Gynecology

The tumor suppressor gene p53 has been implicated in the regulation of epithelial-mesenchymal transition (EMT) and tumor metastasis by regulating microRNA (miRNA) expression. Here, we report that mutant p53 exerts oncogenic functions and promotes EMT in endometrial cancer (EC) by directly binding to the promoter of miR-130b (a negative regulator of ZEB1) and inhibiting its transcription. We transduced p53 mutants into p53-null EC cells, profiled the miRNA expression by miRNA microarray and identified miR-130b as a potential target of mutant p53. Ectopic expression of p53 mutants repressed the expression of miR-130b and triggered ZEB1-dependent EMT and cancer cell invasion. Loss of an endogenous p53 mutation increased the expression of miR-130b, which resulted in reduced ZEB1 expression and attenuation of the EMT phenotype. Furthermore, re-expression of miR-130b suppressed mutant p53-induced EMT and ZEB1 expression. Importantly, the expression of miR-130 was significantly reduced in EC tissues, and patients with higher expression levels of miR-130b survived longer. These data provide a novel understanding of the roles of p53 gain-of-function mutations in accelerating tumor progression and metastasis through modulation of the miR-130b-ZEB1 axis.Oncogene advance online publication, 30 July 2012; doi:10.1038/onc.2012.334.

IQGAP1 is a multifunctional protein involved in actin cytoskeleton assembly and E-cadherin-mediated cell adhesion. To determine the role of IQGAP1 in ovarian tumors, we evaluated IQGAP1 expression by immunohistochemistry in 17 adenomas, 30 borderline tumors and 80 adenocarcinomas and its relation with patient survival. IQGAP1 was overexpressed in adenocarcinomas compared with adenomas and borderline tumors. Enhanced immunostaining in invasive tumor fronts was categorized as focal or diffuse. The diffuse expression pattern correlated with high histological grade and clinicopathological stages. IQGAP1 overexpression and diffuse invasion pattern were significantly associated with poor prognosis by multivariate analysis. Our findings suggest the involvement of IQGAP1 in the progression and spread of ovarian adenocarcinomas. Overexpression and diffuse expression pattern of IQGAP1 are potentially useful independent molecular predictors of highly aggressive ovarian carcinomas.

BACKGROUND:

Epithelial-mesenchymal transition (EMT) is the key process driving cancer metastasis. Oncogene/self renewal factor BMI-1 has been shown to induce EMT in cancer cells. Recent studies have implied that noncoding microRNAs (miRNAs) act as crucial modulators for EMT. The aims of this study was to determine the roles of BMI-1 in inducing EMT of endometrial cancer (EC) cells and the possible role of miRNA in controlling BMI-1 expression.

METHODS AND RESULTS:

We evaluated the expression of BMI-1 gene in a panel of EC cell lines, and detected a strong association with invasive capability. Stable silencing of BMI-1 in invasive mesenchymal-type EC cells up-regulated the epithelial marker E-cadherin, down-regulated mesenchymal marker Vimentin, and significantly reduced cell invasion in vitro. Furthermore, we discovered that the expression of BMI-1 was suppressed by miR-194 via direct binding to the BMI-1 3'-untranslated region 3'-UTR). Ectopic expression of miR-194 in EC cells induced a mesenchymal to epithelial transition (MET) by restoring E-cadherin, reducing Vimentin expression, and inhibiting cell invasion in vitro. Moreover, BMI-1 knockdown inhibited in vitro EC cell proliferation and clone growth, correlated with either increased p16 expression or decreased expression of stem cell and chemoresistance markers (SOX-2, KLF4 and MRP-1).

CONCLUSION:

These findings demonstrate the novel mechanism for BMI-1 in contributing to EC cell invasion and that repression of BMI-1 by miR-194 could have a therapeutic potential to suppress EC metastasis.

Dominant negative (DN) mutations of tumor suppressor p53 (TP53) are clinically associated with cancer progression and metastasis of endometrial malignancy. To investigate the DN effect on tumor migration and invasion, we generated cells that stably co-expressed wild-type (wt) and R273H DN mutant TP53 (273H cells), and wt and R213Q recessive mutant TP53 (213Q cells), by transfection in endometrial cancer cells HHUA that expressed wt p53. R273H, but not R213Q, repressed wt p53-stimulated transcription of p21, Bax, and MDM2. 273H cells also showed markedly increased in vitro invasion and migration potentials, and displayed reduced Maspin, PAI-1, and KAI1 mRNA expressions as compared with 213Q and wt cells. The induction of wt p53 function by use of Adriamycin resulted in the inhibition of the invasion/migration capacity in association with the up-regulation of p53 target genes to a far greater degree in 213Q and wt cells than in 273H cells. R273H expression in p53-null cancer cell SK-OV-3 and Saos-2 did not significantly affect cell invasion and migration activities. Taken together, these results suggest that transdominance of R273H mutant over wt p53 rather than a gain-of-function promotes tumor metastasis by increasing invasion and migration in HHUA cells.

BACKGROUND:

p53 is the most commonly mutated tumor suppressor gene in human cancers. In addition to the loss of tumor suppression function and exertion of dominant-negative effects over the remaining wild-type protein, several p53 mutants can gain novel oncogenic functions (gain-of-function, GOF) that actively regulate cancer development and progression. In human endometrial cancer, p53 mutation is more often associated with aggressive nonendometrioid cancer. However, it was unknown if p53 mutants contributed to endometrial cancer progression through the GOF properties.

METHODS:

To clarify the relationship between expression of p53 GOF mutation (p53-R175H) and invasive potential of human endometrial cancer KLE cells, we tested the consequences of up-regulation and down-regulation of p53-R175H in KLE cells by inducing p53-R175H expression vector or suppressing the p53 gene with short hairpin RNA.

RESULTS:

We found that forced over-expression of p53-R175H significantly promoted cell migration and invasion, and induced activation of the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Conversely, suppression of p53-R175H with short hairpin RNA significantly inhibited cell migration and invasion, and resulted in attenuation of EGFR/PI3K/AKT pathway.

CONCLUSION:

These findings show for the first time that elevated expression of p53-R175H mutant may exert gain-of-function activity to activate the EGFR/PI3K/AKT pathway and thus may contribute to the invasive phenotype in endometrial cancer.

AIMS:

Oxidative stress and fibrosis is implicated in cardiac remodeling and failure. We tested whether allopurinol could decrease myocardial oxidative stress and attenuate cardiac fibrosis and left ventricular diastolic dysfunction in angiotensin II (AngII)-induced hypertensive mice.

METHODOLOGY:

We used 8-week-old male C57BL/6J mice, in which angiotensin II was subcutaneously infused for 4 weeks to mimic cardiac remodeling and fibrosis. They were treated with either normal saline or allopurinol in daily doses, which did not lower blood pressure.

RESULTS:

Allopurinol improved diastolic dysfunction in angiotensin II-induced hypertensive mice, which was associated with the amelioration of cardiac fibrosis. However, allopurinol showed no effect on the increased systolic blood pressure by angiotensin II infusion. The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) [GSH/GSSG] was decreased and malondialdehyde levels were increased in the hearts of AngII-treated mice. Allopurinol also inhibited both the decrease in the GSH/GSSG ratio and the increase in malondialdehyde levels in the heart. Infusion of AngII-induced upregulation of transfer growth factor (TGF)-β1, Smad3 expression and downregulation of Smad7 expression. Treatment with allopurinol reduced cardiac levels of TGF-β1, Smad3, and increased Smad7 expression.

CONCLUSIONS:

These results suggest that allopurinol prevents pathological remodeling of the heart in AngII-induced hypertensive mice. The antioxidative effect of allopurinol contributes to the regression of AngII-induced cardiac diastolic dysfunction. These effects of allopurinol to prevent cardiac fibrosis are mediated at least partly through modulation of the TGF-β1/Smad signaling pathway.

1. Granulocyte colony stimulating factor (G-CSF) is reported to have a beneficial effect on cardiac dysfunction in postinfarction and doxorubicin-induced cardiomyopathy. Thus, the aim of the present study was to investigate the effects of G-CSF on cardiac remodelling in angiotensin (Ang) II-induced hypertrophy. 2. Four groups of mice were investigated. The first group served as a control group. The second group was injected with recombinant human G-CSF (10 microg/kg per day, s.c.) on the first 5 days of each week and treatment was continued for 4 weeks. An osmotic minipump was implanted subcutaneously into each mouse in the third group so that pressor doses of AngII (2.88 mg/kg per day) or saline could be administered over a period of 4 weeks. The fourth group was infused with AngII (2.88 mg/kg per day) and injected with G-CSF (10 microg/kg per day, s.c.) for 4 weeks. 3. Angiotensin II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in mice. Treatment of mice with G-CSF did not reduce the AngII-induced increase in blood pressure, but ameliorated the development of cardiac fibrosis and hypertrophy. Infusion of AngII induced upregulation of angiotensin-converting enzyme (ACE) expression and downregulation of ACE2 expression. Treatment with G-CSF reduced cardiac levels of ACE and increased ACE2 expression. In addition, G-CSF treatment reduced the expression of osteopontin (OPN) and phospho-p70S6 kinase, which were upregulated by AngII infusion. 4. These results suggest that G-CSF reduces AngII-induced hypertrophy. Modulation of the expression of the ACE isoforms contributes to regression of AngII-induced cardiac hypertrophy. The effect of G-CSF to prevent cardiac fibrosis and hypertrophy may be mediated, in part, via inhibition of OPN expression and p70S6 kinase phosphorylation.

Background and ObjectivesThe aim of this study was to analyze the stage migration and survival of endometrial cancer by the revised FIGO 2008 staging system compared with the 1988 staging system.The aim of this study was to analyze the stage migration and survival of endometrial cancer by the revised FIGO 2008 staging system compared with the 1988 staging system.MethodsA total of 355 patients with endometrial cancer, who underwent complete surgical staging, were enrolled. We compared the surgical stages and survival by FIGO 1988 staging system with those by FIGO 2008 staging system.A total of 355 patients with endometrial cancer, who underwent complete surgical staging, were enrolled. We compared the surgical stages and survival by FIGO 1988 staging system with those by FIGO 2008 staging system.Results2008 FIGO staging system resulted in an increase of stage I patients and decrease of stage II and IIIa patients. The 5-year overall survival (OS) rates for patients with 2008 FIGO stage IA and IB disease were 98.2% and 91.9%, respectively (P = 0.004). Five-year OS rate of new stage II (82.6%) was significantly worse than that of new stage IA (98.2%, P = 0.003). Patients with positive washing cytology alone revealed a 5-year OS rate similar to that of patients with new stage IIIA disease (96.2% vs. 90.9%, respectively; P = 0.53). The 5-year OS rate for patients with stage IIIC1 disease was improved compared with that for patients with stage IIIC2 disease (85.7% vs. 63.0%, respectively; P = 0.08).2008 FIGO staging system resulted in an increase of stage I patients and decrease of stage II and IIIa patients. The 5-year overall survival (OS) rates for patients with 2008 FIGO stage IA and IB disease were 98.2% and 91.9%, respectively (P = 0.004). Five-year OS rate of new stage II (82.6%) was significantly worse than that of new stage IA (98.2%, P = 0.003). Patients with positive washing cytology alone revealed a 5-year OS rate similar to that of patients with new stage IIIA disease (96.2% vs. 90.9%, respectively; P = 0.53). The 5-year OS rate for patients with stage IIIC1 disease was improved compared with that for patients with stage IIIC2 disease (85.7% vs. 63.0%, respectively; P = 0.08).ConclusionNew revised FIGO 2008 staging system for endometrial cancer produced better discrimination in OS outcomes compared with the 1988 system. J. Surg. Oncol. 2012; 106: 938–941. © 2012 Wiley Periodicals, Inc.New revised FIGO 2008 staging system for endometrial cancer produced better discrimination in OS outcomes compared with the 1988 system. J. Surg. Oncol. 2012; 106: 938–941. © 2012 Wiley Periodicals, Inc.

Endometrial cancer (EC) is the leading malignant tumor occurring in the female genital tract and some subtypes are highly invasive and metastatic. miRNAs are small non-coding RNAs that have a broad impact on cancer progression. In particular, miR-194 regulates epithelial to mesenchymal transition (EMT) by suppressing the expression of BMI-1 in EC. In this retrospective study, the clinical significance of miR-194 was investigated in archival EC specimens. We extracted total RNA from thirty-two EC samples and quantified the expression level of miR-194. We discovered that the expression level of miR-194 was significantly (P = 0.03) lower in type I EC patients with more advanced stage. In addition, patients with higher miR-194 levels have better prognosis than those with lower miR-194 levels (P = 0.0067; Cut-off value of miR-194 = 0.3). These results indicate that miR-194 has potential to serve as prognostic biomarker for EC patients.

Krüppel-like factor 17 (KLF17), a member of the KLF transcription factor family, has been shown to inhibit the epithelial-mesenchymal transition (EMT) and tumor growth. However, the expression, the cellular function and the mechanism of KLF17 in endometrioid endometrial cancer (EEC; a dominant type of endometrial cancer) remain elusive. Here, we report that among the KLF family members, KLF17 was consistently upregulated in EEC cell lines compared with immortalized endometrial epithelial cells. Overexpression of KLF17 in EEC cell lines induced EMT and promoted cell invasion and drug resistance, resulting in increased expression of TWIST1. In contrast, KLF17 suppression reversed EMT, diminished cell invasion, restored drug sensitivity and suppressed TWIST1 expression. Luciferase assays, site-directed mutagenesis and transcription factor DNA-binding analysis demonstrated that KLF17 transactivates TWIST1 expression by directly binding to the TWIST1 promoter. Knockdown of TWIST1 prevented KLF17-induced EMT. Consistent with these results, both KLF17 and TWIST1 levels were found to be elevated in EECs compared with normal tissues. KLF17 expression positively correlated with tumor grade but inversely correlated with estrogen and progesterone receptor expression. Thus, KLF17 may have an oncogenic role during EEC progression via initiating EMT through the regulation of TWIST1.

Purpose The aim of this study was to demonstrate the precise mapping of lymph node metastasis (LNM) sites in endometrial cancer. Methods A total of 266 patients who underwent primary radical surgery including systematic pelvic and para-aortic lymphadenectomy for endometrial cancer from 1993 to 2010 were enrolled in this study. We removed lymph nodes from the femoral ring to the para-aortic node up to the level of renal veins. We analyzed the distribution of positive-node sites according to their anatomical location. Results Overall, 42 of 266 patients (15.8 %) showed LNM. The median number of nodes harvested was 62.5 (range 40–119) in pelvic nodes (PLN), and 20 (range 3–47) in para-aortic nodes (PAN). Among 42 cases with positive-nodes, 16 cases (38.1 %) showed positive PLN alone, 7 cases (16.7 %) in PAN alone, and 19 cases (45.2 %) in both PLN and PAN. The most prevalent site of positive-nodes was PAN (9.8 %) followed by obturator nodes (9.4 %), internal iliac nodes (7.1 %), and common iliac nodes (5.6 %). Six of 19 cases (31.6 %) of positive PAN above the inferior mesenteric artery (IMA) showed negative PAN below IMA. Metastasis to the deep inguinal nodes was found to be extremely rare (0.38 %). Single-site LNM was the most frequently observed in obturator nodes, followed by PAN above IMA. Conclusion Routine resection of deep inguinal nodes is not recommended, whereas para-aortic lymphadenectomy should be extended up to the level of renal veins for endometrial cancer.

Type II endometrial carcinoma is an aggressive subtype of endometrial cancer (EC). TWIST1, a helix-loop-helix transcription regulator, is known to induce epithelial–mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) also serve as important regulators of EMT and metastasis by regulating EMT-related genes. In this study, we sought to explore the role of TWIST1 in inducing EMT in representative type II EC cell lines, and to determine the miRNAs involved in regulating TWIST1 gene expression. Functional analysis suggested that TWIST1 contributes to the EMT phenotypes of EC cells, as evidenced by the acquisition of fibroblast-like properties, enhanced invasiveness, and induction of an EN-switch (downregulation of epithelial marker E-cadherin and upregulation of mesenchymal marker N-cadherin). Conversely, silencing of TWIST1 by siRNA inhibited cell invasion and the mesenchymal phenotype, which was accompanied by a reversion of the EN-switch. We also observed a novel post-transcriptional regulatory mechanism of TWIST1 expression mediated by miR-106b via its direct interaction with TWIST1 mRNAs at the 3′-untranslated region. Our data suggest that TWIST1 is a critical inducer of EMT in invasive EC cells and that miR-106b could suppress EC cell invasion by downregulating TWIST1 expression. © 2013 Wiley Periodicals, Inc.Type II endometrial carcinoma is an aggressive subtype of endometrial cancer (EC). TWIST1, a helix-loop-helix transcription regulator, is known to induce epithelial–mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) also serve as important regulators of EMT and metastasis by regulating EMT-related genes. In this study, we sought to explore the role of TWIST1 in inducing EMT in representative type II EC cell lines, and to determine the miRNAs involved in regulating TWIST1 gene expression. Functional analysis suggested that TWIST1 contributes to the EMT phenotypes of EC cells, as evidenced by the acquisition of fibroblast-like properties, enhanced invasiveness, and induction of an EN-switch (downregulation of epithelial marker E-cadherin and upregulation of mesenchymal marker N-cadherin). Conversely, silencing of TWIST1 by siRNA inhibited cell invasion and the mesenchymal phenotype, which was accompanied by a reversion of the EN-switch. We also observed a novel post-transcriptional regulatory mechanism of TWIST1 expression mediated by miR-106b via its direct interaction with TWIST1 mRNAs at the 3′-untranslated region. Our data suggest that TWIST1 is a critical inducer of EMT in invasive EC cells and that miR-106b could suppress EC cell invasion by downregulating TWIST1 expression. © 2013 Wiley Periodicals, Inc.

Although clinical trials of molecular therapies targeting critical biomarkers (mTOR, epidermal growth factor receptor/epidermal growth factor receptor 2, and vascular endothelial growth factor) in endometrial cancer show modest effects, there are still challenges that might remain regarding primary/acquired drug resistance and unexpected side effects on normal tissues. New studies that aim to target both genetic and epigenetic alterations (noncoding microRNA) underlying malignant properties of tumor cells and to specifically attack tumor cells using cell surface markers overexpressed in tumor tissue are emerging. More importantly, strategies that disrupt the cancer stem cell/epithelial-mesenchymal transition-dependent signals and reactivate antitumor immune responses would bring new hope for complete elimination of all cell compartments in endometrial cancer. We briefly review the current status of molecular therapies tested in clinical trials and mainly discuss the potential therapeutic candidates that are possibly used to develop more effective and specific therapies against endometrial cancer progression and metastasis.

0 0.2 0.4 0.6 0.8 1 1.2 1 2 3 4 0 0.2 0.4 0.6 0.8 1 1.2 1 2 3 4 9.3 6.8 0 hsa-let-7b hsa-miR-26b hsa-miR-199a hsa-miR-27b hsa-miR-143 hsa-miR-194 hsa-miR-106b hsa-miR-301 hsa-miR-130b hsa-miR-30e-5p hsa-miR-424 hsa-miR-191* hsa-miR-369-5p hsa-miR-431 hsa-miR-433 hsa-miR-329 hsa-miR-451 hsa-miR-409-3p hsa-miR-146b hsa-miR-193b hsa-miR-512-5p hsa-miR-519e* hsa-miR-656 A Vector R273H R175H C135Y HEC-50 B 0 0.2 0.4 0.6 0.8 1 1.2 1 2 3 4 Vector R273H C135Y R175H Figure S4. The p53 GOF mutants contribute to global repression of miRNA expression. (A) Heat map showing the expression of miRNAs that are downregulated in mutant p53s expressing HEC-50 cells when compared with control cells. The bar code on the bottom represents the color scale of the log 2 values. Dark green denotes high signal and light green denotes low signal. (B) Relative miRNA expression (normalized to GAPDH) in HEC-50 cells transfected with mutant p53 constructs or control construct, determined by qRT-PCR (mean ± s.d.; n = 3; * P < 0.01).

Invasion assays were performed were performed using Transwell system (8-μm pore size, matrigel-coated polycarbonate membrane BD Biosciences, Bedford, MA), as described previously [Citation]. Briefly, 5 × 10 4 cells resuspended in serum-free medium was added to the upper inserts. In the lower chamber, 750 μl medium supplemented with 10% FBS served as a chemoattractant. After incubation for 24 hours, the number of cells adhering to the lower surface of membrane was counted under the microscope. Citation: Dong PX, Jia N, Xu ZJ, Liu YT, Li DJ, Feng YJ. Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro. J Exp Clin Cancer Res. 2008 Nov 27;27:77.

MicroRNA-101 has been implicated as a tumor suppressor miRNA in human tumors. However, its potential functional impact and the underlying mechanisms in endometrial cancer progression have not been determined. Here, we report that in aggressive endometrial cancer cells, re-expression of microRNA-101 leads to inhibition of cell proliferation and induction of apoptosis and senescence. Ectopic overexpression of microRNA-101 attenuates the epithelial-mesenchymal transition-associated cancer cell migration and invasion, abrogates the sphere-forming capacity and enhances chemosensitivity to paclitaxel. Algorithm and microarray-based strategies identifies potential microRNA-101 targets. Among these, we validated EZH2, MCL-1 and FOS as direct targets of miR-101 and silencing of these genes mimics the tumor suppressive effects observed on promoting microRNA-101 function. Importantly, further results suggest an inverse correlation between low miR-101 and high EZH2, MCL-1 and FOS expression in EC specimens. We conclude that, as a crucial tumor suppressor, microRNA-101 suppresses cell proliferation, invasiveness and self-renewal in aggressive endometrial cancer cells via modulating multiple critical oncogenes. The microRNA-101-EZH2/MCL-1/FOS axis is a potential therapeutic target for endometrial cancer.

Activation of the PI3K/AKT pathway, a common mechanism in all subtypes of endometrial cancers (endometrioid
and non-endometrioid tumors), has important roles in contributing to epithelial-mesenchymal transition (EMT) and
cancer stem cell (CSC) features. MicroRNAs (miRNAs) are small non-coding RNA molecules that concurrently affect
multiple target genes, and regulate a wide range of genes involved in modulating EMT and CSC properties. Here
we overview the recent advances revealing the impact of miRNAs on EMT and CSC phenotypes in tumors including
endometrial cancer via regulating PI3K/AKT pathway. MiRNAs are crucial mediators of EMT and CSC through
targeting PTEN-PI3K-AKT-mTOR axis. In endometrial cancer cells, miRNAs can activate or attenuate EMT and CSC
by targeting PTEN and other EMT-associated genes, such as Twist1, ZEB1 and BMI-1. More detailed studies of miRNAs
will deepen our understanding of the molecular basis underlying PI3K/AKT-induced endometrial cancer initiation
and progression. Targeting key signaling components of PI3K/AKT pathway by restoring or inhibiting miRNA
function holds promise as a potential therapeutic approach to suppress EMT and CSC in endometrial cancer