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Progress in aromatase research and identification of key future directions
Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 311–315
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Progress in aromatase research and identification of key future directions夽 Anita K. Dunbier a,∗, Yanyan Hong b, Selma Masri b, Kristy A. Brown c, Gauri J. Sabnis d, Melanie R. Palomares e a
Academic Department of Biochemistry, Royal Marsden Hospital and Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JJ, United Kingdom Division of Tumor Cell Biology, Beckman Research Institute of the City of Hope, Duarte, CA, United States c Prince Henry’s Institute of Medical Research, Clayton, Victoria, Australia d Department of Pharmacology and Experimental Therapeutics, University of Maryland Baltimore, Baltimore, MD, United States e City of Hope Medical Center, Duarte, CA, United States b
a r t i c l e
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Article history: Received 27 August 2009 Accepted 11 September 2009 Keywords: Meeting report Aromatase
a b s t r a c t The IX International Aromatase Conference focused upon key developments in research related to the aromatase enzyme that had occurred since the last meeting. A session took place at the conclusion of conference discussing key areas for future research and issues currently facing researchers in the field. While significant progress on understanding structural elements of the enzyme and regulatory mechanisms of both the gene and protein provides an excellent basis for development of improved aromatase inhibitors and exploration of the important problem of aromatase inhibitor resistance, significant challenges remain. Increasing the speed with which findings are translated into clinical practice and finding an appropriate balance between basic and translational research were identified as areas which require further attention before the next meeting in 2010. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The IX International Aromatase Conference was held in Shanghai in October 2008 and focused upon key developments in research related to the aromatase enzyme that had occurred since the last meeting in 2006. A session took place at the conclusion of conference discussing key areas for future research. Professors Shiuan Chen, William Miller and John Forbes gave presentations covering their thoughts on the current state and future of basic, translational and clinical research in the field. This article summarizes the major advances in these areas and the key points of ensuing discussion from both the speakers in the session and the audience. 2. Aromatase 2008: major new basic science discoveries 2.1. Structural characterization of aromatase One of the key advances presented at the conference was the crystal structure of aromatase from human placenta. Much effort has previously been focused on understanding the structure–function relationship of aromatase, including molecu-
夽 Special Issue selected article from the IX International Aromatase Conference (Aromatase 2008) held at Shanghai, China, on October 13th–16th, 2008. ∗ Corresponding author. Tel.: +44 207 808 2883; fax: +44 207 376 3918. E-mail address: [email protected] (A.K. Dunbier). 0960-0760/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2009.09.005
lar characterization of purified aromatase [1–3] and structural modeling [4–6]. This major breakthrough in aromatase research, presented by Dr. Debashis Ghosh, provides significant insight into the active site of aromatase [7]. The crystal structure of full-length aromatase in complex with androstenedione solved at 2.9 Å resolution marks a major milestone in structure determination of the cytochrome P450 proteins (CYP450s), as this is the first crystal of full-length transmembrane CYP450s, although the structure of the N-terminal transmembrane domain was not well defined. The presence of detergent molecules within the crystal reveals residues located at the transmembrane surface, including tryptophan and arginine within the -domain and the F-G loop. The active-site cleft of the complex is relatively small (<400 Å3 ) when compared with other CYP450 s, thus an androstenedione molecule fits snugly into this androgen-specific cleft. This crystal structure confirms several key active-site residues predicted from previous site-directed mutagenesis and structure modeling, including D309 and T310 (I helix), F134 (B-C loop), S478 (-4 sheet), and V370-M374 (3 -flanking loop of the K helix) as presented at the VIII International Aromatase Conference [3], and additionally identifies active-site residues F221, W224, M447, and S470. Since the conference, this X-ray crystal structure of aromatase with bound androstenedione has been published [8], allowing subsequent crystallization efforts to focus on the co-crystallization with aromatase inhibitors (AIs), including exemestane, letrozole, and anastrozole. Crystal structures of aromatase in complex with AIs will provide structural basis for inhibitor recognition
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and binding. However, a crystal structure only captures a single state of a protein and provides a static snapshot of one conformation. Thus, additional biochemical studies are required to address the catalytic mechanism of aromatase and the inhibition mechanism by AIs. Based on enzyme kinetic analysis, computer modeling, and mutagenesis, Dr. Yanyan Hong presented a binding model of aromatase with its electron-transfer partner NADPHcytochrome P450 reductase [9], in which the FMN-binding domain of reductase undergoes a structural rearrangement, allowing the proximal surface of aromatase to fit in the cleft between the FMN- and FAD-binding domains of reductase. This model also predicts the electrostatic interactions between K108 of aromatase and N175/T177 of reductase. Future directions may include detailed studies of the interaction between aromatase and reductase and the mechanism of exemestane-mediated irreversible inhibition of aromatase. Structural and functional characteristics of aromatase are critical for the development of new aromatase inhibitors as well as for the identification of novel anti-aromatase phytochemicals for the prevention and treatment of hormone-dependent breast cancers. 2.2. Targeting the tissue-specific expression of aromatase as a novel cancer therapy While current aromatase inhibitors are very efficient adjuvant therapies, they are also accompanied by a number of undesirable side effects, including bone loss, hot flushes and arthralgia [10]. This arises mainly due to the mechanism of action of aromatase inhibitors which target aromatase activity throughout the body, leading to a state of estrogen-deficiency. The expression of the aromatase gene, however, is under the control of several tissue-specific promoters, allowing a fine-tuned regulation of aromatase expression in various tissues. Dysregulated expression of aromatase has been implicated in a variety of diseases, including prostate cancer [11], the metabolic syndrome with male-specific fatty-liver [12], and a number of women’s cancers [13]. In breast, ovarian, uterine, and prostate cancer, activation of PI.3/II leads to the overexpression of aromatase within the tissues themselves, and this has been associated with an increase in tumor growth [13]. Previous research has demonstrated that a number of proteins interact with promoter I.3/II in response to tumor-derived factors, such as PGE2, and are important modulators of aromatase expression within the breast. These include the cAMP response element binding protein (CREB) [14,15], Snail/Slug proteins [16], and the orphan nuclear receptor liver receptor homologue-1 (LRH-1) [17,18]. Consistent with this, LRH-1 antagonizing peptides have previously been shown to inhibit aromatase expression [19], and current research is aiming to identify pharmacological compounds that would target LRH-1 interactions specifically [20]. More recently, peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) [19], BRCA1 [21], C/EBP, ATF-2, and CREB binding protein [22] were identified as being involved in regulating PI.3/II-driven aromatase expression within the breast. At the IXth International Aromatase Conference, PELP1 was shown to activate PI.3/PII in a Src/PI3K-derpendent manner and its recruitment to the promoter was also affected by HER2 [23]. The CREB regulated transcription coactivator 2 (CRTC2) was also demonstrated to be important in the CREB-mediated expression of aromatase in breast adipose stromal cells [24]. The next generation of aromatase inhibitors may need to rely on methods of targeting tissuespecific aromatase transcripts either using antisense technology and/or developing drugs that will prevent interacting proteins from activating specific aromatase promoters. Interestingly, N-methyl NS-398, an analogue of the cyclooxygenase-2 (COX-2) inhibitor NS398, was also demonstrated to inhibit aromatase mRNA expression, independent of COX-2 activity [25,26] and elucidating the mecha-
nisms by which this occurs may result in the identification of new targets. Although there is still some uncertainty regarding the source of the steroids that initiate tumor growth, the idea that local estrogen biosynthesis plays an important role in breast cancer proliferation is well accepted, and inhibiting aromatase expression specifically within the breast or tissues involved in disease progression would allow the therapeutic benefits to outweigh to side effects associated with inhibiting aromatase activity globally.
3. Translational research: bridging the gap between the laboratory and the clinic Based on discussion at the conference, the consensus from both basic researchers as well as clinicians was that there is still a great need to bridge the gap between the laboratory bench and the clinic. In the aromatase field, translational research needs to make a more robust impact, especially because ideas from the laboratory are not being translated into effective clinical practice. For instance, both researchers and clinicians were in agreement that acquired as well as de novo resistance to aromatase inhibitors (AIs) are a major hindrance in the clinic. Translational research is imperative to bridge this gap in our lack of understanding in how to treat AI-resistant breast cancers, and even more importantly, how to prevent the onset of AI-resistance. Over the last decade AIs have made significant advances in the treatment of breast cancer and have become the new standard of care [27,28]. An intratumoral aromatase model system has been developed to mimic the postmenopausal hormone responsive breast cancer [29,30]. The results from this model system have also predicted outcomes of several clinical trials [30]. However, not all patients respond to the AIs in same way and others may eventually develop resistance. Studies using model systems have suggested a gradual adaptation of tumor cells during treatment to finally acquire resistance [31]. Experimental data using model systems and microarray profiling suggests differences in the resistance mechanisms of different AIs [32]. In addition, clinical data suggests heterogeneity within responders or non-responders based on microarray profiling [33]. These observations show heterogeneity in clinical material and also suggest heterogeneity in AI-resistance mechanisms in patients. Based on this idea, heterogeneity in the clinic would subsequently necessitate multiple animal models and/or cell culture model systems, and these model systems need to be selective toward a specific question. Currently, animal model systems as well as cell culture systems have been geared toward understanding acquired resistance in an MCF-7 background only [34–36]. Yet, from a single cell line, both animal models as well as cell culture systems have generated different results. Attendees at the conference suggested that translational researchers should agree on a subset of genes that they believe would be crucial biomarkers for AI-resistance, and provide these target genes to their clinical colleagues. An important aspect of AI-resistance that was discussed at the conference is scheduling of endocrine therapy to combat acquired resistance. It is imperative that we determine which biomarkers would enable us to correctly pinpoint the right time for starting new therapy. Also, tumor markers for these studies should be easy to measure in a clinical setting. Currently, multiple laboratories as well as clinical trial efforts are looking at sequencing and scheduling of endocrine therapy. Clinicians are also asking from these researchers to come to a consensus agreement on what therapeutic options are most effective, in order to translate these results into the clinic. Given the “plasticity” of cancer cells, the aim should be to completely destroy all cancer cells. Any remaining cancer cells can adapt
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to endocrine therapy and can give rise to secondary tumors or metastases. This leads to the concept of cancer stem cells (CSC). The involvement and importance of CSC in relapse or metastatic growth needs to be studied and newer agents need to be discovered that target the CSC. In addition, it was discussed by Dr. Richard Santen that pathways to effectively treat breast cancer must focus on “killing pathways” or the apoptotic response. Currently, many groups are focusing on inhibiting growth factor and signal transduction pathways [36]. While these efforts are valid, due to experimental as well as clinical data that suggests these pathways are involved in breast cancer and AI-resistance, cancer cells can easily adapt and utilize a different growth pathway. Translational research efforts not only are needed to provide experimental data to clinicians, but also laboratory researchers are in great need of tumor specimens from non-responders or patients that have relapsed on AIs. Clinicians and researchers should collaborate to construct appropriate biorepositories of tumor samples, in order to provide clinical material that can address a specific scientific question. Studies of tumor specimens would help generate new models, which in turn would translate easily into clinical practice. Hence, molecular profiling and pathological analysis of relapse should be an important step. Related to molecular profiling, discussion at the conference focused on the need for streamlining bioinformatics methodologies, in order to generate more uniform data sets. High throughput genomic analysis technology (microarrays) has provided an opportunity to study thousands of genes and gene products at a time, which has the potential for identifying responders or nonresponders [37,38]. However, findings from microarrays depict changes at the gene level, and results in the pre-clinical setting suggest changes in the activation of proteins [31,39,40]. These key activated proteins (p-MAPK, p-Akt, etc.) would not be detected by microarray and may represent a large group of potential biomarkers which would not be detected on a genome-wide scale. However, investigating one pathway at a time is quite arduous and also does not consider new types of pathways that have currently not been elucidated. Thus, innovative and efficient yet comprehensive high throughput technologies are needed. Basic and translational research focuses on hypothesis-driven model systems. On the other hand, clinical research has focused on clinical outcomes and problems. Although both areas of research have provided substantial information, disconnect between results obtained by each of these scientific groups necessitates a need for more collaborative translational research. Basic and clinical investigators must work together at all phases of drug development, from preclinical study to clinical trial, in order to be most effective in producing results that will ultimately make an impact on breast cancer patient management.
4. From bench to bedside and beyond: future research directions for clinical application Acknowledging that the ultimate goal of research is to benefit patients, presentations of disciplines in the population sciences were included in the IXth International Aromatase Conference that have the potential to guide basic, clinical, and translational research in aromatase. While we often consider the gap between bench research and clinical trials, we sometimes forget that the gap between clinical trial and clinical practice can also be quite large. Taking advantage of the location of the 2008 conference in Shanghai, China, several regional speakers presented comparisons and contrasts in breast cancer epidemiology between Western and Eastern populations [41–43]. Improving our understanding population differences in breast cancer development and progression across cultures where both exposures and practices vary widely
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will deepen our understanding of both breast cancer etiology and treatment. For instance, as presented by Dr. Chen-Yan Shen from China Medical University in Taiwan [41], Asian breast cancer is has been observed to be generally early-onset, which was attributed to perhaps a higher frequency of the basal-like tumor phenotype. However, recent findings have not supported this hypothesis. Now it is thought that perhaps functional variants involved in estrogen metabolism and the DNA repair pathway may be more common in Asian populations, and this may influence susceptibility to breast cancer as well as differential response to therapy [44,45]. Along these lines, Dr. Richard Weinshilboum from Mayo Clinic gave a presentation on the Pharmacogenetics Research Network (PGRN), a collaborative effort across 12 centers in the United States working with NCIC, Tel Aviv University in Israel, and most recently the RIKEN Center for Genomic Medicine in Japan. The aim of the PGRN is to conduct large-scale pharmacogenetic studies to understand the genetic basis for variable drug responses across populations. Variable responses of interest include both those related to efficacy and those related to toxicity. Genomewide association studies (GWAS) being performed within the PGRN are amassing large amounts of data, and information gained is being organized into a Pharmacogenomics Knowledge base (PharmGKB). This information may be used to build upon our current understanding of how to best use aromatase inhibitors to optimize benefit against risk, with the goal to achieve personalized medicine in the future [46]. Dr. Joseph Ragaz from McGill University presented Chinese–Canadian collaborative research that elucidated interesting trends in breast cancer incidence and mortality related to degrees of westernization. While breast mortality has decreased in Western countries, such as Canada, United States, and the United Kingdom over the past two decades, the opposite trend has been observed in Shanghai. Dr. Ragaz attributes this observation to differing timelines with regards to westernization of these countries. Similar trends are apparent even within China and Canada, when health care services in urban communities are compared to rural ones, with more breast cancer incidence and decreased breast cancer mortality observed in urban areas [47]. These opposing trends likely reflect the interaction of diagnostic and therapeutic advances against lifestyle alterations such as westernized diet and exercise [48]. A better understanding of these individual effects and their interaction, though international research collaborations could lead to opportunities for prevention in rapidly westernizing Asian countries. As oncology becomes more subspecialized into programs that concentrate on specific tumor types, we were reminded to not only bridge cultural divides in our future research but also to bridge subspecialty divides. While past Aromatase Conferences have focused on breast cancer, Dr. Gail Risbridger of Monash University in Australia was invited to speak at the IXth International Aromatase Conference about aromatase research in prostate cancer [49]. By understanding aromatase applications in other hormonally related cancers, particularly those that occur in males rather than postmenopausal females, we may learn more about the effect of a different hormonal milieu on aromatase function. Discussion in the future directions session of the IXth International Aromatase Conference also pointed out that clinical trials, particularly those conducted in the adjuvant and preventive settings, which have the greatest potential for public health impact, are limited by the fact that many study participants and long follow-up times are needed in order to demonstrate an effect that may lead to a new drug indication. Indeed, it was acknowledged that access to cancer patient populations and to cancer research funding are the greatest bottlenecks in aromatase research advancement. Therefore, future research must focus on the development of biomarkers
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that can be used in clinical trials. Important specific applications for such biomarkers include study population selection, surrogate measurement of cancer risk modification, and objective measures of early toxicity. As in the examples discussed above, the population sciences of cancer epidemiology and pharmacogenetics will contribute knowledge to improve our selection of more homogeneous study populations in the future, those who are both most at risk for a cancer outcome and least at risk for developing toxicities to the intervention being studied. As far as surrogate endpoint biomarkers (SEBs) for cancer risk modification, Dr. Hironobu Sasano of Tohoku University in Japan presented his work related to two potential SEBs used in breast cancer chemoprevention trials, Ki67 index and mammographic breast density [50,51]. One presentation focused on the use of Ki67 and aromatase immunohistochemical staining to understand molecular changes within the tumor microenvironment. He was able to show that epithelial proliferation, measured by Ki67, is associated with upregulation of aromatase expression by fibroblasts. He also presented some work which aimed to understand the correlation between breast histology and mammographic density, a non-invasive biomarker of breast cancer risk. He demonstrated that mammographically dense breasts have proportionately more stromal tissue and observed that fibroblasts within the stroma express aromatase more strongly by immunohistochemistry than do fibroblasts within breasts that are not mammographically dense. These findings may lead to a better understanding of the molecular biology behind risk modification SEBs, which have been observed to correlate with cancer risk, but the mechanisms behind these observations have been poorly understood. Lastly, as far as objective measures of early toxicity, this year’s meeting included two good examples. Dr. Leilani Morales of the Katholieke Universiteit in Belgium presented her work establishing MRI and grip strength as objective measures of aromatase inhibitor related arthralgias [52], and Dr. Laura Coker of Wake Forest University presented work within the Women’s Health Initiative (WHI) on standardized measures for measuring cognitive effects of estrogen-deficiency [53–55]. More work along these lines is needed to improve the efficiency of clinical trials. More importantly, there is a need to include biomarkers within ongoing clinical trials in order to validate such measures against clinical outcomes. In the meantime, as discussed at the close of the IXth International Aromatase Conference, improvement of clinical trial efficiency can only be part of the answer. There is a great need for prioritization of research questions to be addressed by clinical trials. While developing multiple model systems to increase our understanding of aromatase resistance can lead to new potential targets, and developing new drugs to specifically target aromatase expression can lead to new interventions, we still do not fully understand how to best schedule drugs that we already have. Should we be focused on intermittent dosing, or novel combinations? Or perhaps, rather than combination therapy, which may lead to greater toxicity, shall we focus on ideal sequencing regimens? These are questions that must be addressed by the large international consortia of trialists so that we may best utilize our resources to maximize patient benefit in clinical practice.
5. Conclusions Presentations and discussion sessions at the IX International Aromatase Conference provided considerable opportunity for debate on the issues currently facing researchers in the field. Within the past 2 years, considerable progress has been made in relation to understanding structural elements of the enzyme and regulatory mechanisms of both the gene and protein. While these findings provide an excellent basis for development of improved aromatase
inhibitors and exploration of the important problem of aromatase inhibitor resistance, there still remain a number of discrepancies between different model systems and also between these models and what has been observed in the clinical setting. Although the conference generally acknowledged that more of these findings need to be translated into clinical practice, there was lack of agreement regarding the appropriate balance between basic and translational research and the speed with which new findings should be translated into the clinic. Nonetheless, the meeting and discussion sessions provided considerable impetus for future research which can to be pursued before the next meeting scheduled for 2010.
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Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
Progress in aromatase research and identification of key future directions夽 Anita K. Dunbier a,∗, Yanyan Hong b, Selma Masri b, Kristy A. Brown c, Gauri J. Sabnis d, Melanie R. Palomares e a
Academic Department of Biochemistry, Royal Marsden Hospital and Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JJ, United Kingdom Division of Tumor Cell Biology, Beckman Research Institute of the City of Hope, Duarte, CA, United States c Prince Henry’s Institute of Medical Research, Clayton, Victoria, Australia d Department of Pharmacology and Experimental Therapeutics, University of Maryland Baltimore, Baltimore, MD, United States e City of Hope Medical Center, Duarte, CA, United States b
a r t i c l e
i n f o
Article history: Received 27 August 2009 Accepted 11 September 2009 Keywords: Meeting report Aromatase
a b s t r a c t The IX International Aromatase Conference focused upon key developments in research related to the aromatase enzyme that had occurred since the last meeting. A session took place at the conclusion of conference discussing key areas for future research and issues currently facing researchers in the field. While significant progress on understanding structural elements of the enzyme and regulatory mechanisms of both the gene and protein provides an excellent basis for development of improved aromatase inhibitors and exploration of the important problem of aromatase inhibitor resistance, significant challenges remain. Increasing the speed with which findings are translated into clinical practice and finding an appropriate balance between basic and translational research were identified as areas which require further attention before the next meeting in 2010. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The IX International Aromatase Conference was held in Shanghai in October 2008 and focused upon key developments in research related to the aromatase enzyme that had occurred since the last meeting in 2006. A session took place at the conclusion of conference discussing key areas for future research. Professors Shiuan Chen, William Miller and John Forbes gave presentations covering their thoughts on the current state and future of basic, translational and clinical research in the field. This article summarizes the major advances in these areas and the key points of ensuing discussion from both the speakers in the session and the audience. 2. Aromatase 2008: major new basic science discoveries 2.1. Structural characterization of aromatase One of the key advances presented at the conference was the crystal structure of aromatase from human placenta. Much effort has previously been focused on understanding the structure–function relationship of aromatase, including molecu-
夽 Special Issue selected article from the IX International Aromatase Conference (Aromatase 2008) held at Shanghai, China, on October 13th–16th, 2008. ∗ Corresponding author. Tel.: +44 207 808 2883; fax: +44 207 376 3918. E-mail address: [email protected] (A.K. Dunbier). 0960-0760/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2009.09.005
lar characterization of purified aromatase [1–3] and structural modeling [4–6]. This major breakthrough in aromatase research, presented by Dr. Debashis Ghosh, provides significant insight into the active site of aromatase [7]. The crystal structure of full-length aromatase in complex with androstenedione solved at 2.9 Å resolution marks a major milestone in structure determination of the cytochrome P450 proteins (CYP450s), as this is the first crystal of full-length transmembrane CYP450s, although the structure of the N-terminal transmembrane domain was not well defined. The presence of detergent molecules within the crystal reveals residues located at the transmembrane surface, including tryptophan and arginine within the -domain and the F-G loop. The active-site cleft of the complex is relatively small (<400 Å3 ) when compared with other CYP450 s, thus an androstenedione molecule fits snugly into this androgen-specific cleft. This crystal structure confirms several key active-site residues predicted from previous site-directed mutagenesis and structure modeling, including D309 and T310 (I helix), F134 (B-C loop), S478 (-4 sheet), and V370-M374 (3 -flanking loop of the K helix) as presented at the VIII International Aromatase Conference [3], and additionally identifies active-site residues F221, W224, M447, and S470. Since the conference, this X-ray crystal structure of aromatase with bound androstenedione has been published [8], allowing subsequent crystallization efforts to focus on the co-crystallization with aromatase inhibitors (AIs), including exemestane, letrozole, and anastrozole. Crystal structures of aromatase in complex with AIs will provide structural basis for inhibitor recognition
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and binding. However, a crystal structure only captures a single state of a protein and provides a static snapshot of one conformation. Thus, additional biochemical studies are required to address the catalytic mechanism of aromatase and the inhibition mechanism by AIs. Based on enzyme kinetic analysis, computer modeling, and mutagenesis, Dr. Yanyan Hong presented a binding model of aromatase with its electron-transfer partner NADPHcytochrome P450 reductase [9], in which the FMN-binding domain of reductase undergoes a structural rearrangement, allowing the proximal surface of aromatase to fit in the cleft between the FMN- and FAD-binding domains of reductase. This model also predicts the electrostatic interactions between K108 of aromatase and N175/T177 of reductase. Future directions may include detailed studies of the interaction between aromatase and reductase and the mechanism of exemestane-mediated irreversible inhibition of aromatase. Structural and functional characteristics of aromatase are critical for the development of new aromatase inhibitors as well as for the identification of novel anti-aromatase phytochemicals for the prevention and treatment of hormone-dependent breast cancers. 2.2. Targeting the tissue-specific expression of aromatase as a novel cancer therapy While current aromatase inhibitors are very efficient adjuvant therapies, they are also accompanied by a number of undesirable side effects, including bone loss, hot flushes and arthralgia [10]. This arises mainly due to the mechanism of action of aromatase inhibitors which target aromatase activity throughout the body, leading to a state of estrogen-deficiency. The expression of the aromatase gene, however, is under the control of several tissue-specific promoters, allowing a fine-tuned regulation of aromatase expression in various tissues. Dysregulated expression of aromatase has been implicated in a variety of diseases, including prostate cancer [11], the metabolic syndrome with male-specific fatty-liver [12], and a number of women’s cancers [13]. In breast, ovarian, uterine, and prostate cancer, activation of PI.3/II leads to the overexpression of aromatase within the tissues themselves, and this has been associated with an increase in tumor growth [13]. Previous research has demonstrated that a number of proteins interact with promoter I.3/II in response to tumor-derived factors, such as PGE2, and are important modulators of aromatase expression within the breast. These include the cAMP response element binding protein (CREB) [14,15], Snail/Slug proteins [16], and the orphan nuclear receptor liver receptor homologue-1 (LRH-1) [17,18]. Consistent with this, LRH-1 antagonizing peptides have previously been shown to inhibit aromatase expression [19], and current research is aiming to identify pharmacological compounds that would target LRH-1 interactions specifically [20]. More recently, peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) [19], BRCA1 [21], C/EBP, ATF-2, and CREB binding protein [22] were identified as being involved in regulating PI.3/II-driven aromatase expression within the breast. At the IXth International Aromatase Conference, PELP1 was shown to activate PI.3/PII in a Src/PI3K-derpendent manner and its recruitment to the promoter was also affected by HER2 [23]. The CREB regulated transcription coactivator 2 (CRTC2) was also demonstrated to be important in the CREB-mediated expression of aromatase in breast adipose stromal cells [24]. The next generation of aromatase inhibitors may need to rely on methods of targeting tissuespecific aromatase transcripts either using antisense technology and/or developing drugs that will prevent interacting proteins from activating specific aromatase promoters. Interestingly, N-methyl NS-398, an analogue of the cyclooxygenase-2 (COX-2) inhibitor NS398, was also demonstrated to inhibit aromatase mRNA expression, independent of COX-2 activity [25,26] and elucidating the mecha-
nisms by which this occurs may result in the identification of new targets. Although there is still some uncertainty regarding the source of the steroids that initiate tumor growth, the idea that local estrogen biosynthesis plays an important role in breast cancer proliferation is well accepted, and inhibiting aromatase expression specifically within the breast or tissues involved in disease progression would allow the therapeutic benefits to outweigh to side effects associated with inhibiting aromatase activity globally.
3. Translational research: bridging the gap between the laboratory and the clinic Based on discussion at the conference, the consensus from both basic researchers as well as clinicians was that there is still a great need to bridge the gap between the laboratory bench and the clinic. In the aromatase field, translational research needs to make a more robust impact, especially because ideas from the laboratory are not being translated into effective clinical practice. For instance, both researchers and clinicians were in agreement that acquired as well as de novo resistance to aromatase inhibitors (AIs) are a major hindrance in the clinic. Translational research is imperative to bridge this gap in our lack of understanding in how to treat AI-resistant breast cancers, and even more importantly, how to prevent the onset of AI-resistance. Over the last decade AIs have made significant advances in the treatment of breast cancer and have become the new standard of care [27,28]. An intratumoral aromatase model system has been developed to mimic the postmenopausal hormone responsive breast cancer [29,30]. The results from this model system have also predicted outcomes of several clinical trials [30]. However, not all patients respond to the AIs in same way and others may eventually develop resistance. Studies using model systems have suggested a gradual adaptation of tumor cells during treatment to finally acquire resistance [31]. Experimental data using model systems and microarray profiling suggests differences in the resistance mechanisms of different AIs [32]. In addition, clinical data suggests heterogeneity within responders or non-responders based on microarray profiling [33]. These observations show heterogeneity in clinical material and also suggest heterogeneity in AI-resistance mechanisms in patients. Based on this idea, heterogeneity in the clinic would subsequently necessitate multiple animal models and/or cell culture model systems, and these model systems need to be selective toward a specific question. Currently, animal model systems as well as cell culture systems have been geared toward understanding acquired resistance in an MCF-7 background only [34–36]. Yet, from a single cell line, both animal models as well as cell culture systems have generated different results. Attendees at the conference suggested that translational researchers should agree on a subset of genes that they believe would be crucial biomarkers for AI-resistance, and provide these target genes to their clinical colleagues. An important aspect of AI-resistance that was discussed at the conference is scheduling of endocrine therapy to combat acquired resistance. It is imperative that we determine which biomarkers would enable us to correctly pinpoint the right time for starting new therapy. Also, tumor markers for these studies should be easy to measure in a clinical setting. Currently, multiple laboratories as well as clinical trial efforts are looking at sequencing and scheduling of endocrine therapy. Clinicians are also asking from these researchers to come to a consensus agreement on what therapeutic options are most effective, in order to translate these results into the clinic. Given the “plasticity” of cancer cells, the aim should be to completely destroy all cancer cells. Any remaining cancer cells can adapt
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to endocrine therapy and can give rise to secondary tumors or metastases. This leads to the concept of cancer stem cells (CSC). The involvement and importance of CSC in relapse or metastatic growth needs to be studied and newer agents need to be discovered that target the CSC. In addition, it was discussed by Dr. Richard Santen that pathways to effectively treat breast cancer must focus on “killing pathways” or the apoptotic response. Currently, many groups are focusing on inhibiting growth factor and signal transduction pathways [36]. While these efforts are valid, due to experimental as well as clinical data that suggests these pathways are involved in breast cancer and AI-resistance, cancer cells can easily adapt and utilize a different growth pathway. Translational research efforts not only are needed to provide experimental data to clinicians, but also laboratory researchers are in great need of tumor specimens from non-responders or patients that have relapsed on AIs. Clinicians and researchers should collaborate to construct appropriate biorepositories of tumor samples, in order to provide clinical material that can address a specific scientific question. Studies of tumor specimens would help generate new models, which in turn would translate easily into clinical practice. Hence, molecular profiling and pathological analysis of relapse should be an important step. Related to molecular profiling, discussion at the conference focused on the need for streamlining bioinformatics methodologies, in order to generate more uniform data sets. High throughput genomic analysis technology (microarrays) has provided an opportunity to study thousands of genes and gene products at a time, which has the potential for identifying responders or nonresponders [37,38]. However, findings from microarrays depict changes at the gene level, and results in the pre-clinical setting suggest changes in the activation of proteins [31,39,40]. These key activated proteins (p-MAPK, p-Akt, etc.) would not be detected by microarray and may represent a large group of potential biomarkers which would not be detected on a genome-wide scale. However, investigating one pathway at a time is quite arduous and also does not consider new types of pathways that have currently not been elucidated. Thus, innovative and efficient yet comprehensive high throughput technologies are needed. Basic and translational research focuses on hypothesis-driven model systems. On the other hand, clinical research has focused on clinical outcomes and problems. Although both areas of research have provided substantial information, disconnect between results obtained by each of these scientific groups necessitates a need for more collaborative translational research. Basic and clinical investigators must work together at all phases of drug development, from preclinical study to clinical trial, in order to be most effective in producing results that will ultimately make an impact on breast cancer patient management.
4. From bench to bedside and beyond: future research directions for clinical application Acknowledging that the ultimate goal of research is to benefit patients, presentations of disciplines in the population sciences were included in the IXth International Aromatase Conference that have the potential to guide basic, clinical, and translational research in aromatase. While we often consider the gap between bench research and clinical trials, we sometimes forget that the gap between clinical trial and clinical practice can also be quite large. Taking advantage of the location of the 2008 conference in Shanghai, China, several regional speakers presented comparisons and contrasts in breast cancer epidemiology between Western and Eastern populations [41–43]. Improving our understanding population differences in breast cancer development and progression across cultures where both exposures and practices vary widely
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will deepen our understanding of both breast cancer etiology and treatment. For instance, as presented by Dr. Chen-Yan Shen from China Medical University in Taiwan [41], Asian breast cancer is has been observed to be generally early-onset, which was attributed to perhaps a higher frequency of the basal-like tumor phenotype. However, recent findings have not supported this hypothesis. Now it is thought that perhaps functional variants involved in estrogen metabolism and the DNA repair pathway may be more common in Asian populations, and this may influence susceptibility to breast cancer as well as differential response to therapy [44,45]. Along these lines, Dr. Richard Weinshilboum from Mayo Clinic gave a presentation on the Pharmacogenetics Research Network (PGRN), a collaborative effort across 12 centers in the United States working with NCIC, Tel Aviv University in Israel, and most recently the RIKEN Center for Genomic Medicine in Japan. The aim of the PGRN is to conduct large-scale pharmacogenetic studies to understand the genetic basis for variable drug responses across populations. Variable responses of interest include both those related to efficacy and those related to toxicity. Genomewide association studies (GWAS) being performed within the PGRN are amassing large amounts of data, and information gained is being organized into a Pharmacogenomics Knowledge base (PharmGKB). This information may be used to build upon our current understanding of how to best use aromatase inhibitors to optimize benefit against risk, with the goal to achieve personalized medicine in the future [46]. Dr. Joseph Ragaz from McGill University presented Chinese–Canadian collaborative research that elucidated interesting trends in breast cancer incidence and mortality related to degrees of westernization. While breast mortality has decreased in Western countries, such as Canada, United States, and the United Kingdom over the past two decades, the opposite trend has been observed in Shanghai. Dr. Ragaz attributes this observation to differing timelines with regards to westernization of these countries. Similar trends are apparent even within China and Canada, when health care services in urban communities are compared to rural ones, with more breast cancer incidence and decreased breast cancer mortality observed in urban areas [47]. These opposing trends likely reflect the interaction of diagnostic and therapeutic advances against lifestyle alterations such as westernized diet and exercise [48]. A better understanding of these individual effects and their interaction, though international research collaborations could lead to opportunities for prevention in rapidly westernizing Asian countries. As oncology becomes more subspecialized into programs that concentrate on specific tumor types, we were reminded to not only bridge cultural divides in our future research but also to bridge subspecialty divides. While past Aromatase Conferences have focused on breast cancer, Dr. Gail Risbridger of Monash University in Australia was invited to speak at the IXth International Aromatase Conference about aromatase research in prostate cancer [49]. By understanding aromatase applications in other hormonally related cancers, particularly those that occur in males rather than postmenopausal females, we may learn more about the effect of a different hormonal milieu on aromatase function. Discussion in the future directions session of the IXth International Aromatase Conference also pointed out that clinical trials, particularly those conducted in the adjuvant and preventive settings, which have the greatest potential for public health impact, are limited by the fact that many study participants and long follow-up times are needed in order to demonstrate an effect that may lead to a new drug indication. Indeed, it was acknowledged that access to cancer patient populations and to cancer research funding are the greatest bottlenecks in aromatase research advancement. Therefore, future research must focus on the development of biomarkers
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that can be used in clinical trials. Important specific applications for such biomarkers include study population selection, surrogate measurement of cancer risk modification, and objective measures of early toxicity. As in the examples discussed above, the population sciences of cancer epidemiology and pharmacogenetics will contribute knowledge to improve our selection of more homogeneous study populations in the future, those who are both most at risk for a cancer outcome and least at risk for developing toxicities to the intervention being studied. As far as surrogate endpoint biomarkers (SEBs) for cancer risk modification, Dr. Hironobu Sasano of Tohoku University in Japan presented his work related to two potential SEBs used in breast cancer chemoprevention trials, Ki67 index and mammographic breast density [50,51]. One presentation focused on the use of Ki67 and aromatase immunohistochemical staining to understand molecular changes within the tumor microenvironment. He was able to show that epithelial proliferation, measured by Ki67, is associated with upregulation of aromatase expression by fibroblasts. He also presented some work which aimed to understand the correlation between breast histology and mammographic density, a non-invasive biomarker of breast cancer risk. He demonstrated that mammographically dense breasts have proportionately more stromal tissue and observed that fibroblasts within the stroma express aromatase more strongly by immunohistochemistry than do fibroblasts within breasts that are not mammographically dense. These findings may lead to a better understanding of the molecular biology behind risk modification SEBs, which have been observed to correlate with cancer risk, but the mechanisms behind these observations have been poorly understood. Lastly, as far as objective measures of early toxicity, this year’s meeting included two good examples. Dr. Leilani Morales of the Katholieke Universiteit in Belgium presented her work establishing MRI and grip strength as objective measures of aromatase inhibitor related arthralgias [52], and Dr. Laura Coker of Wake Forest University presented work within the Women’s Health Initiative (WHI) on standardized measures for measuring cognitive effects of estrogen-deficiency [53–55]. More work along these lines is needed to improve the efficiency of clinical trials. More importantly, there is a need to include biomarkers within ongoing clinical trials in order to validate such measures against clinical outcomes. In the meantime, as discussed at the close of the IXth International Aromatase Conference, improvement of clinical trial efficiency can only be part of the answer. There is a great need for prioritization of research questions to be addressed by clinical trials. While developing multiple model systems to increase our understanding of aromatase resistance can lead to new potential targets, and developing new drugs to specifically target aromatase expression can lead to new interventions, we still do not fully understand how to best schedule drugs that we already have. Should we be focused on intermittent dosing, or novel combinations? Or perhaps, rather than combination therapy, which may lead to greater toxicity, shall we focus on ideal sequencing regimens? These are questions that must be addressed by the large international consortia of trialists so that we may best utilize our resources to maximize patient benefit in clinical practice.
5. Conclusions Presentations and discussion sessions at the IX International Aromatase Conference provided considerable opportunity for debate on the issues currently facing researchers in the field. Within the past 2 years, considerable progress has been made in relation to understanding structural elements of the enzyme and regulatory mechanisms of both the gene and protein. While these findings provide an excellent basis for development of improved aromatase
inhibitors and exploration of the important problem of aromatase inhibitor resistance, there still remain a number of discrepancies between different model systems and also between these models and what has been observed in the clinical setting. Although the conference generally acknowledged that more of these findings need to be translated into clinical practice, there was lack of agreement regarding the appropriate balance between basic and translational research and the speed with which new findings should be translated into the clinic. Nonetheless, the meeting and discussion sessions provided considerable impetus for future research which can to be pursued before the next meeting scheduled for 2010.
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