Normal Cervical Epithelium Research Articles

Abstract 4037: Downregulation of ZNF516 and INTS1 is related with promoter methylation in cervical uterine cancer cell lines

Abstract Introduction: One of the most common neoplasia in developing countries is cervical uterine carcinoma (CC). HPV infection is recognized as the major risk factor for carcinogenesis of cervix. However, not all infected women will develop a cervical cancer. Several studies have proposed DNA methylation as early biomarker in CC. A higher promoter methylation is associated with lower gene expression. Aim: To evaluate the association between DNA methylation and gene expression of ZNF516 and INTS1 in cervical cancer cell lines. Materials and Methods: DNA isolated from 12 normal and 7 cervical cancer tissue samples was enriched by Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K CpG Islands plus Promoter arrays (MeDIP-chip). Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions by Methylation Specific PCR in a cohort of 221 normal, premalignant, and cervical cancer samples. Relative expression analysis was performed in a normal cervical epithelium cell line (ECT1 E6/E7) and three cervical cancer cell lines (C-4I, SiHa and C-33A) using real time PCR. MSP was also used to examine promoter methylation in normal cervical and cervical cancer cells lines. Results: ZNF516 and INTS1 were differential methylated in the promoter region of normal and cervical cancer samples in the MeDIP-chip discovery project. The methylation frequency was 100% in cervical cancer cell lines. Relative expression of ZNF516 was significantly lower in C-4I and SiHa (p <0.05) in relation with the normal cell line. C-33A showed elevated promoter methylation and a low expression of ZNF516, however, this result was not significant. INTS1 expression was significantly lower in C-33A and SiHa (p <0.05) in relation with the normal cell line. C-4I showed a high promoter methylation and a low expression of INTS1, however, this result was not significant. Conclusions: We have identified ZNF516 and INTS1 as potentials biomarkers for cervical cancer. These genes may be a useful tool for diagnosis and clinical management of cervical cancer. These results need to be confirmed in clinical samples, from an independent laboratory before the gene panel is examined in a Phase II Biomarker Development Trial. Work sponsored by CORFO Project 09CN14-5960 and Postdoctoral Fondecyt Research Project 3120141. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4037. doi:1538-7445.AM2012-4037

Expression of the carbohydrate tumour marker Sialyl Lewis A, Sialyl Lewis X, Lewis Y and Thomsen-Friedenreich antigen in normal squamous epithelium of the uterine cervix, cervical dysplasia and cervical cancer.

The carbohydrate molecules Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA), Lewis Y (LeY) and Thomsen-Friedenreich antigen (TF) are known to mediate the adhesion between tumor cells and endothelium. They are used as serum markers in diagnosis and treatment in a broad spectrum of human carcinomas, but their expression profile and role in the development of cervical cancer remains unclear. The aim of this study was to investigate the expression of SLeX, SLeA, LeY and TF in normal cervical squamous epithelium, cervical dysplasia and cervical cancer. Slides of paraffin-embedded tissue were fixed and incubated with monoclonal antibodies against SLeX, SLeA, LeY and TF. Immunohistochemical staining was evaluated by using a semi-quantitative score (IRS Score). We found a significant difference of SLeA expression in invasive cervical cancer compared to normal epithelium (p=0.006) and all grades of dysplasia (p=0.002). The expression of SLeX in normal epithelium was less intense than in carcinoma in situ (p=0.036). Staining for LeY showed the weakest results of the investigated markers. Significant differences were found when normal epithelium was compared to CIN I (p=0.011), to CIN II (p=0.013) and to invasive cervical cancer (p=0.005). For TF, significant differences were found in normal epithelium compared to CIN I (p=0.011), CIN II (p=0.013) and compared to invasive cervical cancer (p=0.005). This is the first study on the expression of SLeA, SLeX, LeY and TF in normal cervical endothelium, cervical dysplasia, carcinoma in situ and invasive cervical cancer. Further studies and higher numbers are desirable.

Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis

Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability. In this study, miRNA expression in normal cervical squamous epithelium, high-grade precancerous lesions (cervical intraepithelial neoplasia (CIN2-3)), squamous cell carcinomas (SCCs) and adenocarcinomas (AdCAs) was integrated with previously generated chromosomal profiles of the same samples. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Of these differentially expressed miRNAs, 27 showed early transiently altered expression in CIN2-3 lesions only, 46 miRNAs showed late altered expression in SCCs only and 33 showed continuously altered expression in both CIN2-3 and SCCs. Altered expression of five significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1), was directly linked to frequent chromosomal alterations. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain of 1q. Hsa-miR-9 overexpression was found to increase cell viability, anchorage-independent growth and migration in vitro. Upon organic raft culturing, hsa-miR-9 hampered differentiation and induced proliferation in all strata of the epithelial layer. These findings support a potential oncogenic function of hsa-miR-9 in cervical cancer. In summary, differential expression of 106 miRNAs, partly associated with chromosomal alterations, was observed during cervical SCC development. Altered expression of hsa-miR-9 associated with a chromosomal gain of chromosome 1q was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis.

Reduced expression of tissue factor pathway inhibitor-2 contributes to apoptosis and angiogenesis in cervical cancer

BackgroundTissue factor pathway inhibitor-2 (TFPI-2) is an extracellular matrix associated broad-spectrum Kunitz-type serine proteinase inhibitor. Recently, down regulation of TFPI-2 was suggested to be involved in tumor invasion and metastasis in some cancers.MethodsThis study involved 12 normal cervical squamous epithelia, 48 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer. The expression of TFPI-2, Ki-67 and vascular endothelial growth factor (VEGF) were investigated by immunohistochemistry staining. The apoptolic index(AI) was determined with an in situ end-labeling assay(TUNEL). And the marker of CD34 staining was used as an indicator of microvessel density (MVD).ResultsTFPI-2 expression has a decreasing trend with the progression of cervical cancer and was significantly correlated with FIGO stage, lymph node metastasis and HPV infection. In addition, there were significant positive correlations between the grading of TFPI-2 expression and AI(P = 0.004). In contrast, the expression of TFPI-2 and VEGF or MVD was negatively correlated (both p < 0.001). However, we did not establish any significant correlation between Ki-67 and TFPI-2 expression in cervical cancer.ConclusionsThe results suggested that the expression of TFPI-2 had a decreasing trend with tumor progression of cervical cancer. There was a close association between the expression of TFPI-2 and tumor cell apoptosis and angiogenesis in patients with cervical cancer. TFPI-2 may play an inhibitive role during the development of cervical cancer.

The Mitochondrial Antisense ncRNAs are Down-Regulated in Early Cervical Carcinoma

Background: Cervical cancer is the third most common disease and the fourth leading cause of death in female’s worldwide. Treatment of this disease is successful when detection is at early neoplasic stages. Hence, multiple efforts have been pursued for early detection of cervical neoplasia. The aim of this study was to analyze the differential expression of mitochondrial non-coding RNAs or ncRNA (sense and antisense) in normal and neoplastic cervical biopsies as a potential tool for diagnostic of cervical cancer. Methods: The expression pattern of the sense and the antisense mitochondrial ncRNAs in cervical biopsies was carried out by chromogenic in situ hybridization (ISH). We examined 17 normal cervical tissues, 108 early and late neoplasias and 24 invasive cervical carcinomas. The hybridization results were compared with the diagnostic of each specimen carried out by pathologists. Results: Like normal human keratinocytes, normal cervical epithelium expresses the sense and the antisense mitochondrial ncRNAs at the basal layer of the epithelium. Interestingly, ISH reveals that the antisense transcripts are always localized in the nucleus of basal cells in normal cervical epithelium. Early and late cervical intraepithelial neoplasia as well as invasive cervical carcinoma expresses the sense transcript. In contrast, the antisense transcripts are down-regulated in early and late neoplasia and in invasive cervical carcinoma. Conclusions: This pilot study indicates that down-regulation of the antisense mitochondrial transcripts at early stages of cervical neoplasia can be explored as a diagnostic tool for early cervical neoplasia.

High ezrin immunoreactivity is an indicator of poor survival in squamous cell carcinoma of uterine cervix

Context: Ezrin, the membrane-linking protein, is highly expressed in several types of human cancers, and correlations between its immunoreactivity and histopathological data as well as patient outcome have previously been shown. Aims: The aim of our study was to investigate the expression of ezrin in cases of cervical squamous cell carcinoma, then correlate the data with survival and other clinicopathological variables. Materials and Methods: We carried out immunohistochemical analysis of ezrin in 64 cases of cervical cancer and correlated it to clinicopathologic variables and disease outcome. Results: Ezrin was identified in 50 (78.1%) of 64 studied cervical cancer cases, which was classified as weak, moderate and marked in 16%, 28% and 56%, respectively. In contrast to the predominantly membranous immunoreactivity in normal cervical epithelium, it was cytoplasmic in cancer cells. Ezrin expression significantly correlated with lymph node metastasis and development of distant metastasis (P<0.001 and 0.0006, respectively), as well as with tumor grade and stage (P=0.02 and 0.05, respectively), but not with other variables such as age, tumor size, and histological type. Regarding outcome, high ezrin expression was associated with short disease-specific survival (DSS) and disease-free survival (DFS) and poor metastasis survival (MS); (P<0.001). Ezrin expression significantly predicted both the 5-year MS, DSS, and DFS, recording P value<0.001 in each. Conclusions: Our results suggested that ezrin expression may represent an effective prognostic marker and a potential target for treatment of invasion and metastasis in cervical cancer.

Two novel unbalanced whole arm translocations are frequently detected in cervical squamous cell carcinoma

Chromosomal aberrations are a hallmark of human papillomavirus (HPV)-induced cervical carcinogenesis. The aim of this project was to identify structural chromosomal aberrations which may be characteristic for intraepithelial neoplasias (CIN) and cervical carcinomas (CxCa). Two independent HPV16 immortalized keratinocyte cell lines (HPKIA, HPKII) were used as a cell culture model system for cervical carcinogenesis. Different passages of HPKIA and HPKII were analyzed by multicolor spectral karyotyping. Several chromosomal translocations were identified in HPK cells and were validated by interphase fluorescence in situ hybridization (I-FISH). Three unbalanced whole chromosome arm translocations, der(10;14), der(7;21), and der(7;12), were cell line specific. The presence and frequency of these translocations were then examined by I-FISH in frozen tissue sections from normal cervical epithelia (n=6), CIN2/3 (n=15), and CxCa (n=15). The der(10;14) and der(7;21) were detected in 80% and 53.3% of CIN2/3, and in 60% and 46.7% of CxCa, respectively. The percentage of nuclei with translocations in individual lesions was significantly higher among CxCa. The der(7;12) could only be detected in 27% of CIN2/3. None of the translocations were detected in normal cervical epithelia. The translocated chromosomes may contribute to the clonal expansion of subpopulations in these cases and may thus be of diagnostic relevance.

Hox B4 as potential marker of non-differentiated cells in human cervical cancer cells

Cervical cancer (CC) is a common malignancy in women worldwide. Cervical tumorigenesis involves a multistep process in which accumulations of genetic alterations are present. Homeotic genes, such as HOX gene re-expression, have been reported in a wide variety of tumors. In order to know the role of HOX B4 gene expression in CC, in the present study, two-dimensional polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization, and time-of-flight mass spectrometry were used for differential screening of protein expression in CC. Immunohistochemical analysis was performed on the cervical tissue microarray (TMA) to detect the Hox B4 protein. Hox B4 peptide was detected among 15 increased spots differentially observed in CC. Using TMA, Hox B4 protein was also immunodetected in the nuclei of cervical epithelial tumor cells, while in normal cervical epithelium, it was absent. Interestingly, it was possible to detect the Hox B4 protein in the precursor lesions. Hox B4 protein is present in the precursor lesions as CC cells, suggesting that Hox B4 could be a protein related to the neoplastic state (non-differentiated cells) of human cervical epithelium.

Abstract A10: A new in silico physical optical model (iPOM) for the study of cervical intraepithelial neoplasia

Abstract Background: Cervical cancer is the second leading cause of deaths in women worldwide. More than 80% of cervical cancers and deaths occur in the developing world. New optical and imaging technologies (confocal imaging, fluorescence/absorbance spectroscopy, OCT, etc…) are being developed and tested as potential keys components of a necessary global solution to this problem. The success of these new technologies requires a better understanding of the precise interactions between the incident light, the underlying biological events and the physical structure of the cervical tissue. Herein, we present a new platform based on an in silico Physical Optical Model (iPOM), which is designed to simulate, analyze, and quantify the cell-and tissue-level biological changes that are associated with cervical preneoplastic growths. By reproducing these features, iPOM also gives insights into the ways in which dysplastic changes will be detected by different optical technologies being developed for cervical cancer screening, detection, diagnosis or treatment. Methods: The physical module of this platform is based on a database of more than 4000 cervical histological intraepithelial neoplastic specimens, collected from about 1800 women. As part of previous multicenters clinical trials (NIH funded Po1 project), Feulgen-stained sections were quantitatively analyzed by an high-resolution imaging software. Features describing the nuclear phenotype such as shape, size, orientation, nuclear membrane irregularities, DNA amount and DNA chromatin organization (ratio and spatial organization of euchromatin and heterochromatin) were calculated for each nucleus and the position of each of them relatively to the basal membrane automatically recorded. At the tissue level, epithelial thickness, cell density and tissue organization were assessed using sophisticated graph-theory algorithms measuring the intrinsic order/disorder and the loss of differentiation, trademarks of the dysplastic growth process. On a subset of 350 biopsies representing the entire spectrum of dysplastic and non-dysplastic abnormalities, p16 protein and Ki-67 protein expression were precisely evaluated by measuring the spatial distribution of Ki67-positive and p16-positive cells in the each individual epithelial layer. HPV typing and ploidy analysis were also performed for each patient. All these data were extrapolated to build a realistic (real metric dimension) cell-based physical model of the stratified normal cervical epithelium and of epithelium from each pathological grade. In the optical module, we used Finite-Difference Time-Domain (FDTD) modeling to simulate light scattering from normal, hyperplastic, metaplastic, CIN 1, CIN2, and CIS cervical cell nuclei at different epithelial depths and we constructed models of basal, parabasal, and other layers nuclei up to the last superficial layer for computational analysis. Simulation results give significant insights into the depth-dependent scattering profile of cervical epithelium as dysplasia progresses. We also intend to carry out meticulous investigation that will also enable mapping of azimuthal asymmetry in angle-dependent scattering patterns; this may lead to observation of fine pattern changes that can further be linked to precancer progression. Results: We will be presenting this novel conceptual framework illustrated by some preliminary data of our ongoing analyses using iPOM predicted results to guide acquisition of confocal imaging as well as spectroscopy probes from clinical cervical tissues. Ultimately, this strategy will allow us to identify the most robust in vivo macroscopic imaging approaches for detection of cervical neoplasia and aid in determining which image features must be acquired to achieve accurate diagnoses (e.g. variations in excitation and emission wavelength, numerical aperture, polarization, etc.). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A10.

Transcriptional analysis of CXCR4, DNMT3A, DNMT3B and DNMT1 gene expression in primary advanced uterine cervical carcinoma

The development of cervical cancer requires genetic and epigenetic factors which result in the persistence of a malignant phenotype. Cervical cancer exhibits also some unique differences from other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue with over-expression of HIF-1 (hypoxia-inducible factor-1) transcription factor, which targets the transcription of over 70 genes involved in many aspects of cancer biology. One of the genes, which could be induced by HIF-1 is chemokine (C-X-C motif) receptor 4 (CXCR4). CXCR4 could also be epigenetically regulated by methylation of CpG dinucleotides located in the promoter region. Here, we examined the CXCR4, DNMT3A, DNMT3B and DNMT1 transcript levels in cancer tissue (n=30) and non-cancer, normal uterine cervical tissue (n=30) from a Polish cohort. We also compared the methylation status of CXCR4 promoter region in cancer and normal tissue samples. Our result showed significantly higher levels of CXCR4, DNMT3A, DNMT3B and DNMT1 transcript (p=0.0058, 0.0163, 0.0003 and <0.0001, respectively) levels in cancer tissue as compared to normal samples. We did not observe DNA methylation in the CXCR4 promoter region in either control or cancer tissue samples. CXCR4 has a functional hypoxia response element (HRE) in the promoter region, located -1.3 kb from the transcription start site. Our work shows for the first time that HIF-1A could promote the induction of CXCR4 gene expression (Spearman's correlation coefficient = 0.515, p=0.003) in patients with primary advanced uterine cervical carcinoma.

Expression of Macrophage Migration Inhibitory Factor and CD74 in Cervical Squamous Cell Carcinoma

Macrophage migration inhibitory factor (MIF) and CD74 emerge as important players in pathogenesis and angiogenesis of several types of malignant tumors. The purpose of this study was to evaluate the expression of MIF and CD74 in cervical squamous cell carcinoma and explore the potential roles they play in cervical tumor angiogenesis. Macrophage migration inhibitory factor and CD74 expression was assessed by immunohistochemistry in 209 cases with various degrees of cervical epithelial lesions, including 40 normal cervical epithelia, 43 mild cervical intraepithelial neoplasia 1 (CIN 1), 41 moderate-severe cervical intraepithelial neoplasia (CIN 2 to 3), and 85 cervical squamous cell carcinomas (SCCs). CD34 staining was used for counting microvessel density. Semiquantitative reverse transcription polymerase chain reaction and Western blot were used to detect messenger RNA and protein levels of MIF and CD74 in normal and malignant cervical tissues and cervical cancer cell lines SiHa and C-33A. The concentration of vascular endothelial growth factor (VEGF) in the conditioned media of cervical cancer cells was analyzed by enzyme-linked immunosorbent assay. Immunohistochemical analysis showed that MIF and CD74 expression was significantly higher in CIN than in the normal samples and higher in SCC than in CIN. The overexpression of MIF was correlated with deep stromal infiltration but not with the other clinicopathologic features of SCC. Correlation analyses revealed that MIF was positively related to CD74, and both protein levels were associated with microvessel density. Exogenous MIF induced VEGF secretion in SiHa and C-33A cells in a dose-dependent manner, which can be inhibited by MIF-specific inhibitor (ISO-1) or anti-CD74 antibody. Overexpression of MIF and CD74 in SCC and its precancerous lesions and the up-regulation of VEGF secretion in cervical cancer cells indicate that MIF and CD74 may play critical roles in the pathogenesis and angiogenesis of cervical cancer.

Increased expression of HIF-1A and its implication in the hypoxia pathway in primary advanced uterine cervical carcinoma

The development of cervical cancer exhibits some unique differences compared to other solid tumors. Normal cervical stratified epithelia have characteristics of hypoxic tissue. Lack of oxygen (hypoxia) induces the HIF-1 (hypoxia-inducible factor-1) transcription factor, which is a heterodimer composed of a constitutively expressed β subunit and a hypoxia-inducible α-subunit. HIF-1A targets the transcription of over 70 genes involved in many aspects of cancer biology. In well-oxygenated environments, the HIF-1A subunit is hydroxylated, recognized and marked for proteosomal destruction by an E3 ubiquitin ligase, the von Hippel-Lindau protein (pVHL) complex. Under hypoxic stress, proline hydroxylase (PHD) activity is diminished, and stabilized HIF-1A is involved in the activation of the tissue response to hypoxia. Here, we examined the HIF-1A and VHL transcript levels and HIF-1A protein levels in cancerous tissue (n=30) and non-cancerous, normal uterine cervical tissue (n=30). We also compared the methylation status of HIF-1A and of the VHL promoter regions in cancerous and normal tissue samples. Significantly higher levels of HIF-1A and VHL transcripts (p<0.0001 and p=0.0042, respectively) and of HIF-1A protein (p=0.0037) were detected in cancerous tissue compared to normal samples. We did not observe DNA methylation in the HIF-1A and VHL promoter region in either control or cancerous tissue samples. VHL has a functional hypoxia response element (HRE) in the promoter region, and the induction of this HRE acts within a negative feedback loop to limit the hypoxic HIF-1A response. Our findings may suggest that HIF-1A could promote its own degradation by the induction of VHL gene expression (Spearman correlation coefficient, 0.515; p=0.003). Our study shows for the first time that this increase in VHL expression could be HIF-1A-dependent and serves within a negative feedback pathway during hypoxia to regulate the cell-specific oxygen threshold for HIF-1A activation.

Decreased D2-40 and increased p16INK4A immunoreactivities correlate with higher grade of cervical intraepithelial neoplasia

BackgroundD2-40 has been shown a selective marker for lymphatic endothelium, but also shown in the benign cervical basal cells. However, the application of D2-40 immunoreactivity in the cervical basal cells for identifying the grade of cervical intraepithelial neoplasia (CIN) has not been evaluated.MethodsIn this study, the immunoreactive patterns of D2-40, compared with p16INK4A, which is currently considered as the useful marker for cervical cancers and their precancerous diseases, were examined in total 125 cervical specimens including 32 of CIN1, 37 of CIN2, 35 of CIN3, and 21 of normal cervical tissue. D2-40 and p16INK4A immunoreactivities were scored semiquantitatively according to the intensity and/or extent of the staining.ResultsDiffuse D2-40 expression with moderate-to-strong intensity was seen in all the normal cervical epithelia (21/21, 100%) and similar pattern of D2-40 immunoreactivity with weak-to-strong intensity was observed in CIN1 (31/32, 97.2%). However, negative and/or focal D2-40 expression was found in CIN2 (negative: 20/37, 54.1%; focal: 16/37, 43.2%) and CIN3 (negative: 22/35, 62.8%; focal: 12/35, 34.3%). On the other hand, diffuse immunostaining for p16INK4A was shown in 37.5% of CIN1, 64.9% of CIN2, and 80.0% of CIN3. However, the immunoreactive pattern of D2-40 was not associated with the p16INK4A immunoreactivity.ConclusionsImmunohistochemical analysis of D2-40 combined with p16INK4A may have a significant implication in clinical practice for better identifying the grade of cervical intraepithelial neoplasia, especially for distinguishing CIN1 from CIN2/3.

Expression and Significance of SFRP1 and Wnt-1 in Cervical Squamous Cell Carcinoma

Objective To explore the expression and significance of SFRP1 and Wnt-1 in cervical squamous cell carcinoma.Methods SFRP1 and Wnt-1 were detected by immunohistochemistry(SP) in 20 cases of normal cervical squamous epithelium,30 cases of CINⅠ,40 cases of CINⅡ/Ⅲ and 50 cases of cervical squamous cell carcinoma.Results(1)In normal cervical squamous epithelium,CINⅠ,CINⅡ/Ⅲ and cervical squamous cell carcinoma,the positive expression rate of SFRP1 were 100%,73.5%,67.5% and 34% respectively.The positive expression rate of Wnt-1 were 25%,60%,82.5% and 98% respectively.There was significantly different among four groups(P0.05).(2)There was no correlation between SFRP1 and Wnt-1 in cervical squamous cell carcinoma.(3)In cervical squamous cell carcinoma group,there was no significant difference for SFRP1 and Wnt-1 expression in different age patient groups,and there was no difference among different tumor size,and clinical stages.However,there was significant difference among different pathological grades(P0.05).Conclusion SFRP1 and Wnt-1 were related to the process of cancerating of cervical squamous epithelium,which maybe play important roles in the development from cervical intraepithelium neoplasis to invasive cervical squamous cell carcinoma.

Identification of High-Risk Human Papillomavirus (hrHPV)-Associated Genes in Early Stage Cervical Squamous Cell Carcinomas

This retrospective study investigated gene expression in tumour samples from 38 patients with early stage human papillomavirus (HPV)-associated cervical squamous cell carcinoma (CSCC). The patients were divided into two groups based on the presence of viral markers of HPV16 or HPV18 infection. Gene expression profiles of tumour samples and the corresponding normal cervical epithelium were analysed using cDNA microarrays. Several genes showed differential expression between the two groups of HPV-infected CSCC patients, although seven genes showed similar changes in both groups. The four genes encoding cyclin-dependent kinase inhibitor 2A, matrix metallopeptidase 9, laminin γ-1, and epidermal growth factor receptor were up-regulated, and the three genes encoding transforming growth factor β receptor 1, interleukin-1α and insulin-like growth factor-binding protein 6 were down-regulated, in both HPV16(+) and HPV18(+) CSCC. These proteins are involved in cell proliferation, cell structure and cell attachment, so their expression might be involved in the mechanism of HPV-induced carcino genesis. A clearer understanding of HPV type-specific gene expression might aid diagnosis and treatment.

Reduced miR-100 expression in cervical cancer and precursors and its carcinogenic effect through targeting PLK1 protein

AimAlthough aberrant miRNAs expression has been documented, altered miR-100 expression in cervical cancer and precursor tissues and its carcinogenic effect and mechanism remain unexplored. The aim of our study was to investigate the role of miR-100 alteration in cervical carcinogenesis. MethodsThe expression of miR-100 was examined by quantitative real-time reverse transcriptase PCR (qRT-PCR) in 125 cervical tissues including normal cervical epithelium, cervical intraepithelial neoplasia (CIN), and cervical cancer, as well as in five cervical cell lines. Through modulating miR-100 expression using miR-100 inhibitor or mimic in vitro, cell growth, cycle and apoptosis were tested separately by MTT or flow cytometry and meanwhile Polo-like kinase1 (PLK1) mRNA and protein expressions were detected by qRT-PCR and immunoblotting. The expression of PLK1 in 125 cervical tissues was also examined by immunohistochemical staining and the correlation between miR-100 and PLK1 expression in the same tissues was analysed. Finally, HPV-16 E6/E7 expression was modulated by gene transfection and subsequently the level of miR-100 was examined by qRT-PCR. ResultsThe miR-100 expression showed a significantly and gradually reduced tendency from low-grade CIN, high-grade CIN to cervical cancer tissues and a significant decrease in HPV positive cervical cancer cell lines. The modulation of miR-100 expression remarkably influenced cell proliferation, cycle and apoptosis, as well as the level of PLK1 protein, but not mRNA, in vitro experiments. PLK1 expression was negatively correlated with miR-100 expression in CIN3 and cervical cancer tissues. The modulation of HR-HPV E6/E7 expression did not change miR-100 level. ConclusionsThe reduced miR-100 expression participates in the development of cervical cancer at least partly through loss of inhibition to target gene PLK1, which probably occurs in a relative late phase of carcinogenesis. HR-HPV E6/E7 may not directly regulate miR-100 expression in cervical cells.

Correlation between LRIG1 and LRIG2 expressions and expression of 11 tumor markers, with special reference to tumor suppressors, in CIN and normal cervical epithelium

Objective Novel biological markers LRIG1 and LRIG2 have been associated with favorable as well as poor prognosis, respectively, in different cancer types, including cervical cancer. The aim of this study was to investigate possible interactions between these proteins and other tumor markers, and as diagnostic adjuncts in CIN. Methods Cervical biopsies from 171 women, with normal epithelium, and low-grade and high-grade CIN were stained for LRIG1 and LRIG2, and 11 additional tumor markers. The tumor markers were chosen to be relevant in cervical neoplasms. Staining was evaluated semiquantitatively. Results Expression of LRIG1 and LRIG2 was found to correlate with increasing CIN grade, as well as with expression of tumor suppressor FHIT, independent of histological grade. In addition, tumor promoter LRIG2 expression correlated negatively with expression of tumor suppressor retinoblastoma protein and positively with IL-10. The latter correlation did not however remain after adjustment for CIN grade. p53 and p16 expressions correlated positively with LRIG1 expression in univariate analyses, but significance did not hold after adjustment for CIN grade. Conclusion LRIG1 and LRIG2 expressions were seen in precancerous cervical epithelium and found to increase with increasing grade. There was an association between expression of these glycoproteins and FHIT tumor suppressor protein, independently of histological grade.

A protein profile study to discriminate CIN lesions from normal cervical epithelium.

BackgroundCervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.MethodsCervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model.ResultsThe age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%.ConclusionsWater-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.

Abstract 4959: Deregulation of microRNA expression patterns during cervical carcinogenesis

Abstract At present little is known about the involvement of alterations in microRNA (miRNA) expression patterns during the development of cervical cancer. In this study we investigated genome-wide miRNA expression profiles in normal cervical squamous epithelium, high-grade precancerous lesions (CIN2/3), squamous cell carcinomas (SCC) and adenocarcinomas (AdCAs), all of which were high-risk HPV positive. In addition, miRNA expression profiles were integrated with previously generated genome-wide chromosomal profiles of the same samples. Unsupervised hierarchical clustering showed that miRNA expression profiling enables the distinction of normal cervical squamous epithelium, CIN2/3 lesions, SCCs and AdCAs. Differential miRNA expression analysis comparing normals, CIN2/3 lesions and SCCs identified early (temporal), late, and continuously differentially expressed miRNAs in cervical carcinogenesis (n=106). Differential expression of selected miRNAs could be validated by qRT-PCR in the same samples as well as in cervical scrapes of women with abnormal cytology and underlying high-grade CIN disease compared to control scrapes. In general, miRNAs located in the same cluster showed similar expression profiles, suggesting common regulatory mechanisms. For 5 of the 106 differentially expressed miRNAs, altered expression could be directly linked to underlying chromosomal alterations. Finally, cell viability assays indicated that ectopic expression of 2 selected miRNAs influenced cell proliferation in vitro, indicating functional relevance of differential miRNA expression during cervical carcinogenesis. In conclusion, deregulation of miRNA expression patterns occurs during cervical carcinogenesis and has functional relevance for cervical cancer development. Since deregulated miRNA expression in cervical (pre)malignant disease is reflected and detectable in cervical scrapes, miRNAs represent a highly promising class of disease markers for further improvement of the triage of high-risk HPV positive women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4959. doi:10.1158/1538-7445.AM2011-4959

FTS (fused toes homolog) a novel oncoprotein involved in uterine cervical carcinogenesis and a potential diagnostic marker for cervical cancer

The high incidence and fatality rate of uterine cervical cancer warrant effective diagnostic and therapeutic target identification for this disease. Here, we have found a novel oncoprotein FTS (Fused Toes Homolog), which is involved in cervical cancer pathogenesis. Immunohistochemical analysis of human cervical biopsy samples revealed that the expression of FTS is absent in normal cervical epithelium but progressively overexpressed in human cervical intraneoplastic lesions (CIN-I to CIN-III), this characteristic phenomenon put this protein, a potential diagnostic marker for the screening of early neoplastic changes of cervix. Using FTS-specific small hairpin RNA (shRNA) in cervical cancer cells, we determined a specific role for FTS protein in, cervical neoplasia. Targeted stable knock down of FTS in HeLa cells led to the growth inhibition, cell-cycle arrest, and apoptosis with concurrent increase in p21 protein. FTS effectively represses the p21 mRNA expression in dual luciferase assay which indicates that p21 is transcriptionally regulated by this oncoprotein which in turn affect the regular cell-cycle process and its components. Consistent with this we found a reciprocal association between these proteins in early cervical neoplastic tissues. These data unraveled the involvement of new oncoprotein FTS in cervical cancer which plays a central role in carcinogenesis. Targeted inhibition of FTS lead to the shutdown of key elemental characteristics of cervical cancer and could lead to an effective therapeutic strategy for cervical cancer.