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4-hydroxytamoxifen (OHT) is an anti-cancer drug that induces apoptosis in breast cancer cells. Although changes in lipid levels and mitochondrial respiration have been observed in OHT-treated cells, the overall mechanisms underlying these metabolic alterations are poorly understood. In this study, time-series metabolomics and lipidomics were used to analyze the changes in metabolic profiles induced by OHT treatment in the MCF-7 human breast cancer cell line. Lipidomic and metabolomic analyses revealed increases in ceramide, diacylglycerol and triacylglycerol, and decreases in citrate, respectively. Gene expression analyses revealed increased expression of ATP-dependent citrate lyase (ACLY) and subsequent fatty acid biosynthetic enzymes, suggesting that OHT-treated MCF-7 cells activate citrate-to-lipid metabolism. The significance of the observed metabolic changes was evaluated by co-treating MCF-7 cells with OHT and ACLY or a diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor. Co-treatment ameliorated cell death and reduced mitochondrial membrane potential compared to that in OHT treatment alone. The inhibition of cell death by co-treatment with an ACLY inhibitor has been observed in other breast cancer cell lines. These results suggest that citrate-to-lipid metabolism is critical for OHT-induced cell death in breast cancer cell lines., (© 2023. The Author(s).)

Tamoxifen-based endocrine therapy remains a major adjuvant therapy for estrogen receptor (ER)-positive breast cancer (BC). However, many patients develop tamoxifen resistance, which results in recurrence and poor prognosis. Herein, we show that fatty acid oxidation (FAO) was activated in tamoxifen-resistant (TamR) ER-positive BC cells by performing bioinformatic and functional studies. We also reveal that CPT1A, the rate-limiting enzyme of FAO, was significantly overexpressed and that its enzymatic activity was enhanced in TamR cells. Mechanistically, the transcription factor c-Jun was activated by JNK kinase-mediated phosphorylation. Activated c-Jun bound to the TRE motif in the CPT1A promoter to drive CPT1A transcription and recruited CBP/P300 to chromatin, catalysing histone H3K27 acetylation to increase chromatin accessibility, which ensured more effective transcription of CPT1A and an increase in the FAO rate, eliminating the cytotoxic effects of tamoxifen in ER-positive BC cells. Pharmacologically, inhibiting CPT1A enzymatic activity with the CPT1 inhibitor etomoxir or blocking c-Jun phosphorylation with a JNK inhibitor restored the tamoxifen sensitivity of TamR cells. Clinically, high levels of phosphorylated c-Jun and CPT1A were observed in ER-positive BC tissues in patients with recurrence after tamoxifen therapy and were associated with poor survival. These results indicate that the assessment and targeting of the JNK/c-Jun-CPT1A-FAO axis will provide promising insights for clinical management, increased tamoxifen responses and improved outcomes for ER-positive BC patients., (© 2023. The Author(s).)

Background: The mechanism of acquired resistance of tamoxifen in endocrine therapy of breast cancer is not fully understood. In this study, we investigated the genomic changes in acquired tamoxifen-resistant cell lines., Methods: Tamoxifen-resistant subclones (MCF-7R) derived from parent MCF-7 cells, which is an ER(+) breast cancer cell line, cultured with 4-hydrotamoxifen more than 6 months were used to obtain genomic alterations. Cell growth, microarray, and quantitative real-time PCR (q-RTPCR) assays were conducted. Additionally, the ITGB1 function was investigated in MCF-7R cells and MCF-7R ITGB1-silenced subclones using MTT and Transwell assays. Online pathway analysis was performed to assess the genetic characteristics of tamoxifen resistance., Results: The gene expression profile of the tamoxifen-resistant cell line was considerably changed compared to the tamoxifen-sensitive cell line. Of 4102 genes with altered expressions, 1986 genes were upregulated, whereas 2116 were downregulated. The ITGB1 expression in MCF-7R cells was higher than that in MCF-7 cells. Interestingly, ITGB1 silencing partially rescued the sensitivity of MCF-7R cells to tamoxifen and reduced their motility. The activation of the β1-integrin signaling pathway was probably responsible for this phenomenon., Conclusions: Our data confirm the presence of alterations in the genes of tamoxifen-resistance breast cancer cells. ITGB1 probably partially contributes to tamoxifen resistance and cell motility via the β1-integrin signaling pathway. Thus, ITGB1 may be a potential target for the improvement of anti-hormone therapy reaction in ER(+) breast cancer patients., (© 2022 The Authors. Asia-Pacific Journal of Clinical Oncology published by John Wiley & Sons Australia, Ltd.)

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vestigial like family member 4 (VGLL4), a member of the Hippo pathway, is a transcriptional cofactor involved in many biological processes, such as tumor progression, postnatal heart growth, and muscle regeneration. However, the VGLL4 expression pattern in vivo remains unclear. To detect and trace Vgll4-expressing cells and their progeny, we generated and characterized a new tamoxifen-inducible Dre knock-in mouse line, Vgll4-DreER. This mouse line expressed DreER (Dre recombinase fused to the estrogen receptor) under the control of the endogenous Vgll4 promoter. After crossing the Vgll4-DreER mouse line with the Dre-responsive reporter H11-rRFP, Dre-mediated recombination in the tissue was monitored on the basis of red fluorescent protein (RFP) signals, which indicated the distribution of VGLL4-positive cells in vivo. Our data revealed that VGLL4 is widely expressed in various cell types at embryonic and neonatal stages. After comparison with our previously reported Vgll4-GFP mouse, we found that the RFP signal profile was wider than the green fluorescent protein (GFP) pattern, indicating that Vgll4-DreER is more sensitive for labeling VGLL4-expressing cells. We next used a dual-recombination system to simultaneously label VGLL4- and keratin 5 (KRT5)-positive cell populations, and no crosstalk was observed in the Krt5-CreER;Vgll4-DreER;R26-rGlR mice. Taken together, the Vgll4-DreER mouse line is a valuable new tool for examining the precise VGLL4 expression profile and conditional manipulating of VGLL4-expressing cells and their progeny., (© 2023 Wiley Periodicals LLC.)

The chemokine stromal cell-derived-factor 1 (SDF)-1/CXCL12 acts by binding to its receptors, the CXC-4 chemokine receptor (CXCR4) and the CXC-7 chemokine receptor (CXCR7). The binding of CXCL12 to its receptors results in downstream signaling that leads to cell survival, proliferation and migration of tumor cells. CXCL12 and CXCR4 are highly expressed in breast cancer (BC) and glioblastoma (GBM) compared to normal cells. High expression of this chemokine axis correlates with increased therapy resistance and grade, tumor spread and poorer prognosis in these tumors. Tamoxifen (TMX) is a selective estrogen receptor modulator (SERM) that inhibits the expression of estrogen-regulated genes, including growth and angiogenic factors secreted by tumor cells. Additionally, TMX targets several proteins, such as protein kinase C (PKC), phospholipase C (PLC), P-glycoprotein (PgP), phosphatidylinositol-3-kinase (PI3K) and ion channels. This drug showed promising antitumor activity against both BC and GBM cells. In this review, we discuss the role of the CXCL12-CXCR4-CXCR7 chemokine axis in BC and GBM tumor biology and propose TMX as a potential modulator of this axis in these tumors. TMX modulates the CXCL12-CXCR4-CXCR7 axis in BC, however, there are no studies on this in GBM. We propose that studying this axis in GBM cells/patients treated with TMX might be beneficial for these patients. TMX inhibits important signaling pathways in these tumors and the activation of this chemokine axis is associated with increased therapy resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Despite the successful combination of therapies improving survival of estrogen receptor α (ER+) breast cancer patients with metastatic disease, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1 (A2B1), a reader of N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. The miRNA-seq transcriptome of MCF-7 cells overexpressing A2B1 identified the serine metabolic processes pathway. Increased expression of two key enzymes in the serine synthesis pathway (SSP), phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH), correlates with poor outcomes in ER+ breast patients who received tamoxifen (TAM). We reported that PSAT1 and PHGDH were higher in LCC9 and LY2 cells compared to MCF-7 cells and their knockdown enhanced TAM sensitivity in these-resistant cells. Here we demonstrate that stable, modest overexpression of A2B1 in MCF-7 cells increased PSAT1 and PHGDH and endocrine resistance. We identified four miRNAs downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3'UTR (miR-145-5p and miR-424-5p), and the PHGDH 3'UTR (miR-34b-5p and miR-876-5p) in dual luciferase assays. Lower expression of miR-145-5p and miR-424-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PSAT1 and lower expression of miR-34b-5p and miR-876-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PHGDH. Transient transfection of these miRNAs restored endocrine-therapy sensitivity in LCC9 and ZR-75-1-4-OHT cells. Overall, our data suggest a role for decreased A2B1-regulated miRNAs in endocrine resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.

Themis is important in regulating positive selection of thymocytes during T cell development, but its role in peripheral T cells is less understood. Here, we investigated T cell activation and its sequelae using a tamoxifen-mediated, acute Themis deletion mouse model. We find that proliferation, effector functions including anti-tumor killing, and up-regulation of energy metabolism are severely compromised. This study reveals the phenomenon of peripheral adaptation to loss of Themis, by demonstrating direct TCR-induced defects after acute deletion of Themis that were not evident in peripheral T cells chronically deprived of Themis in dLck-Cre deletion model. Peripheral adaptation to long-term loss was compared using chronic versus acute tamoxifen-mediated deletion and with the (chronic) dLck-Cre deletion model. We found that upon chronic tamoxifen-mediated Themis deletion, there was modulation in the gene expression profile for both TCR and cytokine signaling pathways. This profile overlapped with (chronic) dLck-Cre deletion model. Hence, we found that peripheral adaptation induced changes to both TCR and cytokine signaling modules. Our data highlight the importance of Themis in the activation of CD8 + T cells., (© 2023 Gautam et al.)

Background: Peritoneal dialysis (PD) remains limited due to dialysis failure caused by peritoneal fibrosis. Tamoxifen (TAM), an inhibitor of estrogen receptor 1 (ESR1), has been reported to treat fibrosis, but the underlying mechanism remains unknown. In this study, we sought to explore whether tamoxifen played an anti-fibrotic role by affecting transcription factor ESR1., Methods: ESR1 expression was detected in the human peritoneum. Mice were daily intraperitoneally injected with 4.25% glucose PD dialysate containing 40 mM methylglyoxal for 2 weeks to establish PD-induced peritoneal fibrosis. Tamoxifen was administrated by daily gavage, at the dose of 10 mg/kg. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay were performed to validate ESR1 bound H19 promoter. Gain-of-function and loss-of-function experiments were performed to investigate the biological roles of H19 on the mesothelial-mesenchymal transition (MMT) of human peritoneal mesothelial cells (HPMCs). Intraperitoneal injection of nanomaterial-wrapped 2'-O-Me-modified small interfering RNA was applied to suppress H19 in the mouse peritoneum. RNA immunoprecipitation and RNA pull-down assays demonstrated binding between H19 and p300. Exfoliated peritoneal cells were obtained from peritoneal dialysis effluent to analyze the correlations between ESR1 (or H19) and peritoneal solute transfer rate (PSTR)., Results: ESR1 was increased significantly in the peritoneum after long-term exposure to PD dialysate. Tamoxifen treatment ameliorated high glucose-induced MMT of HPMCs, improved ultrafiltration rate, and decreased PSTR of mouse peritoneum. Tamoxifen reduced the H19 level by decreasing the ESR1 transcription of H19. Depletion of H19 reversed the pro-fibrotic effect of high glucose while ectopic expression of H19 exacerbated fibrotic pathological changes. Intraperitoneal injection of nanomaterial-wrapped 2'-O-Me-modified siRNAs targeting H19 mitigated PD-related fibrosis in mice. RNA immunoprecipitation (RIP) and RNA pull-down results delineated that H19 activated VEGFA expression by binding p300 to the VEGFA promoter and inducing histone acetylation of the VEGFA promoter. ESR1 and H19 were promising targets to predict peritoneal function., Conclusions: High glucose-induced MMT of peritoneal mesothelial cells in peritoneal dialysis via activating ESR1. In peritoneal mesothelial cells, ESR1 transcribed the H19 and H19 binds to transcription cofactor p300 to activate the VEGFA. Targeting ESR1/H19/VEGFA pathway provided new hope for patients undergoing peritoneal dialysis., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Tamoxifen is a drug used for treating breast cancer (BC), especially for individuals diagnosed with estrogen receptor-positive (ER+) BC. Its prolonged use could reduce the risk of recurrence and significantly lengthen the survival rate of BC patients. However, an increasing number of patients developed resistance to tamoxifen treatment, which reduced therapeutic efficiency and caused substandard prognosis. Therefore, the exploration of the molecular processes involved in tamoxifen resistance (TR) is urgently required. This investigation aimed to elucidate the relationship of microRNA-330 (miR-330-3p) with the TR of BC. There is little information on miR-330-3p's link with drug-resistant BC, although it is well known to regulate cell proliferation and apoptosis. Primarily, miR-330-3p expression in parental BC (MCF7/T47D), TR (MCF7-TR), and T47D/TR cell lines was detected by qRT-PCR. Then, the impact of miR-330-3p on the TR of BC cells was assessed by a cell proliferation assay. Lastly, dual-luciferase reporter, qRT-PCR, and western blot assessments were carried out to identify histone deacetylase 4 (HDAC4) as the potential miR-330-3p target gene. The data indicated that miRNA-330 was overexpressed in TR ER+ BC cells and its overexpression could induce TR. Furthermore, miRNA-330 could also reduce the expression of HDAC4, which is closely linked to TR, and overexpression of HDAC4 could reverse miRNA-330-induced drug resistance. In summary, miR-330-3p could induce TR of ER+ BC cells by downregulating HDAC4 expression, which might be a novel marker of TR and a possible treatment target against BC patients who are tamoxifen-resistant., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Meng Zhang et al.)

Atherosclerosis, a leading cause of cardiovascular disease, remains a significant global health concern. Tamoxifen and raloxifene, selective estrogen receptor modulators (SERMs), have demonstrated potential cardioprotective effects. However, the underlying molecular mechanisms by which these SERMs modulate Transforming Growth Factor-β (TGF-β) signaling in human vascular smooth muscle cells (VSMCs) remain largely unexplored. This study sought to investigate the impact of tamoxifen and raloxifene on TGF-β-induced CHSY1 expression and Smad2 linker region phosphorylation in VSMCs and to elucidate the role of reactive oxygen species (ROS), NADPH oxidase (NOX), and kinase pathways in mediating these effects. Employing a comprehensive experimental strategy, VSMCs were treated with TGF-β in the presence or absence of tamoxifen, raloxifene, and various pharmacological inhibitors. Subsequently, CHSY1 mRNA expression, Smad2C and Smad2L phosphorylation, ROS production, p47phox and ERK 1/2 phosphorylation were assessed. Our results revealed that tamoxifen and raloxifene significantly attenuated TGF-β-mediated CHSY1 mRNA expression and Smad2 linker region phosphorylation, without affecting the canonical TGF-β-Smad2C pathway. Furthermore, these compounds effectively inhibited ROS production, p47phox and ERK 1/2 phosphorylation, implicating the involvement of the TGF-β-NOX-ERK-Smad2L signaling cascade in their cardioprotective properties. This study provides a comprehensive understanding of the molecular mechanisms underlying the cardioprotective effects of tamoxifen and raloxifene in VSMCs, offering valuable insights for the development of targeted therapeutic strategies aimed at atherosclerosis prevention and the promotion of cardiovascular health., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Known for a long time, well-tested substances are still finding new applications in science, medicine and industry. This is a popular and cost-effective strategy because, when searching for new applications, effective methods of their large-scale production and pharmacological activity, and the results of pharmacokinetic and toxicological studies are usually already known. Tamoxifen is known mainly as a drug used in the treatment of estrogen receptor-dependent breast cancer. Despite the discovery of this effective and profitable property many years ago and the constant expansion of related applications and patents, completely new ways of using tamoxifen and its derivatives in various fields continue to appear, and the number of patents for novel applications unrelated to breast cancer remains high. The aim of this article is to illustrate drug repositioning on the example of tamoxifen and to bring the ever-developing story of discoveries related to it to a wider audience.

Objectives: Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF-β1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis., Methods: In vitro, vocal fold fibroblasts were treated with TAM (10 -8 or 10 -9  M) ± TGF-β1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real-time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre-injury day 5 to post-injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture., Results: TAM (10 -8  M) did not affect Smad3, Smad7, Acta2, or genes related to extracellular matrix metabolism. TAM (10 -8 or 10 -9  M) + TGF-β1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF-β1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment., Conclusions: TAM has antifibrotic potential via the regulation of TGF-β1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis., Level of Evidence: NA Laryngoscope, 133:2248-2254, 2023., (© 2022 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: Breast cancer is the second most common malignancy worldwide and 70% of all breast cancer cases are estrogen receptor-positive (ER+). Endocrine therapy, Tamoxifen (TAM), is a popular treatment for ER+ breast cancer patients; however, despite its success in reducing breast cancer mortality, cancer drug resistance remains a significant challenge. A major contributor to this resistance is the dysregulation of cholesterol homeostasis, where breast cancer cells have elevated cholesterol levels. MicroRNAs (miRNAs) are master regulators of cholesterol-related and cancer drug resistance pathways, and their aberrant expression often confers resistance. Therefore, we aimed to investigate the roles of miRNA-128 and miRNA-223 in cholesterol-mediated TAM resistance., Methods: Three breast cancer cell lines were treated with a combination of 1 μM TAM and 10 μM of a cholesterol depleting agent (Acetyl Plumbagin: AP) following transfection with a miR-128 inhibitor or a miR-223 mimic. Cell viability and cholesterol levels were assessed using an MTT assay and fluorescence staining, respectively. In addition, expression levels of several genes and proteins involved in cancer drug resistance and cholesterol homeostasis were also assessed using RT-qPCR and western blotting., Results: The combination treatment with altered miRNA expression led to reduced cell viability due to a reduction in free cholesterol and lipid rafts in MCF-7, MDA-MB-231, and long-term estrogen-deprived cells (resistant breast cancer cells). Moreover, reduced miR-128 expression was favoured in all breast cancer cell lines as this alteration lowered the expression of genes involved in cholesterol synthesis and transport, drug resistance, and cell signalling., Conclusions: Investigating the gene expression profiles in different breast cancer cell lines was important to elucidate further the molecular mechanisms involved in miRNA-regulated cholesterol homeostasis and cancer drug resistance. Therefore, our findings demonstrated that miR-128 and miR-223 could be potential targets in reducing TAM resistance through the depletion of excess cholesterol., (© 2023 The Authors. IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Oxysterols play significant roles in many physiological and pathological processes including cancer. They modulate some of the cancer hallmarks pathways, influence the efficacy of anti-cancer drugs, and associate with patient survival. In this study, we aimed to analyze the role of 7-ketocholesterol (7-KC) in breast carcinoma cells and its potential modulation of the tamoxifen effect. 7-KC effects were studied in two estrogen receptor (ER)-positive (MCF-7 and T47D) and one ER-negative (BT-20) breast cancer cell lines. First, we tested the viability of cells in the presence of 7-KC. Next, we co-incubated cells with tamoxifen and sublethal concentrations of 7-KC. We also tested changes in caspase 3/7 activity, deregulation of the cell cycle, and changes in expression of selected genes/proteins in the presence of tamoxifen, 7-KC, or their combination. Finally, we analyzed the effect of 7-KC on cellular migration and invasion. We found that the presence of 7-KC slightly decreases the efficacy of tamoxifen in MCF-7 cells, while an increased effect of tamoxifen and higher caspase 3/7 activity was observed in the BT-20 cell line. In the T47D cell line, we did not find any modulation of tamoxifen efficacy by the presence of 7-KC. Expression analysis showed the deregulation in CYP1A1 and CYP1B1 with the opposite trend in MCF-7 and BT-20 cells. Moreover, 7-KC increased cellular migration and invasion potential regardless of the ER status. This study shows that 7-KC can modulate tamoxifen efficacy as well as cellular migration and invasion, making 7-KC a promising candidate for future studies., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Breast cancer is the leading cause of cancer deaths for women worldwide. Endocrine therapies represent the cornerstone for hormone-dependent breast cancer treatment. However, in many cases, endocrine resistance is induced with poor prognosis for patients. In the current study, we have developed MCF-7 cell lines resistant to fulvestrant (MCF-7Fulv) and tamoxifen (MCF-7Tam) aiming at investigating mechanisms underlying resistance. Both resistant cell lines exerted lower proliferation capacity in two-dimensional (2-D) cultures but retain estrogen receptor α (ERα) expression and proliferate independent of the presence of estrogens. The established cell lines tend to be more aggressive exhibiting advanced capacity to form colonies, increased expression of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and heterodimerization of ERBB family receptors and activation of EGFR downstream pathways like MEK/ERK1/2 and PI3K/AKT. Tyrosine kinase inhibitors tested against resistant MCF-7Fulv and MCF-7Tam cells showed moderate efficacy to inhibit cell proliferation, except for lapatinib, which concomitantly inhibits both EGFR and HER2 receptors and strongly reduced cell proliferation. Furthermore, increased autophagy was observed in resistant MCF-7Fulv and MCF-7Tam cells as shown by the presence of autophagosomes and increased Beclin-1 levels. The increased autophagy in resistant cells is not associated with increased apoptosis, suggesting a cytoprotective role for autophagy that may favor cells' survival and aggressiveness. Thus, by exploiting those underlying mechanisms, new targets could be established to overcome endocrine resistance. NEW & NOTEWORTHY The development of resistance to hormone therapy caused by both fulvestrant and tamoxifen promotes autophagy with concomitant apoptosis evasion, rendering cells capable of surviving and growing. The fact that resistance also triggers ERBB family signaling pathways, which are poorly inhibited by tyrosine kinase inhibitors might attribute to cells' aggressiveness. It is obvious that the development of endocrine therapy resistance involves a complex interplay between deregulated ERBB signaling and autophagy that may be considered in clinical practice.

Aims: In this study, we aimed to investigate previously unrecognized lipid metabolic perturbations in tamoxifen-resistant breast cancer (BC) by conducting comprehensive metabolomics and transcriptomics analysis. We identified the role of 3-hydroxy-3-methylglutary-coenzyme-A-synthase 2 (HMGCS2), a key enzyme responsible for ketogenesis, in tamoxifen-resistant BC growth., Main Methods: Comprehensive metabolomics (CE-TOFMS, LC-TOFMS) and transcriptiomics analysis were performed to characterize metabolic pathways in tamoxifen-resistant BC cells. The upregulation of HMGCS2 were verified thorugh immunohistochemistry (IHC) in clinical samples obtained from patients with recurrent BC. HMGCS2 inhibitor was discovered through surface plasmon resonance analysis, enzyme assay, and additional molecular docking studies. The effect of HMGCS2 suppression on tumor growth was studied thorugh BC xenograft model, and intratumoral lipid metabolites were analyzed via MALDI-TOFMS imaging., Key Findings: We revealed that the level of HMGCS2 was highly elevated in both tamoxifen-resistant T47D sublines (T47D/TR) and clinical refractory tumor specimens from patients with ER+ breast cancer, who had been treated with adjuvant tamoxifen. Suppression of HMGCS2 in T47D/TR resulted in the accumulation of mitochondrial reactive oxygen species (mtROS) and apoptotic cell death. Further, we identified alphitolic acid, a triterpenoid natural product, as a novel HMGCS2-specific inhibitor that elevated mtROS levels and drastically retarded the growth of T47D/TR in in vitro and in vivo experiments., Significance: Enhanced ketogenesis with upregulation of HMGCS2 is a potential metabolic vulnerability of tamoxifen-resistant BC that offers a new therapeutic opportunity for treating patients with ER+ BC that are refractory to tamoxifen treatment., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Raloxifene belongs to the family of Selective Estrogen Receptor Modulators (SERMs), which are drugs widely prescribed for Estrogen Receptor alpha (ERα)-related pathologies. Recently, SERMs are being tested in repurposing strategies for ERα-independent clinical indications, including a wide range of microbial infections. Macrophages are central in the fight against pathogen invasion. Despite estrogens have been shown to regulate macrophage phenotype, SERMs activity in these cells is still poorly defined. We investigated the activity of Raloxifene in comparison with another widely used SERM, Tamoxifen, on immune gene expression in macrophages obtained from mouse and human tissues, including mouse peritoneal macrophages, bone marrow-derived macrophages, microglia or human blood-derived macrophages, assaying for the involvement of the ERα, PI3K and NRF2 pathways also under inflammatory conditions. Our data demonstrate that Raloxifene acts by a dual mechanism, which entails ERα antagonism and off-target mediators. Moreover, micromolar concentrations of Raloxifene increase the expression of immune metabolic genes, such as Vegfa and Hmox1, through PI3K and NRF2 activation selectively in peritoneal macrophages. Conversely, Il1b mRNA down-regulation by SERMs is consistently observed in all macrophage subtypes and unrelated to the PI3K/NRF2 system. Importantly, the production of the inflammatory cytokine TNFα induced by the bacterial endotoxin, LPS, is potentiated by SERMs and paralleled by the cell subtype-specific increase in IL1β secretion. This work extends our knowledge on the biological and molecular mechanisms of SERMs immune activity and indicate macrophages as a pharmacological target for the exploitation of the antimicrobial potential of these drugs., Competing Interests: Declaration of Competing Interest The authors declare that are no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Tamoxifen (TMX), a selective estrogen receptor modulator, is commonly used in the treatment of hormone-responsive cancers. However, the effects of TMX in anabolic tissues harboring estrogen receptors, such as skeletal muscle, are poorly understood. We report a tandem mass-tag approach to TMX-treated myogenesis in C 2 C 12 cells, a well-characterized model of in vitro murine skeletal muscle differentiation. A longitudinal analysis of >10,000 proteins identified in untreated C 2 C 12 myogenesis revealed a novel subset of 1,062 myogenically regulated proteins. These proteins clustered into five distinct longitudinal expression trends which significantly overlap those obtained in similar analyses performed in human myocytes. We document a specific functional enrichment for adiponectin-signaling unique to TMX-treated myogenesis, as well as a subset of 198 proteins that are differentially expressed in TMX-treated cells relative to controls at one or more stages of myogenesis, the majority of which were involved in steroid and lipid metabolism. Further analysis highlights metallothionein-1 as a novel target of TMX treatment at each stage of C 2 C 12 myogenesis. Finally, we present a powerful, self-validating pipeline for analyzing the total proteomic response to in vitro treatment across every stage of muscle cell development which can be easily adapted to study the effects of other drugs on myogenesis.

Gene knockout is a technique routinely used in basic experimental research, particularly in mouse skeletal and developmental studies. Tamoxifen-induced Cre/loxp system is known for its temporal and spatial precision and commonly utilized by researchers. However, tamoxifen has been shown its side effects on affecting the phenotype of mouse bone directly. This review aimed to optimize tamoxifen administration regimens including its dosage and duration, to identify an optimal induction strategy that minimizes potential side effects while maintaining recombination efficacy. This study will help researchers in designing gene knockout experiments in bone when using tamoxifen., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interests., (Copyright © 2023. Published by Elsevier Masson SAS.)

CKD progression depends on the activation of an intricate set of hemodynamic and inflammatory mechanisms, promoting renal leukocyte infiltration, inflammation and fibrosis, leading to renal function loss. There are currently no specific drugs to detain renal fibrogenesis, which is a common end-point for different nephropathies. Clinical therapy for CKD is mostly based on the management of hypertension and proteinuria, partially achieved with renin-angiotensin-aldosterone system (RAAS) blockers, and the control of inflammation by immunosuppressive drugs. The aim of the present study was to verify if the administration of tamoxifen (TAM), an estrogen receptor modulator, clinically employed in the treatment of breast cancer and predicted to exert antifibrotic effects, would promote additional benefits when associated to a currently used therapeutic scheme for the conservative management of experimental CKD. Wistar rats underwent the NAME model of hypertensive nephrosclerosis, obtained by daily oral administration of a nitric oxide synthesis inhibitor, associated to dietary sodium overload. The therapeutic association of TAM to losartan (LOS), and mofetil mycophenolate (MMF) effectively reduced the severe hypertension, marked albuminuria and glomerular damage exhibited by NAME animals. Moreover, the association also succeeded in limiting renal inflammation in this model, and promoted further reduction of ECM interstitial accumulation and renal fibrosis, compared to the monotherapies. According to our results, the association of TAM to the currently used conservative treatment of CKD added significant antifibrotic effects both in vivo and in vitro, and may represent an alternative to slow the progression of chronic nephropathy., (© 2023. Springer Nature Limited.)

GPER is a seven transmembrane G-protein-coupled estrogen receptor that mediates rapid estrogen actions. Large volumes of data have revealed its association with clinicopathological variables in breast tumors, role in epidermal growth factor (EGF)-like effects of estrogen, potential as a therapeutic target or a prognostic marker, and involvement in endocrine resistance in the face of tamoxifen agonism. GPER cross-talks with estrogen receptor alpha (ERα) in cell culture models implicating its role in the physiology of normal or transformed mammary epithelial cells. However, discrepancies in the literature have obfuscated the nature of their relationship, its significance, and the underlying mechanism. The purpose of this study was to assess the relationship between GPER, and ERα in breast tumors, to understand the mechanistic basis, and to gauge its clinical significance. We mined The Cancer Genome Atlas (TCGA)-BRCA data to examine the relationship between GPER and ERα expression. GPER mRNA, and protein expression were analyzed in ERα-positive or -negative breast tumors from two independent cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter (KM) was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17β-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ERα agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ERα knockdown. ERα-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation (ChIP) assay. Clinical data revealed significant positive association between GPER and ERα expression in breast tumors. The median GPER expression in ERα-positive tumors was significantly higher than ERα-negative tumors. High GPER expression was significantly associated with longer overall survival (OS) of patients with ERα-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ERα-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ERα occupancy in the upstream region of GPER. Furthermore, treatment with 17β-estradiol or PPT significantly reduced the IC 50 of the GPER agonist (G1)-mediated loss of MCF-7 or T47D cell viability. In conclusion, GPER is positively associated with ERα in breast tumors, and induced by estrogen-ERα signalling axis. Estrogen-mediated induction of GPER makes the cells more responsive to GPER ligands. More in-depth studies are warranted to establish the significance of GPER-ERα co-expression, and their interplay in breast tumor development, progression, and treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The cytosolic dipeptidyl-aminopeptidases 8 (DPP8) and 9 (DPP9) belong to the DPPIV serine proteases with the unique characteristic of cleaving off a dipeptide post-proline from the N -termini of substrates. To study the role of DPP8 and DPP9 in breast cancer, MCF-7 cells (luminal A-type breast cancer) and MDA.MB-231 cells (basal-like breast cancer) were used. The inhibition of DPP8/9 by 1G244 increased the number of lysosomes in both cell lines. This phenotype was more pronounced in MCF-7 cells, in which we observed a separation of autophagosomes and lysosomes in the cytosol upon DPP8/9 inhibition. Likewise, the shRNA-mediated knockdown of either DPP8 or DPP9 induced autophagy and increased lysosomes. DPP8/9 inhibition as well as the knockdown of the DPPs reduced the cell survival and proliferation of MCF-7 cells. Additional treatment of MCF-7 cells with tamoxifen, a selective estrogen receptor modulator (SERM) used to treat patients with luminal breast tumors, further decreased survival and proliferation, as well as increased cell death. In summary, both DPP8 and DPP9 activities confine macroautophagy in breast cancer cells. Thus, their inhibition or knockdown reduces cell viability and sensitizes luminal breast cancer cells to tamoxifen treatment.

Drug repositioning is an alternative to overcome the complexity of the drug discovery and approval procedures for the treatment of Mycobacterium abscessus Complex (MABSC) infections that are increasing globally due to the emergency of antimicrobial resistance mechanisms. Here, an in silico chemogenomics approach was performed to compare the sequences from 4942 M. abscessus subsp. abscessus (M. abscessus) proteins with 5258 or 3473 therapeutic targets registered in the DrugBank or Therapeutic Target Database, respectively. This comparison identified 446 drugs or drug candidates whose targets were homologous to M. abscessus proteins. These identified drugs were considered potential inhibitors of MABSC (anti-MABSC activity). Further screening and inspection resulted in the selection of ezetimibe, furosemide, itraconazole, miconazole (MCZ), tamoxifen (TAM), and thiabendazole (THI) for experimental validation. Among them, MCZ and TAM showed minimum inhibitory concentrations (MIC) of 32 and 24 µg mL -1 against M. abscessus, respectively. For M. bolletii and M. massiliense strains, MCZ and TAM showed MICs of 16 and 24 µg mL -1 , in this order. Subsequently, the antibacterial activity of MCZ was confirmed in vivo, indicating its potential to reduce the bacterial load in the lungs of infected mice. These results show that MCZ and TAM can serve as molecular scaffolds for the prospective hit-2-lead optimization of new analogs with greater potency, selectivity, and permeability., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Endocrine therapies targeting estrogen signaling, such as tamoxifen, have significantly improved management of estrogen receptor alpha (ERα)-positive breast cancers. However, their efficacy is limited by intrinsic and acquired resistance to treatment, and there is currently no predictive marker of response to these anti-estrogens to guide treatment decision. Here, using two independent cohorts of breast cancer patients, we identified nuclear PRMT5 expression as an independent predictive marker of sensitivity to tamoxifen. Mechanistically, we discovered that tamoxifen stimulates ERα methylation by PRMT5, a key event for its binding to corepressors such as SMRT and HDAC1, participating in the inhibition of the transcriptional activity of ERα. Although PRMT5 is mainly localized in the cytoplasm of tumor cells, our analyses show that tamoxifen triggers its nuclear translocation in tamoxifen-sensitive tumors but not in resistant ones. Hence, we unveil a biomarker of sensitivity to tamoxifen in ERα-positive breast tumors that could be used to enhance the response of breast cancer patients to endocrine therapy, by fostering its nuclear expression., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Casein kinase 1 plays a crucial role in carcinogenesis. 4-Hydroxytamoxifen (4-OHT), which is widely used to treat breast cancer, often leads to the development of endometrial carcinoma with poor prognosis, particularly among women who receiving long-term treatment. This study was performed to elucidate whether specific inhibition of casein kinase 1 (CK1) controls 4-OHT-mediated Ishikawa cell carcinogenesis. 4-OHT significantly stimulated the activity of estrogen receptor alpha (ERα) and nuclear translocation and expression of epidermal growth factor receptor (EGFR) from the plasma membrane to perinuclear or nuclear regions, as well as the activities of G-protein-coupled estrogen receptor 1 (GPER1) and Src in Ishikawa cells. However, inhibition of EGFR by Gefitinib blocked all these events, and inhibition of GPER1 or Src produced a partial block. GPER1 and Src controlled Ishikawa cell carcinogenesis in different manners: GPER1 accelerated EGFR mobility without affecting ERα activity, while Src activated ERα and EGFR without any change in GPER1 expression. EGFR and GPER1 performed reciprocal regulation in endometrial cell carcinogenesis via direct interaction in 4-OHT-treated Ishikawa cells, implying a possible key role of GPER1 in these events. Inhibition of CK1 by CKI-7 and IC261, however, impeded all changes beginning with EGFR translocation and activity in 4-OHT-treated Ishikawa cells. These findings indicate that inhibition of CK1 could control 4-OHT-mediated activation and translocation of ER/EGFR and GPER1/Src expression, inhibiting 4-OHT-triggered endometrial carcinogenesis. Therefore, targeting of CK1 by CKI-7 and IC261 could be a prospective adjuvant therapy for breast cancer patients taking tamoxifen., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tamoxifen (TAM) is an accredited drug used for treatment and prevention of breast cancer. Due to the long-term taking and the trend for women to delay childbearing, inadvertent conception occasionally occurs during TAM treatment. To explore the effects of TAM on a fetus, pregnant mice at gestation day 16.5 were orally administrated with different concentrations of TAM. Molecular biology techniques were used to analyze the effects of TAM on primordial follicle assembly of female offspring and the mechanism. It was found that maternal TAM exposure affected primordial follicle assembly and damaged the ovarian reserve in 3 dpp offspring. Up to 21 dpp, the follicular development had not recovered, with significantly decreased antral follicles and decreased total follicle number after maternal TAM exposure. Cell proliferation was significantly inhibited; however, the cell apoptosis was induced by maternal TAM exposure. Epigenetic regulation was also involved in the process of TAM induced abnormal primordial follicle assembly. The changed levels of H3K4me3, H3K9me3, and H3K27me3 presented the function of histone methylation in the regulation of the effects of maternal TAM exposure on the reproduction of female offspring. Moreover, the changed level of RNA m6A modification and the changed expression of genes related to transmethylation and demethylation proved the role of m6A in the process. Maternal TAM exposure led to abnormal primordial follicle assembly and follicular development by affecting cell proliferation, cell apoptosis, and epigenetics., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Tamoxifen, a triphenylethylene-based selective estrogen-receptor modulator, is a landmark drug for the treatment of breast cancer and is also used for treating liver cancer and osteoporosis. Structural studies of tamoxifen have led to the synthesis of more than 20 novel tamoxifen analogs as receptor modulators, including 16 ERα modulators 2-17 , an ERRβ inverse agonist 19 and six ERRγ inverse agonists 20 - 25 . This paper summarizes the research progress and structure-activity relationships of tamoxifen analogs modulating these three nuclear receptors reported in the literature, and introduces the relationship between these three nuclear receptor-mediated diseases and tamoxifen analogs to guide the research of novel tamoxifen analogs.

Dysregulated ERα signaling is responsible for endocrine resistance and eventual relapse in patients with estrogen receptor-positive (ER + ) breast cancer. Thus, identifying novel ERα regulators is necessary to fully understand the mechanisms of endocrine resistance. Here, we identified circRNA-SFMBT2 to be highly expressed in ER + breast cancer cells in comparison to ER - cells and found that high circRNA-SFMBT2 levels were related to larger tumor size and poor prognosis in patients with ER + breast cancer. In vitro and in vivo experiments confirmed that the circRNA-SFMBT2 level was positively correlated with the ERα protein level, implying a regulatory role for circRNA-SFMBT2 in ERα signaling. Moreover, we found that circRNA-SFMBT2 biogenesis could be facilitated via RNA-binding protein quaking (QKI), and biologically elevated circRNA-SFMBT2 expression promoted cell growth and tamoxifen resistance in ER + breast cancer. Mechanistically, circRNA-SFMBT2 exhibits a specific tertiary structure that endows it with a high binding affinity for ERα and allows it to interact with the AF2 and DBD domains of ERα, enforcing recruitment of RNF181 to the AF1 domain of ERα. Furthermore, the circRNA-SFMBT2/RNF181 axis differentially regulated K48-linked and K63-linked ubiquitination of ERα to enhance ERα stability, resulting in increased expression of ERα target genes and tumor progression. In summary, circRNA-SFMBT2 is an important regulator of ERα signaling, and antagonizing circRNA-SFMBT2 expression may constitute a potential therapeutic strategy for breast cancer., (© 2023. The Author(s).)

In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.

Specific and efficient smooth muscle cell-targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLSP2A or CreERT2-P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2-P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

Background: Numerous studies have reported the anti-cancer effects of different parts of Annona muricata Linn, however ; most of them focused on the in vitro evaluation of isolates. In vivo evidence on which part is best suited for breast cancer chemoprevention remains to be demonstrated. This is a comparative study of the effects of A. muricata fruit and leaves extracts on DMBA induced-breast cancer in rats., Methods: Rats exposed to DMBA (50 mg/kg, s.c.), were treated with A. muricata fruit aqueous extract at 200 mg/kg BW (3 days/week or daily) and A. muricata Linn leaves ethanolic extract at 200 mg/kg daily. Positive control group received tamoxifen at 3.3 mg/kg, while the normal and diseased controls received vehicle. After 20 weeks of treatment, the tumor incidence, tumor burden, tumor volume, histopathology, protein and CA 15 - 3 levels as well as antioxidant status, pro-inflammatory cytokines were assessed., Results: Thus, 100% of diseased rats presented cribriform ductal carcinoma of SBR grade III. A. muricata extracts (leaves and fruit) and tamoxifen significantly reduced death and tumor incidences, volume and weight of the tumors, total protein and CA15-3 levels compared to the DMBA group. They exhibited antioxidant activity, through an increase in the GSH level and SOD and catalase activities with reduced levels of MDA compared to DMBA group. TNF-α, IL-6 and INF-γ levels reduced with regards to A. muricata treatment., Conclusion: These results confirm the anti-breast cancer effect of A. muricata, however, the aqueous fruit extract was more potent than the ethanolic leaves extract., (© 2023. The Author(s).)

Estrogen receptor alpha (ERα) is a well-established therapeutic target for the treatment of ER-positive (ER+) breast cancers. Despite the tremendous successes achieved with tamoxifen, a selective ER modulator, and aromatase inhibitors (AIs), resistance to these therapies is a major clinical problem. Therefore, induced protein degradation and covalent inhibition have been pursued as new therapeutic approaches to target ERα. This Perspective summarizes recent progress in the discovery and development of oral selective ER degraders (SERDs), complete estrogen receptor antagonists (CERANs), selective estrogen receptor covalent antagonists (SERCAs), and proteolysis targeting chimera (PROTAC) ER degraders. We focus on those compounds which have been advanced into clinical development.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach., Competing Interests: Conflict of interest The authors declare the following competing interest(s): L. B. and C. O. are scientific co-founders of Ambagon Therapeutics. C. O. and E. S. are employees of Ambagon Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Breast cancer one of the most common diseases in women, has a high death and morbidity rate. Tamoxifen being very much effective in the chemoprevention of breast cancer has been shown to develop resistance during the course of treatment making it difficult for patient's survival. By combining tamoxifen with naturally occurring substances having similar activities, might control the toxicity and increase the susceptibility towards the treatment. As a natural compound, D-limonene has been reported to inhibit the growth of certain malignancies significantly. The main goal of our work is to investigate the combinatorial antitumor effects of D-limonene and tamoxifen in MCF-7 cells, as well as understand the potential underlying anticancer mechanism. MTT assays, colony formation assays, DAPI and Annexin V-FITC labeling, flow cytometer analysis, and western blot analysis were used to explore the details of anticancer mechanism. The combined effects of tamoxifen with D-limonene have shown significant decrease in the cell viability of MCF 7 cells. According to flow cytometer analyses and Annexin V/PI staining, D-limonene has been found to increase tamoxifen-mediated apoptosis as compared to the treatment alone in these cells. Additionally, cell growth has been found to be arrested at G1 phase by regulating cyclin D1 and cyclin B1. Our research consequently provided the first evidence that combining D-limonene and tamoxifen might increase the anticancer efficacy by inducing apoptosis in MCF 7 breast cancer cells. This combinatorial treatment strategy demands more research which might fulfill the need for improved treatment efficacy in controlling breast cancer., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Peyronie's disease (PD) is a fibrotic disorder characterized by plaque formation in the tunica albuginea (TA) of the penis, and we have previously shown that inhibition of transformation of TA-derived fibroblasts to myofibroblasts using a combination phosphodiesterase type 5 (PDE5) inhibitors and selective estrogen receptor modulators (SERMs) is effective in slowing the progression of early PD., Aim: The study sought to investigate whether combinations of statins with PDE5 inhibitors or SERMs would affect myofibroblast transformation in vitro., Methods: Primary fibroblasts were isolated from TA of patients with PD and stimulated with transforming growth factor β1 in the absence and presence of a range of concentrations of statins, PDE5 inhibitors, SERMs, and their combinations for 72 hours before quantifying α-smooth muscle actin using in-cell enzyme-linked immunosorbent assay., Outcomes: The prevention of transforming growth factor β1-induced transformation of TA-derived fibroblasts to myofibroblasts was measured in vitro., Results: Statins (simvastatin, lovastatin) inhibited myofibroblast transformation in a concentration-dependent manner with half maximal inhibitory concentration values of 0.77 ± 0.07 μM and 0.8 ± 0.13 μM, respectively. Simvastatin inhibited myofibroblast transformation in a synergistic fashion when combined with vardenafil (a PDE5 inhibitor; log alpha >0). Combination of tamoxifen (a SERM) and simvastatin did not show synergy (log alpha <0). When 3 drugs (simvastatin, vardenafil, and tamoxifen) were combined, the effect was not synergistic, but rather was additive., Clinical Implications: A combination of a statin with a PDE5 inhibitor might be useful in the clinic to slow the progression of the disease in patients with early PD; however, caution should be taken with such a combination because of the reported myopathy as a side effect., Strengths and Limitations: The use of primary human cells from patients with PD is a strength of this study. The mechanisms by which these drug classes exert synergy when used in combination was not investigated., Conclusion: This is the first demonstration of an antifibrotic synergy between statins and PDE5 inhibitors., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society of Sexual Medicine.)

Insulin-like growth factor-I (IGF-I) levels, which are reduced by age, and cortical bone dimensions are major determinants of fracture risk in elderly subjects. Inactivation of liver-derived circulating IGF-I results in reduced periosteal bone expansion in young and older mice. In mice with lifelong depletion of IGF-I in osteoblast lineage cells, the long bones display reduced cortical bone width. However, it has not previously been investigated whether inducible inactivation of IGF-I locally in bone in adult/old mice affects the bone phenotype. Adult tamoxifen-inducible inactivation of IGF-I using a CAGG-CreER mouse model (inducible IGF-IKO mice) substantially reduced IGF-I expression in bone (-55%) but not in liver. Serum IGF-I and body weight were unchanged. We used this inducible mouse model to assess the effect of local IGF-I on the skeleton in adult male mice, avoiding confounding developmental effects. After tamoxifen-induced inactivation of the IGF-I gene at 9 months of age, the skeletal phenotype was determined at 14 months of age. Computed tomography analyses of tibia revealed that the mid-diaphyseal cortical periosteal and endosteal circumferences and calculated bone strength parameters were decreased in inducible IGF-IKO mice compared with controls. Furthermore, 3-point bending showed reduced tibia cortical bone stiffness in inducible IGF-IKO mice. In contrast, the tibia and vertebral trabecular bone volume fraction was unchanged. In conclusion, inactivation of IGF-I in cortical bone with unchanged liver-derived IGF-I in older male mice resulted in reduced radial growth of cortical bone. This suggests that not only circulating IGF-I but also locally derived IGF-I regulates the cortical bone phenotype in older mice., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Estrogen receptor positive (ER + ) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER + BCa cells and is a novel, tractable therapeutic target for the treatment of ER + BCa patients. Analyses of publicly available clinical datasets indicate that ER + BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER + BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER + BCa cells, implicating a growth dependency of ER + BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER + breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER + breast cancer signaling and growth that suggests ER + BCa patients may benefit from SEMA3C-targeted therapy.

The Insulin-like Growth Factor (IGF) system in breast cancer progression has been a matter of interest for decades, but targeting this system did not result in a successful clinical strategy. The system's complexity and homology of its two receptors-insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R)-are possible causes. The IGF system maintains cell proliferation and also regulates metabolism, making it a pathway to explore. To understand the metabolic phenotype of breast cancer cells, we quantified their real-time ATP production rate upon acute stimulation with ligands-insulin-like growth factor 1 (1GF-1) and insulin. MCF-7L cells express both IGF-1R and IR, while tamoxifen-resistant MCF-7L (MCF-7L TamR) cells have downregulated IGF-1R with unchanged IR levels. Treating MCF-7L cells with 5 nM IGF-1 increased the glycolytic ATP production rate, while 10 nM insulin did not affect metabolism when compared with the control. Neither treatment altered ATP production in MCF-7L TamR cells. This study provides evidence of the relationship between metabolic dysfunction, cancer, and the IGF axis. In these cells, IGF-1R, and not IR, regulates ATP production.

Fibroadenomas (FAs) are the most common breast tumors in women. No pharmacological agents are currently approved for FA intervention owing to its unclear mechanisms and a shortage of reproducible human models. Here, using single-cell RNA sequencing of human FAs and normal breast tissues, we observe distinct cellular composition and epithelial structural changes in FAs. Interestingly, epithelial cells exhibit hormone-responsive functional signatures and synchronous activation of estrogen-sensitive and hormone-resistant mechanisms (ERBB2, BCL2 and CCND1 pathways). We develop a human expandable FA organoid system and observe that most organoids seem to be resistant to tamoxifen. Individualized combinations of tamoxifen with ERBB2, BCL2 or CCND1 inhibitors could significantly suppress the viability of tamoxifen-resistant organoids. Thus, our study presents an overview of human FA at single-cell resolution that outlines the structural and functional differences between FA and normal breast epithelium and, in particular, provides a potential therapeutic strategy for breast FAs., (© 2023. The Author(s).)

Objective: To investigate CITED1 as a potential biomarker of anti-endocrine response and breast cancer recurrence, given its previously determined role in mediating estrogen-dependant transcription. The study is a continuation of earlier work establishing the role of CITED1 in mammary gland development., Results: CITED1 mRNA is associated with estrogen-receptor positivity and selectively expressed in the GOBO dataset of cell lines and tumours representing the luminal-molecular subtype. In patients treated with tamoxifen, higher CITED1 correlated with better outcome, suggesting a role in anti-estrogen response. The effect was particularly evident in the subset of estrogen-receptor positive, lymph-node negative (ER+/LN-) patients although noticeable divergence of the groups was apparent only after five years. Tissue microarray (TMA) analysis further validated the association of CITED1 protein, by immunohistochemistry, with favourable outcome in ER+, tamoxifen-treated patients. Although we also found a favourable response to anti-endocrine treatment in a larger TCGA dataset, the tamoxifen-specific effect was not replicated. Finally, MCF7 cells overexpressing CITED1 showed selective amplification of AREG but not TGFα suggesting that maintenance of specific ERα-CITED1 mediated transcription is important for the long-term response to anti-endocrine therapy. These findings together confirm the proposed mechanism of action of CITED1 and support its potential use as a prognostic biomarker., (© 2023. The Author(s).)

Background: Premenopausal women diagnosed with breast cancer often face aggressive chemotherapy resulting in infertility. Tamoxifen (TAM) is a selective estrogen receptor modulator that was previously suggested as a protective agent against chemotherapy-induced ovarian failure. In the current study, we examined mechanisms of the protective action of TAM in the ovaries of tumor-bearing rats treated with the chemotherapy drug cyclophosphamide (CPA)., Results: TAM prevented CPA-induced loss of ovarian follicular reserves. The protective TAM effect in the rat ovary partially resulted from decreased apoptosis. In addition, transcriptomic and proteomic screening also implicated the importance of DNA repair pathways as well as cell adhesion and extracellular matrix remodeling in the protective ovarian actions of TAM., Conclusions: Tamoxifen shielded the ovary from the side effects of chemotherapy without lessening the tumoricidal actions of mammary cancer treatment., (© 2023. The Author(s).)

Label-free electrochemical visualization of cancer cell apoptosis is essential for cancer therapies. In this work, we proposed a noninvasive imaging method using a light-addressable electrochemical sensor (LAES) for label-free imaging of drug-induced tumor cell apoptosis. The dynamic AC photocurrent changes on MCF-7 human breast adenocarcinoma cells after inducing by tamoxifen were imaged. And the reasons for photocurrent changes on the cells were explored by monitoring the changes in the ζ potentials of cells and Faradic impedance. The results demonstrated that the AC photocurrent on apoptotic MCF-7 cells increased, and the apoptosis degree of each cell was heterogeneous. Moreover, the AC photocurrent increase was attributed to the increased cell membrane permeability and the increased gap between the cell basal surface and the substrate caused by cell apoptosis. This study provides a brand new approach for label-free visualizing cell apoptosis heterogeneity, which has great potential in apoptosis-associated drug screening or drug efficacy evaluation.

Introduction: Breast cancer is the most common malignancy in women, and it is linked to several risk factors including genetic alterations, obesity, estrogen signaling, insulin levels, and glucose metabolism deregulation. Insulin and Insulin-like growth factor signaling exert a mitogenic and pro-survival effect. Indeed, epidemiological and pre-clinical studies have shown its involvement in the development, progression, and therapy resistance of several cancer types including breast cancer. Insulin/Insulin-like growth factor signaling is triggered by two insulin receptor isoforms identified as IRA and IRB and by Insulin-like growth factor receptor I. Both classes of receptors show high homology and can initiate the intracellular signaling cascade alone or by hybrids formation. While the role of Insulin-like growth factor receptor I in breast cancer progression and therapy resistance is well established, the effects of insulin receptors in this context are complex and not completely elucidated., Methods: We used estrogen-dependent insulin-like growth factor receptor I deleted gene (MCF7 IGFIRKO ) breast cancer cell models, lentivirally transduced to over-express empty-vector (MCF7 IGFIRKO/EV ), IRA (MCF7 IGFIRKO/IRA ) or IRB (MCF7 IGFIRKO/IRB ), to investigate the role of insulin receptors on the antiproliferative activity of tamoxifen in presence of low and high glucose concentrations. The tamoxifen-dependent cytotoxic effects on cell proliferation were determined by MTT assay and clonogenic potential measurement. Cell cycle and apoptosis were assessed by FACS, while immunoblot was used for protein analysis. Gene expression profiling was investigated by a PCR array concerning genes involved in apoptotic process by RT-qPCR., Results: We found that glucose levels played a crucial role in tamoxifen response mediated by IRA and IRB. High glucose increased the IC50 value of tamoxifen for both insulin receptors and IRA-promoted cell cycle progression more than IRB, independently of glucose levels and insulin stimulation. IRB, in turn, showed anti-apoptotic properties, preserving cells' survival after prolonged tamoxifen exposure, and negatively modulated pro-apoptotic genes when compared to IRA., Discussion: Our findings suggest that glucose levels modify insulin receptors signaling and that this event can interfere with the tamoxifen therapeutic activity. The investigation of glucose metabolism and insulin receptor expression could have clinical implications in Estrogen Receptor positive breast cancer patients receiving endocrine treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stella, Massimino, Manzella, Parrinello, Vitale, Martorana and Vigneri.)

Globally, breast cancer is among the most frequently diagnosed and common cause of death among women. Aromatase inhibitors, such as anastrozole, are one of the first-line therapies used in the treatment of breast cancer in postmenopausal women; however, thromboembolic complications are common. Thus, this study investigated the combined effects of anastrozole and antiplatelet therapies, aspirin and clopidogrel, on breast cancer cytotoxicity and survival in vitro. Breast cancer cell lines (MCF-7 and T47D) were treated with varying Cmax concentrations of anastrozole and/or antiplatelet therapies for 24 h. A wound-healing scratch assay was used to measure migration and the WST-1 assay for cellular proliferation. An autophagy/cytotoxicity dual staining kit was used to assay cell death and survival. Changes in cell morphology were assessed using scanning electron microscopy. Data were analyzed with Statistica software. Our findings showed that sub-phenotypic differences exist between the luminal-A breast cancer cell lines, with T47D cells being more aggressive than MCF-7 cells. Cellular proliferation and migration responded in a dose-dependent manner for the different treatment groups. Notably, anastrozole combined with aspirin and clopidogrel mediated higher levels of cell survival than each agent individually, with autophagy levels being significantly increased in comparison to that induced with antiplatelet therapy alone., Competing Interests: Conflict of Interest The authors declare that they have no competing interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.

Pancratistatin (PST) and narciclasine (NRC) are natural therapeutic agents that exhibit specificity toward the mitochondria of cancerous cells and initiate apoptosis. Unlike traditional cancer therapeutic agents, PST and NRC are effective, targeted, and have limited adverse effects on neighboring healthy, noncancerous cells. Currently, the mechanistic pathway of action for PST and NRC remains elusive, which in part inhibits PST and NRC from becoming efficacious therapeutic alternatives. Herein, we use neutron and x-ray scattering in combination with calcein leakage assays to characterize the effects of PST, NRC, and tamoxifen (TAM) on a biomimetic model membrane. We report an increase in lipid flip-flop half-times (t 1/2 ) (≈12.0%, ≈35.1%, and a decrease of ≈45.7%) with 2 mol percent PST, NRC, and TAM respectively. An increase in bilayer thickness (≈6.3%, ≈7.8%, and ≈7.8%) with 2 mol percent PST, NRC, and TAM, respectively, was also observed. Lastly, increases in membrane leakage (≈31.7%, ≈37.0%, and ≈34.4%) with 2 mol percent PST, NRC, and TAM, respectively, were seen. Considering the maintenance of an asymmetric lipid composition across the outer mitochondrial membrane (OMM) is crucial to eukaryotic cellular homeostasis and survival, our results suggest PST and NRC may play a role in disrupting the native distribution of lipids within the OMM. A possible mechanism of action for PST- and NRC-induced mitochondrial apoptosis is proposed via the redistribution of the native OMM lipid organization and through OMM permeabilization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. All rights reserved.)

Mouse atonal homolog 1 ( Math1/Atoh1 ) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1 lox/lox VilCreER T2 ( Math1 ΔIEC ) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1 ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1 ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1 ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1 ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells. NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1 lox/lox VilCreER T2 mouse model.