Genome-wide CRISPR/Cas9 library screen identifies PCMT1 as a critical driver of ovarian cancer metastasis

Background The development of lethal cancer metastasis depends on the dynamic interactions between cancer cells and the tumor microenvironment, both of which are embedded in the extracellular matrix (ECM). The acquisition of resistance to detachment-induced apoptosis, also known as anoikis, is a critical step in the metastatic cascade. Thus, a more in-depth and systematic analysis is needed to identify the key drivers of anoikis resistance. Methods Genome-wide CRISPR/Cas9 knockout screen was used to identify critical drivers of anoikis resistance using SKOV3 cell line and found protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) as a candidate. Quantitative real-time PCR (qRT-PCR) and immune-histochemistry (IHC) were used to measure differentially expressed PCMT1 in primary tissues and metastatic cancer tissues. PCMT1 knockdown/knockout and overexpression were performed to investigate the functional role of PCMT1 in vitro and in vivo. The expression and regulation of PCMT1 and integrin-FAK-Src pathway were evaluated using immunoprecipitation followed by mass spectrometry (IP-MS), western blot analysis and live cell imaging. Results We found that PCMT1 enhanced cell migration, adhesion, and spheroid formation in vitro. Interestingly, PCMT1 was released from ovarian cancer cells, and interacted with the ECM protein LAMB3, which binds to integrin and activates FAK-Src signaling to promote cancer progression. Strikingly, treatment with an antibody against extracellular PCMT1 effectively reduced ovarian cancer cell invasion and adhesion. Our in vivo results indicated that overexpression of PCMT1 led to increased ascites formation and distant metastasis, whereas knockout of PCMT1 had the opposite effect. Importantly, PCMT1 was highly expressed in late-stage metastatic tumors compared to early-stage primary tumors. Conclusions Through systematically identifying the drivers of anoikis resistance, we uncovered the contribution of PCMT1 to focal adhesion (FA) dynamics as well as cancer metastasis. Our study suggested that PCMT1 has the potential to be a therapeutic target in metastatic ovarian cancer.

Nimesulide derivatives reduced cell proliferation against breast and ovarian cancer: synthesis, characterization, biological assessment, and crystal structure.

New nimesulide derivatives (A1-A6) were synthesized and investigated by IR, 1H NMR, 13C NMR, melting point, elemental analysis, mass spectra, and DSC analysis. Agent A3 single crystal was grown and solved in a monoclinic crystal system with Cc. Heat shock protein 27 (HSP27) and tubulin are essential cellular proteins for normal cell division and growth. In addition, these proteins are expressed highly in cancer cells. Breast cancer (SKBR3) and ovarian cancer (SKOV3) cell lines are our models for biological assessment. The data revealed that nimesulide analogs showed high cytotoxicity when treated with SKBR3 cell line ranges from 0.22 µM to 12.0 µM, while SKOV3 cell line from 0.1 µM to 16.0 µM. In-depth, structure-activity relationship applied on nimesulide lead structure highlights the importance of a bulk moiety on position two that reduces cell proliferation in both cell lines.

Chitosan nanoparticle-mediated effect of antimiRNA-324-5p on decreasing the ovarian cancer cell proliferation by regulation of GLI1 expression

Introduction: MicroRNAs (miRNAs) are short-sequence RNAs that regulate gene expression by targeting messenger RNAs (mRNAs). Recent studies reveal that miRNA-324-5p plays an important role in worsening the ovarian cancer prognosis when the expression is very high. This study aimed to develop a miRNA targeted therapy by targeting the miRNA-324-5p function as a miRNA-324-5p inhibitor. Methods: Chitosan nanoparticles were used for antimiRNA-324-5p delivery into SKOV3 cell lines formulated by ionic gelation method. Antiproliferative effect of CS-NPs-antimiRNA was assessed by the MTT Assay. A mechanism study assessed the anticancer effect of the formula. In silico analysis used miRTar.Human and StarmiRDB combined with Genecard to predict the target genes of antimiR. Hawkdock web server was used to analyze protein-protein interactions that were further validated by quantitative polymerase chain reaction (qPCR). Results: The results of qPCR analysis showed endogenous miRNA-324-5p decreased after 24-hour transfection of antagonist miRNA. Furthermore, the MTT assay results showed that antimiRNA was able to inhibit SKOV3 cell proliferation (80 nM 68.13%, P<0.05). In silico analysis found miRNA-324-5p can regulate MEN1 and indirectly repress Gli1 mRNA. Validation results confirmed antimiR can decrease GLI1 mRNA expression. Conclusion: Our results showed antimiRNA-324-5p can act as a microRNA-based therapy to inhibit ovarian cancer proliferation by the reduction of GLI1 expression.

Long Non-Coding RNA F11-Antisense 1 (F11-AS1) Suppresses Ovarian Cancer Biological Activity by Regulating Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN)

Aim: To discuss F11-AS1’s effects and mechanisms in ovarian cancer development. Methods: Evaluating F11-AS1 expression by ISH assay and F11-AS1 mRNA level in difference cell lines by RT-qPCR assay. Using MTT, flow cytometry, transwell and wound healing assay to evaluate SKOV3 cell proliferation, cell apoptosis, invasion and migration. And using WB assay to measure PTEN, p-PI3K, AKT, P53 and MMP-9 proteins expressions. Results: F11-AS1 was significantly down-regulation with stage increasing in cancer tissues (P <0.01, respectively). With F11-AS1 transfection, the SKOV3 cell proliferation rate was significantly depressed with cell apoptosis and G1 phase rate significantly increasing (P <0.001, respectively). And then, invasion cell number and wound healing rate of lncRNA group which transfected with F11-AS1 significantly down-regulation (P <0.001). By WB assay, PTEN and P53 proteins expressions significantly up-regulation and p-PI3K, AKT and MMP-9 proteins expressions were significantly down-regulation (P <0.001). Conclusion: F11-AS1 depresses ovarian cancer biological activity by regulating PTEN by vitro study.

miR-486 Promotes the Invasion and Cell Cycle Progression of Ovarian Cancer Cells by Targeting CADM1

Objective. To explore the role and possible underlying mechanism of miR-486 in ovarian cancer (OC) cells. Methods. The expression of miR-486 and CADM1 was detected by qRT-PCR in OC tissues and adjacent nontumor tissues and OC cell lines. The dual-luciferase reporter gene system was used to determine the targeting relationship between miR-486 and CADM1. CCK-8, colony formation assay, Transwell, and flow cytometry were performed to detect cell proliferation, cell invasion, cell cycle progression, and the apoptotic cell death, respectively. Western blot was carried out to detect the expression of CADM1 protein and the proteins associated with cell cycle progression. Results. miR-486 was significantly upregulated in OC tissues and cells, while CADM1 expression was significantly downregulated. Dual-luciferase reporter assays further confirmed that CADM1 was a target gene of miR-486. Interference with miR-486 could inhibit the proliferation and invasion and promoted the apoptosis of SKOV3 cells. Knocking down both miR-486 and CADM1 significantly increased the SKOV3 cell proliferation, invasion, and the number of cells transitioning from the G0/G1 phase into the S phase of cell cycle and reduced the cellular apoptosis. Western blot analysis revealed that the expression of cell cycle progression-related proteins (CyclinD1, CyclinE, and CDK6) was significantly reduced, and the p21 expression was increased when interfering with both miR-486 and CADM1 expression. Conclusion. Our results suggested that miR-486 could act as a tumor promoter by targeting CADM1 and be a potential therapeutic target for the treatment of OC.

Oncogenic Role of miR-200c-3p in High-Grade Serous Ovarian Cancer Progression via Targeting the 3′-Untranslated Region of DLC1

High-grade serous ovarian cancer (HGSC) is the most common ovarian cancer with highly metastatic properties. A small non-coding RNA, microRNA (miRNA) was discovered to be a major regulator in many types of cancers through binding at the 3′-untranslated region (3′UTR), leading to degradation of the mRNA. In this study, we sought to investigate the underlying mechanisms involved in the dysregulation of miR-200c-3p in HGSC progression and metastasis. We identified the upregulation of miR-200c-3p expression in different stages of HGSC clinical samples and the downregulation of the tumor suppressor gene, Deleted in Liver Cancer 1 (DLC1), expression. Over expression of miR-200c-3p in HGSC cell lines downregulated DLC1 but upregulated the epithelial marker, E-cadherin (CDH1). Based on in silico analysis, two putative binding sites were found within the 3′UTR of DLC1, and we confirmed the direct binding of miR-200c-3p to the target binding motif at position 1488–1495 bp of 3′UTR of DLC1 by luciferase reporter assay in a SKOV3 cell line co-transfected with vectors and miR-200c-3p mimic. These data showed that miR-200c-3p regulated the progression of HGSC by regulating DLC1 expression post-transcription and can be considered as a promising target for therapeutic purposes.

P53 Gain-of-Function Mutants and Steroids in Ovarian Cancer Cell Metastasis

High-grade serous ovarian cancer (HGSOC) is a heterogeneous disease for which there currentlyis no cure. Because p53 is mutated in &gt;90% of all ovarian cancer, we studied specific gain-of-function (GOF) p53 mutants and steroid hormones for tumor morphology and metastasis in vivo. For this, we analyzed ALST (WT p53), SKOV3 (p53 null), TYK-NU (p53-R175H), OVCAR3 (p53-R248Q) and OVCA420 (p53-R273H) cell line xenografts in Foxn1-/- mice. ALST cells failed tometastasize, likely due to the known apoptotic effects of WT p53. SKOV3 and the p53-GOF celllines metastasized to the omentum and exhibited distinct morphologies: SKOV3, epithelial-like;TYK-Nu, vascular-like; OVCAR3, epithelial/mesenchymal; and OVCA420 epithelial exclusive. Despite different morphologies and p53 status, each tumor type contained large, Polyploid GiantCancer Cells (PGCCs) that are stem-like cells undergoing endoreplication. A specificphosphorylated, active form of β−catenin (pCTNNB1-S31/S37/T41; pCTNNB1) co-localizedselectively with GOF p53 and the mitotic stress regulatory kinase pMSK1-T581 in mitotic cells andPGCCs, indicating that in addition to GOF p53 mutants, pCTNNB1 and pMSK1 play a role intumor progression. To determine if ALST cells could be rendered metastatic, the p53 GOFmutants R175H and R273H were stably expressed in these cells. Remarkably, the ALST(WT/R273H) cells, but not the ALST (WT/R175H) cells, formed solid tumors on the ovary, visceralfat and uterus; but not on the omentum where OVCA420 (p53-R273H) cells formed tumors. TheALST (WT/R273H) tumors harbored discreet populations of pCTNNB1+ mitotic cells and PGCCsand exhibited distinct growth-promoting responses to estradiol while showing growth-inhibitoryeffects of DHT and nuclear AR in the tumor-associated stromal cells. Stably expressing p53-R175H or p53-R273H GOF mutants in SKOV3 (p53 null) cells enhanced tumor progression withthe R273H mutant being most aggressive. The parental SKOV3 tumors also contained pCTNNB1that was associated with mitotic cells and PGCCs. In the SKOV3 p53-R175H cells - and moreimpressively in the p53-R273H cells - pCTNNB1 co-localized with p53 in PGCCs. Whereas ESR1staining was diffuse in the ALST-R273H cells, it was nuclear in the SKOV3 cell lines. However,tumor promotion by estrogen and inhibition by DHT were observed in each SKOV3 cell line,suggesting that stromal cells may also contribute to the steroid dependent effects. Collectively, 1)elevated levels of specific phosphorylated forms of CTNNB1 and MSK1 identify mitotic cells andPGCCs within the tumors and new targets for therapeutic approaches; 2) knowing the status ofp53, and the presence and localization of pCTNNB1 and pMSK1 and steroid hormone receptorsin ovarian tumors suggest combinatorial approaches should be considered to combat the mostlethal gynecological cancer. NIH-CA181808; NIH-HD097321 (JSR).

Radix Tetrastigma Hemsleyani Flavone Inhibits the Occurrence and Development of Ovarian Cancer Cells by Regulating miRNA-4458 Expression

Ovarian cancer (OC) has been identified to have the highest mortality rate among gynecological tumors. Most patients are diagnosed at an advanced stage because of its asymptomatic nature and a lack of effective early diagnostic methods. Advanced-stage cancer cells are prone to metastasis which reduces the efficacy of standard therapies. Thus, we evaluated the effect of different concentrations of radix tetrastigma hemsleyani flavone (RTHF) on SKOV3 OC cells. Our findings indicated a significant inhibition in cell proliferation, migration, and invasion. RTHF treatment resulted in a significant increase in p21 protein expression, whereas the expression of cyclin D1, MMP-2, and MMP-9 has reportedly decreased. In addition, the expression of miRNA-4458 expression increased significantly in a dose-dependent manner. Co-transfection of miRNA-4458 mimics into SKOV3 cells revealed that overexpressed miRNA-4458 can increase SKOV3 cell proliferation and p21 protein expression. Reduced cell migration and invasion were also observed along with decreased expression of cyclin D1, MMP-2, and MMP-9. Furthermore, inhibition of miRNA-4458 expression reversed the RTHF effect on SKOV3 cell proliferation, migration, invasion, and cyclin D1, MMP-2, and MMP-9 expression. These results indicate that RTHF reduces the proliferation, migration, and invasion of OC cells, and the underlying mechanism is associated with the upregulation of miRNA-4458 expression. These findings provide a new treatment strategy for advanced OC.