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.

Ovarian Cancer Cells in Ascites Form Aggregates That Display a Hybrid Epithelial-Mesenchymal Phenotype and Allows Survival and Proliferation of Metastasizing Cells

Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Cancer cells float in peritoneal fluid, named ascites, together with a definitely higher number of non neo-neoplastic cells, as single cells or multicellular aggregates. The aim of this work is to uncover the features that make these aggregates the metastasizing units. Immunofluorescence revealed that aggregates are made almost exclusively of ovarian cancer cells expressing the specific nuclear PAX8 protein. The same cells expressed epithelial and mesenchymal markers, such as EPCAM and αSMA, respectively. Expression of fibronectin further supported a hybrid epithelia-mesenchymal phenotype, that is maintained when aggregates are cultivated and proliferate. Hematopoietic cells as well as macrophages are negligible in the aggregates, while abundant in the ascitic fluid confirming their prominent role in establishing an eco-system necessary for the survival of ovarian cancer cells. Using ovarian cancer cell lines, we show that cells forming 3D structures neo-expressed thoroughly fibronectin and αSMA. Functional assays showed that αSMA and fibronectin are necessary for the compaction and survival of 3D structures. Altogether these data show that metastasizing units display a hybrid phenotype that allows maintenance of the 3D structures and the plasticity necessary for implant and seeding into peritoneal lining.

PVT1, a YAP1 dependent stress responsive lncRNA drives ovarian cancer metastasis and chemoresistance

Metastatic growth of ovarian cancer cells into the peritoneal cavity requires adaptation to various cellular stress factors to facilitate cell survival and growth. Here we demonstrate the role of PVT1, one such stress induced long non-coding RNA, in ovarian cancer growth and metastasis. PVT1 is an amplified and overexpressed lncRNA in ovarian cancer with strong predictive value for survival and response to targeted therapeutics. We find that expression of PVT1 is regulated by ovarian tumor cells in response to cellular stress, particularly loss of cell-cell contacts and changes in matrix rigidity occurring in a YAP1 dependent manner. Induction of PVT1 promotes tumor cell survival, growth, and migration. Conversely, reducing PVT1 levels robustly abrogates metastatic behavior and tumor cell dissemination in cell lines and syngeneic transplantation models in vivo. We find that reducing PVT1 causes widespread transcriptome changes leading to alterations in cellular stress response and metabolic pathways including doxorubicin metabolism, which directly impacts chemosensitivity. Together, these findings implicate PVT1 as a promising therapeutic target to suppress metastasis and avoid chemoresistance in ovarian cancer.

Anticancer effect of 7-hydroxycoumarin in cisplatin-resistant ovarian cancer cell is mediated via apoptosis induction, caspase activation and cell cycle arrest at G2M phase

Purpose: To investigate the anticancer effects of 7-hydroxycoumarin against cisplatin-resistant ovarian cancer cell line, and the underlying mechanism(s). Methods: Cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The 4’,6-diamidino-2-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) dual staining methods were used for measuring cell apoptosis in terms of DNA damage. Flow cytometry was used for analysis of mitosis of cancer cells, while protein expression levels were assayed with western blotting. Results: The 7-hydroxycoumarin preferentially inhibited the proliferation of the ovarian cancer cells, but had significantly less prominent effects on normal cells (p < 0.05). The decrease in cell proliferation was due to induction of cell apoptosis via caspase-linked apoptotic pathway. Treatment with 7- hdoxycoumarin further led to the arrest of cancer cell cycle at G2/M stage (p < 0.05) via down-regulation of the expressions of regulatory proteins that promote mitotic entry. Conclusion: 7-Hydroxycoumarin exerts significant anticancer effect against cisplatin-resistant ovarian cancer cells via decrease in cell proliferation, induction of apoptosis and mitotic cell cycle arrest. Thus, the compound could emerge as a vital lead molecule in the treatment of cisplatin-resistant type of human ovarian cancer.

The metabolic stress-activated checkpoint LKB1-MARK3 axis acts as a tumor suppressor in high-grade serous ovarian carcinoma

High-grade serous ovarian carcinoma (HGSOC) is the most aggressive gynecological malignancy, resulting in approximately 70% of ovarian cancer deaths. However, it is still unclear how genetic dysregulations and biological processes generate the malignant subtype of HGSOC. Here we show that expression levels of microtubule affinity-regulating kinase 3 (MARK3) are downregulated in HGSOC, and that its downregulation significantly correlates with poor prognosis in HGSOC patients. MARK3 overexpression suppresses cell proliferation and angiogenesis of ovarian cancer cells. The LKB1-MARK3 axis is activated by metabolic stress, which leads to the phosphorylation of CDC25B and CDC25C, followed by induction of G2/M phase arrest. RNA-seq and ATAC-seq analyses indicate that MARK3 attenuates cell cycle progression and angiogenesis partly through downregulation of AP-1 and Hippo signaling target genes. The synthetic lethal therapy using metabolic stress inducers may be a promising therapeutic choice to treat the LKB1-MARK3 axis-dysregulated HGSOCs.

Wnt5A and TGFβ1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation

In this paper, we investigate whether Wnt5A is associated with the TGF-β1/Smad2/3 and Hippo-YAP1/TAZ-TEAD pathways, implicated in epithelial to mesenchymal transition (EMT) in epithelial ovarian cancer. We used 3D and 2D cultures of human epithelial ovarian cancer cell lines SKOV-3, OVCAR-3, CAOV-4, and different subtypes of human serous ovarian cancer compared to normal ovary specimens. Wnt5A showed a positive correlation with TAZ and TGFβ1 in high- and low-grade serous ovarian cancer specimens compared to borderline serous and normal ovaries. Silencing Wnt5A by siRNAs significantly decreased Smad2/3 activation and YAP1 expression and nuclear shuttling in ovarian cancer (OvCa) cells. Furthermore, Wnt5A was required for TGFβ1-induced cell migration and invasion. In addition, inhibition of YAP1 transcriptional activity by Verteporfin (VP) altered OvCa cell migration and invasion through decreased Wnt5A expression and inhibition of Smad2/3 activation, which was reverted in the presence of exogenous Wnt5A. We found that the activation of TGFβ1 and YAP1 nuclear shuttling was promoted by Wnt5A-induced integrin alpha v. Lastly, Wnt5A was implicated in activating human primary omental mesothelial cells and subsequent invasion of ovarian cancer cells. Together, we propose that Wnt5A could be a critical mediator of EMT-associated pathways.

Short-term starvation synergistically enhances cytotoxicity of Niraparib via Akt/mTOR signaling pathway in ovarian cancer therapy

Background Short-term starvation (STS) has gradually been confirmed as a treatment method that synergistically enhances the effect of chemotherapy on malignant tumours. In clinical applications, there are still some limitations of poly (ADP-ribose) polymerase inhibitors (PARPi), including understanding their effectiveness and side effects. Here, we sought to investigate the effect and mechanism of the combined use of STS and niraparib in the treatment of ovarian cancer. Methods In in vitro experiments, SKOV3 and A2780 ovarian cancer cells were treated with STS and niraparib alone or in combination. Cell viability was assessed with CCK-8, and cell cycle, apoptosis, DNA damage repair and autophagy were examined to explore the molecular mechanisms. Akt and mTOR inhibitors were used to examine any changes in DNA damage repair levels. Xenograft animal models were treated with STS and niraparib, and HE staining and immunohistochemistry were performed to examine the effects. Results The combined use of STS and niraparib inhibited cell proliferation and increased apoptosis more than niraparib application alone. In addition, compared with the niraparib group, the STS + niraparib group had increased G2/M arrest, DNA damage and autophagy, which indicated that STS pretreatment enhanced the cytotoxicity of niraparib. In animal experiments, STS did not affect the growth of transplanted tumours, but the combined treatment synergistically enhanced the cytotoxicity of niraparib. In in vivo experiments, STS did not affect the growth of transplanted tumours, but the combined treatment synergistically enhanced the cytotoxicity of niraparib and reduced the small intestinal side effects caused by niraparib chemotherapy. Conclusion STS pretreatment can synergistically enhance the cytotoxicity of niraparib. STS + niraparib is a potentially effective strategy in the maintenance therapy of ovarian cancer.

Enhanced Anti-Proliferative Effect of Carboplatin in Ovarian Cancer Cells Exploiting Chitosan – Poly (lactic glycolic acid) Nanoparticles.

Objective: The aim of the present article was to enhance the therapeutic efficacy of carboplatin (CP) using the formulation of chitosan – poly (lactic glycolic acid) nanoparticles (CS-PLGA NPs). Methods: Nanoparticles were synthesized by an ionic gelation method and were characterized for their morphology, particle size, and surface potential measurements by TEM and zeta sizer. This study was highlighted for the evaluation of drug entrapment, loading and in vitro drug release capabilities of the prepared nanoparticles by spectrophotometric analysis. The stability study was also conducted after 3 months for their particle size, zeta potential, drug loading and encapsulation efficiencies. Further, ovarian cancer cell line PEO1 were used to evaluate the toxicity and efficacy of nano-formulation by MTT assay. Further, the study was evaluated for apoptosis using flow cytometric analysis. Result: The CS-PLGA-CP NPs were uniform and spherical in shape. The particle size and zeta potential of CS-PLGA-CP NPs were measured 156 ± 6.8 nm and +52 ± 2.4 mV, respectively. High encapsulation (87.4 ± 4.5 %) and controlled retention capacities confirmed the efficiency of the prepared nanoparticles in a time and dose dependant manner. The cytotoxicity assay results also showed that CS-PLGA-CP NPs has high efficiency on PEO1 cells compared to the free drug. The flow cytometric result showed 64.25 % of the PEO1 cells were apoptotic and 8.42 % were necrotic when treated with CS-PLGA-CP NPs. Conclusion: Chitosan-PLGA combinational polymeric nanoparticles were not only steady but also non-toxic. Our experiments revealed that the chitosan- PLGA nanoparticles could be used as a challenging vehicle candidate for drug delivery for the therapeutic treatment of ovarian cancer.