The interaction between C35 and ΔNp73 promotes chemo-resistance in ovarian cancer cells.

BACKGROUND: The purpose of this study was to characterise the oncogenic roles of C35, a novel protein binding partner of ΔNp73, in ovarian cancer and to investigate the functional significance of C35-ΔNp73 interaction in the regulation of chemo-resistance. METHODS: C35 expression was evaluated by quantitative real-time PCR in human ovarian cancer tissues and cell lines. The aggressiveness of ovarian cancer cells overexpressing C35 was examined by cell proliferation, migration, soft agar and nude mouse xenograft. The significance of C35-ΔNp73 interaction in chemo-resistance was evaluated by apoptosis assays and cell viability after cisplatin treatment. RESULTS: The expression of C35 was significantly enhanced in human ovarian cancer tissues. Overexpression of C35 augmented proliferation, migration and tumourigenicity in ovarian cancer cell lines. C35 knockdown inhibited cell motility and cell growth. The co-expression of C35 and ΔNp73 by transient or stable transfection in ovarian cancer cells induced greater resistance to cisplatin treatment than did transfection with C35 or ΔNp73 alone. The cisplatin resistance was demonstrated to be caused by increased AKT and NFκB activity induced by C35-ΔNp73. CONCLUSION: Our results suggest that ΔNp73 might cooperate with C35 to promote tumour progression and contribute to cisplatin resistance in ovarian cancer cells. Future studies of the functional roles of ΔNp73 and C35 will provide insight that will aid in the establishment of new strategies and more effective therapies.

Possible role of adrenomedullin in the pathogenesis of tubal ectopic pregnancy.

CONTEXT: Tubal ectopic pregnancy (tEP) is currently the leading cause of pregnancy-related deaths during the first trimester. Our current knowledge on the molecular pathogenesis is limited. OBJECTIVE: The objective of the study was to find out the possible role of adrenomedullin (ADM) in the pathogenesis of tEP. DESIGN: This was an experimental in vitro study on oviductal tissue. SETTING: The study was conducted at a university teaching hospital. PATIENTS AND INTERVENTIONS: Patients included those having oviducts removed surgically during salpingectomy for tEP or hysterectomy for benign gynecological conditions. Oviductal tissues were incubated in hormonal condition mimicking early pregnancy before used for in vitro experiments. MAIN OUTCOME MEASURES: Plasma ADM concentration, oviductal expression of ADM and its receptors, ciliary beat frequency, smooth muscle contraction were measured. RESULTS: The ciliary beat frequency and frequency of muscle contraction were lower in the oviducts from patients with tEP than those from simulated normal pregnancy. The plasma and oviductal tissue ADM levels were also lower. The decreases in ciliary beat and frequency of contraction were restored to normal after ADM treatment. CONCLUSIONS: The results suggest that the lower ADM level in the oviducts of tEP may lead to the decrease in ciliary beating and muscle contraction, with the result that the embryo is retained and implanted in the oviduct. Our findings explain for the first time the etiology of tubal pregnancy on the basis of an impairment of the transport of the fertilized ovum resulting from an ADM deficiency and raise the possibility of using the plasma ADM level as a predictor for tubal ectopic pregnancy.

P-cadherin cooperates with insulin-like growth factor-1 receptor to promote metastatic signaling of gonadotropin-releasing hormone in ovarian cancer via p120 catenin.

Gonadotropin-releasing hormone (GnRH) is a potent prometastatic factor in ovarian cancer, but the intracellular signaling events are not well understood. The classical Gα(q)-phospholipase C signal transduction pathway known to operate in the pituitary is not involved in GnRH actions at non-pituitary targets. Here we showed that GnRH treatment of ovarian cancer cells led to a rapid and remarkable tyrosine phosphorylation of p120 catenin (p120(ctn)), which was mediated by P-cadherin. The use of P-cadherin small interfering RNA or neutralizing antibodies to inhibit P-cadherin expression and function resulted in diminished p120(ctn) activation, confirming that the effect was P-cadherin specific. On exploring how P-cadherin, which lacks intrinsic kinase activity, might regulate the activation of p120(ctn), we found that P-cadherin could induce the ligand-independent activation of insulin-like growth factor-1 receptor (IGF-1R). Inhibition of IGF-1R expression or its activity significantly inhibited GnRH-induced p120(ctn) activation, and the subsequent cell migration and invasion. In addition, we showed that IGF-1R regulation by P-cadherin was associated with complex formation between IGF-1R and P-cadherin, and this regulation was also observed to be in vivo correlated with metastasis. Furthermore, using a mouse model of ovarian cancer metastasis, GnRH receptor knockdown was shown to diminish peritoneal dissemination of tumors and ascites formation. These findings suggest for the first time that GnRH can initiate an outside-in p120(ctn) signal transduction through the cross-talk between P-cadherin and IGF-1R, thus providing a novel molecular mechanism by which GnRH may control the high level of aggressiveness and invasion and metastasis potential that are characteristic of ovarian cancer.

p70 S6 kinase in the control of actin cytoskeleton dynamics and directed migration of ovarian cancer cells.

Ovarian cancer is highly metastatic with a poor prognosis. The serine/threonine kinase, p70 S6 kinase (p70(S6K)), which is a downstream effector of phosphatidylinositol 3-kinase/Akt pathway, is frequently activated in ovarian cancer. Here, we show that p70(S6K) is a critical regulator of the actin cytoskeleton in the acquisition of the metastatic phenotype. This regulation is through two important activities: p70(S6K) acts as an actin filament cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70(S6K) in ovarian cancer cells induced a marked reorganization of the actin cytoskeleton and promoted directional cell migration. Using cosedimentation and differential sedimentation assays, p70(S6K) was found to directly bind to and cross-link actin filaments. Immunofluorescence studies showed p70(S6K) colocalized with cytochalasin D-sensitive actin at the leading edge of motile cells. The p70(S6K) did not affect the kinetics of spontaneous actin polymerization, but could stabilize actin filaments by the inhibition of cofilin-induced actin depolymerization. In addition, we showed that p70(S6K) stimulated the rapid activation of both Rac1 and Cdc42, and their downstream effector p21-activated kinase (PAK1), but not RhoA. Depletion of p70(S6K) expression or inhibition of its activity resulted in significant inhibition of actin cytoskeleton reorganization and reduced migration, with a concomitant reduction in Rac1, Cdc42 and PAK1 activation, confirming that the effect was p70(S6K) specific. Similarly, the actin cytoskeleton reorganization/migratory phenotype could be reversed by expression of dominant negative Rac1 and Cdc42, or inhibition of PAK1. These results reveal a new direction for understanding the oncogenic roles of p70(S6K) in tumor progression.

Atypical glandular cells diagnosed during pregnancy and the postpartum period: a retrospective analysis.

OBJECTIVES: The objective of the study was to determine the prevalence and clinical significance of atypical glandular cells (AGC) or atypical glandular cells of undetermined significance (AGUS) diagnosed in pregnant and postpartum women. STUDY DESIGN: Smears having a diagnosis of AGC or AGUS, taken from pregnant and postpartum (within six weeks after delivery) women between 1995 and 2008 were reviewed and subclassified according to the Bethesda 2001 classification. Case records were then reviewed and a second cytology review was performed after disclosure of the follow-up data. RESULTS: Among 91,133 smears taken from pregnant and postpartum women, 70 had AGC or AGUS (0.07%) diagnosed. Follow-up data were available in 40 cases, with mean duration of follow-up being 43 months. Among the 40 patients with follow-up data, nineteen had smears with coexisting squamous abnormalities. Thirty patients had positive pathology, including 18 (45%) cervical intraepithelial neoplasia III (CIN III), four (10%) cervical adenocarcinoma-in situ, three (7.5%) squamous cell carcinoma of cervix, four (10%) condylomas and one (2.5%) hydatidiform mole. On review, 24 out of 32 smears with AGC 'not otherwise specified' ('NOS') had significant pathology. CONCLUSIONS: AGC found on cervical smears during pregnancy and the postpartum period is uncommon. The chance of having significant cervical pathology, however, is high and colposcopy should be performed.

Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer.

BACKGROUND: Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. METHODS: FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. RESULTS: Quantitative RT-PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21(WAF1/CIP1) in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-beta-induced p21(WAF1/CIP1) expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20-26% decrease in cell proliferation together with 16-33% increase in p21(WAF1/CIP1) expression. Notably, FOXG1 was able to inhibit the p21(WAF1/CIP1) promoter activity in a p53-independent manner by transient reporter assays. CONCLUSION: Our results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-beta-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21(WAF1/CIP1) transcription.

Acceptability of human papillomavirus vaccination among Chinese women: concerns and implications.

OBJECTIVE: To explore Chinese women's perceptions of human papillomavirus (HPV) vaccination and their intention to be vaccinated. DESIGN: A cross-sectional community-based survey study. SETTING: Thirteen community women's health centres of The Family Planning Association of Hong Kong. SAMPLE: A total of 1450 ethnic Chinese women aged 18 or above who attended the health centres. METHODS: Participants completed a written consent and an anonymous questionnaire onsite. MAIN OUTCOME MEASURES: Knowledge and beliefs about HPV and HPV vaccination against cervical cancer and participants' own intention to be vaccinated. RESULTS: About 38% of the participants (n = 527) had heard of HPV and 50% (n = 697) had heard of vaccination against cervical cancer. HPV infection was perceived to be stigmatising and detrimental to intimate, family and social relationships. Despite misconceptions and a grossly inadequate knowledge about HPV and HPV vaccination, 88% of the participants (n = 1219) indicated that they would likely be vaccinated. Majority of the participants believed that sexually experienced women should be vaccinated, while 27% opposed vaccinating sexually naive women. Younger age women who perceived a disruptive impact of HPV infection on intimate relationship and their partners' approval were significantly associated with a positive intention to be HPV vaccinated. CONCLUSIONS: The easy acceptability of HPV vaccination among the mostly sexually experienced Chinese participants and their knowledge deficit on the subject may implicate potential misuse of the vaccines and a false sense of security against cervical cancer. There is a dire need for culturally sensitive and tailored education for the public, women of different ages and their partners about HPV and HPV vaccination. Emphasis must be placed on the prophylactic nature of the current vaccines, the uncertain effects when given to sexually experienced women, the importance of adolescent vaccination and the need for continued cervical screening whether vaccinated or not.

Functional polymorphisms in the BRCA1 promoter influence transcription and are associated with decreased risk for breast cancer in Chinese women.

BACKGROUND: The BRCA1 gene is an important breast-cancer susceptibility gene. Promoter polymorphisms can alter the binding affinity of transcription factors, changing transcriptional activity and may affect susceptibility to disease. METHODS AND RESULTS: Using direct sequencing of the BRCA1 promoter region, we identified four polymorphisms c.-2804T-->C (rs799908:T-->C), c.-2265C-->T (rs11655505:C-->T), c.-2004A-->G (rs799906:A-->G) and c.-1896(ACA)(1)-->(ACA)(2) (rs8176071:(ACA)(1)-->(ACA)(2)) present in Hong Kong Chinese. Each polymorphism was studied independently and in combination by functional assays. Although all four variants significantly altered promoter activity, the c.-2265T allele had stronger binding than the C allele, and the most common mutant haplotype, which contains the c.-2265T allele, increased promoter activity by 70%. Risk association first tested in Hong Kong Chinese women with breast cancer and age-matched controls and replicated in a large population-based study of Shanghai Chinese, together totalling >3000 participants, showed that carriers of the c.-2265T allele had a reduced risk for breast cancer (combined odd ratio (OR) = 0.80, 95% CI 0.69 to 0.93; p = 0.003) which was more evident among women aged >or=45 years at first diagnosis of breast cancer and without a family history of breast cancer (combined OR = 0.75, 95% CI 0.61 to 0.91; p = 0.004). The most common haplotype containing the c.-2265T allele also showed significant risk association for women aged >or=45 years without a family history of breast cancer (OR = 0.64, 95% CI 0.46 to 0.89; p = 0.008). CONCLUSION: This comprehensive study of BRCA1 promoter polymorphisms found four variants that altered promoter activity and with the most significant contribution from c.-2265C-->T, which could affect susceptibility to breast cancer in the Chinese population. Its significance in other populations remains to be investigated.

Activated Stat3 expression in gestational trophoblastic disease: correlation with clinicopathological parameters and apoptotic indices.

AIMS: To assess the expression profile of the activated form of signal transducer and activator of transcription (Stat)3 in gestational trophoblastic disease (GTD) and correlate the findings with clinicopathological parameters. METHODS AND RESULTS: By immunohistochemistry, both cytoplasmic and nuclear expression of p-Stat3-Ser(727) was demonstrated in 88 trophoblastic tissues, including placentas and GTD. Nuclear immunoreactivity of p-Stat3-Ser(727) was significantly higher in hydatidiform mole (HM) (P < 0.001) and choriocarcinoma (P = 0.009) when compared with normal placentas. Placental site trophoblastic tumours (PSTT) and epithelioid trophoblastic tumours (ETT) also demonstrated higher nuclear p-Stat3-Ser(727) expression than their normal trophoblast counterparts. Higher p-Stat3-Ser(727) expression was confirmed in choriocarcinoma cell lines, JEG-3 and JAR, than in a normal trophoblast cell line, with both nuclear and cytoplasmic fractions demonstrated by immunoblotting. Spontaneously regressed HM showed significantly increased nuclear and cytoplasmic p-Stat3-Ser(727) immunoreactivity over those that developed gestational trophoblastic neoplasia (GTN) (P = 0.013, P = 0.039). There was a significant positive and inverse correlation between nuclear p-Stat3-Ser(727) immunoreactivity and apoptotic indices [terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling and M30 CytoDeath antibody] (P = 0.001, P < 0.001, Spearman's rho test) and Bcl-2 expression (P = 0.034), respectively. CONCLUSIONS: p-Stat3-Ser(727) plays a role in the pathogenesis of GTD, probably through the regulation of apoptosis. p-Stat3-Ser(727) immunoreactivity is a potential marker in predicting GTN in HM.

Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis.

The Forkhead Box M1 (FOXM1) transcription factor plays a crucial role in regulating expression of cell cycle genes which are essentially involved in cell proliferation, differentiation and transformation. Recent studies have reported that aberrant expression of FOXM1 in a variety of human cancers is associated with their aggressive behaviour. However, the functional significance of FOXM1 in human cervical cancer is not known. We have shown that FOXM1 was significantly over-expressed in cervical squamous cell carcinoma (SCC) compared to normal cervical epithelium immunohistochemically (p < 0.001). In addition, intratumoural FOXM1 positivity was increased in cervical intraepithelial neoplasia (CIN) and carcinoma, compared with that in normal epithelium, indicating that FOXM1 is involved in tumour progression. Indeed, this is supported by clinicopathological analysis that the over-expression of FOXM1 was significantly associated with tumour late stage (p = 0.012) and cell proliferation marker, Ki67 (p < 0.001). Functionally, enforced expression of FOXM1c in FOXM1-deficient cervical cancer cells (C33A) remarkably enhanced cell proliferation and anchorage-independent growth ability. Conversely, depletion of FOXM1 by RNA interference in FOXM1-over-expressing cervical cancer cells (SiHa) caused significant inhibition on cell proliferation and anchorage-independent growth ability on soft agar. This inhibitory phenomenon was associated with the reduced expressions of cyclin B1, cyclinD1 and cdc25B but increased expression of p27(Kip1) and p21(Cip1). Our findings suggest a role for FOXM1 in the development and pathogenesis of human cervical SCC.

Oct4 is epigenetically regulated by methylation in normal placenta and gestational trophoblastic disease.

Oct4 is a transcription factor that plays a crucial role in maintaining pluripotency of embryonic stem cells. Down-regulation of Oct4 is associated with the differentiation of trophectoderm cell lineage, from which the normal placenta derives. We investigated the methylation and expression status of Oct4 in normal placenta and gestational trophoblastic disease (GTD) as attempts to investigate the role of Oct4 in the pathogenesis of GTD. By methylation-specific PCR, we observed both methylated and unmethylated Oct4 alleles in all 25 first trimester and 10 term placentas while 33% (18/54) of hydatidiform moles, and two choriocarcinoma cell line (JEG3 and JAR), only displayed methylated Oct4 allele. By quantitative TaqMan real-time PCR, Oct4 mRNA was significantly reduced in hydatidiform moles (P=0.04), JEG3 and JAR (P=0.024) when compared with normal placentas. Oct4 methylation was significantly correlated with Oct4 mRNA expression in placenta and GTD (P=0.012). Hypermethylation in minimal promoter and exon 1 region of Oct4 were confirmed in JEG3 and JAR by bisulfite genomic sequencing. The Oct4 mRNA expression in JEG3 and JAR increased after treatment with 5-aza-2'-deoxycytidine and/or trichostatin A. Our findings suggest that Oct4 is down-regulated by hypermethylation in normal placenta and GTD and such process is important in pathogenesis of GTD.

Barriers and facilitators to human papillomavirus vaccination among Chinese adolescent girls in Hong Kong: a qualitative-quantitative study.

OBJECTIVES: To explore perceptions towards cervical cancer, human papillomavirus (HPV) infection and HPV vaccination and to identify factors affecting the acceptability of HPV vaccination among Chinese adolescent girls in Hong Kong. METHODS: Six focus groups were conducted with Chinese adolescent girls (median age 16 years, age range 13-20, n = 64) in Hong Kong in April 2007. Thematic analysis was employed to identify major themes related to cervical cancer and HPV vaccination. A supplementary questionnaire was administered to all participants before and after group discussion to assess their knowledge, attitudes and intention to be vaccinated and to collect demographic information. RESULTS: Participants' knowledge on cervical cancer was limited and HPV was largely unheard of. They had difficulty understanding the mechanism linking cervical cancer with HPV infection. Participants held a favourable attitude towards HPV vaccination but the perceived timing of vaccination varied. Barriers to vaccination include high monetary cost, uncertain length of vaccine effectiveness, low perceived risk of HPV infection, no immediate perceived need of vaccination, anticipated family disapproval and fear of the pain of injection. Factors conducive to vaccination include perceived family and peer support and medical reassurance on safety and efficacy of vaccine. The differences on knowledge, attitudes, intention to be vaccinated now and willingness to conform to significant others before and after the discussion were statistically significant, with an increased tendency towards favouring vaccination after the focus group. CONCLUSIONS: Participants favoured HPV vaccination despite not feeling an immediate need to be vaccinated. Interventions could focus on providing professional information on HPV vaccination and raising adolescents' perceived need to take preventive measures against HPV infection.

Overexpression of prostate stem cell antigen is associated with gestational trophoblastic neoplasia.

AIMS: Hydatidiform mole (HM) is the most common type of gestational trophoblastic disease. A proportion of patients with HM develop gestational trophoblastic neoplasia (GTN) requiring chemotherapy. The aim was to identify differentially expressed genes that are associated with development of GTN. METHODS AND RESULTS: Using cDNA microarray, differential expression of prostate stem cell antigen (PSCA) was identified in HMs that developed GTN compared with those that spontaneously regressed. Significant overexpression of PSCA RNA (P = 0.037) and protein (P < 0.05) in aggressive HM was verified by real-time polymerase chain reaction (PCR) and immunohistochemical analysis in 10 first-trimester placentas, 36 HM that subsequently regressed and 11 HM that developed GTN. A high level of PSCA expression was also found in three choriocarcinomas and three placental site trophoblastic tumours. A positive correlation was observed between PSCA expression and proliferation and apoptotic indices as assessed by Ki67 (P = 0.01), mcm7 (P = 0.001) and M30 (P = 0.016), as well as p53 (P < 0.01), p21(WAF1/CIP1) (P < 0.01) and mdm2 (P = 0.002) expression. CONCLUSIONS: Overexpression of PSCA is associated with development of GTN in HM. PSCA probably plays a role in the regulation of cell growth through p53-related signaling pathways.

Methylation profile in benign, borderline and malignant ovarian tumors.

PURPOSE: Promoter hypermethylation is a common phenomenon in neoplasm. The aims of this study were (a) to compare the methylation profiles in different types of ovarian tumors and (b) to determine the possible relationship between the methylation status and different clinicopathologic characteristics. METHODS: We examined the promoter methylation status of 9 tumor suppressor genes (RARbeta2, TMS1, RIZ1, P15, P16, PTEN, MINT31, APC and HIC1) in 89 ovarian cancers, 16 borderline ovarian tumors, 19 benign ovarian tumors, 16 normal ovarian tissue and 5 ovarian cancer cell lines. The methylation status was examined with respect to clinicopathologic characteristics of the ovarian cancer patients. RESULTS: Methylation indices for ovarian cancer, borderline ovarian tumor, benign ovarian tumor, normal ovarian tissue and ovarian cancer cell lines were 28.8, 20.1, 10.5, 11.8 and 42.2%, respectively. It was significantly higher in ovarian cancer, borderline ovarian tumor and ovarian cancer cell lines (X (2) test, P < 0.001, P = 0.01 and P < 0.001, respectively) than benign or normal ovarian tissues. In ovarian cancer, concurrent methylation of at least two genes (CM2) was associated with early stage disease (X (2) test, P = 0.035) and less recurrence (X (2) test, P = 0.020). When the methylation statuses of the nine genes as well as CM2 were included in multivariate Cox Regression analysis, CM2 was the only independent predictor for survival (P = 0.013). CONCLUSION: CM2 was an independent predictor for survival in ovarian cancer.

Association of decreased mitochondrial DNA content with ovarian cancer progression.

Mitochondrial DNA (mtDNA) content in ovarian carcinomas was assessed by quantitative PCR. Results show that mtDNA content in tumour cell was significantly higher than that in normal ovary. Change in mtDNA content was not related with patients' age or tumour stages. However, the average mtDNA copy number in pathological low-grade tumours was over two-fold higher than that in high-grade carcinomas (P=0.012). Moreover, type I carcinomas also had a significantly higher mtDNA copy number than in type II carcinomas (P=0.019). Change in mtDNA content might be an important genetic event in the progression of ovarian carcinomas.