The TFDP1 gene coding for DP1, the heterodimeric partner of the transcription factor E2F, is a target of deregulated E2F.

The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1∼E2F8), expression of activator E2Fs (E2F1∼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known. We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5' and 3' deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs. a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.

Inhibiting Wnt/beta-catenin in CTNNB1-mutated endometrial cancer.

The role of ß-catenin/TCF transcriptional activity in endometrial cancer (EC) recurrence is not well understood. We assessed the impact of Wnt/ß-catenin inhibition in EC models. In an analysis of the Cancer Genome Atlas, we confirmed that CTNNB1 mutations are enriched in recurrent low-risk EC and showed that aberrant Wnt/ß-catenin pathway activation is associated with recurrence. We studied CTNNB1-wildtype (HEC1B, Ishikawa) and CTNNB1-mutant (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y) EC cell lines. Dose response curves were determined for 5 Wnt/ß-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). XAV939, Wnt-C59 and PyrPam inhibited function upstream of ß-catenin transcriptional activity and were ineffective at inhibiting cell viability. In contrast, PRI724 and SM04690 indirectly inhibited ß-catenin transcriptional activity and significantly reduced cell viability in CTNNB1-mutant cell lines. Treatment with SM04690 reduced cell viability (Licor Cell stain) in all EC cell lines, but viability was significantly lower in CTNNB1-mutant cell lines (p < 0.01). Mechanistically, SM04690 significantly inhibited proliferation measured via 5'-bromo-2'-deoxyuridine incorporation and reduced T cell factor (TCF) transcriptional activity. HEC1B, HEC1B-S33Y and HEC265 tumor-bearing mice were treated with vehicle or SM04690. Tumors treated with SM04690 had smaller mean volumes than those treated with vehicle (p < 0.001, p = 0.014, p = 0.06). In HEC1B-S33Y and HEC265 tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (p = 0.035, p = 0.024). Targeting the Wnt/ß-catenin pathway in CTNNB1-mutant EC effectively inhibited proliferation and ß-catenin/TCF transcriptional activity and blunted tumor progression in in vivo models. These studies suggest ß-catenin transcriptional inhibitors are effective in EC and particularly in CTNNB1-mutant EC, highlighting a potential therapeutic vulnerability for treatment of CTNNB1-mutant EC.

Identification of a novel diagnostic gene expression signature to discriminate uterine leiomyoma from leiomyosarcoma.

Leiomyosarcomas are rare, aggressive tumors, which exhibit a poor prognosis regardless of stage. Pre-operative diagnosis can be difficult as leiomyosarcoma can mimic features of the more common, benign uterine leiomyoma. The goal of this study was to identify specific molecular markers to discriminate between uterine leiomyosarcomas and leiomyomas to facilitate timely, accurate diagnosis and treatment. Gene expression profiles of three leiomyosarcomas, leiomyomas, and normal myometrial tissue samples were analyzed using the Affymetrix Human Gene 1.0 ST Array. GC-robust multiarray average calculation and ANOVA statistical testing were used to identify differentially expressed genes. Sixty genes, with functional roles in tumor progression or suppression, exhibited divergent expression profiles in leiomyosarcomas and leiomyomas, compared to normal myometrium. Differential RNA and protein levels of seven genes, with the most discriminatory expression patterns, were confirmed by RTPCR and immunohistochemistry in an additional 10 leiomyosarcoma and 20 leiomyoma independent samples. CHI3L1, MELK, PRC1, TOP2A, and TPX2 were overexpressed in leiomyosarcomas, while HPGD and TES were overexpressed in leiomyomas. Distinguishing leiomyosarcomas from leiomyomas represents a diagnostic challenge, particularly in the context of minimally invasive surgery. The unique gene expression signatures identified in this study may accurately differentiate between these tumor types at the earliest stage and provides potential prognostic factors and novel therapeutic targets for the treatment of leiomyosarcoma.

Evidence for disruption of normal circadian cortisol rhythm in women with obesity.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis may play a role in the pathogenesis of comorbidities encountered in obesity, including the relative hypogonadotropic hypogonadism that we and others have observed. We sought to examine serum cortisol profiles throughout the day and evening in a sample of normal weight women and women with obesity. In this cross-sectional study, regularly cycling obese (n = 12) and normal weight (n = 10) women were recruited. Mean serum cortisol was measured by frequent blood sampling for 16 h (8am-midnight) in the luteal phase of the menstrual cycle. Women with obesity had significantly higher overall cortisol levels when compared to normal weight women (6.2 [4.3, 6.6] vs. 4.7 [3.7, 5.5] ug/dl, p = .04). Over the two-hour postprandial period, obese women displayed an almost two-fold greater (7.2 [6.5, 8.6] ug/dl) rise in cortisol than normal weight controls (4.4 [3.7, 6.2] ug/dl, p < .01). In addition, obese women demonstrated a sustained evening cortisol elevation compared to normal weight women, who displayed the typical decline in cortisol (3.2 [2.3, 4] vs. 2 [1.5, 3.2] ug/dl, p < .05). Changes in the HPA axis in the setting of obesity may be related to risks of obesity-associated metabolic comorbidities and reproductive dysfunction often seen in these women.

The phosphatidyl inositol 3 kinase pathway does not suppress activation of the ARF and BIM genes by deregulated E2F1 activity.

The transcription factor E2F plays crucial roles in tumor suppression by activating pro-apoptotic genes such as the tumor suppressor ARF. The regulation of the ARF gene is distinct from that of growth-related E2F targets, in that it is specifically activated by deregulated E2F activity, induced by over-expression of E2F or forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. The phosphatidyl inositol 3 kinase (PI3K) pathway was reported to suppress expression of some atypical pro-apoptotic genes by over-expressed E2F1. However, the effects of the PI3K pathway on the distinct regulation of typical pro-apoptotic E2F targets have not been elucidated. We examined whether the PI3K pathway suppressed activation of the typical pro-apoptotic E2F targets ARF and BIM. Activation of the PI3K pathway by growth stimulation or introduction of a constitutively active Akt/PKB did not reduce induction of ARF or BIM gene expression or activation of their promoters by over-expressed E2F1. These results suggest that the PI3K pathway does not suppress induction of typical pro-apoptotic genes that are selectively activated by deregulated E2F1.

Ectopic expression of the CDK inhibitor p21Cip1 enhances deregulated E2F activity and increases cancer cell-specific cytotoxic gene expression mediated by the ARF tumor suppressor promoter.

In cancer treatment, specifically targeting cancer cells is important for optimal therapeutic efficacy. One strategy is to utilize a cancer specific promoter to express a cytotoxic gene or a viral gene required for replication. In this approach, the therapeutic window is dependent on the relative promoter activity in cancer cells versus normal cells. Therefore, a promoter with optimal cancer cell-specificity should be used. The tumor suppressor ARF promoter, which specifically responds to deregulated E2F activity, is a potent candidate. Defects in the RB pathway resulting in deregulated E2F activity are observed in almost all cancers. Furthermore, the ARF promoter exhibits greater cancer cell specificity than the E2F1 promoter and consequently, adenovirus expressing HSV-TK under the control of the ARF promoter (Ad-ARF-TK) has more selective cytotoxicity in cancer cells than the analogous E2F1 construct. Ideally, cancer specific gene expression driven by the ARF promoter could be enhanced for optimal therapeutic efficacy, with minimal side effects. We show here that ectopic expression of the CDK inhibitor p21Cip1 enhanced deregulated E2F activity and pro-apoptotic E2F target gene expression in cancer cells. Moreover, ectopic expression of p21Cip1 augmented cancer specific cytotoxicity of Ad-ARF-TK, and apoptosis induced by p21Cip1 was dependent on deregulated E2F activity. These results suggest that p21Cip1 specifically enhances deregulated E2F activity and that a combination of the CDK inhibitor with Ad-ARF-TK could be effectively employed for cancer therapy.

Identification of novel target genes specifically activated by deregulated E2F in human normal fibroblasts.

The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth-related genes but also in tumor suppression by activating pro-apoptotic and growth-suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27(Kip1) and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth-related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F-responsive promoter elements and to contribute to E2F1-mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function.

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs.

In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.

Luteal phase dynamics of follicle-stimulating and luteinizing hormones in obese and normal weight women.

OBJECTIVES: Female obesity is a state of relative hypogonadotrophic hypogonadism. The aim of this study is to examine gonadotrophin secretion and response to gonadotrophin-releasing hormone (GnRH) in the luteal phase of the menstrual cycle and to investigate the pharmacodynamics and pharmacokinetics of endogenous and exogenous luteinizing hormone (LH) in obese women. DESIGN: Participants underwent a luteal phase frequent blood sampling study. Endogenous LH pulsatility was observed, gonadotrophin-releasing hormone (GnRH) was given in two weight-based doses, and GnRH antagonist was administered followed by recombinant LH. PATIENTS: Regularly menstruating obese (n = 10) and normal weight (n = 10) women. MEASUREMENTS: Endogenous hypothalamic-pituitary function (as measured by LH pulsatility), pituitary sensitivity (GnRH-induced LH secretion), pharmacodynamics of endogenous LH and pharmacokinetics of exogenous LH were compared between the obese and normal weight groups. RESULTS: There were no statistically significant differences in endogenous LH pulsatility or pituitary responses to two weight-based doses of GnRH between the obese and normal weight women. There were no differences in the pharmacodynamics of endogenous LH or the pharmacokinetics of exogenous LH between the groups. FSH dynamics did not differ between the groups throughout the study. CONCLUSIONS: The relative hypogonadotrophic hypogonadism of obesity cannot be explained by differences in LH and FSH luteal phase dynamics or differences in endogenous LH pharmacodynamics or exogenous LH pharmacokinetics.

Acceptability and Feasibility of Initiating a Low Fat Eating Plan in Reproductive Aged Women with Obesity.

Adverse effects of obesity on reproduction are believed in part due to diet related factors leading to hyperlipidemia and hyperinsulinemia. It is unknown whether administration of a low fat eating plan, regardless of weight loss, will improve reproductive axis function in women with obesity. To develop an acceptable and feasible low fat eating plan for a diverse group of reproductive aged women with obesity. Focus groups to determine preferences and barriers to a planned dietary intervention providing very low fat (22% daily calories from fat) eucaloric food to control fat exposure, but not cause weight loss. Logistics of the intervention and monitoring over three menstrual cycles were discussed. Eighteen women enrolled into 4 different focus groups both live and video, 2 at the University of Colorado and 2 at the Morehouse School of Medicine. All participants expressed interest in implementing a low fat dietary intervention and were further interested in instruction on how to maintain healthy eating habits for future fertility. Provision of ethnically appropriate foods, social support to avoid lapses, and tasty alternatives to high fat foods were considered ideal aspects of a feasible intervention. Incentives and graduated compensation for adherence were considered desirable features. Women with obesity are interested in implementing dietary interventions that may improve their health and fertility. Given the diversity of responses based upon the demographics of our sample, it is important to assess geographical and cultural preferences prior to implementing of a dietary strategy.

Analysis of Inflammatory Markers in Response to Induction of Reprometabolic Syndrome by a Eucaloric High Fat Diet in Normal Weight Women.

Obesity is associated with chronic low-level inflammation and is known to contribute to metabolic dysfunction and hypogonadotropic hypogonadism, which we have previously termed the 'Reprometabolic Syndrome.' To investigate potential factors involved in obesity-related reproductive endocrine dysfunction, we conducted a secondary analysis of inflammatory markers in a sample of normal weight women exposed to a one-month eucaloric high-fat diet (HFD), which, as reported earlier, induced the relative hypogonadotropic hypogonadism characteristic of Reprometabolic Syndrome. Eighteen healthy women with a BMI between 18.0-24.9 kg/m2 and regular menstrual cycles participated in the study. Frequent blood sampling was performed during the early follicular phase before and after the one-month eucaloric HFD intervention (48% of calories from fat). Serum samples pooled from each participant were analyzed using immunoassay to measure levels of 30 cytokines, interleukins, and chemokines. Differences between pre- and post-HFD intervention measures were examined by one-sample t-tests. Exposure to the eucaloric HFD did not result in changes in body weight. HFD-induction of Reprometabolic Syndrome in normal weight women was associated with a significant elevation only in the anti- inflammatory cytokine IL-10 (p = 0.04). Eotaxin, IL-6 and MIP-1ß also increased in response to the HFD, but not statistically significantly (p = 0.07). Results suggest that the increase in multiple inflammatory markers, typically associated with obesity, are not primary mediators of the relative hypogonadotropic hypogonadism of Reprometabolic Syndrome. Clinical Trials Registration Number: NCT02653092; Date of Registration: January 6, 2016.

Activation of the CDK7 Gene, Coding for the Catalytic Subunit of the Cyclin-Dependent Kinase (CDK)-Activating Kinase (CAK) and General Transcription Factor II H, by the Trans-Activator Protein Tax of Human T-Cell Leukemia Virus Type-1.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

A high-fat eucaloric diet induces reprometabolic syndrome of obesity in normal weight women.

We examined the effects of 1 month of a eucaloric, high-fat (48% of calories) diet (HFD) on gonadotropin secretion in normal-weight women to interrogate the role of free fatty acids and insulin in mediating the relative hypogonadotropic hypogonadism of obesity. Eighteen eumenorrheic women (body mass index [BMI] 18-25 kg/m2) were studied in the early follicular phase of the menstrual cycle before and after exposure to an HFD with frequent blood sampling for luteinizing hormone (LH) and follicle-stimulating hormone (FSH), followed by an assessment of pituitary sensitivity to gonadotropin-releasing hormone (GnRH). Mass spectrometry-based plasma metabolomic analysis was also performed. Paired testing and time-series analysis were performed as appropriate. Mean endogenous LH (unstimulated) was significantly decreased after the HFD (4.3 ± 1.0 vs. 3.8 ± 1.0, P < 0.01); mean unstimulated FSH was not changed. Both LH (10.1 ± 1.0 vs. 7.2 ± 1.0, P < 0.01) and FSH (9.5 ± 1.0 vs. 8.8 ± 1.0, P < 0.01) responses to 75 ng/kg of GnRH were reduced after the HFD. Mean LH pulse amplitude and LH interpulse interval were unaffected by the dietary exposure. Eucaloric HFD exposure did not cause weight change. Plasma metabolomics confirmed adherence with elevation of fasting free fatty acids (especially long-chain mono-, poly-, and highly unsaturated fatty acids) by the last day of the HFD. One-month exposure to an HFD successfully induced key reproductive and metabolic features of reprometabolic syndrome in normal-weight women. These data suggest that dietary factors may underlie the gonadotrope compromise seen in obesity-related subfertility and therapeutic dietary interventions, independent of weight loss, may be possible.

Effectiveness of preconception weight loss interventions on fertility in women: a systematic review and meta-analysis.

IMPORTANCE: Weight loss before conception is recommended for women with overweight or obesity to improve fertility outcomes, but evidence supporting this recommendation is mixed. OBJECTIVE: To examine the effectiveness of weight loss interventions using lifestyle modification and/or medication in women with overweight or obesity on pregnancy, live birth, and miscarriage. DATA SOURCES: An electronic search of MEDLINE, Embase, Cochrane Library, including Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials, and Cumulative Index to Nursing and Allied Health Literature was conducted through July 6, 2022, via Wiley. STUDY SELECTION AND SYNTHESIS: Randomized controlled trials examining weight loss interventions through lifestyle and/or medication in women with overweight or obesity planning pregnancy were included. Random-effects meta-analysis was conducted, reporting the risk ratio (RR) for each outcome. Subgroup analyses were conducted by intervention type, type of control group, fertility treatment, intervention length, and body mass index (BMI). MAIN OUTCOME(S): Clinical pregnancy, live birth, and miscarriage events. RESULT(S): A narrative review and meta-analysis were possible for 16 studies for pregnancy (n = 3,588), 13 for live birth (n = 3,329), and 11 for miscarriage (n = 3,248). Women randomized and exposed to a weight loss intervention were more likely to become pregnant (RR = 1.24, 95% CI 1.07-1.44; I2 = 59%) but not to have live birth (RR = 1.19, 95% CI 0.97-1.45; I2 = 69%) or miscarriage (RR = 1.17, 95% CI 0.79-1.74; I2 = 31%) compared with women in control groups. Subgroup analyses revealed women randomized to weight loss interventions lasting 12 weeks or fewer (n = 9, RR = 1.43; 95% CI 1.13-1.83) and women with a BMI ≥ 35 kg/m2 (n = 7, RR = 1.54; 95% CI, 1.18-2.02) were more likely to become pregnant compared with women in the control groups. Miscarriage was higher in intervention groups who underwent fertility treatment (n = 8, RR 1.45; 95% CI 1.07-1.96). CONCLUSION(S): Pregnancy rates were higher in women undergoing preconception weight loss interventions with no impact on live birth or miscarriage rates. Findings do not support one-size-fits-all recommendation for weight loss through lifestyle modification and/or medication in women with overweight or obesity immediately before conception to improve live birth or miscarriage outcomes.

Characterization of the uterine leiomyoma microRNAome by deep sequencing.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, which are negative regulators of gene expression. Many genes in human uterine leiomyoma (ULM) are aberrantly expressed and in some cases this can be due to dysregulation of miRNAs. Here we present the first study to determine genome-wide miRNA expression patterns in uterine leiomyoma and myometrium using Solexa high-throughput sequencing. We found more than 50 miRNAs, which were differentially expressed, and furthermore we extend the list of putative new miRNA genes. The top five significantly de-regulated miRNAs in ULMs that we found in our libraries were miR-363, miR-490, miR-137, miR-217 and miR-4792. We also observed "isomiRs" with higher copy number than referenced mature miRNA specific for the leiomyoma libraries, which have a potential role in tumorigenesis. The microRNA transcriptomes obtained in this study deliver insights and further expand our understanding the role of small RNAs in uterine leiomyoma development.

Expanding Roles of the E2F-RB-p53 Pathway in Tumor Suppression.

The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology.

Deregulated E2F Activity as a Cancer-Cell Specific Therapeutic Tool.

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells.

Effects of pulsatile intravenous follicle-stimulating hormone treatment on ovarian function in women with obesity.

OBJECTIVE: To establish conditions for effective hypothalamic suppression in women with normal and high body mass index (BMI) and test the hypothesis that intravenous (IV) administration of pulsatile recombinant follicle-stimulating hormone (rFSH) can overcome the clinically evident dysfunctional pituitary-ovarian axis in women with obesity. DESIGN: Prospective interventional study. SETTING: Academic medical center. PATIENT(S): Twenty-seven normal-weight women and 27 women with obesity, who were eumenorrheic and aged 21-39 years. INTERVENTION(S): Two-day frequent blood sampling study, in early follicular phase, before and after cetrorelix suppression of gonadotropins and exogenous pulsatile IV rFSH administration. MAIN OUTCOME MEASURE(S): Serum inhibin B and estradiol (E2) levels (basal and rFSH stimulated). RESULT(S): A modified gonadotropin-releasing hormone antagonism protocol effectively suppressed production of endogenous gonadotropins in women with normal and high BMIs, providing a model to address the functional role of FSH in the hypothalamic-pituitary-ovarian axis. The IV rFSH treatment resulted in equivalent serum levels and pharmacodynamics in normal-weight women and those with obesity. However, women with obesity exhibited reduced basal levels of inhibin B and E2 and a significantly decreased response to FSH stimulation. The BMI was inversely correlated with serum inhibin B and E2. In spite of this observed deficit in ovarian function, pulsatile IV rFSH treatment in women with obesity resulted in E2 and inhibin B levels comparable with those in normal-weight women, in the absence of exogenous FSH stimulation. CONCLUSION(S): Despite normalization of FSH levels and pulsatility by exogenous IV administration, women with obesity demonstrate ovarian dysfunction with respect to E2 and inhibin B secretion. Pulsatile FSH can partially correct the relative hypogonadotropic hypogonadism of obesity, thereby providing a potential treatment strategy to mitigate some of the adverse effects of high BMI on fertility, assisted reproduction, and pregnancy outcomes. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov #NCT02478775.

Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways.

INTRODUCTION: Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. METHODS: We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. RESULTS: High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-ß) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers. CONCLUSIONS: Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-ß and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.