Primary Cervical Cancer Cell Research Articles

Elucidating the anticancerous efficacy of genistein via modulating HPV (E7 and E6) oncogenes expression and apoptotic induction in cervical cancer cells.

In recent years, genistein has garnered increased interest for its ability to inhibit numerous deregulated targets associated with cancer progression and induction of programmed cell death and antiproliferative activities in human carcinoma cells. Cancer etiology is influenced via multiple disrupted signaling pathways. This study therefore directed toward investigating genistein efficacy in modulating mRNA expression levels of two crucial Human Pappiloma Virus (HPV) (E7 and E6) oncogenes for cancer treatment. Moreover, the inhibitory effects of genistein for HPV (E7 and E6) oncogenes in cervical carcinoma have not yet been reported. Current study investigated inhibitory potential of genistein in HPV (E7 and E6) oncogenes in HeLa cells. These oncogenes are known to deactivate many tumor suppressor proteins (p53 and pRB). Genistein therapy resulted in decreased cell proliferation and increased cell accumulation in the G (G0/G1) phase in HeLa cell lines. In addition, genistein therapy has resulted in the suppression of HPV (E7 and E6) gene expression and simultaneously increasing expression levels of p53 and pRB mRNA levels. As a consequence, there has been an activation of a series of caspases (3, 8, and 9), resulting in their cleavage. Consequently, our data suggests that genistein could be a powerful candidate for treating cervical cancer by targeting two important oncogenes involved in viral development. However, more in vitro research on primary cervical cancer cells is required to validate the clinically relevant efficacy of genistein against cervical cancer.

Effect of trichostatin A on biological characteristics of side population cells of cervical cancer cell line (HeLa)

Purpose: To investigate the effect of trichostatin A (TSA) on biological characteristics of side population (SP) cells of cervical cancer cell line (HeLa)Methods: Side population (SP) and NSP cells were obtained from 6th generation of primary cervical cancer cells and cervical cancer cell line (HeLa). Cell surface markers (ATP-binding membrane transporter superfamily G member 2 (ABCG2), CD133, CD43, p63, Ki67, multidrug resistance (MDR) as well as cell cycle phase distribution and apoptosis, were determined. SP cells in HeLa were divided into 3 groups that received TSA at doses of 0.01, 0.05, and 0.2 μM. Untreated cells served as control. Cell growth inhibition rates of different dose groups were compared. SP cells in HeLa were divided into simple irradiation group and combined irradiation group TSA (10 % inhibition concentration (IC10) + irradiation, and values of SP cell survival fraction (SF) of the two groups under different irradiation conditions were compared.Results: The content of SP in HeLa cell line was significantly higher than in cervical cancer tissue (p > 0.05). There were no significant differences in expression levels of ABCG2, CD133, CD43, p63, Ki67, and MDR, as well as G0/G1, S, and G2/M phase distributions and apoptosis rate between HeLa cell line and cervical cancer tissue (p > 0.05). On the 3rd, 5th, and 7th day, SF value of SP cells was significantly higher in NSP cells (p > 0.05). With increasing concentration of TSA, SF value of NSP cells gradually decreased, while that of SP cells did not change significantly.Conclusion: Cervical cancer cell line (HeLa) contains SP cells which have biological characteristics of tumor stem cells. Moreover, TSA exerts good cytotoxicity and radio-sensitization effect on SP cells.

A laser free self-luminous nanosystem for photodynamic therapy of cervical cancer cells

Photodynamic therapy is a tumor treatment strategy. However, most of the photodynamic therapies rely on laser irradiation triggering, which limits their application in deep tissues. This study designed a self-luminescent nano system, hybrid protein oxygen nanocarrier coated graphene quantum dots (GQDs@HPOC) and mesoporous silica nanoparticles coated Luminol (L@MSNs), which self-assembled into GQDs@HPOC/L@MSNs without laser irradiation. The system utilized the weak acidic environment of tumors to trigger the release of Luminol and the chemiluminescence was catalyzed by HPOC. Next CRET occurred between Luminol and GQDs, producing 1O2, which could generate photodynamic damage to cervical cancer cells without the need for external laser irradiation. The system achieved the peak uptake in primary cervical cancer cells in 3 h, and had good biosafety before self-assembly. The system could significantly kill cells at a concentration of 16 μg/ml. The system will be further applied in in vivo experiments to investigate its therapeutic ability, providing a new strategy for the clinical treatment of cervical cancer.

Insights into the characteristics of primary radioresistant cervical cancer using single-cell transcriptomics

Radioresistance is a major cause of radiotherapy failure among patients with cervical cancer (CC), the fourth most common cause of cancer mortality in women worldwide. Traditional CC cell lines lose intra-tumoral heterogeneity, posing a challenge for radioresistance research. Meanwhile, conditional reprogramming (CR) maintains intra-tumoral heterogeneity and complexity, as well as the genomic and clinical characteristics of original cells and tissues. Three radioresistant and two radiosensitive primary CC cell lines were developed under CR conditions from patient specimens, and their characteristics were verified via immunofluorescence, growth kinetics, clone forming assay, xenografting, and immunohistochemistry. The CR cell lines had homogenous characteristics with original tumor tissues and maintained radiosensitivity in vitro and in vivo, while also maintaining intra-tumoral heterogeneity according to single-cell RNA sequencing analysis. Upon further investigation, 20.83% of cells in radioresistant CR cell lines aggregated in the G2/M cell cycle phase, which is sensitive to radiation, compared to 38.1% of cells in radiosensitive CR cell lines. This study established three radioresistant and two radiosensitive CC cell lines through CR, which will benefit further research investigating radiosensitivity in CC. Our present study may provide an ideal model for research on development of radioresistance and potential therapeutic targets in CC.

Endothelial cell-specific molecule 1 drives cervical cancer progression

The expression, biological functions and underlying molecular mechanisms of endothelial cell-specific molecule 1 (ESM1) in human cervical cancer remain unclear. Bioinformatics analysis revealed that ESM1 expression was significantly elevated in human cervical cancer tissues, correlating with patients’ poor prognosis. Moreover, ESM1 mRNA and protein upregulation was detected in local cervical cancer tissues and various cervical cancer cells. In established and primary cervical cancer cells, ESM1 shRNA or CRISPR/Cas9-induced ESM1 KO hindered cell proliferation, cell cycle progression, in vitro cell migration and invasion, and induced significant apoptosis. Whereas ESM1 overexpression by a lentiviral construct accelerated proliferation and migration of cervical cancer cells. Further bioinformatics studies and RNA sequencing data discovered that ESM1-assocaited differentially expressed genes (DEGs) were enriched in PI3K-Akt and epithelial-mesenchymal transition (EMT) cascades. Indeed, PI3K-Akt cascade and expression of EMT-promoting proteins were decreased after ESM1 silencing in cervical cancer cells, but increased following ESM1 overexpression. Further studies demonstrated that SYT13 (synaptotagmin 13) could be a primary target gene of ESM1. SYT13 silencing potently inhibited ESM1-overexpression-induced PI3K-Akt cascade activation and cervical cancer cell migration/invasion. In vivo, ESM1 knockout hindered SiHa cervical cancer xenograft growth in mice. In ESM1-knockout xenografts tissues, PI3K-Akt inhibition, EMT-promoting proteins downregulation and apoptosis activation were detected. In conclusion, overexpressed ESM1 is important for cervical cancer growth in vitro and in vivo, possibly by promoting PI3K-Akt activation and EMT progression. ESM1 represents as a promising diagnostic marker and potential therapeutic target of cervical cancer.

Triptonide Inhibits the Cervical Cancer Cell Growth via Downregulating the RTKs and Inactivating the Akt-mTOR Pathway.

The high incidence and mortality of cervical cancer (CC) require an urgent need for exploring novel valuable therapeutics. Triptonide (TN) is a small molecule monomer extracted from the Chinese herb Tripterygium wilfordii Hook. Our results showed that TN, at only nanomolar concentrations, strongly inhibited growth, colony formation, proliferation, migration, and invasion of established and primary human cervical cancer cells. TN induced apoptosis and cell cycle arrest in cervical cancer cells. Moreover, cervical cancer cell in vitro migration and invasion were suppressed by TN. It was however noncytotoxic and proapoptotic to normal cervical epithelial cells and human skin fibroblast cells. Gene set enrichment analysis (GSEA) of RNA sequencing data of differentially expressed genes (DEGs) in TN-treated cervical cancer cells implied that DEGs were enriched in the receptor tyrosine kinase (RTK) signaling and PI3K-Akt-mTOR cascade. In cervical cancer cells, RTKs, including EGFR and PDGFRα, were significantly downregulated and Akt-mTOR activation was largely inhibited after TN treatment. In vivo, oral administration of TN significantly inhibited subcutaneous cervical cancer xenograft growth in nude mice. EGFR and PDGFRα downregulation as well as Akt-mTOR inactivation was detected in TN-treated HeLa xenograft tumor tissues. Thus, TN inhibits human cervical cancer cell growth in vitro and in vivo. Its anticervical cancer activity was associated with RTK downregulation and Akt-mTOR inactivation.

The sphingosine kinase inhibitor SKI-V suppresses cervical cancer cell growth.

Overexpression and/or overactivation of sphingosine kinase 1/2 (SphK1/2) is important for tumorigenesis and progression of cervical cancer. The current study examined the potential activity and signaling mechanisms of SKI-V, a non-lipid small molecule SphK inhibitor, against cervical cancer cells. In different primary and immortalized cervical cancer cells, SKI-V exerted significant anti-cancer activity by inhibiting cell viability, colony formation, proliferation, cell cycle progression and cell migration. Significant apoptosis activation was detected in SKI-V-treated cervical cancer cells. Significantly, SKI-V also provoked programmed necrosis cascade in cervical cancer cells, as it induced mitochondrial p53-cyclophilin-D-adenine nucleotide translocator-1 (ANT1) complexation, mitochondrial membrane potential collapse, reactive oxygen species production and the release of lactate dehydrogenase into the medium. Further, SKI-V blocked SphK activation and induced ceramide accumulation in primary cervical cancer cells, without affecting SphK1/2 expression. SKI-V-induced cytotoxicity in cervical cancer cells was largely inhibited by sphingosine-1-phosphate or the SphK1 activator K6PC-5, but was sensitized by adding the short-chain ceramide C6. Moreover, SKI-V inhibited Akt-mTOR (mammalian target of rapamycin) activation in primary cervical cancer cells, and its cytotoxicity was mitigated by a constitutively-active Akt. In vivo, daily intraperitoneal injection of SKI-V significantly inhibited subcutaneous primary cervical cancer xenograft growth in nude mice. Together, the SphK inhibitor SKI-V suppresses cervical cancer growth in vitro and in vivo.

Identification of Gαi3 as a novel molecular therapeutic target of cervical cancer.

Here we studied expression and potential functions of Gαi3 in cervical cancer. The bioinformatics analysis together with the results from local patients' tissues revealed that Gαi3 expression was remarkably elevated in human cervical cancer tissues and different cervical cancer cells, and was associated with poor overall survival and poor disease-specific survival of patients. Gαi3 depletion resulted in profound anti-cervical cancer activity. In primary or immortalized cervical cancer cells, Gαi3 shRNA or CRISPR/Cas9-caused Gαi3 knockout/KO largely hindered cell proliferation and migration, and provoked apoptosis. On the contrast, ectopic Gαi3 overexpression further enhanced cervical cancer proliferation and migration. Akt-mTOR activation in primary cervical cancer cells was significantly reduced after Gαi3 silencing or KO, but was augmented following Gαi3 overexpression. Further studies revealed that the transcription factor GATA4 binding to Gαi3 promoter region was significantly enhanced in cervical cancer tissues and cells. Gαi3 expression was decreased by GATA4 shRNA, but upregulated following GATA4 overexpression. In vivo, the growth of cervical cancer xenografts was robustly suppressed after Gαi3 silencing or KO. Gαi3 depletion and Akt-mTOR inactivation were detected in Gαi3-silenced/-KO cervical cancer xenograft tissues. Together, upregulated Gαi3 is a valuable oncotarget of cervical cancer.

Biodegradable and Dual-Responsive Polypeptide-Shelled Cyclodextrin-Containers for Intracellular Delivery of Membrane-Impermeable Cargo.

The transport of membrane impermeable compounds into cells is a prerequisite for the efficient cellular delivery of hydrophilic and amphiphilic compounds and drugs. Transport into the cell's cytosolic compartment should ideally be controllable and it should involve biologically compatible and degradable vehicles. Addressing these challenges, nanocontainers based on cyclodextrin amphiphiles that are stabilized by a biodegradable peptide shell are developed and their potential to deliver fluorescently labeled cargo into human cells is analyzed. Host–guest mediated self‐assembly of a thiol‐containing short peptide or a cystamine‐cross‐linked polypeptide shell on cyclodextrin vesicles produce short peptide‐shelled (SPSVss) or polypeptide‐shelled vesicles (PPSVss), respectively, with redox‐responsive and biodegradable features. Whereas SPSVss are permeable and less stable, PPSVss effectively encapsulate cargo and show a strictly regulated release of membrane impermeable cargo triggered by either reducing conditions or peptidase treatment. Live cell experiments reveal that the novel PPSVSS are readily internalized by primary human endothelial cells (human umbilical vein endothelial cells) and cervical cancer cells and that the reductive microenvironment of the cells’ endosomes trigger release of the hydrophilic cargo into the cytosol. Thus, PPSVSS represent a highly efficient, biodegradable, and tunable system for overcoming the plasma membrane as a natural barrier for membrane‐impermeable cargo.

Abstract 5341: Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma

Abstract Purpose: The prognosis for women with advanced or recurrent cervical cancer (CC) no longer responsive to radiation and/or chemotherapy remains poor. Whole-exome-sequencing (WES) studies have recently reported c-MYC gene amplification and HUWE1 gene mutations in a significant number of CC suggesting the HUWE1/c-MYC pathway as potential therapeutic target. We investigated HUWE1 and c-MYC expression in fresh-frozen CC and the potential activity of the novel BET bromodomain inhibitor GS-626510 (Gilead-Science-Inc.) against primary WES CC-cultures and CC-xenografts. Experimental Design: HUWE1 and c-MYC expression were evaluated by qRT-PCR in a total of 23 CC including 12 fresh frozen tumor tissues and 11 primary cell lines. c-MYC expression was also evaluated by Western-Blot (WB) and fluorescence in situ hybridization (FISH) experiments in all 11 fully sequenced primary CC cell lines. Primary tumors were evaluated for sensitivity to GS-626510 in-vitro using proliferation and viability-assays. siRNA experiments were used to evaluate the effect of HUWE1 silencing on primary CC cell line growth and sensitivity to GS-626510. Finally, the in-vivo activity of GS-626510 was studied in CC-CVX8 mouse-xenografts. Results: Fresh-frozen CC and primary CC cell lines overexpressed c-MYC when compared to normal control tissues (mean ± SEM mRNA values = 7.06 ± 1.17 vs a mean ± SEM = 2.96 ± 0.97, p = 0.01). FISH experiments demonstrated amplification of c-MYC in 9 out of 11 (82%) of the primary CC cell lines. Primary CC cell lines with derangements in the HUWE1/c-MYC pathway were highly sensitive to GS-626510, with a dose-response decrease in cell proliferation and IC50 values ranging from 3 to 57 nM. Silencing of HUWE1 by siRNA significantly increased c-MYC expression as well as CC cell proliferation and enhanced the in vitro sensitivity to GS-626510. Twice daily oral doses of GS-626510 (10 mg/kg) were well tolerated in vivo in animals and highly effective in decreasing tumor-growth (p = 0.004) and increasing survival (p value = 0.004) of mice harboring CC-CVX8 xenografts. Conclusions: Downregulation/inactivation of HUWE1 may increase c-MYC expression and proliferation in primary CC cell lines. GS-626510 may represent a novel, potentially highly effective therapeutic agent against CC overexpressing c-MYC and/or harboring HUWE1 mutations. Clinical studies with BET inhibitor in CC-patients harboring radiation/chemotherapy-resistant disease are warranted. Citation Format: Elena Bonazzoli, Stefania Bellone, Luca Zammataro, Barbara Gnutti, Adele Guglielmi, Silvia Pelligra, Paola Manara, Joan Tymon-Rosario, Burak Zeybek, Chanhee Han, Alessandro D. Santin. Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma [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 5341.

Application of CircEIF4G2 in Screening of Cervical Lesions.

The aim was to investigate the expression of circulating RNA EIF4G2 (CircEIF4G2) in cervical cancer and its correlation with clinicopathological features. Cervical tissue and peripheral blood serum samples were collected from 148 patients with cervical lesions, including 30 patients with low-grade squamous intraepithelial lesions (LSIL group), 35 patients with high squamous intraepithelial lesion (HSIL group), 28 patients with atypical squamous cells (ASC group), and 55 patients with cervical cancer (CC group). At the same time, cervical biopsy specimens and peripheral blood serum were collected from 40 healthy women (Normal group). RT-PCR was used to detect the expression of CircEIF4G2 in cervical tissues and peripheral blood of each group. Electron microscopy was used to observe the distribution of exosomes CircEIF4G2 in cervical tissues. Meanwhile, the correlation between the expression level of CircEIF4G2 and clinical pathological data of patients was analyzed. In vitro, HeLa cells and primary cervical epithelial cells were cultured for 24 hours. Then, the expression levels of CircEIF4G2 in the two kinds of cells were detected by RT-PCR in medium. Furthermore, primary cervical epithelial cells were co-cultured with HeLa cells to observe the effect of exosome CircEIF4G2 on primary cervical epithelial cells. The expression of CircEIF4G2 in the cervical tissue and serum of the normal group was significantly lower than that in the CC group (p < 0.05), but there was no significant difference between the LSIL group and the HSIL group in the cervical tissue and serum (p < 0.05). The distribution and expression of exosomes CircEIF4G2 in each group were consistent with RT-PCR results under an electron microscope. The results of experiments in vitro showed that the expression level of CircEIF4G2 in HeLa cells was significantly higher than that in primary cervical epithelial cells (p < 0.05). The medium in which Hela cells were cultured for 24 hours was added to the culture medium of primary cervical epithelial cells. The process of exosomes CircEIF4G2 entering primary cervical cancer cells was observed by electron microscopy. The increased expression of CircEIF4G2 in tissues and serum of cervical lesions may be caused by the secretion of exosomes containing CircEIF4G2 by cervical cancer cells. Therefore, CircEIF4G2 can be used as a marker for the diagnosis of cervical lesions.

Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells

BackgroundLactobacillus plantarum, a major species of Lactic Acid Bacteria (LAB), are capable of producing postbiotic metabolites (PM) with prominent probiotic effects that have been documented extensively for rats, poultry and pigs. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on cytotoxic and antiproliferative activity of PM produced by L. plantarum. Therefore, the cytotoxicity of PM produced by six strains of L. plantarum on various cancer and normal cells are yet to be evaluated.MethodsPostbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses.ResultsUL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis.ConclusionIn conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer.

Cervical carcinomas that overexpress human trophoblast cell-surface marker (Trop-2) are highly sensitive to the antibody-drug conjugate sacituzumab govitecan.

e17028 Background: Recurrent disseminated cervical cancer is a discouraging clinical entity with 1-year survival rates between 10-15%, and development of novel, effective treatments remains an unmet medical need. We investigated the preclinical efficacy of IMMU-132 (sacituzumab govitecan), an antibody-drug conjugate (ADC) comprised of a humanized anti-trophoblast-cell-surface-antigen (Trop-2) antibody (hRS7), conjugated with a hydrolysable linker to the active metabolite of irinotecan (SN-38), against Trop-2 positive primary cervical cancer cell lines and mouse xenografts. Methods: Trop-2 expression was evaluated in 151 cervical tumors and 5 primary tumor cell lines by immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and flow cytometry. Cervical cancer cell viability against IMMU-132, non-targeting control ADC, and naked antibody (hRS7 IgG) was evaluated using flow-cytometry-based assays after 48-72 hours of incubation at a concentration of 0.05, 0.5, 1, 2, 4, and 10 nM of each agent. In vivo antitumor activity was tested in xenograft models with 3+ Trop-2 expression. Results: Out of 151 cervical tumors, 123 were squamous cell carcinoma (SCC), 26 were adenosquamous/adenocarcinoma, 1 was neuroendocrine carcinoma, and 1 was clear cell carcinoma. Strong diffuse staining was seen in 61% (75/123) of SCCs, and 92% (24/26) of adenocarcinoma/adenosquamous cancers. The neuroendocrine carcinoma was also strongly positive, while there was no staining in the clear cell carcinoma. Four out of five (80%) primary cervical cancer cell lines evaluated overexpressed Trop-2 as determined by RT-PCR and flow cytometry. Trop-2 positive cell lines showed high sensitivity to IMMU-132 in vitro, with IC50-values in the range of 0.18 to 0.26 nM ( P= 0.02). In contrast, a Trop-2 negative cervical cell line was highly resistant to the ADC with similar IC50 for IMMU-132 and control ADC. In xenografts, twice-weekly intravenous administration of IMMU-132 for three weeks demonstrated significant tumor growth inhibition compared to controls ( P≤0.001). Overall survival was significantly increased in mice treated with IMMU-132 ( P= 0.014) when compared to naked antibody and ADC-control groups. Conclusions: Sacituzumab govitecan may represent a novel class of active drugs for cervical cancers overexpressing Trop-2. Clinical trials are warranted.

Abstract LB-A01: Yes-associated protein 1 regulates cervical cancer initiation and progression via suppressing innate immunity and increasing cell susceptibility to human papillomavirus infection

Abstract Introduction: Although human Papillomavirus (HPV) has been identified as a causative reagent for cervical cancer (CVC), accumulating evidence indicates that HPV alone is insufficient to induce CVC. The molecular mechanisms underlying CVC development are largely unknown. Our previous in vitro studies indicated that YAP1, the major effector of the Hippo pathway, interacts with HPV to drive the progression of CVC. However, whether the Hippo/YAP1 pathway also regulates cervical carcinogenesis in vivo is unknown. Aims: To examine the molecular mechanism(s) by which the Hippo/YAP pathway interacts with HPV to regulate the cervical carcinogenesis. Methods: The cBioportal for Cancer Genomics, an online tool developed to visualize The Cancer Genomic Atlas (TCGA) studies, was used to analyze alterations of the Hippo pathway in human CVC. Primary cervical epithelial cells and cervical cancer cell lines were used to determine effects of YAP1 on cell susceptibility to HPV infection. KRT14-YAPS127A mice, which express constitutively active YAP1 (YAPS127A) in cervical epithelial cells under doxycycline (Dox) induction, and KRT14-E6/E7-YAPS127A mice, which express both HPV E6/E7 and YAPS127A in cervical epithelial cells under Dox induction, were used to examine the role of YAP1 gene in cervical carcinogenesis. Results: Multidimensional cancer genomic data showed that disruption of the Hippo signaling pathway is a common event in human CVC, which is featured by frequent amplification and up-regulation of oncogenes and deletion/mutation of tumor suppressors. Moreover, the disrupted Hippo pathway is associated with poor prognosis of CVC patients. Dox-induced expression of YAPS127A in KRT14-YAPS127A mouse cervical epithelial cells resulted in neoplasia of cervical epithelium within 2 months. Most KRT14-YAPS127A mice developed invasive CVC after induction with dox for 6 months. Metastatic spread of CVC induced urethral and outlet obstruction in some KRT14-YAPS127A mice. Around 50% of KRT14-YAPS127A mice died of CVC within 16 months of Dox induction. Histological analysis revealed that KRT14-YAPS127A mice developed squamous cell carcinomas (SCC). Although KRT14-E6/E7 mice (only expressing E6/E7) rarely developed CVC after induction with Dox for 12 months, KRT14-E6/E7-YAPS127A mice developed cervical cancer within 4 months of Dox induction. Compared to KRT14-YAPS127A mice, cancer cells in the cervical tissue of KRT14-E6/E7-YAPS127A mice were more proliferative and invasive. Mechanistic studies showed that ectopic expression of YAPS127A in human cervical epithelial cells resulted in a significant increase in cell susceptibility to HPV infection, while inhibition of YAP1 activity reduced the acceptability of cervical epithelial cells to HPV infection. Intriguingly, we found that activation of YAP induced up-regulation of the putative HPV cellular receptor and disrupted innate immunity in cervical epithelial cells. Conclusion: Our research demonstrates that constitutive activation of YAP1 is sufficient to induce cervical SCC. Hyper-activation of YAP increases the susceptibility of cervical epithelial cells to HPV infection. HPV, in turn, synergistically promotes YAP1-induced initiation and progression of cervical cancer. The Hippo/YAP signaling pathway may represent a promising target for developing novel strategies to prevent and treat cervical cancer. [C.H., X.L., and C.H. contributed equally to this work.] Citation Format: Chunbo He, Xiangmin Lv, Cong Huang, Guohua Hua, Jin Zhou, Zhengfeng Wang, Jixin Dong, Peter C Angeletti, Paul F. Lambert, Bo R Rueda, John S Davis, Cheng Wang. Yes-associated protein 1 regulates cervical cancer initiation and progression via suppressing innate immunity and increasing cell susceptibility to human papillomavirus infection [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A01.

Characteristics of primary side population cervical cancer cells.

The aim of the present study was to identify and characterize side population (SP) cells in primary cervical cancer. A primary culture was successfully established, and the SP cells were isolated via fluorescence-activated cell sorting. Subsequently, in vitro analysis of clonogenic capacity by soft agar assay and in vivo analysis of tumorigenicity were performed. The isolated SP cells accounted for ~4.73% of the total primary culture cells. The SP cells had a decreased proliferation rate and an increased distribution in G0/G1 compared with non-SP (NSP) cells. Following isolation, SP cells exhibited increased proliferative and self-renewal potency compared with NSP cells. Furthermore, significant ATP binding cassette subfamily G member 2 (ABCG2) expression was detected in SP cells but not in NSP cells. The tumor formation rate of SP cells was longer, and the tumor size and tumor formation rate of SP cells were increased in non-obese diabetic/severe combined immunodeficiency mice. In conclusion, the present study demonstrated that SP cells can be isolated from primary cervical cancer cell culture, and SP cells are enriched with stem cell-like cells that have a high capacity for colony formation and tumorigenesis.

Recurrence of cervical cancer and its resistance to progestin therapy in a mouse model.

Studies using K14E6/K14E7 transgenic mice expressing E6 and E7 oncoprotein of human papillomavirus type 16 (HPV16) have demonstrated that estrogen (E2) is required for the genesis and growth of cervical cancer. Our prior study using the same mouse model has showed that progestin drug medroxyprogesterone acetate (MPA) promotes regression of primary cervical cancer. In the present study, we use the same transgenic mouse model to determine whether the cancer recurs after MPA therapy. Cervical cancer recurred even if MPA treatment was continued. Unlike primary cervical cancer, the cancer recurred even in the absence of exogenous E2 when MPA treatment was ceased. Furthermore, recurrent cervical cancer did not fully regress upon MPA treatment. Our results support that MPA fails to completely eliminate primary cervical cancer cells and that remaining cancer cells grow independent of exogenous E2 and are refractory to MPA.

Neratinib, an irreversible pan-ErbB receptor inhibitor, is highly effective against primary cervical cancer cell lines harboring HER2/neu gene mutations

(OS) and progression-free survival (PFS) were estimated using the Kaplan–Meier method. The associations of smoking and of smoking + treatment (i.e., bevacizumab vs. no bevacizumab) with PFS and OS were evaluated with the Cox proportional hazards model. Results: Smoking questionnaires were available for analysis in 99% of study participants. Two hundred eighty-six women (64%) were active or former smokers. In the entire study population, this group had significantly worse OS than never smokers (14.2 vs. 16.8 months, HR 1.29, 95% CI 1.03–1.61, P= 0.026). Smokers who received chemotherapy plus bevacizumab (n= 148, 52%) experienced significantly longer PFS (median 7.9 vs. 5.7 months, log-rank P b 0.01) and significantly longer OS (median 16.7 vs. 12.0 months, log rank P= 0.027) than smokers who received chemotherapy alone (n= 138, 48%). Overall response rate among smokers was 49.3% (chemotherapy plus bevacizumab) vs. 29% (chemotherapy alone) (P= 0.002). The median number of cycles of therapy was 6 for smokers and nonsmokers (interquartile range, 4–10, smokers vs. 2–9, nonsmokers) (P=ns). Smokers receiving bevacizumab experienced the following adverse events: grade 2+ fistula (16.2%), grade 3+ venous thromboembolism (9.5%), grade 2+hypertension (27%), and grade 2+proteinuria (2.7%). Cervical cancer was the reported cause of death in 74.5% of smokers. Conclusions: Smoking is common and associated with a 29% increased risk of death among women with advanced or recurrent cervical cancer. The survival benefits conferred by antiangiogenesis therapy are sustained, even among smokers in this population. Smokers with CxCA should be informed of these results and efforts at smoking cessation strongly encouraged.

Bee venom inhibits growth of human cervical tumors in mice.

We studied whether bee venom (BV) inhibits cervical tumor growth through enhancement of death receptor (DR) expressions and inactivation of nuclear factor kappa B (NF-κB) in mice. In vivo study showed that BV (1 mg/kg) inhibited tumor growth. Similar inhibitory effects of BV on cancer growth in primary human cervical cancer cells were also found. BV (1-5 μg/ml) also inhibited the growth of cancer cells, Ca Ski and C33Aby the induction of apoptotic cell death in a dose dependent manner. Agreed with cancer cell growth inhibition, expression of death receptors; FAS, DR3 and DR6, and DR downstream pro-apoptotic proteins including caspase-3 and Bax was concomitantly increased, but the NF-κB activity and the expression of Bcl-2 were inhibited by treatment with BV in tumor mice, human cancer cell and human tumor samples as well as cultured cancer cells. In addition, deletion of FAS, DR3 and DR6 by small interfering RNA significantly reversed BV-induced cell growth inhibitory effects as well as NF-κB inactivation. These results suggest that BV inhibits cervical tumor growth through enhancement of FAS, DR3 and DR6 expression via inhibition of NF-κB pathway.

MicroRNA-218 enhances the radiosensitivity of human cervical cancer via promoting radiation induced apoptosis.

We previously reported frequent loss of microRNA-218 (miR-218) in cervical cancer, which was associated with tumor progression and poor prognosis. As microRNAs were found invovled in the regulation of radiosensitivity in various human cancers, we therefore aim to investigate the effects of miR-218 on radiosensitivity of cervical cancer in the present study. The clonogenic survival assay demonstrated that loss of miR-218 could predict radioresistance in the primary cervical cancer cells (R2=0.6516, P<0.001). In vitro, abundant miR-218 increased the radiosensitivity in cervical cancer cells (P<0.001 for HeLa, P=0.009 for SiHa, P=0.016 for C33A and P=0.01 for CaSki). Upregulation of miR-218 significantly enhanced the radiation-induced apoptosis, which was further enhanced by the combination of miR-218 overexpression and radiation In xenograft growth assay, combination of miR-218 overexpression and radiation notably induced cellular apoptosis and suppressed tumor growth. In conclusion, we demonstrated that miR-218 resensitized cervical cancer cells to radiation via promoting cellular apoptosis. Moreover, we proved that miR-218 as a potent predictor of radiosensitivity in cervical cancer, especially for those patients with loss of miR-218.

Time‐varying magnetic fields of 60 Hz at 7 mT induce DNA double‐strand breaks and activate DNA damage checkpoints without apoptosis

The potential genotoxic effect of a time-varying magnetic field (MF) on human cells was investigated. Upon continuous exposure of human primary fibroblast and cervical cancer cells to a 60 Hz MF at 7 mT for 10-60 min, no significant change in cell viability was observed. However, deoxyribonucleic acid (DNA) double-strand breaks (DSBs) were detected, and the DNA damage checkpoint pathway was activated in these cells without programmed cell death (called apoptosis). The exposure of human cells to a 60 Hz MF did not induce intracellular reactive oxygen species (ROS) production, suggesting that the observed DNA DSBs are not directly caused by ROS. We also compared the position and time dependency of DNA DSBs with numerical simulation of MFs. The Lorentz force and eddy currents in these experiments were numerically calculated to investigate the influence of each factor on DNA DSBs. The DNA DSBs mainly occurred at the central region, where the MF was strongest, after a 30-min exposure. After 90 min, however, the amount of DNA DSBs increased rapidly in the outer regions, where the eddy current and Lorentz force were strong.