Tumor-derived small extracellular vesicles facilitate omental metastasis of ovarian cancer by triggering activation of mesenchymal stem cells.

BACKGROUND: Omental metastasis is the major cause of ovarian cancer recurrence and shortens patient survival, which can be largely attributed to the dynamic evolution of the fertile metastatic microenvironment driven by cancer cells. Previously, we found that adipose-derived mesenchymal stem cells (ADSCs) undergoing a phenotype shift toward cancer-associated fibroblasts (CAFs) participated in the orchestrated omental premetastatic niche for ovarian cancer. Here, we aim to elucidate the underlying mechanisms. METHODS: Small extracellular vesicles were isolated from ovarian cancer cell lines (ES-2 and its highly metastatic subline, ES-2-HM) and patient ascites using ultracentrifugation. Functional experiments, including Transwell and EdU assays, and molecular detection, including Western blot, immunofluorescence, and RT-qPCR, were performed to investigate the activation of ADSCs in vitro. High-throughput transcriptional sequencing and functional assays were employed to identify the crucial functional molecules inducing CAF-like activation of ADSCs and the downstream effector of miR-320a. The impact of extracellular vesicles and miR-320a-activated ADSCs on tumor growth and metastasis was assessed in subcutaneous and orthotopic ovarian cancer xenograft mouse models. The expression of miR-320a in human samples was evaluated using in situ hybridization staining. RESULTS: Primary human ADSCs cocultured with small extracellular vesicles, especially those derived from ES-2-HM, exhibited boosted migration, invasion, and proliferation capacities and elevated α-SMA and FAP levels. Tumor-derived small extracellular vesicles increased α-SMA-positive stromal cells, fostered omental metastasis, and shortened the survival of mice harboring orthotopic ovarian cancer xenografts. miR-320a was abundant in highly metastatic cell-derived extracellular vesicles, evoked dramatic CAF-like transition of ADSCs, targeted the 3'-untranslated region of integrin subunit alpha 7 and attenuated its expression. miR-320a overexpression in ovarian cancer was associated with omental metastasis and shorter survival. miR-320a-activated ADSCs facilitated tumor cell growth and omental metastasis. Depletion of integrin alpha 7 triggered CAF-like activation of ADSCs in vitro. Video Abstract CONCLUSIONS: miR-320a in small extracellular vesicles secreted by tumor cells targets integrin subunit alpha 7 in ADSCs and drives CAF-like activation, which in turn facilitates omental metastasis of ovarian cancer.

Unveiling a Virulence-Regulating Mechanism in Aeromonas hydrophila: a Quantitative Exoproteomic Analysis of an AraC-Like Protein.

Bacterial AraC is a transcription factor family that initiates transcription by recruiting RNA polymerase to the promoter and directly regulating various bacterial phenotypes. It also directly regulates various bacterial phenotypes. However, how this transcription factor regulates bacterial virulence and affects host immunity is still largely unknown. In this study, deleting the orf02889 (AraC-like transcription factor) gene in virulent Aeromonas hydrophila LP-2 affected several important phenotypes, such as increasing biofilm formation and siderophore production abilities. Moreover, Δorf02889 also significantly decreased the virulence of A. hydrophila and has promising attenuated vaccine potential. To better understand the effects of orf02889 on biological functions, a data independent acquisition (DIA)-based quantitative proteomics method was performed to compare the differentially expressed proteins between Δorf02889 and the wild-type strain in extracellular fractions. The following bioinformatics analysis suggested that ORF02889 may regulate various metabolic pathways, such as quorum sensing and ATP binding cassette (ABC) transporter metabolism. Moreover, 10 selected genes from the top 10 decreasing abundances in proteomics data were deleted, and their virulence to zebrafish was evaluated, respectively. The results showed that ΔcorC, Δorf00906, and Δorf04042 significantly reduced bacterial virulence. Finally, the following chromatin immunoprecipitation and polymerase chain reaction (ChIP-PCR) assay validated that the promoter of corC was directly regulated by ORF02889. Overall, these results provide insight into the biological function of ORF02889 and demonstrate its inherent regulatory mechanism for the virulence of A. hydrophila.

Omental cancer-associated fibroblast-derived exosomes with low microRNA-29c-3p promote ovarian cancer peritoneal metastasis.

Ovarian cancer (OC) is characterized by frequent widespread peritoneal metastasis. Cancer-associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote omentum metastasis in OC patients. However, the role of exosomes derived from omental CAFs in metastasis remains unclear. We isolated exosomes from primary omental normal fibroblasts (NFs) and CAFs from OC patients (NF-Exo and CAF-Exo, respectively) and assessed their effect on metastasis. In mice bearing orthotopic OC xenografts, CAF-Exo treatment led to more rapid intraperitoneal tumor dissemination and shorter animal survival. Similar results were observed in mice undergoing intraperitoneal injection of tumor cells. Among the miRNAs downregulated in CAF-Exo, miR-29c-3p in OC tissues was associated with metastasis and survival in patients. Moreover, increasing miR-29c-3p in CAF-Exo significantly weakened the metastasis-promoting effect of CAF-Exo. Based on RNA sequencing, expression assays, and luciferase assays, matrix metalloproteinase 2 (MMP2) was identified as a direct target of miR-29c-3p. These results verify the significant contribution of exosomes from omental CAFs to OC peritoneal metastasis, which could be partially due to the relief of MMP2 expression inhibition mediated by low exosomal miR-29c-3p.

Identification of miR-30c-5p as a tumor suppressor by targeting the m6 A reader HNRNPA2B1 in ovarian cancer.

BACKGROUND: microRNAs (miRNAs) and N6-methyladenosine (m6 A) play important roles in ovarian cancer (OvCa). However, the mechanisms by which miRNAs regulate m6 A in OvCa have not been elucidated so far. METHODS: To screen m6 A-related miRNAs, Pearson's correlation analysis of miRNAs and m6 A regulators was implemented using The Cancer Genome Atlas database (TCGA). To determine the level of m6 A, RNA m6 A quantitative assays were used. Then, colony formation assays, EdU assays, wound healing assays, and Transwell assays were performed. The dual-luciferase reporter assay was used to confirm the miRNA target genes. Protein-protein interaction (PPI) analysis of the target genes was performed, and hub genes were discovered using the cytoHubba/Cytoscape software. The underlying molecular mechanisms were explored by bioinformatics and RNA stability assays. RESULTS: A total of 126 miRNAs were identified as m6 A-related miRNAs by Pearson's correlation analysis. Among them, the high level of miR-30c-5p was associated with good prognosis in OvCa patients. In vitro, the miR-30c-5p agomir lowered the m6 A level and inhibited OvCa cell proliferation, migration, and invasion. The hub target genes of miR-30c-5p were identified as (i) XPO1, (ii) AGO1, (iii) HNRNPA2B1, of which m6 A reader HNRNPA2B1 was highly expressed in OvCa tissues and related with poor prognosis. In vitro, knockdown of HNRNPA2B1 significantly reduced m6 A level and hampered the proliferation and migration of OvCa cells. The inhibition of m6 A reader HNRNPA2B1 attenuated the suppression of proliferation and migration and the low m6 A level induced by the miR-30c-5p downregulation. Mechanistically, m6 A reader HNRNPA2B1 might regulate CDK19 mRNA stability to alter m6 A level. CONCLUSIONS: miR-30c-5p inhibits OvCa progression and reduces the m6 A level by inhibiting m6 A reader HNRNPA2B1, thus providing new insights into the m6 A regulatory mechanism in OvCa.

Tumour-derived exosomal piR-25783 promotes omental metastasis of ovarian carcinoma by inducing the fibroblast to myofibroblast transition.

Ovarian carcinoma inherently possesses a distinct metastatic organotropism for the adipose-rich omentum, contributing to disease progression. Although the premetastatic microenvironment (PMM) has been known to often play a prometastatic role during the process, incomplete mechanistic insight into PMM formation has prevented its therapeutic targeting. Omental fibroblasts can be activated by tumour cells to differentiate into myofibroblasts, termed the fibroblast-to-myofibroblast transition (FMT), which, in turn, enhances cancer aggressiveness. Here, we report crosstalk between cancer cells and omental fibroblasts through exosomal piR-25783, which fuels tumour metastasis. Tumour cell-secreted exosomal piR-25783 activates the TGF-ß/SMAD2/SMAD3 pathway in fibroblasts and promotes the FMT in the omentum along with the secretion of various cytokines and elevation of proliferative, migratory, and invasive properties, contributing to the formation of PMMs. Furthermore, piR-25783-induced myofibroblasts promote tumour implantation and growth in the omentum. In addition, the overexpression of piR-25783 in ovarian carcinoma is associated with unfavourable clinicopathological characteristics and shorter survival. In this study, we provide molecular, functional, and translational evidence suggesting that exosomal piR-25783 plays an important role in the formation of PMMs and the development of metastatic diseases in vitro and in vivo and may serve as a potential therapeutic target for ovarian carcinoma with metastasis.

FABP4 is an independent risk factor for lymph node metastasis and poor prognosis in patients with cervical cancer.

BACKGROUND: Pelvic lymph node metastasis (LNM) is a crucial independent prognostic factor in cervical cancer (CCa) and serves as an indicator for radiation therapy as the primary or an adjuvant treatment option. However, preoperative diagnosis of LNM remains challenging. Thus, we aimed to identify biomarkers of LNM in patients with presumed early-stage CCa. METHODS: The differentially expressed genes (DEGs) between tumours with different lymph node statuses were identified by using The Cancer Genome Atlas database. Then, univariate Cox regression analysis and Kaplan-Meier analyses were utilized to screen overall survival (OS)-associated genes. Multivariate Cox analysis and logistical analysis were utilized to evaluate independent risk factors for OS and LNM, respectively. Subsequently, the protein level of fatty acid binding protein 4 (FABP4) was detected in normal cervical and CCa tissues by immunohistochemistry assays. EdU assays were performed to determine whether FABP4 altered the proliferation of cervical cancer cells. Wound healing and Transwell assays were conducted to explore the effects of FABP4 depletion on migratory and invasive abilities of cervical cancer cells. F-actin fluorescence staining were performed to investigate morphological change and Western blotting analyses were performed to determine epithelial mesenchymal transition-related marker expression and downstream signalling pathways. RESULTS: A total of 243 DEGs, including 55 upregulated and 188 downregulated DEGs, were found in CCa patients with LNM versus those without LNM. Among these, FABP4 was found to be closely associated with poor OS. Multivariate analysis uncovered that FABP4 was an independent risk factor for OS and LNM in patients with CCa. The immunohistochemical results verified dramatically increased FABP4 expression in CCa tissues compared to normal cervical epithelia and its association with poor OS and LNM. In vitro, The proliferation, migration and invasion of cervical cancer cells were significantly inhibited after knocking down of FABP4, which was accompanied by elevated expression of E-cadherin and downregulated expression of N-cadherin, Vimentin and p-AKT. CONCLUSIONS: FABP4 might be a promising biomarker of LNM and survival in patients with early-stage CCa and therefore could significantly contribute to the development of personalized prognosis prediction and therapy optimization.

Exosomes in ovarian cancer ascites promote epithelial-mesenchymal transition of ovarian cancer cells by delivery of miR-6780b-5p.

The poor prognosis of ovarian cancer is mainly due to metastasis, and the specific mechanism underlying ovarian cancer metastasis is not clear. Ascites-derived exosomes (ADEs) play an important role in the progression of ovarian cancer, but the mechanism is unknown. Here, we found that ADEs promoted ovarian cancer metastasis not only in vitro but also in vivo. This promotive function was based on epithelial-mesenchymal transition (EMT) of ovarian cancer cells. Bioinformatics analysis of RNA sequencing microarray data indicated that miR-6780b-5p may be the key microRNA (miRNA) in ADEs that facilitates cancer metastasis. Moreover, the expression of exosomal miR-6780b-5p correlated with tumor metastasis in ovarian cancer patients. miR-6780b-5p overexpression promoted and miR-6780b-5p downregulation suppressed EMT of ovarian cancer cells. These results suggest that ADEs transfer miR-6780b-5p to ovarian cancer cells, promoting EMT and finally facilitating ovarian cancer metastasis.

GADD45B Facilitates Metastasis of Ovarian Cancer Through Epithelial-Mesenchymal Transition.

BACKGROUND: Growth arrest and DNA-damage-inducible 45 beta (GADD45B) is overexpressed and is associated with poor clinical outcomes in many human cancers, but the clinical implication of GADD45B in epithelial ovarian cancer (EOC) remains unclear. METHODS: Bioinformatics analysis of The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) cohorts was used to illustrate the relationship between GADD45B expression and metastasis, as well as the survival time of EOC. GADD45B was downregulated by siRNAs in EOC cells, and migration ability was determined by a transwell assay and wound-healing assay. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene set enrichment analysis (GSEA) were conducted to discover the downstream pathway of GADD45B. The regulation of epithelial-mesenchymal transition (EMT) by GADD45B was verified by Western blotting and qRT-PCR. Finally, the correlation of GADD45B expression with EOC metastasis was investigated in EOC tissues by immunohistochemistry. RESULTS: Overexpression of GADD45B indicates shorter overall survival time and progression-free survival time, and it is an independent risk factor for poor survival in EOC patients. Elevated GADD45B is related to venous invasion, lymphatic invasion and peritoneal carcinomatosis. Downregulation of GADD45B decreases the migration of ES2 and SKOV3 cells. Further KEGG enrichment analysis and GSEA revealed that EMT may be the downstream pathway of GADD45B. In addition, reduced GADD45B increases the expression of E-cadherin and decreases that of N-cadherin and vimentin. Finally, immunohistochemical analysis of GADD45B expression revealed that the expression of GADD45B in omental metastatic tissues was higher than that in matched primary ovarian cancer tissues. These results suggest that elevated GADD45B promotes the motility of ovarian cancer cells through EMT and is associated with EOC metastasis. CONCLUSION: GADD45B can promote the motility of ovarian cancer cells through EMT, is associated with EOC metastasis, and may be a new biomarker of metastasis and prognosis.

Circulating Tie2-Expressing Monocytes: A Potential Biomarker for Cervical Cancer.

BACKGROUND: Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2)-expressing monocytes (TEMs) are a highly proangiogenic subset of myeloid cells, which are characterized by expressing the angiopoietin receptor Tie2 with pro-tumor activity. PURPOSE: The present study aimed to determine the clinical value of circulating TEMs (cTEMs) for cervical cancer. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 7 healthy volunteers, 17 uterine fibroid patients, 24 cervical intraepithelial neoplasia (CIN) II patients, 31 CIN III patients and 99 patients with cervical cancer. The cTEMs were evaluated by the ratio of Tie2+ CD14+ cells to all CD14+ monocytes in the PBMCs through flow cytometry. The diagnostic value of cTEM was assessed by receiver operating characteristic (ROC) curves and the correlation between cTEM and clinicopathological characters in cervical cancer patients was analyzed. RESULTS: The proportion of cTEMs was gradually increasing from healthy volunteers to patients with non-invasive lesions, then to cervical cancer patients. The area under the ROC curve was 0.913 when the level of cTEMs was used to distinguish cervical cancer from all the other women ranging from healthy volunteers to CIN III patients. In cervical cancer, an increased cTEM fraction was significantly correlated with advanced tumor stage, larger tumor size, lymph node metastasis (LNM), deep stromal infiltration, parametrial involvement and lymph-vascular space invasion and was an independent risk factor for LNM. CONCLUSION: The cTEM proportion might be a promising biomarker for the malignant transformation of cervical lesions and the progression of cervical cancer.

Let-7e Suppresses DNA Damage Repair and Sensitizes Ovarian Cancer to Cisplatin through Targeting PARP1.

Increased DNA damage repair is one of the mechanisms implicated in cisplatin resistance. Our previous study indicated that the deregulation of let-7e promoted cisplatin resistance and that let-7e could suppress DNA double-strand break repair in ovarian cancer. In this study, we further characterized the role of let-7e in DNA damage repair and cisplatin resistance in ovarian cancer, and investigated the underlying mechanisms. The alkaline and neutral comet assay indicated that let-7e impeded both DNA single- and double-strand break repairs through downregulating its target gene PARP1. In vitro and in vivo experiments provided evidence that the let-7e-PARP1-DNA repair axis was involved in the modulation of cisplatin sensitivity in ovarian cancer. Contrary to let-7e, PARP1 was overexpressed in cisplatin-resistant ovarian cancer tissues, and patients with high PARP1 expression exhibited poor progression-free survival (PFS) and overall survival (OS). Multivariate logistic and Cox regression analyses showed that let-7e and FIGO stage were independent prognostic factors for PFS and OS, whereas let-7e and PARP1 were able to independently predict chemotherapy response. Taken together, our results indicated that low expression of let-7e promoted DNA single- and double-strand break repairs and subsequently contributed to cisplatin resistance by relieving the suppression on PARP1 in ovarian cancer. IMPLICATIONS: Targeting the let-7e-PARP1-DNA repair axis might be an effective strategy for the treatment of chemoresistant ovarian cancer.

Tumor cell­fibroblast heterotypic aggregates in malignant ascites of patients with ovarian cancer.

Ascitic multicellular aggregates (MCAs) promote peritoneal metastasis of ovarian cancer. The aim of the present study was to elucidate the role of cancer­associated fibroblasts (CAFs) in MCA formation and metastasis in patients with high­grade serous ovarian cancer (HGSOC). Immunohistochemistry was used to identify the cell phenotypes and the presence of CAFs in ascitic MCAs. The role of CAFs in tumor­cell MCA formation was assessed by co­culture in suspension. Primary ascitic tumor cells and omental CAFs were used to generate ex vivo MCAs in hanging drops, and the invasiveness of MCAs was evaluated by mesothelial clearance and adhesion assays in vitro and in vivo. MCAs containing CAFs and tumor cells were identified in the ascitic fluid. CAFs facilitated tumor cell aggregation and compaction to form MCAs, and enhanced the mesothelial clearance and adhesion abilities of tumor­cell MCAs. These findings suggest that ascitic CAFs promote peritoneal metastasis by forming heterotypic aggregates with tumor cells, and that they may serve as potential targets for the treatment of HGSOC.