Therapeutic Strategies for Targeting Ovarian Cancer Stem Cells.

Ovarian cancer is a fatal gynecological malignancy. Although first-line chemotherapy and surgical operation are effective treatments for ovarian cancer, its clinical management remains a challenge owing to intrinsic or acquired drug resistance and relapse at local or distal lesions. Cancer stem cells (CSCs) are a small subpopulation of cells inside tumor tissues, and they can self-renew and differentiate. CSCs are responsible for the cancer malignancy involved in relapses as well as resistance to chemotherapy and radiation. These malignant properties of CSCs are regulated by cell surface receptors and intracellular pluripotency-associated factors triggered by internal or external stimuli from the tumor microenvironment. The malignancy of CSCs can be attenuated by individual or combined restraining of cell surface receptors and intracellular pluripotency-associated factors. Therefore, targeted therapy against CSCs is a feasible therapeutic tool against ovarian cancer. In this paper, we review the prominent roles of cell surface receptors and intracellular pluripotency-associated factors in mediating the stemness and malignancy of ovarian CSCs.

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.

Prognostic implication of programmed cell death 1 protein and its ligand expressions in endometrial cancer.

OBJECTIVE: Monoclonal antibodies targeting programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1) demonstrated promising clinical response. The predictive/prognostic value of PD-1/PD-L1 immunohistochemistry (IHC) has been evaluated in many cancer types. However, the prognostic value of PD-1/PD-L1 IHC has not been evaluated in endometrial cancer. METHODS: We conducted a retrospective study to quantify the IHC CD8, PD-1, and PD-L1 expressions in immune cells at center of tumor (CT), invasive margin (IM), and/or tumor cell in 183 primary endometrial cancer samples from a single cohort, followed by their reciprocal combinations, including compartmental differences, and correlated them with overall survival (OS) and progression-free survival (PFS). RESULTS: In repeated Cox multivariable models adjusted by clinicoimmunopathologic factors, high CT-PD-L1 was an independent adverse prognostic factor for PFS in all patients and in the microsatellite-stable subgroup. Immune marker ratios revealed independently shorter PFS for high CT-PD-L1/CT-CD8 and CT-PD-L1/CT-PD-1 ratios. Classification of endometrial cancer into four groups based on CT-CD8 and CT-PD-L1 revealed significantly different survival among groups. CONCLUSIONS: The high PD-L1/CD8 ratio and the high expression of PD-L1 on immune cells were independent poor prognostic factors for PFS in endometrial cancer, providing insights into the tumor microenvironment.

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.

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.

Inhibition of MEK-ERK pathway enhances oncolytic vaccinia virus replication in doxorubicin-resistant ovarian cancer.

Oncolytic vaccinia virus (OVV) has been reported to induce cell death in various types of cancer; however, the oncolytic activity of OVV in drug-resistant ovarian cancer remains limited. In the present study, we established doxorubicin-resistant ovarian cancer cells (A2780-R) from the A2780 human ovarian cancer cell line. Both A2780 and A2780-R cells were infected with OVV to explore its anticancer effects. Interestingly, OVV-infected A2780-R cells showed reduced viral replication and cell death compared with A2780 cells, suggesting their resistance against OVV-induced oncolysis; to understand the mechanism underlying this resistance, we explored the involvement of protein kinases. Among protein kinase inhibitors, PD0325901, an MEK inhibitor, significantly augmented OVV replication and cell death in A2780-R cells. PD0325901 treatment increased the phosphorylation of STAT3 in A2780-R cells. Moreover, cryptotanshinone, a STAT3 inhibitor, abrogated PD0325901-stimulated OVV replication. Furthermore, trametinib, a clinically approved MEK inhibitor, increased OVV replication in A2780-R cells. Transcriptomic analysis showed that the MEK inhibitor promoted OVV replication via increasing STAT3 activation and downregulating the cytosolic DNA-sensing pathway. Combined treatment with OVV and trametinib attenuated A2780-R xenograft tumor growth. These results suggest that pharmacological inhibition of MEK reinforces the oncolytic efficacy of OVV in drug-resistant ovarian cancer.

Incorporation of SKI-G-801, a Novel AXL Inhibitor, With Anti-PD-1 Plus Chemotherapy Improves Anti-Tumor Activity and Survival by Enhancing T Cell Immunity.

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86+ macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.

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.

Transcriptional activation of HIF-1 by RORalpha and its role in hypoxia signaling.

OBJECTIVE: Hypoxia-inducible factor 1alpha (HIF-1alpha) is primarily involved in the adapting of cells to changes in oxygen levels, which is essential for normal vascular function. Recently, physiological roles for retinoic acid-related orphan receptor alpha (RORalpha) have been implicated in cardiovascular diseases such as atherosclerosis. In this study, we have investigated the potential roles of RORalpha in the hypoxia signaling pathway in connection with activation of HIF-1alpha. METHODS AND RESULTS: Under hypoxic conditions, expression of RORalpha was induced. When RORalpha was introduced exogenously, protein level as well as transcriptional activity of HIF-1alpha was enhanced. Putative ligands of RORalpha, such as melatonin and cholesterol sulfate, induced transcriptional activity for HIF-1alpha, which was abolished by RNA interference against RORalpha. RORalpha was physically associated with HIF-1alpha through DNA binding domain, which was required to the RORalpha-induced stabilization and transcriptional activation of HIF-1alpha. Finally, either infection with adenovirus encoding RORalpha or treatment with ROR ligands enhanced the formation of capillary tubes by human umbilical vascular endothelial cells. CONCLUSIONS: Our results provide a new insight for the function of RORalpha in amplification of hypoxia signaling and suggest a potential application of RORalpha ligands for the therapy of hypoxia-associated vascular diseases.

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.

DNA Mismatch Repair Protein Immunohistochemistry and MLH1 Promotor Methylation Testing for Practical Molecular Classification and the Prediction of Prognosis in Endometrial Cancer.

The incidence of endometrial cancer is rapidly increasing worldwide, and its molecular classification has gained importance for new therapeutic approaches. This study sought to examine the clinicopathologic features and immune markers associated with the DNA mismatch repair (MMR) status and MLH1 promoter methylation status of endometrial cancer patients. A total of 173 patients with primary endometrial cancer who had received a hysterectomy were evaluated for four MMR proteins (MLH1, MSH2, MSH6, and PMS2), immune markers (CD8, programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1)) and p53 by immunohistochemistry (IHC), followed by an MLH1 methylation test. Patients were classified into MMR deficiency or proficiency, sporadic cancer, or probable Lynch syndrome (PLS), and the clinicopathologic features (including the expression of peritumoral immune markers) and prognosis of each group were compared. Patients with MMR deficiency or PLS showed an increase in immune markers compared those with MMR proficiency or sporadic cancer, respectively, and PLS demonstrated higher immune marker expression than MLH1 promoter methylation. Regarding prognosis, patients with MMR deficiency showed significant adverse overall survival (OS) when in stages I and II. Practical molecular classifications based on p53 staining results, in addition to MMR or PLS status, revealed an increased predictive ability for OS compared with the European Society of Medical Oncologists (ESMO) risk groups. The results of this study suggest that PLS may be a better candidate for an immune checkpoint inhibitor than MMR deficiency. The practical molecular classification contributes not only to the screening of Lynch syndrome, but also assists in predicting the prognosis in endometrial cancer.

Comparison of Clinical Features and Outcomes in Epithelial Ovarian Cancer according to Tumorigenicity in Patient-Derived Xenograft Models.

PURPOSE: Although the use of xenograft models is increasing, few studies have compared the clinical features or outcomes of epithelial ovarian cancer (EOC) patients according to the tumorigenicity of engrafted specimens. The purpose of this study was to evaluate whether tumorigenicity was associated with the clinical features and outcomes of EOC patients. MATERIALS AND METHODS: Eighty-eight EOC patients who underwent primary or interval debulking surgery from June 2014 to December 2015 were included. Fresh tumor specimens were implanted subcutaneously on each flank of immunodeficient mice. Patient characteristics, progression-free survival (PFS), and germline mutation spectra were compared according to tumorigenicity. RESULTS: Xenografts were established successfully from 49 of 88 specimens. Tumorigenicity was associated with lymphovascular invasion and there was a propensity to engraft successfully with high-grade tumors. Tumors from patientswho underwent non-optimal (residual disease ≥ 1 cm) primary orinterval debulking surgery had a significantly greater propensity to achieve tumorigenicity than those who received optimal surgery. In addition, patients whose tumors became engrafted seemed to have a shorter PFS and more frequent germline mutations than patients whose tumors failed to engraft. Tumorigenicity was a significant factor for predicting PFS with advanced International Federation of Gynecology and Obstetrics stage and high-grade cancers. CONCLUSIONS: Tumorigenicity in a xenograft model was a strong prognostic factor and was associated with more aggressive tumors in EOC patients. Xenograft models can be useful as a preclinical tool to predict prognosis and could be applied to further pharmacologic and genomic studies on personalized treatments.

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.