Infection trains the host for microbiota-enhanced resistance to pathogens.

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.

Deletion of PD-1 destabilizes the lineage identity and metabolic fitness of tumor-infiltrating regulatory T cells.

Regulatory T (Treg) cells have an immunosuppressive function and highly express the immune checkpoint receptor PD-1 in the tumor microenvironment; however, the function of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Here, we showed that conditional deletion of PD-1 in Treg cells delayed tumor progression. In Pdcd1fl/flFoxp3eGFP-Cre-ERT2(+/-) mice, in which both PD-1-expressing and PD-1-deficient Treg cells coexisted in the same tissue environment, conditional deletion of PD-1 in Treg cells resulted in impairment of the proliferative and suppressive capacity of TI Treg cells. PD-1 antibody therapy reduced the TI Treg cell numbers, but did not directly restore the cytokine production of TI CD8+ T cells in TC-1 lung cancer. Single-cell analysis indicated that PD-1 signaling promoted lipid metabolism, proliferation and suppressive pathways in TI Treg cells. These results suggest that PD-1 ablation or inhibition can enhance antitumor immunity by weakening Treg cell lineage stability and metabolic fitness in the tumor microenvironment.

Chemically Driven Clearance of Amyloid Aggregates by Polyfunctionalized Furo[2,3-b:4,5-b']dipyridine-Chalcone Hybrids to Ameliorate Memory in an Alzheimer Mouse Model.

The aberrant assembly of amyloid-ß (Aß) is implicated in Alzheimer's disease (AD). Recent clinical outcomes of Aß-targeted immunotherapy reinforce the notion that clearing Aß burden is a potential therapeutic approach for AD. Herein, to develop drug candidates for chemically driven clearance of Aß aggregates, we synthesized 51 novel polyfunctionalized furo[2,3-b:4,5-b']dipyridine-chalcone hybrid compounds. After conducting two types of cell-free anti-Aß functional assays, Aß aggregation prevention and Aß aggregate clearance, we selected YIAD-0336, (E)-8-((1H-pyrrol-2-yl)methylene)-10-(4-chlorophenyl)-2,4-dimethyl-7,8-dihydropyrido[3',2':4,5]furo[3,2-b]quinolin-9(6H)-one, for further in vivo investigations. As YIAD-0336 exhibited a low blood-brain barrier penetration profile, it was injected along with aggregated Aß directly into the intracerebroventricular region of ICR mice and ameliorated spatial memory in Y-maze tests. Next, YIAD-0336 was orally administered to 5XFAD transgenic mice with intravenous injections of mannitol, and YIAD-0336 significantly removed Aß plaques from the brains of 5XFAD mice. Collectively, YIAD-0336 dissociated toxic aggregates in the mouse brain and hence alleviated cognitive deterioration. Our findings indicate that chemically driven clearance of Aß aggregates is a promising therapeutic approach for AD.

Regulation of sexually dimorphic placental adaptation in LPS exposure-induced intrauterine growth restriction.

BACKGROUND: Sexual dimorphism in placental physiology affects the functionality of placental adaptation during adverse pregnancy. Defects of placental function compromise fetal programming, affecting the offspring's adult life. However, studies focusing on the relationship between sex-specific placental adaptation and consequent fetal maldevelopment under sub-optimal uterus milieu are still elusive. METHODS: Here, we investigated the effects of maternal lipopolysaccharide (LPS) exposure between placental sex. Pregnant ICR mice received intraperitoneal injection of phosphate-buffered saline or 100, 200, and 400 µg/kg LPS on the gestational day (GD) 15.5. To determine whether prenatal maternal LPS exposure resulted in complicated pregnancy outcomes, survival rate of embryos was calculated and the growth of embryos and placentas was examined. To elucidate global transcriptomic changes occurring in the placenta, total RNA-sequencing (RNA-seq) was performed in female and male placentas. RESULTS: LPS administration induced placental inflammation in both sexes at GD 17.5. Prenatal infection resulted in growth retardation in both sexes of embryos, and especially more prevalently in male. Impaired placental development was observed in a sex-specific manner. LPS 400 µg/kg reduced the percentage area of the labyrinth in females and junctional zone in males, respectively. RNA-sequencing revealed widespread sexually dimorphic transcriptional changes in placenta. In particular, representative changes were involved in biological processes such as trophoblast differentiation, nutrient/ion transporter, pregnancy, and immune system. CONCLUSIONS: Our results present the sexually dimorphic responses of placental physiology in intrauterine growth restriction model and provide tentative relationship further to be elucidated between sex-biased placental functional change and long-term effects on the offspring's later life.

Natural versus Laboratory World: Incorporating Wild-Derived Microbiota into Preclinical Rodent Models.

Advances in data collection (high-throughput shotgun metagenomics, transcriptomics, and metabolomics) and analysis (bioinformatics and multiomics) led to the realization that all mammals are metaorganisms, shaped not only by their own genome but also by the genomes of the microbes that colonize them. To date, most studies have focused on the bacterial microbiome, whereas curated databases for viruses, fungi, and protozoa are still evolving. Studies on the interdependency of microbial kingdoms and their combined effects on host physiology are just starting. Although it is clear that past and present exposure to commensals and pathogens profoundly affect human physiology, such exposure is lacking in standard preclinical models such as laboratory mice. Laboratory mouse colonies are repeatedly rederived in germ-free status and subjected to restrictive, pathogen-free housing conditions. This review summarizes efforts to bring the wild microbiome into the laboratory setting to improve preclinical models and their translational research value.

Identification of MYC as an antinecroptotic protein that stifles RIPK1-RIPK3 complex formation.

The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1-RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1-RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

Biosynthesis of Nonimmunosuppressive ProlylFK506 Analogues with Neurite Outgrowth and Synaptogenic Activity.

Separating the immunosuppressive activity of FK506 (1) from its neurotrophic activity is required to develop FK506 analogues as drugs for the treatment of neuronal diseases. Two new FK506 analogues, 9-deoxo-36,37-dihydro-prolylFK506 (2) and 9-deoxo-31-O-demethyl-36,37-dihydro-prolylFK506 (3) containing a proline moiety instead of the pipecolate ring at C-1 and modifications at the C-9/C-31 and C-36-C-37 positions, respectively, were biosynthesized, and their biological activities were evaluated. The proline substitution in 9-deoxo-36,37-dihydroFK506 and 9-deoxo-31-O-demethyl-36,37-dihydroFK506 reduced immunosuppressive activity by more than 120-fold, as previously observed. Compared with FK506 (1), 2 and 3 exhibited ∼1.2 × 105- and 2.2 × 105-fold reductions in immunosuppressive activity, respectively, whereas they retained almost identical neurite outgrowth activity. Furthermore, these compounds significantly increased the strength of synaptic transmission, confirming that replacement of the pipecolate ring with a proline is critical to reduce the strong immunosuppressive activity of FK506 (1) while enhancing its neurotrophic activity.

Biosynthesis of Nonimmunosuppressive FK506 Analogues with Antifungal Activity.

A reduction in the strong immunosuppressive activity of FK506 (1) is essential for developing this compound as an antifungal agent. Seven new FK506 analogues modified at both the FK506-binding protein 12- and the calcineurin-binding regions were biosynthesized. 9-DeoxoFK520 (7) exhibited a >900-fold reduction in the in vitro immunosuppressive activity but maintained significant antifungal activity, indicating that the C-9 and C-21 positions are critical for separation of immunosuppressive and antifungal activities. 7 exhibited robust synergistic antifungal activity with fluconazole. FK506 (1) is a 23-membered macrolide produced by several Streptomyces species and is used as an immunosuppressive drug to prevent the rejection of transplanted organs. FK506 has also exhibited antifungal, neuroprotective, and neuroregenerative activities. In humans, FK506 binds to FK506-binding protein (FKBP) 12, and the resulting FKBP12-FK506 complex interacts with a Ca2+-calmodulin-dependent phosphatase, calcineurin (CaN). Inactivation of CaN by forming the FKBP12-FK506-CaN ternary complex prevents the activation of nuclear factor of activated T cells (NF-AT), inhibiting the production of interleukin-2 and subsequent T-cell proliferation. This CaN signaling pathway also plays a critical role in the growth and pathogenesis of major fungal pathogens such as Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Therefore, the synthesis of FK506 analogues that can discriminate human FKBP12/CaN from its fungal counterparts may separate antifungal activity from the immunosuppressive activity, thereby allowing the development of a novel antifungal agent.

Retinoic acid and CTCF play key roles in inducing the collinear expression of the Hoxa cluster.

During the development of an embryo, the initiation of the collinear expression of Hox genes is essential for the proper formation of the anteroposterior body axis. Retinoic acid (RA), a natural derivative of vitamin A, plays a role in vertebrate development by regulating Hox gene expression. CCCTC-binding factor (CTCF), an insulator protein that controls gene transcription, also regulates the expression of Hox genes by binding to the CTCF-binding sites (CBSs). It has been reported that upon RA signaling, retinoic acid response elements (RAREs) located in the Hox clusters become occupied. Interestingly, RAREs exist in close proximity with CBSs, and therefore when RA is bound, CTCF cannot bind. Without CTCF and its insulator activities, the repressive domain in the chromatin becomes open for gene transcription. Here, we examine the relationship between RA and CTCF during the RA-induced expression of the Hoxa cluster genes, using F9 murine embryonic teratocarcinoma cells as a model system. We treated F9 cells with RA for different time, confirmed the collinear expression of Hoxa genes, and validated CTCF-binding in F9 cells as well as in CTCF-overexpressing F9 cells, in the presence of RA. The present study suggests that RA and CTCF pose antagonistic effects on each other during vertebrate development to attain Hox gene collinearity.

The histone acetylation mediated by Gcn5 regulates the Hoxc11 gene expression in MEFs.

Hox genes are responsible for encoding transcription factors that are essential for anterior-posterior body patterning at early stages of embryogenesis. However, detailed mechanisms of Hox genes are yet to be defined. Protein kinase B alpha (Akt1) was previously identified as a possible upstream regulator of Hox genes. Furthermore, the Hoxc11 gene has been upregulated in Akt1 null (Akt1-/-) mouse embryonic fibroblasts (MEFs), while repressed in wild-type MEFs. In this study, we propose to investigate the role of Gcn5, a histone acetyltransferase, in the regulation of Hoxc11 expression in MEFs. We showed that the H3 lysine 9 acetylation (H3K9ac) status has the same correlation with Hoxc11 expression and reported that Gcn5 is associated with the upregulation of Hoxc11 expression through H3K9ac in Akt1-/- MEFs. Since Hoxc11 was upregulated through histone acetylation in Akt1-/- MEFs, a functional role of Gcn5 on Hoxc11 expression was analyzed in Akt1-/- MEFs treated with Gcn5 specific inhibitor or transfected with Gcn5-small interfering RNA (Gcn5-siRNA). When the expression of Hoxc11 was analyzed using RT-PCR and real-time PCR, the Hoxc11 mRNA level was found to be similar in both Akt1-/- MEFs and control-siRNA transfected Akt1-/- MEFs. However, the Hoxc11 expression level was decreased in Gcn5-inhibited or Gcn5-knockdown Akt1-/- MEFs. Additionally, to analyze Gcn5-mediated histone acetylation status, chromatin immunoprecipitation assay was carried out in Gcn5-siRNA-transfected Akt1-/- MEFs. The H3K9ac at the Hoxc11 locus was decreased in Gcn5-knockdown Akt1-/- MEFs compared to controls. Based on these findings, we conclude that Gcn5 regulates Hoxc11 gene expression through mediating site-specific H3K9 acetylation in Akt1-/- MEFs.

Akt1 mediates the posterior Hoxc gene expression through epigenetic modifications in mouse embryonic fibroblasts.

The evolutionarily conserved Hox genes are organized in clusters and expressed colinearly to specify body patterning during embryonic development. Previously, Akt1 has been identified as a putative Hox gene regulator through in silico analysis. Substantial upregulation of consecutive 5' Hoxc genes has been observed when Akt1 is absent in mouse embryonic fibroblast (MEF) cells. In this study, we provide evidence that Akt1 regulates the 5' Hoxc gene expression by epigenetic modifications. Enrichment of histone H3K9 acetylation and a low level of the H3K27me3 mark were detected at the posterior 5' Hoxc loci when Akt1 is absent. A histone deacetylase (HDAC) inhibitor de-repressed 5' Hoxc gene expression when Akt1 is present, and a DNA demethylating reagent synergistically upregulated HDAC-induced 5' Hoxc gene expression. A knockdown study revealed that Hdac6 is mediated in the Hoxc12 repression through direct binding to the transcription start site (TSS) in the presence of Akt1. Co-immunoprecipitation analysis revealed that endogenous Akt1 directly interacted with Hdac6. Furthermore, exogenous Akt1 was enriched at the promoter region of the posterior Hoxc genes such as Hoxc11 and Hoxc12, not the Akt1-independent Hoxc5 and Hoxd10 loci. The regulation of the H3K27me3 mark by Ezh2 and Kdm6b at the 5' Hoxc gene promoter turned out to be Akt1 dependent. Taken together, these results suggest that Akt1 mediates the posterior 5' Hoxc gene expression through epigenetic modification such as histone methylation and acetylation, and partly through a direct binding to the promoter region of the 5' Hoxc genes and/or Hdac6 in mouse embryonic fibroblast cells.

QSOX2 Upregulated in triple-negative breast cancer exacerbates patient prognosis by stabilizing integrin ß1.

Breast cancer (BC) remains a significant global health threat, with triple-negative breast cancer (TNBC) standing out as a particularly aggressive subtype lacking targeted therapies. Addressing this gap, we propose Quiescin Q6 sulfhydryl oxidase 2 (QSOX2) as a potential therapeutic target, a disulfide bond-forming enzyme implicated in cancer progression. Using publicly available datasets, we conducted a comprehensive analysis of QSOX2 expression in BC tumor and non-tumor tissues, assessing its specificity across different molecular subtypes. We further explored correlations between QSOX2 expression and patient outcomes, utilizing datasets like TCGA and METABRIC. In addition, we performed in vitro experiments to evaluate QSOX2 expression in BC cell lines and investigate the effects of QSOX2 knockdown on various TNBC cellular processes, including cell proliferation, apoptosis resistance, migration, and the epithelial-to-mesenchymal transition (EMT). Our results reveal significantly elevated QSOX2 expression in BC tumor tissues, particularly in TNBC, and establish an association between high QSOX2 expression and increased patient mortality, cancer progression, and recurrence across various BC subtypes. Notably, QSOX2 knockdown in TNBC cell lines reduces cell proliferation, enhances apoptosis, and suppresses migration, potentially mediated through its influence on the EMT process. Furthermore, we identify a significant link between QSOX2 and integrin ß1 (ITGB1), suggesting that QSOX2 enhances ITGB1 stability, subsequently exacerbating the malignancy of TNBC. In conclusion, elevated QSOX2 expression emerges as a key factor associated with adverse patient outcomes in BC, particularly in TNBC, contributing to disease progression through various mechanisms, including the modulation of ITGB1 stability. Our findings underscore the potential of targeting QSOX2 as a therapeutic strategy for improving patient prognoses not only in TNBC but also in other BC subtypes.

The homeoprotein HOXB2 limits triple-negative breast carcinogenesis via extracellular matrix remodeling.

Homeobox genes and their encoded DNA-binding homeoproteins are master regulators of development. Consequently, these homeotic elements may regulate key steps in cancer pathogenesis. Here, using a combination of in silico analyses of large-scale patient datasets, in vitro RNAi phenotyping, and in vivo validation studies, we investigated the role of HOXB2 in different molecular subtypes of human breast cancer (BC). The gene expression signatures of HOXB2 are different across distinct BC subtypes due to various genetic alterations, but HOXB2 was specifically downregulated in the aggressive triple-negative subtype (TNBC). We found that the reduced expression of HOXB2 was correlated with the metastatic abilities (epithelial-to-mesenchymal transition) of TNBC cells. Further, we revealed that HOXB2 restrained TNBC aggressiveness by ECM organization. HOXB2 bound to the promoter regions of MATN3 and ECM2 and regulated their transcription levels. Forced expression of HOXB2 effectively prevented TNBC progression and metastasis in a mouse xenograft model. Reduction of HOXB2 and the HOXB2/MATN3/ECM2 transcriptional axis correlated with poor survival in patients with various cancers. Further, we found the long non-coding RNA HOXB-AS1 in complex with SMYD3, a lysine methyltransferase, as an epigenetic switch controlling HOXB2 expression. Overall, our results indicate a tumor-suppressive role of HOXB2 by maintaining ECM organization and delineate potential clinical utility of HOXB2 as a marker for TNBC patients.

Comparative oncology: overcoming human cancer through companion animal studies.

Comparative oncology is a field of study that has been recently adopted for studying cancer and developing cancer therapies. Companion animals such as dogs can be used to evaluate novel biomarkers or anticancer targets before clinical translation. Thus, the value of canine models is increasing, and numerous studies have been conducted to analyze similarities and differences between many types of spontaneously occurring cancers in canines and humans. A growing number of canine cancer models as well as research-grade reagents for these models are becoming available, leading to substantial growth in comparative oncology research spanning from basic science to clinical trials. In this review, we summarize comparative oncology studies that have been conducted on the molecular landscape of various canine cancers and highlight the importance of the integration of comparative biology into cancer research.

Increase in convective extreme El Niño events in a CO2 removal scenario.

Convective extreme El Niño (CEE) events, characterized by strong convective events in the eastern Pacific, are known to have a direct link to anomalous climate conditions worldwide, and it has been reported that CEE will occur more frequently under greenhouse warming. Here, using a set of CO2 ramp-up and ramp-down ensemble experiments, we show that frequency and maximum intensity of CEE events increase further in the ramp-down period from the ramp-up period. These changes in CEE are associated with the southward shift of the intertropical convergence zone and intensified nonlinear rainfall response to sea surface temperature change in the ramp-down period. The increasing frequency of CEE has substantial impacts on regional abnormal events and contributed considerably to regional mean climate changes to the CO2 forcings.

Akt1 Decreases Gcn5 Protein Stability through Regulating The Ubiquitin-Proteasome Pathway in Mouse Embryonic Fibroblasts.

General control non-derepressible 5 (Gcn5) is a member of histone acetyltransferase (HAT) that plays key roles during embryogenesis as well as in the development of various human cancers. Gcn5, an epigenetic regulator of Hoxc11, has been reported to be negatively regulated by Akt1 in the mouse embryonic fibroblasts (MEFs). However, the exact mechanism by which Akt1 regulates Gcn5 is not well understood. Using protein stability chase assay, we observed that Gcn5 is negatively regulated by Akt1 at the post-translational level in MEFs. The stability of Gcn5 protein is determined by the competitive binding with the protein partner that interacts with Gcn5. The interaction of Gcn5 and Cul4a-Ddb1 complex predominates and promotes ubiquitination of Gcn5 in the wild-type MEFs. On the other hand, in the Akt1-null MEFs, the interaction of Gcn5 and And-1 inhibits binding of Gcn5 and Cul4a-Dbd1 E3 ubiquitin ligase complex, thereby increasing the stability of the Gcn5 protein. Taken together, our study indicates that Akt1 negatively controls Gcn5 via the proteasomal degradation pathway, suggesting a potential mechanism that regulates the expression of Hox genes.

HOXA5 confers tamoxifen resistance via the PI3K/AKT signaling pathway in ER-positive breast cancer.

Tamoxifen is a commonly used drug to treat estrogen receptor-positive patients with breast cancer. Despite the outstanding efficacy of tamoxifen, approximately one-third of patients develop resistance toward it, thereby presenting a therapeutic challenge. HOX genes may be involved in the acquisition of tamoxifen resistance. In this study, we identified HOXA5, a member of the HOX gene family, as a marker of tamoxifen resistance. Using ChIP assay, we found that HOXA5 expression was significantly overexpressed in tamoxifen-resistant MCF7 (TAMR) breast cancer cells because of reduced H3K27me3 binding. HOXA5 upregulation resulted in activation of the PI3K/AKT signaling cascade, which in turn, led to p53 and p21 reduction, ultimately making the TAMR cells less apoptotic. Furthermore, elevated HOXA5 expression resulted in breast cancer cells acquiring more mesenchymal-like and stem cell traits associated with aggressive breast cancer phenotypes. In conclusion, our results delineate a mechanism by which HOXA5 promotes tumorigenesis, cancer progression, and tamoxifen resistance in breast cancer cells.

NR4A1 Regulates Tamoxifen Resistance by Suppressing ERK Signaling in ER-Positive Breast Cancer.

Endocrine therapy is used to treat estrogen receptor (ER)-positive breast cancer. Tamoxifen is effective against this cancer subtype. Nonetheless, approximately 30% of patients treated with tamoxifen acquire resistance, resulting in therapeutic challenges. NR4A1 plays key roles in processes associated with carcinogenesis, apoptosis, DNA repair, proliferation, and inflammation. However, the role of NR4A1 in tamoxifen-resistant ER-positive breast cancer has not yet been elucidated. Here, we propose that NR4A1 is a promising target to overcome tamoxifen resistance. NR4A1 gene expression was downregulated in tamoxifen-resistant MCF7 (TamR) cells compared to that in MCF7 cells. Kaplan-Meier plots were used to identify high NR4A1 expression correlated with increased survival rates in patients with ER-positive breast cancer following tamoxifen treatment. Gain and loss of function experiments showed that NR4A1 restores sensitivity to tamoxifen by regulating cell proliferation, migration, invasion, and apoptosis. NR4A1 localized to the cytoplasm enhanced the expression of apoptotic factors. In silico and in vitro analyses revealed that NR4A1 enhanced responsiveness to tamoxifen by suppressing ERK signaling in ER-positive breast cancer, suggesting that the NR4A1/ERK signaling axis modulates tamoxifen resistance. These results indicate that NR4A1 could be a potential therapeutic target to overcome tamoxifen resistance in ER-positive breast cancer.

HOXB5 Confers Tamoxifen Resistance in Breast Cancer Cells and Promotes Tumor Aggression and Progression.

BACKGROUND/AIM: ER-positive breast cancer patients commonly undergo endocrine therapy with drugs such as tamoxifen. Despite tamoxifen being a highly effective drug, long-term treatment results in resistance in one-third of the patients. Although many explanations for the development of tamoxifen resistance have been put forward, a clearly defined underlying mechanism is still lacking. MATERIALS AND METHODS: The expression level of HOXB5 was evaluated between MCF7 breast cancer cells and tamoxifen-resistant MCF7 (TAMR) cells by RT-PCR. Then, the effect of HOXB5 on invasion and migration abilities as well as on cancer stemness were investigated through 3D culture and spheroid formation assay. RESULTS: In this study, we provide evidence that HOXB5 is up-regulated in TAMR cells. EGFR is concurrently overexpressed, and the EGFR signaling cascade is activated, resulting in migratory and invasive phenotypes in TAMR cells compared to MCF7 cells. However, HOXB5 knockdown in TAMR cells resulted in the de-activation of the EGFR signaling pathway, less aggressive phenotypes and restoration of sensitivity to tamoxifen treatment. More interestingly, TAMR cells expressed higher levels of stem cell markers, and as a result, their enhanced stemness allowed for a better formation of spheroids than MCF7 cells. When HOXB5 was overexpressed in MCF7 cells, they were able to form a larger number of spheroids as in TAMR cells. CONCLUSION: HOXB5 is one of the key factors involved in tumor aggression and progression in tamoxifen-resistant breast cancer cells.