Different Expressions and Methylation Patterns of cGAS and STING in Cervical Cancer.

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.

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

Emerging studies have clarified the critical role of LncRNA MALAT1 in various pathological progressions. Here, we identified its positive relationship with cervical carcinoma proliferation. Cervical carcinoma has been considered as one of the most malignant tumors among female. Thus, our study was designed to investigate the underlying mechanism of LncRNA MALAT1 on cervical tumor cell proliferation. We observed that miR-124 was the potential target of LncRNA MALAT1 in cervical tumor cell lines (Hela, C-33A, Caski, and SiHa), the expression level of which is negatively correlated with LncRNA MALAT1 in cervical tumor cells, tissues of cervical patients, and mice. Gain- or loss-of-function analyses in cervical tumor cells have further verified the regulatory role of MALAT1 on miR-124. Additionally, the proliferation of cervical carcinoma was inhibited by miR-124 overexpression, whereas it was blocked by LV-MALAT1 transfection. In vivo assays, overexpression of miR-124, or knockdown of MALAT1 exhibited beneficial effects on tumor weight, size, and volume, together with elevating the survival rate, tightly related with the progression of cervical cancer. In conclusion, LncRNA MALAT1 disabled the effects of miR-124 as an inhibitory sponge, accelerating the progression of cervical carcinoma.

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.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

BackgroundImmune checkpoint inhibitors have aroused great expectation of tumor eradication. However, the effect of anti-PD-L1 treatment for cervical cancer is unsatisfactory and the underlying antagonist to anti-PD-L1 efficacy is remained to be studied. Here, we investigated the anti-tumor effect of anti-PD-L1 treatment in cervical tumor model and identified the antagonist to the therapeutic efficacy of anti-PD-L1 treatment.ResultsWe found that PD-L1 exhibited a moderate expression in both cervical tumor cell lines and clinical samples compared to other tumor types and the para-tumor tissue respectively. Interestingly, our results showed that the anti-PD-L1 treated mice were dichotomously divided into responsive and unresponsive group after five cycles of anti-PD-L1 treatment although all the mice had the same genome background. In addition, the unresponsive tumors showed less tumor necrosis area and higher immunosuppression activity induced by regulatory T cells (Tregs) population than the responsive ones. Furthermore, we found that anti-PD-L1 treatment autonomously upregulated Tregs proliferation and frequency in multiple immune organs, and, most importantly, Tregs depletion significantly depressed the tumor growth rate and tumor weight compared with either anti-PD-L1 or anti-CD25 treatment alone. Finally, we observed that the upregulating effector CD8+ T cell is associated with the better therapeutic effect of anti-PD-L1 therapy post Tregs depletion.ConclusionsAnti-PD-L1 treatment upregulates Tregs frequency and proliferation in tumor model, and the depletion of Tregs may be a useful adjuvant strategy for anti-PD-L1 therapy of cervical cancer.

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

BackgroundTo explore the diagnostic value of FAM19A4 methylation in high-risk human papilloma virus (hrHPV)-positive cervical samples from Chinese women for estimating cervical cancer or its precancerous lesions.MethodsCervical samples from 215 women infected with high-risk HPV were collected by smear testing. We purposely chose 61 patients with cervical cancer, 57 with high-grade squamous intraepithelial lesions (HSIL), 31 with low-grade squamous intraepithelial lesions (LSIL), and 66 without cervical intraepithelial neoplasia (CIN) after histological confirmation. Taqman probe-based quantitative PCR (qPCR) was utilized to detect the methylation status of FAM19A4 in the cervical samples and further evaluate the use of this gene in the diagnosis of cervical cancer.Results(1) An increasing level of FAM19A4 methylation was detected with increasing progression of cervical lesions, with methylation rates of 10.61%(7/66), 35.48%(11/31), 56.14%(32/57) and 93.44%(57/61) in no CIN, LSIL, HSIL and cervical carcinoma samples respectively. (2) In all hrHPV-positive samples, the levels of FAM19A4 methylation in HPV16/18 groups were higher than that in 12 other hrHPV groups (P < 0.05), but there was no significant difference between two groups after grouping cervical lesions into cervical cancer, HSIL, LSIL and no CIN groups (P>0.05). (3)There were no significant differences of FAM19A4 methylation in different clinicopathological parameters of cervical cancer. (4) Though the sensitivity of FAM19A4 methylation test was inferior to that of cytology and FAM19A4 combining with HPV16/18 genotyping, but showed the best specificity with 81.44% both for detection HSIL alone and ≥ HSIL, with favorable youden index (YI) and area under curve (AUC).ConclusionFAM19A4 is a specific biomarker of cancerous lesions of the cervix. FAM19A4 methylation analysis may serve as an auxiliary screening method for diagnosis of cervical (pre)cancer. However, in consideration of the limitations of this retrospective study, prospective population-based studies are necessary for further confirmation of the diagnostic value of FAM19A4 methylation for detection of cervical (pre)cancer in Chinese women.

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines

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

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.

We have recently demonstrated that overexpression of dihydrodiol dehydrogenase (DDH) in human ovarian carcinoma cells (2008/C13*) is associated with cisplatin and carboplatin resistance. Furthermore, we have also elucidated that transfection of parental human ovarian carcinoma cells with a full-length DDH1 cDNA leads to induction of resistance to the platinum drugs. The development of cisplatin resistance in the transfected cells is associated with an increase in DDH enzyme activity. Previous studies have identified several different mechanisms for development of cisplatin resistance, including altered DNA repair capacity, increased GSH-based detoxification, and increased metallothionein content. However, none of these mechanisms has been found to be universally associated with the development of cisplatin resistance in tumor cells from different tissue sources. The present study was undertaken to assess whether overexpression of DDH1 or DDH2 (in human ovarian, cervical, lung and germ-cell tumor cell lines) could specifically induce resistance to the platinum drugs in these cell lines. We demonstrated a ubiquitous association of increased expression of DDH1 or DDH2 (as judged by increased enzyme activity in transfected clones) with development of resistance to cisplatin and carboplatin. Moreover, we also found a lack of cross-resistance to anticancer drugs that have a different mode of action including paclitaxel, vincristine, doxorubicin hydrochloride, and melphalan. Although at present it is not clear how DDH is involved in platinum drug resistance, the identification of this gene as a causal factor in a series of cell lines derived from different tumors with different intracellular compositions indicates the importance of deciphering this hitherto undefined pathway which can produce resistance to platinum drugs.

MicroRNA-138 is a potential biomarker and tumor suppressor in human cervical carcinoma by reversely correlated with TCF3 gene

e17506 Background: Human papillomavirus infections often resolve on their own; however, ongoing high risk HPV infections can progress to cervical cancer, a significant global health issue responsible for approximately 660,000 new cases and 350,000 deaths in 2022. Current screening methods emphasize cytology or HPV DNA detection, with a limited focus on protein biomarkers. Thus, identifying sensitive and specific cervical cancer protein biomarkers is essential, as this analysis provides opportunities for developing affordable point-of-care screening tests. Methods: This study optimized a unified protocol for cell lysis and protein extraction to validate biomarkers using cervical cell lines and cervical swab samples. Four proteins—Topoisomerase II Alpha (TOP2A), Minichromosome Maintenance Complex Component 2 (MCM2), Valosin Containing Protein (VCP), and Cyclin-Dependent Kinase Inhibitor 2A (p16INK4a)—were quantified using enzyme-linked immunosorbent assays. Furthermore, the expression of target biomarkers in cervical tumors (G1-G3) and squamous intraepithelial lesions (SIL) was evaluated using immunohistochemistry. Cytological evaluation of swab samples categorized them as normal, reactive cellular changes (RCC), atypical squamous cells of undetermined significance (ASC-US), low grade SIL (LSIL), or high grade SIL (HSIL). The study utilized cervical cancer cell lines HeLa, Ca Ski, HT-3, C-33A, and PCS. Statistical analysis was performed using a gamma-generalized linear mixed model, and imaging tools (Aperio and Visiopharm) were employed for tissue analysis. Results: RIPA buffer and acetone precipitation achieved optimal cell isolation and high-yield protein recovery. Biomarker protein concentrations were normalized to Beta-actin. Compared to normal samples, HSIL, LSIL, and ASC-US samples showed overexpression of p16INK4a, while HSIL samples exhibited higher levels of VCP. VCP and p16INK4a were overexpressed across all cancer cell lines compared to primary cells. MCM2 was overexpressed in HT-3 and HeLa, while TOP2A was underexpressed in Ca Ski. Further tissue analysis confirmed strong staining of all target biomarkers in precancerous and cancerous tissues, with significant differences in VCP, TOP2A, and p16INK4a expression observed across tumor grades and SIL. Conclusions: Through a simplified sample preparation and protein extraction protocol, four protein biomarkers were effectively identified and validated for cervical cancer screening. This research emphasizes the importance of detecting a panel of proteins expressed in both HPV-positive and HPV-negative cervical cancers. The results highlight the promise of these target proteins in recognizing precancerous lesions and the potential benefits of incorporating protein biomarker-based screening for improving early detection and cervical cancer outcomes, especially in resource limited settings.

Functional defects in mitochondria are involved in the induction of cell death in cancer cells. We assessed the toxic effect of camptothecin against the human cervical and uterine tumor cell line SiHa with respect to the mitochondria-mediated cell death process, and examined the combined effect of camptothecin and anticancer drugs. Camptothecin caused apoptosis in SiHa cells by inducing mitochondrial membrane permeability changes that lead to the loss of mitochondrial membrane potential, decreased Bcl-2 levels, cytochrome c release, caspase-3 activation, formation of reactive oxygen species and depletion of GSH. Combination of camptothecin with other anticancer drugs (carboplatin, paclitaxel, doxorubicin and mitomycin c) or signaling inhibitors (farnesyltransferase inhibitor and ERK inhibitor) did not enhance the camptothecin-induced cell death and caspase-3 activation. These results suggest that camptothecin may cause cell death in SiHa cells by inducing changes in mitochondrial membrane permeability, which leads to cytochrome c release and activation of caspase-3. This effect is also associated with increased formation of reactive oxygen species and depletion of GSH. Combination with other anticancer drugs (or signaling inhibitors) does not appear to increase the anti-tumor effect of camptothecin against SiHa cells, but rather may reduce it. Combination of camptothecin with other anticancer drugs does not seem to provide a benefit in the treatment of cervical and uterine cancer compared with camptothecin monotherapy.