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Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully re-acquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally non-functional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a sub-classification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.

Epithelial–mesenchymal transition (EMT) is a critical process involved in cancer metastasis and chemoresistance. Twist1 is a key EMT-inducing transcription factor, which is upregulated in multiple types of cancers and has been shown to promote tumor cell invasiveness and support tumor progression. Conversely, p53 is a tumor suppressor gene that is frequently mutated in cancers. This study demonstrates the ability of wild-type (WT) p53 to promote the degradation of Twist1 protein. By forming a complex with Twist1 and the E3 ligase Pirh2, WT p53 promotes the ubiquitination and proteasomal degradation of Twist1, thus inhibiting EMT and maintaining the epithelial phenotype. The ability of p53 to induce Twist1 degradation is abrogated when p53 is mutated. Consequently, the loss of p53-induced Twist1 degradation leads to EMT and the acquisition of a more invasive cancer phenotype. Implication: These data provide new insight into the metastatic process at the molecular level and suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to curtail cancer progression.

Anoikis is a type of programmed cell death triggered by the loss of cellular interaction with the extracellular matrix (ECM) and culminates in the activation of caspases. Specific interaction between cellular receptors such as integrins and the ECM is important to maintain cellular homeostasis in normal tissues through multiple cascades. This interaction provides not only physical attachment, but more importantly, vital interaction with the actin cytoskeleton and growth factors. Normal epithelial and endothelial cells require this interaction with ECM to survive. In cancer, the acquisition of anoikis resistance is a hallmark of malignant transformation and is required in the process of metastasis formation. As such, strategies to inhibit and/or counteract anoikis resistance are important in controlling cancer progression. In this chapter, we describe the method for detecting anoikis using cell viability and caspase activity assays.

Background: Metabolic pathways are non-canonical targets of the EMT program. The relevance of re-programming metabolism during the metastasis cascade is underscored in ovarian cancer cells, which primarily metastasizes to the adipocyte-rich omentum. Ovarian cancer-adipocyte interaction has been shown to promote fatty acid (FA) uptake into the cancer cells. These FA are then catabolized via β-oxidation thus providing the energy required for rapid tumor growth. β-oxidation of FA requires efficient mitochondria and oxygen processing. The objective of this study is to determine if E/M status determine adaptation and survival in adipocyte-rich microenvironment. Materials and Methods: RNA seq was performed in Ck18+/Twist- epithelial and Ck18-/Twist+ mesenchymal human ovarian cancer cells. Pathway and Gene Ontology analyses were performed on differentially expressed (DEX) genes (FC>2 and p Results: Pathway analysis of DEX genes with focus on metabolic pathways showed the following DEX pathways: glycolysis/gluconeogenesis (p=0.008); pyruvate metabolism (0.019); fatty acid degradation (p=0.01); fatty acid elongation (0.008). Gene ontology analysis showed significant difference in GO:0006979: response to oxidative stress (p=0.007). Further analysis of DEX genes within these pathways showed upregulation in mesenchymal cancer cells of both pyruvate dehydrogenase subunits, PDHA1 (p = 0.011) and PDHB (p=0.014) and upregulation of carnitine palmitoyltransferase (CPT1; p=0.011), the rate limiting enzyme in FA β-oxidation. The upregulation of these genes was validated at the protein level by western blot analysis, which also showed increased levels of COX-IV in mesenchymal cancer cells compared to epithelial cancer cells. Moreover, mesenchymal cells exhibited enhanced maximal respiration and enhanced spare respiratory capacity. Finally, viability studies conclusively demonstrate the metabolic flexibility in mesenchymal cancer cells, which maintained viability upon nutrient deprivation suggesting metabolic plasticity. In contrast, cell death was observed epithelial cancer cells upon nutrient deprivation, which was rescued by the addition of glucose but not the FA, oleic acid. Conclusion: Ovarian cancer cells acquire enhanced pyruvate shuttling to the TCA cycle, a more efficient FA β-oxidation machinery, as well as more efficient oxygen processing during EMT. These phenotypes are relevant in supporting survival in the adipocyte microenvironment. Further elucidation of EMT related pathways that re-program lipid metabolism may lead to better control of carcinomatosis in ovarian cancer patients. Citation Format: Hussein Chehade, Alexandra Fox, Roslyn Tedja, Radhika Gogoi, Gil Mor, Ayesha B. Alvero. EMT programs ovarian cancer cells to survive the adipocyte-rich microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2333.

Introduction: Ovarian cancer metastasizes via the transcoelomic route and yields overwhelmingly numerous micrometastases. This clinical presentation limits the value of standard of care and contributes to patient mortality. Whereas EMT is required in the acquisition of a migratory phenotype, MET has been shown to occur on secondary sites leading to the establishment of metastasis. The nature of the ovarian cancer cells that result after MET is, however, yet to be fully characterized. Whether these secondary epithelial ovarian cancer cells (sEOC) are required to repeat EMT in order to generate additional metastatic sites is not known. The objectives of this study are twofold: to determine if full MET occurs on secondary sites, and to determine mechanisms that confer to disseminated ovarian cancer cells the ability to rapidly spread and form the characteristic micrometastatic disease. Materials and Methods: Ovarian cancer spheroids were derived from three epithelial ovarian cancer (EOC) cell lines (OVCAR3, R182, R2615). sEOC were derived from the spheroids by plating in tissue culture-treated plates. Gene signature pattern was determined by RNAseq. mRNA and protein levels were quantified by RT-qPCR and Western blot, respectively. TWIST-1 activity was measured by a luciferase reporter system. Promoter binding was determined by anti-CBX7 chromatin IP followed by PCR. CBX7 was knocked out (KO) using CRISPR-Cas9. Tumorigenic potential was quantified by measuring resulting intraperitoneal burden (g) in nude mice. Results: PCA and clustering analysis of RNA-seq data showed sEOC do not fully recapitulate the molecular signature of the primary EOC cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintain a high expression of the classical mesenchymal effector, TWIST-1. TWIST-1 is, however, transcriptionally inactive in these cells as it is inhibited by the PcG protein, CBX7. KO of CBX7 in sEOC was sufficient to promote a mesenchymal morphology, significantly promote TWIST-1-induced transcription of mir-199A (p = 0.004, wt vs. CBX7KO), and induce higher protein levels of FOXC2. CBX7 precludes TWIST-1 from accessing its promoter by binding at or near the E box. Finally, KO of CBX7 was able to significantly enhance tumorigenicity and increase tumor load and metastatic potential in vivo (p=0.033, wt vs CBX7 KO). Conclusion: We demonstrate that full MET does not occur in ovarian cancer cells at secondary metastatic sites. Although sEOC display an epithelial morphology, they do not achieve the full molecular signature of the primary EOC cells from which they are derived. sEOC maintain high levels of TWIST-1 whose activity is inhibited by CBX7. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage to achieve a prompt reversal to a mesenchymal morphology and gain in tumorigenic potential upon perturbation of CBX7. This may explain ovarian cancer’s rapid progression and characteristic micrometastatic disease. Citation Format: Juanni Li, Ayesha B Alvero, Sudhakar Nuti, Roslyn Tedja, Cai M. Roberts, Mary Pitruzzello, Yimin Li, Qing Xiao, Sai Zhang, Yaqi Gan, Gil Mor, Gang Yin. CBX7 binds TWIST-1’s E-box to inhibit TWIST-1 function and curtail tumorigenecity and metastatic potential in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A02.

BACKGROUND: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism underlying metastasis is still not clear. Twist1 is a transcriptional factor that promotes epithelial-mesenchymal transition (EMT) and enhances the migration and tumor-initiation of ovarian cancer cells. It also has been associated with cancer stem cells and chemoresistance. The purpose of our study is to better understand the regulation of Twist1 and to identify molecular pathways that can be targeted in order to control metastatic diseases. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We hypothesize that through their interaction, p53 promotes the proteasomedependent degradation of Twist1 and consequently inhibits EMT in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. METHOD: In ubiquitin assay p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Coimmunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Plasmid constructs containing different Twist1 mutants were created to select the degradation resistant mutation. ΔC-Twist1 (C-terminal deletion) was used in in vivo study. RESULT: 1) In EOC cells, proteasome-dependent degradation suppressed Twist1 protein. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In ovarian cancer cells, wild type p53 overexpression reduced Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to enhance Twist1 degradation. E3 ligase, Pirh2, formed complex with p53 and Twist1 to induce Twist1 degradation. 2) ΔC-Twist1 was resistant to p53-regulated degradation. In xenograft mouse model, EOC cells overexpressing ΔC-Twist1 could form more aggressive tumors with increased numbers of metastatic lesions than wild type Twist1 group. 3) In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. Nine samples with high levels of wild type p53 all showed low or no Twist1 expression. CONCLUSION: Our data revealed a mechanism by which Twist1 is regulated through p53-promoted proteasome-dependent degradation. p53 facilitates the formation of a Twist1-p53-Pirh2 complex and the Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP16.

Background: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism behind ovarian cancer metastasis is still not clear. Twist1 is associated with the increased ability of migration and tumor-initiation of ovarian cancer cells; therefore, understanding the regulation of Twist is critical for the prevention of metastatic disease. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We propose that p53 functions as a normal inhibitor of Twist1. We hypothesize that through their interaction, p53 promotes the proteasome-dependent degradation of Twist1 and consequently inhibits epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis. Method: p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Co-immunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Result: Wild type p53 overexpression reduces Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to regulate Twist1 degradation. E3 ligase, Pirh2, forms complex with p53 and Twist1 to induce Twist1 degradation. In EOC cells, Twist1 is suppressed by proteasome-dependent degradation. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. The 9 samples with high levels of wild type p53 all showed low or no Twist1 expression. Conclusion: Our data revealed a mechanism by which a crucial EMT inducer, Twist1, is regulated by wild type p53 through enhancing its proteasome-dependent degradation. p53 promotes the formation of a Twist1-p53-Pirh2 complex and facilitates Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-352.

Exposure to fetal bovine serum (FBS) is shown herein to reduce the aggregate size of titanium dioxide (TiO(2)) nanoparticles, affecting uptake and consequent effect on A549 and H1299 human lung cell lines. Initially, the cellular uptake of the FBS-treated TiO(2) was lower than that of non-FBS-treated TiO(2). Expulsion of particles was then observed, followed by a second phase of uptake of FBS-treated TiO(2), resulting in an increase in the cellular content of FBS-treated TiO(2), eventually exceeding the amount by cells exposed to non-FBS-treated TiO(2). Surface adsorbed vitronectin and the clathrin-mediated endocytosis pathway were shown to regulate the uptake of TiO(2) into A549 cells, while the endocytosis mechanism responsible remains elusive for H1299. Intriguingly, nystatin treatment was shown to have the unexpected effect of increasing nanoparticle uptake into the A549 cells via an alternate endocytic pathway. The surface adsorbed serum components were found to provide some protection from the cytotoxic effect of endocytosed TiO(2) nanoparticles.