PRKDC-Mediated NHEJ May Play a Crucial Role in Aneuploidy of Chromosome 8-Driven Progression of Ovarian Cancer.

High malignancy is a prominent characteristic of epithelial ovarian cancer (EOC), emphasizing the necessity for further elucidation of the potential mechanisms underlying cancer progression. Aneuploidy and copy number variation (CNV) partially contribute to the heightened malignancy observed in EOC; however, the precise features of aneuploidy and their underlying molecular patterns, as well as the relationship between CNV and aneuploidy in EOC, remain unclear. In this study, we employed single-cell sequencing data along with The Cancer Genome Atlas (TCGA) to investigate aneuploidy and CNV in EOC. The technique of fluorescence in situ hybridization (FISH) was employed using specific probes. The copy number variation within the genomic region of chromosome 8 (42754568-47889815) was assessed and utilized as a representative measure for the ploidy status of individual cells in chromosome 8. Differential expression analysis was performed between different subgroups based on chromosome 8 ploidy. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and hub-gene analyses were subsequently utilized to identify crucial genes involved. By classifying enriched tumor cells into distinct subtypes based on chromosome 8 ploidy combined with TCGA data integration, we identified key genes driving chromosome 8 aneuploidy in EOC, revealing that PRKDC gene involvement through the mediated non-homologous end-joining pathway may play a pivotal role in disease progression. Further validation through analysis of the GEO and TCGA database and survival assessment, considering both mRNA expression levels and CNV status of PRKDC, has confirmed its involvement in the progression of EOC. Further functional analysis revealed an upregulation of PRKDC in both ovarian EOC cells and tissues, with its expression showing a significant correlation with the extent of copy number variation (CNV) on chromosome 8. Taken together, CNV amplification and aneuploidy of chromosome 8 are important characteristics of EOC. PRKDC and the mediated NHEJ pathway may play a crucial role in driving aneuploidy on chromosome 8 during the progression of EOC.

Characterization of candidate factors associated with the metastasis and progression of high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is the biggest cause of gynecological cancer-related mortality because of its extremely metastatic nature. This study aimed to explore and evaluate the characteristics of candidate factors associated with the metastasis and progression of HGSOC. METHODS: Transcriptomic data of HGSOC patients' samples collected from primary tumors and matched omental metastatic tumors were obtained from three independent studies in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were selected to evaluate the effects on the prognosis and progression of ovarian cancer using data from The Cancer Genome Atlas (TCGA) database. Hub genes' immune landscapes were estimated by the Tumor Immune Estimation Resource (TIMER) database. Finally, using 25 HGSOC patients' cancer tissues and 10 normal fallopian tube tissues, immunohistochemistry (IHC) was performed to quantify the expression levels of hub genes associated with International Federation of Gynecology and Obstetrics (FIGO) stages. RESULTS: Fourteen DEGs, ADIPOQ , ALPK2 , BARX1 , CD37 , CNR2 , COL5A3 , FABP4 , FAP , GPR68 , ITGBL1 , MOXD1 , PODNL1 , SFRP2 , and TRAF3IP3 , were upregulated in metastatic tumors in every database while CADPS , GATA4 , STAR , and TSPAN8 were downregulated. ALPK2 , FAP , SFRP2 , GATA4 , STAR , and TSPAN8 were selected as hub genes significantly associated with survival and recurrence. All hub genes were correlated with tumor microenvironment infiltration, especially cancer-associated fibroblasts and natural killer (NK) cells. Furthermore, the expression of FAP and SFRP2 was positively correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage, and their increased protein expression levels in metastatic samples compared with primary tumor samples and normal tissues were confirmed by IHC ( P = 0.0002 and P = 0.0001, respectively). CONCLUSIONS: This study describes screening for DEGs in HGSOC primary tumors and matched metastasis tumors using integrated bioinformatics analyses. We identified six hub genes that were correlated with the progression of HGSOC, particularly FAP and SFRP2 , which might provide effective targets to predict prognosis and provide novel insights into individual therapeutic strategies for HGSOC.

Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer.

OBJECTIVE: Hematogenous metastasis is essential for the progression of ovarian cancer (OC), and circulating tumor cells (CTCs) are part of the metastatic cascade. However, the detection rate of CTC is low due to the use of less sensitive detection methods. Therefore, this study aimed to detect CTCs and circulating tumorigenic endothelial cells (CTECs) in patients with OC using subtraction enrichment and immunostaining and fluorescence in situ hybridization (SE-iFISH). METHODS: We enrolled a total of 56 subjects, including 20 OC patients and 36 ovarian benign tumor patients. CTCs and CTECs were captured by subtraction enrichment (SE) and counted and classified according to immunofluorescence staining of tumor markers (TMs) carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4) combined with fluorescence in situ hybridization (iFISH) of chromosome 8 (Chr8) aneuploidy. The diagnostic value and subtype characteristics of CTCs and CTECs were investigated. RESULTS: The detection rate of CTCs by SE-iFISH was high. Compared with CA125 and HE4, Chr8 aneuploidy was the major identification feature of CTC. CTC counts in OC were statistically higher than those in benign groups. CTC and CTEC with ≥pentaploidy were detected in both groups, illustrating the poor diagnostic value of CTC or CTEC. Distributions of triploid and tetraploid CTC subtypes were significantly different, and combined detection of triploid and tetraploid CTCs showed the best diagnostic value. In contrast, the distribution of CTECs in the OC and benign groups had no statistically significant difference. Small CTCs accounted for over 1/3 of the total CTC count. We also found that small CTCs and CTECs primarily comprised triploid cells, while large CTCs and CTECs mainly comprised pentaploidy and beyond. CONCLUSIONS: The application of SE-iFISH offered a more comprehensive understanding of heterogeneous CTCs and CTECs in OC. Analysis of subclass characteristics of the CTCs and CTECs according to Chr8 aneuploidy and cell size may broaden their potential clinical utility and deepen mechanistic studies in OC.

Oncogenic Role of NUPR1 in Ovarian Cancer.

BACKGROUND: Nuclear protein 1 (NUPR1) plays a critical role in the development and progression of various types of human cancers. However, the role and mechanism of NUPR1 in ovarian cancer have not been elucidated. The purpose of this study was to investigate the effect of NUPR1 on ovarian cancer in vivo and in vitro. MATERIALS AND METHODS: Through the pretreatment of ovarian cancer cell lines, including A2780 and SKOV3 cells, the expression of NUPR1 was detected by RT-PCR and Western blot assays. When NUPR1 was overexpressed and knocked down in A2780 cells and overexpressed in SKOV3 cells, the MTT assays, colony formation assays and EdU assays were used to detect cell proliferation. Furthermore, cell invasion and migration ability were detected with the transwell assays. Cell cycle and apoptosis of A2780 cells after small interfering RNA-NUPR1 (siRNA-NUPR1) were detected by flow cytometry assays. Finally, the effect of NUPR1 gene silencing on the growth of ovarian cancer was evaluated by tumor xenograft experiment in vivo. RESULTS: The expression of NUPR1 protein in A2780 cells was significantly higher than that in ovarian surface epithelium (OSE) cells (P < 0.05). The results showed that downregulation of NUPR1 gene expression significantly inhibited the proliferation, migration and invasion ability of A2780 cells, and increased apoptosis of A2780 cells, which expressed relatively high levels of NUPR1. And the expression of apoptosis-related proteins caspase 3, caspase 9 and Bax was upregulated when NUPR1 was knocked out, while the expression of anti-apoptotic proteins of Bcl-2 and Bcl-xl was downregulated. At the same time, the opposite results were observed when NUPR1 was overexpressed in A2780 and SKOV3 cells. Notably, the effect of NUPR1 overexpression in A2780 cells could be partially or completely eliminated by treatment with the AKT inhibitor LY294002. In addition, NUPR1 knockdown could effectively inhibit tumor growth of mice in vivo. CONCLUSION: In summary, NUPR1 has a carcinogenic effect in ovarian cancer, and the oncogenic effect of NUPR1 in ovarian cancer may be achieved by the AKT pathway.

Effects of tumor origins and therapeutic options on the prognosis of hepatic neuroendocrine tumors: A retrospective study.

ABSTRACT: Hepatic neuroendocrine tumors (HNETs) are uncommon neoplasms that can be subdivided into 2 types: primary and metastatic HNETs. Due to its rarity, heterogeneity and complexity, the diagnosis, treatment modalities and prognosis are still controversial.This retrospective study reviewed the effects of tumor origins and therapeutic options on the prognosis of gastroenteropancreatic neuroendocrine tumors with liver metastasis (GEP-NETLM) and primary hepatic neuroendocrine tumors (PHNETs), providing additional evidence for clinicians evaluating patients.HNETs consisted of PHNETs and GEP-NETLM. GEP-NETLM (76.2%, 112/147) was more common, which was mainly manifested as multiple lesions in both lobes of the liver. PHNETs were relatively rare (23.8%, 35/147) and were mainly single lesion located in the right lobe of the liver. In patients with GEP-NETLM, primary tumor resection could prolong survival (P = .044). As the most widely used treatment method, systematic therapy alone could not achieve a satisfactory survival. However, the combination with hepatectomy or liver-directed therapy improved the prognosis (P = .023). As the main treatment, patients with PHNETs treated with local therapy could achieve a better prognosis (P = .049). Compared with PHNETs patients, GEP-NETLM patients with higher ki-67 index showed higher mortality and poorer prognosis (P = .006).Therefore, patients with PHNETs can be distinguished from GEP-NETLM by comprehensive imaging examinations and long-term follow-ups. The choice of appropriate treatment strategies can improve the prognosis of HNETs patients.

Long non-coding RNA PTPRG-AS1 promotes cell tumorigenicity in epithelial ovarian cancer by decoying microRNA-545-3p and consequently enhancing HDAC4 expression.

BACKGROUND: Long non-coding RNA PTPRG antisense RNA 1 (PTPRG-AS1) deregulation has been reported in various human malignancies and identified as an important modulator of cancer development. Few reports have focused on the detailed role of PTPRG-AS1 in epithelial ovarian cancer (EOC) and its underlying mechanism. This study aimed to determine the physiological function of PTPRG-AS1 in EOC. A series of experiments were also performed to identify the mechanisms through which PTPRG-AS1 exerts its function in EOC. METHODS: Reverse transcription-quantitative polymerase chain reaction was used to determine PTPRG-AS1 expression in EOC tissues and cell lines. PTPRG-AS1 was silenced in EOC cells and studied with respect to cell proliferation, apoptosis, migration, and invasion in vitro and tumor growth in vivo. The putative miRNAs that target PTPRG-AS1 were predicted using bioinformatics analysis and further confirmed in luciferase reporter and RNA immunoprecipitation assays. RESULTS: Our data verified the upregulation of PTPRG-AS1 in EOC tissues and cell lines. High PTPRG-AS1 expression was associated with shorter overall survival in patients with EOC. Functionally, EOC cell proliferation, migration, invasion in vitro, and tumor growth in vivo were suppressed by PTPRG-AS1 silencing. In contrast, cell apoptosis was promoted by loss of PTPRG-AS1. Regarding the mechanism, PTPRG-AS1 could serve as a competing endogenous RNA in EOC cells by decoying microRNA-545-3p (miR-545-3p), thereby elevating histone deacetylase 4 (HDAC4) expression. Furthermore, rescue experiments revealed that PTPRG-AS1 knockdown-mediated effects on EOC cells were, in part, counteracted by the inhibition of miR-545-3p or restoration of HDAC4. CONCLUSIONS: PTPRG-AS1 functioned as an oncogenic lncRNA that aggravated the malignancy of EOC through the miR-545-3p/HDAC4 ceRNA network. Thus, targeting the PTPRG-AS1/miR-545-3p/HDAC4 pathway may be a novel strategy for EOC anticancer therapy.

ROR2 induces cell apoptosis via activating IRE1α/JNK/CHOP pathway in high-grade serous ovarian carcinoma in vitro and in vivo.

BACKGROUND: Epithelial ovarian cancer (EOC) is the most lethal cancer in female genital tumors. New disease markers and novel therapeutic strategies are urgent to identify considering the current status of treatment. Receptor tyrosine kinases family plays critical roles in embryo development and disease progression. However, ambivalent research conclusions of ROR2 make its role in tumor confused and the underlying mechanism is far from being understood. In this study, we sought to clarify the effects of ROR2 on high-grade serous ovarian carcinoma (HGSOC) cells and reveal the mechanism. METHODS: Immunohistochemistry assay and western-blot assay were used to detect proteins expression. ROR2 overexpression adenovirus and Lentivirus were used to create ROR2 overexpression model in vitro and in vivo, respectively. MTT assay, colony formation assay and transwell assay were used to measure the proliferation, invasion and migration ability of cancer cells. Flow cytometry assay was used to detect cell apoptosis rate. Whole transcriptome analysis was used to explore the differentially expressed genes between ROR2 overexpression group and negative control group. SiRNA targeted IRE1α was used to knockdown IRE1α. Kira6 was used to inhibit phosphorylation of IRE1α. RESULTS: Expression of ROR2 was significantly lower in HGSOC tissues compared to normal fallopian tube epithelium or ovarian surface epithelium tissues. In HGSOC cohort, patients with advanced stages or positive lymph nodes were prone to express lower ROR2. Overexpression of ROR2 could repress the proliferation of HGSOC cells and induce cell apoptosis. RNA sequencing analysis indicated that ROR2 overexpression could induce unfold protein response. The results were also confirmed by upregulation of BIP and phosphorylated IRE1α. Furthermore, pro-death factors like CHOP, phosphorylated JNK and phosphorylated c-Jun were also upregulated. IRE1α knockdown or Kira6 treatment could reverse the apoptosis induced by ROR2 overexpression. Finally, tumor xenograft experiment showed ROR2 overexpression could significantly repress the growth rate and volume of transplanted tumors. CONCLUSIONS: Taken together, ROR2 downregulation was associated with HGSOC development and progression. ROR2 overexpression could repress cell proliferation and induce cell apoptosis in HGSOC cells. And the underlying mechanism might be the activation of IRE1α/JNK/CHOP pathway induced by ROR2.

Akt/mTOR-Mediated Autophagy Confers Resistance To BET Inhibitor JQ1 In Ovarian Cancer.

BACKGROUND: Bromodomain and extra-terminal domain inhibitors like JQ1 have proved to be promising epigenetic agents for the treatment of malignant ovarian carcinoma. However, the resistance of ovarian cancer cells to BET inhibitors has not been elucidated. In this study, we investigated the potential mechanisms underlying the resistance of ovarian cancer cell lines to the BET inhibitor JQ1. MATERIALS AND METHODS: We evaluated the apoptotic and proliferative response of four ovarian cancer cell lines to JQ1. The cell lines were designated as resistant (A2780 and HO-8910) and sensitive groups (SKOV-3 and HEY). Further experiments detected the different levels of JQ1-induced autophagy. Anti-tumour effect of the combination of JQ1 and autophagy inhibitors was tested both in vitro and in vivo. RESULTS: In the JQ1-sensitive group, JQ1 effectively inhibited proliferation and apoptosis in a concentration-dependent manner. Conversely, JQ1 showed modest inhibition of proliferation and negligible apoptosis in the resistant group. We detected increased LC3-II lipidation, autophagosome formation, upregulation of Beclin-1 and ATG5, and downregulation of P62/SQSTM1 in the resistant group. Inhibition of JQ1-induced autophagy by pharmacologic inhibitors 3-MA and CQ enhanced the inhibition of proliferation and significantly increased the apoptosis in the JQ1-resistant group, which was also verified by in vivo experiments, indicating that JQ1-induced autophagy played a cytoprotective role. Inactivation of Akt (Ser473)/mTOR(Ser2448) pathway was associated with JQ1-induced autophagy in the resistant group. Overexpression of Akt1 suppressed autophagy and increased the anti-tumour effect of JQ1. CONCLUSION: These findings revealed that JQ1-induced pro-survival autophagy might be a potential mechanism in the resistance of ovarian cancer cells to BET inhibition by JQ1. Combination of JQ1 and autophagy inhibitors could be an effective therapeutic strategy for overcoming BET inhibitor resistance in ovarian cancer.

Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways.

BACKGROUND Hypoxia induces cell apoptosis in the uterosacral ligaments of patients with pelvic organ prolapse by upregulation of hypoxia-inducible factor-1α (HIF-1α). This study aimed to investigate the effects of HIF-1α on human uterosacral ligament fibroblasts (hUSLFs) following treatment with the chemical inducer of hypoxia, cobalt chloride (CoCl2), and to explore the underlying mechanisms. MATERIAL AND METHODS Ten women who underwent hysterectomy for benign disease provided uterosacral ligament tissue for cell extraction. Following CoCl2 treatment, cell viability of isolated and cultured hUSLFs was evaluated by the MTT assay. JC-1 fluorescence mitochondrial imaging was used to study the change in mitochondrial membrane potential. Cell apoptosis and expression of apoptosis-associated proteins and collagen type I alpha 1 (COL1A1) were measured by flow cytometry, TUNEL and Western blot, respectively. RESULTS Hypoxia increased the expression of HIF-1a and increased cell apoptosis, decreased cell viability and expression levels of COL1A1. The JC-1 assay showed that the mitochondrial membrane potential was reduced and caspase-8, and -9 inhibitors partly reduced hUSLF apoptosis. HIF-1α treatment downregulated the expression of cellular FLICE inhibitory protein (c-FLIP), decoy receptor 2 (DcR2), and the ratio of Bcl-2 to Bax, and upregulated the expression tumor necrosis factor related apoptosis-inducing ligand (TRAIL), death receptor 5 (DR5) or TRAIL-R2, Fas, Bcl-2 interacting protein 3 (BNIP3), and cytochrome C, and increased the activation of caspase-3, caspase-8, and caspase-9, all of which were reversed by knockdown of HIF-1α. CONCLUSIONS HIF-1α significantly induced apoptosis of hUSLFs through both the cell death receptor and the mitochondrial-associated apoptosis pathways.