Human Uterine Cervical Cancer Research Articles

Proteogenomic Analysis of Human Uterine Cervical Cancer (UCC) Reveals Treatment-Resistant Subtypes of UCC

Locally advanced uterine cervical cancer (UCC) is treated by radiotherapy with concurrent chemotherapy, but heterogenous treatment responses are frequently observed. To better optimize therapeutic options based on molecular signatures, we performed proteogenomic analysis of UCC. UCC tissue and blood samples were collected from patients who underwent primary radiotherapy ± chemotherapy at the National Cancer Center (NCC) in Korea from July 2004 to March 2020. Most samples were obtained via biopsy. Genomic DNA for WES was isolated from frozen biopsy tumor tissues and peripheral blood buffy-coat of patients. Both global proteome and phosphoproteome were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Whole exome sequencing, RNA sequencing, global proteomics and phosphoproteomics were performed using 253, 337, and 147 cervical cancer samples, respectively. Patient-derived xenograft were established using intratongue implantation using 2 primary cell lines from sub3 and sub5, and flow cytometric analysis was performed. Fourteen significantly mutated genes (SMG) were found in our study cohort which include 5 newly identified SMGs. Mutation-phosphorylation analysis revealed association with apoptosis and actin cytoskeleton pathway. Proteogenomic analysis defined 6 molecular subtypes of UCC. Of those, 3 subtypes (i.e., Sub3, Sub5 and Sub6) were associated with treatment-resistant phenotypes. The cell-type deconvolution analysis suggested activated stroma with activation of cancer-associated fibroblast in Sub 3, while Sub5 showed low levels of activated stroma and high levels of myeloid immune cells. FACS analysis of UCC mouse models established from these 2 radio-resistant primary cell lines showed high component of PDGFRA+CAF infiltration in Sub 3, and high level of PVR+CD45+ immune cells mainly composed of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in Sub5. For Sub6, genes and/or protein signatures represented mucin-related processes (e.g., mucin glycosylation/sugar metabolism), which are linked to metastasis-associated Tn antigen production. The proteogenomic analysis thus suggests potential targets for radiotherapy-resistant subtypes of UCC; secretory factors from activated stroma and cancer-associated fibroblast (Sub3); RHOA signaling, PVR, and PMN-MDSCs (Sub5), and keratin/chondroitin sulfate proteoglycan and Tn antigen production (Sub6). Our study shows the importance of proteogenomic analysis in unveiling the subtype specific molecular pathways of UCC that are beyond reach by genomic data alone. The validity of our molecular pathway and cellular signatures linking these pathways should be further validated through detailed functional experiments and in larger UCC cohorts.

Primary and secondary prevention of uterine cervical cancer: Up-To-Date Information, Guidelines, and the need for their application in Greece

Background/Aim: Human papilloma virus (HPV) and uterine cervical cancer (UCC) are causatively related. In Greece, evidence-based guidelines for primary and secondary prevention of UCC have yet to be developed and applied in clinical practice. Our purpose here is to present such guidelines together with up-to-date data on HPV screening protocols. Methods: We surveyed current literature on UCC prevention, national and international data on UCC, and national guidelines on UCC preventive measures in various countries, as well as their role in UCC control. Results: It is well known that clinical practice guidelines optimize patient care. Introduction of guidelines regarding primary and secondary prevention of UCC express a favorable outcome of control and elimination of the disease. This is outlined in screening protocols of several countries worldwide. The lack of such guidelines in Greece in previous years led to misinformation of both healthcare professionals and general population. The development of clear and up-to-date guidelines in Greece during 2021 constitutes an important step that will play a pivotal role in UCC prevention in Greece. What needs to be ensured is on the one hand that all healthcare professionals are informed about these guidelines and on the other hand the application of these guidelines an everyday clinical practice. Conclusions: Systematic use of primary and secondary preventive measures and screening protocols are efficient in both control and elimination of UCC. Based on our search and our clinical experience, it is imperative for all Obstetrics and Gynecology clinicians in Greece to be familiar with all relevant screening and vaccination programs available.

USP18-deficiency in cervical carcinoma is crucial for the malignant behavior of tumor cells in an ERK signal-dependent manner.

Ubiquitin-specific peptidase (USP)18 belongs to the USP family, and is involved in cleaving and removing ubiquitin or ubiquitin-like molecules from their target molecules. Recently, increasing evidence has suggested that USP18 is constitutively expressed in different types of human tumors, and ectopic expression or downregulation of USP18 expression may contribute to tumorigenesis. However, the role of USP18 in uterine cervical cancer (UCC) remains unclear. Thus, the present study aimed to investigate USP18 expression in a human tissue microarray constructed using UCC and non-cancer cervical tissues, and to determine the potential role and molecular mechanism by which USP18 is implicated in the tumor biology of human UCC HeLa cells. Microarray analysis demonstrated that USP18 protein expression was downregulated in tumor tissues compared with in normal tissues. In addition, in vitro analysis revealed that USP18-knockdown markedly promoted the proliferation, colony formation, migration and aggressiveness of HeLa cells. Mechanistic analysis demonstrated that USP18-knockdown increased the levels of Bcl-2, STAT3 and phosphorylated-ERK in HeLa cells. Notably, USP18 silencing-induced malignant phenotypes were interrupted following exogenous administration of the ERK1/2 inhibitor PD98059. Overall, the results of the present study suggested that USP18 may be a potent inhibitor involved in UCC tumor-associated biological behaviors, which are associated with the ERK signaling pathway.

Cell-type-specific Modulation of Non-homologous End Joining of Gamma Ray-induced DNA Double-strand Breaks by cAMP Signaling in Human Cancer Cells.

BackgroundCyclic AMP (cAMP) signaling is activated by various hormones and neurotransmitters and regulates numerous physiological phenomena, including energy metabolism, gene expression, and proliferation. cAMP signaling plays a role in the repair of DNA damage, but its specific function is inconsistent in the literature. The present study aimed to investigate the mechanism of the different roles of cAMP signaling in DNA repair by analyzing the cell-type differences in the modulation of DNA repair by cAMP signaling following γ-ray irradiation.MethodscAMP signaling was activated in human malignant melanoma cells (SK-MEL-2 and SK-MEL-28), human uterine cervical cancer cells (HeLa and SiHa) and human non-small cell lung cancer cells (H1299 and A549) by expressing a constitutively active mutant of the long-form stimulatory α subunit of GTP-binding protein or by treating with isoproterenol and prostaglandin E2 before γ-ray irradiation. DNA damage was quantitated by western blot analysis of γ-H2AX, and non-homologous end joining (NHEJ) was assessed by fluorescent reporter plasmid repair assay and immunofluorescence of microscopic foci of XRCC4 and DNA-ligase IV.ResultscAMP signaling modulated DNA damage, apoptosis and the NHEJ repair following γ-ray irradiation differently depending upon the cell type. cAMP signaling regulated the phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at Ser2056 and Thr2609 in cell-type-specific manners following γ-ray irradiation, an activity that was mediated by protein kinase A.ConclusioncAMP signaling modulates the NHEJ repair of γ-ray-induced DNA damage in melanoma cells, uterine cervical cancer cells and lung cancer cells in a cell-type-specific manner, and the modulation is likely mediated by protein kinase A-dependent phosphorylation of DNA-PKcs. This study suggests that cell- and tissue-specific modulation of DNA damage repair by cAMP signaling may contribute to improve the therapeutic efficiency of radiation therapy.

DNA binding interaction studies of flavonoid complexes of Cu(II) and Fe(II) and determination of their chemotherapeutic potential

The present study is based on molecular level investigation of DNA binding of three flavonoids (Fls), morin (mor), quercetin (quer) and primuletin (prim) and their metal (Cu (II) and Fe (III)) complexes through UV–Vis spectroscopy, fluorescence spectroscopy, thermal analysis and cyclic voltammetry (CV). The spectroscopic experiments were performed by titrating compounds with DNA and the results were in good agreement with those obtained from titration of DNA by the compounds. The Fe-mor, Cu-quer, Fe-quer and prim which exhibited hypsochromic/hyperchromic shifts in UV–Vis spectra, showed negligible effects over florescent spectra of NR-DNA (intercalating Neil red dye–DNA complex), intact Tm (thermal melting temperature) of DNA and the shift of peak potential (Ep) towards less positive value were designated to exhibit electrostatic mode of binding with dsDNA having binding constant, Kb ≈ 102 M−1. The mor, Cu-mor, Cu-prim and Fe-prim showed intercalating mode of interaction with dsDNA which was recognized from hypochromic/bathochromic shifts in the spectra, Ep shifts towards higher potential in CV, significant decay in fluorescence intensity, distinct shift in Tm of DNA and binding constant (Kb) of the order of 104 M−1. The mor exhibited mixed binding mode (intercalation and groove binding) at different concentrations. The quer displayed increase in peak current and hyperchromism thus depicted groove binding into dsDNA (Kb ≈ 103 M−1). The binding modes were found to be correlated with the apoptotic activity of the Fls and M-Fls (flavonoids and metal-flavonoids) assayed against human breast cancer cell lines (MCF-7) and human uterine cervical cancer cell lines (HeLa). The correlation is interpreted in terms of, the intercalation mode as cytostatic activity and the groove binding with no apoptosis. However, electrostatic binding of (M-)Fls to dsDNA did not produce significant anti-proliferation activity against Hela cell lines with exception of Fe-mor (cytostatic activity) and Cu-quer (cytotoxicity) against MCF-7 cell line.

UCP2 expression may represent a predictive marker of neoadjuvant chemotherapy effectiveness for locally advanced uterine cervical cancer.

Concurrent chemoradiotherapy is the standard treatment for locally advanced uterine cervical cancer. However, effective neoadjuvant chemotherapy (NAC) can reduce tumor size and facilitate hysterectomy for locally advanced uterine cervical cancer. NAC treatment could improve the prognosis of patients with locally advanced cervical cancer. However, if NAC is ineffective, radiotherapy must be pursued. This causes a delay in initiating the core treatment and results in a worse prognosis. Therefore, the identification of predictive markers of whether NAC is likely to be effective for the treatment of locally advanced uterine cervical cancer could improve patient prognosis. Uncoupling protein 2 (UCP2) is broadly expressed in cancer cells, and suppresses mitochondrial reactive oxygen species (ROS) production. UCP2 contributes to both carcinogenesis and chemoresistance by reducing ROS. Downregulation of UCP2 results in significantly increased cell death following chemotherapy. The present study investigated the association between UCP2 expression and NAC effectiveness. A total of 58 patients with locally advanced uterine cervical cancer (stage IIIA or IIIB) treated at Osaka City University Hospital between April 1995 and March 2010 were examined. Tumor tissue samples were obtained by punch biopsy prior to NAC. UCP2 expression was examined immunohistochemically and scored using a weighted scoring system. Patients were divided into NAC effective (n=34) and ineffective (n=24) groups. Furthermore, UCP2 expression in human uterine cervical cancer cells was inhibited by genipin, and changes in cisplatin sensitivity were examined. UCP2 weighted score was higher in the NAC ineffective group than in the NAC effective group (P=0.038). Additionally, the low UCP2 expression group was more sensitive to NAC than the high UCP2 expression group (P=0.041). Sensitivity to cisplatin was significantly increased when UCP2 was inhibited in human uterine cervical cancer cells in vitro. UCP2 expression may become a predictive marker of whether NAC is effective for patients with locally advanced uterine cervical cancer, which could improve patient prognosis.

Cordycepin increases radiosensitivity in cervical cancer cells by overriding or prolonging radiation-induced G2/M arrest

Cordycepin (3-deoxyadenosine) has many pharmacological activities. We studied the radiosensitising effect of cordycepin and the underlying mechanisms relating to cell cycle changes in two human uterine cervical cancer cell lines, ME180 and HeLa cells. Cordycepin produced concentration- and time-dependent reductions in cell viability with more pronounced effects in ME180 cells. Cells pre-treated with cordycepin showed lower cell survival than those exposed to irradiation only. Radiation-induced expression of the histone, γ-H2AX, and apoptosis were also increased following cordycepin pre-treatment. In ME180 cells, pre-treatment with cordycepin reduced radiation-induced G2/M arrest and this G2/M checkpoint override was sustained for longer than in HeLa cells, where G2/M arrest was observed earlier and more briefly, the number of HeLa cells in the G2/M phase was subsequently increased. Cordycepin produced different effects on the expression of p53 and cell cycle checkpoint proteins in these two cell lines. It can be assumed that the mechanism underlying cordycepin-mediated radiosensitisation involves multiple effects that are primarily based on the induction of p53-mediated apoptosis and modulation of the expression of cell cycle checkpoint molecules.

Anthocyanins fromVitis coignetiaePulliat Inhibit Cancer Invasion and Epithelial-Mesenchymal Transition, but These Effects Can Be Attenuated by Tumor Necrosis Factor in Human Uterine Cervical Cancer HeLa Cells

Recently we have demonstrated that anthocyanins from fruits of Vitis coignetiae Pulliat (AIMs) have anticancer effects. Here, we investigate the effects of AIMs on cell proliferation and invasion as well as epithelial-mesenchymal transition (EMT) which have been linked to cancer metastasis in human uterine cervical cancer HeLa cells. AIMs inhibited the invasion of HeLa cells in a dose-dependent manner. AIMs inhibited MMP-9 expression in a dose-dependent manner. AIMs inhibited the motility of HeLa cells in a wound healing test. AIMs still suppressed NF-κB activation induced by TNF. AIMs also inhibited EMT in HeLa cells. AIMs suppressed vimentin, N-cadherin, and β-catenin expression and induced E-cadherin. AIMs also suppressed expression of β-catenin and Snail, which was regulated by GSK-3. These effects of AIMs were also limited in the HeLa cells treated with TNF. In conclusion, this study indicates that AIMs have anticancer effects by suppressing NF-κB-regulated genes and EMT, which relates to suppression of IκBα phosphorylation and GSK-3 activity, respectively. However, the effects of AIMs were attenuated in the TNF-high condition.

Abstract 414: Role of stem cell marker, nestin in human uterine cervical intraepithelial neoplasia and cervical cancer

Abstract BACKGROUND: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell marker expressed during central nervous system development. Increased nestin expression has been reported in various tumor cells, including central nervous system tumors, gastrointestinal stromal tumors, pancreatic cancers, prostate cancers, breast cancers and malignant melanomas. Recent studies have shown that nestin is a cancer stem cell (CSC) marker in glioblastoma and malignant melanoma, and its expression correlates with aggressive growth, metastasis, and poor prognosis in the tumors. However, expression of nestin and its role in cervical cancer cells have not been well identified. In the present study, we used gene transfection to clarify nestin expression levels in cervical intraepithelial neoplasia (CIN) and cervical cancer and determine the role of nestin in cervical cancer. METHODS: To determine nestin expression patterns, we performed immunohistochemical analysis of nestin in CIN (26 cases) and cervical cancer tissues (55 cases). Furthermore, nestin expression was examined in human cervical cancer cell lines (ME-180 and CaSki). To examine the role played by nestin in cervical cancer cells, we transfected a stable expression vector containing nestin cDNA into ME-180 cells. Empty vector transfected ME-180 cells were prepared as the negative control (mock cells). We then studied the effects of increased nestin expression on cell proliferation, cell motility, migration, invasion, and sphere and soft agar formations. RESULTS: Nestin was not localized in the squamous epithelium in normal cervical tissue, but it was weakly expressed in the basal squamous epithelium of CIN 1 tissue. In CIN 2 tissue, nestin was found to be localized in the basal to lower two-third section of the squamous epithelium, whereas in CIN 3, it was localized in the majority of the squamous epithelium. Nestin was detected in all cases of invasive cervical cancer examined. Using in situ hybridization analysis, we confirmed that the expression patterns of nestin mRNA were similar to those of nestin protein. Nestin mRNA was expressed in both ME-180 and CaSki cells, with the expression level lower in ME-180 than in CaSki cells. The growth rates and migration ability of stable nestin-transfected ME-180 cells were lower than those of the mock cells, but cell motility and invasion ability were no different from the mock cells. However, the nestin-transfected ME-180 cells formed more spheres and colonies than the mock cells. CONCLUSION: There is a positive correlation between increased nestin expression and advanced grades of CIN. In cervical cancer, nestin expression correlates with the property of CSC. These findings suggest that nestin plays an important role in the carcinogenesis and maintainance of cervical CSC functions. Nestin may be a novel therapeutic target for CIN and cervical cancer. 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 414. doi:1538-7445.AM2012-414

The activity of G-ROS and the predominant role of Type II reaction in the photodynamic therapy using 9-hydroxypheophorbide-α for HeLa cell lines

Photodynamic therapy (PDT) is a treatment modality that destroys the tumor. It activates the photosensitizer with the light of a specific wavelength, where the light is well absorbed by the photosensitizer, thus causing a fatal injury and thereby leading to a tumor necrosis. To date, a hematoporphyrin-derived photosensitizer has been widely used. It is disadvantageous, however, in that it causes a long-term photo-toxicity and has a poor selectivity for the tumor. This had led to the development of a chlorophyll-derived photosensitizer. We conducted this study to elucidate the mechanisms by which the activity of ROS is involved in the PDT using a novel type of chlorophyll-derived photosensitizer, 9-hydroxypheophorbide-α (9-HpbD-α), for the HeLa cell lines. Besides, we also attempted to determine which reaction plays a predominant role in the synthesis of ROS, either Type I reaction or Type II one, when both reactions are involved in the synthesis of ROS during the PDT using 9-HpbD-α. Our results showed not only that the activity of ROS is involved in the PDT using 9-HpbD-α in human uterine cervical cancer cell lines but also that the mechanisms of PDT are based on Type II reaction where the singlet oxygen is involved.

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Immunolabelling of the inhibin/activin-βC subunit in normal and malignant human uterine cervical tissue and cervical cancer cell lines

Inhibins are dimeric glycoproteins, composed of an α-subunit and one of two possible β-subunits (βA or βB), with substantial roles in human reproduction and in endocrine-responsive tumours. Recently a novel β subunit named βC was described, although it is still unclear if normal or cancerous cervical epithelial cells as well as cervical cancer cell lines can synthesise the inhibin-βC subunit. Four normal cervical tissue samples together with specimens of well-differentiated squamous cervical cancer and adenocarcinoma of the cervix were immunohistochemically analyzed. Additionally, two cervical carcinoma cell lines (HeLa and CaSKi) were analyzed by immunofluorescence for the expression of this novel subunit. We demonstrated for the first time an immunolabelling of the inhibin-βC subunit in normal and malignant cervical tissue, as well as cervical cancer cells. Although the physiological role is still unclear in cervical tissue, the inhibin-βC subunit might play important roles in carcinogenesis. Moreover, the synthesis of this subunit in cervical carcinoma cell lines of squamous and epithelial origins allows the use of these cell lines in elucidating its functions in cervical pathogenesis.

Human papillomavirus genotyping of cervical uterine preneoplastic lesions in a high risk area

The relationship between human papillomavirus (HPV) and uterine cervical cancer (UCC) is widely known and accepted. To determine the frequency of genotypes of HPV in cervical preneoplastic lesions in a high risk area of UCC. Using a combination of PCR and Reverse Line Blot technique, 235 formalin fixed paraffin embedded samples, with diagnosis of low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL) were genotyped. HPV was detected in 61.2% of LSIL and 78.1% of HSIL. The main genotypes found were HPV 16, 18, 31, 45, 56 y 58. HPV 16 was the most common in both LSIL (18.1%) and HSIL (36.9%). HPV 16 or 18 were present in 25.1% and 47.1% of the LSIL and HSIL respectively. In both LSIL and HSIL, the predominant viral genotypes were those types classified as with a high oncogenic risk. HPV genotypes 16, 18, 31, 45, 56 y 58 were the most common in our series. HPV 16 and 18, viral types with high oncogenic risk and included in commercial vaccines, were found in 25.1% and 47.1% of LSIL and HSIL, respectively.

Detección y tipificación de virus papiloma humano en lesiones preneoplásicas de cuello uterino

The relationship between human papillomavirus (HPV) and uterine cervical cancer (UCC) is widely known and accepted.To determine the frequency of genotypes of HPV in cervical preneoplastic lesions in a high risk area of UCC.Using a combination of PCR and Reverse Line Blot technique, 235 formalin fixed paraffin embedded samples, with diagnosis of low-grade squamous intraepithelial lesion (LSIL) or high-grade squamous intraepithelial lesion (HSIL) were genotyped.HPV was detected in 61.2% of LSIL and 78.1% of HSIL. The main genotypes found were HPV 16, 18, 31, 45, 56 y 58. HPV 16 was the most common in both LSIL (18.1%) and HSIL (36.9%). HPV 16 or 18 were present in 25.1% and 47.1% of the LSIL and HSIL respectively. In both LSIL and HSIL, the predominant viral genotypes were those types classified as with a high oncogenic risk.HPV genotypes 16, 18, 31, 45, 56 y 58 were the most common in our series. HPV 16 and 18, viral types with high oncogenic risk and included in commercial vaccines, were found in 25.1% and 47.1% of LSIL and HSIL, respectively.

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.

Expression of fibroblast growth factor receptor 2 IIIc in human uterine cervical intraepithelial neoplasia and cervical cancer

Fibroblast growth factor receptors (FGFRs) 1-3 have IIIb and IIIc isoforms, and we reported that FGFR2 IIIb is highly expressed in cervical keratinizing squamous cell carcinoma (SCC). In this study, we determined the expression and roles of FGFR2 IIIc in cervical intraepithelial neoplasia (CIN) and cervical cancer. In CINs 1 and 2, FGFR2 IIIc was found to be localized at the basal to lower two-thirds of the squamous epithelium, whereas it was localized in most of the squamous epithelium, except for the superficial layer in CIN 3. In situ hybridization (ISH) analysis showed that the expression patterns of FGFR2 IIIc mRNA are similar to those of FGFR2 IIIc protein in CINs. The FGFR2 IIIc protein was detected in all invasive cervical cancer patients (29 cases) and its mRNA was found to be strongly expressed in the invasive front of cancer cell nests. FGFR2 IIIc cDNA was stably transfected into CaSki cells, which are derived from a cervical SCC. The growth rates of the CaSki cells were higher than those of Mock cells in vitro, and the CaSki cells tended to form larger subcutaneous tumors in nude mice. These findings suggest that FGFR2 IIIc plays important roles in carcinogenesis and growth of cervical cancer cells. Anti-FGFR2 IIIc therapies may represent therapeutic strategies for inhibiting the growth of CIN and cervical cancer.

Enhanced suicide gene therapy using a tumor-specific promoter in combination with cisplatin

Human telomerase reverse transcriptase (hTERT) promoter has been proposed in cancer-targeted gene therapy. However, this promoter has not been strong enough to achieve therapeutic levels of transgene expression. We favor the hypothesis that telomerase may participate in the process of DNA repair and that the up-regulation of hTERT promoter activity may be a reaction to DNA damage. In previous investigations, we tested an 'indirected-activator' strategy that utilizes radiation to increase the activity of the hTERT promoter. The purpose of the present study was to implement a strategy using cisplatin to enhance hTERT promoter activity. The results indicate that, in human uterine cervical cancer (HeLa) cells exposed to 5 µg/ml cisplatin, the hTERT promoter had 3.1-fold increased activity compared to untreated cells. In addition, the combination of cisplatin and hTERT promoter-mediated horseradish peroxidase/indole-3-acetic acid gene-directed enzyme prodrug therapy induced cell cycle arrest at the S phase and apoptosis leading to a more significant reduction in cell viability. These findings suggest that hTERT promoter-mediated gene therapy is improved when combined with cisplatin.

Dehydroepiandrosterone inhibits the proliferation and induces the death of HPV‐positive and HPV‐negative cervical cancer cells through an androgen‐ and estrogen‐receptor independent mechanism

Dehydroepiandrosterone (DHEA) has a protective role against epithelial-derived carcinomas; however, the mechanisms remain unknown. We determined the effect of DHEA on cell proliferation, the cell cycle and cell death in three cell lines derived from human uterine cervical cancers infected or not with human papilloma virus (HPV). We also determined whether DHEA effects are mediated by estrogen and androgen receptors. Proliferation of C33A (HPV-negative), CASKI (HPV16-positive) and HeLa (HPV18-positive) cells was evaluated by violet crystal staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction. Flow cytometry was used to evaluate the phases of the cell cycle, and cell death was detected using a commercially available carboxyfluorescein apoptosis detection kit that determines caspase activation. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Flutamide and ICI 182,780 were used to inhibit androgen and estrogen receptors, respectively, and letrozol was used to inhibit the conversion of DHEA to estradiol. Our results show that DHEA inhibited cell proliferation in a dose-dependent manner in the three cell lines; the DHEA IC(50) doses were 50, 60 and 70 mum for C33A, CASKI and HeLa cells, respectively. The antiproliferative effect was not abrogated by inhibitors of androgen and estrogen receptors or by an inhibitor of the conversion of testosterone to estradiol, and this effect was associated with an increase in necrotic cell death in HPV-negative cells and apoptosis in HPV-positive cells. These results suggest that DHEA strongly inhibits the proliferation of cervical cancer cells, but its effect is not mediated by androgen or estrogen receptor pathways. DHEA could therefore be used as an alternative in the treatment of cervical cancer.

Distinct DNA Methylation Profiles Between Adenocarcinoma and Squamous Cell Carcinoma of Human Uterine Cervix

Alterations in DNA methylation offer unique prospects as tumor markers. The big limitation in cervical cancer research is that it is too hard to obtain the pure normal tissue from a cervical cancer mass. So, we first profile type-specific DNA methylation of major two types of human uterine cervical cancer, adenocarcinoma (ACA) and squamous cell carcinoma (SCC), to establish a precise source of marker research. To assess the DNA methylation status of promoter regions in human uterine cervical ACAs and SCCs, fresh frozen tissues were obtained from bulky tumor masses to minimize the contamination from normal tissues and two array platforms using digestion with methylation-sensitive restriction-enzyme HpaII, ligation, and PCR were performed: an array of 11,994 (approximately 1.5 kb) PCR products from 10,445 promoter regions, and an array of 355,264 oligonucleotides for 18,212 HpaII fragments in 12,617 promoter regions. Loci near 21 genes showed significant differences between six ACA and four SCC from the analysis of two array data. Real-time PCR-based validation was performed on 13 loci using other nearby candidate methylation targets in the same promoter. Methylation patterns of 11 of 13 linked loci concurred with the microarray results. Four loci were further studied using tissues from additional patients (23 ACA and 24 SCC). Hypermethylation of loci in PAK6 and NOGOR most strongly correlated with ACA. Therefore, we have identified the 21 genes with differential methylation pattern between ACA and SCC and, furthermore, we found that PAK6 and NOGOR could be useful markers of ACA to be distinct from SCC.

Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia-mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute-2 (MDM2) interaction. In addition, ISL-mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL-mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl-2 and Bcl-X(L), and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase-9 inhibitor blocked ISL-induced apoptosis, indicating that caspase-9 activation is involved in ISL-mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.