Active Metabolite Of Tamoxifen Research Articles

Abstract LB051: Protein kinase C beta 1 (PKCβ1) is allosterically inhibited and paradoxically translocated to the membrane by z-endoxifen

Abstract The protein kinase C (PKC) family of enzymes are indispensable serine-threonine kinases involved in signal pathways implicated in many different cellular processes. Dysregulation of PKC isoforms may contribute to the development and progression of breast cancer. Z-Endoxifen (Z-ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM) exhibited potent antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancers. Mechanistically, we discovered that ENDX inhibits PKCβI and downstream AKT signaling. Based on insights from our recently determined crystal structure of PKCβI, we investigated the effects of ENDX on PKCβI in estrogen receptor-positive (ER+) breast cancer. In vitro FRET-based biochemical assays revealed that ENDX inhibits the kinase activity of conventional (PKCβI) as well as a novel (PKC;) PKC isoform with a similar IC50. However, the PKCβI catalytic domain was found to be less sensitive to ENDX compared to full-length PKCβI. We identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory model using X-ray crystallography. Since the spatial assembly of PKC is crucial for its kinase activity, we assessed the effects of ENDX on the intracellular localization of PKCβI using live cell confocal imaging. Sub-cellular tracking of YFP-tagged PKCβI in ER+ MCF7 cells paradoxically revealed that ENDX promoted the translocation of PKCβI to the plasma membrane in both a dose and time-dependent manner. Furthermore, co-treatment with inhibitors of PHLPP phosphatases, which regulate PKC, alleviated this translocation. Based on our prior data demonstrating greater antitumor activity of the Z vs E isomer of ENDX, we discovered that Z-ENDX inhibits PKCβI kinase activity more effectively than its E-isomer using in vitro kinase assays. Further, E-ENDX failed to promote PKCβI translocation to the membrane.Taken together, these results suggest that allosteric effects of Z-ENDX trigger PKCβI recruitment to the plasma membrane where it is normally active yet suppresses its kinase activity. These findings indicate that Z-ENDX induces a non-productive structure of the enzyme and "breaks" the well-known mechanism of PKC activation upon binding to the cell membrane. Moreover, Z-ENDX likely triggers PHLPP-mediated PKCβI dephosphorylation, and ultimately PKCβI degradation, suggesting that Z-ENDX represents a new mechanistic basis for targeting and downregulating PKCβI, and potentially other PKC family members, in cancer cells. This newly discovered function of Z-ENDX likely contributes to its superior anti-cancer effects when compared to TAM and other endocrine therapies. Citation Format: Sayantani Sarkar Bhattacharya, Huy V. Huynh, Jasmin K. Farmakes, Taylor L. Witter, Elizabeth S. Bruinsma, Swaathi Jayaraman, Anh T. Cong, John R. Hawse, Matthew P. Goetz, Matthew J. Schellenberg. Protein kinase C beta 1 (PKCβ1) is allosterically inhibited and paradoxically translocated to the membrane by z-endoxifen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB051.

Whole mount of adult ear skin as a model to study vascular malformations.

Genetic analysis in human patients has linked mutations in PIK3CA, the catalytic subunit of PI-3'Kinase, to sporadic incidences of vascular malformations. We have developed a mouse model with inducible and endothelial-specific expression of PIK3CAH1047R , resulting in the development of vascular malformations. Systemic induction of this mutation in adult mice results in rapid lethality, limiting our ability to track and study these lesions; therefore, we developed a topical and local induction protocol using the active metabolite of tamoxifen, 4OH-T, on the ear skin of adults. This approach allows us to successfully model the human disease in a mature and established vascular bed and track the development of vascular malformations. To validate the utility of this model, we applied a topical rapamycin ointment, as rapamycin is therapeutically beneficial to patients in clinical trials. We found that the induced ear lesions showed significant attenuation after treatment, which was easily quantified. These data collectively provide evidence of a new model to study vascular malformations in adult tissues, which should be particularly useful in environments lacking specialized small-animal imaging facilities.

Evaluation of a polymeric topical formulation of Endoxifen in an estrogen receptor positive breast cancer murine model

Breast cancer (BC) has surpassed lung cancer as the most diagnosed cancer and, in terms of mortality, is the fifth leading cause with 684,996 new deaths (6.7% of all cancer-related deaths) and the highest mortality amongst all cancers (15.5%) in women. Selective estrogen-receptor modulators (SERMs) have been used for the last thirty years for estrogen receptor-positive (ER+) BC prevention and treatment. Tamoxifen (TAM), the most widely used SERM, is orally administered and its long-term oral administration has been associated to toxicity and adverse side effects. Endoxifen (EDX) is one of the known active metabolites of TAM, with an affinity to ERα 100 times higher than TAM. Furthermore, EDX has shown antiproliferative activity against the ER+ BC cell line MCF-7. Alternative administration routes that avoid the metabolic processing of TAM seem an appealing alternative to its oral administration. With this aim, we have prepared a polymeric gel-like solution of Pluronic® F127 as vehicle for topical administration of EDX. In order to shed light on the potential clinical use of this formulation, we have compared it with the standard pharmaceutical form, i.e. orally administered TAM. The biodistribution, antitumor efficacy and toxic effects of topical EDX and oral TAM were evaluated in ER+ tumor xenograft athymic nu/nu mouse models. The results showed a statistically significant antitumor effect and reduced toxicity of topical EDX as compared to oral TAM or empty F127 gel. This novel administration route of SERMs could also have a strong impact in the prevention of BC at early development stages and could help to ameliorate the mortality and morbidity related to this disease.

A randomized phase 2 non-inferiority trial of (Z)-endoxifen and exemestane + goserelin as neoadjuvant treatment for premenopausal women with ER+/HER2- breast cancer (EVANGELINE).

TPS633 Background: Ovarian function suppression (OFS) when combined with tamoxifen or an aromatase inhibitor is the standard of care for premenopausal estrogen receptor positive (ER+) human epidermal growth factor receptor 2 negative (HER2-) breast cancer (BC) based on SOFT (Francis 2022) and TEXT (Pagani 2022) clinical trials and confirmed in a metanalysis (EBCTCG 2022). However, estrogen deprivation in premenopausal women is associated with significant morbidity and up to 40% of premenopausal women with ER+/HER2- BC are intolerant of OFS. For these women, tamoxifen monotherapy is the only FDA approved option. Therefore, there is substantial room to improve endocrine therapy for premenopausal women with ER+/HER2- BC. (Z)-endoxifen is one of the most active anti-estrogen tamoxifen metabolites, with preclinical data demonstrating superiority to tamoxifen and letrozole in aromatase expressing xenograft models and evidence for antitumor activity in early phase clinical trials in patients with prior progression on endocrine therapy including tamoxifen. In addition to its ability to potently block ERa, (Z)-endoxifen at clinically achievable concentrations inhibits protein kinase C beta 1 (PKCβ1), resulting in downstream AKT inhibition and apoptosis. It is hypothesized that (Z)-endoxifen, when delivered as monotherapy at doses that dually target both ERα and PKCβ1, will potently inhibit BC proliferation while obviating the need for OFS in premenopausal women. If successful, this innovative approach could spare women with endocrine sensitive BC from the side effects of OFS. Methods: To further study (Z)-endoxifen in premenopausal women, a multicenter study in the United States, with possible expansion to other countries was designed (EVANGELINE; NCT05607004). This is a Phase 2, open-label, randomized, neoadjuvant study in premenopausal women with Stage IIA or IIB ER+/HER2- BC. Subjects are randomized 1:1 to either exemestane plus goserelin or (Z)-endoxifen. The primary objective is to determine whether the endocrine sensitive disease (ESD) rate (defined as Ki-67 ≤ 10% after 4 weeks of neoadjuvant therapy) with (Z)-endoxifen is non-inferior to exemestane plus goserelin. Subjects will be enrolled with the intent of surgical treatment in the involved breast(s) after completing 6 months of neoadjuvant treatment. Safety, tolerability, response rates and surgical outcomes will be assessed. Disease progression will be monitored throughout study participation. Paired tumor biopsies and blood sampling will allow for a robust biomarker evaluation. Prior to initiating the Treatment Cohort (162 subjects), up to 12 subjects will be enrolled in a pharmacokinetic (PK) Run-in Cohort to identify the optimal dose for the Treatment Cohort. Enrolment in the PK Run-in cohort began in February 2023. Clinical trial information: NCT05607004 .

Development and validation of a liquid chromatography-mass spectrometry assay for quantification of Z- and E- isomers of endoxifen and its metabolites in plasma from women with estrogen receptor positive breast cancer

The selective estrogen receptor modifier tamoxifen (TAM) is widely used for the treatment of women with estrogen receptor positive (ER+ ) breast cancer. Endoxifen (ENDX) is a potent, active metabolite of TAM and is important for TAM’s clinical activity. While multiple papers have been published regarding TAM metabolism, few studies have examined or quantified the metabolism of ENDX. To quantify ENDX and its metabolites in patient plasma samples, we have developed and validated a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of the E- and Z-isomers of ENDX (0.5–500 ng/ml) and the ENDX metabolites norendoxifen (1–500 and 0.5–500 ng/ml E and Z, respectfully), ENDX catechol (3.075–307.5 and 1.92–192 ng/ml E and Z, respectfully), 4′-hydroxy ENDX (0.33–166.5 and 0.33–333.5 ng/ml E and Z, respectfully), ENDX methoxycatechol (0.3–300 and 0.2–200 ng/ml E and Z, respectfully), and ENDX glucuronide (2–200 and 3–300 ng/ml E and Z, respectfully) in human plasma. Chromatographic separation was accomplished on a HSS T3 precolumn attached to an Poroshell 120 EC-C18 analytical column using 0.1 % formic acid/water and 0.1 % formic acid/methanol as eluents followed by MS/MS detection. The analytical run time was 6.5 min. Standard curves were linear (R2 ≥ 0.98) over the concentration ranges. The intra- and inter-day precision and accuracy, determined at high-, middle-, and low-quality control concentrations for all analytes, were within the acceptable range of 85 % and 115 %. The average percent recoveries were all above 90 %. The method was successfully applied to clinical plasma samples from a Phase I study of daily oral Z-ENDX.

Abstract P2-24-03: Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

Value of therapeutic drug monitoring of endoxifen in Egyptian premenopausal patients with breast cancer given tamoxifen adjuvant therapy: A pilot study.

The complex metabolic profile of tamoxifen anticancer drug and polymorphism in its metabolizing enzymes particularly CYP2D6 contribute to the high-observed inter-individual variability in its main active metabolite endoxifen. Therapeutic drug monitoring of endoxifen may play a key role in optimizing tamoxifen therapy, and control of both adverse effects and cancer recurrence. This pilot study aims to assess the clinical benefits of applying endoxifen measurement during tamoxifen therapy in patients with breast cancer. Adult premenopausal breast cancer patients ≥ 18 years who received tamoxifen at a fixed dose of 20 mg daily were included. The primary endpoint was to identify the inter-subject variability in serum concentration of the drug and its metabolites especially endoxifen, through fixation of the tamoxifen dose. The secondary endpoint was to check the correlation between endoxifen metabolite concentration and the development of tamoxifen's adverse effects and cancer recurrence. Sixty patients were included in the study with a mean age of 38.4 ± 0.6 years (range: 26-50). The mean concentration of tamoxifen and endoxifen was 181 ± 9.6 ng/mL and 31.49 ng/mL, respectively. The inter-individual variability in concentrations for the drug and its active metabolite as estimated by the coefficient of variation percentage was in 41% and 31%, respectively. Cancer recurrence was observed in a group of patients (n = 16) with an average endoxifen level of 24.48 ng/mL. Another group of patients (n = 25) developed different tamoxifen adverse effects including hot flashes, vaginal bleeding, endometrial thickness, and ovarian cysts with the average endoxifen level of 38.61 ng/mL. The rest of the patients (n = 19) who responded smoothly to the drug with no complications had an average endoxifen level of 31.37 ng/mL. Analysis of variance test showed a significant difference in endoxifen levels between the three groups (p = 0.002). The measurement of the endoxifen active metabolite of tamoxifen in breast cancer patients can help dose optimization in light of the observed wide inter-individual variability in drug fixed-dose related concentration of the metabolite. Monitoring of serum concentration of endoxifen can help to reveal, reduce and control tamoxifen's adverse effects and cancer recurrence.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

Bioavailability and Pharmacokinetics of Endoxifen in Female Rats and Dogs: Evidence to Support the Use of Endoxifen to Overcome the Limitations of CYP2D6-Mediated Tamoxifen Metabolism.

Endoxifen (ENDX) is an active metabolite of tamoxifen (TAM), a drug commonly used for the treatment of estrogen receptor-positive breast cancer and metabolized by CYP2D6. Genetic or drug-induced reductions in CYP2D6 activity decrease plasma ENDX concentrations and TAM efficacy. It was proposed that direct oral administration of ENDX would circumvent the issues related to metabolic activation of TAM by CYP2D6 and increase patient response. Here, we characterized the pharmacokinetics and oral bioavailability of ENDX in female rats and dogs. Additionally, ENDX exposure was compared following equivalent doses of ENDX and TAM. ENDX exposure was 100-fold and 10-fold greater in rats and dogs, respectively, with ENDX administration compared with an equivalent dose of TAM. In single-dose administration studies, the terminal elimination half-life and plasma clearance values were 6.3 hours and 2.4 L/h per kg in rats given 2 mg/kg i.v. ENDX and 9.2 hours and 0.4 L/h/kg in dogs given 0.5 mg/kg i.v. ENDX, respectively. Plasma concentrations above 0.1 µM and 1 µM ENDX were achieved with 20-mg/kg and 200-mg/kg doses in rats, and concentrations above 1 µM and 10 µM were achieved with 15-mg/kg and 100-mg/kg doses in dogs. Oral absorption of ENDX was linear in rats and dogs, with bioavailability greater than 67% in rats and greater than 50% in dogs. In repeated-dose administration studies, ENDX peak plasma concentrations reached 9 µM in rats and 20 µM in dogs following four daily doses of 200 mg/kg or 30 mg/kg ENDX, respectively. The results indicate that ENDX has high oral bioavailability, and therapeutic concentrations were maintained after repeated dosing. Oral dosing of ENDX resulted in substantially higher ENDX concentrations than a similar dose of TAM. These data support the ongoing development of ENDX to overcome the limitations associated with CYP2D6-mediated metabolism of TAM in humans. SIGNIFICANCE STATEMENT: This study presents for the first time the pharmacokinetics and bioavailability of endoxifen and three key tamoxifen metabolites following repeated oral dosing in female rats and dogs. This study reports that endoxifen has high oral bioavailability, and therapeutic concentrations were maintained after repeated dosing. On the basis of these data, Z-endoxifen (Z-ENDX) was developed as a drug based upon the hypothesis that oral administration of Z-ENDX would overcome the limitations of CYP2D6 metabolism required for full metabolic activation of tamoxifen.

Combinatorial treatments of tamoxifen and SM6Met, an extract from Cyclopia subternata Vogel, are superior to either treatment alone in MCF-7 cells.

Synergistic drug combinations are not only popular in antibiotic, anti-microbial, immune disease (i.e., AIDS) and viral infection studies, but has also gained traction in the field of cancer research as a multi-targeted approach. It has the potential to lower the doses needed of standard of care (SOC) therapeutic agents, whilst maintaining an effective therapeutic level. Lower dosages could ameliorate the fundamental problems such as drug resistance and metastasis associated with current SOC therapies. In the current study, we show that the combination of SM6Met with (2)-4-hydroxytamoxifen (4-OH-Tam, the active metabolite of tamoxifen) produces a strong synergistic effect in terms of inhibiting MCF7 ER-positive (ER+) breast cancer cell proliferation and that a 20 times lower dose of 4-OH-Tam in combination with SM6Met is required to produce the same inhibitory effect on cell proliferation as 4-OH-Tam on its own. Cell cycle analyses of the best combination ratios of SM6Met and 4-OH-Tam also suggests that the combination results in increased accumulation of cells in the S-phase and in the apoptotic phase. Moreover, the best combination ratio (20:1) of SM6Met with 4-OH-Tam displayed greater anti-metastatic potential in terms of inhibiting ER+ breast cancer cell migration, invasion, and colony formation than the SOC therapy alone, suggesting that SM6Met together with 4-OH-Tam could be a viable drug combination for not only delaying resistance and ameliorating the negative side-effects associated with current SOC therapies, like tamoxifen, but could also provide a novel, more affordable therapeutic alternative for treating or preventing ER+ breast cancer metastasis.

Cdh5-lineage-independent origin of dermal lymphatics shown by temporally restricted lineage tracing.

The developmental origins of lymphatic endothelial cells (LECs) have been under intense research after a century-long debate. Although previously thought to be of solely venous endothelial origin, additional sources of LECs were recently identified in multiple tissues in mice. Here, we investigated the regional differences in the origin(s) of the dermal lymphatic vasculature by lineage tracing using the pan-endothelial Cdh5-CreER T2 line. Tamoxifen-induced labeling of blood ECs at E9.5, before initiation of lymphatic development, traced most of the dermal LECs but with lower efficiency in the lumbar compared with the cervical skin. By contrast, when used at E9.5 but not at E11.5, 4-hydroxytamoxifen, the active metabolite of tamoxifen that provides a tighter window of Cre activity, revealed low labeling frequency of LECs, and lymphvasculogenic clusters in the lumbar skin in particular. Temporally restricted lineage tracing thus reveals contribution of LECs of Cdh5-lineage-independent origin to dermal lymphatic vasculature. Our results further highlight Cre induction strategy as a critical parameter in defining the temporal window for stage-specific lineage tracing during early developmental stages of rapid tissue differentiation.

(Z)-Endoxifen and Early Recurrence of Breast Cancer: An Explorative Analysis in a Prospective Brazilian Study.

Adherence to treatment and use of co-medication, but also molecular factors such as CYP2D6 genotype, affect tamoxifen metabolism, with consequences for early breast cancer prognosis. In a prospective study of 149 tamoxifen-treated early-stage breast cancer patients from Brazil followed up for 5 years, we investigated the association between the active tamoxifen metabolite (Z)-endoxifen at 3 months and event-free survival (EFS) adjusted for clinico-pathological factors. Twenty-five patients (16.8%) had recurred or died at a median follow-up of 52.3 months. When we applied a putative 15 nM threshold used in previous independent studies, (Z)-endoxifen levels below the threshold showed an association with shorter EFS in univariate analysis (p = 0.045) and after adjustment for stage (HR 2.52; 95% CI 1.13–5.65; p = 0.024). However, modeling of plasma concentrations with splines instead of dichotomization did not verify a significant association with EFS (univariate analysis: p = 0.158; adjusted for stage: p = 0.117). Hence, in our small exploratory study, the link between impaired tamoxifen metabolism and early breast cancer recurrence could not be unanimously demonstrated. This inconsistency justifies larger modeling studies backed up by mechanistic pharmacodynamic analyses to shed new light on this suspected association and the stipulation of an appropriate predictive (Z)-endoxifen threshold.

Tamoxifen treatment ameliorates contractile dysfunction of Duchenne muscular dystrophy stem cell-derived cardiomyocytes on bioengineered substrates

Duchenne muscular dystrophy (DMD) is a progressive genetic myopathy that leads to heart failure from dilated cardiomyopathy by early adulthood. Recent evidence suggests that tamoxifen, a selective estrogen receptor modulator widely used to treat breast cancer, ameliorates DMD cardiomyopathy. However, the mechanism of action of 4-hydroxytamoxifen, the active metabolite of tamoxifen, on cardiomyocyte function remains unclear. To examine the effects of chronic 4-hydroxytamoxifen treatment, we used state-of-the-art human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and a bioengineered platform to model DMD. We assessed the beating rate and beating velocity of iPSC-CMs in monolayers and as single cells on micropatterns that promote a physiological cardiomyocyte morphology. We found that 4-hydroxytamoxifen treatment of DMD iPSC-CMs decreased beating rate, increased beating velocity, and ameliorated calcium-handling deficits, leading to prolonged viability. Our study highlights the utility of a bioengineered iPSC-CM platform for drug testing and underscores the potential of repurposing tamoxifen as a therapy for DMD cardiomyopathy.

Metformin reverses tamoxifen resistance through the lncRNA GAS5-medicated mTOR pathway in breast cancer

BackgroundMetformin (Met) has antitumor effects on various cancers, but it is still unclear whether it exerts a reversible effect on endocrine resistance in breast cancer (BC) patients. In the present survey, metformin’s effects on tamoxifen-resistant MCF cells (The cell line was established at the Michigan Cancer Foundation, hence the name MCF cell) were evaluated and the molecular mechanism was explored.MethodsWe constructed a tamoxifen-resistant BC cell line MCF-7R, then applied Cell Counting Kit-8 (CCK-8), flow cytometry, and EdU assessments to determine the growth and apoptosis of MCF-7R cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the expression level of lncRNA GAS5 and the mTOR-associated proteins. Overexpression and downregulation of lncRNA GAS5 were carried out to explore the molecular mechanism of Met.ResultsThe combined effect of Met and OHT (4-hydroxytamoxifen, main active metabolite of tamoxifen) was to remarkably reduce the growth and increase the apoptosis of MCF-7R cells compared with OHT alone. The levels of the mTOR, p-mTOR and p-P70S6K proteins in MCF-7R cells decreased after Met treatment, but the p-AMPK2 and PTEN proteins significantly increased. The overexpression of lncRNA GAS5 in MCF-7R cells inhibited cell growth with decreased mTOR, p-mTOR, PCNA, and Bcl-2 proteins. Metformin had no significant effect on MCF-7R cells with lncRNA GAS5 knockdown but contrarily activated p-mTOR.ConclusionsMetformin can inhibit overactivation of the mTOR signaling pathway through upregulating lncRNA GAS5 expression, thereby inhibiting the growth and inducing the apoptosis of BC cells, providing a new clinical treatment for BC.

Abstract P1-07-06: Endoxifen exposure after 5 weeks of preoperative tamoxifen is predictive of long-term outcome of operable hormone receptor positive (HR+) HER2 - early breast cancer

Abstract Introduction: Endoxifen exposure, the most active metabolite of tamoxifen, may be a better predictor of tamoxifen efficacy, than CYP2D6 genotypes. However, conflicting results have been reported so far. We evaluated prospectively the impact of 5 weeks of pre-operative tamoxifen on Ki-67 levels in early-stage breast cancer (EBC) patients. We correlated CYP2D6 genotype, exposure of tamoxifen and its metabolites with long-term outcome. Patients and methods: TAM pre-op was an open-label, multicentre, phase 2 trial in which pre and postmenopausal women with WHO performance status 0-1 and HR+ HER2-, operable breast cancer were given tamoxifen (20mg per day orally) for 5 weeks before surgery. Adjuvant treatments were given as standard practice. CYP2D6 genotype was determined at initiation. Plasma concentrations of tamoxifen and its main metabolites (N-desmethyltamoxifen, Z-endoxifen, and Z-4-hydroxytamoxifen) were assessed at baseline (b), week 2 (w2) and week 5 (w5) of treatment, using a validated performed LC-MS/MS method. AUCs, used as marker of exposure, were computed with Monolix© (Lixoft, Antony, France). Tumour Ki-67 variations between baseline (Ki-67b) and w5 (Ki-67w5) were determined to assess the sensitivity to tamoxifen. We used a pre-defined 10% cut-off to dichotomize low versus high Ki-67. All these parameters were associated with disease free survival by Cox regression analyses. Results: Out of the 136 patients enrolled, 98 pts had paired Ki-67 values evaluable. With a median follow up of 78 months (IQR 58-95), 14/136 women had cancer recurrence (12 distant and 2 contralateral) with the proportion free from cancer recurrence at 5 years of 90% (95% CI 83-94). Ki-67b, Ki-67w5 and variations between baseline and week 5 were not associated with DFS. Endoxifen AUC was correlated with CYP2D6 genotype (normal/hyper metabolizer versus low metabolizer) but CYP2D6 genotype was not predictive for DFS (adjusted hazard ratio, 2.24; 95% CI, 0.29 to 17.37; P = 0.44). Endoxifen AUCs at w2 and w5 were similar in patients with low Ki67b and low Ki67w5 (low-low) (n=36), low Ki67b and high Ki67w5 (n=12), high Ki67b and low Ki67w5 (n=14) and high Ki67b and high Ki67w5 (n=30). However, low endoxifen AUC at w5 was a pejorative factor of disease free survival: 5 years PFS 81.8% vs 90.3% (HR 2.70, CI 95% [1.04-7.06], p=0.04). Conclusion: This prospective clinical study shows that endoxifen exposure at w5 of pre-operative tamoxifen is associated with DFS in patients with HR+/HER2- EBC. Translationnal studies are ongoing. N°EUDRACT 2008-007652-10. Citation Format: Jean-Sebastien Frenel, Loic Campion, Antonin Schmitt, Patricia De Cremoux, Elsa Rossignol, Celine Renaudeau, Marie Robert, Jean-Marie Bard, Christine Bobin-Dubigeon, Mario Campone. Endoxifen exposure after 5 weeks of preoperative tamoxifen is predictive of long-term outcome of operable hormone receptor positive (HR+) HER2 - early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-06.

Abstract P1-07-08: Endoxifen predicts early recurrence in breast cancer: A Brazilian prospective study

Abstract Tamoxifen is widely used in adjuvant therapy for breast cancer patients, but up to one-third of patients recur within 15 years after surgery even with its regular use. Low plasma levels of (Z)-endoxifen, the most active tamoxifen metabolite, have been studied as a possible predictor of treatment failure. Here, we evaluated plasma (Z)-endoxifen as a predictor of response to adjuvant treatment in breast cancer patients. Methods: One hundred forty nine patients were selected from a prospective cohort of consecutive newly diagnosed breast cancer patients recruited at a national referral center for treatment of cancer in Curitiba, Southern Brazil. Inclusion criteria were breast cancer women on stage I - III, positive hormone receptor tumors, operable, undergoing or not (neo)adjuvant chemotherapy, and signed for adjuvant tamoxifen for at least five years. At diagnosis, peripheral blood samples were collected for CYP2D6 genotyping for *1 *2, *3, *4, *5, *6, *7, *8, *9, *10, *17, *29 and *41 and subsequent metabolic activity scores according to phenotypes assigned for each allele (poor metabolizer PM; intermediate, IM; extensive, EM and ultrarapid metabolizer, UM). After the tamoxifen initiation, patients were evaluated at months 3, 6, and 12 for adherence and drug interactions, and plasma (Z)-endoxifen quantification. They were followed from April 2014 to April 2019. The study's primary objective was to evaluate whether (Z)-endoxifen plasma levels predict early recurrence in patients on adjuvant tamoxifen. The secondary aim was to evaluate an association between (Z)-endoxifen levels and clinical and phenotypic CYP2D6 metabolism variables. A Cox model estimated hazard ratios (HR) comparing recurrence free survival (RFS) according to (Z)-endoxifen plasma levels. The survival was compared using the Kaplan-Meier method and the log-rank test. Results: The median follow-up period was 52.3 months and at that point, there were 20 recurrences of 149 patients. At 3 months there were no cases of low adherence to tamoxifen. The (Z)-endoxifen levels were established by quartiles, with the first lower quartile being used as a cutoff (14.72nM). High (Z)-endoxifen levels at 3 months (>14.72nM) were detected in 75.8% (n = 113) of cases and low levels (≤14.72nM) in 24.2% (n = 36). There was no statistically significant difference between the groups with low or high (Z)-endoxifen levels regarding clinical and pathological variables, but there was a higher CYP2D6 PM/IM frequency in the low (Z)-endoxifen levels group (p<0.001). High (Z)-endoxifen levels were associated with lower RFS compared to low (Z)-endoxifen levels (HR 2.5, 95% CI 1.00-6.22, p=0.041). Low (Z)-endoxifen levels increased the recurrence risk even when adjusted for staging and molecular subtypes (HR 2.55, 95% CI 1.00-6.48, p<0.001) or staging and CYP2D6 phenotype (UM/EM vs IM/PM; HR 4.38, 95% CI 1.66-11.59, p=0.003), or prior chemotherapy exposure (HR 2.65, 95% CI 1.06-6.61, p=0.036). Conclusion: This is the first Brazilian prospective trial evaluating the association of plasma (Z)-endoxifen levels, with the clinical outcome in breast cancer women treated with adjuvant tamoxifen, controlled by predictable confounding factors of plasma (Z)-endoxifen levels, such as tamoxifen adherence, and drug interaction with CYP2D6 inhibitors. In our study, a low plasma level of (Z)-endoxifen (≤ 14.72nM) after 3 months of tamoxifen treatment was a predictor of early recurrence in patients under adjuvant tamoxifen (p=0.041). A longer follow-up and a larger cohort may be needed to confirm the (Z)-endoxifen cutoff value that is a predictor of long-term recurrence. Citation Format: Thais Almeida, Werner Schroth, Jeanine Marie Nardin, Thomas Mürdter, Solane Picolotto, Reiner Hoppe, Jenifer Primon Kogin, Elisa Daniele Gaio, Angela Dasenbrock, Raquel Cristina Skrsypcsak, Lucia Noronha, Hiltrud Brauch, José Claudio Casali-da-Rocha. Endoxifen predicts early recurrence in breast cancer: A Brazilian prospective study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-08.

Beneficial effects of transdermal administration of tamoxifen on capsular contracture after breast implantation in murine models.

Capsular contracture is the most common complication with smooth-type silicone implants. We investigated the preventive effect of an active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OH TAM), on capsular contracture. A silicone sheet was implanted into the back of 28 female ICR mice. Mixtures of gel with 0.2% 4-OH TAM and 0.1% 4-OH TAM were administered transdermally once a day for 4weeks. Saline was administered to the control. After killing the mice, capsular thickness was measured in H&E-stained specimens. Estrogen receptor (ER), α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) expressions were immunohistochemically investigated in the capsules. The capsule was thinner in the 0.2% 4-OH TAM gel group than in the control group (control, 0.1% 4-OH TAM gel, 0.2% 4-OH TAM gel: 52.8 ± 3.4µm, 54.2 ± 6.8µm, 46.4 ± 3.3µm, respectively). ER was found in most fibroblasts of all samples. α-SMA expression in the capsule was significantly lower in the 4-OH TAM gel groups than in the control group (control = 70.0 ± 3.4%, 0.1% 4-OH TAM = 57.0 ± 3.4%, 0.2% 4-OH TAM = 49.4 ± 4.9%). TGF-β expression was significantly reduced by the 4-OH TAM gel injections dose-dependently (control = 67.3 ± 2.2%, 0.1% 4-OH TAM = 52.4 ± 3.1%, 0.2% 4-OH TAM = 45.1 ± 2.4%). The transdermal administration of 0.1% and 0.2% 4-OH TAM gels inhibited capsule development. The inhibition of TGF-β expression is a mechanism by which 4-OH TAM suppresses fibroblast growth, preventing capsular formation.

Evaluating the Risk of Breast Cancer Recurrence and Metastasis After Adjuvant Tamoxifen Therapy by Integrating Polymorphisms in Cytochrome P450 Genes and Clinicopathological Characteristics.

Tamoxifen (TAM) is the most commonly used adjuvant endocrine drug for hormone receptor-positive (HR+) breast cancer patients. However, how to accurately evaluate the risk of breast cancer recurrence and metastasis after adjuvant TAM therapy is still a major concern. In recent years, many studies have shown that the clinical outcomes of TAM-treated breast cancer patients are influenced by the activity of some cytochrome P450 (CYP) enzymes that catalyze the formation of active TAM metabolites like endoxifen and 4-hydroxytamoxifen. In this study, we aimed to first develop and validate an algorithm combining polymorphisms in CYP genes and clinicopathological signatures to identify a subpopulation of breast cancer patients who might benefit most from TAM adjuvant therapy and meanwhile evaluate major risk factors related to TAM resistance. Specifically, a total of 256 patients with invasive breast cancer who received adjuvant endocrine therapy were selected. The genotypes at 10 loci from three TAM metabolism-related CYP genes were detected by time-of-flight mass spectrometry and multiplex long PCR. Combining the 10 loci with nine clinicopathological characteristics, we obtained 19 important features whose association with cancer recurrence was assessed by importance score via random forests. After that, a logistic regression model was trained to calculate TAM risk-of-recurrence score (TAM RORs), which is adopted to assess a patient’s risk of recurrence after TAM treatment. The sensitivity and specificity of the model in an independent test cohort were 86.67% and 64.56%, respectively. This study showed that breast cancer patients with high TAM RORs were less sensitive to TAM treatment and manifested more invasive characteristics, whereas those with low TAM RORs were highly sensitive to TAM treatment, and their conditions were stable during the follow-up period. There were some risk factors that had a significant effect on the efficacy of TAM. They were tissue classification (tumor Grade < 2 vs. Grade ≥ 2, p = 2.2e−16), the number of lymph node metastases (Node-Negative vs. Node < 4, p = 5.3e−07; Node < 4 vs. Node ≥ 4, p = 0.003; Node-Negative vs. Node ≥ 4, p = 7.2e−15), and the expression levels of estrogen receptor (ER) and progesterone receptor (PR) (ER < 50% vs. ER ≥ 50%, p = 1.3e−12; PR < 50% vs. PR ≥ 50%, p = 2.6e−08). The really remarkable thing is that different genotypes of CYP2D6*10(C188T) show significant differences in prediction function (CYP2D6*10 CC vs. TT, p < 0.019; CYP2D6*10 CT vs. TT, p < 0.037). There are more than 50% Chinese who have CYP2D6*10 mutation. So the genotype of CYP2D6*10(C188T) should be tested before TAM therapy.

Targeting Estrogen Receptor-Positive Breast Microtumors with Endoxifen-Conjugated, Hypoxia-Sensitive Polymersomes.

Endoxifen is the primary active metabolite of tamoxifen, a nonsteroidal-selective estrogen receptor modulator (SERM) and widely used medication to treat estrogen receptor-positive (ER+) breast cancer. In this study, endoxifen was conjugated to the surface of polymeric nanoparticles (polymersomes) for targeted delivery of doxorubicin (DOX) to estrogen receptor-positive breast cancer cells (MCF7). Rapid cell growth and insufficient blood supply result in low oxygen concentration (hypoxia) within the solid breast tumors. The polymersomes developed here are prepared from amphiphilic copolymers of polylactic acid (PLA) and poly(ethylene glycol) (PEG) containing diazobenzene as the hypoxia-responsive linker. We prepared two nanoparticle formulations: DOX-encapsulated hypoxia-responsive polymersomes (DOX-HRPs) and endoxifen-conjugated, DOX-encapsulated hypoxia-responsive polymersomes (END-DOX-HRPs). Cellular internalization studies demonstrated eight times higher cytosolic and nuclear localization after incubating breast cancer cells with END-DOX-HRPs (targeted polymersomes) in contrast to DOX-HRPs (nontargeted polymersomes). Cytotoxicity studies on monolayer cell cultures exhibited that END-DOX-HRPs were three times more toxic to ER+ MCF7 cells than DOX-HRPs and free DOX in hypoxia. The cell viability studies on three-dimensional hypoxic cultures also demonstrated twice as much toxicity when the spheroids were treated with targeted polymersomes instead of nontargeted counterparts. This is the first report of surface-decorated polymeric nanoparticles with endoxifen ligands for targeted drug delivery to ER+ breast cancer microtumors. The newly designed endoxifen-conjugated, hypoxia-responsive polymersomes might have translational potential for ER+ breast cancer treatment.

Pharmacological Inhibition of MCT4 Reduces 4-Hydroxytamoxifen Sensitivity by Increasing HIF-1α Protein Expression in ER-Positive MCF-7 Breast Cancer Cells.

The rate of glycolysis in cancer cells is higher than that of normal cells owing to high energy demands, which results in the production of excess lactate. Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, play a critical role in maintaining an appropriate pH environment through lactate transport, and their high expression is associated with poor prognosis in breast cancer. Thus, we hypothesized that inhibition of MCTs is a promising therapeutic target for adjuvant breast cancer treatment. We investigated the effect of MCT inhibition in combination with 4-hydroxytamoxifen (4-OHT), an active metabolite of tamoxifen, using two estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. Lactate transport was investigated in cellular uptake studies. The cytotoxicity of 4-OHT was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In both cell lines evaluated, MCT1 and MCT4 were constitutively expressed at the mRNA and protein levels. [14C]-L-lactate uptake by both cells was significantly inhibited by bindarit, a selective MCT4 inhibitor, but weakly affected by 5-oxoploline (5-OP), a selective MCT1 inhibitor. The results of the MTT assay showed that combination with bindarit, but not 5-OP, decreased 4-OHT sensitivity. Bindarit significantly increased the levels of hypoxia-inducible factor-1α (HIF-1α) in MCF-7 cells. Moreover, HIF-1α knockdown significantly increased 4-OHT sensitivity, whereas induction of HIF-1α by hypoxia decreased 4-OHT sensitivity in MCF-7 cells. In conclusion, pharmacological MCT4 inhibition confers resistance to 4-OHT rather than sensitivity, by increasing HIF-1α protein levels. In addition, HIF-1α inhibition represents a potential therapeutic strategy for enhancing 4-OHT sensitivity.