Philipp Y. Maximov

The discovery and development of selective estrogen receptor modulators (SERMs) for clinical practice.

Selective estrogen receptor modulators (SERMs) are structurally different compounds that interact with intracellular estrogen receptors in target organs as estrogen receptor agonists or antagonists. These drugs have been intensively studied over the past decade and have proven to be a highly versatile group for the treatment of different conditions associated with postmenopausal women’s health, including hormone responsive cancer and osteoporosis. Tamoxifen, a failed contraceptive is currently used to treat all stages of breast cancer, chemoprevention in women at high risk for breast cancer and also has beneficial effects on bone mineral density and serum lipids in postmenopausal women. Raloxifene, a failed breast cancer drug, is the only SERM approved internationally for the prevention and treatment of postmenopausal osteoporosis and vertebral fractures. However, although these SERMs have many benefits, they also have some potentially serious adverse effects, such as thromboembolic disorders and, in the case of tamoxifen, uterine cancer. These adverse effects represent a major concern given that long-term therapy is required to prevent osteoporosis or prevent and treat breast cancer. The search for the ‘ideal’ SERM, which would have estrogenic effects on bone and serum lipids, neutral effects on the uterus, and antiestrogenic effects on breast tissue, but none of the adverse effects associated with current therapies, is currently under way. Ospemifene, lasofoxifene, bazedoxifene and arzoxifene, which are new SERM molecules with potentially greater efficacy and potency than previous SERMs, have been investigated for use in the treatment and prevention of osteoporosis. These drugs have been shown to be comparably effective to conventional hormone replacement therapy in animal models, with potential indications for an improved safety profile. Clinical efficacy data from ongoing phase III trials are available or are awaited for each SERM so that a true understanding of the therapeutic potential of these compounds can be obtained. In this article, we describe the discovery and development of the group of medicines called SERMs. The newer SERMs in late development: ospemifene, lasofoxifene, bazedoxifene, are arzoxifene are described in detail.

The Paradox of Oestradiol-Induced Breast Cancer Cell Growth and Apoptosis.

High dose oestrogen therapy was used as a treatment for postmenopausal patients with breast cancer from the 1950s until the introduction of the safer antioestrogen, tamoxifen in the 1970s. The anti-tumour mechanism of high dose oestrogen therapy remained unknown. There was no enthusiasm to study these signal transduction pathways as oestrogen therapy has almost completely been eliminated from the treatment paradigm. Current use of tamoxifen and the aromatase inhibitors seek to create oestrogen deprivation that prevents the growth of oestrogen stimulated oestrogen receptor (ER) positive breast cancer cells. However, acquired resistance to antihormonal therapy does occur, but it is through investigation of laboratory models that a vulnerability of the cancer cell has been discovered and is being investigated to provide new opportunities in therapy with the potential for discovering new cancer-specific apoptotic drugs. Laboratory models of resistance to raloxifene and tamoxifen, the selective oestrogen receptor modulators (SERMs) and aromatase inhibitors demonstrate an evolution of drug resistance so that after many years of oestrogen deprivation, the ER positive cancer cell reconfigures the survival signal transduction pathways so oestrogen now becomes an apoptotic trigger rather than a survival signal. Current efforts are evaluating the mechanisms of oestrogen-induced apoptosis and how this new biology of oestrogen action can be amplified and enhanced, thereby increasing the value of this therapeutic opportunity for the treatment of breast cancer. Several synergistic approaches to therapeutic enhancement are being advanced which involve drug combinations to impair survival signaling with the use of specific agents and to impair bcl-2 that protects the cancer cell from apoptosis. We highlight the historical understanding of oestrogens role in cell survival and death and specifically illustrate the progress that has been made in the last five years to understand the mechanisms of oestrogen-induced apoptosis. There are opportunities to harness knowledge from this new signal transduction pathway to discover the precise mechanism of this oestrogen-induced apoptotic trigger. Indeed, the new biology of oestrogen action also has significance for understanding the physiology of bone remodeling. Thus, the pathway has a broad appeal in both physiology and cancer research.

The Conformation of the Estrogen Receptor Directs Estrogen-Induced Apoptosis in Breast Cancer: A Hypothesis.

Abstract Background: Estrogens are classified as type I (planar) and type II (angular) based on their structures. In this study, we used triphenylethylenes (TPEs) compounds related to 4-hydroxytamoxifen 4OHT to address the hypothesis that the conformation of the liganded estrogen receptor (ERα) can dictate the E2-induced apoptosis of the ER+ breast cancer cells. Materials and methods: ERα positive MCF7:5C cells were used to study apoptosis induced by E2, 4OHT and TPEs. Growth and apoptosis assays were used to evaluate apoptosis and the ability to reverse E2-induced apoptosis. ERα protein was measured by Western blotting to investigate the destruction of ERα by TPEs in MCF7 cells. Chromatin immunoprecipitation (ChIP) assays were performed to study the in vivo recruitment of ERα and SRC3 at classical E2-responsive promoter TFF1 (PS2) by TPEs. Molecular modeling was used to predict the binding mode of the TPE to the ERα. Results: TPEs were not only unable to induce efficient apoptosis in MCF7:5C cells but also reversed the E2-induced apoptosis similar to 4OHT. Furthermore, the TPEs and 4OHT did not reduce the ERα protein levels unlike E2. ChIP assay confirmed very weak recruitment of SRC3 despite modest recruitment of ERα in the presence of TPEs. Mole-ular modeling suggests that TPE would bind in antagonistic mode with ERα. Conclusion: Our results advances the hypothesis that the TPE liganded ERα complex structurally resembles the 4OHT bound ERα and cannot efficiently recruit co-activator SRC3. As a result, the TPE complex cannot induce apoptosis of ER+ breast cancer cells, although it can cause growth of the breast cancer cells. The conformation of the estrogen-ER complex differentially controls growth and apoptosis.

Estrogen Receptor Mutations Found in Breast Cancer Metastases Integrated With the Molecular Pharmacology of Selective ER Modulators

The consistent reports of mutations at Asp538 and Tyr537 in helix 12 of the ligand-binding domain (LBD) of estrogen receptors (ERs) from antihormone-resistant breast cancer metastases constitute an important advance. The mutant amino acids interact with an anchor amino acid, Asp351, to close the LBD, thereby creating a ligand-free constitutively activated ER. Amino acids Asp 538, Tyr 537, and Asp 351 are known to play a role in either the turnover of ER, the antiestrogenic activity of the ER complex, or the estrogen-like actions of selective ER modulators. A unifying mechanism of action for these amino acids to enhance ER gene activation and growth response is presented. There is a range of mutations described in metastases vs low to zero in primary disease, so the new knowledge is of clinical relevance, thereby confirming an additional mechanism of acquired resistance to antihormone therapy through cell population selection pressure and enrichment during treatment. Circulating tumor cells containing ER mutations can be cultured ex vivo, and tumor tissues can be grown as patient-derived xenografts to add a new dimension for testing drug susceptibility for future drug discovery.

Estrogen-Mediated Mechanisms to Control the Growth and Apoptosis of Breast Cancer Cells. A Translational Research Success Story.

The treatment and prevention of solid tumors have proved to be a major challenge for medical science. The paradigms for success in the treatment of childhood leukemia, Hodgkins disease, Burketts lymphoma, and testicular carcinoma with cytotoxic chemotherapy did not translate to success in solid tumors--the majority of cancers that kill. In contrast, significant success has accrued for patients with breast cancer with antihormone treatments (tamoxifen or aromatase inhibitors) that are proved to enhance survivorship, and remarkably, there are now two approved prevention strategies using either tamoxifen or raloxifene. This was considered impossible 40 years ago. We describe the major clinical advances with nonsteroidal antiestrogens that evolved into selective estrogen receptor modulators (SERMs) which successfully exploited the ER target selectively inside a womans body. The standard paradigm that estrogen stimulates breast cancer growth has been successfully exploited for over 4 decades with therapeutic strategies that block (tamoxifen, raloxifene) or reduce (aromatase inhibitors) circulating estrogens in patients to stop breast tumor growth. But this did not explain why high-dose estrogen treatment that was the standard of care to treat postmenopausal breast cancer for 3 decades before tamoxifen caused tumor regression. This paradox was resolved with the discovery that breast cancer resistance to long-term estrogen deprivation causes tumor regression with physiologic estrogen through apoptosis. The new biology of estrogen action has been utilized to explain the findings in the Womens Health Initiative that conjugated equine estrogen alone given to postmenopausal women, average age 68, will produce a reduction of breast cancer incidence and mortality compared to no treatment. Estrogen is killing nascent breast cancer cells in the ducts of healthy postmenopausal women. The modulation of the ER using multifunctional medicines called SERMs has provided not only significant improvements in womens health and survivorship not anticipated 40 years ago but also has been the catalyst to enhance our knowledge of estrogens apoptotic action that can be further exploited in the future.

Integration of Downstream Signals of Insulin-like Growth Factor-1 Receptor by Endoplasmic Reticulum Stress for Estrogen-Induced Growth or Apoptosis in Breast Cancer Cells

Estrogen (E2) exerts a dual function on E2-deprived breast cancer cells, with both initial proliferation and subsequent induction of stress responses to cause apoptosis. However, the mechanism by which E2 integrally regulates cell growth or apoptosis-associated pathways remains to be elucidated. Here, E2 deprivation results in many alterations in stress-responsive pathways. For instance, E2-deprived breast cancer cells had higher basal levels of stress-activated protein kinase, c-Jun N-terminal kinase (JNK), compared with wild-type MCF-7 cells. E2 treatment further constitutively activated JNK after 24 hours. However, inhibition of JNK (SP600125) was unable to abolish E2- induced apoptosis, whereas SP600125 alone arrested cells at the G2 phase of the cell cycle and increased apoptosis. Further examination showed that inhibition of JNK increased gene expression of TNFα and did not effectively attenuate expression of apoptosis-related genes induced by E2. A notable finding was that E2 regulated both JNK and Akt as the downstream signals of insulin-like growth factor-1 receptor (IGFIR)/PI3K, but with distinctive modulation patterns: JNK was constitutively activated, whereas Akt and Akt-associated proteins, such as PTEN and mTOR, were selectively degraded. Endoplasmic reticulum–associated degradation (ERAD) was involved in the selective protein degradation. These findings highlight a novel IGFIR/PI3K/JNK axis that plays a proliferative role during the prelude to E2-induced apoptosis and that the endoplasmic reticulum is a key regulatory site to decide cell fate after E2 treatment. Implications: This study provides a new rationale for further exploration of E2-induced apoptosis to improve clinical benefit. Mol Cancer Res; 13(10); 1367–76. ©2015 AACR.

Simulation with cells in vitro of tamoxifen treatment in premenopausal breast cancer patients with different CYP2D6 genotypes

Tamoxifen is a prodrug that is metabolically activated by 4‐hydroxylation to the potent primary metabolite 4‐hydroxytamoxifen (4OHT) or via another primary metabolite N‐desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti‐oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment).

Influence of the Length and Positioning of the Antiestrogenic Side Chain of Endoxifen and 4-Hydroxytamoxifen on Gene Activation and Growth of Estrogen Receptor Positive Cancer Cells

Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.

Novel Selective Estrogen Mimics for the Treatment of Tamoxifen-Resistant Breast Cancer

Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2. Mol Cancer Ther; 13(11); 2515–26. ©2014 AACR.

Pharmacological Relevance of Endoxifen in a Laboratory Simulation of Breast Cancer in Postmenopausal Patients

BACKGROUND Tamoxifen is metabolically activated via a CYP2D6 enzyme system to the more potent hydroxylated derivatives 4-hydroxytamoxifen and endoxifen. This study addresses the pharmacological importance of endoxifen by simulating clinical scenarios in vitro. METHODS Clinical levels of tamoxifen metabolites in postmenopausal breast cancer patients previously genotyped for CYP2D6 were used in vitro along with clinical estrogen levels (estrone and estradiol) in postmenopausal patients determined in previous studies. The biological effects on cell growth were evaluated in a panel of estrogen receptor-positive breast cancer cell lines via cell proliferation assays and real-time polymerase chain reaction (PCR). Data were analyzed with one- and two-way analysis of variance and Students t test. All statistical tests were two-sided. RESULTS Postmenopausal levels of estrogen-induced proliferation of all test breast cancer cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 11 ± 1.74, P < .001; T47D = 7.52 ± 0.72, P < .001; BT474 = 1.75 ± 0.23, P < .001; ZR-75-1 = 5.5 ± 1.95, P = .001. Tamoxifen and primary metabolites completely inhibited cell growth regardless of the CYP2D6 genotype in all cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 1.57 ± 0.38, P = .54; T47D = 1.17 ± 0.23, P = .79; BT474 = 0.96 ± 0.2, P = .98; ZR-75-1 = 0.86 ± 0.67, P = .99). Interestingly, tamoxifen and its primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. CONCLUSIONS The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting.