Contributions of the Department of Defense Congressionally Directed Medical Research Programs to Advances in Cancer Therapeutics.

Nearly a billion dollars is spent annually in the Military Health System (MHS) on cancer diagnosis and treatment, with a large portion of that directed toward breast, prostate, and ovarian cancers. Multiple studies have demonstrated the impact of specific cancers on MHS beneficiaries and Veterans, highlighting the fact that active duty and retired military members have a higher incidence than the general public for many chronic diseases and certain forms of cancer. The Congressionally Directed Medical Research Programs have supported research that has contributed to the development, clinical testing, and commercialization of 11 cancer drugs approved by the Food and Drug Administration to treat breast, prostate, or ovarian cancers. In addition to hallmark funding mechanisms that prioritize innovative, groundbreaking ideas, the Congressionally Directed Medical Research Program's cancer programs continue to identify new approaches to fill critical gaps across the full research spectrum, including bridging the translational research gap toward developing new treatments for cancer patients in the MHS and in the general American public.

Factors associated with delay in treatment-seeking behaviour for fever cases among caregivers of under-five children in India: Evidence from the National Family Health Survey-4, 2015-16.

BACKGROUND: Fever is one of the common clinical symptoms found among children suffering from various illnesses. India carries a substantial burden of febrile illness among under-five children which heighten the risk of malnutrition, mortality and morbidity. This study aims to determine the factors associated with delay in treatment-seeking for fever among under-five children in India. METHODS: A cross-sectional study was carried out using the large-scale nationally representative data from the National Family Health Survey (NFHS-4), conducted in 2015-2016. The data were collected by using four survey questionnaires i.e., Household Questionnaire, Woman's Questionnaire, Man's Questionnaire, and Biomarker Questionnaire. Delay in treatment-seeking was defined as taking a child for treatment after 24 hours of fever onset. Bivariate and multivariate logistic regression models were performed to assess the factors associated with delay in treatment-seeking behaviour for fever in under-five children. RESULTS: In India, 31.12% (n = 7229) of the caregivers sought treatment for children after 24 hours of the onset of fever. Findings show no significant differences in delay in treatment-seeking behaviour by age groups and sex of children. Multivariate analysis revealed that the odds of delay in treatment-seeking behaviour of fever were higher among children from the poorest wealth quintile (AOR: 2.06; 95% CI: 1.85, 2.31), belonging to the scheduled tribe (AOR: 1.35; 95% CI: 1.24, 1.48), children who resided in rural areas (AOR: 1.14; 95% CI: 1.07, 1.22), children from the northeast region (AOR: 1.29; 95% CI: 1.14, 1.46), and children of caregivers who perceived distance to health facilities as a 'big problem' (AOR: 1.16; 95% CI: 1.09, 1.23). CONCLUSION: The study shows a high prevalence of delay in seeking treatment for fever among caregivers of under-five children in India. Delay in seeking treatment is associated with socio-demographic and socio-economic factors. Therefore, there is a need for intensified health promotion programs to sensitize caregivers on the importance of early health-seeking behaviour.

Experimental and theoretical exploration of magnetic exchange interactions and single-molecule magnetic behaviour of bis(η1:η2:µ2-carboxylate)Gd/Dy systems.

The present report deals with the syntheses, crystal structures, dc/ac magnetic properties and DFT/ab initio CASSCF calculations of two isomorphous bis(η1:η2:µ2-acetate)GdIII2/DyIII2 compounds of the formula [LnIII2L2(acetate)4(MeOH)2] (1, Ln = Gd; 2, Ln = Dy), where HL is (E)-N'-(3-ethoxysalicylidene)acetohydrazide. The two lanthanide(iii) centres in each compound are symmetry-related owing to the presence of an inversion centre. Both compounds exhibit intramolecular ferromagnetic exchange interactions. The DyIII2 analogue is a single-molecule magnet (SMM) with Ueff = 52.8 cm-1 and τ0 = 1.52 × 10-6 s. DFT calculations for 1 and ab initio calculations for 2 also reveal ferromagnetic interactions. Ab initio calculations of the SMM behaviour of 2 and two other reported and structurally related compounds reveal the importance of the weak exchange interaction present between the two DyIII ions, and a relaxation mechanism has been developed to take into account the magnetic exchange interaction and to rationalize the observed difference in the Ueff values.

A nickel(ii)-manganese(ii)-azido layered coordination polymer showing a three-dimensional ferrimagnetic order at 35 K.

The work in this report describes the synthesis, characterization, crystal structure and magnetic properties of a two-dimensional azido-bridged NiIIMnII heterometallic coordination polymer, [{(NiIILMnII)2(µ1,1,3-N3)2}(µ1,3-N3)2]n (1), in which the H2L Schiff base is the [2 + 1] condensation product of 3-methoxysalicylaldehyde and 1,3-diaminopropane. In 1, two bis(µ-phenoxo)-µ1,1,3-azido-NiIIMnII dinuclear units are interlinked to form a dimer-of-dinuclear moiety {NiIIMnII}2; the {NiIIMnII}2 structural units are further interlinked with each other by µ1,3-azido ligands to form a layered coordination polymer. Upon cooling, the χMT product slowly decreases from 300 K to about 100 K, then increases abruptly from 3.74 cm3 K mol-1 at 100 K to 113.2 cm3 K mol-1 at 30 K, indicating a ferrimagnetic behaviour; a three-dimensional magnetic order below 37 K was determined by the divergence in the ZFC-FC measurements and ac measurements, in which the peak temperature 35 K in the in-phase at different frequencies suggests that the Curie temperature is 35 K, whereas the out-of-phase is somewhat frequency dependent, due to the glassy magnet behaviour. The title compound is among the rare examples of metallo-organic systems having a magnetic ordering Curie temperature above 30 K. Other interesting aspects in 1 regarding magnetic properties and structural features are discussed.

Effects of six months of Yoga on inflammatory serum markers prognostic of recurrence risk in breast cancer survivors.

Yoga-based exercise has proven to be beneficial for practitioners, including cancer survivors. This study reports on the effect on inflammatory biological markers for 20 breast cancer survivors who participated in a six-month yoga-based (YE) exercise program. Results are compared to a comprehensive exercise (CE) program group and a comparison (C) exercise group who chose their own exercises. "Pre" and "post" assessments included measures of anthropometrics, cardiorespiratory capacity, and inflammatory markers interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor alpha (TNFα) and C-reactive protein (CRP). Descriptive statistics, effect size (d), and dependent sample 't' tests for all outcome measures were calculated for the YE group. Significant improvements were seen in decreased % body fat, (-3.00%, d = -0.44, p = <.001) but not in cardiorespiratory capacity or in inflammatory serum markers. To compare YE outcomes with the other two groups, a one-way analysis of co-variance (ANCOVA) was used, controlling for age, BMI, cardiorespiratory capacity and serum marker baseline values. We found no differences between groups. Moreover, we did not see significant changes in any inflammatory marker for any group. Our results support the effectiveness of yoga-based exercise modified for breast cancer survivors for improving body composition. Larger studies are needed to determine if there are significant changes in inflammatory serum markers as a result of specific exercise modalities.

Genome-wide DNA methylation profiling reveals parity-associated hypermethylation of FOXA1.

Early pregnancy in women by the age of 20 is known to have a profound effect on reduction of lifelong breast cancer risk as compared to their nulliparous counterparts. Additional pregnancies further enhance the protection against breast cancer development. Nationwide trend of delayed pregnancy may contribute to the recently reported increase in the incidence of advanced breast cancer among young women in this country. The underlying mechanism for the parity-associated reduction of breast cancer risk is not clearly understood. The purpose of the current study is to use whole-genome DNA methylation profiling to explore a potential association between parity and epigenetic changes in breast tissue from women with early parity and nulliparity. Breast tissue was collected from age-matched cancer-free women with early parity (age < 20; n = 15) or nulliparity (n = 13). The methyl-CpG binding domain-based capture-sequencing technology was used for whole-genome DNA methylation profiling. Potential parity-associated hypermethylated genes were further verified by locus-specific pyrosequencing, using an expanded cohort of parous (n = 19) and nulliparous (n = 16) women that included the initial samples used in the global analysis. Our study identified six genes that are hypermethylated in the parous group (P < 0.05). Pyrosequencing confirmed parity-associated hypermethylation at multiple CpG islands of the FOXA1 gene, which encodes a pioneer factor that facilitates chromatin binding of estrogen receptor α. Our work identifies several potential methylation biomarkers for parity-associated breast cancer risk assessment. In addition, the results are consistent with the notion that parity-associated epigenetic silencing of FOXA1 contributes to long-term attenuation of the estrogenic impact on breast cancer development.

Association of obesity and circulating adipose stromal cells among breast cancer survivors.

A positive association of obesity with breast cancer incidence and mortality is well established. Recent reports indicate that adipose stromal cells (ASCs) play an important role in breast cancer development and progression by producing estrogens and tumor-promoting cytokines. Furthermore, circulating ASCs have been uniquely detected in obese individuals, which is likely due to increased tissue remodeling and cell mobilization. The number of circulating ASCs is even more prominent in obese patients with colon and prostate cancers, both of which are exacerbated by obesity. To determine whether a similar association exists for breast cancer, we collected blood samples from a cohort of breast cancer survivors and enumerated circulating ASCs by flow cytometry on the basis of the previously established ASC-associated immunophenotype (CD34+/CD31-/CD45-). We found significantly higher levels of circulating ASCs (p<0.001) in breast cancer survivors with body mass index (BMI)≥30 kg/m2 than their non-obese counterparts (BMI<30). We also compared circulating ASCs before and after exercise of only the obese subjects enrolled in a 6-month individualized exercise program, but found no statistically significant difference, likely due to limited number of subjects in the study. Our findings suggest that circulating ASCs can serve as a potential biomarker for future studies of the impacts of obesity and physical activity on breast cancer recurrence and survival.

Motivation, exercise, and stress in breast cancer survivors.

PURPOSE: Reduced stress and reduced risk of cancer recurrence are among the many benefits of physical activity (PA) for cancer survivors. Exercise behaviors are linked to motivational factors. We investigated the associations between motivational profile, self-reported levels of PA and stress, and mental functioning in 94 posttreatment breast cancer survivors who voluntarily enrolled in an exercise program. METHODS: Participants completed the Apter Motivational Style Profile (AMSP), Lifetime of Physical Activity (LTPA) Questionnaire, International Physical Activity Questionnaire (IPAQ), Medical Outcomes Short Form SF-36® (SF-36), and Perceived Stress Scale (PSS) and provided 10 saliva specimens (to measure cortisol levels). PA levels were calculated in metabolic equivalent hours per week (MET-hours/week). RESULTS: Participants reported high levels of current and historical PA (M = 39.2 MET-h/week, SD = 39.7; M = 14.2 MET-h/week, SD = 15.4, respectively). They also reported high levels of stress (M = 33.6, SD = 4.5) coupled with low mental functionality as measured by SF-36 mental component scale (MCS) (M = 44.4, SD = 8.8). PSS was negatively associated with MCS (r = -0.27, p = 0.009). Salivary cortisol was not associated with any measure. Participants had a conformist ("follow rules") and alloic ("about others") motivational profile. No motivational, exercise history, or stress variables were associated with current PA. CONCLUSIONS: As expected, participants reported higher levels of stress and lower mental functioning. Participants presented a unique motivational profile relative to the general population. Further research into the associations of motivation, exercise behaviors, and stress is warranted.

Regulation of adipose oestrogen output by mechanical stress.

Adipose stromal cells are the primary source of local oestrogens in adipose tissue, aberrant production of which promotes oestrogen receptor-positive breast cancer. Here we show that extracellular matrix compliance and cell contractility are two opposing determinants for oestrogen output of adipose stromal cells. Using synthetic extracellular matrix and elastomeric micropost arrays with tunable rigidity, we find that increasing matrix compliance induces transcription of aromatase, a rate-limiting enzyme in oestrogen biosynthesis. This mechanical cue is transduced sequentially by discoidin domain receptor 1, c-Jun N-terminal kinase 1, and phosphorylated JunB, which binds to and activates two breast cancer-associated aromatase promoters. In contrast, elevated cell contractility due to actin stress fibre formation dampens aromatase transcription. Mechanically stimulated stromal oestrogen production enhances oestrogen-dependent transcription in oestrogen receptor-positive tumour cells and promotes their growth. This novel mechanotransduction pathway underlies communications between extracellular matrix, stromal hormone output, and cancer cell growth within the same microenvironment.

An IL-6 link between obesity and cancer.

Obesity is a growing epidemic all over the world that by virtue of inducation of a chronic, low-grade, and systemic inflammation leads to an increased risk of a number of diseases, including cancer. IL-6 an important cytokine in the increased risk to cancer in obese patients mainly because of its pro-inflammatory activity. Some data suggest that IL-6 might increase the risk of certain cancers such as those that originate from breast, liver, prostate, colon, and esophagus. A better understanding of the regulation and role of IL-6 in obesity-associated cancer is required to develop effective therapeutic approaches.

Mechanical phenotype is important for stromal aromatase expression.

Evidence that aromatase expression in tumor-associated breast stroma is elevated, provides a rationale for use of aromatase inhibitors (AIs) in breast cancer treatment. However, regulation of local aromatase expression in cancer-free breast stroma is poorly understood. Recent clinical work indicates that stromal cells in dense breast tissue tend to express higher levels of aromatase than their counterpart from non-dense tissue. Consistent with the clinical observation, our cell culture-based study indicated that cell density, cell shape, and extracellular matrix (ECM) significantly induced stromal aromatase expression via a distinct signal transduction pathway. In addition, we identified a number of cell surface markers that are commonly associated with aromatase-expressing stromal cells. As mammographic density is one of the strongest and most prevalent risk factors for breast cancer, these findings provide a potential mechanistic link between alterations in tissue composition of dense breast tissue and increased stromal aromatase expression. Further exploration of the in vitro model system may advance understanding of an important problem in breast cancer biology.

Cell density-dependent transcriptional activation of endocrine-related genes in human adipose tissue-derived stem cells.

Adipose tissue is recognized as an endocrine organ that plays an important role in human diseases such as type II diabetes and cancer. Human adipose tissue-derived stem cells (ASCs), a distinct cell population in adipose tissue, are capable of differentiating into multiple lineages including adipogenesis. When cultured in vitro under a confluent condition, ASCs reach a commitment stage for adipogenesis, which can be further induced into terminally differentiated adipocytes by a cocktail of adipogenic factors. Here we report that the confluent state of ASCs triggers transcriptional activation cascades for genes that are responsible for the endocrine function of adipose tissue. These include insulin-like growth factor 1 (IGF-1) and aromatase (Cyp19), a key enzyme in estrogen biosynthesis. Despite similar adipogenic potentials, ASCs from different individuals display huge variations in activation of these endocrine-related genes. Bioinformatics and experimental data suggest that transcription factor Foxo1 controls a large number of "early" confluency-response genes, which subsequently induce "late" response genes. Furthermore, siRNA-mediated knockdown of Foxo1 substantially compromises the ability of committed ASCs to stimulate tumor cell migration in vitro. Thus, our work suggests that cell density is an important determinant of the endocrine potential of ASCs.

Cell density is a critical determinant of aromatase expression in adipose stromal cells.

Obesity is associated with an increased risk of breast cancer among postmenopausal women. This is at least partly due to excessive estrogen production in adipose tissue of obese women. Aromatase, the key enzyme in estrogen biosynthesis, is an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. In this study we show that high confluency of human adipose stromal cells (ASCs) cultured in vitro can significantly stimulate aromatase gene expression and reduce the expression of breast tumor suppressor BRCA1 and members of the NR4A orphan nuclear family. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Nurr1, a member of the NR4A family, substantially increased aromatase expression. Lastly, we found that the cell density-triggered inducibility of aromatase expression varies in ASCs isolated from different disease-free individuals. Our finding highlights the impact of increased cell number on estrogen biosynthesis as in the case of excessive adiposity.

IKKbeta mediates cell shape-induced aromatase expression and estrogen biosynthesis in adipose stromal cells.

Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in breast cancer therapy. Within tumor microenvironment, tumor cells stimulate aromatase expression in adipose stromal cells (ASCs), which in turn promotes estrogen-dependent growth of estrogen receptor (ER)-positive tumor cells. However, it is not clear how aromatase transcription and estrogen biosynthesis are regulated in ASCs under a precancerous condition. Here we demonstrate that cell shape change alone is sufficient to induce aromatase expression in primary ASCs from cancer-free individuals. The activation of aromatase transcription is mediated by IkappaB kinase-beta (IKKbeta), a kinase previously known for its cancer-promoting activity in tumor cells. Activation of IKKbeta leads to elevated expression of transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta), which binds to and stimulates two breast cancer-associated promoters of the aromatase gene. We also show that shape-induced estrogen production in ASCs can stimulate estrogen-dependent transcription in ER-positive breast tumor cells. We suggest that IKKbeta-dependent aromatase induction due to changes in cellular architecture in adipose tissue may contribute to the breast cancer risks associated with high mammagraphic density and obesity.

Deregulation of cofactor of BRCA1 expression in breast cancer cells.

Cofactor of BRCA1 (COBRA1) is an integral component of the human negative elongation factor (NELF), a four-subunit protein complex that inhibits transcription elongation. Previous in vivo work indicates that COBRA1 and the rest of the NELF complex repress estrogen-dependent transcription and the growth of breast cancer cells. In light of the COBRA1 function in breast cancer-related gene expression, we sought to examine regulation of COBRA1 expression in both established breast cancer cell lines and breast carcinoma tissues. We found that COBRA1 expression was inversely correlated with breast cancer progression, as tumor samples of patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 mRNA when compared to those who were disease free for over 10 years (P = 0.0065 and 0.0081, respectively). Using both breast and prostate cancer cell lines, we also explored the possible mechanisms by which COBRA1 expression is regulated. Our results indicate that the protein abundance of COBRA1 and the other NELF subunits are mutually influenced in a tightly coordinated fashion. Small interfering RNA (siRNA) that targeted at one NELF subunit dampened the protein levels of all four subunits. Conversely, ectopic expression of COBRA1 in the knockdown cells partially rescues the co-depletion of the NELF subunits. In addition, our study suggests that a post-transcriptional, proteasome-independent mechanism is involved in the interdependent regulation of the NELF abundance. Furthermore, a lack of COBRA1 expression in breast carcinoma may serve as a useful indicator for poor prognosis.

A Role of CREB in BRCA1 Constitutive Promoter Activity and Aromatase Basal Expression.

Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Its ovary-specific promoter, PII, is induced in response to protein kinase A (PKA) activation. It has been proposed that breast cancer susceptibility gene 1, BRCA1, is involved in negative regulation of aromatase PII activity. Surprisingly, inhibition of PKA pathway by inhibitor H89 elevates basal aromatase expression while abolishes cAMP-mediated aromatase induction in an ovarian granulosa cell line, KGN. In this report, we decipher the mechanism by which the PKA pathway negatively regulates aromatase basal expression. We show that PKA pathway plays a positive role in the expression of BRCA1. H89 effectively reduces endogenous BRCA1 mRNA levels as well as reporter gene expression from a BRCA1 promoter. Mutation of a cAMP-responsive element (CRE) in the BRCA1 promoter reduces BRCA1 expression. Chromatin immunoprecipitation (ChIP) shows that CRE-binding protein, CREB, binds to the BRCA1 promoter. Furthermore, knockdown of CREB in KGN cells leads to decreased BRCA1 level as well as elevated basal aromatase mRNA expression. These data demonstrate that both the CRE site in the BRCA1 promoter and CREB are required for BRCA1 constitutive expression. Our study suggests that PKA pathway exerts its negative impact on basal aromatase expression indirectly by contributing to the constitutive expression of BRCA1.

Tumor suppressor BRCA1 inhibits a breast cancer-associated promoter of the aromatase gene (CYP19) in human adipose stromal cells.

Adipose tissue provides an important extragonadal source of estrogen. Obesity-associated elevation of estrogen production increases risk of breast cancer in postmenopausal women. Aromatase (CYP19), which converts androgen to estrogen, is a key enzyme in estrogen biosynthesis. In normal adipose tissue, transcription of the aromatase gene is initiated from a relatively weak adipose-specific promoter (I.4). However, in breast cancer, a switch of promoter utilization from I.4 to a strong ovary-specific promoter, PII, leads to increased aromatase expression and, hence, elevated estrogen production. Here, we report an intriguing relationship between the breast cancer susceptibility gene BRCA1 and aromatase expression in human adipose stromal cells (ASCs). Upon stimulation by phorbol ester or dexamethasone, increased aromatase expression in ASCs was accompanied by significant reduction of the BRCA1 level. In addition, adipogenesis-induced aromatase expression was also inversely correlated with BRCA1 abundance. Downregulation of BRCA1 expression in response to various stimuli was through distinct transcription or posttranscription mechanisms. Importantly, siRNA-mediated knockdown of BRCA1 led to specific activation of the breast cancer-associated PII promoter. Therefore, in addition to its well-characterized activities in breast epithelial cells, a role of BRCA1 in modulation of estrogen biosynthesis in ASCs may also contribute to its tissue-specific tumor suppressor function.

Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression.

Mutations in BRCA1 increase risks of familial breast and ovarian cancers, particularly among premenopausal women. While BRCA1 plays an active role in DNA repair, this function alone may not be sufficient to explain why BRCA1-associated tumors predominantly occur in estrogen-responsive tissues. Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Aromatase expression in ovarian granulosa cells dictates levels of circulating estrogen in premenopausal women, and its aberrant overexpression in breast adipose tissues promotes breast cancer growth. Here, we show that BRCA1 modulates aromatase expression in ovarian granulosa cells and primary preadipocytes. The cyclic AMP-dependent expression of aromatase in ovarian granulosa cells is inversely correlated with the protein level of BRCA1. Importantly, transient knockdown of BRCA1 enhances aromatase expression in both ovarian granulosa cells and primary preadipocytes. We propose that BRCA1 deficiency in epithelial and certain nonepithelial cells may result in combined effects of aberrant estrogen biosynthesis and compromised DNA repair capability, which in turn may lead to specific cancers in the breast and ovary.

Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

Estrogen is critical to both normal mammary gland and breast cancer development. Circulating levels of estrogen in premenopausal women are primarily determined by the action of aromatase in ovarian granulosa cells that converts testosterone to estradiol. In the current study, we unraveled an important role of Jun proteins in modulating ovary-specific aromatase expression. Ectopic expression of the Jun proteins in a human granulosa cell line significantly inhibited an ovary-specific promoter (PII) of the aromatase gene, whereas expression of dominant-negative mutants of Jun led to increased promoter activity. The Jun-mediated repression was specific to the aromatase promoter, as Jun proteins stimulated known AP1-responsive promoters in the same cellular context. Both the activation and basic leucine zipper domains of Jun were required for the transcriptional repression. Electrophoretic gel mobility assay showed that endogenous Jun proteins bound to a functionally important cAMP-responsive element (CRE) in the PII promoter-proximal region. Alteration of the CRE-like site impaired both the cAMP-responsive transcriptional activation and Jun-mediated repression. Furthermore, chromatin immunoprecipitation indicated the presence of cJun at the proximal region of the native PII promoter. Taken together, our work suggests that Jun proteins may attenuate estrogen biosynthesis by directly downregulating transcription of the aromatase gene in ovarian granulosa cells.

The orphan nuclear receptors NURR1 and NGFI-B modulate aromatase gene expression in ovarian granulosa cells: a possible mechanism for repression of aromatase expression upon luteinizing hormone surge.

Ovarian granulosa cells play pivotal roles in many aspects of ovary functions including folliculogenesis and steroidogenesis. In response to FSH and LH, the elevation of intracellular cAMP level in granulosa cells leads to activation of multiple ovarian genes. Here, we report findings from a genome-wide study of the cAMP-responsive gene expression profiles in a human granulosa-like tumor cell line, KGN. The study identified 140 genes that are either activated or repressed by 2-fold or greater after stimulation by the adenylyl cyclase activator forskolin. The induction patterns of some cAMP-responsive genes were further analyzed by quantitative real-time PCR. Consistent with previous observations, the LH-responsive genes, such as the nuclear receptor 4A subfamily (NURR1, NGFI-B, and NOR-1), were rapidly but transiently induced, whereas the FSH-responsive gene CYP19 encoding aromatase was induced in a delayed fashion. Interestingly, ectopic expression of NURR1 or NGFI-B severely attenuated the cAMP-responsive activation of the ovary-specific aromatase promoter. Reduction of the endogenous NURR1 or NGFI-B by small interfering RNA significantly elevated aromatase gene expression. The cis-elements responsible for NURR1/NGFI-B-mediated repression were mapped to the minimal aromatase promoter sequence that confers camp responsiveness. Furthermore, the DNA-binding domain of NURR1 was required for the repression. Taken together, these results strongly suggest a causal relationship between the rapid decline of aromatase mRNA and induction of nuclear receptor subfamily 4A expression, which concomitantly occur upon LH surge at the later stages of ovarian follicular development.