Inhibition of Plasminogen Activator Inhibitor-1 (PAI-1) by Tiplaxtinin Reduces Aggressiveness of Cervical Carcinoma Cells.

G protein-coupled estrogen receptor 1 (GPER1) appears to play a tumor-suppressive role in cervical squamous cell carcinoma (CSCC)GPER1 suppression leads to significantly increased expression of serpin family E member 1 (SERPINE1)/protein plasminogen activator inhibitor type 1 (PAI-1). The question arises, what role does SERPINE1/PAI-1 play in GPER1-dependent tumorigenic potential of CSCC. SiHa and C33A CSCC cells were treated with GPER1 agonist G1 or antagonist G36. SERPINE1/PAI-1 expression was suppressed by RNAi and success was confirmed by RT-qPCR. Protein expression of PAI-1 was quantified by Western blot. Viability was analyzed using resazurin assay, while migration was investigated using gap closure. Colony and tumor sphere formation were used to test clonogenicity. After G1 treatment, viability of SiHa and C33A cells remained unchanged. Cell migration was dose-dependently reduced. SiHa and C33A cells formed significantly fewer and smaller colonies as well as spheroids. Furthermore, treatment with G1 led to decreased expression of SERPINE1/PAI-1, while blockade of GPER1 with G36 resulted in significantly increased SERPINE1/PAI-1 expression. After suppression of SERPINE1/PAI-1 in SiHa cells using RNAi, cell viability remained unaffected; however, significantly smaller colonies were formed, and fewer and smaller spheroids were developed. Cell migration remained unaffected. Activation of GPER1 reduces clonogenicity and migration of CSCC cells and suppresses expression of SERPINE1/PAI-1. Suppression of SERPINE1/PAI-1 in CSCC cells reduces tumorigenic potential. GPER1 may be a suitable target for suppression of SERPINE1/PAI-1 in CSCC. However, SERPINE1/PAI-1 does not appear to be the decisive factor for GPER1-regulated cell migration.

BackgroundSirtuin 7 (SIRT7) is an oncogene that promotes tumor progression in various malignancies, however, its role and regulatory mechanism in cervical squamous cell carcinoma (CSCC) is unknown. Herein, we attempted to investigate the functional role and molecular mechanism of SIRT7 underlying CSCC progression.MethodsSIRT7 expression was evaluated in CSCC cells using various assays. We then used a series of function gain-and-loss experiments to determine the role of SIRT7 in CSCC progression. Furthermore, mechanism experiments were conducted to assess the interaction between SIRT7/USP39/FOXM1 in CSCC cells. Additionally, rescue assays were conducted to explore the regulatory function of USP39/FOXM1 in CSCC cellular processes.ResultsSIRT7 was highly expressed in CSCC patient tissues and cell lines. SIRT7 deficiency showed significant repression on the proliferation, and autophagy of CSCC cells in vitro and tumorigenesis in vivo. Similarly, apoptosis and ROS production in CSCC cells were accelerated after the SIRT7 knockdown. Moreover, SIRT7 and USP39 were found colocalized in the cell nucleus. Interestingly, SIRT7 was revealed to deacetylate USP39 to promote its protein stability in CSCC cells. USP39 protein was also verified to be upregulated in CSCC tissues and cells. USP39 silencing showed suppressive effects on CSCC cell growth. Mechanistically, USP39 was revealed to upregulate SIRT7 by promoting the transcriptional activity of FOXM1. Rescue assays also indicated that SIRT7 promoted autophagy and inhibited ROS production in CSCC cells by regulating USP39/FOXM1.ConclusionThe SIRT7/USP39/FOXM1 positive feedback network regulates autophagy and oxidative stress in CSCC, thus providing a new direction for CSCC-targeted therapy.Graphical

Cervical squamous cell carcinoma (CSCC), the most common cervical malignancy, is more likely to invade and metastasize than other cervical cancers. miR-125a, a tumor suppressor gene, has been confirmed to be associated with cancer metastasis. However, the role of miR-125a in CSCC and the underlying mechanism are unknown. miR-125a expression was confirmed by real-time quantitative PCR (RT–qPCR), and the Rad51 expression level was measured by western blotting analysis. CSCC cell proliferation, migration and invasion were assessed with functional assays, including CCK-8, colony formation, wound healing and Transwell assays. Our data confirmed that miR-125a is expressed at low levels in CSCC tissues and cells. Functionally, the overexpression of miR-125a greatly prevented the proliferation, migration and invasion of CSCC cells, and the inhibition of miR-125a expression strongly enhanced these behaviors in CSCC cells. Moreover, the expression of Rad51, a miR-125a target gene, greatly reversed the miR-125-mediated inhibition of CSCC cell proliferation, migration and invasion. In addition, we discovered that miR-125a downregulated the levels of phosphorylated PI3K, AKT and mTOR through Rad51 in CSCC cells. miR-125a, a tumor suppressor, can attenuate the malignant behaviors of CSCC cells by targeting Rad51. Therefore, the miR-125a/Rad51 axis might be a target for CSCC therapy.

BackgroundCervical squamous cell carcinoma (CSCC) is responsible for 80–85% of cervical cancer. Cyclin B1 (CCNB1) represents a hub gene during the development of cervical cancer. However, the oncogenic role of CCNB1 in CSCC remains unclear. Our study aims to explore the mechanism underlying CCNB1 regulation on cell cycle progression in CSCC cells.MethodsFirst, we analyzed differentially expressed genes from CSCC dataset GSE63678 and conducted gene function enrichment analysis. Subsequently, CCNB1 expression was knocked down in CSCC cell lines to assess cell proliferation, apoptosis, and cell cycle distribution. After the validation of the binding relationship between forkhead box protein M1 (FOXM1) and the promoter of CCNB1, the effect of FOXM1 on CCNB1 expression and on CSCC cell growth and apoptosis was verified. We further analyzed the histone ChIP-Seq data of CCNB1 in CSCC cells and measured the acetylation levels of the CCNB1 promoter histones.ResultsCCNB1 was overexpressed in CSCC tissues and cells, and CCNB1 silencing inhibited the growth of CSCC cells, and promoted cell cycle arrest and apoptosis. FOXM1 potentiated CCNB1 transcription by binding to its promoter and recruiting CBP/P300, a histone acetyltransferase. Further increasing FOXM1 expression or increasing P300 activity in CSCC cells with CCNB1 knockdown elevated CCNB1 expression and proliferation and cell cycle progression of CSCC cells. Knockdown of CCNB1 activated the p53 pathway in cells.ConclusionFOXM1 inhibited the activation of the p53 pathway by recruiting CBP/P300, which promoted the transcription of CCNB1, resulting in the growth and cell cycle progression of CSCC cells.

BackgroundThe dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear.MethodsExosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed.ResultsOur previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα+ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα+ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression.ConclusionsOur results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.

IntroductionThe incidence of cervical squamous cell carcinoma (CSCC) has expanded in recent years. However, the function of long non-coding RNA (lncRNA) MAGI2-AS3 in the occurrence and progression of CSCC remains unclear. Therefore, the role of lncRNA MAGI2-AS3 in cervical squamous cell carcinoma (CSCC) was investigated in our study.MethodsWe used qRT-PCR analysis to identify the level of MAGI2-AS3 mRNA expression in CSCC clinical samples and cell lines. We investigated cell migration and invasion of CSCC cells transfected with MAGI2-AS3, miR-233 mimic, or EPB41L3 with transwell assays. Bioinformatics analysis and a luciferase reporter assay were employed to predict the interaction between MAGI2-AS3 and miR-233.ResultsWe found that MAGI2-AS3 and EPB41L3 were both downregulated in CSCC and the expression of this two was positively correlated. Bioinformatics analysis showed that MAGI2-AS3 might bind to miR-233, which could directly target EPB41L3. In CSCC cells, overexpression of MAGI2-AS3 led to upregulated, while overexpression of miRNA-233 led to downregulated expression of EPB41L3. However, MAGI2-AS3 and miR-233 did not affect the expression of each other. In addition, overexpression of MAGI2-AS3 and EPB41L3 led to inhibited cancer cell invasion and migration, while overexpression of miR-233 played an opposite role and attenuated the effects of overexpressing MAGI2-AS3.ConclusionMAGI2-AS3 may sponge miR-233 to upregulate EPB41L3, thereby inhibiting CSCC cell invasion and migration.

Cervical cancer (CC) is the most common malignancy of the female reproductive system, among which cervical squamous cell carcinoma (CESC) is the most common type. The demethylase ALKBH5 has been previously revealed to be downregulated in CC tissue. N6 methyladenine (m6A) is the most common modification in eukaryotic RNAs and is involved in modulating tumour progression. Therefore, we attempted to clarify the ALKBH5 role and mechanism underlying CESC progression. In CESC, patient tissue and control tissue m6A levels were measured. Reverse transcription quantitative real-time polymerase chain reaction, western blotting and immunochemistry were used to measure ALKBH5 levels. A correlation between CESC patient survival and ALKBH5 levels was evaluated. Wound healing, transwell and colony formation assays were used to detect CESC cellular behaviours. Corresponding kits and BODIPY staining were used to detect CESC lipid metabolism. Bioinformatics, immunoprecipitation, RNA pulldown and RNA immunoprecipitation assays as well as half-life measurements were used to assess the association and mechanism of ALKBH5 with silent mating type information regulation 2 homologue 3 (SIRT3), acetyl-CoA carboxylase 1 (ACC1) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1). The m6A demethylase ALKBH5 was depleted in CESC tissue and cells, and a low level of ALKBH5 predicted an unfavourable prognosis in CESC patients. ALKBH5 overexpression suppressed CESC growth and lipid metabolism in vitro and CESC tumour growth in vivo, and ACC1 overexpression rescued these changes. ALKBH5 downregulated ACC1 levels in CESC cells by facilitating SIRT3 methylation to repress ACC1 deacetylation. ALKBH5 destabilized SIRT3 to downregulate SIRT3 levels in CESCs in an m6A-IGF2BP1-dependent manner. ALKBH5 demethylates and destabilizes SIRT3 in an m6A-IGF2BP1-dependent manner, repressing CESC growth, lipid metabolism and tumorigenesis by downregulating ACC1.

Invasion of uterine cervical squamous cell carcinoma cells is facilitated by locoregional interaction with cancer-associated fibroblasts via activating transforming growth factor-beta

Long non-coding RNA (lncRNA) LINC00514 is a cancer-related lncRNA that has been proven to be implicated in the progression of several cancers. However, the biological function of LINC00514 in cervical squamous cell carcinoma (CSCC) remains unclear. Thus, we aimed to identify the LINC00514 expression profile in CSCC and determine its exact mechanism. Our results showed that the expression of LINC00514 was up-regulated in human CSCC tissues and cell lines. Knockdown of LINC00514 significantly inhibited the proliferation and invasion of CSCC cells, as well as suppressed tumorigenesis of CSCC in vivo. In addition, LINC00514 was found to work as a miRNA sponge for miR-708-5p and suppressed the expression of miR-708-5p in CSCC cells. Homeobox B3 (HOXB3) was found to be a target gene of miR-708-5p. Rescue assays demonstrated that miR-708-5p inhibitor attenuated the effects of LINC00514 knockdown on cell proliferation and invasion in CSCC cells. In addition, overexpression of HOXB3 reversed the inhibitory effects of miR-708-5p mimics on cell proliferation and invasion. Taken together, our findings for the first time elucidated that lncRNA LINC00514 promotes the proliferation and invasion through the miR-708-5p/HOXB3 axis in CSCC. Thus, LINC00514/miR-708-5p/HOXB3 axis might be a promising therapeutic target for the treatment of CSCC.

Background/AimCervical cancer (CC) is the fourth most common cancer in women worldwide. There are two main histological subtypes of CC: the more common cervical squamous cell carcinoma (CSCC) and the rarer cervical adenocarcinoma (CAC), which has a poorer prognosis. Unlike estrogen receptor (ER) α and ERβ, G-protein-coupled estrogen receptor 1 (GPER1) is recognized as a rapid mediator of cellular estrogenic action and tends to have tumor suppressive properties in CC. Since a clinical study showed that an elevated GPER1 expression is associated with a worse prognosis, we investigated the effects of stable GPER1 overexpression (GPER1-OE) on SiHa CSCC and HeLa CAC cells.Materials and MethodsSiHa CSCC and HeLa CAC cells with stable GPER1-OE were generated. GPER1-OE was tested by RT-qPCR, western blot and fluorescence-activated cell analysis (FACS). The effects of GPER1-OE on proliferation, migration, invasion, apoptosis and stem cell properties (colony and sphere formation) were then examined.ResultsSuccessful GPER1-OE in SiHa CSCC and HeLa CAC cells was confirmed. The cell characterization experiments showed that SiHa CSCC cells with stable GPER1-OE had faster proliferation and migration, and increased stem cell properties with larger and more numerous colonies and larger tumor spheres. In HeLa CAC cells, on the other hand, GPER1-OE resulted in slower cell proliferation, migration and invasion, reduced colony formation and tumor sphere formation. An increased rate of apoptosis was also observed.ConclusionGPER1-OE resulted in a more aggressive tumor behavior of SiHa CSCC cells and a less aggressive tumor behavior of HeLa CAC cells, due to a different effect of GPER1 overexpression depending on the respective histological subtypes of CC. This underlines the need for personalized medicine and a precise differentiation of subtypes in CC-related research.

BackgroundThis study aimed to investigate the expression of long noncoding RNA (lncRNA) loc285194 in cervical squamous cell carcinoma (CSCC) biopsies that were positive and negative for human papillomavirus (HPV) and in human CSCC cell lines SiHa and C33A and to investigate the overexpression of lncRNA loc285194.Material/MethodsCervical biopsy tissue and plasma samples from 66 patients with histologically confirmed CSCC, that were HPV16-positive (N=22), HPV18-positive (N=27), and HPV-negative (N=17), and healthy controls (N=20) and human CSCC cell lines SiHa (HPV16-positive) and C33A (HPV-negative) were studied. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the expression of lncRNA loc285194 in cervical biopsies and plasma. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to measure levels of transforming growth factor-β1 (TGF-β1). A lncRNA loc285194 expression vector was constructed and transfected into SiHa and C33A cells that underwent a transwell assay for cell migration.ResultsExpression of lncRNA loc285194 was downregulated in HPV-positive and HPV-negative tissue samples and plasma from patients with CSCC and distinguished between patients and healthy controls. Plasma levels of loc285194 and TGF-β1 were significantly correlated with the presence of CSCC. In SiHa and C33A cells, TGF-β1 expression was downregulated, and cell migration was inhibited following lncRNA loc285194 overexpression. Although lncRNA loc285194 expression was not affected by TGF-β1 treatment, its effects on cell migration were reduced by TGF-β1.ConclusionsThe expression of lncRNA loc285194 inhibited the migration of CSCC cells in vitro through the inactivation of TGF-β1.

FHIT suppresses cervical squamous cell carcinoma progression by negatively regulating UBE2I-mediated SUMO modification of NOTCH1.

An important feature of cervical squamous carcinogenesis is genomic instability caused by deregulated expression of HPV oncogenes in proliferating epithelial cells. A genomic imbalance that is commonly selected in advanced cervical squamous cell carcinoma (SCC) is copy number gain of chromosome 5p. One gene that is likely to drive selection of this abnormality is the oncostatin-M receptor (OSMR; located at 5p13.1), which is commonly up-regulated in cervical SCC and produces a significantly worse clinical outcome when over-expressed. Cervical SCC cells that over-expressed OSMR showed enhanced responsiveness to the major ligand OSM, which induced multiple pro-malignant effects, including increased cell migration and invasiveness. We now show that transglutaminase-2 (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in cervical carcinoma cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of SCC of the cervix and head and neck. TGM2 depletion in cervical SCC cells abrogated the increased migration and invasiveness induced by OSM. TGM2 was shown to interact with integrin-α5β1 and fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin-α5β1 and fibronectin were over-expressed in cervical SCC samples, where levels correlated with those of OSMR. We conclude that an OSMR-TGM2-integrin-α5β1 pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting. Citation Format: Maria M. Caffarel, Anasuya Chattopadhyay, Cinzia G. Scarpini, Nicholas Coleman. Transglutaminase-2 mediates the pro-malignant effects of oncostatin-M receptor overexpression in cervical squamous cell carcinoma. [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 4383. doi:10.1158/1538-7445.AM2013-4383

BackgroundCell division cycle 25A (CDC25A) is a well-recognized regulator of cell cycle progression and is involved in cancer development. This work focused on the function of CDC25A in cervical cancer cell growth and the molecules involved.MethodsA GEO dataset GSE63514 comprising data of cervical squamous cell carcinoma (CSCC) tissues was used to screen the aberrantly expressed genes in cervical cancer. The CDC25A expression in cancer and normal tissues was predicted in the GEPIA database and that in CSCC and normal cells was determined by RT-qPCR and western blot assays. Downregulation of CDC25A was introduced in CSCC cells to explore its function in cell growth and the cell cycle progression. The potential regulators of CDC25A activity and the possible involved signaling were explored.ResultsCDC25A was predicted to be overexpressed in CSCC, and high expression of CDC25A was observed in CSCC cells. Downregulation of CDC25A in ME180 and C33A cells reduced cell proliferation and blocked cell cycle progression, and it increased cell apoptosis. ALX3 was a positive regulator of CDC25A through transcription promotion. It recruited a histone demethylase, lysine demethylase 2B (KDM2B), to the CDC25A promoter, which enhanced CDC25A expression through demethylation of H3k4me3. Overexpression of ALX3 in cells blocked the inhibitory effects of CDC25A silencing. CDC25A was found as a positive regulator of the PI3K/Akt signaling pathway.ConclusionThis study suggested that the ALX3 increased CDC25A expression through KDM2B-mediated demethylation of H3K4me3, which induced proliferation and cell cycle progression of cervical cancer cells.