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.

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.

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.

Microsatellite instability in mitochondrial genome of common female cancers.

To investigate the occurrence of mitochondrial genome instability in primary cervical, endometrial, ovarian, and breast carcinomas, we analyzed 12 microsatellite regions in mitochondrial DNA (mtDNA) of tumor tissues and their matched normal controls. Four of the 12 microsatellite markers starting at nucleotide position (np) 303, 514, 956, and 16184, respectively, exhibited instability as indicated by the change in length of short base-repetitive sequences of mtDNA in cancer tissue relative to that in control normal tissue from the same patient. About 25.4% of cervical cancers, 48.4% of endometrial cancers, 21.9% of ovarian cancers, and 29.4% of breast cancers carried one or more mitochondrial microsatellite instability (mtMSI). mtMSI was frequently detected in the D-loop region but rarely occurred in the coding region. A relatively long C tract interrupted by a T residue is the mtMSI hot spot in all four types of cancer studied. Different tumors have different mtMSI profiles. In particular, the frequency of mtMSI in endometrial cancer was significantly higher than in the other three types of cancer. Furthermore, carriers of a germ-line T to C polymorphism at np 16189 could be more susceptible to breast cancer development in light of the higher frequency detected in cancer patients than in normal individuals.

Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes.

Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors' age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors' age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 +/- 265 862. Women >or=35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor's age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.

Quantification of human papillomavirus DNA in the plasma of patients with cervical cancer.

Plasma human papillomavirus (HPV)-DNA level was measured to evaluate the clinical usefulness of circulating DNA for cervical cancer management. DNA extracted from pretreatment plasma of 50 cervical cancer patients and from serial longitudinal plasma of 21 patients was quantified for HPV16/HPV18 by means of quantitative polymerase chain reaction. Another 15 patients with low-grade lesion (LG), 18 patients with high-grade lesion (HG), and 96 normal individuals were studied as controls. Plasma HPV16-DNA was detectable in 50% of cancer patients. The incidence and median level were statistically higher than those in LG patients and normal, but similar to HG patients. Plasma HPV18-DNA was only detected in 6% of cancer patients and 1% of normal. Same type of HPV present in plasma was also detected in its primary tumor; and the level of plasma HPV16-DNA was dependent on the viral load in primary tumor. Plasma HPV-DNA was not detected in 16 of 21 patients after treatment, and those patients had complete response to therapy. HPV-DNA persisted or reappeared in five patients after treatment (one had persistent disease and another had recurrence). Plasma HPV-DNA might be a valuable marker for monitoring therapeutic response and disease progression in cervical cancer.

Detection of hypermethylated genes in tumor and plasma of cervical cancer patients.

OBJECTIVE: The aim of this study is to investigate the prevalence of promotor CpG island methylation of the death-associated protein kinase (DAPK), p16, and O(6)-methylguanine-DNA methyltransferase (MGMT) genes in both tumor and plasma samples of cervical cancers. METHODS: Methylation-specific PCR (MSP) was employed to detect promotor CpG island methylation of the DAPK, p16, and MGMT genes in 85 surgical tumor tissue samples and 40 pretreatment plasma samples from cervical cancers. RESULTS: Promotor CpG island methylation of DAPK, p16, and MGMT was detectable, respectively, in 60%, 28.2%, and 18.8% of cases of cervical tumor DNA; and in 40%, 10%, and 7.5% of cases of patients' plasma DNA. Moreover, at least one of the three methylated genes was detected in 75.3% (64/85) of cases of tumor and in 55% (22/40) of cases of plasma. Higher prevalence of methylation of DAPK was found in squamous cell carcinoma than in adenocarcinoma in both univariate and multivariate analysis. Methylation of p16 was significantly associated with that of MGMT in both univariate and multivariate analysis. The methylation pattern in primary tumor and plasma was found to be concordant in 23 patients with matched tissue and plasma samples. In cases positive for DAPK and p16 methylation in tumor, detection in the paired plasma sample was 64.3% (9/14) and 33.3% (3/9), respectively. CONCLUSIONS: Promotor CpG island methylation is a frequent event in cervical carcinogenesis. Detection of the methylated sequences in the circulation suggests that plasma DNA methylation warrants further study to determine its potential role in cancer management.

High frequency of mitochondrial genome instability in human endometrial carcinomas.

To investigate the occurrence of somatic mitochondrial DNA (mtDNA) mutations in human primary endometrial carcinomas, we sequenced the D-loop region, the 12S and 16S rRNA genes of mtDNA of cancer tissues and their matched normal controls. About 56% (28 out of 50) of cases carry one or more somatic changes in mtDNA including deletion, point mutation and mitochondrial microsatellite instability (mtMSI), namely the change in length of short base-repetitive sequences of mtDNA. In particular, mtMSI was frequently detected in 89% (25 out of 28) of all the cases carrying somatic changes followed by point mutations (25%; seven out of 28) and deletion (3.5%; one out of 28). The CCCCCTCCCC sequences located in the Hypervariable Regions I and II of the D-loop and 12S rRNA gene are instability hot spot regions in endometrial carcinomas. It is suggested that errors in replication may account for the high frequency of mtMSI in human endometrial carcinomas. The relatively high prevalence of mtMSI may be a potential new tool for detection of endometrial cancer.

Molecular analysis of the androgen receptor gene in Hong Kong Chinese infertile men.

PURPOSE: To investigate the relationship between CAG repeat length in the androgen receptor gene and impaired spermatogenesis in Hong Kong Chinese population. METHODS: The CAG repeat region was amplified by polymerase chain reaction (PCR) in 85 nonobstructive azoospermic or severe oligozoospermic men, and 45 fertile males. The number of CAG repeat was analyzed by DNA sequencing. Serum FSH, LH, and testosterone levels were also determined in these men. RESULTS: Among nonobstructive azoospermic males, three men (5.7%) possessed short CAG repeats (< 16), and three (5.7%) other men possessed long CAG repeats (> 30). Short CAG repeats (< 16) were also found in two severe oligozoospermic males (6.3%). The incidence of infertile men with short or long CAG repeats is significantly higher in the azoospermic group (p = 0.03) but not in the severe oligozoospermic group (p = 0.17) when compared with the fertile controls CONCLUSION: Our data suggest an association between CAG repeat lengths and impaired spermatogenesis in azoospermic males in our population.

Low incidence of HPV DNA in sera of pretreatment cervical cancer patients.

OBJECTIVE: The aim of this study was to investigate the feasibility of using DNA in the circulation as a diagnostic tool for cervical cancer. METHODS: We used PCR followed by Southern hybridization to detect human papillomavirus (HPV) DNA in serum samples taken from patients of cervical cancer before treatment. RESULTS: A total of 60 samples were analyzed. In a set of 40 samples, without knowledge of the HPV DNA status in the corresponding cervical carcinomas, we could detect 8 (20%) positive samples, of which 7 (17.5%) were HPV 16 and 1 (2.5%) was HPV 18. In another set of 20 samples, known to be HPV 16 infected in the corresponding cervical carcinomas, we detected only 4 (20%) HPV-16-positive samples. The occurrence of HPV DNA in sera of cervical cancer patients seems sporadic. CONCLUSION: The low incidence indicates that serum HPV DNA has limited application in the diagnosis of cervical cancer.

High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas.

To investigate the potential role of somatic mitochondrial DNA (mtDNA) mutations in tumorigenesis, the occurrence of mutations in mtDNA of ovarian carcinomas was studied. We sequenced the D-loop region of mtDNA of 15 primary ovarian carcinomas and their matched normal controls. Somatic mtDNA mutations were detected in 20% (3 of 15) tumor samples carrying single or multiple changes. Complete sequence analysis of the mtDNA genomes of another 10 pairs of primary ovarian carcinomas and control tissues revealed somatic mtDNA mutations in 60% (6 of 10) of tumor samples. Most of these mutations were homoplasmic, and most were T-->C or G-->A transitions, but one represented a differential length within a run of identical C residues. A region of mtDNA sequence including the 16S and 12S rRNA genes, the D-loop and the cytochrome b gene, may represent the zone of preferred mtDNA mutation in ovarian cancer. The high incidence of mtDNA mutations found in ovarian carcinomas and other human cancers suggests that genetic instability of mtDNA might play a significant role in tumorigenesis.

Primary peritoneal malignant mixed Müllerian tumors. A clinicopathologic, immunohistochemical, and genetic study.

BACKGROUND: Primary peritoneal malignant mixed Müllerian tumors (MMMTs) are rarely reported in the literature. METHODS: The clinical, pathologic, and immunohistochemical features of five cases of MMMT of female peritoneum were analyzed. The tumors were also investigated for expression of hormone receptors, specific BRCA-1 mutations, and clonality. RESULTS: The patients' ages ranged from 33 to 67 years. They presented with abdominal pain or mass. One case of peritoneal MMMT was associated with a synchronous endometrial carcinoma whereas another case was detected 2 years after the diagnosis of a primary adenocarcinoma of the fallopian tube. One patient died 1 month after diagnosis whereas 2 patients died with disease within 1 year. Both carcinomatous and sarcomatous elements are present in all the tumors. Squamous differentiation was noted in two cases. Heterologous elements, including chondroid, rhabodomyoblastic, and osteoid differentiation were detected in all tumors. Immunohistochemical studies confirm the biphasic differentiation with variable demonstration of neural and smooth muscle differentiation. All five MMMTs were negative for estrogen and progestogen receptors although the related endometrial and tubal carcinomas were positive. Heteroduplex analysis used to screen for specific BRCA-1 mutations were negative in all five MMMTs. Clonality study of the two MMMTs found in association with endometrial carcinoma and tubal carcinoma was inconclusive. CONCLUSIONS: Our study confirmed that primary peritoneal MMMTs were aggressive tumors with poor prognosis. The presence of synchronous or metachronous genital carcinomas suggests multifocal tumorigenesis from tissue of same embryologic origin. The lack of hormone receptor in these tumors indicates deviation from hormonal control. Specific BRCA-1 mutations found in ovarian carcinoma in Chinese patients could not be detected in our series.

Homozygous arginine at codon 72 of p53 has no prognostic significance in cervical cancer.

Mutation of p53, a tumour suppressor gene, is uncommon in cervical cancer but the detection of human papillomavirus (HPV) DNA in cervical cancer is common. The findings of increased susceptibility to degradation of p53 by E6 protein of HPV16/18 in cervical cancer with homozygous arginine at codon 72 (HA72) of p53 led to this study on whether cervical cancers with HA72 were more aggressive with the increase in the rate of loss of p53 function. In 102 cervical cancers, 76.5% were HPV16/18 positive and 30% had HA72. No survival difference was detected between HA72 and non-HA72 tumours irrespective of HPV16/18 status. Furthermore, the detection of HPV16/18 in cervical cancer was found not to be of prognostic significance in this study.

Risk of cervical cancer is not increased in Chinese carrying homozygous arginine at codon 72 of p53.

Homozygous arginine at codon 72 (HA72) of p53 was found in 22% of normal cervices and 30.0% of cervical cancers and no significant difference was detected between normal and cervical cancer with or without HPV 16/18. There was no correlation between HA72 and risk of cervical cancer in Chinese.

Concurrent increase of oxidative DNA damage and lipid peroxidation together with mitochondrial DNA mutation in human lung tissues during aging--smoking enhances oxidative stress on the aged tissues.

Although mutation of mitochondrial DNA (mtDNA) in human tissues has been established to associate with intrinsic aging, the impact of environmental factors on the formation and accumulation of mtDNA mutations and oxidative DNA damage in human tissues is poorly understood. We have investigated the levels of mtDNA with the 4977-bp deletion and A3243G point mutation, oxidative DNA damage (indicated by the formation of 8-hydroxy-2'-deoxyguanosine, 8-OH-dG), and lipid peroxides in lung tissues from smokers and nonsmokers of subjects of different ages. The results showed concurrent age-dependent increase of the 4977-bp deleted mtDNA (P < 0.001), 8-OH-dG (P < 0.05), and lipid peroxides (P < 0.05) in the human lung. In the group of subjects above 60 years old, smokers had more extensive DNA damage and lipid peroxidation than did the nonsmokers. However, the levels of mtDNA with the 4977-bp deletion and A3243G point mutation in the lung of smokers were not significantly different from those of the age-matched nonsmokers. Taken together, these results suggest that accumulation of mtDNA with the 4977-bp deletion together with oxidative DNA damage and lipid peroxides is associated with aging and that smoking enhances oxidative damage in human lung tissues.

Mitochondrial DNA deletions in human cardiac tissue show a gross mosaic distribution.

The variability of mitochondrial DNA (mtDNA) deletional patterns has been investigated in adjacent slices of human heart atrium. Using quantitative PCR we found differential abundances of one particular mtDNA deletion, that of 4977 bp (mtDNA4977), among sets of adjacent slices of right atrial trabeculae pectinatae from 10 subjects. Some subjects had relatively constant abundance of mtDNA4977 among the tissue slices, while others covered a wide range. A qualitative PCR procedure was used to visualize the patterns of multiple deletions within an 8.64-kb segment of the mtDNA genome, in the same set of atrial trabeculae samples. Some subjects showed completely different multiple deletional patterns in each of the trabeculae slices analyzed. There was no correlation between the variation of the abundance of mtDNA4977 and that of the multiple deletions. The results are consistent with the notion that the occurrence of mtDNA deletions during aging is a random process, involving their production throughout the lifetime of an individual. In this view, the patterns of new deletions are superimposed on those already accumulated by propagation and segregation of mutations formed earlier in life.

Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging.

Seven mtDNA mutations (five base substitutions and two deletions) were studied in skeletal muscle samples of 18 human subjects aged 1 hr to 90 years. Quantitative PCR procedures were applied to determine the incidence (frequency of occurrence) and abundance (percentage of mutant mtDNA out of total mtDNA). The base substitutions, in general, showed a very early onset, three such mutations being detectable in the muscles of infants aged 1 hr and 5 weeks. Of two disease-associated point mutations studied, 3243 A-->G showed significant accumulation with age (P < 0.05), while 8993 T-->G showed no significant age accumulation (P > 0.1). Moreover, three arbitrarily chosen mutations (not disease-associated) showed no age-associated accumulation: two (7029 C-->T and 7920 A-->G) showed little change over the years (P > 0.1), while the other (13167 A-->G) showed a significant decrease (P < 0.05). both the 4,977-bp and 7,436-bp deletions showed a significant age-associated occurrence (P < 0.01 and P < 0.05, respectively). The age of onset of detectable deletions is about 20-40 years; thereafter, the incidence and abundance of deletions tend to increase as a function of advancing age. The seven specific mutations were found to occur independent of each other, indicating the random nature of mtDNA mutations in skeletal muscle. Moreover, the age-associated accumulation of multiple deletions was observed in the same set of muscle tissues, each extract displaying a unique set of multiple PCR products. Thus, mutations in mtDNA occur differentially in human skeletal muscle during aging.

Independent occurrence of somatic mutations in mitochondrial DNA of human skin from subjects of various ages.

The incidence (frequency of occurrence) and abundance (percentage of mutant out of total mtDNA population) of two different somatic mtDNA mutations in human skin were investigated in 44 subjects ranging from 19 to 87 years of age. Using quantitative allele-specific polymerase chain reaction (AS-PCR) to analyse the A-->G base substitution at nucleotide 3243, 50% of the samples showed detectable levels of that particular mutation, with abundances ranging from 0.01% to 0.12%. In the same set of skin samples, the overall incidence of the 4977 bp "common" deletion was also approximately 50%. Where detected, the abundance of this deletion ranged from 0.0002% to 0.1%. Comparative analyses of the incidence and abundance of these two mutations, collectively and in individual skin samples, led to these two conclusions: (1) there is independent occurrence of these two mtDNA mutations in human skin, and (2) whereas the 4977 bp deletion shows an age-associated accumulation in human skin, no age association is apparent for the 3243 A-->G base substitution. Furthermore, in general, there is a much lower incidence of somatic mutations in mtDNA of human skin as compared to that in postmitotic tissues such as skeletal muscle.