Uterine Cervical Cancer Tissues 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.

MicroRNA-101 Promotes the Proliferation, Migration, and Invasion of Uterine Cervix Cancer Cells by Targeting Cyclin-Dependent Kinase 8

Uterine cervical cancer (UCC), or carcinoma of the uterine cervix, is a familiar malignancy in gynecology. The expression of multiple microRNAs is abnormal in UCC. Research has demonstrated that the level of microRNA-101 (miR-101) was decreased in UCC, but the mechanism by which miR-101 regulates UCC is still unclear. The TargetScan software predicted that one of the target genes of miR-101 was CDK8. This study aims to explore whether miR-101 affects the migration, invasion, and proliferation of UCC cells through CDK8. First, nanoparticle-assisted PCR was used to determine the levels of miR-101 and CDK8 in UCC tissues, normal adjacent tissues, and two UCC cell lines, C-33A and Siha. Compared to the normal tissues, the level of miR-101 was decreased in UCC tissues, while the level of CDK8 was increased. The dual-luciferase reporter experiments confirmed that miR-101 directly interacted with the binding site in CDK8 3’UTR to regulate luciferase activity. The UCC cells were transfected with the MIR101 mimic construct to overexpress miR-101. It was demonstrated that cell migration, invasion, and proliferation was reduced in C-33A cells overexpressing miR-101. In the cells co-transfected with CDK8 and MIR101, the overexpression of CDK8 reversed the effect of MIR101 overexpression on cell migration, invasion, and proliferation. Therefore, miR-101 can regulate the migration, invasion, and proliferation of UCC cells by targeting CDK8. Therefore, miR-101 has potential applications in the treatment of UCC.

CXCL3 overexpression promotes the tumorigenic potential of uterine cervical cancer cells via the MAPK/ERK pathway.

CXCL3 belongs to the CXC-type chemokine family and is known to play a multifaceted role in various human malignancies. While its clinical significance and mechanisms of action in uterine cervical cancer (UCC) remain unclear. This investigation demonstrated that the UCC cell line HeLa expressed CXCL3, and strong expression of CXCL3 was detected in UCC tissues relative to nontumor tissues. In addition, CXCL3 expression was strongly correlated with CXCL5 expression in UCC tissues. In vitro, HeLa cells overexpressing CXCL3, HeLa cells treated with exogenous CXCL3 or treated with conditioned medium from WPMY cells overexpressing CXCL3, exhibited enhanced proliferation and migration activities. In agreement with these findings, CXCL3 overexpression was also associated with the generation of HeLa cell tumor xenografts in athymic nude mice. Subsequent mechanistic studies demonstrated that CXCL3 overexpressing influenced the expression of extracellular signal-regulated kinase (ERK) signaling pathway associated genes, including ERK1/2, Bcl-2, and Bax, whereas the CXCL3-induced proliferation and migration effects were attenuated by exogenous administration of the ERK1/2 blocker PD98059. The data of the current investigation support that CXCL3 appears to hold promise as a potential tumor marker and interference target for UCC.

Sensitive molecular detection of small nodal metastasis in uterine cervical cancer using HPV16-E6/CK19/MUC1 cancer biomarkers.

Metastatic nodal involvement is a critical prognostic factor in uterine cervical cancer (UCC). To improve current methods of detecting UCC metastases in lymph nodes (LNs), we used quantitative PCR (qPCR) to assess mRNA expression of potential metastatic biomarkers. We found that expression of HPV16-E6, cytokeratin19 (CK19), and mucin1 (MUC1) is consistently upregulated in tumors and metastatic tissues, supporting a role for these genes in UCC progression. These putative biomarkers were able to predict the presence of histologically positive metastatic LNs with respective sensitivities and specificities of 82% and 99% (CK19), 76% and 95% (HPV16-E6), and 76% and 78% (MUC1). While the biomarkers failed to detect 1.7% to 2.2% of the histologically positive LNs when used individually, combining CK19 and HPV16-E6 enhanced sensitivity and specificity to 100% and 94%, respectively. To explore the sensitivity of qPCR-based detection of varying proportions of invading HPV16-positive UCC cells, we designed a LN metastasis model that achieved a fresh cell detection limit of 0.008% (1:12500 HPV16-positive to HPV16-negative cells), and a paraffin-embedded, formalin-fixed (PEFF) detection limit of 0.02% (1:5000 HPV16-positive to HPV16-negative cells), both of which are within the theoretical detection limit for micrometastasis. Thus, HPV E6/E7 oncogenes may be useful targets for the ultrasensitive detection of nodal involvements like micrometastases in fresh or archived tissue samples. Moreover, our results suggest that the biomarker combination of CK19/HPV-E6 could support a real-time intraoperative strategy for the detection of small, but potentially lethal, metastatic nodal involvements in fresh UCC tissues.

Anti-glypican-1 antibody-drug conjugate exhibits potent preclinical antitumor activity against glypican-1 positive uterine cervical cancer.

Glypican-1 (GPC1) is highly expressed in solid tumors, especially squamous cell carcinomas (SCCs), and is thought to be associated with disease progression. We explored the use of a GPC1-targeted antibody-drug conjugate (ADC) as a novel treatment for uterine cervical cancer. On immunohistochemical staining, high expression levels of GPC1 were detected in about 50% of uterine cervical cancer tissues and also in a tumor that had relapsed after chemoradiotherapy. Novel anti-GPC1 monoclonal antibodies were developed, and clone 01a033 was selected as the best antibody for targeted delivery of the cytotoxic agent monomethyl auristatin F (MMAF) into GPC1-positive cells. The anti-GPC1 antibody was conjugated with MMAF. On flow cytometry, HeLa and ME180 cervical cancer cells highly expressed GPC1, however, RMG-I ovarian clear cell cancer cell line showed weak expression. The GPC1-ADC was rapidly internalized into GPC1-expressing cells in vitro and was potently cytotoxic to cancer cells highly expressing GPC1. There were no inhibitory effects on cancer cells with low expression of GPC1. In a murine xenograft model, GPC1-ADC also had significant and potent tumor growth inhibition. GPC1-ADC-mediated G2/M phase cell cycle arrest was detected, indicating that the dominant antitumor effect in vivo was MMAF-mediated. The toxicity of GPC-ADC was tolerable within the therapeutic dose range in mice. Our data showed that GPC1-ADC has potential as a promising therapy for uterine cervical cancer.

Tc17 Cells in Patients with Uterine Cervical Cancer

BackgroundThe existence of Tc17 cells was recently shown in several types of infectious and autoimmune diseases, but their distribution and functions in uterine cervical cancer (UCC) have not been fully elucidated.MethodsThe frequency of Tc17 cells in peripheral blood samples obtained from UCC patients, cervical intraepithelial neoplasia (CIN) patients and healthy controls was determined by flow cytometry. Besides, the prevalence of Tc17 cells and their relationships to Th17 cells and Foxp3-expressing T cells as well as microvessels in tissue samples of the patients were assessed by immunohistochemistry staining.ResultsCompared to controls, patients with UCC or CIN had a higher proportion of Tc17 cells in both peripheral blood and cervical tissues, but the level of Tc17 cells in UCC tissues was significantly higher than that in CIN tissues. Besides, the increased level of Tc17 in UCC patients was associated with the status of pelvic lymph node metastases and increased microvessel density. Finally, significant correlations of infiltration between Tc17 cells and Th17 cells or Foxp3-expressing T cells were observed in UCC and CIN tissues.ConclusionsThis study indicates that Tc17 cell infiltration in cervical cancers is associated with cancer progression accompanied by increased infiltrations of Th17 cells and regulatory T cells as well as promoted tumor vasculogenesis.

Development of Image Processing Algorithm for Cytological Diagnosis of Uterine Cervical Cancer Tissue Examination

In this study is relevant to cell area extraction for cytological diagnosis of liquid-based cell examination being used for early diagnosis of uterine cervical cancer by utilizing image processing. As existing cytological diagnosis process has been performed manually by cytotechnologist, the amount of cell image that could be processed was limited. Therefore, in this study, cellular domain extraction through which automatic processing of cytological diagnosis process is enabled was performed and its condition was established depending on the size of cell nucleus based on cytological diagnosis standard of uterine cervical cancer cell. Obtained cell image was processed by hough transform matching with cell image by using matlab through preprocessing phase. As a result, 28 sample images among total 30 sample images were succeeded in finding out target cell and two sample images failed to find it. In the future, modification and verification for such failure case may be required to be performed. It is expected that the result of this study could be utilized for diagnosis process automation of liquid-based cell examination.

Intravenous Paclitaxel Is Specifically Retained in Human Gynecologic Carcinoma Tissues In Vivo

Paclitaxel and carboplatin are commonly used and well-tolerated agents for gynecologic malignancies. The persistence of platinum in human tissues for 14 days and the long-term retention of platinum in tissues for up to 17 months have been reported. Paclitaxel remains in human uterine cervical cancer tissues for 6 days. These findings prompted us to determine the retention of paclitaxel and carboplatin in human uterine cervical carcinoma, endometrial carcinoma, ovarian carcinoma, and pelvic lymph nodes to establish baseline parameters and guide the development of more effective treatment interventions. Thirty patients with uterine or ovarian carcinomas were treated with intravenous weekly paclitaxel-carboplatin chemotherapy before surgery. The concentrations of these agents in carcinoma tissue, normal cervical, myometrial and ovarian tissues, and pelvic lymph nodes were measured 5 days after the final administration. Paclitaxel was specifically retained in cervical, endometrial, and ovarian carcinoma tissues but was not detected in lymph nodes. In contrast to paclitaxel, carboplatin was readily detectable with similar levels in all tumor-associated and normal host tissues. In addition, a low paclitaxel concentration in cervical carcinoma tissue was significantly associated with short progression-free survival and overall survival. Further studies are needed to clarify the tissue distribution of anticancer drugs in humans and promote optimal treatment strategies enhancing paclitaxel lymphatic targeting.

Coexpression of EphB4 and ephrinB2 in tumor advancement of uterine cervical cancers

Objective Receptor EphB4 and the corresponding ligand ephrinB2 contribute to tumor growth in various human tumors. This prompted us to study the expression and localization of EphB4 and ephrinB2 in uterine cervical cancers to analyze the EphB4/ephrinB2 functions against clinical backgrounds. Methods Immunohistochemistry and real-time RT-PCR have been done to determine the histoscores and mRNA levels of EphB4 and ephrinB2, respectively, in sixty-two uterine cervical cancer tissue samples. Patient prognoses were analyzed with a 36-month survival rate. Results The localization of EphB4 and ephrinB2 was dominantly in the cancer cells of uterine cervical cancers of all cases given. Both the histoscores and mRNA levels of EphB4 and ephrinB2 significantly increased with clinical stages (I < II < III+IV, p < 0.001) in uterine cervical cancers. The tumor sizes significantly correlated with the histoscore and mRNA levels of EphB4 and ephrinB2. There were significant differences in histoscores and mRNA levels of EphB4 and ephrinB2 in accordance with lymph node metastasis, but not according to histopathological types. The 36-month survival rates of the 31 patients with high EphB4 and ephrinB2 expression were poor (31% and 19%, respectively), while survival rates for the other 31 patients with low EphB4 and ephrinB2 expression were significantly higher (72% and 73%, respectively). Conclusion Coexpression of EphB4 and ephrinB2 increased with the disease advancement based on clinical stage, lymph node metastasis, tumor size and with poor patient prognoses. Therefore, EphB4/ephrinB2 expression might work on tumor advancement and coexpression of the Eph/ephrin system may potentiate tumor progression leading to poor survival, thus can be recognized as a novel prognostic indicator in the primary tumors of uterine cervical cancers.

Calpain 6 supports tumorigenesis by inhibiting apoptosis and facilitating angiogenesis

Since calpain 6 is overexpressed in uterine cervical cancer tissue compared to normal tissue, we sought to define the role of calpain 6 during tumorigenesis. We overexpressed calpain 6 or inhibited calpain 6 in human cervical cancer cells (HeLa cells) and human umbilical vein endothelial cells (HUVECs), and measured cisplatin-mediated apoptosis and VEGF-mediated angiogenesis. The results indicated that calpain 6 supported tumorigenesis by inhibiting apoptosis and facilitating angiogenesis. To our knowledge, this result is the first evidence implicating calpain 6 in tumorigenesis, and it reveals calpain 6 as a novel therapeutic target for certain types of cancers.

Expression of cyclooxygenase-2 related to angiogenesis in uterine cervical cancers

Angiogenesis is essential for development, growth and advancement of solid tumors. Cyclooxygenase (COX)-2 is recognized as an angiogenic factor in various tumors. This prompted us to study the clinical implications of COX-2 expression related to angiogenesis in uterine cervical cancers. There was a significant correlation between microvessel counts and COX-2 levels in uterine cervical cancers. COX-2 localized in the cancer cells, but not in the stromal cells of uterine cervical cancer tissues. COX-2 levels increased with advancement, and the prognosis of the 30 patients with high COX-2 expression in uterine cervical cancers was poor (60%), while the 24-month survival rate of the other 30 patients with low COX-2 expression was 90%. Furthermore, COX-2 levels significantly correlated with VEGF levels in uterine cervical cancers. VEGF associated with COX-2 might work on angiogenesis in advancement. Therefore, long-term administration of COX-2 inhibitors might be effective on the suppression of regrowth or recurrence after intensive treatment for advanced uterine cervical cancers.

Tumor-specific gene therapy for uterine cervical cancer using MN/CA9-directed replication-competent adenovirus.

Although gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers, few studies have explored this possibility for uterine cervical cancer. MN/CA9 is a transmembrane glycoprotein that was first identified in the human cervical carcinoma cell line, HeLa. Since MN/CA9 protein is highly expressed in uterine cervical cancer tissues, but not in normal cervix, we constructed a tumor-specific replication-competent adenoviral vector utilizing MN/CA9 promoter (Ad-MN/CA9-E1a), which can replicate only in MN/CA9-expressing cells. Infection of Ad-MN/CA9-E1a to MN/CA9-positive uterine cervical cancer cells (HeLa, C-33 A and SiHa) resulted in much stronger Ad5 E1a protein expressions compared with MN/CA9-negative cells (SK-RC-29), suggesting a tissue-specific replication of this recombinant adenovirus. In vitro cytotoxicity assay revealed that the growth of MN/CA9-positive cells was significantly inhibited with 0.01-1 MOI of Ad-MN/CA9-E1a, but the growth of MN/CA9-negative cells (SK-RC-29) could only be inhibited by as many as 100 MOI. Intratumoral injection of Ad-MN/CA9-E1a effectively induced growth delay of HeLa tumors in nude mice. These results suggest that a novel replication-competent adenoviral vector mediated by MN/CA9 promoter, Ad-MN/CA9-E1a, can selectively replicate in MN/CA9-expressing tumors with cytotoxic effects and may be utilized for the treatment of uterine cervical cancer.

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.

The Thomsen-Friedenreich antigen in uterine cervical cancer tissue and culture cells, and thier alteration by drugs

The Thomsen-Friedenreich antigen in uterine cervical cancer tissue and culture cells, and thier alteration by drugs

Detection of herpes simplex virus tumor-associated antigen in uterine cervical cancer tissue: Five case studies

Biopsies from five gynecologic cancer patients were examined for the presence of herpes simplex virus tumor-associated antigen (HSV-TAA) using the highly sensitive peroxidase-antiperoxidase technique. The results demonstrate that two of three patients with squamous cell carcinoma of the uterine cervix that were serologically positive for HSV-TAA, determined by complement fixation, contained HSV-TAA in the cytoplasm of the malignant cells of the biopsy. Tissue from one squamous cell carcinoma of the uterine cervix patient, as well as tissue from an adenoacanthoma patient, was negative for tissue HSV-TAA. However, both of these patients were also serologically negative for HSV-TAA. All patients, regardless of cancer type, had serum antibodies to herpes simplex virus types 1 and 2. These studies continue to suggest a role for HSV in certain gynecologic cancers.