Noncancerous Cervical Tissues Research Articles

Elevated expression of protein-L-isoaspartate O-methyltransferase-1 (PCMT1) in cervical cancer.

BackgroundProtein-L-isoaspartate O-methyltransferase-1 (PCMT1) is a protein carboxyl methyltransferase enzyme, which has been found to play roles in cancers. However, no clinical information about the correlation between cervical cancer and PCMT1 expression has been reported.MethodsWe used immunohistochemistry (IHC) to characterize the protein level of PCMT1 in human cervical intraepithelial neoplasia and cervical cancer specimens. The mRNA expression profile of PCMT1 in cervical cancer was also analyzed by using Gene Expression Omnibus (GEO) databases. The prognostic value of PCMT1 in patients with cervical cancer was evaluated by using the Kaplan-Meier plotter. Gene set enrichment analysis (GSEA) was conducted by using The Cancer Genome Atlas (TCGA) cervical cancer dataset.ResultsThe protein level of PCMT1 was increased in cervical high-grade squamous intraepithelial lesion (HSIL) (7.40±0.42) and cervical cancer tissues (10.70±0.54), compared to normal cervix (5.00±0.86) and low-grade squamous intraepithelial lesion (LSIL) (6.22±0.57) (P<0.05). the immunoreactivity score (IRS) of PCMT1 was also higher in cervical cancer tissues than in paired adjacent non-cancerous cervical tissues (9.03±0.52 vs. 6.32±0.46) (P<0.05). High expression of PCMT1 was associated with decreased overall survival (OS) of patients with cervical cancer (P=0.0022). GSEA demonstrated that cervical cancer patients with high expression of PCMT1 were enriched in the various cancer-related signaling pathways.ConclusionsThese results suggest that PCMT1 might be a diagnostic and prognostic biomarker for cervical cancer, and further validation studies should be performed.

Expression Analysis of MIST1 and EMT Markers in Primary Tumor Samples Points to MIST1 as a Biomarker of Cervical Cancer.

BackgroundMist1 is a basic transcription factor, which plays an important role in the development of multiple organs, and may also regulate tumor progression by mediating epithelial-mesenchymal transformation. However, there is lack of research on its role of squamous cell carcinoma, especially in cervical squamous cell carcinoma.MethodsBioinformatic methods were used to analyze gene expression, correlation, and patient survival according to the TCGA database. Thirty pairs of cancer tissues and distal cancer tissues from cervical cancer patients who received radical surgery were enrolled in the study. The expression of Mist1 was analyzed using Western blot. Furthermore, the potential associations among Mist1 expression, EMT biomarkers and various clinicopathological characteristics were investigated. All statistical tests employed in this study were two-sided, and P values <0.05 were deemed statistically significant.ResultsOverall survival data were obtained from TCGA-CESC dataset, containing 3 control samples and 305 tumor samples. The expression of Mist1 was significantly higher in primary tumor than in normal tissues (P<0.001). The samples were divided into a low Mist1 expression group (n=144) and a high Mist1 expression group (n=146) according to the median expression level. Kaplan–Meier survival analysis revealed that high expression of Mist1 was significantly correlated with poor overall survival (P=0.032). We further explored the relationships between Mist1 and EMT. Among the 30 primary cervical cancer specimens investigated, the difference in Mist1 expressed statuses between cervical cancer tissues and distal noncancerous cervical tissues was significant (P=0.001). And the epithelial cell marker E-cadherin was downregulated in Mist1 overexpressed cervical cancer cells; however, the mesenchymal marker N-Cadherin and Twist was upregulated.ConclusionOur study found that Mist1 seemed to play the role of oncogene in cervical squamous cell carcinoma and could be a potential biomarker.

Oxidative stress markers in patient-derived non-cancerous cervical tissues and cells

High-risk human papillomaviruses (HPV) are the causative agents of cervical cancer. However, not all infected women develop cervical cancer. Cervical tumorigenesis is characterized by a multifactorial etiology, with oxidative stress (OS) likely playing a major role. In addition to exogenous sources, metabolic processes also contribute to OS. In principle, variability in levels of cervical OS has the potential to influence the likelihood of conversion to cervical cancer. To ask whether such variability indeed existed, we assessed the levels of ROS and the oxidative DNA damage biomarker 8-oxodG in normal non-cancerous cervical tissues and cells obtained from women with uterovaginal pelvic organ prolapse following vaginal hysterectomy. We demonstrated five and ten-fold variability between tissues isolated from the transformation zone (TZ) and ectocervix (EC) of different women, respectively. Despite the greater variability (likely due to differences in tissue composition), the overall pattern of ROS levels in EC tissues mirrored those obtained in their corresponding TZ tissues. Our results also show that the levels of ROS in TZ tissues were always higher than or equal to those found in the respective EC tissues, providing a possible explanation for TZ tissue being the primary target for HPV infection and cervical carcinogenesis. Interestingly, primary keratinocytes isolated and cultured from these cervical specimens also displayed high variability in ROS levels, with some strongly mirroring the levels of ROS observed in their corresponding tissues, while others were less closely associated. Finally, we demonstrated that the levels of DNA damage mirrored the levels of ROS in the cultured primary cells. Understanding the factors and mechanisms that dispose certain individuals to develop cervical cancer has the potential to enable the development of approaches that make the conversion of HPV infection to cancer development even more rare.

S100 Calcium Binding Protein A11 (S100A11) Promotes The Proliferation, Migration And Invasion Of Cervical Cancer Cells, And Activates Wnt/β-Catenin Signaling.

PurposeThis study is aimed to investigate the specific regulatory role of S100 calcium binding protein A11 (S100A11) on cervical cancer (CC), and reveal the potential mechanisms relating to Wnt/β-catenin signaling.Patients and methodsThe expression of S100A11 in cervical squamous cell carcinoma (CSCC), adjacent non-cancerous, cervical intraepithelial neoplasia (CIN), and normal cervical tissues was detected by quantitative real-time PCR and/or immunohistochemistry. After transfection of pENTER-S100A11 or sh-S100A11-1/sh-S100A11-2, the viability, cell cycle, migration and invasion of C33A or SiHa cells were detected. The tumor volume and tumor weight were measured after injection of transfected C33A cells into mice. The expression of E-caherin (CDH2), N-caherin (CDH1), β-catenin (CTNNB1), and c-Myc (MYC) in C33A and SiHa cells was detected by Western blot.ResultsThe expression of S100A11 was significantly higher in CSCC tissues than in adjacent non-cancerous, CIN, and normal cervical tissues (P < 0.05). S100A11 expression was positively correlated with the FIGO stage and lymph node metastasis of CSCC patients (P < 0.05). The transfection of pENTER-S100A11 into C33A cells significantly increased the cell viability, the percentage of cells in G2/M phase, the numbers of migratory and invasive cells, as well as the tumor volume and weight in mice (P < 0.05). Overexpression of S100A11 also significantly downregulated E-caherin, and upregulated N-caherin, β-catenin, and c-Myc in C33A cells (P < 0.05). The transfection of sh-S100A11-1/sh-S100A11-2 exhibited the opposite results to that of pENTER-S100A11 on SiHa cells.ConclusionOverexpression of S100A11 promotes the proliferation, migration, invasion, and epithelial-mesenchymal transition of CC cells, and activates Wnt/β-catenin signaling.

Long noncoding RNA HOTAIR knockdown inhibits autophagy and epithelial-mesenchymal transition through the Wnt signaling pathway in radioresistant human cervical cancer HeLa cells.

Cervical cancer is one of the most common female malignancies around the world, and radiation resistance is a major obstacle to cancer therapy. Previously, overexpression of the long noncoding ribonucleic acid (RNA) (lncRNA) HOX transcript antisense RNA (HOTAIR) has been found to be associated with the invasion and metastasis capacities of several epithelial cancers, including cervical cancer. To gain insights into the molecular mechanisms of HOTAIR in cervical cancer resistance to radiotherapy, we investigated cellular autophagy and epithelial-to-mesenchymal transition (EMT) in radioresistant human cervical cancer HeLa cells when HOTAIR was suppressed.HOTAIR levels were quantified in cancerous and noncancerous cervical tissues obtained from 108 patients with cervical cancer. Next, we inhibited HOTAIR by RNA interference and activated the Wnt signaling pathway by LiCl in radioresistant HeLa cells to investigate the regulatory mechanisms for the HOTAIR mediating Wnt signaling pathway.We determined that the upregulated HOTAIR may contribute to cervical cancer progression. We found that the short interfering ribonucleic acid (siRNA)-mediated knockdown of HOTAIR disturbed the Wnt signaling pathway, reduced autophagy, inhibited EMT, decreased cell proliferation, and induced apoptosis in radioresistant HeLa cells. It is worthy to note that the combination treatment of siRNA-HOTAIR and LiCl demonstrated that the activation of the Wnt signaling pathway is responsible for the beneficial effect of HOTAIR knockdown in enhancing sensitivity to radiotherapy in radioresistant HeLa cells.Together, our results revealed an important role of HOTAIR in regulating cervical cancer resistance to radiotherapy. Functional suppression of HOTAIR could enhance sensitivity to radiotherapy by reduction of autophagy and reversal of EMT through the suppression of the Wnt signaling pathway.

The association of CCAT2 rs6983267 SNP with MYC expression and progression of uterine cervical cancer in the Polish population

PurposePrevious studies have reported a significant contribution of NC_000008.10:g.128413305 G>T (rs6983267) single-nucleotide polymorphism (SNP) in the MYC enhancer region to the susceptibility of various cancers. However, the role of rs6983267 SNP in cervical cancer (CC) development and progression has not been demonstrated to date. Therefore, we evaluated the role of rs6983267 SNP in MYC expression in cervical cancers and non-cancerous cervical tissues. In addition, we assessed the role of this SNP in the development and progression of CC.MethodsUsing high-resolution melting analysis, we evaluated rs6983267 SNP frequency in women diagnosed with cervical squamous cell carcinoma (SCC) (n = 481) and controls (n = 502) in a Polish Caucasian population. Logistic regression analysis was employed to adjust for the effects of age, parity, oral contraceptive use, tobacco smoking, and menopausal status.ResultsDividing patients based on clinical characteristics demonstrated an association of the rs6983267 genotype with tumor stage III and grade of differentiation G2 and G3. The p trend value calculated for the rs6983267 SNP in patients with stage III was 0.0006. We also observed a significant contribution of rs6983267 SNP to tumor grade of differentiation G2 and G3. Additional contributors were oral contraceptive use, smoking, and postmenopausal age. We found statistically significant increase of MYC transcript levels in cervical SCC tissues from carriers of the GG vs. T/T (p < 0.00001), G/T vs. T/T (p = 0.0002), and in the non-cancerous cervical tissues from carriers of the GG vs. T/T (p = 0.00046).ConclusionThe rs6983267 SNP may contribute to the increased MYC expression as well as the spread and rapid growth of cervical SCC as compared to lower grade carcinomas.

Expression of KATP channels in human cervical cancer: Potential tools for diagnosis and therapy.

Various ion channels, including ATP-sensitive potassium (KATP) channels, are expressed in cancer and have been suggested as potential tumor markers and therapeutic targets. KATP channels are composed of at least two types of subunit, an inwardly rectifying K+ channel (Kir6.x) and a sulfonylurea receptor (SUR). However, the association between KATP channels and cervical cancer remains elusive. The present study determined that the Kir6.2, SUR1 and SUR2 subunits are expressed in cervical cancer cell lines and/or human biopsies. The potential association of subunit expression with tumor differentiation and invasion was analyzed. The effect of the KATP channel blocker glibenclamide on the proliferation of cervical cancer cell lines was also studied. Five cervical cancer cell lines, two primary cultures of cervical cancer cells, one normal keratinocyte cell line and 74 human biopsies were used in the experiments. The mRNA and protein levels of the Kir6.2 subunit were assessed by reverse transcription-polymerase chain reaction and immunochemistry, respectively. Cell proliferation was evaluated by MTT assay. Kir6.2 subunit overexpression compared with control, was observed in some cervical cancer cell lines and cervical tumor tissues. Additionally, increased KATP channel expression was observed in high-grade, poorly differentiated and invasive human cervical cancer biopsies. Kir6.2 subunit expression was not observed in the majority of the non-cancerous cervical tissues. The effect of the KATP channel blocker glibenclamide on the proliferation of five different cervical cancer cell lines was studied, revealing that as Kir6.2 mRNA expression increased, the inhibitory effect of glibenclamide also increased. The results of the present study suggest, for the first time to the best of our knowledge, that the KATP channel subunits, Kir6.2 and SUR2, could potentially represent tools for diagnosing and treating cervical cancer.

High expression of FUNDC1 predicts poor prognostic outcomes and is a promising target to improve chemoradiotherapy effects in patients with cervical cancer.

FUN14 domain containing 1 (FUNDC1) is an important molecule in receptor‐dependent mitophagy. However, the roles of FUNDC1 in human cancer biology remain unknown. The aim of this study was to explore the expression and roles of FUNDC1 in cervical cancer. Immunohistochemistry and Western blotting were applied to detect the expression of FUNDC1, and small‐hairpin RNA was applied to inhibit the expression of endogenous FUNDC1 in cervical cancer cells. MTT assays and Flow cytometric analysis were applied to examine cell proliferation and apoptosis. Immunofluorescence was used to detect the formation of γH2AX foci and evaluate the extent of DNA damage. Compared with corresponding adjacent noncancerous cervical tissues, the expression of FUNDC1 in cervical cancer cells was significantly increased. High expression of FUNDC1 and the prognosis of patients with cervical cancer were correlated negatively, which could be used as an independent prognostic factor for overall survival and disease‐free survival. Depletion of FUNDC1 significantly inhibited the proliferation of tumor cells, induced apoptosis, and enhanced cell sensitivity to cisplatin and ionizing radiation (IR). Our data suggested that FUNDC1 can be used as a prognostic biomarker in patients with cervical cancer, and may be a new therapeutic target to improve the antitumor effects of chemoradiotherapy.

Role of rs13117307 single nuclear polymorphism in the risk of uterine cervical cancer from Polish population and its impact on exocyst complex component 1 expression

We evaluated the role of NM_001024924.1:c.1330+1646C>T (rs13117307) single nucleotide polymorphism (SNP), situated in the intronic region of exocyst complex component 1 (EXCO1), in the development and spreading of cervical squamous cell carcinoma (SCC). Utilizing high resolution melting curve analysis, we analyzed this polymorphism in patients with cervical SCC (n=485) and controls (n=509) in the Polish Caucasian population. Logistic regression analysis was used to adjust for age, parity, oral contraceptive use, tobacco smoking, and menopausal status. The influence of this polymorphism on the expression of EXCO1 was assessed by reverse transcription and real-time quantitative PCR analysis. For all patients with SCC, the p trend value calculated for rs13117307 was statistically significant (ptrend=0.0158). The adjusted odds ratio (OR) for T/T vs. C/C was 1.434 (95 % CI 1.105–1.861, p=0.007). We also found a significant contribution of rs13117307 to tumor stages III, IV and grade of differentiation G3. Other contributors are parity, oral contraceptive use, smoking, and women of postmenopausal age. We observed significant upregulation of EXCO1 transcript levels in the non-cancerous cervical tissues in carriers of the T/T vs. C/C (p=0.016), as well as an increase in the EXCO1 transcript levels in the cervical SCC tissue in carriers of the T/T vs. C/C (p=0.029) and for T/T vs C/T (p=0.0032). The rs13117307 SNP variants may upregulate the transcription of EXCO1, as well as the risk of development and spreading of cervical SCC.

Transcript levels of ten-eleven translocation type 1-3 in cervical cancer and non-cancerous cervical tissues.

Decreased expression of ten-eleven translocation (TET1, TET2 and TET3) proteins has been reported in various types of cancer. However, the expression levels of TET proteins in cervical cancer (CC) remain to be elucidated. The present study determined the levels of TET1, TET2 and TET3 transcripts in cancerous (n=80) and non-cancerous cervical tissues (n=41). The results revealed a significant reduction in TET1 transcripts (P=0.0000001) in cervical tissue samples from patients with primary CC compared with samples from control patients. Significantly decreased TET1 transcript levels, as compared to non-cancerous cervical tissues, were also observed in tissue samples with the following characteristics: Stage I (P=0.016), II (P<0.0001), III (P=0.00007) and grade of differentiation G1 (P=0.026), G2 (P=0.00006), G3 (P=0.0007) and Gx (P=0.0004) and squamous histological type (P<0.00001). TET1 transcript levels were significantly lower in patients aged 45-60 years (P=0.0002) and patients age >60 years (P=0.003), as compared with non-cancerous cervical tissues. TET2 transcript levels were lower in cervical cancer tissues classified as stage II (P=0.043) and TET3 transcript levels were lower in stage III samples (P=0.010), tissue samples with a grade of differentiation of G3 (P=0.025) and tissue with squamous type histology (P=0.047), all compared with non-cancerous cervical tissues. The present study demonstrated a significantly reduced level of TET1 transcripts in cancerous cervical tissues, as compared with non-cancerous tissues. Furthermore, decreased TET1-3 transcript levels were identified when patients with CC were stratified by clinicopathological variables, as compared with non-cancerous cervical tissues.

MicroRNA-145 modulates epithelial-mesenchymal transition and suppresses proliferation, migration and invasion by targeting SIP1 in human cervical cancer cells.

Previously, it has been reported that microRNA-145 (miR-145) is lowly expressed in human cervical cancers and that its putative tumour suppressive role may be attributed to epithelial-mesenchymal transition (EMT) regulation. Here, we aimed to assess whether miR-145 may affect EMT-associated markers/genes and suppress cervical cancer growth and motility, and to provide a mechanistic basis for these phenomena. The identification of the SMAD-interacting protein 1 (SIP1) mRNA as putative miR-145 target was investigated usinga 3' untranslated region (3'UTR) luciferase assay and Western blotting, respectively. The functional effects of exogenous miR-145 expression, miR-145 suppression or siRNA-mediated SIP1 expression down-regulation in cervical cancer-derived C33A and SiHa cells were analysed using Western blotting, BrdU incorporation (proliferation), transwell migration and invasion assays. In addition, the expression levels of miR-145 and SIP1 were determined in primary human cervical cancer and non-cancer tissue samples using qRT-PCR. We found that miR-145 binds to the wild-type 3'UTR of SIP1, but not to its mutant counterpart, and that, through this binding, miR-145 can effectively down-regulate SIP1 expression. In addition, we found that exogenous miR-145 expression or siRNA-mediated down-regulation of SIP1 expression attenuates the proliferation, migration and invasion of C33A and SiHa cells and alters the expression of the EMT-associated markers CDH1, VIM and SNAI1, whereas inhibition of endogenous miR-145 expression elicited the opposite effects. The expression of miR-145 in cervical cancer tissue samples was found to be low, while that of SIP1 was found to be high compared to non-cancerous cervical tissues. An inverse expression correlation between the two was substantiated through the anlaysis of data deposited in the TCGA database. Our data indicate that low miR-145 expression levels in conjunction with elevated SIP1 expression levels may contribute to cervical cancer development. MiR-145-mediated regulation of SIP1 provides a novel mechanistic basis for its tumour suppressive mode of action in human cervical cancer cells.

NR2F6 Expression Correlates with Pelvic Lymph Node Metastasis and Poor Prognosis in Early-Stage Cervical Cancer.

Background: There is an abnormal expression of nuclear receptor subfamily 2 group F member 6 (NR2F6) in human cancers such as breast cancer, colon cancer, and acute myelogenous leukemia. However, its clinical significance in cervical cancer has not been established. We explored NR2F6 expression and its clinicopathological significance in early-stage cervical cancer. Methods: NR2F6 expression in cervical cancer cell lines and cervical cancer tissues was determined by Western blotting, real-time PCR, and immunochemistry (IHC). NR2F6 expression in 189 human early-stage cervical cancer tissue samples was evaluated using IHC. The relevance between NR2F6 expression and early-stage cervical cancer prognosis and clinicopathological features was determined. Results: There was marked NR2F6 mRNA and protein overexpression in the cervical cancer cells and clinical tissues compared with an immortalized squamous cell line and adjacent noncancerous cervical tissues, respectively. In the 189 cervical cancer samples, NR2F6 expression was positively related to International Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.006), squamous cell carcinoma antigen (p = 0.006), vital status (p < 0.001), tumor recurrence (p = 0.001), chemotherapy (p = 0.039), and lymph node metastasis (p < 0.001). Overall and disease-free survival was shorter in patients with early-stage cervical cancer and higher NR2F6 levels than in patients with lower levels of NR2F6. Univariate and multivariate analysis determined that NR2F6 was an independent prognostic factor of survival in early-stage cervical cancer. Conclusions: Taken together, our findings suggest that high NR2F6 expression predicts pelvic lymph node metastasis, tumor recurrence and poor prognosis in early-stage cervical cancer. NR2F6 might be a novel prognostic biomarker and potential therapeutic target of cervical cancer.

Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells.

The expression of immediate early response 3 (IER3), a protein with a short half-life, is rapidly induced by various cellular stimuli. We recently reported that IER3 induces the apoptosis of cervical cancer cells and that its expression is downregulated in patients with cervical cancer. However, the molecular mechanism involved in the rapid degradation of IER3 remains unknown. Here, we demonstrate that MDM2 is an E3 ligase that interacts with IER3 and promotes its ubiquitination, followed by proteasomal degradation. Polyubiquitination of the conserved lysine 60 of IER3 is essential for its degradation. In addition, four and a half LIM domains protein 2 (FHL2) binds to both IER3 and MDM2, allowing for efficient MDM2-mediated IER3 degradation by facilitating an association between MDM2 and IER3. Moreover, IER3 induces cell cycle arrest in cervical cancer cells and its activity is further enhanced in cells in which FHL2 or MDM2 was silenced, thereby preventing IER3 degradation. The E6 and E7 oncoproteins of human papilloma virus 18 regulated IER3 expression. FHL2 expression was significantly higher in the squamous epithelium of cervical carcinoma tissues than in non-cancerous cervical tissues, whereas cervical carcinoma expression of IER3 was downregulated in this region. Thus, we determined the molecular mechanism responsible for IER3 degradation, involving a ternary complex of IER3, MDM2 and FHL2, which may contribute to cervical tumor growth. Furthermore, we demonstrated that FHL2 serves as a scaffold for E3 ligase and its substrate during the ubiquitination reaction, a function that has not been previously reported for this protein.

B3GNT3 Expression Is a Novel Marker Correlated with Pelvic Lymph Node Metastasis and Poor Clinical Outcome in Early-Stage Cervical Cancer.

BackgroundThe β1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) encodes a member of the B3GNT family that functions as the backbone structure of dimeric sialyl-Lewis A and is involved in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. B3GNT3 has been implicated as an important element in the development of certain cancers. However, the characteristics of B3GNT3 in the development and progression of cancer remain largely unknown. Thus, our study aimed to investigate the expression pattern and the prognostic value of B3GNT3 in patients with early-stage cervical cancer.MethodsThe mRNA and protein levels of B3GNT3 expression were examined in eight cervical cancer cell lines and ten paired cervical cancer tumors, using real-time PCR and western blotting, respectively. Immunohistochemistry (IHC) was used to analyze B3GNT3 protein expression in paraffin-embedded tissues from 196 early-stage cervical cancer patients. Statistical analyses were applied to evaluate the association between B3GNT3 expression scores and clinical parameters, as well as patient survival.Results B3GNT3 expression was significantly upregulated in cervical cancer cell lines and lesions compared with normal cells and adjacent noncancerous cervical tissues. In the 196 cases of tested early-stage cervical cancer samples, the B3GNT3 protein level was positively correlated with high risk TYPES of human papillomavirus (HPV) infection (P = 0.026), FIGO stage (P < 0.001), tumor size (P = 0.025), tumor recurrence (P = 0.004), vital status (P < 0.001), concurrent chemotherapy and radiotherapy (P = 0.016), lymphovascular space involvement (P = 0.003) and most importantly, lymph node metastasis (P = 0.003). Patients with high B3GNT3 expression had a shorter overall survival (OS) and disease-free survival (DFS) compared with those with low expression of this protein. Multivariate analysis suggested that B3GNT3 expression is an independent prognostic indicator for cervical cancer patients.ConclusionsOur study demonstrated that elevated B3GNT3 expression is associated with pelvic lymph node metastasis and poor outcome in early-stage cervical cancer patients. B3GNT3 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer.

URG4 overexpression is correlated with cervical cancer progression and poor prognosis in patients with early-stage cervical cancer.

BackgroundUpregulator of cell proliferation 4 (URG4) has been implicated in the oncogenesis of certain cancers. However, the correlation between URG4 expression and clinicopathological significance in human cancer remains unclear. Therefore, this study investigated its expression and clinicopathological significance in cervical cancer patients.MethodsURG4 expression was examined using quantitative PCR (qPCR) and western blotting in normal cervical epithelial cells, cervical cancer cells, and eight matched pairs of cervical cancer tissues and adjacent noncancerous tissues from the same patient. In addition, immunohistochemistry (IHC) was used to examine URG4 expression in paraffin-embedded tissues from 167 cervical cancer patients (FIGO stages Ib1-IIa2). Statistical analyses were performed to evaluate associations between URG4 expression and prognostic and diagnostic factors.ResultsURG4 was significantly upregulated in the cervical cancer cell lines and tissues compared with the normal cells and adjacent noncancerous cervical tissues. IHC revealed high URG4 expression in 59 out of the 167 (35.13%) cervical cancer specimens. Its expression was significantly correlated with clinical stage (P < 0.0001), tumour size (P = 0.012), T classification (P = 0.023), lymph node metastasis (P = 0.001) and vaginal involvement (P = 0.002). Patients with high URG4 expression, particularly those who received concurrent chemotherapy and radiotherapy (P < 0.0001), showed a shorter overall survival (OS) and disease-free survival (DFS) compared to those with the low expression of this protein. Multivariate analysis revealed that URG4 expression is an independent prognostic factor for cervical cancer patients.ConclusionsOur results demonstrated that elevated URG4 protein expression is associated with a poor outcome in patients with early-stage cervical cancer. URG4 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-885) contains supplementary material, which is available to authorized users.

Abstract 286: Expression and role of fibroblast growth factor receptor2 IIIc in human uterine cervical intraepithelial neoplasia (CIN) and invasive cervical cancer

Abstract Fibroblast growth factor receptors (FGFR) FGFR1 through FGFR3 possess IIIb and IIIc isoforms as the result of alternative splicing of the immunoglobulin-like domain. The IIIb isoforms are mainly expressed in normal epithelial cells, and IIIc isoforms are expressed in mesenchymal cells. Single nucleotide polymorphisms (SNPs) of the FGFR2 gene are associated with endometrial cancer, and missense mutations or copy number gains of the FGFR2 gene occur in breast cancer. In uterine cervical cancer, FGFR2 IIIb, also known as keratinocyte growth factor receptor (KGFR), was reported to be highly expressed in squamous cell carcinoma (SCC), which accounts for 90% of all cervical cancer patients. However, there has been no report on the expression and roles of FGFR2 IIIc in cervical cancer. In the present study, we determined the expression and roles of FGFR2 IIIc in CIN and cervical SCC. In noncancerous cervical tissues, FGFR2 IIIc immunoreactivity was either not present or very faintly localized in squamous epithelial cells. In CINs 1 and 2, FGFR2 IIIc was localized at the basal to lower two-thirds of squamous epithelium, whereas FGFR2 IIIc was strongly expressed in most of the squamous epithelium, except for the superficial layer in CIN 3. FGFR2 IIIc protein was detected in keratinizing and non-keratinizing types of SCC in all cervical cancer patients examined (8 and 21 cases, respectively). In situ hybridization (ISH) analysis using an FGFR2 IIIc-specific antisense RNA probe showed that the expression patterns of FGFR2 IIIc mRNA are similar to those of FGFR2 IIIc protein in noncancerous and CIN tissues. In addition, FGFR2 IIIc mRNA was strongly expressed in the invasive fronts of cancer cell nests in cancer tissues. Moreover, full-length FGFR2 IIIc cDNA, subcloned to pIRES2-EGFP vector, was stably transfected into CaSki cells, which are derived from a well-differentiated type of SCC. The growth rates of the FGFR2 IIIc-transfected CaSki cells were higher than those of mock cells in vitro. Cell migration assays did not show any differences between FGFR2 IIIc-transfected CaSki cells and mock cells. To assess the effect of FGFR2 IIIc expression on tumorigenicity, the CaSki cells were injected into nude mice. The FGFR2 IIIc-transfected CaSki cells tended to form larger subcutaneous tumors than mock cells in nude mice. These findings suggest that FGFR2 IIIc plays important roles in the carcinogenesis and proliferation of cervical cancer cells. FGFR2 IIIc is considered to be a novel therapeutic target for inhibiting the growth of CIN and cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 286.

Epidermal Growth Factor-activated Aryl Hydrocarbon Receptor Nuclear Translocator/HIF-1β Signal Pathway Up-regulates Cyclooxygenase-2 Gene Expression Associated with Squamous Cell Carcinoma

Hypoxia-inducible factor (HIF) accumulates when tumors grow under hypoxic conditions. The genesis of tumors, however, usually involves normoxic conditions. In this study, we were interested in examining the potential role of aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta in tumor growth under normoxic conditions, specifically when cells are treated with epidermal growth factor (EGF), which is known to affect the gene expression of tumor growth-related protein COX-2 (cyclooxygenase-2). The results showed that EGF receptor inhibitor, AG1478, abolished EGF-induced nuclear accumulation of ARNT as well as the expression of COX-2. ARNT small interfering RNA inhibited the promoter activity, mRNA level, and protein expression of COX-2 in cells treated with EGF. In contrast, CoCl(2)-induced HIF-1alpha exhibited no effect on COX-2 expression. EGF also stimulated the formation of the ARNT.c-Jun complex as well as the complex binding to the COX-2 promoter. ARNT small interfering RNAs blocked EGF-activated cell migration. Moreover, COX-2 and ARNT were cohorts present distinctively in clinical specimens of human cervical squamous cell carcinoma and were almost nondetectable in adjacent normal or noncancerous cervical tissues. Our results revealed that ARNT plays an important role in EGF-regulated COX-2 gene expression and may thus be related to either a cause or a consequence of tumorigenesis in cervical cancer.

EGF upregulates Na+/H+ exchanger NHE1 by post‐translational regulation that is important for cervical cancer cell invasiveness

Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.

Functional Analysis of Bladder Cancer-Related Protein Gene: A Putative Cervical Cancer Tumor Suppressor Gene in Cervical Carcinoma

Our previous study has suggested thatthe bladder cancer-associated protein gene (BLCAP) was among the differentially expressed genes in cervical cancer. We confirm here that BLCAP is expressed in all noncancerous cervical tissues (10/10), but it is greatly lost in primary cervical cancer tissue (31/39). In order to further investigate the functional roles of BLCAP, we stably transfected BLCAP cDNA into HeLa cells. The HeLa cells expressing BLCAP show reduced cell growth and clone genicity compared to the vector-transfected cognate cells. BLCAP expression in HeLa cells leads to growth arrest and significantly enhanced apoptosis in vitro and reduced tumor formation in vivo. Thus, BLCAP might be a potential tumor suppressor gene in cervical carcinoma.

Expression and accumulation of lumican protein in uterine cervical cancer cells at the periphery of cancer nests

Lumican is a member of a small leucine-rich proteoglycan (SLRP) family and is reported to be overexpressed during the wound healing process of the cornea, and ischemic and reperfused heart. In the carcinomatous tissues, lumican is overexpressed in human breast and pancreatic cancer tissues. In the present study, we aimed to clarify the expression of lumican mRNA and its protein in human cervical cancer cell lines (CaSki, ME-180 and HeLa cells) and their localization in normal and cancerous human cervical tissues. Reverse transcription-polymerase chain reaction and Western blot analysis revealed the expression of lumican mRNA and its protein in CaSki, ME-180 and HeLa cells. No or weak immunoreactivity of the lumican protein was observed in stroma but not in squamous and ductal cells of non-cancerous uterine cervical tissues. In 21 of 28 (75%) cervical cancer cases, the lumican protein was strongly expressed in cancer cells, and accumulated particularly in cancer cells at the periphery of the cancer nests. It was also expressed in the fibroblasts adjacent to the cancer cells. In situ hybridization analysis revealed that lumican mRNA was not expressed in squamous or ductal epithelial cells in non-cancerous tissues, but was expressed in most cancer cells and stromal fibroblasts in uterine cervical cancer tissues. The lumican protein was not localized in normal squamous or ductal cells close to cancer cells, but its mRNA was strongly expressed in the same cells. To our knowledge, this is the first report on lumican synthesized by squamous cell carcinomas. These findings may indicate that the accumulated lumican protein in cancer cells at the periphery of cancer nests may play roles in the growth or invasion of human cervical cancer cells.