-7, but induce a paradoxical increase of VEGF mRNA synthesis. This study of VEGF hormonal regulation has been continued [11]: hormonal regulation of VEGF has been studied in vivo (on mammary tumors of CD1-NU mice), ex vivo (on mouse tumor preparations), and in vitro (on MCF-7 MC cells). A 2-week tamoxifen course has caused an increase of microcapillary permeability in mammary tumors. Increase of vascular permeability, in turn, is one of the main physiological effects of VEGF. Immunohistochemical analysis has shown a 37-fold higher level of VEGF protein in tamoxifentreated tumors in comparison with estrogen-treated ones. The final stage of this study has detected a reduction of VEGF mRNA level in MCF-7 cells cultured with estradiol (which contradicts the data obtained by [93] and [61]), while tamoxifen has failed to change VEGF mRNA level (also contradicting the data on the stimulatory effect of tamoxifen on VEGF level [93]). Hence, the data on the effects of estrogens and antiestrogens on VEGF synthesis are ambiguous. Attempts at summing up the data on tamoxifen regulation of VEGF expression in BC have been made [35]. Some previous studies [11,93] indicate a stimulatory effect of tamoxifen on VEGF synthesis in receptor-positive BC. Clinical studies have demonstrated a relationship between VEGF level and efficiency of
hormone therapy in BC patients (high level of VEGF correlating with inefficiency of first-line hormone therapy in BC [72]). Another study [35] has shown that intracellular and extracellular concentrations of VEGF in BC are differently regulated by tamoxifen: the drug increases intracellular VEGF content (acting as estrogen agonist) and reduces VEGF concentration in culture medium in vitro, and also reduces the extracellular content of this protein in vivo in nude mice with MCF-7 tumors. This model partially explains the relationship between VEGF level and hormone therapy efficiency. The authors suggest using in situ measurement of extracellular tumor VEGF level (microdialysis analysis) in a clinical setting as an additional method for evaluation of VEGF biological activity. Let us note once more than tamoxifen reduces extracellular VEGF level in BC (and it is the extracellular content that reflects the proangiogenic biological activity of VEGF). Some characteristics of molecular mechanism of the VEGF synthesis hormone regulation have been described [79]. Normally and in human endometrial tumor cells the hormone regulation of VEGF synthesis is mediated by ERE sites (estrogen-sensitive elements) in VEGF promoter (DNA sequences interacting with ER. Endometrial cells have been transfected by a luciferase gene containing construction under VEGF promotor control and standard analysis of luciferase activity has been carried out. In the resultant cells, estradiol has increased luciferase activity (a 3-4-fold increase). Similar effects have been described later for BC cells [54]. The data indicate that VEGF expression regulation in BC cells is mediated by ER [54] by the “classical” genome pathway estrogen/ER/ERE sites of VEGF gene (Fig. 1). The results of another study [61] also indirectly indicate the “classical” hormone regulation of VEGF expression. Estradiol enhanced VEGF expression in MCF-7 BC cells (ER+/ER+), while tamoxifen reduces it. Estradiol and tamoxifen do not modify VEGF mRNA level in MB-MDA-231 BC cells not expressing ER. As mentioned above, hypoxia is the central regulator of VEGF synthesis [81]. In addition, VEGF expression is regulated by steroid hormones [61]. An in vitro study [22] of the relationship between hypoxia, estrogens, antiestrogens, and ER expression profile, on the one hand, and VEGF levels in BC cells MCF7 (ER+/ER+) and MB-MDA-231 (ER–/ER+), on the other, has been carried out. Hypoxia significantly increased VEGF level in MCF-7 and MB-MDA-231 cells, while culturing with antiestrogens (tamoxifen and Faslodex) did not prevent the increase in VEGF level under conditions of hypoxia. Estradiol, in turn, induced an increase of VEGF level in MCF-7. By contrast, in MB-MDA-231 cells (ER–/ER+) estradiol reduced VEGF level, this indirectly indicating
A. M. Scherbakov, M. A. Krasil’nikov, and N. E. Kushlinskii
negative regulation of ER-mediated VEGF synthesis. Interestingly, Faslodex antiestrogen reduced the estradiol-induced VEGF level in MCF-7 cells only under conditions of normoxia. Hence, hormone regulation of VEGF synthesis in BC is less effective in hypoxia and hence, the efficiency of VEGF synthesis suppression by antiestrogens decreases. These observations partially explain inefficiency of hormone therapy in BC patients with high VEGF levels in the tumor. Different roles of ER and ER in the regulation of VEGF synthesis have been confirmed [16]. Dimerization of ER with ER can inhibit the stimulatory effect of estradiol on VEGF synthesis. Transfection of MCF-7 cells (high level of ER; low content of ER) by a plasmid containing ER cDNA leads to inhibition of estradiol stimulatory effect on VEGF synthesis. A similar effect is observed in cotransfection of MB-MDA-231 cells (ER–/ER+) by ER cDNA and ER cDNA. Hence, estrogens are involved in regulation of VEGF expression in BC cells, ER and ER playing a different role in this process. In addition, the findings of some authors indicate that antiestrogens modify the expression of VEGF in BC cells. Hormone resistance of BC and VEGF. The involvement of VEGF in the maintenance of hormoneindependent BC growth is intensely studied. In vitro studies of MCF-7 cells [37], transfected by VEGF121 (V121 substrain) or VEGF165 cDNA (substrain V165) or treated by recombinant VEGF, gave interesting results. Cell substrains with VEGF hyperexpression are characterized by higher growth rate than the parental MCF-7 strain (V121 by 43%, V165 by 57% higher than the parental cell strain growth rate). Culturing of V121 and V165 substrains with specific antibodies to VEGF led to inhibition of cell growth rate. This proves the direct involvement of VEGF in regulation of these substrains proliferation. Culturing of the parental MCF-7 strain with recombinant VEGF (VEGF121 and VEGF165) also increased cell growth rate (by 28% and 50%, respectively). Parallel culturing of MCF-7 with VEGF121 and estradiol and with VEGF165 and estradiol has demonstrated an additive effect of the hormone and VEGF on cell growth: 269% (VEGF121 and estradiol), 275% (VEGF165 and estradiol) of the control level. The effect of VEGF hyperexpression on hormoneindependent growth of MCF-7 tumors in ovariectomied nude mice has been studied [37]. In order to evaluate the impact of VEGF level for tumor growth, ovariectomied nude mice were implanted with MCF7 (control), substrains V121, or V165. The incidence of tumor formation after implantation of cells with VEGF hyperexpression was much higher than after implantation of parental cells (0% MCF-7 (control), 81% sub-
389 strain V121, 90% substrain V165). Additional injection of estradiol to mice stimulated tumor growth, but had no effect on the incidence of the substrain tumor formation. Hence, mammary tumors with VEGF hyperexpression in vivo are characterized by rather high growth rate in the presence of estrogens and without them. The results of in vivo and in vitro experiments indicate that VEGF hyperexpression leads to hormone-independent growth of BC and is therefore a factor maintaining the tumor hormone resistance. The majority of authors attribute VEGF gene to classical gene targets for estrogens (genes containing the estrogensensitive elements in the promotor). Numerous studies have shown that estradiol increased the content of VEGF mRNA and VEGF protein in cultured BC cells. The level of VEGF in BC cells cultured with antiestrogens increases or decreases. In vitro studies have shown that tamoxifen does not modify the level of VEGF in BC cells [11]. It seems that VEGF level regulation by tamoxifen is determined by several factors: the presence of ER and ER in the cells, the ratio of ER to ER expression, levels of estrogens, and hypoxia (the main regulator of VEGF expression). Experimental data on VEGF regulation in BC by estrogens/antiestrogens and the role of VEGF in hormone-independent growth of BC suggest a relationship between VEGF signal pathwaysand the level of steroid hormone receptors (as the main prognostic factors of endocrine therapy efficiency) and efficiency of hormone therapy for BC. The findings indicate a relationship between VEGF level and content of steroid hormone receptors in the tumor. Inverse correlations between tumor levels of VEGF and ER [73] and between VEGF and PR [71,73] have been detected in a group of 1127 patiens. Another study has confirmed the inverse correlation between VEGF and ER levels [5] for free and total (bound and free) VEGF levels in a group of 202 patients with BC. Inverse correlation between tumor level of VEGF and PR has also been confirmed. Similar correlations between the content of VEGF and ER, VEGF and PR have been confirmed for 845 patients with disseminated BC (VEGF was measured by enzyme immunoassay) [30]. However, one study has failed to confirm the relationship between VEGF level and receptor status of the tumor in postmenopausal patients with BC: VEGF content, measured by the immunohistochemical method, did not correlate with ER and PR levels. Another study has failed to detect appreciable differences in VEGF levels (measured by enzyme immunoassay) in BC patients with different steroid hormone receptor status [116]. The results of two studies [5,73] do not confirm the findings of a study [1] demonstrating a weak direct correlation between tumor level of VEGF and ER (r=0.366; p=0.019) and no correlation between
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tumor VEGF and PR status. Direct correlation between tumor VEGF level and ER status has also been confirmed in one more study [69]. Hence, opinions on the relationships between VEGF level in BC and the steroid hormone receptor status vary. The relationship between VEGF receptor (VEGFR) level and content of steroid hormone receptors in the tumor has been studied [95]. A significant inverse correlation between VEGFR-2 and PR has been detected. Another study [5] has failed to confirm the relationship between the levels of VEGFR-2 (measured by enzyme immunoassay) and PR in 202 patients with BC; no correlation between VEGFR-2 and ER levels has been detected either. According to one more study [80], VEGFR-2 level measured by the immunohistochemical method does not correlate with tumor levels of steroid hormone receptors, p53, and Bcl-2 antiapoptotic protein. VEGF and hormone therapy for BC. The relationship between tumor level of VEGF and response of patients with disseminated BC to first-line hormone therapy has been studied [30]. Tumor level of VEGF has been measured by enzyme immunoassay in a group of 618 patients with disseminated BC. The results indicate a lower efficiency of tamoxifen in the patients with high and medium VEGF levels in the tumor than in those with its low levels. The authors suggest that VEGF is an independent marker of endocrine therapy efficiency in disseminated BC. Another study [72] has evaluated the relationship between tumor level of VEGF and efficiency of firstline endocrine therapy in 96 patients with disseminated BC. Similar results have been attained in 82 patients, receiving tamoxifen as the first-line drug [72]. The authors claim that VEGF is an independent prognostic factor for response to hormone therapy in BC patients. However, a more representative study [73], carried out by the same team, has failed to confirm the significance of VEGF in the prognosis of endocrine therapy for BC: VEGF level has not been related to hormone therapy and chemotherapy efficiency in a group of 1127 patients. The pattern of VEGF/VEGFR-2 relationship with hormone therapy efficiency seems to be determined by the menstrual status and level of steroid hormone receptor expression in the tumor. No relationship between the tumor VEGF level, measured by the immunohistochemical method, and response to tamoxifen therapy has been detected in 460 postmenopausal patients with BC [94]. According to these data, the tumor content of VEGFR-2 correlates with response to tamoxifen therapy. Adjuvant tamoxifen therapy has prolonged significantly the relapse-free survival in the group of steroid hormone receptor-positive patients with a negligible level of VEGFR-2 in the tumor,
while in patients with higher levels of VEGFR-2 in tumors tamoxifen therapy has been ineffective [94]. It seems that VEGF is essential for endocrine therapy prognosis in menopausal patients [95]: women with steroid hormone receptor-positive tumors (more than 10% tumor cells containing ER), containing no VEGF, have developed a significant response to tamoxifen therapy. However, VEGF+ tumors are insensitive to tamoxifen. In addition, high level of VEGFR-2 reflects low tamoxifen sensitivity of these patients. However, VEGFR-2 level correlates with response to endocrine therapy only for tumors with high content of ER (more than 90% stained cells, according to the immunohistochemical method) [95]. Hence, the role of VEGF in BC is not confined to regulation of tumor angiogenesis processes, and experimental data on hormone-independent growth of tumors with high levels of VEGF and clinical data on VEGF relationship with the steroid hormone receptor status and the significance of VEGF and its receptors for endocrine therapy prognosis in BC suggest regarding the activity of VEGF signal pathways as a prospective marker of BC hormone resistance. The above-mentioned growth factor signal pathways, supporting hormone-independent BC growth, have common proteins (PI3K, Akt, mTOR, MAPK, NF-B, etc.), through which the signal is transmitted in the cells. Disorders in the activity/expression of these signal-transmitting proteins leads to formation of BC resistance to hormone therapy. PI3K/Akt signal pathway. Akt (proteinkinase B, PKB, RAC-PK) is a serine/threonine kinase, playing an important role in cell metabolism regulation. Protein Akt is the main sublying effector of PI3K (enzyme regulating cell proliferation, apoptosis, annoykis, protein synthesis, glycolysis, and survival [15,112]), and disorders in the PI3K/Akt signal pathway regulation are closely connected to various mechanisms of tumor transformation and malignant cell resistance to some antitumor agents [48,50]. Interactions between SH2 domains of p85-PI3K regulatory subunit with cell tyrosine kinases – nonreceptor (Src) and receptor (EGFR, VEGFR, HER-2/ neu, PDGFR, FGFR) ones – are assummed to be the classical mechanisms of PI3K-Akt signal pathway activation [90]. Active PI3K phosphorylates phosphatidylinositol in the inositol ring 3-OH (D3) position, this later leading to Akt phosphorylation. Proteinkinases PDK1 and ILK, Rictor protein complex with mTOR proteinkinase, PDK2 kinase, and steroid hormones are also involved in Akt activation [2,3,32,34,99]. It is assumed that the main suppressor molecules, modulating the PI3K-Akt signal pathway, are PTEN phosphatase and proteinphosphatase 2A (PP2A) [20,58,115].
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Hormone regulation of Akt activity in BC cells has been demonstrated [2]. In MCF-7 cells, estradiol activated Akt and increased cell growth rate. Estradiolinduced activity of Akt was suppressed by Faslodex antiestrogen and vortmannin (PI3K specific inhibitor). The mechanism of inverse regulation, signal transmission from PI3K/Akt pathway to ER, is also described for hormone-dependent BC cells: PI3K and Akt cause ER activation [17]. The PI3K regulates ER activities by two modes: by signal transmission through Akt and by directly modulating the PI3K–ER interactions. This signal mechanism seems to play an important role in the development of hormone resistance. Disputable, but no doubts, interesting data are presented in this paper [113]. The Akt in MCF-7 cells is involved in regulation of ER activity and expression. On the other hand, 17-estradiol modulates the activity of Akt. A new nongenomic effect of estrogens is described in this study. In BC cells 17-estradiol binds to estrogen membrane receptor, a component of a heterocomplex with receptor tyrosine kinases from ErbB-1 and ErbB-3 epidermal growth factor receptor family. The resultant complex, in turn, activates the PI3K/Akt signal pathway. This seems to lead to the formation of the ER-ErbB-2/ErbB-3-PI3K-Akt-ER regulatory loop in BC cells. These intricate signal mechanisms may be involved in the regulation of hormone-independent BC growth. On the other hand, the question is: can Akt hyperactivity lead to hormone-independent BC growth? An experimental study [28] has been carried out, in which mice were implanted with MCF-7 cells with Akt hyperexpression. These cells formed tumors similar by size to estrogen-stimulated tumors from parental cells. Faslodex antiestrogen has not changed the growth of tumors with Akt hyperexpression, this indirectly indicating inefficiency of antiestrogens in BC patients with disorders in Akt synthesis. Constitutive activation of Akt is regarded [50] as a factor maintaining the growth of tamoxifen-resistant substrain. A cell subpopulation, resistant to the cytostatic effect of tamoxifen, has been derived from parental MCF-7 hormone-resistant BC. One of Akt isoforms, Akt1, is activated in the tamoxifen-resistant substrain. The EGF/EGFR and TGF cause AKT hyperactivation in the tamoxifen-resistant substrain in comparison with the parental strain. Hence, BC cells acquire hypersensitivity to growth factors in some forms of hormone resistance, and Akt seems to be one of the key factors determining this sensitivity. Hence, some nongenome effects of estradiol consist in activation of the PI3K/Akt signal pathway, while PI3K and Akt hyperactivity in BC cells can lead to hormone-independent growth. On the whole, activation of this molecular mechanism is one of the
391 probable causes of antiestrogen inefficiency in BC. A new target for therapy of hormone-resistant BC: mTOR. The next key molecular step in the PI3K/ Akt signal pathway is mTOR, the enzyme regulating protein synthesis and proliferation. For this reason these targets are united in the same group in clinical studies. Good prospects of this approach to cancer therapy are confirmed by the possibility of developing inhibitors with the double activity, suppressing PI3K and mTOR. High efficiency of NVP-BEZ235, a double PI3K and mTOR inhibitor, overcoming acquired resistance to letrosole, has been demonstrated on the MCF-7/AROM-1 BC model [19]. Clinical trials of this drug in patients with disseminated receptor-positive BC, receiving endocrine therapy, are now in progress. The results of studies of combined use of everolimus (mTOR inhibitor) with aromatase inhibitors in patients with disseminated ER+ BC (BOLERO-2) have been published in 2012 [8]. The relapse-free survival median in the groups of BC patients receiving everolimus combined with exemestane reached 10.6 months, i.e. by 2.6 times longer than in the group receiving exemestane with placebo. Sensitization of BC cells to the apoptotic effect of estrogens, a factor accompanying the development of hormone resistance. Modern concepts on the role of estrogens in BC are not confined to the data on their growth-stimulating effect on the cells. Studies carried out by many teams, including N. N. Blokhin Oncological Center, have described estrogen-induced cell death [66,102,109,110]. It seems that ER can play a dual role: stimulate tumor growth and under certain conditions trigger apoptosis mechanisms in the target cells. Published data and our results indicate that a shift of the balance towards the apoptotic effect of estrogens is as a rule conjugated with the development of BC hormone resistance [102,109]. It has been demonstrated [6] that transfection of ER– BC strain MDA-MB-231 with constructions carrying wild type ER reduced proliferative activity of cells cultured with estradiol. Hence, estrogens play the cytostatic role in the studied system; the level of estrogen-induced apoptosis has not been measured. The role of ER in estrogen-induced apoptosis remains disputable. Persistence of a high level of ER in the cells is an obligatory condition for cell sensitization to the apoptotic effect of estradiol. Direct evidence of ER involvement in the apoptotic signal transmission from estradiol [109] and arrest in the cell cycle G(2)/M phase has been obtained [6]. However, estradiol also caused apoptosis and arrest in the cell cycle G(2)/M phase in the Jurkat human acute leukemia cells, despite the absence of ER [46,51]. Paradoxical induction of apoptosis by estrogens has been demonstrated in cells of “nonendocrine” origin [104]: estra-
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diol induced apoptosis in aortic endothelial cells from LDL-R(–/–) C57Bl/6 mice. It seems that the origin of the tumor/normal tissue is essential for estrogen-induced apoptosis, and ER expression in BC is an obligatory condition for estrogen induction of cell death. The form of ER, inducing cell death, also remains unclear. According to one report [47], it is not the “classical” form of ER, ER with the molecular weight of 66 kDa, that induces cell death, but a reduced ER form with a molecular weight of 46 kDa, lacking the AF-1 transactivation domain. Estrogen-induced apoptosis develops in ER+ cells with participation of the Fas signal pathway with subsequent activation of caspase-7 and caspase-9 [111]. Estradiol-induced apoptosis in ER– Jurkat acute leukemia cells includes cytochrome C release, activation of caspases (9, 3, 8), and PARP degradation [51]. Sensitization of MCF-7 cells to estrogen-induced apoptosis in our study has developed under conditions of activation of estrogen-independent mitogen pathways (EGF/EGFR, VEGF/VEGFR-2) [102]. Cell sensitization to estrogen-induced apoptosis developed after the cells acquired capacity to estrogen-independent growth. This fact suggested regarding activation of mitogen signal pathways and sensitization to estrogen-induced apoptosis as two successive stages in the development of BC cell hormone resistance. Summing up our findings and published data, we should like to note that paradoxical sensitivity to the apoptotic effect of estrogens is as a rule observed in the cells cultured for a long time in steroid-free medium, insensitive to antiestrogens and growth-stimulating effects of estrogens, but retaining a high level of ER expression [62,65,102,109,110]. Some observations demonstrating the efficiency of estrogen therapy in resistant BC confirm the possibility of tumor cell sensitization to apoptotic effect of estradiol [62,65,68,85-87,89,109,110]. One of the key proteins involved in the development of hormone resistance and in cell sensitization to estrogen-induced apoptosis is NF-B transcription factor [66,67,118]. NF-B transcription factor (nuclear factor kappalight-chain-enhancer of activated B cells) is a protein responsible for cell reaction to cytokines, growth factors, destructive exposure, and other factors [12,107]. Inert NF-B is present in the cytoplasm in complex with IB inhibitor. Exposure of a cell to a specific stimulus leads to phosphorylation and degradation of inhibitors and NF-B transition into an active state. Activated NF-B is translocated into the cell nucleus, where it stimulates the expression of genes with antiapoptotic and mitogenic characteristics [12,121]. The NF-B proteins are homologous to v-Rel retroviral protein, and are therefore classified as NF-B/
Rel proteins. Five proteins of the family have been found in mammals: NF-B1/p50 (NFKB1 gene), NFB2/p52 (NFKB2 gene), RelA/p65 (RELA gene), RelB (RELB gene), and c-Rel (REL gene) [12]. Proteins of NF-B family form hetero- and homodimers, bound (in inert state) to IB. Kinases are responsible for IB phosphorylation and NF-B activation, which can be divided into groups: NF-B main regulators (IKK, IKK, and IKK/NEMO) and proteins modulating NF-B activity under the effects of specific stimuli (directly or indirectly, through IKK) NIK, GSK3, CKII, PKC, PKA, PKR, etc. [12,60,64]. Studies of interactions between ER and NF-B indicate pronounced antagonism of these proteins [27,67,97]. Estradiol capacity to suppress the transcription activity and, according to some data, expression of NF-B is sufficiently well known and has been described for many experimental models [10,27,111]. Moreover, many authors have noted mutual antagonism between NF-B and ER, caused by the formation of inert complexes between these proteins [41] and/or competition for binding to common activator proteins, such as CBP/p300 [10,27,119]. Studies of NF-B role in the development of BC have shown that ER– BC forms are characterized by a high constitutive level of NF-B [108]. The authors claim that NF-B activation can be one of the factors determining high malignancy of estrogen-independent tumors and their low sensitivity to chemotherapy. Several NF-B inhibitors have been found by the present time, mainly blocking phosphorylation and/or proteosome degradation of IB. Drugs based on NF-B inhibitors induce apoptosis and cause tumor cell sensitization to Herceptin, which is extremely important in therapy of HER-2+ BC [18]. The group of signal molecules, linked with NF-B transcription factor, can be regarded as prospective targets for the development of approaches to BC therapy. We have studied the NF-B signal pathway in vitro in cultured MCF-7 estrogen-dependent BC cells and MCF-7/LS resistant substrain, obtained by longterm (60 days) culturing of MCF-7 cells in steroid-free medium [66,67]. The MCF-7/LS substrain is characterized by resistance to the proliferative effect of estradiol and rather high growth rate in steroid-free medium in the presence of active ER system [66,67]. Measurement of the basal level of TNF-R1, one of the main NF-B inductors (by immunoblotting) and of Fas expression (by the immunofluorescent method) has failed to detect any appreciable differences in the levels of these proteins in MCF-7/LS substrain cells in comparison with the MCF-7 strain. Cell culturing with estradiol (10–9 M) has caused no appreciable changes in the expression of TNF-R1 and Fas [67]. Analysis of NF-B transcription activity has shown that it was low
A. M. Scherbakov, M. A. Krasil’nikov, and N. E. Kushlinskii
in MCF-7/LS cells (only 25-30% of NF-B activity in the parental strain). Estradiol causes a significant suppression of NF-B, the MCF-7/LS cells fully retaining the capacity to NF-B inhibition in response to estradiol. Analysis of IkBa gene (regulated by NF-B) mRNA level in MCF-7/LS cells by the PCR method has shown a similar trend [66]. Antagonism of the NF-B and ER signal pathways has not been confirmed [33]. One of the main inductors of NF-B, TNF, enhanced the expression of about 30% estradiol-activated genes [33]. Estradiol, in turn, enhanced the expression of about 15% TNFdependent genes. Hence, interactions between NF-B and ER should be regarded as an intricate molecular mechanism, exhibiting under different conditions antagonistic or synergic effects. Studies of the effects of long-term estradiol treatment on cell growth and survival have shown a significant inhibition of cell growth rate after 8-day culturing of resistant MCF-7/LS substrain with estradiol (10–9 M) [67]. Evaluation of apoptosis level by flow cytofluorometry has shown that MCF-7/LS cell growth inhibition under the effect of estradiol is due to more intense apoptosis in these cells, while the parental MCF-7 cells are insensitive to the apoptotic effect of estradiol [67]. Further experiments have shown that acquisition of capacity to estrogen-independent growth by BC cells is insufficient for sensitization to estradiol apoptotic activity. For example, substrain MCF-7/T2, obtained by long culturing of MCF-7 cells with tamoxifen, is characterized by a stable estrogen-independent growth but is insensitive to estrogen-induced apoptosis [66]. Only transfer of MCF-7/T2 cells into steroidfree medium and long-term culturing without estradiol leads to cell sensitization to the apoptotic effect of estradiol, similar to sensitization of MCF-7/LS. It is noteworthy that activity of NF-B gradually decreases during culturing of MCF-7/T2 cells in steroid-free medium, reaching the minimum by the end of culturing. In order to evaluate the role of NF-B in regulation of cell sensitivity to the apoptotic effect of estrogens, MCF-7/T2 substrain cells were transfected by pMIG plasmid containing NFKB gene dominantnegative variant. Study of MCF-7/T2 cell growth and survival after transfection has shown that pMIG transfectants, in contrast to the cells transfected by the control vector, acquire capacity to induce apoptosis in response to estradiol [66]. Transfection of pMIG in the parental estrogen-dependent MCF-7 cells has not led to cell sensitization to the apoptotic effect of estradiol. On the whole, our results demonstrate the important role of NF-B in sensitization of resistant BC cells to the apoptotic effect of estradiol. Presumably, constitutive reduction of NF-B activity and imbalance of the pro- and antiapoptotic pathways are the
393 key factors responsible for acquisition of the cell sensitivity to estrogen-induced apoptosis, while estrogen-dependent suppression of NF-B provokes further shift of this balance and eventually stimulates the onset of apoptosis. Further experiments have been aimed at the analysis of the sensitivity of hormone-resistant MCF-7/LS cells to doxorubicin and evaluation of NF-B role in estrogen-induced apoptosis. Studies of combined effects of estradiol and doxorubicin on MCF-7/LS substrain have shown that as early as just 3 days after addition to the cells estradiol potentiated apoptotic activity of doxorubicin [101]. In the absence of doxorubicin, estradiol did not induce apoptosis during this period. Analysis of transcription activity showed that the effect of doxorubicin alone was associated with an appreciable increase of NF-B activity, while addition of estradiol to cells with doxorubicin prevents NF-B activation. Hence, the results of these experiments indicate that suppression of NF-B in the presence of estradiol presumably determines its capacity to stimulate the apoptotic effect of doxorubicin in resistant cells. The key role of NF-B in regulation of resistant cell response to apoptotic stimulus was confirmed in our experiments on transfection of MCF-7/LS substrain by NF-B siRNA (small interfering RNA), specifically inhibiting NF-B activity. Suppression of NF-B led to a significant increase of doxorubicin sensitivity of resistant cells [101]. An important role of NF-B in the realization of the cytostatic and apoptotic effects of doxorubicin, docetaxel, and cisplatin has been noted [63]. Genistein (phytoestrogen) pretreatment of tumor cells blocks NF-B and potentiates the effect of antitumor compounds [63]. Our findings and published data attest to direct involvement of NF-B in the regulation of resistant BC cell growth and survival. Its suppression largely determines cell sensitization to the apoptotic effect of antitumor compounds. Hence, the main factors determining BC resistance to endocrine therapy are high activity and/or expression of receptor tyrosine kinases (FGFR, EGFR, HER-2/neu, VEGFR, etc.), changes in the activities of regulator proteins of cellular defense mechanisms (NF-B, PI3K, Akt, mTOR), and high activities of cell cycle regulator proteins (Myc, c-Fos, Cyclin D1). The data indicate good prospects of using the HER-2/neu, EGFR, mTOR, PI3K/Akt molecular pathways as targets for therapy of BC, including the resistant forms.
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