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To outline the progress being made in the understanding of acquired resistance to long term therapy with the selective oestrogen receptor modulators (SERMs, tamoxifen and raloxifene) and aromatase inhibitors. The question to be addressed is how we can amplify the new biology of oestrogen-induced apoptosis to create more complete responses in exhaustively antihormone treated metastatic breast cancer.
Three questions are posed and addressed.
1.) Do we know how oestrogen works? 2.) Can we improve adjuvant antihormonal therapy? 3.) Can we enhance oestrogen-induced apoptosis?
The new player in oestrogen action is GPR30 and there are new drugs specific for this target to trigger apoptosis. Similarly, anti-angiogenic drugs can be integrated into adjuvant antihormone therapy or to enhance oestrogen-induced apoptosis in Phase II antihormone resistant breast cancer. The goal is to reduce the development of acquired antihormone resistance or undermine the ability of breast cancer cells to undergo apoptosis with oestrogen respectively. Finally, drugs to reduce the synthesis of glutathione, a subcellular molecule compound associated with drug resistance, can enhance oestradiol-induced apoptosis.
We propose an integrated approach for the rapid testing of agents to blunt survival pathways and amplify oestrogen-induced apoptosis and tumour regression in Phase II resistant metastatic breast cancer. This Pharma platform will provide rapid clinical results to predict efficacy in large scale clinical trials.
Tamoxifen (ICI46,474) was not hailed as an impressive breakthrough in the early 1970’s when it was marketed as an orphan drug that produced modest responses in the treatment of metastatic breast cancer in post menopausal women (1). Only one in three tumours responded to treatment for about a year. Nevertheless, side effects with tamoxifen were less than other available endocrine therapies (diethylstilboestrol (DES) or androgens) (2-4).
Despite initial disinterest in endocrine therapy, significant progress was subsequently made in the treatment and chemoprevention of breast cancer through the clinical application of laboratory principles for the antihormonal therapy of breast cancer (5). Today antihormonal therapies (tamoxifen and aromatase inhibitors) target the oestrogen receptor (ER) present in the majority of breast cancers and long term adjuvant therapy with tamoxifen increases patient survival (6, 7). Aromatase inhibitors that are used exclusively in postmenopausal patients improve disease-free survival when compared to tamoxifen, and reduced the risk of endometrial cancer and blood clots noted with tamoxifen (8). Additionally, the application of SERMs for the chemoprevention of breast cancer either directly with tamoxifen and raloxifene (9, 10) or indirectly with raloxifene for the prevention of osteoporosis (11-13) will surely reduce the incidence of breast cancer in select populations over the next decade.
The critical strategy that led to the success of endocrine therapy for the treatment and prevention of breast cancer was the implementation of the laboratory principle of extended durations of treatment (14, 15). However, the consequence of long term treatment is the development of antihormonal drug resistance. Numerous laboratory models of antihormonal drug resistance have been developed over the past 20 years and several valuable principles have emerged. Drug resistance with SERMs evolves through at least two distinct phases: Phase I and Phase II (16) (Figure 1). Phase I resistance to tamoxifen treatment is characterized by either tamoxifen or oestradiol-stimulated growth. Both ligands can exploit the ER signal transduction pathway to aid tumour cell survival. This phase of drug resistance has a clinical equivalent in metastatic breast cancer. When treatment fails during tamoxifen therapy, the tumour has a withdrawal response or regression upon withdrawal of tamoxifen treatment (17). Second line therapy following tamoxifen treatment failure is with either an aromatase inhibitor or fulvestrant (18).
The description of Phase II resistance to tamoxifen was first presented at the St. Gallen Breast Cancer Conference in 1992 (19). Re-transplantation of tamoxifen-resistant MCF-7 breast tumours into tamoxifen treated athymic mice for 5 or more years causes the signaling networks through the ER, that normally act as a survival network, to become reconfigured to be activated by physiological oestradiol that causes rapid apoptosis and triggers tumour regression (20). The fact that these laboratory data pertaining to the evolution of drug resistance to tamoxifen also applies to antihormonal resistance to raloxifene (21), and oestrogen withdrawal (22-24) creates a valid general principle in breast cancer that can now be exploited in the clinic to enhance patient survivorship (25, 26). Indeed, it has been suggested that these current data (22, 27), can explain the effectiveness of high dose oestrogen therapy to treat metastatic breast cancer in post menopausal women before tamoxifen was available (28). High dose DES produces a 35% response rate in unselected patients (29) and interestingly enough Lonning and colleagues (30) reported a 30% response rate for high dose DES in a population of women who have received exhaustive anti-hormonal therapy to treat metastatic breast cancer. Remarkably, one woman has had a complete remission for more than 8 years after starting DES treatment (Per Lonning, personal communication).
We now choose to amplify the clinical potential of short term low oestradiol therapy to treat breast cancer in those patients whose ER positive tumour has responded and failed at least two consecutive antihormonal therapies. Based on the emerging clinical experience and on an expanding laboratory data base we anticipate a 30% clinical benefit (30) We address the question of why tumour cell survival signaling prevents 70% of Phase II tumours from responding to low dose oestradiol and we will advance short and long term solutions to apply pharmacological interventions to sensitize refractory breast cancers to oestradiol’s apoptotic actions.
We have previously proposed a clinical test bed to define the molecular biology and breast tumour responsiveness to both high doses (30 mg daily) and low dose (6 mg daily) oestradiol. That strategy is based on the translation of our laboratory description of the evaluation of anti-hormone resistance through phase I to phase II resistance where oestradiol switches from being a survival signal to an apoptotic trigger (16, 27, 31) The schema for the trial is illustrated in Figure 2. Breast cancer patients who are eligible for recruitment to the trial must have responded and failed two consecutive anti-hormonal therapies e.g. fail tamoxifen adjuvant therapy during year 3-5 and subsequently respond and fail an aromatase inhibitor during the treatment of metastatic breast cancer. In our first protocol of high dose oestradiol therapy (30 mg daily), we are treating for 12 weeks and then responding patients will be treated with an aromatase inhibitor (anastrazole 1 mg daily) until progression (32) Several other investigators have initiated similar clinical trials but with less rigorous entry criteria concerning failure of repeated endocrine therapies. Nevertheless, we contend that the moment is right to address the issue of the regulation of apoptosis and advance the idea that other agents may be synergistic with oestradiol to trigger apoptosis in the predicted 70% of patients that do not respond to short term oestradiol therapy.
Naturally, it is not possible to consider all of the opportunities that could be exploited for patient benefit but we pose three questions that will be answered with an example of current research from our laboratory. The questions will be addressed as an integrated translational research scheme summarized in Figure 3.
The ER with its modulation through co-activators and co-repressors (33) has been investigated extensively through the structure function relationships of ligands that create novel folding of the receptor complex (34). However the array of SERMs is only able to add marginally to advancing cancer therapeutics. We are beginning to understand the cross talk between the ER pathway and growth factor receptor pathways but our basic knowledge is in its infancy. There is an increasing menu of medicines to block growth factor pathways, but the challenge is to place the right targeted agent in the endocrine treatment paradigms. We will return to this challenge in our summary.
Our confidence in the position that “we understand how oestradiol works” has been challenged twice in recent times firstly with the discovery of a second ER referred to as ER-β (ER-α is the classical ER), and secondly with the discovery of the G protein-coupled receptor GPR30, an oestrogen-, SERM- and fulvestrant-binding protein. The role of ER-β in breast cancer is controversial but there is evidence that overexpression of ER-β can inhibit proliferation (35) and cause apoptosis (36). However, ER-β specific ligands have yet to find a role in cancer therapeutics. The G protein-coupled receptor GPR30 is the latest putative receptor that can modulate oestrogen action specifically (37). The molecule, a seven-pass transmembrane receptor located in the endoplasmic reticulum, mediates rapid non-genomic actions of oestradiol to initiate mobilization of intracellular Ca ++ stores.
Based on high through put screening assays a new class of molecules specific for GPR30 has been identified (38), and one compound G1 (Figure 4) is available for laboratory investigations. We have addressed the question of whether the GPR30 agonist G1 is a stimulator or blocker of oestradiol-stimulated growth in the wild-type ER-positive MCF-7 breast cancer cell line and whether G1 can provoke apoptosis in our oestrogen deprivation-resistant cell lines MCF-7:5C (23) and MCF-7:2A (39) data shown in Figure 5 illustrates the fascinating pharmacology of the new drug group. G1 is anti-oestrogenic in the wild type MCF-7 cell line, and enhances apoptosis in both the MCF-7:5C and MCF-7:2A cell lines. Most importantly, G1 induces apoptosis in the MCF-7:2A cells more rapidly than oestradiol. This is important as it provides evidence that in endocrine resistant breast cancer cells, which are initially refractory to the immediate apoptotic actions of oestradiol, there is the potential to circumvent survival and initiate apoptosis quickly via a new pathway. The mechanism of action of G1 in all breast cancer cell lines is the rapid mobilization of high levels of Ca++ from intracellular stores. This increase of Ca++ is cytotoxic, thus, the new drug group has potential to enhance apoptosis in anti-hormone resistant cell lines and further development of these agents may find an application for short term treatment of patients whose tumours are Phase II anti-hormone resistant.
We have probably reached a zenith with what can be achieved with adjuvant antihormonal therapy. Nevertheless, significant increases in efficacy can be achieved by improving compliance for long term adjuvant therapy or selecting out those patients that have variant CYP2D6 that does not metabolize tamoxifen to the active metabolite endoxifen (40). What is required is a new initiative that can significantly enhance the effectiveness of antihormonal therapy and reduce the development of acquired drug resistance and possibly block intrinsic resistance. It could be, that the 40% of ER positive breast cancers that do not respond initially to antihormones could be encouraged to do so by pharmacologic intervention.
Angiogenesis is critical for the growth of tumours and the establishment of metastatic lesions (41). However, antiangiogenic drugs must be integrated into the cancer treatment plan as there are no advantages to monotherapy. As a result there is increasing interest in combining antiangiogenic drugs with cytotoxic chemotherapy with the goal of achieving better tumour responses (42). There has however, been little interest in combining antiangiogenic agents with antihormonal therapy primarily because such long term treatments are required and the effective doses of antiangiogenic drugs have significant side effects that are often life threatening.
The development of acquired resistance to SERMs implies that angiogenic mechanisms must be activated in cancer cells to permit SERM stimulated growth. Indeed, recent research has demonstrated that an autocrine Vascular Endothelial Growth Factor (VEGF) VEGF receptor 2 (VEGFR2) and P38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells (43). Thus, the rationale of combining antihormonal therapy with antiangiogenic therapy has conceptual merit.
We have addressed the idea that low doses of an inhibitor of the VEGFR2 tyrosine kinase could be synergistic with tamoxifen to enhance the control of tumour cell growth in vivo. There is merit to using low doses of small molecule inhibitors of VEGFR2 in treatment regimens as side effects will be reduced and the drug may be sufficient to block the modest, but significant, angiogenic action of tamoxifen. In preliminary studies, we show (Figure 6) that a combination of tamoxifen and a VEGFR2 inhibitor brivanib alaninate is superior to tamoxifen alone at inhibiting oestradiol induced tumour growth in athymic animals. The low dose of brivanib alaninate used does not significantly affect oestradiol-stimulated tumour growth when used alone. We conclude that the angiogenic signal from oestradiol is too strong but that the inhibition of the cell cycle with tamoxifen and the antiangiogenic brivanib alaninate in combination is synergistic.
The issue to be addressed is how to test the concept before committing to large scale adjuvant trials. One approach would be to evaluate efficacy and safety in our proposed model of oestradiol induced apoptosis in Phase II resistant breast cancer (Figure 2). The goal would be to evaluate short term antiangiogenesis treatment by limiting toxicity during the 12 week treatment period and to assess improvements in response rates to physiologic (6mg dose) oestradiol treatment alone. This would also address the third question we pose.
An effective treatment strategy for breast cancer must have a clear goal with the aim of enhancing patient survivorship. The progress (44) being made by translating the laboratory studies(45) of low dose apoptotic oestradiol therapy into clinical practice must be amplified to bring further benefits to a select group of patients. Those patients with Phase II resistant metastatic breast cancer are a significant proportion of all those who respond initially to adjuvant endocrine therapy. The goal is to harness the apoptotic trigger and create an enhanced sensitivity to oestrogen so that a higher proportion of tumours have a complete response to treatment. An application of general pharmacologic principles can be seen as a first step in amplifying oestrogen-induced apoptosis. Inhibitors of angiogenesis would be a logical innovation to aid oestrogen-induced apoptosis. By denying the ability of resistant cells to grow by restricting angiogenesis, may result in cellular instability and to enhance sensitivity to apoptosis. However, it is the critical players in the inhibition of apoptosis that need to be targeted in a broad strategy of combination therapy. It is generally agreed that Bcl-2 plays a central role in preventing the intrinsic apoptosis trigger through the mitochondrial pathway of cytochrome C release. One mechanism by which Bcl-2 may function is as an anti-oxidant by up-regulation of glutathione leading to rapid detoxification of reactive oxygen species and inhibition of free-radical mediated mitochondrial damage.
Glutathione is a water soluble tripeptide composed of glutamine, cysteine, and glycine. Elevated levels prevent apoptotic cell death whereas depletion of glutathione facilitates apoptosis (46) L-Buthionine sulfoximine (BSO) (Figure 7) is a specific inhibitor of glutanylcysteine synthase that blocks the rate limiting step of glutathione biosyntheses.
Recent laboratory studies demonstrate (39) that oestrogen deprived MCF7 cells that are initially refractory to oestradiol induced apoptosis are sensitized to the immediate apoptotic action of oestrogen by BSO at concentrations that can be achieved clinically (47) (Figure 7) Since there is an extensive clinical experience with BSO it would not be unreasonable to integrate the antioxidant concept into the clinical test model (48).
Overall, we are making significant progress towards understanding oestrogen-induced apoptosis and there is evidence that a new drug group based on GPR30 agonists could be developed to provide additional specificity and induce apoptosis in breast cancer. This approach could overcome some resistance in tumour cells observed with oestradiol alone. This, however, is a long term goal and builds on an evolving understanding of the complexities of oestrogen action in cancer. Similarly the anti-angiogenic drugs that block the tyrosine kinase activity of VEGFR2 could undermine the survival of tumours that are refractory to the apoptotic oestrogen trigger. But in practical terms, the application of BSO with an apoptotic oestrogen trigger has immediate clinical applications in our clinical test model (Figure 2). We have presented developing laboratory evidence to support each of these pharmacologic strategies to amplify the apoptotic oestrogen therapy in Phase II resistant breast cancer. However, the mechanism based clinical test model is more than a translational research tool.
Rapid clinical results can be developed through mechanism based targeting of several pathways that have the potential to amplify the apoptotic oestrogen trigger to create a significant increase in complete tumour responses. In Figure 8 are examples of agent classes that could potentially be tested rapidly in the 12 week model against oestrogen alone. This will establish efficacy of a new targeted agent as a clinically useful drug.
The hypothetical, yet systematic, strategy to evaluate selectively the inhibition of survival signals has a foundation in laboratory science. The obvious strategy of blocking the growth factor receptor signal cascade using either trastuzumab or the tyrosine kinase inhibitor lapatinib as an immediate practical approach in the 12 week test model. Recent studies demonstrate that antihormone responsiveness can be restored by aromatase resistant cells using trastuzumab (49) and lapatinib (50) is showing promise in clinical trials of breast cancer with chemotherapy (51).
The mammalian target-of-rapamycin (mTOR) is emerging as an important target for therapeutic intervention in multiple cancer tissue types including breast cancer. mTOR integrates signals from multiple pathways to sense cellular nutrient and energy levels. mTOR is a serine/threonine kinase downstream of PI3K/Akt that, in the presence of mitogenic stimulation and sufficient nutrients, promotes protein translation by activating 40S ribosomal protein S6 kinases (S6K1-2) and inhibiting the eukaryotic initiation factor 4E binding proteins (4E-BP1-3) (52). RAD001 (everolimus) is an orally available mTOR inhibitor that alone and synergistically in combination with the aromatase inhibitor letrozole (53) blocks proliferation and induced apoptosis in MCF-7 and T47D breast cancer cells stably expressing aromatase (53). These and other data have lead to the evaluation of RAD001 in combination with letrozole in a recently completed Phase I clinical trial in patients with advanced breast cancer (54). RAD001 is also currently under evaluation in 15 other breast cancer clinical trials (search of clinicaltrials.gov on 2/26/2009) either as a single agent or in combination with various chemotherapeutics, fulvestrant, aromatase inhibitors, and agents which target EGFR and HER2/ErbB2.
We evaluated RAD001 in an MCF-7 breast cancer xenograft tumour model (MCF-7/E2) grown in athymic mice (Figure 9A) that represents the therapeutic stage of antihormone-based therapy, and in a SERM-resistant (MCF-7/RAL1) xenograft tumour model (Figure 9B) that was selected in vivo by continuous treatment with the SERM raloxifene for greater than 3 years (31, 55). RAD001 inhibited MCF-7/E2 tumour growth in the presence of E2 (E2 + RAD001 vs E2 alone). Additionally, RAD001 in the absence of E2, a situation comparable to combination therapy of RAD001 plus an aromatase inhibitor in the clinic, further reduced MCF-7/E2 growth (RAD001 alone vs. E2 + RAD001). These MCF-7/RAL1 tumours can be considered cross-resistant to oestrogen deprivation (or aromatase inhibitors). However, RAD001 was still effective at blocking growth despite resistance to oestrogen deprivation (RAD001 vs. vehicle). Fulvestrant can be used clinically as a second-line therapy after failure of a first-line antihormone therapy, as illustrated here by fulvestrant inhibiting growth in the MCF-7/RAL1 tumours (fulvestrant vs. vehicle or RAL). Yet the combination of RAD001 plus fulvestrant was superior at blocking growth than either agent alone (Fulvestrant + RAD001 vs. fulvestrant alone and vs. RAD001 alone). Taken together, RAD001 represents a promising therapeutic for use in antihormone-sensitive, and importantly, in antihormone-resistant breast cancer, especially in combination with fulvestrant.
It is clear that other inhibitors of signal transduction pathways (Figure 8)may be useful to enhance estrogen-induced apoptosis such as the MEK inhibitor CI-1040 (56), the farnesyl transferase inhibitor lonafarnib (57) and the cyclin-dependent kinase inhibitor flavopiridol (58). Indeed, inhibitors of CDK may have merit as a short term blocking strategy to enhance apoptosis. The cyclin-dependent kinase inhibitory drugs such as flavopiridol that have been tested clinically and causes apoptosis through an intrinsic pathway dependent on BAX and BAK (58) would be of significant interest in combination with oestradiol to amplify apoptosis.
In summary a whole spectrum of new compounds can now be tested to enhance tumour response to oestrogen with the added advantage that this testing platform can document rapid tumour responses. Combinations could create an optimal cocktail for individual tumours to predict a complete response triggered by oestrogen.
We thank Marybeth Jannotti for preparing the manuscript. Supported by the Department of Defense Breast Program under Award No. BC050277 (VCJ), Center of Excellences SPORE in Breast Cancer CA 89018 (VCJ), R01 GM067156 (VCJ), Fox Chase Cancer Center Core Grant No. NIH P30 CA006927, BMS research grant on angiogenesis (VCJ) the Avon Foundation (VCJ), the Genuardi’s Fund (VCJ), the Weg Fund of the Fox Chase Cancer Center (VCJ), the American Cancer Society Grant IRG-92-027-14 (JSLW), the Hollenbach Family Fund (JSLW), the NIH Career Development Grant K01CA120051-01A2 (JSLW), and 5T32CA10365-03 (RRP).