FEBS Open Bio

Reduced MAGI3 level by HPV18E6 contributes to Wnt/β‐catenin signaling activation and cervical cancer progression

Human papillomavirus type 18 (HPV18) has high carcinogenic power in invasive cervical cancer (ICC) development. However, the underlying mechanism remains elusive. The carcinogenic properties of HPV18 require the PDZ‐binding motif of its E6 oncoprotein (HPV18 E6) to degrade its target PSD95/Dlg/ZO‐1 (PDZ) proteins. In this study, we demonstrated that the PDZ protein membrane‐associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3) inhibited the Wnt/β‐catenin pathway, and subsequently cervical cancer (CC) cell migration and invasion, via decreasing β‐catenin levels. By reducing MAGI3 protein levels, HPV18 E6 promoted CC cell migration and invasion through activation of Wnt/β‐catenin signaling. Furthermore, HPV18 rather than HPV16 was preferentially associated with the downregulation of MAGI3 and activation of the Wnt/β‐catenin pathway in CC. These findings shed light on the mechanism that gives HPV18 its high carcinogenic potential in CC progression.

Indoleamine 2,3‐dioxygenase 1 inhibition reverses cancer‐associated fibroblast‐mediated immunosuppression in high‐grade serous ovarian cancer

Cancer‐associated fibroblasts (CAFs) contribute to immunosuppression in the ovarian cancer microenvironment, partly through upregulation of indoleamine 2,3‐dioxygenase 1 (IDO1). This study examined CAF‐mediated suppression of T‐cell function and the potential of IDO1 inhibition to reverse these effects. CAFs from high‐grade serous ovarian cancer (HGSOC) patients exhibited increased IDO1, COX2, and PD‐L1 expression upon interaction with activated T cells, along with elevated immunosuppressive cytokines. CAFs suppressed T‐cell proliferation and induced PD‐1 expression in CD4+ and CD8+ T cells, effects reversed by epacadostat. IDO1 inhibition enhanced T‐cell proliferation via AKT signaling, restored T‐cell cytotoxicity, and increased ovarian cancer cell apoptosis. These findings suggest that targeting IDO1 may help counteract CAF‐mediated immunosuppression and enhance antitumor immunity in HGSOC.

High LRIG1 expression predicts lymph node metastasis in patients with uterine cervical cancer

Fifteen percent of patients with preoperative stage IA2‐IB1 uterine cervical cancer are diagnosed with lymph node metastasis (LNM) following surgery. They must be treated with both surgery and radiotherapy, a combination associated with severe side effects. Since current diagnostic methods have limitations, biomarkers are urgently needed to improve staging. Leucine‐rich repeats and immunoglobulin‐like domains protein 1 (LRIG1) is a regulator of growth factor signaling and a prognostic factor in cervical cancer. This study investigates whether LRIG1 expression could predict LNM in cervical cancer. Sixty‐seven patients were included: 31 without LNM and 36 with LNM. Tumor blocks were retrieved, and clinical data were collected. Immunohistochemical analysis of LRIG1 expression was performed, and LRIG1 immunoreactivity was correlated with lymph node status and clinicopathological prognostic factors, such as human papillomavirus status and smoking status. High LRIG1 expression (> 25% positive cells) was significantly associated with an increased risk of LNM (odds ratio 9.49, 95% CI: 1.80–50.05, P  = 0.008, adjusted for age, smoking status, and BMI), suggesting the potential of LRIG1 as a biomarker. Larger, multicenter studies are needed to validate our results.

Knockdown of RNF6 inhibits HeLa cervical cancer cell growth via suppression of MAPK/ERK signaling

Ring finger protein 6 (RNF6) is implicated in various human malignancies, but its function in cervical cancer (CC) is incompletely understood. Here, we explored the biological significance of RNF6 in HeLa CC cells and the underlying regulatory mechanisms. The expression of RNF6 was observed to be high in both primary tissues and CC cells. RNF6 promoted HeLa CC cell growth. Knockdown of RNF6 in CC cells resulted in suppression of proliferation and promotion of apoptosis. Moreover, elevation of RNF6 had an adverse effect on the prognosis of CC. Subsequent analyses showed that these effects may be mediated via activation of ERK signaling. These findings provide evidence that the knockdown of RNF6 suppresses the MAPK/ERK pathway to regulate the growth of CC cells, which suggests that RNF6 may have potential as a target for diagnosis and treatment for CC.

Microbiological investigation of pregnancies following vaginal radical trachelectomy using 16S rRNA sequencing of FFPE placental specimens

This study examined the risk of intrauterine infection associated with radical trachelectomy (RT) in early‐stage cervical cancer patients. This procedure preserves fertility but is linked to increased risk of intrauterine infection due to cervical defects during pregnancy. DNA was extracted from the formalin‐fixed paraffin‐embedded (FFPE) placental specimens of 23 pregnant post‐RT patients and 16S rRNA gene sequencing was used for bacterial identification. The prevalence of Lactobacillus crispatus and Burkholderia stabilis was significantly higher in the non‐chorioamnionitis group. In contrast, alpha diversity analysis using the PD index showed significantly higher diversity in the chorioamnionitis group ( P  = 0.04). The demonstrated relationship between chorioamnionitis and microbial diversity affirms the importance of controlling the genital bacterial flora in pregnancies following RT.

Chromatin modified protein 4C (CHMP4C) facilitates the malignant development of cervical cancer cells

Despite improvements in prevention and treatment, cervical cancer (CC) still poses a serious threat to women’s health. CHMP4C (chromatin modified protein 4C) is a subunit of the endosomal sorting complex required for transport, which is expressed in both nucleus and cytoplasm. Here, we examined the effect of CHMP4C on the biological behavior of CC cells and the underlying mechanisms. We report that CHMP4C expression is higher in CC tissues, and high CHMP4C expression is associated with lower survival. Up‐regulation of CHMP4C in C‐33A cells accelerates cell proliferation, migration and invasion, whereas down‐regulation of CHMP4C in Ca Ski cells had the opposite effect. Moreover, overexpression of CHMP4C induced activation of the epithelial–mesenchymal transition pathway, whereas depletion of CHMP4C inhibited activation. Our results suggest that CHMP4C contributes to the viability and motility of CC cells by modulating epithelial–mesenchymal transition and may facilitate the identification of novel biomarkers for CC therapy.

IFNγ induces Bcl3 expression by JAK1/STAT1/p65 signaling, resulting in increased IL‐8 expression in ovarian cancer cells

We have recently shown that IFNγ, produced during cancer therapy, induces expression of the Bcl3 proto‐oncogene in ovarian cancer (OC) cells, resulting in their increased proliferation, migration, and invasion, but the mechanisms are unknown. Here, we demonstrate that the IFNγ‐induced Bcl3 expression is dependent on JAK1 and STAT1 signaling, and on p65 NFκB. Furthermore, the IFNγ‐induced Bcl3 expression is associated with an increased occupancy of Ser‐727 phosphorylated STAT1 and acetylated histone H3 at the Bcl3 promoter. Our data indicate that Bcl3 promotes expression of the pro‐inflammatory chemokine interleukin‐8 (IL‐8) in OC cells. These findings identify Bcl3 as a novel target of IFNγ/JAK1/STAT1 signaling and suggest that targeting the JAK1/STAT1 pathway may suppress IFNγ‐induced Bcl3 expression in OC.

Nuclear endonuclease G controls cell proliferation in ovarian cancer

Ovarian cancer is characterized by a high degree of genetic heterogeneity. Platinum‐based chemotherapy and some gene‐targeted therapies have shown limited treatment efficacy due to toxicity and recurrence, and thus, it is essential to identify additional therapeutic targets based on an understanding of the pathological mechanism. Here, we report that endonuclease G, which exhibits altered expression in ovarian cancer, does not function as a cell death effector that digests chromosomal DNA in ovarian cancer. Endonuclease G is modulated by intracellular reactive oxygen species dynamics and plays a role in cell proliferation in ovarian cancer, suggesting that targeting endonuclease G alone or in combination with other antitumor agents may have the potential for development into a treatment for endonuclease G‐overexpressing cancers, including ovarian cancer.

Multi‐omics‐based analysis of high grade serous ovarian cancer subtypes reveals distinct molecular processes linked to patient prognosis

Despite advancements in treatment, high‐grade serous ovarian cancer (HGSOC) is still characterized by poor patient outcomes. To understand the molecular heterogeneity of this disease, which underlies the challenge in selecting optimal treatments for HGSOC patients, we have integrated genomic, transcriptomic, and epigenetic information to identify seven new HGSOC subtypes using a multiscale clustering method. These subtypes not only have significantly distinct overall survival, but also exhibit unique patterns of gene expression, microRNA expression, DNA methylation, and copy number alterations. As determined by our analysis, patients with similar clinical outcomes have distinct profiles of activated or repressed cellular processes, including cell cycle, epithelial‐to‐mesenchymal transition, immune activation, interferon response, and cilium organization. Furthermore, we performed a multiscale gene co‐expression network analysis to identify subtype‐specific key regulators and predicted optimal targeted therapies based on subtype‐specific gene expression. In summary, this study provides new insights into the cellular heterogeneity of the HGSOC genomic, epigenetic, and transcriptomic landscapes and provides a basis for future studies into precision medicine for HGSOC patients.

BNIP3 contributes to cisplatin‐induced apoptosis in ovarian cancer cells

BNIP3 is a proapoptotic protein that mediates apoptosis, necrosis and autophagy. However, the involvement of BNIP3 in cisplatin‐induced apoptosis in ovarian cancer is not clear. In this study, we examined the role of BNIP3 in ovarian cancer during cisplatin treatment and its correlation with clinical outcomes. We first measured cisplatin cytotoxicity and BNIP3 levels before and after cisplatin exposure for ovarian cancer cell lines A2780, SKOV3, OVCAR4, OV2008, ES2 and HO8910. BNIP3 was observed to be differentially expressed in these cell lines, and cisplatin induced a significant increase in BNIP3 levels in A2780 and OVCAR4. BNIP3 knockdown with siRNA in A2780 and OVCAR4 significantly reduced cisplatin cytotoxicity in these two cell lines and alleviated cisplatin‐induced apoptosis. We searched the online databases Gene Expression Omnibus and The Cancer Genome Atlas to analyze the correlation between BNIP3 level and overall survival and progression‐free survival in patients with ovarian cancer. Pooled analyses showed that higher BNIP3 level was correlated with poorer overall survival (95% confidence intervals; hazard ratio = 1.18, 1.04–1.34;P = 0.013) and progression‐free survival (95% confidence intervals; hazard ratio = 1.26, 1.10–1.43;P = 0.00049). However, the results of individual datasets and stratification analyses of histology, FIGO (Federation Internationale de Gynecolgie et d’Obstetrique) stage, chemotherapy regimen and P53 mutation status varied. These findings indicate that cisplatin‐induced apoptosis is dependent on BNIP3 level in ovarian cancer cell lines. Targeting BNIP3 may therefore be a potential way of restoring cisplatin sensitivity.

Serum trace element levels and activity of enzymes associated with oxidative stress in endometriosis and endometrial cancer

Endometriosis and endometrial cancer are closely related to oxidative stress. However, the direct relationship between copper and zinc levels and oxidative stress in the extracellular and intracellular space remains unclear. The presented study is focused on the determination of serum Zn and Cu levels, glutathione concentration and enzyme activity in three groups: patients diagnosed with endometrial cancer (EC), patients diagnosed with endometriosis (EM), and a healthy control group. Spectrophotometric determination of trace elements revealed that levels of zinc and copper were lower in blood plasma of patients with endometriosis as compared with the other groups; however, there were no significant differences in the Cu/Zn ratio. Furthermore, significantly increased blood serum glutathione levels were detected in both EM and EC groups compared with the control group. While the activity of superoxide dismutase (SOD) was similar across the studied groups, we observed differences in the activity of other enzymes associated with oxidative stress, including glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S‐transferase (GST), between the control group and the EM and EC patients. Additionally, analysis of gene expression based on free circulating mRNA indicated significant differences in the expression of SOD isoenzymes between the patient groups and the control group; expression of GPx isoenzymes was also altered. Obtained results may have potential application in diagnostics as well as monitoring of endometriosis and endometrial cancer.

MTHFD2 promotes ovarian cancer growth and metastasis via activation of the STAT3 signaling pathway

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a bifunctional enzyme located in the mitochondria. MTHFD2 has been reported to be overexpressed in several malignant tumors and is implicated in cancer development. This study aimed to investigate the effect of MTHFD2 on ovarian cancer progression. The expression of MTHFD2 was detected by bioinformatic analysis, immunohistochemistry, RT‐qPCR (real‐time quantitative PCR analysis), and western blot analysis. The effects of MTHFD2 depletion on cell proliferation, migration, and invasion were determined through in vitro experiments. Cell cycle progression and apoptosis were accessed by flow cytometry. The related signaling pathway protein expression was determined by western blot analysis. We found that MTHFD2 is highly expressed in both ovarian cancer tissues and cell lines. MTHFD2 deletion suppressed cell proliferation and metastasis. Knockdown of MTHFD2 induces cell apoptosis and G2/M arrest, whereas the number of cells in S phase increased with MTHFD2 overexpression. Mechanically, our results indicate that an inhibitory effect of MTHFD2 knockdown may be mediated by the downregulation of cyclin B1/Cdc2 complex and the inhibitory effect on its activity. Additionally, MTHFD2 could regulate cell growth and aggressiveness via activation of STAT3 and the STAT3‐induced epithelial–mesenchymal transition signaling pathway. These findings indicate that MTHFD2 is overexpressed in ovarian cancer and regulates cell proliferation and metastasis, presenting an attractive therapeutic target.

DNA methylation subtypes for ovarian cancer prognosis

Ovarian cancer is one of three major malignancies of the female reproductive system. DNA methylation (MET) is closely related to ovarian cancer occurrence and development, and as such, elucidation of effective MET subtype markers may guide individualized treatment and improve ovarian cancer prognosis. To identify potential markers, we downloaded a total of 571 ovarian cancer MET samples from The Cancer Genome Atlas (TCGA), and established a Cox proportional hazards model using the MET spectrum and clinical pathological parameters. A total of 250 prognosis‐related MET loci were obtained by Cox regression, and six molecular subtypes were screened by consensus clustering of CpG loci with a significant difference in both univariate and multivariate analyses. There was a remarkable MET difference between most subtypes. Cluster 2 had the highest MET level and demonstrated the best prognosis, while Clusters 4 and 5 had MET levels significantly lower than those of the other subtypes and demonstrated very poor prognosis. All Cluster 5 samples were at a high grade, while the percentage of stage IV samples in Cluster 4 was greater than in the other subtypes. We obtained five CpG loci using a coexpression network: cg27625732, cg00431050, cg22197830, cg03152385, and cg22809047. Our cluster analysis showed that prognosis in patients with hypomethylation was significantly worse than in patients with hypermethylation. These MET molecular subtypes can be used not only to evaluate ovarian cancer prognosis, but also to fully distinguish the tumor stage and histological grade in patients with ovarian cancer.

SIRT6 promotes mitochondrial fission and subsequent cellular invasion in ovarian cancer

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a “metabolic disorder,” is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1‐SIRT7) belong to the group of NAD+ deacetylase and ADP‐ribosylation enzymes that function as NAD+ sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non‐malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2‐driven activatory phosphorylation of DRP1 at serine‐616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA‐mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.

O‐GlcNAcylation regulates lysophosphatidic acid‐induced cell migration by regulating ERM family proteins

O‐GlcNAcylation of intracellular proteins (O‐GlcNAc) is a post‐translational modification that often competes with phosphorylation in diverse cellular signaling pathways. Recent studies on human malignant tumors have demonstrated that O‐GlcNAc is implicated in cellular features relevant to metastasis. Here, we report that lysophosphatidic acid (LPA)‐induced ovarian cancer cell (OVCAR‐3) migration is regulated by O‐GlcNAc. We found that O‐GlcNAc modification of ERM family proteins, a membrane‐cytoskeletal crosslinker, was inversely correlated with its phosphorylation status. Moreover, the LPA‐induced formation of membrane protrusion structures, as well as the migration of OVCAR‐3 cells, was reduced by the accumulation of O‐GlcNAc. Collectively, these findings suggest that O‐GlcNAc is an essential signaling element controlling ERM family proteins involved in OVCAR‐3 cell migration.

The splicing factor DHX38/PRP16 is required for ovarian clear cell carcinoma tumorigenesis, as revealed by a CRISPR‐Cas9 screen

Certain cancers, such as ovarian clear cell carcinoma (OCCC), display high levels of genetic variation between patients, making it difficult to develop effective therapies. In order to identify novel genes critical to OCCC growth, we carried out a comprehensive CRISPR‐Cas9 knockout screen against cell growth using an OCCC cell line and a normal ovarian surface epithelium cell line. We identified the gene encoding DHX38/PRP16, an ATP‐dependent RNA helicase involved in splicing, as critical for the growth and tumorigenesis of OCCC. DHX38/PRP16 knockdown in OCCC cells, but not normal cells, induces apoptosis and impairs OCCC tumorigenesis in a mouse model. Our results suggest that DHX38/PRP16 may play a role in OCCC tumorigenesis and could potentially be a promising therapeutic target.

GDF15 knockdown suppresses cervical cancer cell migration in vitro through the TGF‐β/Smad2/3/Snail1 pathway

Growth differentiation factor 15 (GDF15), a member of the transforming growth factor β (TGF‐β) superfamily, is a prognostic biomarker of cervical cancer. In addition, GDF15 has been reported to enhance the migration of colorectal cancer cells and liver cancer stem‐like cells. However, the mechanism by which GDF15 promotes cervical cancer cell migration is not completely understood. Here, we report that GDF15 expression is enhanced in cervical cancer tissues, as well as in cultured cervical cancer cells. ShGDF15 transfection markedly inhibited expression of Vimentin, N‐cadherin and Snail1, and resulted in up‐regulation of E‐cadherin expression in HT‐3 and HeLa cells. Moreover, knockdown of GDF15 suppressed wound healing rate and reduced the number of invasive cells. Furthermore, knockdown of GDF15 significantly suppressed the expression of phosphorylated Smad2 and Smad3. The addition of TGF‐β1 partially abolished the inhibitory effects of GDF15 knockdown on the migration and invasion of cervical cancer cells. In summary, we report here that GDF15 knockdown inhibits migration and invasion of cervical cancer cells in vitro through the TGF‐β/Smad2/3/Snail1 pathway.

SHIP2 inhibition alters redox‐induced PI3K/AKT and MAP kinase pathways via PTEN over‐activation in cervical cancer cells

Phosphatidylinositol (3,4,5)‐trisphosphate (PI(3,4,5)P3) is required for protein kinase B (AKT) activation. The level of PI(3,4,5)P3 is constantly regulated through balanced synthesis by phosphoinositide 3‐kinase (PI3K) and degradation by phosphoinositide phosphatases phosphatase and tensin homologue (PTEN) and SH2‐domain containing phosphatidylinositol‐3,4,5‐trisphosphate 5‐phosphatase 2 (SHIP2), known as negative regulators of AKT. Here, I show that SHIP2 inhibition in cervical cancer cell lines alters H2O2‐mediated AKT and mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway activation. In addition, SHIP2 inhibition enhances reactive oxygen species generation. Interestingly, I found that SHIP2 inhibition and H2O2 treatment enhance lipid and protein phosphatase activity of PTEN. Pharmacological targeting or RNA interference(RNAi) mediated knockdown of PTEN rescues extracellular signal‐regulated kinase and AKT activation. Using a series of pharmacological and biochemical approaches, I provide evidence that crosstalk between SHIP2 and PTEN occurs upon an increase in oxidative stress to modulate the activity of mitogen‐activated protein kinase and phosphoinositide 3/ATK pathways.

Retracted: Reduced levels of miR‐485‐5p in HPV‐infected cervical cancer promote cell proliferation and enhance invasion ability

Cervical cancer (CC) is the most common gynecological malignancy, with high incidence and mortality rates in China. The microRNA miR‐485‐5p has previously been reported to serve as a negative regulator of tumorigenesis in breast cancer and hepatocellular carcinoma, and miR‐485‐5p has been observed to be differentially expressed between CC and normal control tissue. Here, we confirmed that miR‐485‐5p expression is lower in CC than in adjacent normal tissue and proceeded to investigate the effects of miR‐485 on tumor behavior in CC cell lines. We report that miR‐485‐5p transcription is decreased in HPV‐infected CC tissue, and levels of miR‐485 in clinical samples are positively correlated with the 5‐year overall survival rate. The Transwell assay showed that miR‐485‐5p inhibited cell invasion and migration but had no influence on apoptosis and cell proliferation. Using a luciferase reporter assay, we demonstrated that miR‐485‐5p partially abrogated cell migration and proliferation by targeting FLOT‐1 mRNA. Transfection of HPV‐infected cervical carcinoma cells with an adenovirus vector encoding human FLOT‐1 partially diminished the inhibitory effects of miR‐485 on cell invasion. Taken, together, these data demonstrated that miR‐485‐5p suppresses the invasion of cancer cells by targeting FLOT‐1 in HPV‐infected cervical carcinoma cells.