Combined treatment with modulated electro-hyperthermia and an autophagy inhibitor effectively inhibit ovarian and cervical cancer growth.

PURPOSE: Modulated electro-hyperthermia (mEHT), known as oncothermia, is an anticancer therapy that induces radiofrequency thermal damage to the cancer tissues. This study aimed to evaluate the potential effectiveness of mEHT as a therapeutic tool in ovarian and cervical cancer. MATERIALS AND METHODS: We used both tumor-bearing mice and ovarian and cervical OVCAR-3, SK-OV-3, HeLa and SNU-17 cancer cell lines to investigate the effects of mEHT in vivo and in vitro, respectively, and determine whether it was enhanced by cotreatment with an autophagy inhibitor. RESULTS: We discovered that phosphorylation of p38, a stress-dependent kinase, was induced at the Thr180/Tyr182 residue in cancer cells exposed to mEHT. Apoptotic markers such as cleaved caspase-3 and poly-ADP ribose polymerase (PARP) were increased in OVCAR-3 and SNU-17 cells. Fluorescence-activated cell sorting (FACS) analysis showed a significant increase in the population of sub-G1 mEHT-exposed cells, which are dying and apoptotic cells. mEHT also reduced both weight and volume of xenograft tumors in mice transplanted with ovarian and cervical cancer cells and patient-derived cancer tissues. We determined that mEHT-induced cellular damage recovery was mediated by autophagy and, therefore, expectedly, cotreatment with mEHT and 3-methyladenine (3-MA), an autophagy inhibitor, more effectively inhibited cancer cell growth than individual treatment did. CONCLUSIONS: mEHT treatment alone was sufficient to inhibit cancer growth, while a combined treatment with mEHT and an autophagy inhibitor amplified this inhibition effect.

AMP-activated protein kinase α2 and E2F1 transcription factor mediate doxorubicin-induced cytotoxicity by forming a positive signal loop in mouse embryonic fibroblasts and non-carcinoma cells.

Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase α2 (AMPKα2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKα2 in MEFs but slightly reduced AMPKα1 expression. Our data support the previous notion that AMPKα1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkα2(-/-) MEFs, gene knockdown of AMPKα2 by shRNA, and inhibition of AMPKα2 activity with an AMPK inhibitor indicated that AMPKα2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKα2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkα2 gene promoter. In turn, AMPKα2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKα2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKα2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKα2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKα2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.

Expression of stress-induced phosphoprotein1 (STIP1) is associated with tumor progression and poor prognosis in epithelial ovarian cancer.

Stress-induced phosphoprotein1 (STIP1) is a candidate biomarker in epithelial ovarian cancer (EOC). In this study, we investigated in detail the expression of STIP1, as well as its functions, in EOC. STIP1 expression was assessed by immunohistochemistry (IHC) and the results were compared with clinicopathologic factors, including survival data. The effects of STIP1 gene silencing via small interfering RNA (siRNA) were examined in EOC cells and a xenograft model. The expression of STIP1 protein in EOC was significantly higher than in the other study groups (P < 0.001), and this increase of expression was significantly associated with tumor stage (P = 0.005), tumor grade (P = 0.029), and lymph node metastasis (P = 0.020). In multivariate analysis, overall survival in EOC was significantly shorter in cases with high STIP1 expression (HR = 2.78 [1.01-7.63], P = 0.047). STIP1 silencing in EOC cells resulted in inhibition of cell proliferation and invasion. In addition, in vivo experiments using STIP1 siRNA clearly showed a strong inhibition of tumor growth and a modulation of expression of prosurvival and apoptotic genes, further suggesting that STIP1 silencing can prevent cell proliferation and invasion. In conclusion, increased STIP1 expression is associated with poor survival outcome in EOC, and STIP1 may represent a useful therapeutic target in EOC patients.

VGLL3 expression is associated with macrophage infiltration and predicts poor prognosis in epithelial ovarian cancer.

Background/objective: High-grade serous ovarian carcinoma (HGSOC) is the most common histologic type of epithelial ovarian cancer (EOC). Due to its poor survival outcomes, it is essential to identify novel biomarkers and therapeutic targets. The hippo pathway is crucial in various cancers, including gynaecological cancers. Herein, we examined the expression of the key genes of the hippo pathway and their relationship with clinicopathological significance, immune cells infiltration and the prognosis of HGSOC. Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data were curated to analyse the mRNA expression as well as the clinicopathological association and correlation with immune cell infiltration in HGSOC. The protein levels of significant genes in the HGSOC tissue were analysed using Tissue Microarray (TMA)-based immunohistochemistry. Finally, DEGs pathway analysis was performed to find the signalling pathways associated with VGLL3. Results: VGLL3 mRNA expression was significantly correlated with both advanced tumor stage and poor overall survival (OS) (p=0.046 and p=0.003, respectively). The result of IHC analysis also supported the association of VGLL3 protein with poor OS. Further, VGLL3 expression was significantly associated with tumor infiltrating macrophages. VGLL3 expression and macrophages infiltration were both found to be independent prognostic factors (p=0.003 and p=0.024, respectively) for HGSOC. VGLL3 was associated with four known and three novel cancer-related signalling pathways, thus implying that VGLL3 is involved in the deregulation of many genes and pathways. Conclusion: Our study revealed that VGLL3 may play a distinct role in clinical outcomes and immune cell infiltration in patients with HGSOC and that it could potentially be a prognostic marker of EOC.

Prognostic value of ß-Arrestins in combination with glucocorticoid receptor in epithelial ovarian cancer.

Hormones may be key factors driving cancer development, and epidemiological findings suggest that steroid hormones play a crucial role in ovarian tumorigenesis. We demonstrated that high glucocorticoid receptor (GR) expression is associated with a poor prognosis of epithelial ovarian cancer. Recent studies have shown that the GR affects ß-arrestin expression, and vice versa. Hence, we assessed the clinical significance of ß-arrestin expression in ovarian cancer and determined whether ß-arrestin and the GR synergistically have clinical significance and value as prognostic factors. We evaluated the expression of ß-arrestins 1 and 2 and the GR in 169 patients with primary epithelial ovarian cancer using immunohistochemistry. The staining intensity was graded on a scale of 0-4 and multiplied by the percentage of positive cells. We divided the samples into two categories based on the expression levels. ß-arrestin 1 and GR expression showed a moderate correlation, whereas ß-arrestin 2 and GR expression did not demonstrate any correlation. Patients with high ß-arrestin 1 and 2 expression exhibited improved survival rates, whereas patients with low GR expression showed a better survival rate. Patients with high ß-arrestin 1 and low GR levels had the best prognosis among all groups. ß-arrestin is highly expressed in ovarian cancer, suggesting its potential as a diagnostic and therapeutic biomarker. The combination of ß-arrestin and GR demonstrated greater predictive prognostic power than GR expression alone, implicating another possible role in prognostication.

Identification of Prognostic Markers of Gynecologic Cancers Utilizing Patient-Derived Xenograft Mouse Models.

Patient-derived xenografts (PDXs) are important in vivo models for the development of precision medicine. However, challenges exist regarding genetic alterations and relapse after primary treatment. Thus, PDX models are required as a new approach for preclinical and clinical studies. We established PDX models of gynecologic cancers and analyzed their clinical information. We subcutaneously transplanted 207 tumor tissues from patients with gynecologic cancer into nude mice from 2014 to 2019. The successful engraftment rate of ovarian, cervical, and uterine cancer was 47%, 64%, and 56%, respectively. The subsequent passages (P2 and P3) showed higher success and faster growth rates than the first passage (P1). Using gynecologic cancer PDX models, the tumor grade is a common clinical factor affecting PDX establishment. We found that the PDX success rate correlated with the patient's prognosis, and also that ovarian cancer patients with a poor prognosis had a faster PDX growth rate (p < 0.0001). Next, the gene sets associated with inflammation and immune responses were shown in high-ranking successful PDX engraftment through gene set enrichment analysis and RNA sequencing. Up-regulated genes in successful engraftment were found to correlate with ovarian clear cell cancer patient outcomes via Gene Expression Omnibus dataset analysis.

Identification of a novel gene signature in second-trimester amniotic fluid for the prediction of preterm birth.

Preterm birth affects approximately 5% to 7% of live births worldwide and is the leading cause of neonatal morbidity and mortality. Amniotic fluid supernatant (AFS) contains abundant cell-free nucleic acids (cfNAs) that can provide genetic information associated with pregnancy complications. In the current study, cfNAs of AFS in the early second-trimester before the onset of symptoms of preterm birth were analyzed, and we compared gene expression levels between spontaneous preterm birth (n = 5) and term birth (n = 5) groups using sequencing analysis. Differential expression analyses detected 24 genes with increased and 6 genes with decreased expression in the preterm birth group compared to term birth. Upregulated expressions of RDH14, ZNF572, VOPP1, SERPINA12, and TCF15 were validated in an extended AFS sample by quantitative PCR (preterm birth group, n = 21; term birth group, n = 40). Five candidate genes displayed a significant increase in mRNA expression in immortalized trophoblast HTR-8/SVneo cell with H2O2 treatment. Moreover, the expression of five candidate genes was increased to more than twofold by pretreatment with lipopolysaccharide in HTR-8/SVneo cells. Changes in gene expression between preterm birth and term birth is strongly correlated with oxidative stress and infection during pregnancy. Specific expression patterns of genes could be used as potential markers for the early identification of women at risk of having a spontaneous preterm birth.

Tetraspanin 1 promotes endometriosis leading to ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) reportedly develops from endometriosis. However, the molecular mechanism underlying its malignant progression to OCCC remains elusive. This study aimed to identify an essential gene in the malignant transformation of endometriosis to OCCC. We performed RNA sequencing in formalin-fixed, paraffin-embedded (FFPE) tissues of endometriosis (n = 9), atypical endometriosis (AtyEm) (n = 18), adjacent endometriosis to OCCC (AdjEm) (n = 7), and OCCC (n = 17). We found that tetraspanin 1 (TSPAN1) mRNA level was significantly increased by 2.4- (DESeq2) and 3.4-fold (edgeR) in AtyEm and by 80.7- (DESeq2) and 101-fold (edgeR) in OCCC relative to endometriosis. We confirmed that TSPAN1 protein level was similarly overexpressed in OCCC tissues and cell lines. In immortalized endometriosis cell lines, TSPAN1 overexpression enhanced cell growth and invasion. Mechanistically, TSPAN1 triggered AMP-activated protein kinase (AMPK) activity, promoting endometriosis and cell growth. Upregulated levels of TSPAN1 are considered an early event in the development of high-risk endometriosis that could progress to ovarian cancer. Our study suggests the potential of TSPAN1 as a screening candidate for high-risk endometriosis.

TOM40 Inhibits Ovarian Cancer Cell Growth by Modulating Mitochondrial Function Including Intracellular ATP and ROS Levels.

TOM40 is a channel-forming subunit of translocase, which is essential for the movement of proteins into the mitochondria. We found that TOM40 was highly expressed in epithelial ovarian cancer (EOC) cells at both the transcriptional and translational levels; its expression increased significantly during the transformation from normal ovarian epithelial cells to EOC (p < 0.001), and TOM40 expression negatively correlated with disease-free survival (Hazard ratio = 1.79, 95% Confidence inerval 1.16-2.78, p = 0.009). TOM40 knockdown decreased proliferation in several EOC cell lines and reduced tumor burden in an in vivo xenograft mouse model. TOM40 expression positively correlated with intracellular adenosine triphosphate (ATP) levels. The low ATP and high reactive oxygen species (ROS) levels increased the activity of AMP-activated protein kinase (AMPK) in TOM40 knockdown EOC cells. However, AMPK activity did not correlate with declined cell growth in TOM40 knockdown EOC cells. We found that metformin, first-line therapy for type 2 diabetes, effectively inhibited the growth of EOC cell lines in an AMPK-independent manner by inhibiting mitochondria complex I. In conclusion, TOM40 positively correlated with mitochondrial activities, and its association enhances the proliferation of ovarian cancer. Also, metformin is an effective therapeutic option in TOM40 overexpressed ovarian cancer than normal ovarian epithelium.

Establishment of five immortalized human ovarian surface epithelial cell lines via SV40 T antigen or HPV E6/E7 expression.

BACKGROUND: Human ovarian surface epithelial (HOSE) cells are a critical cell source for ovarian cancer research; however, they are difficult to obtain and maintain under standard laboratory conditions in large quantities. The aim of this study was to generate immortalized HOSE (IHOSE) cells with maintained properties to the original cell source, thereby guaranteeing a sufficiently large cell quantity for ovarian cancer research. METHODS: HOSE cells isolated from four non-cancer patients and five IHOSE cell lines were established by induction of HPV-E6/E7 expression or SV40 large T antigen using a lenti-viral system. Each of IHOSE cells was confirmed to be distinct by STR profiling. RNA-sequencing was used to compare gene expression profiles in HOSE, IHOSE and ovarian cancer cells. RESULTS: RNA-sequencing results revealed a stronger linear correlation in gene expression between IHOSE and HOSE cells (R2 = 0.9288) than between IHOSE or HOSE cells and ovarian cancer cells (R2 = 0.8562 and R2 = 0.7982, respectively). The gene expression pattern of 319 differentially expressed genes revealed minimal differences between HOSE and IHOSE cells, while a strong difference between ovarian cancer cells and HOSE or IHOSE cells was observed. Furthermore, the five IHOSE cell lines displayed morphological characteristics typical of epithelial cells but showed a lower level of EpCAM, CD133 and E-cadherin, as cancer stem marker, than ovarian cancer cells. Moreover, unlike cancer cells, IHOSE cells could not form colonies in the anchorage-independent soft agar growth assay. CONCLUSION: These findings demonstrate that five newly established IHOSE cell lines have characteristics of progenitor HOSE cells while exhibiting continuous growth, and thus, should be highly useful as control cells for ovarian cancer research.

Accumulation of cytoplasmic Cdk1 is associated with cancer growth and survival rate in epithelial ovarian cancer.

Cyclin dependent kinase 1 (Cdk1) have previously reported correlation with cancer growth and a key regulator for cell cycle. Mostly, Cdk1's function of nucleus for cell cycle is well known to be associated with cancer, but cytoplasmic Cdk1's traits are not clearly identified, yet. We revealed that tissue microarray blocks of epithelial ovarian cancer (n = 249) showed increased level of cytoplasmic Cdk1 (p < 0.001), but not in nucleus (p = 0.192) of histologic cell type independently. On survival analysis, Cdk1 overexpression conferred a significantly worse prognosis in 5-year overall survival (Log-rank p = 0.028, Hazard ratio = 2.016, 95% CI = 1.097 to 4.635). Also, the expression of Cdk1 was increased in ovarian cancer cell lines and Gene Expression Omnibus datasets. When the expression and activity of Cdk1 were inhibited by si-Cdk1 or RO-3306 which is a potent Cdk1 inhibitor, the growth of ovarian cancer was diminished. Moreover, combined treatment with RO-3306 and cisplatin in ovarian cancer significantly elevated anti-cancer effects than single-agent treatment. In conclusion, cytoplasmic Cdk1 expression which was elevated in ovarian cancer predicts a poor overall survival. The inhibition of Cdk1 expression and activity reduced ovarian cancer growth.

Makorin Ring Finger Protein 1 as Adjunctive Marker in Liquid-based Cervical Cytology.

To assess the utility of makorin ring finger protein 1 (MKRN1) as a marker of cervical pathology.A PROspective specimen collection and retrospective Blinded Evaluation study was conducted. Liquid-based cytology samples were collected from 187 women, embedding all residuals as cell blocks for immunohistochemical staining of MKRN1 and P16 . Results of liquid-based cervical cytology, immunostained cell block sections, and human papillomavirus (HPV) hybrid capture (with real-time polymerase chain reaction) were analyzed. Clinical outcomes were analyzed overall and in subsets of specimens yielding atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions.Makorin ring finger protein 1 positivity and grades (1-3) of cervical intraepithelial neoplasia (CIN) increased in tandem (CIN1, 32.4%; CIN2, 60.0%; and CIN3, 80.0%), reaching 92.3% in invasive cancer. Sensitivity, specificity, positive predictive value, and negative predictive value in detecting CIN2+ via MKRN1 were 73.8%, 76.8%, 75.6%, and 75.0%, respectively. The performance of liquid-based cytology was poorer by comparison (61.3%, 69.5%, 66.2%, and 64.8%, respectively), and HPV assay (versus MKRN1 immunohistochemical staining) displayed lower specificity (67.7%). Combined HPV + MKRN1 testing proved highest in sensitivity, specificity, positive predictive value, and negative predictive value (71.8%, 85.5%, 82.3%, and 76.5%, respectively), whereas corresponding values for cytology + HPV (60.6%, 81.8%, 75.4%, and 69.2%) and cytology + MKRN1 (58.8%, 84.1%, 78.3%, and 67.7%) were all similar. In instances of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions, the HPV + MKRN1 combination performed best by above measures (100%, 72.7%, 73.9%, and 100%), followed by cytology + MKRN1 (100%, 50.0%, 60.7%, and 100%).Makorin ring finger protein 1 displayed greater sensitivity and specificity than liquid-based cytology and proved more specific than HPV assay. In combination testing, MKRN1 + HPV showed the highest sensitivity and specificity levels. The MKRN1 biomarker may be a useful adjunct in primary cervical cytology screening.

The role of S100A14 in epithelial ovarian tumors.

S100A14 is an EF-hand calcium-binding protein that has been reported to be involved in the progression of many malignancies. However, its role in ovarian cancer has not yet been clarified. In this study, we investigated the significance of S100A14 expression in epithelial ovarian cancers (EOCs) as well as it's mechanism of action. On both RNA and protein levels, S100A14 was overexpressed in transformed cells. Immunohistochemical staining demonstrated that S100A14 expression was associated with advanced stage (P<0.001) and poor tumor grade (P<0.001). Moreover, S100A14 overexpression was an independent prognostic factor for overall survival (HR = 4.53, P = 0.029). We also investigated S100A14's functional role by employing lentiviral-mediated overexpression and knockdown in EOC cells. S100A14 overexpression promoted cell proliferation, tumorigenesis, migration, and invasion, whereas S100A14 knockdown inhibited these properties. TOV112D cells that overexpressed S100A14 also exhibited greater tumor growth potential in xenografted mice. S100A14 promoted such a malignant phenotype in EOC cells through the PI3K/Akt pathway. Taken together, our data indicate that S100A14 has a crucial role in EOC progression, and its overexpression is associated with poor prognosis. Further study of S100A14's molecular mechanisms may lead to the development of a novel therapeutic target for ovarian cancer.