IGSF4 promoter methylation and expression silencing in human cervical cancer

OBJECTIVES: Functional assays of tumor suppression and loss of heterozygosity point to a tumor suppressor gene (TSG) for cervical cancer (CC) on chromosome 11q23. We evaluated IGSF4, a putative TSG located in the region, for promoter methylation and gene silencing in CC cell lines and cervical tissues. METHODS: IGSF4 expression was detected by both RT-PCR and Northern blot analysis. Methylation maps of the IGSF4 promoter region were generated for 11 CC cell lines based upon bisulfite-genomic sequencing, using seven nested-PCR primer sets covering 97 CpG sites. Methylation fingerprints in primary cervical tissues were evaluated by denaturing high performance liquid chromatography. RESULTS: A 4.4-kb mRNA was seen in cell lines, consistent with the RT-PCR results for both cell lines and primary cervical tissue. IGSF4 was expressed in 6/11 cell lines, 6/8 CC tissues and in all seven normal cervical epithelia. In the cell lines, IGSF4 silencing was associated with promoter hypermethylation. The methylation status in the region covering the -18 to -2 CpG sites correlated most strongly with expression, pointing to the existence of an unmethylated core in the IGSF4 promoter in cell lines expressing IGSF4. This unmethylated core spans approximately 180 bp and is immediately upstream of the ATG site. In primary tissues, methylation was detected in 15/23 (65%) CC specimens but in none of seven normal cervical epithelia. CONCLUSIONS: Our data strongly suggest that IGSF4 is a TSG and that gene silencing by aberrant hypermethylation may contribute to the development of CC.     

p53 Protein Expression and Gene Analysis in Clear Cell Adenocarcinoma of the Vagina and Cervix

Objective:p53 is the most commonly mutated gene in human cancers. The objective of this study was to determine if clear cell adenocarcinomas (CCAs) of the vagina and cervix are associated with p53 gene mutations or alterations in p53 tumor-suppressor protein expression.Methods:Paraffin-embedded tissue specimens from 21 women (median age 22 years) with clear cell adenocarcinoma of the vagina or cervix were studied. Fifteen women had a prior history ofin uteroexposure to diethylstilbestrol. p53 protein expression was detected by immunohistochemical (IHC) analysis with monoclonal antibody DO-7 (Dako Corp.) which recognizes both wild-type and mutant p53 proteins. For p53 gene analysis, genomic DNA from malignant tissue was isolated and exons 4–10 were amplified by PCR and subjected to mutation screening by single-stranded conformation polymorphism (SSCP) analysis.Results:p53 protein was detected by IHC in tumors from 14 of 21 cases (67%). The observed p53 staining patterns were heterogeneous in both the proportion and intensity of tumor cells stained but were clearly overexpressed relative to the surrounding benign stroma. Metastatic tumors from 3 women with metastatic disease were also positive for p53 staining. SSCP analysis did not identify p53 mutations in any of the cases and strongly suggests that the tumors contained only wild-type p53 alleles.Conclusions:Recent studies have demonstrated that wild-type p53 may accumulate in response to DNA damage which normally leads to growth arrest or programmed cell death. Our observations are consistent with the hypothesis that p53 overexpression in CCAs of the vagina and cervix is a response to generalized DNA damage, rather than a result of p53 protein half-life prolongation resulting from mutational inactivation of p53. Overexpression of wild-type p53 protein in vaginal and cervical CCA may relate to the more favorable prognosis of this subset of tumors in comparison to other gynecologic tumors containing mutated p53 genes.     

The Status of Human Papillomavirus and Tumor Suppressor Genesp53andp16in Carcinomas of Uterine Cervix from India

Objectives.Infection with the high-risk strain of human papillomaviruses (HPVs) and the inactivation of the tumor suppressor genep53through mutation are important factors in cervical carcinogenesis. To know whether such events would occur in cervical carcinomas of Indians, 43 tumors (consisting of 36 of stage III B and 6 of stage II B) were screened forp53andp16gene mutations.Methods.PCR followed by single-strand conformation polymorphism (SSCP) analysis were used to detect mutations inp53andp16genes and PCR for the presence of human papillomavirus genome. HPV status was ascertained by PCR amplification of parts of E6 and E7 genes using primers pU-1M and pU-2R and typing was carried out by restriction analysis.Results.Of the 43 samples analyzed, 4 samples (9%) showed mobility shifts forp53mutations; PCR products of thep16gene did not show band shifts in SSCP analysis. HPV DNA was detected in 70% of the 43 samples analyzed: HPV 16 in 23 cases (53%), HPV 18 in 4 cases (13.3%), and HPV 33 in 1 case (3.3%). Two amplified HPV DNAs that were difficult to type with various restriction enzymes were cloned and the amplified regions were sequenced. One of these was 93% close to HPV 35 and the other was 80% close to HPV 58. Three samples had bothp53mutations and HPV genome.Conclusions.Our results indicate that HPV 16 infection was more common than HPV 18, thep53mutations and HPV infection were not mutually exclusive events in the genesis of carcinoma of uterine cervix among Indian women, andp16gene may not play a role in Indian cervical carcinomas.     

p53Mutations and Presence of HPV DNA Do Not Correlate with Radiosensitivity of Gynecological Cancer Cell Lines

Objective.The correlation betweenp53tumor suppressor gene mutations and the presence of high-risk human papillomavirus (HPV) DNA with thein vitroradiosensitivity of gynecological malignancies was studied in 26 cell lines derived from gynecological cancers of 23 patients.Methods.Comparison of the intrinsic radiosensitivity was performed with mean inactivation dose (D?) determined with the 96-well plate clonogenic assay.p53mutations were investigated with polymerase chain reaction and single-strand conformation polymorphism (PCR–SSCP) analysis and direct DNA sequencing, and the presence of HPV DNA was studied with PCR using HPV consensus primers.Results. p53mutations were found in 6 of 10 vulvar squamous cell carcinoma (SCC) lines. Nine vulvar and 1 vaginal SCC cell lines were HPV DNA negative and 1 vulvar cell line was HPV 16 positive. All 4 cervical SCC lines were HPV positive and possessed the wild-typep53.Three cell lines expressed HPV 16 and 1 HPV 68. Among 10 endometrial cancer cell lines, 2 cell lines with mutantp53and 1 HPV 16 positive cell line were found. No correlation could be demonstrated between inactivation of thep53gene and radiosensitivityin vitro;the cell lines were evaluated as one group or according to their anatomical origin or histology.Conclusion.Our results indicate that inactivation of thep53gene through mutation or binding with HPV DNA does not increase the resistance of gynecological malignancies to ionizing radiationin vitro.     

Low TWEAK expression is correlated to the progression of squamous cervical carcinoma

Objective

To investigate the correlation of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its specific receptor fibroblast growth factor-inducible immediate-early response protein 14 (Fn14) to cervical carcinogenesis by examining TWEAK/Fn14 expression levels or locations in different cervical tissues and cells.

Methods

TWEAK/Fn14 mRNA expressions were detected by quantitative real-time PCR in total of 120 cervical samples including normal, precancerous and cancerous tissues, while protein expressions were detected by immunofluorescent staining and western blot in both tissues and cell lines. Correlation between TWEAK expression levels to cancer progression and clinicopathologic features was statistically analyzed.

Results

The TWEAK expression was significantly decreased while Fn14 expression was increased in carcinoma and cervical intraepithelial neoplasm (CIN) specimens compared with that in normal control specimens. A similar trend of TWEAK/Fn14 expression was also observed in cervical cell lines. In addition, TWEAK expression decreased further along with the interstitial depth of invasion (P < 0.05) and tumor grade (P < 0.05), suggesting that TWEAK acts rather on local cancer tissue infiltration.

Conclusion

TWEAK/Fn14 pathway may play a role in the development of squamous cervical carcinoma, in which the reduced level of TWEAK could promote the progression and invasion of cervical cancer. An increase in Fn14 may reflect a compensatory response to decreased TWEAK and may provide a novel therapeutic target for human cervical cancer treatment or a biomarker for cervical cancer diagnosis.     

Allelic Losses at Chromosome 3p Are Seen in Human Papilloma Virus 16 Associated Transitional Cell Carcinoma of the Cervix

OBJECTIVE: Transitional cell carcinomas (TCCs) of the cervix are rare neoplasms of the female genital tract. Although these tumors display urothelial differentiation, there is controversy regarding their histogenetic relationship to squamous cell carcinomas (SCC) of the cervix versus transitional cell carcinomas of the bladder. METHODS: We performed partial allelotyping of five TCCs of the cervix using 23 polymorphic markers located on chromosomes 3p and 9, which demonstrate frequent and early losses in cervical SCC and urothelial TCC, respectively. Multiplex polymerase chain reaction was used on DNA extracted from archival paraffin-embedded tissue using precise microdissection. Additionally, P53 gene mutation analysis was performed using single-strand confirmation polymorphism (SSCP) and the presence of human papilloma virus (HPV) sequences was analyzed using general and specific (types 16 and 18) primers. RESULTS: General HPV sequences were demonstrated in all cases, but the oncogenic strain HPV 16 was present in only three (60%) of the five tumors; no HPV 18 was detected in any sample. Three of five TCCs, all harboring HPV 16 sequences, demonstrated concurrent allelic losses at several 3p loci (specifically 3p12, 3p14.2 [the FHIT gene locus], 3p21.3, and 3p22-24.2). LOH at a single locus on 9q32-qter was demonstrated in one tumor; no other deletions were seen on chromosome 9. P53 gene mutations in exons 5-8 were absent by SSCP analysis. CONCLUSIONS: The infrequent involvement of chromosome 9 in TCCs of the cervix, along with the concurrent presence of 3p LOH and oncogenic HPV 16 in a subset of tumors, suggests a closer histogenetic relationship of this neoplasm to cervical SCCs rather than urothelial TCCs.     

Tumor Angiogenesis as a Prognostic Factor in Cervical Carcinoma

Angiogenesis, the induction of new capillaries and venules, has been associated with tumor growth. Increased tumor size and new vessel growth may further the opportunity for tumor cells to enter the circulation and potentiate metastatic disease. To investigate if tumor angiogenesis could serve as a prognostic factor in cervical carcinoma, we counted microvessels (capillaries and venules) in 29 patients with squamous cell carcinoma of the cervix. Surgical specimens were stained for endothelial cells specifically with Factor VIII to identify all vessels. The microvessels were counted by light microscopy (per 200× field) in tumor sections with the highest population of microvessels. This was performed by two investigators without knowledge of patient outcome or extent of disease. Microvessel counts in patients with squamous cell carcinoma were significantly different from those of control subjects: 56 ± 28.9 and 16.3 ± 3.3 (P = 0.013). There was no correlation between microvessel count and node status, parametrial involvement, depth of invasion, or gross disease. Microvessel count was significantly correlated with vascular space involvement (P = 0.017). Four patients who developed recurrent disease within 1 year had high microvessel counts and yet were node negative and VSI negative at surgery. As shown by Folkman in breast cancer, angiogenesis may also be an independent predictor for recurrent disease in squamous cell carcinoma of the cervix. Microvessel counts could be of prognostic value in patients who do not have other risk factors for disease recurrence.     

子宫颈癌及其癌前病变组织端粒酶活性的研究

目的研究宫颈癌及其癌前病变组织中端粒酶激活的意义.方法应用端粒酶重复序列扩增-酶联免疫吸附法(TRAP-ELISA)及电泳-银染法,对36例宫颈浸润癌及16例宫颈上皮内瘤变(CIN)组织进行端粒酶活性测定,以吸光度(A)值判断端粒酶活性.同时测定11例正常宫颈、6例慢性宫颈炎症及8例癌旁组织端粒酶活性作为对照.结果CIN、宫颈癌及对照组端粒酶活性A值分别为0.398±0.293、1.580±0.819和0.050±0.012,3组比较,差异有极显著性(P＜0.01).端粒酶活性高低与肿瘤分化程度呈负相关,与淋巴结转移呈正相关;与组织学类型、分期、体积大小无关.结论端粒酶的激活发生在宫颈癌病变的早期,可能成为宫颈癌及癌前病变早期诊断和鉴别诊断的指标.     

Mutations of the TP53 gene in adenocarcinoma and squamous cell carcinoma of the cervix: A systematic review

ObjectiveMutations of the tumor suppressor gene TP53 are the most significant events in several human cancers. Few studies have analyzed the frequency of TP53 alterations in squamous cell carcinoma and adenocarcinoma of the cervix with controversial results. This study provides a detailed analysis of TP53 mutation spectra in cervical squamous cell carcinoma and adenocarcinoma from different geographical regions.MethodsThe analysis of TP53 mutational profiles was performed in 1353 cervical cancers retrieved from the IARC p53 mutation database (R15, 2010) and the COSMIC data along with the literature review of related studies identified by PubMed searching.ResultsThis analysis showed a significant higher mutation frequency of TP53 gene in cervical adenocarcinoma (32 of 241; 13.3%) compared to squamous cell carcinoma (39 of 657; 5.9%; P = 0.0003, χ² test). The proportion of adenocarcinoma with mutated TP53 varied from 4% in North America to 19% in Asia. Among the six hot-spot codons of TP53 gene, three codons (175, 248 and 273) were the most commonly mutated in both types of cervical cancer, one codon (249) mainly in squamous cell carcinoma and one codon (282) only in adenocarcinoma. The G to A and C to T transitions were the prevalent type of mutations in both squamous cell carcinoma and adenocarcinoma (48.7% and 53.5% of all mutations, respectively). The frequency of C to A transversion was relatively high only in adenocarcinoma (25%), while the mirror mutation G to T was comparatively frequent in squamous cell carcinoma (14.6%).ConclusionsDifferent patterns of TP53 mutations occur in squamous cell carcinoma and adenocarcinoma of the cervix in different regions of the world. The highest frequency of mutated TP53 has been observed in cervical adenocarcinoma from Asia. Further studies are needed to better define the role of TP53 alterations in cervical cancer and possibly to understand the impact of mutations on cancer prognosis and outcomes.     

Multiple primaries among gynecologic malignancies

Seventy-eight synchronous or metachronous tumors among 2362 patients followed by the Downstate Gynecologic Tumor Registry are reviewed. Significant synchronous tumor pairs include cervix (invasive and in situ)-ovary, cervix (in situ)-uterus, cervix (in situ)-kidney, endometrium-ovary, endometrium-rectosigmoid and ovary-breast. Significant metachronous pairs include cervix (invasive and in situ combined)-lung, cervix (invasive and in situ combined)-upper alimentary tract, and cervix (invasive)-rectosigmoid. In the case of in situ and invasive cervical cancer-lower genital tract, significance was determined for both synchronous and metachronous pairs. Long survival is an important factor in the appearance of a second tumor as demonstrated in patients with cervical carcinoma. Synchronous data prove to be valuable in assessing risk of second primaries in patients surviving for short periods. The roles of cigarette smoking, hormones, immunosuppression, radiotherapy, and screening are discussed.     

Growth factor progranulin contributes to cervical cancer cell proliferation and transformation in vivo and in vitro

Objective

The growth factor progranulin (PGRN) is overexpressed in a number of tumors. We aimed to investigate the expression and role of PGRN in cervical cancer tumorigenesis.

Methods

PGRN expression and secretion was assessed in cells and normal and cancerous cervical tissues by Western blot analysis, ELISA or immunohistochemistry. The role of PGRN in cervical carcinogenesis was explored by cell-proliferation, colony-formation and tumor-growth assays. We assessed the role of PGRN-mediated signaling in the cervical cell with specific inhibitors.

Results

PGRN expression was upregulated in cervical cancer cell lines and tissue. PGRN promoted the transformation of human cervical mucosa epithelial H8 cells in vitro and tumor formation in vivo. Knockdown of PGRN expression in cervical cancer cells in vivo decreased cell proliferation and slowed tumor growth. PGRN stimulated cervical cell proliferation, and transformation was mediated, at least in part, by Akt and Erk signaling.

Conclusions

PGRN is overexpressed in cervical cancer and promotes the malignant growth and transformation of cervical cells. Therefore, PGRN plays a critical role in carcinogenesis of cervical cancer and shows promise for therapeutic strategies for cervical cancer.     

MiR-31 is an independent prognostic factor and functions as an oncomir in cervical cancer via targeting ARID1A

Objectives

MicroRNAs(miRNAs) play important roles in tumor development and progression. The purposes of this study were to investigate the role of miR-31 in cervical cancer and clarified the regulation of ARID1A by miR-31.

Methods

Quantitative RT-PCR was used to examine miR-31 expression in cervical cancer cell lines and patient specimens. The clinicopathological significance of miR-31 upregulation was further analyzed. The MTT, colony formation, apoptosis, cell cycle, wound healing and Transwell invasion assays, and a xenograft model were performed. A luciferase reporter assay was conducted to confirm the target gene of miR-31, and the results were validated in cell lines and patient specimens.

Results

MiR-31 was significantly up-regulated in cervical cancer cell lines and clinical tissues. The high miR-31 level was significantly correlated with higher FIGO stage, node metastasis, vascular involvement and deep stromal invasion. Patients with high expression of miR-31 had poorer overall survival than patients with low expression. MiR-31 was an independent prognostic factor in cervical cancer in multivariate Cox regression analysis. Down-regulation of miR-31 impaired cell proliferation, colony formation, and cell migration and invasion in vitro, and inhibited xenograft tumor growth in vivo. ARID1A was verified as a direct target of miR-31, which was further confirmed by the inverse expression of miR-31and ARID1A in patient specimens.

Conclusions

The newly identified miR-31/ARID1A pathway provides insight into cervical cancer progression, and may represent a novel therapeutic target.     

survivin、FHIT及bFGF在宫颈鳞癌组织中的表达及意义

目的:研究凋亡抑制基因survivin、抑癌基因FHIT及碱性成纤维细胞生长因子(bFGF)在正常宫颈组织、宫颈上皮内瘤变组织(CIN)及宫颈鳞癌组织中的表达及其与宫颈鳞癌临床分期、病理分级及淋巴结转移的关系,探讨其在宫颈鳞癌发生、发展中的作用。方法:分析郑州大学第二附属医院病理科2008年1月至2009年11月手术切除、病理检查证实为宫颈鳞癌的组织标本45例,另取CIN45例、正常宫颈组织15例(子宫肌瘤行子宫全切除术的宫颈组织)作为对照,应用免疫组化SP法检测survivin、FHIT及bFGF在3组宫颈组织中的表达情况。结果:sur-vivin在正常宫颈组织、CIN及宫颈鳞癌组织中的阳性表达呈上升趋势(χ2=11.429,P<0.05),survivin的表达在宫颈鳞癌不同临床分期、病理分级间比较,差异有统计学意义(P<0.05);FHIT在正常宫颈组织、CIN及宫颈鳞癌组织中的阳性表达呈下降趋势(χ2=24.640,P<0.05),FHIT的表达与宫颈鳞癌病理特征的关系中,仅在淋巴结有无转移方面比较差异有统计学意义(P<0.05);bFGF在正常宫颈组织、CIN及宫颈鳞癌组织中的阳性表达依次呈上升趋势(χ2=17.552,P<0.05),bFGF的表达在宫颈鳞癌不同临床分期、病理分级及淋巴结有无转移方面比较,差异均有统计学意义(P<0.05)。结论:survivin、FHIT、bFGF的表达与宫颈病变的进展有一定的关系,并且可能与宫颈鳞癌的发生、发展及浸润和转移有关。