Osteomyelitis Possibly Caused by Exfoliation of Primary Teeth in a Patient with Osteopetrosis.

Osteopetrosis is a rare inherited skeletal disease characterized by increased density in the bones and can be detected by radiographs. Sclerosis makes the jaw bones extremely susceptible to infections, osteomyelitis, and fractures. Here, we report a case of osteomyelitis possibly caused by spontaneous exfoliation of primary teeth in a patient with osteopetrosis. A 2 years and 9 months (2Y9M)-old boy with osteopetrosis was referred to our clinic for oral management. Only four primary central incisors had erupted, and they all exhibited hypoplasia. The mandibular right central primary incisor suddenly became exfoliated at 4 years and 1 month. The mandibular right lateral primary incisor also became exfoliated at 4 years and 3 months, soon after eruption, and the mandibular left central primary incisor became exfoliated at 4 years and 5 months. Subsequently, we confirmed the eruption of calcified tissue at 4 years and 9 months in the location where the mandibular right lateral primary incisor had become exfoliated. The patient was admitted to the pediatrics clinic for mandibular cellulitis at 5 years and 2 months, then referred to our clinic for the management of osteomyelitis. The patient's acute inflammation was reduced by repeated irrigation and the administration of antibiotics; the inflammation gradually became chronic. When treating patients with osteopetrosis, dentists and oral surgeons should prioritize infection control in the jaw, periodic assessment of dental eruption, and the maintenance of oral hygiene.

GATA6 regulates expression of annexin A10 (ANXA10) associated with epithelial-mesenchymal transition of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) can disturb oral function and quality of life and is associated with poor survival, likely due to the development of cervical lymph node metastases. Epithelial-mesenchymal transition (EMT) is a process in which cells acquire molecular alterations that facilitate cell motility and invasion, and has been associated with tumor metastasis. EMT changes also play important roles in the induction of lymph node metastasis in OSCC. GATA6 is known as the earliest marker of the primitive endoderm lineages. GATA6 inhibits de-differentiation and EMT in human pancreatic ductal adenocarcinoma cells and promotes EMT. However, in OSCC, the expression and function of GATA6 in EMT and lymph node metastasis remains unclear. Therefore, this study aimed to clarify the targets of GATA6 in OSCC cells and whether the change in GATA6 expression affects EMT in OSCC cells, as well as the association between GATA6 and lymph node metastasis. The results showed that GATA6 knockdown OSCC cells promoted EMT and increased lymph node metastasis compared with control cells, whereas the overexpression of GATA6 inhibited the induction of EMT and reduced lymph node metastasis. In addition, annexin A10 (ANXA10) which is the largest type of Ca2+-regulated phospholipid-binding protein in eukaryotic cells was detected as a target gene for GATA6 and ANXA10 suppressed Vimentin expression in EMT in OSCC. Therefore, the GATA6/ANXA10 cascade may be a potential therapeutic approach for the treatment of lymph node metastases in OSCC patients.

Pleomorphic Adenoma of the Salivary Glands and Epithelial-Mesenchymal Transition.

Pleomorphic adenoma (PA) is a localized tumor that presents pleomorphic or mixed characteristics of epithelial origin and is interwoven with mucoid tissue, myxoid tissue, and chondroid masses. The literature reported that PA most often occurs in adults aged 30-60 years and is a female predilection; the exact etiology remains unclear. Epithelial-mesenchymal transition (EMT) is the transdifferentiation of stationary epithelial cells primarily activated by a core set of transcription factors (EMT-TFs) involved in DNA repair and offers advantages under various stress conditions. Data have suggested that EMTs represent the basic principle of tissue heterogeneity in PAs, demonstrating the potential of adult epithelial cells to transdifferentiate into mesenchymal cells. It has also been reported that multiple TFs, such as TWIST and SLUG, are involved in EMT in PA and that SLUG could play an essential role in the transition from myoepithelial to mesenchymal cells. Given this background, this review aims to summarize and clarify the involvement of EMT in the development of PA, chondrocyte differentiation, and malignant transformation to contribute to the fundamental elucidation of the mechanisms underlying EMT.

Differential Diagnosis between Oral Metastasis of Renal Cell Carcinoma and Salivary Gland Cancer.

Renal cell carcinoma, which has clear cells in 70% of cases, has a high frequency of hematogenous distant metastases to lung, bone, liver, and other areas. Metastatic cancer accounts for 1 to 3% of malignant tumors in the stomatognathic region, and the metastasis of renal cell carcinoma to the oral mucosal tissue, though extremely rare, does occur. In addition, clear cells have been observed in some salivary gland cancers in the oral cavity. Therefore, the differential diagnosis of metastatic renal cell carcinoma and salivary gland cancer is important. This review discusses the differential diagnosis between metastatic renal cell carcinoma and malignant tumors of the salivary gland.

Erratum: Oral metastasis of renal cell carcinoma mimicking recurrence of excised malignant myoepithelioma: A case report.

[This corrects the article DOI: 10.3892/mco.2018.1630.].

Fetal Head Growth during Early to Mid-Gestation Associated with Weight Gain in Mothers with Hyperemesis Gravidarum: A Retrospective Cohort Study.

The epigenetic impact of malnutrition in mothers with hyperemesis gravidarum (HG) on their offspring has not been fully elucidated. Recently, several reports have demonstrated that children born to mothers with HG were small for gestational age and had low birth weight, reduced insulin sensitivity, and neurodevelopmental delays during childhood. Therefore, we examined the relationship between fetal growth and changes in the maternal body weight in HG cases. A total of 34 patients with HG were hospitalized and delivered at term between 2009 and 2012. The records of 69 cases of pregnant women without a history of HG were extracted after matching their maternal age, parity, pregestational body mass index (BMI), gestational age, and fetal sex ratio with those of the HG group for comparison. The maternal weight gain at term was less in the HG than in the control group. There was no statistical difference in birth weight, placental weight, and ultrasonic fetometric parameters expressed in standard deviation (SD) scores, including biparietal diameter, abdominal circumference, and femur length, between the HG and the control group. Whereas fetal head growth in the HG group was positively associated with maternal weight gain at 20 weeks of gestation only, this association was not observed in the control group. We herein demonstrate that maternal weight gain from the nadir is associated with fetal head growth at mid-gestation. Thus, maternal undernutrition in the first trimester of pregnancy could affect fetal brain growth and development, leading to an increased risk of neurodevelopmental delays in later life.

Functional Blockade of E-Selectin in Tumor-Associated Vessels Enhances Anti-Tumor Effect of Doxorubicin in Breast Cancer.

Chemotherapy is a mainstay of treatment for solid tumors. However, little is known about how therapy-induced immune cell infiltration may affect therapy response. We found substantial CD45+ immune cell density adjacent to E-selectin expressing inflamed vessels in doxorubicin (DOX)-treated residual human breast tumors. While CD45 level was significantly elevated in DOX-treated wildtype mice, it remained unchanged in DOX-treated tumors from E-selectin null mice. Similarly, intravenous administration of anti-E-selectin aptamer (ESTA) resulted in a significant reduction in CD45+ immune cell density in DOX-treated residual tumors, which coincided with a delay in tumor growth and lung metastasis in MMTV-pyMT mice. Additionally, both tumor infiltrating T-lymphocytes and tumor associated-macrophages were skewed towards TH2 in DOX-treated residual breast tumors; however, ESTA suppressed these changes. This study suggests that DOX treatment instigates de novo intratumoral infiltration of immune cells through E-selectin, and functional blockade of E-selectin may reduce residual tumor burden as well as metastasis through suppression of TH2 shift.

Quantitative analysis of exercise among pregnant women with impaired glucose tolerance using pedometer data: An observational study.

AIMS: The aim of our study was to examine the feasibility of the use of a pedometer to quantify the amount of exercise and the relationship between the amount of exercise and carbohydrate metabolism in pregnant women with impaired glucose tolerance. METHODS: Seven pregnant women with impaired glucose tolerance (gestational diabetes: three, overt diabetes in pregnancy: one, pregestational diabetes type 2: three) were provided with pedometers. The relationship between pedometer data with blood glucose levels, maternal body weight, amount of insulin administered, blood hemoglobin A1c (HbA1c) levels, blood glycoalbumin levels and infant birth weight was investigated. RESULTS: When the 24-h-based data were examined, there was no correlation between the number of steps walked and blood glucose level immediately after walking, nor the average number of steps per day and the fasting blood glucose level in the next day. However, 4-week-based data showed that there was a negative correlation between the number of steps per day and the change in HbA1c level. Moreover, there was a negative correlation between the average number of steps per day and change in the maternal body weight. A 1-week-based data from five participants who were being administered insulin indicated that there was a negative correlation between the average number of steps per day and the total amount of insulin administered per day. CONCLUSION: Active application of pedometers is suggested to be feasible to improve metabolic control in pregnant women with glucose intolerance through the quantification of their exercise.

Oral metastasis of renal cell carcinoma mimicking recurrence of excised malignant myoepithelioma: A case report.

Following breast and lung cancers, renal cell carcinoma (RCC) is the third most frequent cancer to metastasize to the head and neck region, though such cases are rarely reported. Distinguishing between malignant tumors of salivary gland origin and metastatic RCC is very important. The case of a 75-year-old man with an oral cavity lesion in the left buccal submucosa measuring 40×30 mm that had grown substantially over several weeks is presented. His medical history included left kidney cancer 26 years earlier and a malignant myoepithelioma of the left buccal region 7 years earlier. It was suspected that this lesion was a recurrent malignant myoepithelioma as it appeared at the same site as the previous operation. Surgery was performed, and metastatic RCC was confirmed upon pathological examination. The diagnosis of metastatic RCC was made by immunohistochemical examination, which also excluded malignant myoepithelioma and other clear cell carcinomas of salivary gland origin. Metastatic RCC must be considered in the differential diagnosis of a new oral cavity lesion presenting in a patient with a past history of kidney cancer. Thus, immunohistochemical staining is required to distinguish malignancies of salivary gland origin, including malignant myoepithelioma, from metastatic RCC.

Aptamer Therapeutics in Cancer: Current and Future.

Aptamer-related technologies represent a revolutionary advancement in the capacity to rapidly develop new classes of targeting ligands. Structurally distinct RNA and DNA oligonucleotides, aptamers mimic small, protein-binding molecules and exhibit high binding affinity and selectivity. Although their molecular weight is relatively small-approximately one-tenth that of monoclonal antibodies-their complex tertiary folded structures create sufficient recognition surface area for tight interaction with target molecules. Additionally, unlike antibodies, aptamers can be readily chemically synthesized and modified. In addition, aptamers' long storage period and low immunogenicity are favorable properties for clinical utility. Due to their flexibility of chemical modification, aptamers are conjugated to other chemical entities including chemotherapeutic agents, siRNA, nanoparticles, and solid phase surfaces for therapeutic and diagnostic applications. However, as relatively small sized oligonucleotides, aptamers present several challenges for successful clinical translation. Their short plasma half-lives due to nuclease degradation and rapid renal excretion necessitate further structural modification of aptamers for clinical application. Since the US Food and Drug Administration (FDA) approval of the first aptamer drug, Macugen® (pegaptanib), which treats wet-age-related macular degeneration, several aptamer therapeutics for oncology have followed and shown promise in pre-clinical models as well as clinical trials. This review discusses the advantages and challenges of aptamers and introduces therapeutic aptamers under investigation and in clinical trials for cancer treatments.

Pathologic evaluation of tumor-associated macrophage density and vessel inflammation in invasive breast carcinomas.

Tumor-associated macrophages (TAMs) are major constituents of the tumor microenvironment in solid tumors and have been implicated as mediators of tumor progression, invasion and metastasis. Correspondingly, accumulation of TAMs is associated with unfavorable clinical outcomes in numerous types of solid tumors. E-selectin is a hallmark of inflammation and a key adhesion molecule that accommodates the initial contact of circulating immune cells with the inflamed vessel surface. Currently, the association between E-selectin and TAMs is not fully elucidated; therefore, the present study investigated the association between vessel inflammation, TAM infiltration, and clinical outcome in breast cancer. A total of 53 procedure-naïve invasive breast cancer cases were immunohistochemically analyzed for the presence of cluster of differentiation (CD)68+ TAMs, E-selectin+ vessels and tumor inflammation. The association between CD68 and E-selectin expression, and tumor inflammation as well as overall survival was evaluated using Kaplan-Meier survival curves and multivariable Cox's proportional hazards regression analysis. The abundance of TAMs was identified to be positively associated with tumor inflammation, estrogen receptor and E-selectin expression levels. A greater prevalence of TAMs and tumor inflammation was significantly associated with shorter overall survival times. E-selectin expression levels were significantly higher in tumor vessels among elderly patients, but were not associated with overall survival. The abundance of TAMs was associated with the presence of E-selectin-expressing inflamed tumor vessels and tumor inflammation, as well as overall survival in patients with invasive breast carcinoma.

The Emerging Role of FOXL2 in Regulating the Transcriptional Activation Function of Estrogen Receptor ß: An Insight Into Ovarian Folliculogenesis.

Germline mutations of the fork-head transcriptional factor forkhead box L2 (FOXL2) predispose embryos to autosomal-dominant blepharophimosis-ptosis-epicanthus inversus syndrome with primary ovarian insufficiency in female patients, but the mechanisms of FOXL2 in ovarian follicular development remain elusive. Estrogens produced by ovarian granulosa cells and estrogen receptor (ER) α and ERß play fundamental roles in ovarian pathophysiology, and a previous study revealed that ERα and ERß physically interact with FOXL2. However, the underlying functions of these interactions have not been investigated. Herein, we report an ERß-specific repressive function of FOXL2. Histological examination demonstrated that FOXL2 expression tends to be intense during early follicular development. Immunoprecipitation revealed that ERß and FOXL2 interact in a ligand-independent manner. In vitro pull-down assays revealed a direct interaction between FOXL2 and the activation function (AF)-1/2 domain of ERß. The expression of FOXL2 represses the ligand-dependent transcriptional activation of ERß, but FOXL2 does not influence the ligand-dependent transcriptional activation of ERα. Consistent with these results, RNA interference-mediated depletion of FOXL2 stimulates the expression of the ERß-downstream gene p450 aromatase. The convergence between FOXL2 functions and ERß-mediated transcription in the ovary suggests the putative mechanism of FOXL2 in early-phase follicular development, which may be partially attributed to the regulation of ERß-dependent gene expression.

Is the neonatal creatine phosphokinase level a reliable marker for fetal hypoxia?

AIM: The creatine phosphokinase (CPK) level is believed to increase in neonatal peripheral blood after tissue damage, including damage from perinatal hypoxia. However, it is not clear whether it is truly a reliable marker for fetal hypoxia. We investigated the chronological changes in neonatal CPK and the reliability of CPK as a marker for fetal hypoxia. METHODS: Sixty term neonates admitted to the neonatal intensive care unit at Tokyo Women's Medical University Medical Center East from April 2009 to April 2010 were enrolled in this study. We evaluated whether asphyxia and fetal heart rate (FHR) abnormality could predict the neonatal CPK level by using receiver-operator curve analysis. We also compared umbilical cord blood pH levels with neonatal CPK levels. In addition, we investigated factors that influence neonatal CPK in non-asphyxia cases. RESULTS: The median value of CPK peaked on day 1. There were no significant differences in CPK levels regardless of the presence of asphyxia or FHR abnormality. Non-asphyxiated neonates with older gestational ages and amniotic fluid abnormalities had significantly higher levels of CPK. CONCLUSION: Our results indicate that the neonatal CPK level is not an appropriate marker for retrospectively predicting either asphyxia or FHR abnormality. There are influencing factors other than asphyxia that increase neonatal CPK. Therefore, one should be careful when making a diagnosis of perinatal hypoxia based solely on increased levels of neonatal CPK after birth.

E-selectin Targeting PEGylated-thioaptamer Prevents Breast Cancer Metastases.

E-selectin is an adhesion molecule expressed on the luminal surface of inflamed blood vessels that mediates hematogenous metastasis by assisting shear-resistant adhesion of circulating tumor cells to the vessel surface under dynamic blood flow. Previously, we developed an E-selectin antagonistic thioaptamer (ESTA) for the prevention of hematogenous metastasis through the blockade of CD44high breast cancer cells (BCa) adhesion to E-selectin-expressing premetastatic endothelial niche. The current study focuses on developing a PEGylated E-selectin targeting thioaptamer with improved pharmaceutical properties. A serial deletion of stem-loops reveled that loop-1 and -2 (ESTA7) are the minimally effective backbone structure necessary to obtain inhibition of the E-selectin/CD44 interaction and shear resistant adhesion of CD44high BCa to E-selectin-expressing human endothelial cells (HMVECs) at a level equal to ESTA. Chemical conjugation of methoxy-polyethylene-glycol (PEG) at the sizes of 5 and 10 kDa did not interfere with ESTA7-mediated shear-resistant adhesion. However, in vivo study demonstrated that only 10 kDa PEG-conjugated ESTA7 (ESTA7-p10) retains the activity to inhibit metastases at a level equal to parental ESTA. Additionally, a single intravenous injection of ESTA7-p10 inhibited the development of lung, brain, and bone metastases of MDA-MB-231, through the blockade of E-selectin. Moreover, PEGylation led to an extension of elimination half-life and increase of AUC, resulting in superior inhibition of metastasis development compared to parental ESTA with a longer interval between dosing in a spontaneous metastasis model. Lastly, repeated intravenous administration of ESTA7-p10 was tolerated in mice, highlighting the potential prophylactic application of ESTA7-p10 for metastasis prevention.

Acidic microenvironments induce lymphangiogenesis and IL-8 production via TRPV1 activation in human lymphatic endothelial cells.

Local acidosis is one of the characteristic features of the cancer microenvironment. Many reports indicate that acidosis accelerates the proliferation and invasiveness of cancer cells. However, whether acidic conditions affect lymphatic metastasis is currently unknown. In the present study, we focused on the effects of acidosis on lymphatic endothelial cells (LECs) to assess the relationship between acidic microenvironments and lymph node metastasis. We demonstrated that normal human LECs express various acid receptors by immunohistochemistry and reverse transcriptase-polymerase chain reaction (PCR). Acidic stimulation with low pH medium induced morphological changes in LECs to a spindle shape, and significantly promoted cellular growth and tube formation. Moreover, real-time PCR revealed that acidic conditions increased the mRNA expression of interleukin (IL)-8. Acidic stimulation increased IL-8 production in LECs, whereas a selective transient receptor potential vanilloid subtype 1 (TRPV1) antagonist, 5'-iodoresiniferatoxin, decreased IL-8 production. IL-8 accelerated the proliferation of LECs, and inhibition of IL-8 diminished tube formation and cell migration. In addition, phosphorylation of nuclear factor (NF)-κB was induced by acidic conditions, and inhibition of NF-κB activation reduced acid-induced IL-8 expression. These results suggest that acidic microenvironments in tumors induce lymphangiogenesis via TRPV1 activation in LECs, which in turn may promote lymphatic metastasis.

The effect of soluble E-selectin on tumor progression and metastasis.

BACKGROUND: Distant metastasis resulting from vascular dissemination of cancer cells is the primary cause of mortality from breast cancer. We have previously reported that E-selectin expression on the endothelial cell surface mediates shear-resistant adhesion and migration of circulating cancer cells via interaction with CD44. As a result of shedding, soluble E-selectin (sE-selectin) from the activated endothelium is present in the serum. In this study, we aimed to understand the role of sE-selectin in tumor progression and metastasis. METHODS: We investigated the effect of sE-selectin on shear-resistant adhesion and migration of metastatic breast cancer cells and leukocytes in vitro and in vivo. RESULTS: We found that sE-selectin promoted migration and shear-resistant adhesion of CD44(+) (/high) breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to non-activated human microvessel endothelial cells (ES-HMVECs), but not of CD44(-/low) breast cancer cell lines (MCF-7 and T-47D). This endothelial E-selectin independent, sE-selectin-mediated shear-resistant adhesion was also observed in a leukocyte cell line (HL-60) as well as human peripheral blood mononuclear cells (PBMCs). Additionally, the incubation of MDA-MB-231 cells with sE-selectin triggered FAK phosphorylation and shear-resistant adhesion of sE-selectin-treated cells resulted in increased endothelial permeabilization. However, CD44 knockdown in MDA-MB-231 and HL-60 cells resulted in a significant reduction of sE-selectin-mediated shear-resistant adhesion to non-activated HMVECs, suggesting the involvement of CD44/FAK. Moreover, functional blockade of ICAM-1 in non-activated HMVECs resulted in a marked reduction of sE-selectin-mediated shear-resistant adhesion. Finally, the pre-incubation of CD44(+) 4 T1 murine breast cancer cells with sE-selectin augmented infiltration into the lung in E-selectin K/O mice and infusion of human PBMCs pre-incubated with sE-selectin stimulated MDA-MB-231 xenografted breast tumor growth in NSG mice. CONCLUSIONS: Our data suggest that circulating sE-selectin stimulates a broad range of circulating cells via CD44 and mediates pleiotropic effects that promote migration and shear-resistant adhesion in an endothelial E-selectin independent fashion, in turn accelerating tissue infiltration of leukocytes and cancer cells.

Patients with Advanced Ovarian Cancer Administered Oral Etoposide following Taxane as Maintenance Chemotherapy.

INTRODUCTION: The concept of maintenance therapy is one of the highly relevant approaches in the management of advanced ovarian cancer. The fundamental goal of maintenance therapy is to improve survival outcomes. We attempted to reinforce maintenance chemotherapy by adding oral etoposide following taxane administration. CASES: We retrospectively evaluated 14 patients with advanced ovarian cancer who had achieved clinically defined complete response to a primary platinum/taxane chemotherapy regimen and who were administered oral etoposide (50 mg/day × 21 days per cycle monthly for 3-5 cycles) following paclitaxel or docetaxel administration as maintenance chemotherapy. With regard to oral etoposide toxicity, grade 2 oral mucositis and grade 3 anemia were observed in 1 patient each. Three to five cycles of etoposide were administered to all patients, though daily dosage was reduced to 25 mg in 2 patients due to toxicity. The median progression-free survival was 43.5 months, the median overall survival was 86 months, and 5-year overall survival was 77.1%. CONCLUSION: The results from this ovarian cancer treatment evaluation suggest that oral etoposide may be administered safely following paclitaxel or docetaxel as maintenance chemotherapy. We expect this regimen to contribute to the improvement in the survival outcomes of patients with advanced ovarian cancer.

Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma.

Lymph node metastasis (LNM) is associated with poor survival in patients with oral squamous cell carcinoma (OSCC). Vascular endothelial growth factor-C (VEGF-C) is thought to be responsible for increased lymphangiogenesis and LNM. Understanding of the mechanism by which VEGF-C expression is regulated in OSCC is thus important to design logic therapeutic interventions. We showed that inoculation of the SAS human OSCC cells expressing the venus GFP (V-SAS cells) into the tongue in nude mice developed LNM. V-SAS cells in LNM were isolated by FACS and re-inoculated into the tongue. This procedure was repeated eight times, establishing V-SAS-LM8 cells. Differential metastasis PCR array between the parental V-SAS and V-SAS-LM8 was performed to identify a molecule responsible for lymphangiogenesis and LNM. Fibronectin 1 (FN1) expression was elevated in V-SAS-LM8 cells compared to V-SAS-cells. V-SAS-LM8 tongue tumor showed increased expression of FN1 and VEGF-C, and promoted lymphangiogenesis and LNM compared with V-SAS tumor. Further, phosphorylation of focal adhesion kinase (FAK), a main downstream signaling molecule of FN1, was up-regulated, and epithelial-mesenchymal transition (EMT) was promoted in V-SAS-LM8 cells. Silencing of FN1 by shRNA in V-SAS-LM8 cells decreased FAK phosphorylation, VEGF-C expression and inhibited lymphangiogenesis and LNM. EMT was also reversed. The FAK phosphorylation inhibitor PF573228 also decreased VEGF-C expression and reversed EMT in V-SAS-LM8 cells. Finally, we detected intense FN1 expression in some clinical specimens obtained from OSCC patients with LNM. These results demonstrate that elevated expression of cellular FN1 and following activation of FAK lead to increased VEGF-C expression, lymphangiogenesis and LNM and promoted EMT in SAS human OSCC cells and suggest that FN1-phosphorylated FAK signaling cascade is a potential therapeutic target in the treatment of LNM in OSCC.

HAND2-mediated proteolysis negatively regulates the function of estrogen receptor α.

A previous study demonstrated that the progesterone­inducible HAND2 gene product is a basic helix­loop­helix transcription factor and prevents mitogenic effects of estrogen receptor α (ERα) by inhibiting fibroblast growth factor signalling in mouse uteri. However, whether HAND2 directly affects the transcriptional activation function of ERα remains to be elucidated. In the present study, the physical interaction between HAND2 and ERα was investigating by performing an immunoprecipitation assay and an in vitro pull­down assay. The results demonstrated that HAND2 and ERα interacted in a ligand­independent manner. The in vitro pull­down assays revealed a direct interaction between HAND2 and the amino­terminus of ERα, termed the activation function­1 domain. To determine the physiological significance of this interaction, the role of HAND2 as a cofactor of ERα was investigated, which revealed that HAND2 inhibited the ligand­dependent transcriptional activation function of ERα. This result was further confirmed and the mRNA expression of vascular endothelial growth factor, an ERα­downstream factor, was decreased by the overexpression of HAND2. This inhibition of ligand­dependent transcriptional activation function of ERα was possibly attributed to the proteasomic degradation of ERα by HAND2. These results indicate a novel anti­tumorigenic function of HAND2 in regulating ERα­dependent gene expression. Considering that HAND2 is commonly hypermethylated and silenced in endometrial cancer, it is hypothesized that HAND2 may serve as a possible tumor suppressor, particularly in uterine tissue.