UTRN as a potential biomarker in breast cancer: a comprehensive bioinformatics and in vitro study.

Utrophin (UTRN), known as a tumor suppressor, potentially regulates tumor development and the immune microenvironment. However, its impact on breast cancer's development and treatment remains unstudied. We conducted a thorough examination of UTRN using both bioinformatic and in vitro experiments in this study. We discovered UTRN expression decreased in breast cancer compared to standard samples. High UTRN expression correlated with better prognosis. Drug sensitivity tests and RT-qPCR assays revealed UTRN's pivotal role in tamoxifen resistance. Furthermore, the Kruskal-Wallis rank test indicated UTRN's potential as a valuable diagnostic biomarker for breast cancer and its utility in detecting T stage of breast cancer. Additionally, our results demonstrated UTRN's close association with immune cells, inhibitors, stimulators, receptors, and chemokines in breast cancer (BRCA). This research provides a novel perspective on UTRN's role in breast cancer's prognostic and therapeutic value. Low UTRN expression may contribute to tamoxifen resistance and a poor prognosis. Specifically, UTRN can improve clinical decision-making and raise the diagnosis accuracy of breast cancer.

Zona Glomerulosa-Derived Klotho Modulates Aldosterone Synthase Expression in Young Female Mice.

Klotho plays a critical role in the regulation of ion and fluid homeostasis. A previous study reported that haplo-insufficiency of Klotho in mice results in increased aldosterone synthase (CYP11B2) expression, elevated plasma aldosterone, and high blood pressure. This phenotype was presumed to be the result of diminished Klotho expression in zona glomerulosa (zG) cells of the adrenal cortex; however, systemic effects on adrenal aldosterone production could not be ruled out. To examine whether Klotho expressed in the zG is indeed a critical regulator of aldosterone synthesis, we generated a tamoxifen-inducible, zG-specific mouse model of Klotho deficiency by crossing Klotho-flox mice with Cyp11b2-CreERT mice (zG-Kl-KO). Tamoxifen-treated Cyp11b2-CreERT animals (zG-Cre) served as controls. Rosa26-mTmG reporter mice were used for Cre-dependent lineage-marking. Two weeks after tamoxifen induction, the specificity of the zG-Cre line was verified using immunofluorescence analysis to show that GFP expression was restricted to the zG. RNA in situ hybridization revealed a 65% downregulation of Klotho messenger RNA expression in the zG of zG-Kl-KO female mice at age 12 weeks compared to control mice. Despite this significant decrease, zG-Kl-KO mice exhibited no difference in plasma aldosterone levels. However, adrenal CYP11B2 expression and the CYP11B2 promotor regulatory transcription factors, NGFIB and Nurr1, were enhanced. Together with in vitro experiments, these results suggest that zG-derived Klotho modulates Cyp11b2 but does not evoke a systemic phenotype in young adult mice on a normal diet. Further studies are required to investigate the role of adrenal Klotho on aldosterone synthesis in aged animals.

Selective impact of ALK and MELK inhibition on ERα stability and cell proliferation in cell lines representing distinct molecular phenotypes of breast cancer.

Breast cancer (BC) is a leading cause of global cancer-related mortality in women, necessitating accurate tumor classification for timely intervention. Molecular and histological factors, including PAM50 classification, estrogen receptor α (ERα), breast cancer type 1 susceptibility protein (BRCA1), progesterone receptor (PR), and HER2 expression, contribute to intricate BC subtyping. In this work, through a combination of bioinformatic and wet lab screenings, followed by classical signal transduction and cell proliferation methods, and employing multiple BC cell lines, we identified enhanced sensitivity of ERα-positive BC cell lines to ALK and MELK inhibitors, inducing ERα degradation and diminishing proliferation in specific BC subtypes. MELK inhibition attenuated ERα transcriptional activity, impeding E2-induced gene expression, and hampering proliferation in MCF-7 cells. Synergies between MELK inhibition with 4OH-tamoxifen (Tam) and ALK inhibition with HER2 inhibitors revealed potential therapeutic avenues for ERα-positive/PR-positive/HER2-negative and ERα-positive/PR-negative/HER2-positive tumors, respectively. Our findings propose MELK as a promising target for ERα-positive/PR-positive/HER2-negative BC and highlight ALK as a potential focus for ERα-positive/PR-negative/HER2-positive BC. The synergistic anti-proliferative effects of MELK with Tam and ALK with HER2 inhibitors underscore kinase inhibitors' potential for selective treatment in diverse BC subtypes, paving the way for personalized and effective therapeutic strategies in BC management.

Enhancing Sperm Motility Parameters in Patients with Asthenospermia: A Combined Approach of Acupuncture at Fusiguan Point and Tamoxifen Citrate Tablets.

OBJECTIVE: To explore the effect of acupuncture at Fuguan point combined with tamoxifen citrate tablet on sperm motility parameters. METHODS: A total of 115 individuals with asthenospermia were categorized based on different treatment regimens: 53 patients in the control group (receiving tamoxifen citrate tablets) and 62 patients in the observation group (undergoing acupoint acupuncture in conjunction with tamoxifen citrate tablets). Both groups underwent a 3-month treatment period. The computer-assisted sperm analysis system was employed to measure various motility parameters of human sperm, including sperm motility rate, average path velocity (VAP), lateral swing amplitude (ALH), percentage of class a sperm, and percentage of class a + b sperm. RESULTS: Prior to treatment, no statistically significant differences were observed between the two groups in terms of sperm motility rate, VAP, ALH, percentage of class a sperm, and percentage of class a + b sperm (p > 0.05). Following treatment, both groups exhibited significant enhancements in sperm motility rate, VAP, ALH, percentage of class a sperm, and percentage of class a + b sperm compared to pretreatment levels (p < 0.05). Furthermore, all measured indicators in the observation group demonstrated significantly superior improvements than those of the control group, with the differences proving statistically significant (p < 0.05). CONCLUSIONS: The combination of acupuncture at Fusiguan point and tamoxifen citrate tablets exerts a notably positive effect on sperm motility in individuals diagnosed with asthenospermia.

Characterization of a Novel Mouse Model for Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a progressive blinding disorder, characterized by increased corneal endothelial excrescences (guttae), corneal endothelial cell loss, and edema. These symptoms are hypothesized to be caused by changes in the extracellular matrix (ECM) and mitochondrial dysfunction in the corneal endothelium. Despite this clinical and biological relevance, a comprehensive animal model that recapitulates all the major disease characteristics is currently unavailable. In this study, we develop such a model to improve our understanding of the signaling pathways involved in the FECD progression and develop strategies for early intervention. Method: To generate a comprehensive FECD model, we generated a double mutant mouse bearing tamoxifen-inducible knockdown of Slc4a11 and the Col8a2 (Q455K) mutation. We performed optical coherence tomography (OCT) and in vivo confocal microscopy using the Heidelberg Retinal Tomography 3 - Rostock Cornea module (HRT3-RCM) on the mice at 5 weeks of age before tamoxifen feeding to establish baseline values for corneal thickness, endothelial cell density, and test for the presence of guttae. We measured these parameters again post-tamoxifen treatment at 16 weeks of age. We collected corneas at 16 weeks to perform histopathology, immunofluorescence staining for tight junctions, adherens junctions, and oxidative stress. We evaluated endothelial pump function using a lactate assay. Results: The double mutant tamoxifen-fed animals showed the presence of guttae, and displayed increased corneal thickness and decreased endothelial cell density. Endothelial cells showed altered morphology with disrupted adherens junctions and elevated reactive oxygen species (ROS). Finally, we found that stromal lactate concentrations were elevated in the double mutant mice, indicative of compromised endothelial pump function. Conclusions: Overall, this mouse model recapitulates all the important phenotypic features associated with FECD.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Phosphatase and Tensin Homolog Inhibition in Proteolipid Protein 1-Expressing Cells Stimulates Neurogenesis and Gliogenesis in the Postnatal Enteric Nervous System.

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7-12 weeks of age; n = 4-15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3-4 days post-DSS) and (2) late (3-4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).

Developing liver-targeted naringenin nanoparticles for breast cancer endocrine therapy by promoting estrogen metabolism.

Endocrine therapy is standard for hormone receptor-positive (HR+) breast cancer treatment. However, current strategies targeting estrogen signaling pay little attention to estradiol metabolism in the liver and is usually challenged by treatment failure. In a previous study, we demonstrated that the natural compound naringenin (NAR) inhibited HR+ breast cancer growth by activating estrogen sulfotransferase (EST) expression in the liver. Nevertheless, the poor water solubility, low bio-barrier permeability, and non-specific distribution limited its clinical application, particularly for oral administration. Here, a novel nano endocrine drug NAR-cell penetrating peptide-galactose nanoparticles (NCG) is reported. We demonstrated that NCG presented specific liver targeting and increased intestinal barrier permeability in both cell and zebrafish xenotransplantation models. Furthermore, NCG showed liver targeting and enterohepatic circulation in mouse breast cancer xenografts following oral administration. Notably, the cancer inhibition efficacy of NCG was superior to that of both NAR and the positive control tamoxifen, and was accompanied by increased hepatic EST expression and reduced estradiol levels in the liver, blood, and tumor tissue. Moreover, few side effects were observed after NCG treatment. Our findings reveal NCG as a promising candidate for endocrine therapy and highlight hepatic EST targeting as a novel therapeutic strategy for HR+ breast cancer.

Cre-LoxP and tamoxifen-induced deletion of ovarian quiescin sulfhydryl oxidase 2 showed disruption of ovulatory activity in mice.

BACKGROUND: Quiescin sulfhydryl oxidase 2 (QSOX2) is a flavin adenine dinucleotide-dependent sulfhydryl oxidase that is known to be involved in protein folding, cell growth regulation, and redox state modification through oxidative activities. Earlier studies demonstrated the tissue and cellular localization of QSOX2 in the male reproductive tract, as well as the highly-regulated mechanism of QSOX2 protein synthesis and expression through the coordinated action of testosterone and epididymal-enriched amino acid, glutamate. However, the presence and the functions of QSOX2 in female reproduction are unknown. In this study, we applied the Cre-loxP gene manipulation system to generate the heterozygous and homozygous Qsox2 knockout mice and examined its effects on ovarian function. RESULTS: We demonstrated that QSOX2 was detected in the follicle-supporting cells (granulosa and cumulus cells) of ovarian follicles of all stages but was absent in the corpus luteum, suggesting its supportive role in folliculogenesis. In comparison with reproductive organogenesis in wild-type mice, there was no difference in testicular and epididymal structure in male Qsox2 knockout; however, Qsox2 knockout disrupted the regular ovulation process in female mice as a drastic decrease in the formation of the corpus luteum was detected, and no pregnancy was achieved when mating males with homozygous Qsox2 knockout females. RNAseq analyses further revealed that Qsox2 knockout altered critical signaling pathways and genes that are responsible for maintaining ovarian functions. CONCLUSION: Our data demonstrated for the first time that Qsox2 is critical for ovarian function in mice.

Mammalian enabled protein enhances tamoxifen sensitivity of the hormone receptor-positive breast cancer patients by suppressing the AKT signaling pathway.

BACKGROUND: Mammalian enabled (MENA) protein is a member of the enabled/vasodilator stimulated phosphoprotein (Ena/VASP) protein family, which regulates cytoplasmic actin network assembly. It plays a significant role in breast cancer invasion, migration, and resistance against targeted therapy and chemotherapy. However, its role in the efficacy of endocrine therapy for the hormone receptor-positive (HR+) breast cancer patients is not known. This study investigated the role of MENA in the resistance against tamoxifen therapy in patients with HR+ breast cancer and the underlying mechanisms. METHODS: MENA expression levels in the clinical HR+ breast cancer samples (n = 119) were estimated using immunohistochemistry (IHC) to determine its association with the clinicopathological features, tamoxifen resistance, and survival outcomes. Western blotting (WB) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was performed to estimate the MENA protein and mRNA levels in the tamoxifen-sensitive and -resistant HR+ breast cancer cell lines. Furthermore, CCK8, colony formation, and the transwell invasion and migration assays were used to analyze the effects of MENA knockdown on the biological behavior and tamoxifen sensitivity of the HR+ breast cancer cell lines. Xenograft tumor experiments were performed in the nude mice to determine the tumor growth rates and tamoxifen sensitivity of the control and MENA knockdown HR+ breast cancer cells in the presence and absence of tamoxifen treatment. Furthermore, we estimated the growth rates of organoids derived from the HR+ breast cancer patients (n = 10) with high and low MENA expression levels when treated with tamoxifen. RESULTS: HR+ breast cancer patients with low MENA expression demonstrated tamoxifen resistance and poorer prognosis compared to those with high MENA expression. Univariate and multivariate Cox regression analysis demonstrated that MENA expression was an independent predictor of tamoxifen resistance in patients with HR+ breast cancer. MENA knockdown HR+ breast cancer cells showed significantly reduced tamoxifen sensitivity in the in vitro experiments and the in vivo xenograft tumor mouse model compared with the corresponding controls. Furthermore, MENA knockdown increased the in vitro invasion and migration of the HR+ breast cancer cells. HR+ breast cancer organoids with low MENA expression demonstrated reduced tamoxifen sensitivity than those with higher MENA expression. Mechanistically, P-AKT levels were significantly upregulated in the MENA-knockdown HR + breast cancer cells treated with or without 4-OHT compared with the corresponding controls. CONCLUSIONS: This study demonstrated that downregulation of MENA promoted tamoxifen resistance in the HR+ breast cancer tissues and cells by enhancing the AKT signaling pathway. Therefore, MENA is a promising prediction biomarker for determining tamoxifen sensitivity in patients with HR+ breast cancer.

Small molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance.

INTRODUCTION: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. METHODS: A specific inhibitor of Hsp70-Bim protein-protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 µM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. RESULTS: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. CONCLUSIONS: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

BACKGROUND: Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/ß. METHODS: Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17ß-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERß expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERß and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. RESULTS: Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERß ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERß expression and a localization of ERs around the nuclear periphery. CONCLUSIONS: As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERß they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERß is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERß in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

MCM2 is involved in subtyping and tamoxifen resistance of ERα-positive breast cancer by acting as the downstream factor of ERα.

Tamoxifen (TAM) resistance is finally developed in over 40% of patients with estrogen receptor α-positive breast cancer (ERα+ -BC), documenting that discovering new molecular subtype is needed to confer perception to the heterogeneity of ERα+ -BC. We obtained representative gene sets subtyping ERα+ -BC using gene set variation analysis (GSVA), non-negative matrix factorization (NMF), and COX regression methods on the basis of METABRIC, TCGA, and GEO databases. Furthermore, the risk score of ERα+ -BC subtyping was established using least absolute shrinkage and selection operator (LASSO) regression on the basis of genes in the representative gene sets, thereby generating the two subtypes of ERα+ -BC. We further found that minichromosome maintenance complex component 2 (MCM2) functioned as the hub gene subtyping ERα+ -BC using GO, KEGG, and MCODE. MCM2 expression was capable for specifically predicting 1-year overall survival (OS) of ERα+ -BC and correlated with T stage, AJCC stage, and tamoxifen (TAM) sensitivity of ERα+ -BC. The downregulation of MCM2 expression inhibited proliferation, migration, and invasion of TAM-resistant cells and promoted G0/G1 arrest. Altogether, tamoxifen resistance entails that MCM2 is a hub gene subtyping ERα+ -BC, providing a novel dimension for discovering a potential target of TAM-resistant BC.

Cellular and molecular characteristics of stromal Lkb1 deficiency-induced gastrointestinal polyposis based on single-cell RNA sequencing.

Liver kinase B1 (Lkb1), encoded by serine/threonine kinase (Stk11), is a serine/threonine kinase and tumor suppressor that is strongly implicated in Peutz-Jeghers syndrome (PJS). Numerous studies have shown that mesenchymal-specific Lkb1 is sufficient for the development of PJS-like polyps in mice. However, the cellular origin and components of these Lkb1-associated polyps and underlying mechanisms remain elusive. In this study, we generated tamoxifen-inducible Lkb1flox/flox;Myh11-Cre/ERT2 and Lkb1flox/flox;PDGFRα-Cre/ERT2 mice, performed single-cell RNA sequencing (scRNA-seq) and imaging-based lineage tracing, and aimed to investigate the cellular complexity of gastrointestinal polyps associated with PJS. We found that Lkb1flox/+;Myh11-Cre/ERT2 mice developed gastrointestinal polyps starting at 9 months after tamoxifen treatment. scRNA-seq revealed aberrant stem cell-like characteristics of epithelial cells from polyp tissues of Lkb1flox/+;Myh11-Cre/ERT2 mice. The Lkb1-associated polyps were further characterized by a branching smooth muscle core, abundant extracellular matrix deposition, and high immune cell infiltration. In addition, the Spp1-Cd44 or Spp1-Itga8/Itgb1 axes were identified as important interactions among epithelial, mesenchymal, and immune compartments in Lkb1-associated polyps. These characteristics of gastrointestinal polyps were also demonstrated in another mouse model, tamoxifen-inducible Lkb1flox/flox;PDGFRα-Cre/ERT2 mice, which developed obvious gastrointestinal polyps as early as 2-3 months after tamoxifen treatment. Our findings further confirm the critical role of mesenchymal Lkb1/Stk11 in gastrointestinal polyposis and provide novel insight into the cellular complexity of Lkb1-associated polyp biology. © 2024 The Pathological Society of Great Britain and Ireland.

Analysis of TLR2 in Primary Endocrine Resistant of Breast Cancer.

BACKGROUND: Previous clinical studies have suggested that Toll-like receptor (TLR)2 had predictive function for endocrine resistance in HER2-positive breast cancer (BCa). Nevertheless, it remains unclear whether TLR2 would relate to development of endocrine therapy resistance in triple-positive breast cancer (TPBC). METHODS: Bioinformatic analysis of TLR2 was carried out through a database. Ten tumor tissues were obtained from TPBC patients who underwent surgery, with five patients displaying primary resistance to tamoxifen (TAM) with the remaining 5 being sensitive. Different levels of proteins were identified through mass spectrometry analysis and confirmed through reverse transcription polymerase chain reaction (RT-PCR) and western blot. TAM-resistant cell lines (BT474-TAM) were established by continuous exposure to TAM, and TAM resistance was assessed via IC50. Additionally, TLR2 mRNA was analyzed through western blot and RT-PCR in BT474, BT474-TAM, MCF-7, and MCF10A cells. Furthermore, TLR2-specific interference sequences were utilized to downregulate TLR2 expression in BT474-TAM cells to elucidate its role in TAM resistance. RESULTS: TLR2 had a correlation with decreased relapse-free survival in BCa patients from the GSE1456-GPL96 cohort, and it was involved in cancer development predominantly mediated by MAPK and PI3K pathways. TLR2 protein expression ranked in the top 5 proteins within the TAM-resistant group, and was 1.9 times greater than that in the sensitive group. Additionally, TLR2 mRNA and protein expression increased significantly in the established TAM-resistant BT474/TAM cell lines. The sensitivity of TAM was restored upon TLR2 downregulation in BT474/TAM cells. CONCLUSIONS: TLR2 might have a therapeutic value as it was involved in the TAM resistance in TPBC, with potential to be a marker for primary endocrine resistance.

A novel bystander effect in tamoxifen treatment: PPIB derived from ER+ cells attenuates ER- cells via endoplasmic reticulum stress-induced apoptosis.

Tamoxifen (TAM) is the frontline therapy for estrogen receptor-positive (ER+) breast cancer in premenopausal women that interrupts ER signaling. As tumors with elevated heterogeneity, amounts of ER-negative (ER-) cells are present in ER+ breast cancer that cannot be directly killed by TAM. Despite complete remissions have been achieved in clinical practice, the mechanism underlying the elimination of ER- cells during TAM treatment remains an open issue. Herein, we deciphered the elimination of ER- cells in TAM treatment from the perspective of the bystander effect. Markable reductions were observed in tumorigenesis of ER- breast cancer cells by applying both supernatants from TAM-treated ER+ cells and a transwell co-culture system, validating the presence of a TAM-induced bystander effect. The major antitumor protein derived from ER+ cells, peptidyl-prolyl cis-trans isomerase B (PPIB), is the mediator of the TAM-induced bystander effect identified by quantitative proteomics. The attenuation of ER- cells was attributed to activated BiP/eIF2α/CHOP axis and promoted endoplasmic reticulum stress (ERS)-induced apoptosis, which can also be triggered by PPIB independently. Altogether, our study revealed a novel TAM-induced bystander effect in TAM treatment of ER+ breast cancer, raising the possibility of developing PPIB as a synergistic antitumor agent or even substitute endocrine therapy.

The effect of human umbilical cord mesenchymal stem cells conditioned medium combined with tamoxifen drug on BRCA1 and BRCA2 expression in breast cancer mouse models.

BACKGROUND: A growing number of studies has indicated that the expression of Breast Cancer Susceptibility Genes 1 (BRCA1) and BRCA2 contribute to the resistance to DNA-damaging chemotherapies. Tamoxifen induces tumor cell death by suppressing estrogen receptor (ER) signaling and inducing DNA damage, and BRCA1 upregulation causes Tamoxifen chemoresistance in breast cancer cells. Consequently, this research study aimed to investigate the possible therapeutic effect of Human Umbilical Cord Mesenchymal Stem Cells Conditioned Medium (UCMSCs-CM) on sensitizing breast cancer cells to Tamoxifen by regulating BRCA1 and BRCA2 expression in vivo. METHODS: Forty female mice, 4-8 weeks old, with weight of 150 g, were used for this study. Mouse 4T1 breast tumor models were established and then treated with UCMSCs-CM and Tamoxifen alone or in combination. After 10 days, the tumor masses were collected and the expression levels of BRCA1 and BRCA2 were evaluated using qRT-PCR assay. RESULTS: The results obtained from qRT-PCR assay illustrated that UCMSCs-CM, either alone or in combination with Tamoxifen, significantly downregulated the mRNA expression levels of BRCA1 in breast cancer mouse models. However, both UCMSCs-CM and Tamoxifen indicated no statistically significant impact on BRCA2 mRNA expression compared to controls. CONCLUSION: Our findings evidenced that UCMSCs-CM could be considered as a potential therapeutic option to modulate Tamoxifen chemosensitivity by regulating BRCA1 in breast cancer.

Prolonged tamoxifen-enriched diet is associated with cardiomyopathy and nutritional frailty in mice.

Tamoxifen (TAM) is required for gene recombination in the inducible Cre/lox system. The TAM-enriched diet is considered safe, with negligible impact on animal wellbeing. However, studies reporting the long-term effects of the TAM diet and its potential impact on experimental outcomes are scarce. We conducted a longitudinal study on mice exposed to a 4-week dietary TAM citrate supplementation. Several parameters were recorded, such as body weight, body composition, mortality, and cardiac function. The collagen1a2 (Col1a2) transgenic mouse was used to assess TAM-induced recombination in vivo in cardiac fibroblasts followed by myocardial infarction (MI). The impact of TAM on the MI outcome was also evaluated. The recombination efficiency and cytotoxic effect of the TAM active metabolite, 4-hydroxy-tamoxifen (4-OHT), were assessed in vitro. Mice exposed to a TAM diet showed body weight loss and a 10% increase in mortality (P = 0.045). The TAM diet decreased cardiac function and induced cardiac remodeling, indicated by decreased fractional shortening from 32.23% to 19.23% (P = 0.001) and left ventricular (LV) wall thinning. All measured parameters were reversed to normal when mice were returned to a normal diet. Infarcted Col1a2-CreER mice on the TAM regimen showed gene recombination in fibroblasts, but it was associated with a substantial increase in mortality post-surgery (2.5-fold) compared to the controls. In vitro, 4-OHT induced gene editing in fibroblasts; however, cell growth arrest and cytotoxicity were observed at high concentrations. In conclusion, prolonged exposure to the TAM diet can be detrimental and necessitates careful model selection and interpretation of the results.

A comparative evaluation of the strengths and potential caveats of the microglial inducible CreER mouse models.

The recent proliferation of new Cre and CreER recombinase lines provides researchers with a diverse toolkit to study microglial gene function. To determine how best to apply these lines in studies of microglial gene function, a thorough and detailed comparison of their properties is needed. Here, we examined four different microglial CreER lines (Cx3cr1YFP-CreER(Litt), Cx3cr1CreER(Jung), P2ry12CreER, and Tmem119CreER), focusing on (1) recombination specificity, (2) leakiness (the degree of tamoxifen-independent recombination in microglia and other cells), (3) the efficiency of tamoxifen-induced recombination, (4) extraneural recombination (the degree of recombination in cells outside of the CNS, particularly myelo/monocyte lineages), and (5) off-target effects in the context of neonatal brain development. We identify important caveats and strengths for these lines, which will provide broad significance for researchers interested in performing conditional gene deletion in microglia. We also provide data emphasizing the potential of these lines for injury models that result in the recruitment of splenic immune cells.

Endosomal recycling inhibitors downregulate estrogen receptor-alpha and synergise with endocrine therapies.

PURPOSE: Breast cancer (BC) accounts for roughly 30% of new cancers diagnosed in women each year; thus, this cancer type represents a substantial burden for people and health care systems. Despite the existence of effective therapies to treat BC, drug resistance remains a problem and is a major cause of treatment failure. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. Recent research indicates that inhibition of the endosomal recycling pathway, an intracellular membrane trafficking pathway that returns endocytosed proteins back to the plasma membrane, may be a promising strategy to downregulate clinically relevant cell surface proteins such as HER2 and HER3, and to overcome drug resistance. METHODS: To investigate the molecular mechanism of action of an endosomal recycling inhibitor (ERI) called primaquine, we performed a reverse-phase protein array (RPPA) assay using a HER2-positive breast cancer cell line. The RPPA findings were confirmed by Western blot and RT-qPCR in several BC cell lines. Novel drug combinations were tested by MTT cell viability and clonogenic assays. RESULTS: Among the signalling molecules downregulated by ERIs were estrogen receptor-alpha (ER-α) and androgen receptor. We confirmed this finding in other breast cancer cell lines and show that downregulation occurs at the transcriptional level. We also found that ERIs synergise with tamoxifen, a standard-of-care therapy for breast cancer. DISCUSSION: Our data suggest that combining ERIs with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.