Spatial distribution of tumor-resident macrophages as predictive biomarkers in endometrial cancer.

BACKGROUND: To investigate the role of CD47 expression and its relationship with tumor-resident macrophages, specifically at the tumor margin, in patients with type II endometrial cancer. This study aims to elucidate whether CD47 could serve as a prognostic marker and to understand the dynamics between CD47 and macrophages, which could inform new therapeutic strategies. METHODS: A retrospective cohort study was conducted involving 75 patients of type II endometrial. Immunohistochemical analysis was performed to assess CD47 expression and macrophage markers (CD68 and CD163). RESULTS: The study found no direct correlation between CD47 expression levels and overall survival (p = 0.32), challenging its role as an independent prognostic marker in type II endometrial cancer. The higher expression of CD47 had significantly less incidence of endometrioid carcinoma G3 (p = 0.047). The negative correlation between CD47 H-score and the density of CD68-positive macrophages at tumor margin was statistically significant (p = 0.049). A high density of CD68-positive macrophages at the tumor margin but a low density of CD163-positive macrophages at the tumor margin were associated with poorer prognosis (p = 0.036). CONCLUSIONS: The complex interaction between CD47 and macrophages, particularly at the tumor margin, suggests new avenues for targeted therapy in type II endometrial cancer.

Real-world data of poly (ADP-ribose) polymerase inhibitor response in Japanese patients with ovarian cancer.

BACKGROUND: Poly (ADP-ribose) polymerase (PARP) inhibitors have been increasingly used in the treatment of ovarian cancer, with BRCA positivity and homologous recombination deficiency (HRD) being common biomarkers used for predicting their efficacy. However, given the limitations of these biomarkers, new ones need to be explored. METHODS: This retrospective study included 181 ovarian cancer patients who received olaparib or niraparib at two independent hospitals in Japan between May 2018 and December 2022. Clinical information and blood sampling data were collected. Patient characteristics, treatment history, and predictability of treatment duration based on blood data before treatment initiation were examined. RESULTS: High-grade serous carcinoma, BRCA positivity, HRD, and maintenance therapy after recurrence treatment were observed more frequently in the olaparib group than in the niraparib group. The most common reasons for treatment interruption were anemia, fatigue, and nausea in the olaparib group and thrombocytopenia in the niraparib group. Regarding response to olaparib treatment, complete response to the most recent treatment, maintenance therapy after the first chemotherapy, high-grade serous carcinoma, and germline BRCA positivity were observed significantly more frequently among responders than among non-responders. Furthermore, neutrophil counts were significantly higher among responders than among non-responders. CONCLUSIONS: Inflammation-related blood data, such as neutrophil count, obtained at the initial pre-treatment visit might serve as potential predictors for prolonged olaparib treatment. While this study offers valuable insights into potential indicators for prolonged olaparib treatment, it underscores the need for more expansive research to strengthen our understanding of PARP inhibitors and optimize treatment strategies in ovarian cancer.

Small extracellular vesicles in follicular fluids for predicting reproductive outcomes in assisted reproductive technology.

BACKGROUND: Assisted reproductive technology accounts for an increasing proportion of infertility treatments, and assessments to predict clinical pregnancy outcomes are desired. Extracellular vesicles exist in follicular fluid, and small non coding RNAs in extracellular vesicles underline the possibility of reflecting pregnancy potential. METHODS: Follicular fluid samples are collected from 20 ovarian follicles of 15 infertile patients undergoing assisted reproductive technology. Extracellular vesicles are isolated by serial centrifugation and small RNA sequencing is performed to investigate the profiles of microRNAs and P-element-induced wimpy testis-interacting RNAs. RESULTS: Small extracellular vesicles with a size range of approximately 100 nm are successfully isolated, and the small non coding RNA profiles of pregnant samples (n = 8) are different from those of non-pregnant samples (n = 12). Fourteen dysregulated small non coding RNAs are selected to identify the independent candidates [mean read count >100, area under the curve >0.8]. Among them, we find that a specific combination of small non coding RNAs (miR-16-2-3p, miR-378a-3p, and miR-483-5p) can predict the pregnant samples more precisely using a receiver operating characteristics curves analysis (area under the curve: 0.96). Furthermore, even in the same patients, the three microRNAs are differentially expressed between pregnant and non-pregnant samples. CONCLUSIONS: Our results demonstrate that small non coding RNAs derived from small extracellular vesicles in follicular fluid can be potential non-invasive biomarkers for predicting pregnancy, leading to their probable application in assisted reproductive technology. Further large-scale studies are required to validate the clinical usefulness of these small non coding RNAs.

Assisted reproductive technologies (ART) are medical procedures used to address infertility. Follicular fluid (FF)is a liquid that surrounds the immature egg cells (oocytes) and provides hormones and nutrients necessary for their maturation and eventual development into embryos. We analyzed genetic components within the FF as a potential predictor of reproductive outcomes following ART. Here, we show that a specific combination of genetic elements produced by the FF in successful pregnancies did not occur in unsuccessful pregnancies. Our findings may help to provide a non-invasive approach to determining reproductive outcomes during ART procedures.

Uterine leiomyosarcoma cell-derived extracellular vesicles induce the formation of cancer-associated fibroblasts.

OBJECTIVE: Uterine leiomyosarcoma (ULMS) is a rare malignant tumor, which is aggressive, and has a poor prognosis even during its early stages. Extracellular vesicles (EVs) carry cargo, such as microRNAs (miRNAs), which are involved in intercellular communication in the tumor microenvironment and other processes. Because there are no studies on EV-related miRNAs in ULMS, we identified EV-related miRNAs in ULMS and examined their function. METHODS: Small EVs (sEVs) and medium/large EVs (m/lEVs) were extracted from ULMS cells by ultracentrifugation and their basic characteristics were evaluated. Then, small RNA sequencing was done to obtain EV-related miRNA profiles. Next, miRNA expression levels in sera and tissues of ULMS patients were compared with those of myoma patients. RESULTS: miR-654-3p and miR-369-3p were indicated to be highly expressed in both sera and tissues of ULMS patients. These two miRNAs are also highly expressed in ULMS cell lines and ULMS-derived EVs. Some cancer-associated fibroblast (CAF) markers were increased when fibroblasts were treated with ULMS-derived EVs. Furthermore, fibroblasts took up EVs derived from ULMS as determined by confocal laser microscopy. In addition, the transfection of the two candidate miRNAs into fibroblasts significantly increased some CAF markers, particularly ACTA2. CONCLUSION: miR-654-3p and miR-369-3p are highly expressed in ULMS-derived EVs, indicating that these EV-related miRNAs induce the formation of cancer-associated fibroblasts.

Emerging bacterial factors for understanding pathogenesis of endometriosis.

The pathogenesis of endometriosis is a complex process, and recent research has introduced novel hypotheses in this field. This review summarizes recent studies on the pathogenesis of endometriosis. We focused on several classical hypotheses, as well as their interactions with the microenvironment of hormonal dependence and immunosuppression. Furthermore, we highlighted the emergence of bacterial factors associated with endometriosis. Recent advances in next-generation sequencing (NGS) have revealed the presence and detailed distribution of these bacteria as well as the involvement of specific bacteria in pathogenesis. These factors alter the microenvironment in the early stages of endometriosis development, leading to lesion formation. Understanding the mechanisms underlying the early development of endometriosis from a new perspective would be helpful for the development of novel therapeutic agents for endometriosis.

Interleukin-17A stimulation induces alterations in Microglial microRNA expression profiles.

BACKGROUND: Increased maternal interleukin (IL)-17A and activated microglia are pivotal factors contributing to the pathological phenotypes of maternal immune activation (MIA), developing neurodevelopmental disorders in offspring. This study aimed to determine whether IL-17A affects the microglial microRNA (miRNA) profiles. METHODS: The miRNA expression profiles of primary cultured microglia stimulated with recombinant IL-17A were examined comprehensively using miRNA sequencing and validated through qRT-PCR. The expressions of miRNAs target genes identified using bioinformatics, were investigated in microglia transfected with mimic miRNA. The target gene's expression was also examined in the fetal brains of the MIA mouse model induced by maternal lipopolysaccharide (LPS) administration. RESULTS: Primary cultured microglia expressed the IL-17A receptor and increased proinflammatory cytokines and nitric oxide synthase 2 upon treatment with IL-17A. Among the three miRNAs with |log2FC | >1, only mmu-miR-206-3p expression was significantly up-regulated by IL-17A. Transfection with the mmu-miR-206-3p mimic resulted in a significant decrease in the expression of Hdac4 and Igf1, target genes of mmu-miR-206-3p. Hdac4 expression also significantly decreased in the LPS-induced MIA model. CONCLUSIONS: IL-17A affected microglial miRNA profiles with upregulated mmu-miR-206-3p. These findings suggest that targeting the IL-17A/mmu-miR-206-3p pathway may be a new strategy for predicting MIA-related neurodevelopmental deficits and providing preventive interventions. IMPACT: Despite the growing evidence of interleukin (IL)-17A and microglia in the pathology of maternal immune activation (MIA), the downstream of IL-17A in microglia is not fully known. IL-17A altered microRNA profiles and upregulated the mmu-miR-206-3p expression in microglia. The mmu-miR-206-3p reduced autism spectrum disorder (ASD) related gene expressions, Hdac4 and Igf1. The Hdac4 expression was also reduced in the brain of MIA offspring. The hsa-miR-206 sequence is consistent with that of mmu-miR-206-3p. This study may provide clues to pathological mechanisms leading to predictions and interventions for ASD children born to mothers with IL-17A-related disorders.

Serum miRNA as a predictive biomarker for ovarian reserve after endometrioma-cystectomy.

Ovarian endometrioma (OE) is a common gynecological disease that is often treated with surgery and hormonal treatment. However, ovarian cystectomy can impair the ovarian reserve (OR). Previously, we showed that perioperative administration of dienogest (DNG) is an effective option for OR preservation. However, there were differences in the extent of OR preservation among patients following perioperative DNG treatment. In the current study, we performed a global examination of serum microRNAs (miRNAs) to identify accurate biomarkers that predict post-operative restoration of OR following perioperative DNG treatment. We also sought to identify specific miRNAs related to the anti-Müllerian hormone (AMH). miRNA sequencing was performed on serum samples obtained from twenty-seven patients who received perioperative DNG treatment. Candidate miRNAs were selected by comparing patients whose ORs were restored postoperatively (responder group, n = 7) with those whose ORs were not (non-responder group, n = 7). miR-370-3p and miR-1307-3p were significantly upregulated in the responder group, whereas miR-27b-3p was upregulated in the non-responder group. The pretreatment value of each miRNA could predict DNG responsiveness for OR following ovarian cystectomy (area under the curve [AUC] > 0.8). The quantitative polymerase chain reaction (qPCR) revealed only miR-1307-3p was found to be significantly upregulated in the responder group (P < 0.05). In addition, we identified miR-139-3p, miR-140-3p, and miR-629-5p as AMH-associated miRNAs. The transition of AMH showed a correlation with miR-139-3p (P < 0.05, r = -0.76). The miRNAs identified herein represent potential serum biomarkers of clinical value in predicting OR prior to DNG treatment.

An update of oncologic and obstetric outcomes of radical trachelectomy for early-stage cervical cancer: The need for further minimally invasive treatment.

AIMS: To investigate the oncologic and obstetric outcomes of radical trachelectomy (RT) in patients with early-stage cervical cancer and to evaluate the potential role of fertility-preserving treatments in improving pregnancy outcomes while oncologic status is stable. METHODS: In this single-institution study, we analyzed the oncologic and obstetric outcomes of 67 patients with early-stage cervical cancer who underwent RT at Nagoya University Hospital. RESULTS: The cancer recurrence rate (6.0%) and the mortality rate (1.5%) were comparable with those of previous studies. Of the 46 patients who attempted to conceive after RT, 19 (41.3%) became pregnant, and 16 gave birth. Of these 37.5% delivered at term, and delivery at less than 28 weeks of gestation occurred in 31.3% of pregnancies. CONCLUSIONS: RT is a viable treatment option for selected patients with early-stage cervical cancer. However, the use of less invasive techniques, such as conization/simple trachelectomy and pelvic lymph node dissection, may improve pregnancy outcomes while oncologic status is stable.

Effect of radical trachelectomy on ovarian reserve: A single-institute prospective study.

AIM: Reduced responses to controlled ovarian stimulation (COS) after radical trachelectomy (RT) have been previously reported. We aimed to assess the effect of RT on ovarian reserve by measuring anti-Müllerian hormone (AMH) levels before and after the procedure in this prospective study. METHODS: We included 12 patients who underwent RT between September 2019 and December 2021 in this study. Serum AMH levels were measured preoperatively, 1 month postoperatively, and 6 months postoperatively. Differences in the AMH levels were assessed using a paired t-test. RESULTS: The median age of the patients was 30.6 years, and the median follow-up time was 30.1 months. AMH levels at 1 and 6 months postoperatively did not show a consistent trend. At 1 month postoperatively, the average AMH level decreased insignificantly but returned to preoperative levels at 6 months. The differences in AMH levels before and after RT were insignificant. CONCLUSION: Our findings indicate that RT did not affect ovarian reserve as measured by AMH levels. However, the relationship between unchanged ovarian reserve and reduced response to COS remains unclear. Further research with larger sample sizes and additional measures of ovarian function is needed to corroborate these results and investigate the long-term effects of RT on ovarian reserve. Understanding these mechanisms will help guide surgical practices and provide patients with valuable information about their reproductive outcomes after RT.

Establishment and characterization of a non-gestational choriocarcinoma patient-derived xenograft model.

BACKGROUND: Non-gestational choriocarcinoma (NGC) is a rare subtype of malignant germ cell tumour and there is no consensus on its treatment. The lack of suitable preclinical models for NGC is a challenge in drug discovery research. Patient-derived xenograft (PDX) models recapitulate the tumour microenvironment of the original cancer tissue. Therefore, they have received considerable attention for studies on rare cancer. Here, we aimed to establish a PDX model from a patient with recurrent NGC. METHODS: Fresh NGC tumour tissue was immediately transplanted into a severely immune-deficient mouse (NOD.Cg-Prkdcscid1l2rgtm1Wjl/SzJ) and maintained for more than three in vivo passages. Subsequently, we evaluated the molecular characteristics of the PDX model using immunohistochemistry, polymerase chain reaction, and RNA sequencing. Moreover, the PDX tumours were transplanted into BALB/c nude mice, and we evaluated their sensitivity for cisplatin and methotrexate. RESULTS: The PDX tumour maintained the morphological features of NGC. Moreover, Immunohistochemistry revealed that the human chorionic gonadotropin, cytokeratin 7, and EpCAM expression levels were similar to those in the primary tumour. Furthermore, serum human chorionic gonadotropin levels were elevated in both the primary tumour and the PDX models. Additionally, using PCR analysis with species-specific primers, we confirmed that the PDX tumour contained human genes and was derived from human tissue. Moreover, the gene expression profile of the NGC was compared with that of epithelial ovarian cancer samples and cell lines, and 568 dysregulated genes in the NGC were extracted. The expression of the dysregulated genes in PDX was significantly correlated with that in the primary tumour (R2 = 0.873, P < 0.001). Finally, we demonstrated that the PDX tumour was sensitive to cisplatin and methotrexate; therefore, its clinical response to the agents was similar to that of the primary tumour. CONCLUSIONS: We successfully established a PDX model of NGC, to the best of our knowledge, for the first time. The established PDX retained the molecular and transcriptome characteristics of the primary tumour and can be used to predict drug effects. It may facilitate further research and the development of novel therapeutic agents for NGC.

9-oxo-ODAs suppresses the proliferation of human cervical cancer cells through the inhibition of CDKs and HPV oncoproteins.

Mucosal human papillomavirus (HPV) subtypes 16 and 18 are causative agents of cervical cancer, a leading cause of cancer-related deaths among women worldwide. In Japan, eggplant calyx is a folk remedy used to treat common warts. 9-oxo-(10E,12E)-octadecadienoic acid, isolated from eggplant calyx, may have antitumor effects. This study investigated the antitumor effects of 9-oxo-(10E, 12Z)-octadecadienoic acid and 9-oxo-(10E,12E)-octadecadienoic acid (9-oxo-ODAs) on human cervical cancer cells. 9-oxo-ODAs suppressed the proliferation of human cervical cancer cell lines (HeLa, and SiHa) in a concentration-dependent manner (IC50 = 25-50 µM). FCM analysis revealed that 9-oxo-ODAs induced apoptosis. Transcriptome, proteomics, and enrichment analyses revealed that treatment with 9-oxo-ODAs significantly altered the cell cycle and p53 pathways and decreased cyclin-dependent kinase 1 (CDK1) protein expression. Real-time PCR analysis demonstrated that 9-oxo-ODAs reduced CDK1 mRNA expression in a concentration-dependent manner. In vitro, 9-oxo-ODAs reduced the HPV oncoprotein expression. In ex vivo human cervical cancer tissues, 9-oxo-ODAs decreased CDK1 expression and increased cleaved caspase 3, an apoptosis marker. Further, 9-oxo-ODAs showed the potential to suppressed metastatic formation and growth of cervical cancer in vivo. These findings suggest that 9-oxo-ODAs induce cell cycle arrest and apoptosis in HPV-positive human cervical cancer cells, and this process involves CDK1. Consequently, 9-oxo-ODAs may be potential therapeutic agents for cervical cancer.

Spatial exosome analysis using cellulose nanofiber sheets reveals the location heterogeneity of extracellular vesicles.

Extracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 µL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

Mean platelet volume as a potential biomarker for survival outcomes in ovarian clear cell carcinoma.

OBJECTIVE: This study aimed to explore the prognostic value of mean platelet volume (MPV) in patients with ovarian clear cell carcinoma (OCCC) and evaluate the predictive performance of a random forest model incorporating MPV and other key clinicopathological factors. METHODS: A total of 204 patients with OCCC treated between January 2004 and December 2019 were retrospectively analyzed. Clinicopathological characteristics and preoperative laboratory data were collected, and survival outcomes were evaluated using the Kaplan-Meier method and Cox proportional hazards models. An optimal MPV cutoff was determined by receiver operating characteristic (ROC) curve analysis. A random forest model was then constructed using the identified independent prognostic factors, and its predictive performance was evaluated. RESULTS: The ROC analysis identified 9.3 fL as the MPV cutoff value for predicting 2-year survival. The MPV-low group had lower 5-year overall survival and progression-free survival rates than the MPV-high group (p = 0.003 and p = 0.034, respectively). High MPV emerged as an independent prognostic factor (p = 0.006). The random forest model, incorporating the FIGO stage, residual tumors, peritoneal cytology, and MPV, demonstrated robust predictive performance (area under the curve: 0.905). CONCLUSION: MPV is a promising prognostic indicator in OCCC. Lower MPV correlated with worse survival rates, advocating its potential utility in refining patient management strategies. The commendable predictive performance of the random forest model, integrating MPV and other significant prognostic factors, suggests a pathway toward enhanced survival prediction, thereby warranting further research.

Small Extracellular Vesicles from adipose-derived stem cells suppress cell proliferation by delivering the let-7 family of microRNAs in ovarian cancer.

INTRODUCTION: Ovarian cancer is the leading cause of death among women with gynecological cancer, and novel treatment options are urgently needed. Extracellular vesicles (EVs), including exosomes, may be one of the most promising therapeutic tools for various diseases. In this study, we aimed to investigate the therapeutic effects of adipose-derived stem cell-derived EVs (ADSC-EVs) on ovarian cancer cell lines. MATERIALS AND METHODS: ADSCs and the ovarian cancer cell lines SKOV3 and OV90 were used for analysis. ADSC-EVs were isolated through ultracentrifugation and validated using a cryotransmission electron microscope, nanoparticle tracking analysis, and western blotting. Then, the effect of ADSC-EVs on ovarian cancer cells was investigated using IncuCyte and microRNA sequencing. Moreover, the potential functions of miRNAs were evaluated by gain-of function analysis and in silico analysis. RESULTS: ADSC-EVs suppressed SKOV3 and OV90 cell proliferation. In particular, small EVs (sEVs) from ADSCs exhibited a stronger antitumor effect than ADSC-medium/large EVs (m/lEVs). Comparison of the miRNA profiles between ADSC-sEVs and ADSC-m/lEVs, along with downstream pathway analysis, suggested the involvement of the let-7 family. Overexpression of hsa-let-7b-5p and hsa-let-7e-5p significantly suppressed the proliferation of SKOV3 cells. In silico analysis revealed that four potential target genes of hsa-let-7b-5p and hsa-let-7e-5p were significantly associated with the prognoses of the patients. CONCLUSION: ADSC-sEVs had a stronger antitumor effect than ADSC-m/lEVs. Hsa-let-7b-5p and hsa-let-7e-5p, which are highly abundant in ADSC-sEVs, suppressed cell proliferation. These findings may open up new possibilities for therapeutic approaches using ADSC-sEVs.

The sarcopenia index measured using the lumbar paraspinal muscle is associated with prognosis in endometrial cancer.

OBJECTIVE: The number of type-II endometrial cancer patients has been increasing and the prognosis is not favorable. We aim to investigate whether sarcopenia index in any of several different muscles could serve as a novel biomarker of prognosis in patients with type-II endometrial cancer. METHODS: We retrospectively investigated a total of 194 patients at four hospitals. Ninety patients were treated as derivation set and the other 104 patients as validation set. Using preoperative computed tomography images, we measured the horizontal cross-sectional area at the third lumbar spine level: the (i) psoas major, (ii) iliac and (iii) paraspinal muscle. The clinical information including recurrence-free survival and overall survival were retrospectively collected. These results were validated with external data sets of three hospitals. RESULTS: The median values of the sarcopenia index (cm2/m2) ± standard deviation with the first data of 90 patients using the psoas, iliac and paraspinal muscle were 3.4 ± 1.0, 1.7 ± 0.6 and 12.6 ± 3.2, respectively. In univariate analyses, the sarcopenia indexes measured using the psoas or paraspinal muscle were associated with recurrence-free survival and overall survival. On the other hand, in multivariate analyses, only the sarcopenia index using paraspinal muscle was significantly related to recurrence-free survival (hazard ratio = 3.78, 95% confidence intervals = 1.29-5.97, P = 0.009) and overall survival (hazard ratio = 3.13, 95% confidence interval = 1.18-8.26, P = 0.022). Paraspinal sarcopenia index was also related to overall survival (hazard ratio = 3.74, 95% confidence interval = 1.31-10.72, P = 0.014) even in patients with advanced stage. Serum albumin was significantly correlated with the sarcopenia index (P = 0.012). Within the analysis of the validation set, sarcopenia index using paraspinal muscle was related to recurrence-free survival (hazard ratio = 2.06, P = 0.045) in multivariate analysis and recurrence-free survival (P = 0.009) in patients with advanced stage. CONCLUSIONS: The sarcopenia index using the paraspinal muscle, not psoas, could be a suitable index to predict recurrence-free survival and overall survival in patients with type-II endometrial cancer even in advanced stage.

Identifying high-grade serous ovarian carcinoma-specific extracellular vesicles by polyketone-coated nanowires.

Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.

The prognostic significance of DDIT4 in endometrial cancer.

BACKGROUND: Despite extensive research on endometrial cancer and tumor hypoxic microenvironment, there are no reports exploring the role of DDIT4 in endometrial cancer. OBJECTIVE: This study aimed to elucidate the significance of DDIT4, as a prognostic biomarker for endometrial cancer by immunohistochemical staining and statistical analysis. METHODS: Four endometrial cancer cells were cultured under normoxia and hypoxia, and the differentially expressed genes were examined using RNA-seq. Immunohistochemical staining for DDIT4 and HIF1A was performed in 86 patients with type II endometrial cancer treated at our hospital, and their correlation with other clinicopathological factors and the prognostic role was analyzed using statistical methods. RESULTS: The expression analysis of hypoxia-inducible genes using four types of endometrial cancer cells revealed that DDIT4 was among the 28 genes that were upregulated in all cells. Based on our results of immunohistochemistry of DDIT4 expression in endometrial cancer tissues, univariate and multivariate analyses based on COX regression analysis showed that high DDIT4 expression significantly correlated to favorable prognosis in both progression-free survival and overall survival. Limited to recurrent cases, metastasis to only lymph nodes was significantly related to high DDIT4 expression, whereas metastasis to other parenchymal organs was significantly dominant in patients with low DDIT4 expression. CONCLUSIONS: The expression of DDIT4 enables to predict survival and recurrence in type II endometrial cancer.

Downregulating vaccinia-related kinase 1 by luteolin suppresses ovarian cancer cell proliferation by activating the p53 signaling pathway.

OBJECTIVES: Ovarian cancer constitutes one of the most common causes of cancer-related deaths, and preventing chemotherapy resistance and recurrence in patients with ovarian cancer remains a challenge. Herein, we aimed to identify the effect of luteolin, a novel therapeutic agent targeting vaccinia-related kinase 1 (VRK1), on high-grade serous ovarian cancer (HGSOC). METHODS: Phosphokinase array, RNA sequencing, and cell cycle and apoptosis assays were conducted to determine the underlying mechanism of the effect of luteolin on HGSOC cells. The anticancer effects of oral and intraperitoneal luteolin administration were assessed in patient-derived xenograft models via several methods, including the assessment of tumor size and immunohistochemistry of phospho-p53, phosphor-HistoneH3 and cleaved caspase 3. RESULTS: Luteolin reduced HGSOC cell proliferation and increased apoptosis and cell cycle arrest at G2/M. Compared with controls, several genes were dysregulated in luteolin-treated cells, and luteolin activated the p53 signaling pathway. The human phosphokinase array revealed distinct p53 upregulation in luteolin-treated cells, as confirmed by p53 phosphorylation at ser15 and ser46 using western blot analysis. In patient-derived xenograft models, oral or intraperitoneal luteolin administration substantially suppressed tumor growth. Moreover, combination treatment involving luteolin and cisplatin inhibited tumor cell proliferation, especially in cisplatin-resistant HGSOC cell lines. CONCLUSIONS: Luteolin demonstrated considerable anticancer effect on HGSOC cells, reduced VRK1 expression, and activated the p53 signaling pathway, thereby inducing apoptosis and cell cycle arrest in G2/M and inhibiting cell proliferation. Furthermore, luteolin exhibited a synergistic effect with cisplatin both in vivo and in vitro. Thus, luteolin can be considered a promising cotreatment option for HGSOC.

The association of UBAP2L and G3BP1 mediated by small nucleolar RNA is essential for stress granule formation.

Stress granules (SGs) are dynamic, non-membranous structures composed of non-translating mRNAs and various proteins and play critical roles in cell survival under stressed conditions. Extensive proteomics analyses have been performed to identify proteins in SGs; however, the molecular functions of these components in SG formation remain unclear. In this report, we show that ubiquitin-associated protein 2-like (UBAP2L) is a crucial component of SGs. UBAP2L localized to SGs in response to various stresses, and its depletion significantly suppressed SG organization. Proteomics and RNA sequencing analyses found that UBAP2L formed a protein-RNA complex with Ras-GTP-activating protein SH3 domain binding protein 1 (G3BP1) and small nucleolar RNAs (snoRNAs). In vitro binding analysis demonstrated that snoRNAs were required for UBAP2L association with G3BP1. In addition, decreased expression of snoRNAs reduced the interaction between UBAP2L and G3BP1 and suppressed SG formation. Our results reveal a critical role of SG component, the UBAP2L/snoRNA/G3BP1 protein-RNA complex, and provide new insights into the regulation of SG assembly.

DDIT4 Facilitates Lymph Node Metastasis via the Activation of NF-κB Pathway and Epithelial-Mesenchymal Transition.

This study was aimed to identify a novel metastasis-promoting molecule and elucidate its functional and prognostic roles in cervical cancer. DDIT4 (DNA-damage-inducible transcript 4), a hypoxia-inducible gene, was identified by analyzing multiple microarray databases. The correlation between DDIT4 expression in immunohistochemistry and clinicopathological characteristics in the public database and our cohort was evaluated by statistical analysis. Transwell® assay and wound-healing assay to determine cell migration and invasion were performed. DDIT4 was knocked down using siRNA or lentiviral vectors. The potential downstream pathways of DDIT4 were explored and verified by a gene set enrichment analysis and western blotting. The in vivo metastatic capability was determined with the use of an intraperitoneal injection mouse model. In the analysis of the public database and our cohort, DDIT4 high expression was significantly related to short overall survival and lymph node metastasis in patients with early-stage cervical cancer. The knockdown of DDIT4 attenuated the migration and invasion activity of tumor cells in vitro and reduced the expression of epithelial-mesenchymal transition (EMT)-related proteins and the NF-κB pathway in cervical cancer cells. DDIT4 also promoted tumor progression in the mouse model. Our results indicate that DDIT4 can be a prognostic indicator in cervical cancer and promote lymph node metastasis, augmenting malignancy via the EMT and NF-kB pathways.