LncRNA MALAT1 Accelerates Cervical Carcinoma Proliferation by Suppressing miR-124 Expression in Cervical Tumor Cells.

The cGAS-STING pathway has established itself as a critical innate immune pathway that has the ability to significantly affect tumor initiation and progression. The expression, methylation, immunological functions, and prognostic importance of cGAS-STING pathway-related genes in cervical squamous cancer (CESC) patients have not yet been thoroughly elucidated. First, we explored the expression of cGAS and STING in cervical carcinoma samples from TCGA by comparing the mRNA and protein levels of cGAS and STING in both TCGA cervical tumor patient samples and cervical tumor cell lines. Second, we examined the CD4+T and CD8+T cell infiltration in STING high and low samples and made Kaplan-Meier prognosis analysis of STING protein expression. Third, to verify the findings in TCGA public datasets, we retrospectively selected 40 cervical squamous carcinoma patients and 10 normal cervical tissues and evaluated cGAS and STING protein expression using immunohistochemistry (IHC). All patients have detailed clinical information, which includes age, FIGO stage, menstruation status, follow-up time, histology, tumor diameter, and serum tumor markers. In both cervical tumor patient samples and cell lines, we observed that cGAS is increased, whereas STING is decreased in tumors, which leads to decreased CD4+T and CD8+T cell infiltration and poor prognosis. Furthermore, the cGAS mRNA transcript showed a gradual increase and STING mRNA showed a decrease according to the tumor stage, tumor grade, metastasis status, and histology types. We confirmed the expression of cGAS and STING proteins in clinical cervical tumor samples using IHC. Mechanically, cGAS and STING showed different DNA methylation patterns, which might contribute to the differences in cGAS and STING mRNA and protein levels. Our work identified different expressions and methylation patterns of cGAS and STING in cervical cancer and their correlation with immune T cell infiltration and prognosis. More mechanistic study is needed to understand the cGAS-STING pathway in cervical squamous tumor.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

The purpose of this study was to determine if squamous cell carcinoma antigen (SCCA), also known as SERPINB3, protects cervical cancer cells from ionizing radiation (IR)-induced death, and to determine the mechanism(s) of IR-induced cell death with or without SERPINB3. Cervical cancer remains a leading cause of cancer death in women worldwide despite advances in screening, vaccination and treatment. Recurrence after definitive chemoradiation occurs in up to 30-50% of patients with locally advanced disease. We and others have demonstrated that elevated serum SCCA portends poor prognosis in cervical cancer. In addition, CRISPR-Cas9-mediated knock out of SERPINB3 significantly sensitizes cervical tumor cells to IR in vitro. We hypothesize that SERPINB3 promotes radioresistance by protecting cells against lysosome damage and lysosomal mediated necrosis. Two cervical cancer cell lines with high SERPINB3 expression were edited using CRISPR-Cas9 technology at the SERPINB3 locus resulting in knock out (KO). B3-KO cells were significantly more radiation sensitive compared to control cells at all doses and time points evaluated. Cell death morphology in the B3-KO cells was necrotic, with large cytoplasmic single membraned vesicles, many of which were ruptured, consistent with that seen in lysosome-mediated necrosis. Live-cell time-lapse imaging showed loss of lysosome integrity in the hours leading up to cell death (propidium iodide nuclear staining). Western blot analysis showed low levels of caspase-3 and caspase-7 cleavage only at high doses and late time points after IR, with no evidence of phospho-MLKL or phospo-RIPK3 (markers of necroptosis), or gasdermin-D cleavage (marker of pyroptosis). Necrostatin, ferrostatin, liproxstatin and YVAD-Cho had little to no effect on cell death following IR, while pan-caspase inhibitors decreased IR-induced cell death in both control and B3-KO cells, and E64D decreased IR-induced cell death in B3-KO cells to a greater degree than control cells. Additionally, cervical tumor cell lines that had no detectable expression of SERPINB3 were engineered to expressed high levels of SERPINB3 (B3-wt) or SERPINB3 containing the P14 mutation A351R (B3-P14m), which lacks protease inhibitory function. B3-wt expressing cells displayed increased radiation resistance in clonogenic survival assays and mouse tumor models. Growth rate and radiation response of B3-p14m expressing tumors was similar to vector control. These data suggest that SERPINB3 is an important mediator of radiation response in cervical cancer and protects cells by inhibiting cysteine protease-dependent cell death, likely via lysosome-mediated necrosis. These findings support SERPINB3 as a potential target for novel radiosensitizing therapies. Citation Format: Songyan Wang, Clifford Luke, Victoria Shi, Perry Grigsby, Gary Silverman, Stephanie Markovina. SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2426.

Ten ovarian and 2 cervical tumour cell lines were analysed for the production of pregnancy-associated proteins. Pregnancy-associated plasma protein-A (PAPP-A) was detected by radioimmunoassay in culture media of 2 out of 4 (50%) tumour granulosa cell lines (mean = 104 microIU/10(5) cells/24 h) but not in any ovarian (n = 6) or cervical (n = 2) tumour cell lines. By contrast, human chorionic gonadotrophin (hCG), pregnancy specific beta 1-glycoprotein and alpha-fetoprotein (AFP) were not detected in any of the PAPP-A positive media. Only two cell lines produced hCG (58.5 and 25.5 mIU/10(5) cells/24 h). No AFP was produced by any of these 12 cell lines, whereas placental protein 5 was positive in 7. None of these proteins were detected in the culture media of 4 cell lines. In vitro derived PAPP-A was immunologically indistinguishable from either pregnancy or ovarian follicular PAPP-A. All PAPP-A species interacted reversibly with immobilised heparin and were determined by molecular sieve chromatography to have an apparent molecular weight of 820,000 daltons. Cultured tumour granulosa cells specifically synthesised and secreted a large protein which was immunologically and physicochemically indistinguishable from in vivo (pregnancy and ovarian follicular) derived PAPP-A.

The contribution of inflammasoma and its cytokines to Human Papillomavirus (HPV) infection and HPV-associated cervical cancer (CC) (HPV-CC) is not yet fully understood. In vitro HPV infection leads to inhibition of IL-1 production in primary keratinocytes. On the other hand, studies of genetic association and the production of IL-1 and / or IL-18 show a positive correlation with the progression of CC. Based on the data obtained in a study of genetic association in a cohort of women with HPV-CC that pointed to IL-1 as a risk factor for the development of CC, we propose to deepen this finding here. Objective: To investigate the contribution of inflammasome in the tumor microenvironment of HPV-CC. Materials and methods: A genetic association study for inflammasome variants was replicated in a new cohort of 107 women with HPV-CC using allele-specific probes and qPCR. A gene expression analysis was performed using tumor and normal uterine cervix tissue data from public databases (TCGA, GTEX). Cervical tumor cell lines (C33-A, SiHa and HeLa) and monocytes derived from peripheral blood from 39 healthy donors were cultured alone and together (co-culture assay). The activation of the inflammasome was evaluated indirectly through the production of IL-1 and IL-18 measured in the culture supernatant by ELISA. Specific inflammasome inhibitors have been used in some trials. Results: Genetic analysis demonstrated once again the association between the rs16944 variant (which leads to an increase in IL-1) as well as a greater expression of the IL1B gene and a greater risk of developing HPV-CC and a worse prognosis, respectively. The data obtained in the co-culture assays revealed that, only in the spheroid CC cell culture model (not in monolayer), tumor cells do not produce IL-1, but induce the activation of the inflammasome and the release of IL -1 in monocytes. This activation is at least partially dependent on the NLRP3 inflammasome receptor. Conclusion: For the first time, to our knowledge, we demonstrated that the increase in IL-1 observed in the progression of CC is possibly due to the induction of NLRP3 activation of inflammasome tumor cells in the monocytes of the microenvironment. Therefore, both NLRP3 and IL-1 can be considered as possible targets for new therapeutic approaches.

Overexpression of epidermal growth factor type-1 receptor (EGF-R1) in cervical cancer: Implications for Cetuximab-mediated therapy in recurrent/metastatic disease

The goal of immunotherapy is to eliminate tumors by generating tumor-specific cytotoxic T lymphocytes (CTLs) in patients or by adoptively transferring ex vivo-activated CTLs into patients. Clinical trials have shown that tumor-specific CTLs often disappear before tumors are completely eliminated. In this study, the authors show that CTLs specific for cervical tumor cells undergo apoptosis after they are co-cultured with cervical tumor cells. The established cervical tumor cell lines and cervical cancer tissues express CD95 (Fas/Apo-1) ligand. The tumor cell-induced T-cell apoptosis can be blocked by an inhibitory anti-CD95 (APO-1/Fas) antibody, indicating that tumor cells induce apoptosis of CTLs through CD95-CD95 ligand interaction. Addition of interleukin-2 (IL-2) and IL-7 into the culture rescues the CTL from tumor cell-induced apoptosis. The rescued T cells retain their full antitumor cytotoxicity. These data suggest that human cervical tumor cells might actively down-regulate a cellular immune response by inducing apoptosis of specific T cells during immunotherapy. Local use of IL-2 and IL-7 as adjuvants may promote survival of the CTL and, thus, enhance the efficacy of immunotherapy.

BackgroundCervical cancer is one of the most common malignancies in women, leading to major health problems for its high morbidity and mortality. Numerous studies have demonstrated that circular RNAs (circRNAs) could be participated in the progression of multifarious diseases, especially plentiful carcinomas. CircAMOTL1 (angiomotin-like1, ID: hsa_circ_0004214), which is located on human chromosome 11:9 4532555-94533477, is involved in the occurrence of breast cancer, etc. However, the intrinsic and concrete molecular mechanism of circAMOTL1 in cervical carcinomas remained thoroughly unclear, which was also the bottleneck of circRNAs studies in cancer.MethodsThe relative expression levels of circAMOTL1 and miR-526b in cervical carcinoma patients’ specimens and cervical carcinoma cell lines were detected by RT-qPCR. Through experiments including loss-function and overexpression, the biological effects of circAMOTL1 and miR-526b on the proliferation, migration, apoptosis, and tumorigenicity were explored in cervical carcinomas. Dual luciferase reporter gene analysis, western blot, and other methods were adopted to explore the circAMOTL1 potential mechanism in cervical carcinomas.ResultsIn our experiments, our researches displayed that circAMOTL1 was significantly higher expression in cervical carcinomas specimens and cell lines. Further experiments illustrated that the knockdown of circAMOTL1 could restrain the malignant phenotype, AKT signaling, and epithelial–mesenchymal transition (EMT) of in cervical carcinomas cells. Meanwhile miR-526b was downregulated in cervical carcinomas and even miR-526b could partially reverse circAMOTL1 function in malignant cervical tumor cells. CircAMOTL1 acts as a microRNA (miRNA) sponge that actively regulates the expression of salt-inducible kinase 2 (SIK2) to sponge miR-526b and subsequently increases malignant phenotypes of cervical carcinomas cells. In a word, circAMOTL1 acts a carcinogenic role and miR-526b serves as the opposite function of antioncogene in the cervical carcinoma pathogenesis.ConclusionCircAMOTL1-miR-526b-SIK2 axis referred to the malignant progression and development of cervical carcinomas. CircAMOTL1 expression was inversely correlated with miR-526b and positively correlated with SIK2 mRNA in cervical cancer tissues. Thus, circAMOTL1 exerted an oncogenic role in cervical cancer progression through sponging miR-526b. Taken together, our study revealed that circAMOTL1 acted as an oncogene and probably was a potential therapeutic target for the cervical cancer.

Cervical cancer is a human papilloma virus (HPV)-related cancer, but most HPV infections are transient or intermittent and resolve spontaneously. Thus, other factors, such as cervical microflora, which are dominated by lactobacilli, must be involved in invasive cervical carcinoma development after HPV infection. Previous studies have demonstrated that lactobacilli have antitumour effects, and it is possible that vaginal lactobacilli prevent cervical cancer. Here we examined the proliferative and apoptotic responses of normal and tumour cervical cells to common vaginal lactobacilli components by investigating human normal fibroblast-like cervical (normal cervical) and HeLa (cervical tumour) cell responses to Lactobacillus gasseri and Lactobacillus crispatus. The effects of different lactobacilli components, such as culture supernatants, cytoplasmic extracts, cell-wall extracts and live cells, were determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, trypan blue staining, lactate dehydrogenase assay and colorimetric caspase-3 activity assay. Changes in caspase-3 and human chorionic gonadotropin β (hCGβ) expression were analysed by quantitative RT-PCR. Tumour cell growth inhibition by culture supernatants was higher than that by pH- and lactate-adjusted controls. However, the effects of the supernatants on normal cells were similar to those of lactate-adjusted controls. Apoptosis was inhibited by supernatants, which was consistent with higher hCGβ expression since hCG inhibits apoptosis. Our study demonstrated that common vaginal lactobacilli exert cytotoxic effects on cervical tumour cells, but not on normal cells, and that this cytotoxicity is independent of pH and lactate. Our results encourage further studies on the interaction between lactobacilli and cervical cells, and administration of common vaginal lactobacilli as probiotics.

It has been firmly established that high-risk human papillomavirus (HPV) infection is associated with the appearance and persistence of anogenital dysplasia. However, only a small fraction of HPV-infected patients ever develop cervical cancer. Therefore, other factors must contribute for the development of a malignant phenotype in HPV-infected cells. Aberrant microRNA (miRNA) expression is nowadays recognized as a key factor for the development of several pathologies including cancer. Several studies on the miR-125a-5p function have suggested a tumor suppressor role in diverse cell types. Our previous results shown that miR-125a-5p induced cell-cycle arrest, proliferation inhibition and apoptosis in cervical carcinoma cells. Nevertheless, miR-125a-5p role and mRNA targets in cervical cancer are not fully established in cervical cancer. RT-qPCR analysis of miR-125a-5p expression showed differential expression on immortal and tumor cervical cell lines suggesting a role in malignant progression. To probe for possible miR-125a-5p targets we performed in silico analyses using several miRNA target analysis tools and validated the results with a panel of cervical immortal and tumor cell lines. The bio-informatics analysis showed Mark1, bcl-2 and vEGF genes as possible targets for miR-125a-5p. Further RT-qPCR and immunoblot analysis indicated an inverse correlation of miR125a-5p and MARK1 expression in tumor lines but not in immortal lines irrespective of the HPV content. Interestingly, transfection of a pre-miR-125a-5p mimic in C33-A cells (lacking HPV and with low endogenous miR-125a-5p levels), resulted in specific down-regulation of MARK1 and VEGF. Therefore, we suggest a potential role for miR-125a-5p in cervical carcinogenesis through the regulation of MARK1. Citation Format: Natalia Martinez Acuna, Maria L. Benitez Hess, Luis M. Alvarez Salas. Target mRNA analysis for miR-125a-5p in cervical carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4167. doi:10.1158/1538-7445.AM2013-4167

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BackgroundIn this study, we intended to understand the regulatory mechanisms of microRNA-125a-5p (miR-125a-5p) in human cervical carcinoma.MethodsThe gene expressions of miR-125a-5p in seven cervical carcinoma cell lines and 12 human cervical carcinoma samples were evaluated by quantitative real-time reverse transcription polymerase chain reaction. Ca-Ski and HeLa cells were transduced with lentivirus carrying miR-125a-5p mimics, and the effects of lentivirus-induced miR-125a-5p upregulation on cervical carcinoma proliferation and migration were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and transwell assays, respectively. In additional, HeLa cells were inoculated into null mice to evaluate the effect of miR-125a-5p upregulation on in vivo cervical carcinoma growth. The direct regulation of miR-125a-5p on its target gene, ABL proto-oncogene 2 (ABL2), in cervical carcinoma was evaluated by quantitative real-time reverse transcription polymerase chain reaction, Western blotting and luciferase reporter assays, respectively. ABL2 was then downregulated by small interfering RNA to examine its effect on cervical carcinoma proliferation and migration.ResultsmiR-125a-5p was downregulated in both cervical carcinoma cell lines and human cervical carcinomas. In Ca-Ski and HeLa cells, lentivirus-mediated miR-125a-5p upregulation inhibited cancer proliferation and migration in vitro and cervical carcinoma transplantation in vivo. ABL2 was shown to be directly targeted by miR-125a-5p. In cervical carcinoma, ABL2 gene and protein levels were both downregulated by miR-125a-5p. Small interfering RNA-mediated ABL2 downregulation also had tumor-suppressive effects on cervical carcinoma proliferation and migration.ConclusionThe molecular pathway of miR-125a-5p/ABL2 plays an important role in human cervical carcinoma. Targeting miR-125a-5p/ABL2 pathway may provide a new treatment strategy for patients with cervical carcinoma.

BackgroundT cell immunoglobulin mucin-3 (Tim-3) has been identified as a negative regulator of anti-tumor immunity. Recent studies highlight the important role of Tim-3 in the CD8+ T cell exhaustion that takes place in both human and animal cancer models. However, the nature of Tim-3 expression in the tumor cell and the mechanism by which it inhibits anti-tumor immunity are unclear. This present study aims to determine Tim-3 is expressed in cervical cancer cells and to evaluate the role of Tim-3 in cervical cancer progression.MethodologyA total of 85 cervical tissue specimens including 43 human cervical cancer, 22 cervical intraepithelial neoplasia (CIN) and 20 chronic cervicitis were involved. Tim-3 expression in tumor cells was detected and was found to correlate with clinicopathological parameters. Meanwhile, expression of Tim-3 was assessed by RT-PCR, Western Blot and confocal microscopy in cervical cancer cell lines, HeLa and SiHa. The migration and invasion potential of Hela cells was evaluated after inhibiting Tim-3 expression by ADV-antisense Tim-3.ConclusionsWe found that Tim-3 was expressed at a higher level in the clinical cervical cancer cells compared to the CIN and chronic cervicitis controls. We supported this finding by confirming the presence of Tim-3 mRNA and protein in the cervical cell lines. Tim-3 expression in tumor cells correlated with clinicopathological parameters. Patients with high expression of Tim-3 had a significant metastatic potential, advanced cancer grades and shorter overall survival than those with lower expression. Multivariate analysis showed that Tim-3 expression was an independent factor for predicting the prognosis of cervical cancer. Significantly, down-regulating the expression of Tim-3 protein inhibited migration and invasion of Hela cells. Our study suggests that the expression of Tim-3 in tumor cells may be an independent prognostic factor for patients with cervical cancer. Moreover, Tim-3 expression may promote metastatic potential in cervical cancers.

Evaluation of the antitumour activity of Rinvanil and Phenylacetylrinvanil on the cervical cancer tumour cell lines HeLa, CaSKi and ViBo

Aberrant miRNA expression is associated with the development of several diseases including cervical cancer. Dysregulation of miR-125a-5p is present in a plethora of tumors, but its role in cervical cancer is not well understood. The aim was to analyze the expression profile of miR-125a-5p in tumor and immortal cell lines with further target prediction, validation and function analysis. MiR-125a-5p expression was determined by real-time RT-PCR from nine cervical cell lines. In silico tools were used to find target transcripts with an miR-125-5p complementary site within the 3'UTR region. Further target selection was based on gene ontology annotation and ΔG analysis. Target validation was performed by transfection of synthetic miR-125a-5p mimics and luciferase assays. Functional evaluation of miR-125a-5p on migration was performed by transwell migration assays. Differential miR-125a-5p expression was observed between immortal and tumor cells regardless of the human papillomavirus (HPV) content. Thermodynamic and ontological analyses showed Microtubule-Affinity-Regulating Kinase1 (MARK1) as a putative target for miR-125a-5p. An inverse correlation was observed among miR-125a-5p expression and MARK1 protein levels in tumor but not in immortal cells. Luciferase assays showed direct miR-125a-5p regulation over MARK1 through recognition of a predicted target site within the 3'-UTR. HeLa and C-33A cervical tumor cells enhanced migration after transfection with miR-125a-5p mimics and stimulation of cell migration was reproduced by siRNA-mediated inhibition of MARK1. The results showed MARK1 as a novel functional target for miR-125a-5p with implications on cell migration of tumor cervical cancer cells.