Regulation of IDO2 by the Aryl Hydrocarbon Receptor (AhR) in Breast Cancer.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a tryptophan-catabolizing enzyme and a homolog of IDO1 with a distinct expression pattern compared with IDO1. In dendritic cells (DCs), IDO activity and the resulting changes in tryptophan level regulate T-cell differentiation and promote immune tolerance. Recent studies indicate that IDO2 exerts an additional, non-enzymatic function and pro-inflammatory activity, which may play an important role in diseases such as autoimmunity and cancer. Here, we investigated the impact of aryl hydrocarbon receptor (AhR) activation by endogenous compounds and environmental pollutants on the expression of IDO2. Treatment with AhR ligands induced IDO2 in MCF-7 wildtype cells but not in CRISPR-cas9 AhR-knockout MCF-7 cells. Promoter analysis with IDO2 reporter constructs revealed that the AhR-dependent induction of IDO2 involves a short-tandem repeat containing four core sequences of a xenobiotic response element (XRE) upstream of the start site of the human ido2 gene. The analysis of breast cancer datasets revealed that IDO2 expression increased in breast cancer compared with normal samples. Our findings suggest that the AhR-mediated expression of IDO2 in breast cancer could contribute to a pro-tumorigenic microenvironment in breast cancer.

Reduction of urea test ordering in the emergency department: multicomponent intervention including education, electronic ordering, and data feedback.

INTRODUCTION: In the emergency department (ED), laboratory testing accounts for a significant portion of the medical assessment. Although excess laboratory test ordering has been proven to be prevalent, different types of interventions have been used to encourage a behavioural change in how physicians order tests. In one western Canadian hospital medicine program, a quality improvement project aimed to reduce the total monthly blood urea nitrogen (BUN) test ordered by physicians was found to be successful. The objective of this project was to evaluate a similar multicomponent intervention aimed at ED physician ordering, with the primary goal of reducing the number of monthly BUN tests ordered per ED visit. METHODS: A pre post intervention design was conducted over 12-months. The first intervention component was an educational presentation conducted by physician leaders. Second, a regularly used order panel within the ED electronic order system was modified, removing the BUN test. The third component involved audit and feedback; the total monthly BUN test ordered for the ED department post intervention start was shared with all ED physicians twice (at 5 and 12 months).An interrupted time series analysis was completed to evaluate the multicomponent intervention effect. RESULTS: The total monthly ordered BUN test declined from an average of 1905 pre-intervention to 448 post-intervention, and the total monthly BUN test to total ED visit ratio declined from 0.46 to 0.1. These results were a statistically significant reduction in physician BUN test ordering. CONCLUSIONS: Targeted education, order panel design and data feedback interventions can impact physician ordering behaviour in the emergent healthcare context, where diagnostic tests are often over used.

RéSUMé: INTRODUCTION: Dans les services d'urgence (SU), les analyses de laboratoire représentent une part importante de l'évaluation médicale. Bien qu'il ait été prouvé que la prescription excessive d'examens de laboratoire est répandue, différents types d'interventions ont été utilisés pour encourager un changement de comportement dans la façon dont les médecins commandent des examens. Dans un programme de médecine hospitalière de l'Ouest canadien, un projet d'amélioration de la qualité visant à réduire le nombre total de tests mensuels d'azote uréique du sang (BUN) demandés par les médecins s'est avéré fructueux. L'objectif de ce projet était d'évaluer une intervention similaire à plusieurs composantes visant les ordonnances des médecins des urgences, avec pour objectif principal de réduire le nombre d'analyses mensuelles de BUN commandés par visite aux urgences. MéTHODE: Une conception pré-post-intervention a été menée sur 12 mois. Le premier volet de l'intervention consistait en une présentation éducative menée par des médecins chefs de file. Deuxièmement, un panneau de commande régulièrement utilisé dans le système de commande électronique du SU a été modifié, supprimant le test BUN. Le troisième volet concernait l'audit et le retour d'information : le nombre total de tests mensuels d'azote uréique sanguin commandés pour le service des urgences après le début de l'intervention a été communiqué à tous les médecins des urgences à deux reprises (à 5 et 12 mois). Une analyse de séries chronologiques interrompues a été réalisée pour évaluer l'effet de l'intervention multicomposante. RéSULTATS: Le nombre total mensuel d'analyses BUN commandés a baissé d'une moyenne de 1905 avant l'intervention à 448 après l'intervention, et le rapport entre le nombre total mensuel de test BUN et le nombre total de visites aux urgences a baissé de 0,46 à 0,1. Ces résultats représentaient une réduction statistiquement significative des ordonnances de test BUN par les médecins. CONCLUSIONS: Des interventions ciblées en matière d'éducation, de conception de panels de commandes et de retour d'informations peuvent avoir un impact sur le comportement des médecins en matière de commandes dans le contexte des soins de santé émergents, où les tests de diagnostic sont souvent surutilisés.

Aberrant promoter methylation contributes to LRIG1 silencing in basal/triple-negative breast cancer.

BACKGROUND: LRIG1, the founding member of the LRIG (leucine-rich repeat and immunoglobulin-like domain) family of transmembrane proteins, is a negative regulator of receptor tyrosine kinases and a tumour suppressor. Decreased LRIG1 expression is consistently observed in cancer, across diverse tumour types, and is linked to poor patient prognosis. However, mechanisms by which LRIG1 is repressed are not fully understood. Silencing of LRIG1 through promoter CpG island methylation has been reported in colorectal and cervical cancer but studies in breast cancer remain limited. METHODS: In silico analysis of human breast cancer patient data were used to demonstrate a correlation between DNA methylation and LRIG1 silencing in basal/triple-negative breast cancer, and its impact on patient survival. LRIG1 gene expression, protein abundance, and methylation enrichment were examined by quantitative reverse-transcription PCR, immunoblotting, and methylation immunoprecipitation, respectively, in breast cancer cell lines in vitro. We examined the impact of global demethylation on LRIG1 expression and methylation enrichment using 5-aza-2'-deoxycytidine. We also examined the effects of targeted demethylation of the LRIG1 CpG island, and transcriptional activation of LRIG1 expression, using the RNA guided deadCas9 transactivation system. RESULTS: Across breast cancer subtypes, LRIG1 expression is lowest in the basal/triple-negative subtype so we investigated whether differential methylation may contribute to this. Indeed, we find that LRIG1 CpG island methylation is most prominent in basal/triple-negative cell lines and patient samples. Use of the global demethylating agent 5-aza-2'-deoxycytidine decreases methylation leading to increased LRIG1 transcript expression in basal/triple-negative cell lines, while having no effect on LRIG1 expression in luminal/ER-positive cell lines. Using a CRISPR/deadCas9 (dCas9)-based targeting approach, we demonstrate that TET1-mediated demethylation (Tet1-dCas9) along with VP64-mediated transcriptional activation (VP64-dCas9) at the CpG island, increased endogenous LRIG1 expression in basal/triple-negative breast cancer cells, without transcriptional upregulation at predicted off-target sites. Activation of LRIG1 by the dCas9 transactivation system significantly increased LRIG1 protein abundance, reduced site-specific methylation, and reduced cancer cell viability. Our findings suggest that CRISPR-mediated targeted activation may be a feasible way to restore LRIG1 expression in cancer. CONCLUSIONS: Our study contributes novel insight into mechanisms which repress LRIG1 in triple-negative breast cancer and demonstrates for the first time that targeted de-repression of LRIG1 in cancer cells is possible. Understanding the epigenetic mechanisms associated with repression of tumour suppressor genes holds potential for the advancement of therapeutic approaches.

Environmental exposure and the role of AhR in the tumor microenvironment of breast cancer.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to chemicals including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) can lead to severe adverse health effects and increase the risk of breast cancer. This review considers several mechanisms which link the tumor promoting effects of environmental pollutants with the AhR signaling pathway, contributing to the development and progression of breast cancer. We explore AhR's function in shaping the tumor microenvironment, modifying immune tolerance, and regulating cancer stemness, driving breast cancer chemoresistance and metastasis. The complexity of AhR, with evidence for both oncogenic and tumor suppressor roles is discussed. We propose that AhR functions as a "molecular bridge", linking disproportionate toxin exposure and policies which underlie environmental injustice with tumor cell behaviors which drive poor patient outcomes.

Targeting the Aryl Hydrocarbon Receptor Signaling Pathway in Breast Cancer Development.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.

Aryl Hydrocarbon Receptor Signaling Synergizes with TLR/NF-κB-Signaling for Induction of IL-22 Through Canonical and Non-Canonical AhR Pathways.

Interleukin 22 (IL-22) is critically involved in gut immunity and host defense and primarily produced by activated T cells. In different circumstances IL-22 may contribute to pathological conditions or act as a cancer promoting cytokine secreted by infiltrating immune cells. Here we show that bone marrow-derived macrophages (BMM) express and produce IL-22 after activation of the aryl hydrocarbon receptor (AhR) when cells are activated through the Toll-like receptor (TLR) family. The additional activation of AhR triggered a significant induction of IL-22 in TLR-activated BMM. Deletion and mutation constructs of the IL-22 promoter revealed that a consensus DRE and RelBAhRE binding element are necessary to mediate the synergistic effects of AhR and TLR ligands. Inhibitor studies and analysis of BMM derived from knockout mice confirmed that the synergistic induction of IL-22 by AhR and TLR ligands depend on the expression of AhR and Nuclear Factor-kappa B (NF-κB) member RelB. The exposure to particulate matter (PM) collected from traffic related air pollution (TRAP) and wildfires activated AhR as well as NF-κB signaling and significantly induced the expression of IL-22. In summary this study shows that simultaneous activation of the AhR and NF-κB signaling pathways leads to synergistic and prolonged induction of IL-22 by integrating signals of the canonical and non-canonical AhR pathway.

Male-derived copulatory plugs enhance implantation success in female Mus musculus.

Among a wide diversity of sexually reproducing species, male ejaculates coagulate to form what has been termed a copulatory plug. A number of functions have been attributed to copulatory plugs, including the inhibition of female remating and the promotion of ejaculate movement. Here we demonstrate that copulatory plugs also influence the likelihood of implantation, which occurs roughly 4 days after copulation in mice. Using a bead transfer method to control for differences in ejaculate retention and fertilization rates, we show that implantation rates significantly drop among females mated to genetically engineered males incapable of forming plugs (because they lack functional transglutaminase 4, the main enzyme responsible for its formation). Surprisingly, this result does not correlate with differences in circulating progesterone levels among females, an important hormone involved in implantation. We discuss three models that connect male-derived copulatory plugs to implantation success, including the hypothesis that plugs contribute to a threshold amount of stimulation required for females to become receptive to implantation.

Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells.

Although the efficacy of cancer radiotherapy (RT) can be enhanced by targeted immunotherapy, the immunosuppressive factors induced by radiation on tumor cells remain to be identified. Here, we report that CD47-mediated anti-phagocytosis is concurrently upregulated with HER2 in radioresistant breast cancer (BC) cells and RT-treated mouse syngeneic BC. Co-expression of both receptors is more frequently detected in recurrent BC patients with poor prognosis. CD47 is upregulated preferentially in HER2-expressing cells, and blocking CD47 or HER2 reduces both receptors with diminished clonogenicity and augmented phagocytosis. CRISPR-mediated CD47 and HER2 dual knockouts not only inhibit clonogenicity but also enhance macrophage-mediated attack. Dual antibody of both receptors synergizes with RT in control of syngeneic mouse breast tumor. These results provide the evidence that aggressive behavior of radioresistant BC is caused by CD47-mediated anti-phagocytosis conjugated with HER2-prompted proliferation. Dual blockade of CD47 and HER2 is suggested to eliminate resistant cancer cells in BC radiotherapy.

Effect of a patient-led educational session on pre-clerkship students' learning of professional values and on their professional development.

BACKGROUND: While there are several curricula using patients as educators, little has been published on how they affect student learning and professional development. OBJECTIVE: To explore what 1st year medical students learn about professional values from a patient-led educational experience and how it affects their professional development. DESIGN: We piloted a pediatric patient and family-led educational session during the molecular medicine course, with the goal of sharing the experience of caring for a child with a chronic illness. Following the session, students were required to submit a written reflection on what they learned and the impact the session had on them. All reflections from one academic year were qualitatively analyzed by two investigators and organized using HyperRESEARCH software. A content analysis approach was used to generate codes and emergent themes. Two theoretical lenses guided the analyses: Arnold's framework on professional values and the lens of professional identity formation, described as a process by which health care professionals "think, act and feel like a physician. RESULTS: Students gained an appreciation of professional values, especially humanism and excellence, and how clinician role models reinforce these values. Reflective writings demonstrated recognition among learners that their identity involved being active participants in health care delivery and not just as passive classroom learners. Students were motivated to study diligently and be patient advocates; some questioned their skills in dealing with ambiguity and with the health-care system, resulting in a sense of helplessness. CONCLUSION: Students learn the importance of professional attributes and of clinician role models through a pediatric family teaching experience. They are motivated, displaying glimpses of their future role as caregivers and patient advocates; however, some also express fear and doubt their own abilities. Based on this, a debriefing session has been introduced to prevent a negative effect on learner self-efficacy.

Targeting the polyamine pathway-"a means" to overcome chemoresistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by its aggressive biology, early metastatic spread, and poor survival outcomes. TNBC lacks expression of the targetable receptors found in other breast cancer subtypes, mandating use of cytotoxic chemotherapy. However, resistance to chemotherapy is a significant problem, encountered in about two-thirds of TNBC patients, and new strategies are needed to mitigate resistance. In this issue of the Journal of Biological Chemistry, Geck et al. report that TNBC cells are highly sensitive to inhibition of the de novo polyamine synthesis pathway and that inhibition of this pathway sensitizes cells to TNBC-relevant chemotherapy, uncovering new opportunities for addressing chemoresistance.

Novel approaches for efficient in vivo fermentation production of noncoding RNAs.

Genome-derived noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), play an essential role in the control of target gene expression underlying various cellular processes, and dysregulation of ncRNAs is involved in the pathogenesis and progression of various diseases in virtually all species including humans. Understanding ncRNA biology has opened new avenues to develop novel RNA-based therapeutics. Presently, ncRNA research and drug development is dominated by the use of ncRNA mimics that are synthesized chemically in vitro and supplemented with extensive and various types of artificial modifications and thus may not necessarily recapitulate the properties of natural RNAs generated and folded in living cells in vivo. Therefore, there are growing interests in developing novel technologies for in vivo production of RNA molecules. The two most recent major breakthroughs in achieving an efficient, large-scale, and cost-effective fermentation production of recombinant or bioengineered RNAs (e.g., tens of milligrams from 1 L of bacterial culture) are (1) using stable RNA carriers and (2) direct overexpression in RNase III-deficient bacteria, while other approaches offer a low yield (e.g., nano- to microgram scales per liter). In this article, we highlight these novel microbial fermentation-based technologies that have shifted the paradigm to the production of true biological ncRNA molecules for research and development.

Clostridium difficile infection in a children's hospital with specific patterns among pediatric oncology and hematopoietic stem cell transplantation populations.

Background: Clostridium difficile (CD) is often classified as a healthcare-associated infection (HAI) and a hospital-acquired condition (HAC) in the hospital setting. However, pediatric oncology patients comprise a significant portion of Clostridium difficile infections (CDI), with hematopoietic stem cell transplant (HSCT) recipients constituting a major subset of this group due to unique, non-modifiable risk factors. We evaluated patterns of clostridium difficile infections at our institution to provide an accurate evaluation of the vulnerability of pediatric oncology and HSCT patients to clostridium difficile infections in comparison to the general pediatric population and underscore the non-tenability of classifying clostridium difficile infections as a hospital-acquired condition in HSCT patients. Methods: Single-center retrospective review of all clostridium difficile stool tests performed over an 11-year period; data analyzed and statistical comparisons performed between patient groups. Results: 5271 total samples were obtained during the study time period from 3127 patients. At least one positive test result was found in 18.6% of patients. Oncology and HSCT patients (38.2%) were more likely to have a positive test result than hematology (17.5%) and other patients (16.8%) (p < 0.001). Sixty-percent of patients who underwent HSCT were tested during this time frame. Of those, 39.3% had a positive test result and 48.5% of those patients went on to have a subsequent infection that met the criteria to be defined as recurrent. Conclusions: The high incidence rate and frequency of recurrence underscores the current near-inevitable nature of clostridium difficile infections in oncology and HSCT patients. We conclude that a blanket designation of clostridium difficile infections as an hospital-acquired condition is therefore questionable in this population.

Patient-Centered Team-Based Learning in Pre-Clinical Curriculum Supporting the Application of Knowledge to Real-World Clinical Experience.

We report an active learning session which effectively supported 1st year medical students applying their learning experience in a clinical setting. A team-based learning (TBL) on familial hypercholesterolemia (FH) with a live patient was given to deliver basic genetics knowledge in a clinically relevant context. Subsequently, two participating students applied their learning experience by presenting a differential diagnosis of homozygous FH in a patient at a medical mission in Central America. We propose that combining active learning with clinically relevant scenarios effectively fosters student's clinical reasoning skills and can bridge the perceived gap between basic science and clinical education.

A Novel Bioengineered miR-127 Prodrug Suppresses the Growth and Metastatic Potential of Triple-Negative Breast Cancer Cells.

miR-127 is downregulated in breast cancer, where it has been shown to suppress the proliferation, migration, and invasion of breast cancer cells. In triple-negative breast cancer (TNBC), miR-127 downregulation correlates with decreased disease-free and overall patient survival. Tumor suppressor miRNAs may hold therapeutic promise but progress has been limited by several factors, including the lability and high cost of miRNA mimics. Here, we take a novel approach to produce a miR-127 prodrug (miR-127PD), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127PD decreased the viability and motility of TNBC cells, sensitized TNBC cells to chemotherapy, and restricted the TNBC stem cell population. Furthermore, systemic delivery of miR-127PD suppressed tumor growth of MDA-MB-231 and MDA-MB-468 TNBC cells and spontaneous metastasis of MDA-MB-231 cells. In addition, CERK, NANOS1, FOXO6, SOX11, SOX12, FASN, and SUSD2 were identified as novel, functionally important targets of miR-127. In conclusion, our study demonstrates that miR-127 functions as a tumor and metastasis suppressor in TNBC and that delivery of miR-127 may hold promise as a novel therapy. SIGNIFICANCE: Exogenous administration of miR-127, which is functionally activated in target cells, inhibits growth and spontaneous metastasis of triple-negative breast cancer.

A Protective Role of Aryl Hydrocarbon Receptor Repressor in Inflammation and Tumor Growth.

The aryl hydrocarbon receptor (AhR) is known for mediating the toxicity of environmental pollutants such as dioxins and numerous dioxin-like compounds, and is associated with the promotion of various malignancies, including lymphoma. The aryl hydrocarbon receptor repressor (AhRR), a ligand-independent, transcriptionally inactive AhR-like protein is known to repress AhR signaling through its ability to compete with the AhR for dimerization with the AhR nuclear translocator (ARNT). While AhRR effectively blocks AhR signaling, several aspects of the mechanism of AhRR's functions are poorly understood, including suppression of inflammatory responses and its putative role as a tumor suppressor. In a transgenic mouse that overexpresses AhRR (AhRR Tg) we discovered that these mice suppress 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)- and inflammation-induced tumor growth after subcutaneous challenge of EL4 lymphoma cells. Using mouse embryonic fibroblasts (MEF) we found that AhRR overexpression suppresses the AhR-mediated anti-apoptotic response. The AhRR-mediated inhibition of apoptotic resistance was associated with a suppressed expression of interleukin (IL)-1ß and cyclooxygenase (COX)-2, which was dependent on activation of protein kinase A (PKA) and the CAAT-enhancer-binding protein beta (C/EBPß). These results provide mechanistic insights into the role of the AhRR to suppress inflammation and highlight the AhRR as a potential therapeutic target to suppress tumor growth.

A Syngeneic ErbB2 Mammary Cancer Model for Preclinical Immunotherapy Trials.

In order to develop a practical model of breast cancer, with in vitro and syngeneic, immune-intact, in vivo growth capacity, we established a primary cell line derived from a mammary carcinoma in the transgenic FVB/N-Tg(MMTV-ErbB2*)NDL2-5Mul mouse, referred to as "NDLUCD". The cell line is adapted to standard cell culture and can be transplanted into syngeneic FVB/N mice. The line maintains a stable phenotype over multiple in vitro passages and rounds of in vivo transplantation. NDLUCD tumors in FVB/N mice exhibit high expression of ErbB2 and ErbB3 and signaling molecules downstream of ErbB2. The syngeneic transplant tumors elicit an immune reaction in the adjacent stroma, detected and characterized using histology, immunophenotyping, and gene expression. NDLUCD cells also express PD-L1 in vivo and in vitro, and in vivo transplants are reactive to anti-immune checkpoint therapy with responses conducive to immunotherapy studies. This new NDLUCD cell line model is a practical alternative to the more commonly used 4T1 cells, and our previously described FVB/N-Tg(MMTV-PyVT)634Mul derived Met-1fvb2 and FVB/NTg(MMTV-PyVTY315F/Y322F) derived DB-7fvb2 cell lines. The NDLUCD cells have, so far, remained genetically and phenotypically stable over many generations, with consistent and reproducible results in immune intact preclinical cohorts.

The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3.

ErbB3 and ErbB4 are receptor tyrosine kinases that are activated by the neuregulin (NRG) family of growth factors. These receptors govern various developmental processes, and their dysregulation contributes to several human disease states. The abundance of ErbB3 and ErbB4, and thus signaling through these receptors, is limited by the E3 ubiquitin ligase Nrdp1, which targets ErbB3 and ErbB4 for degradation. Reticulons are proteins that influence the morphology of the endoplasmic reticulum (ER) by promoting the formation of tubules, a response of cells to some stressors. We found that the ER structural protein reticulon 4A (Rtn4A, also known as Nogo-A) increased ErbB3 abundance and proliferative signaling by suppressing Nrdp1 function. Rtn4A interacted with Nrdp1 and stabilized ErbB3 in an Nrdp1-dependent manner. Rtn4A overexpression induced the redistribution of Nrdp1 from a cytosolic or perinuclear localization to ER tubules. Rtn4A knockdown in human breast tumor cells decreased ErbB3 abundance, NRG-stimulated signaling, and cellular proliferation and migration. Because proteins destined for the plasma membrane are primarily synthesized in the sheet portions of the ER, our observations suggest that Rtn4A counteracts the Nrdp1-mediated degradation of ErbB3 by sequestering the ubiquitin ligase into ER tubules. The involvement of a reticulon suggests a molecular link between ER structure and the sensitivity of cells to receptor tyrosine kinase-mediated survival signals at the cell surface.

Down-regulatory mechanism of mammea E/BB from Mammea siamensis seed extract on Wilms' Tumor 1 expression in K562 cells.

BACKGROUND: Wilms' tumor 1 (WT1) is a biological marker for predicting leukemia progression. In this study, mammea E/BB, an active compound from Saraphi (Mammea siamensis) seed extract was examined for its effect on down-regulatory mechanism of WT1 gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell line. METHODS: M. siamensis seeds were obtained from the region of Chiang Mai (North of Thailand). Mammea E/BB was extracted from seeds of M. siamensis. WT1 protein expression and stability were evaluated by Western blot analysis. WT1 mRNA stability was assessed by qRT-PCR. WT1-DNA binding and WT1 promoter activity were assayed by ChIP assay and luciferase-reporter assay, respectively. Cell cycle arrest was studied by flow cytometry. RESULTS: Treatment with mammea E/BB led to down-regulation of WT1 expression. The suppression of WT1 expression did not involve protein and mRNA degradation. Rather, WT1 protein was down-regulated through disruption of transcriptional auto-regulation of the WT1 gene. Mammea E/BB inhibited WT1-DNA binding at the WT1 promoter and decreased luciferase activity. It also disrupted c-Fos/AP-1 binding to the WT1 promoter via ERK1/2 signaling pathway and induced S phase cell cycle arrest in K562 cells. CONCLUSION: Mammea E/BB had pleotropic effects on kinase signaling pathways, resulting in inhibition of leukemia cell proliferation.