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The effects of Î"b-Twist1 overexpression on spheroids formation and migration. (A) Representative images of spheroid forming assay. (B) Representative images of the migrated cells in migration assay. (C) Quantification of the migrated cells.*P< 0.05 compared to control group.

Supplementary Tables

The protein levels of Twist1 (A) and p53 (B) in tumor samples. Protein levels were determined by the quantification of western blot band intensity comparing to GAPDH bands as loading control.

Expression of p53-associated E3 ligase in cancer cell lines. (A) RNA expression of MDM2, Pirh2, and E6-AP1, and TOPORS assessed by QPCR in R182 (p53high/Twist-) and Tara R182 (p53low/Twist+) cells. GADPH was used as reference gene. (B) Protein expression of Pirh2 assessed by Western blot. β-actin was used as loading control.

TP53 mutation status and protein levels of p53 and Twist1 ovarian cancer cell lines. * u = unknown.

The tumor microenvironment of ovarian cancer is the peritoneal cavity wherein adipose tissue is a major component. The role of the adipose tissue in support of ovarian cancer progression has been elucidated in several studies from the past decades. The adipocytes, in particular, are a major source of factors, which regulate all facets of ovarian cancer progression such as acquisition of chemoresistance, enhanced metastatic potential, and metabolic reprogramming. In this review, we summarize the relevant studies, which highlight the role of adipocytes in ovarian cancer progression and offer insights into unanswered questions and possible future directions of research.

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.

Purpose: Cancer progression, invasiveness, and metastatic potential have been associated with the activation of the cellular development program known as epithelial-to-mesenchymal transition (EMT). This process is known to yield not only mesenchymal cells, but instead an array of cells with different degrees of epithelial and mesenchymal phenotypes with high plasticity, usually referred to as E/M hybrid cells. The characteristics of E/M hybrid cells, their importance in tumor progression, and the key regulators in the tumor microenvironment that support this phenotype are still poorly understood. Methods: In this study, we established an in vitro model of EMT and characterized the different stages of differentiation, allowing us to identify the main genomic signature associated with the E/M hybrid state. Results: We report that once the cells enter the E/M hybrid state, they acquire stable anoikis resistance, invasive capacity, and tumorigenic potential. We identified the hepatocyte growth factor (HGF)/c-MET pathway as a major driver that pushes cells in the E/M hybrid state. Conclusions: Herein, we provide a detailed characterization of the signaling pathway(s) promoting and the genes associated with the E/M hybrid state.

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.

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.

Epithelial ovarian cancer is the deadliest gynecologic cancer, due in large part to recurrent tumors. Recurrences tend to have metastasized, mainly in the peritoneal cavity and developed resistance to the first line chemotherapy. Key to the progression and ultimate lethality of ovarian cancer is the existence of extensive intra-tumoral heterogeneity (ITH). In this review, we describe the genetic and epigenetic changes that have been reported to give rise to different cell populations in ovarian cancer. We also describe at length the contributions made to heterogeneity by both linear and parallel models of clonal evolution and the existence of cancer stem cells. We dissect the key biological signals from the tumor microenvironment, both directly from other cell types in the vicinity and soluble or circulating factors. Finally, we discuss the impact of tumor heterogeneity on the choice of therapeutic approaches in the clinic. Variability in ovarian tumors remains a major barrier to effective therapy, but by leveraging future research into tumor heterogeneity, we may be able to overcome this barrier and provide more effective, personalized therapy to patients.

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.

Purpose: Mortality in ovarian cancer occurs in the setting of recurrent disease. Recurrent disease is known to arise from residual disease, which persists after the conclusion of standard of care, consequently rebuilding the tumor. Adipose tissue provides cancer cells with a unique microenvironment that can enhance survival by upregulating antiapoptosis genes or by decreasing drug bioavailability. Indeed, adipose-rich organs such as the omentum are not only preferential sites for metastasis but also main sites of residual disease in ovarian cancer. We hypothesized that targeting adipose-rich tissues could limit residual disease and prevent recurrence. Thus, the objective of this study was to develop effective drug delivery platforms that can specifically target residual disease in adipose-rich tissues. Methods: The delivery system is a nanoparticle platform composed of polymerized ursodeoxycholic acid (PUDCA) synthesized by esterification of UDCA monomer. Paclitaxel (PTX) or deep-infrared FL dye (DIR) was encapsulated to a final concentration of 24 ug or 5 mg per mg PUDCA, respectively. In vitro dose response studies were performed using mCherry+ human OCSC1-F2 ovarian cancer cells for 72 h. DMSO-resuspended PTX (DMSO-PTX) was used as control. In vivo studies were performed in athymic nude mice bearing i.p. tumors from mCherry+ OCSC1-F2 cells. For biodistribution studies, DIR-PUDCA was administered as one-time i.p. injection of 1.3 mg of DIR-PUDCA followed by ex vivo measurement of FL intensity after 14 days. DMSO-resuspended DIR (DMSO-DIR) was used as control. For efficacy studies, the groups were: (1) vehicle control; (2) taxol (PTX in Cremaphor; 12mg/kg) + DMSO-PTX (31.2 ug PTX); and (3) taxol + PUDCA-PTX. Treatment was administered i.p. twice a week for a total of 4 doses. Tumor burden and residual disease were determined by quantification of mCherry ROI FL area. Results: PUDCA-PTX demonstrated significantly improved efficacy both in vitro and in vivo. PUDCA-PTX demonstrated a six-fold reduction in in vitro GI50 compared to DMSO-PTX (23.04 nM PUDCA-PTX vs. 137.2 nM DMSO-PTX). Biodistribution studies showed delivery of DIR specifically to adipose-rich tissues (DIR mean FL intensity (MFI) for greater and lesser omentum = 76570±17808 vs. mesentery MFI = 22326±2956; p = 0.0297). Efficacy studies showed that PUDCA-PTX was significantly more effective in inhibiting tumor progression (p = 0.0191) and, more importantly, in preventing recurrent disease compared to DMSO-PTX. Finally, PUDCA-PTX was able to significantly reduce omental-residual disease compared to DMSO-PTX (p=0.049). Conclusion: We demonstrate for the first time that PUDCA is able to target residual disease localized in adipocyte-rich tissues and that this significantly delays tumor progression and prevents recurrence. These results underscore the value of i.p.-delivered PUDCA as a drug delivery platform for enhanced targeting of cancer cells within adipose-rich tissues and its value in the management of ovarian cancer patients. Citation Format: Roslyn Tedja, Ayesha B. Alvero, Jung Seok Lee, Mary Pitruzzello, Tarek Fahmy, Gil G. Mor. Specific targeting of adipose tissue-associated metastasis using bile salt nanoparticles [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 A01.

PURPOSE: Metastasis and carcinomatosis remains a major problem in ovarian cancer. Metastasis requires a process known as Epithelial-to-Mesenchymal Transition (EMT), where by epithelial cancer cells undergo dynamic changes to acquire a mesenchymal and invasive phenotype. Twist1 is a transcription factor with a central role in EMT. We previously showed that in epithelial ovarian cancer (EOC), Twist1 is constitutively ubiquitinated and targeted for proteasomal degradation and inhibition of Twist degradation is associated with mesenchymal phenotype and chemoresistance. Accordingly, identification of mechanisms that promote EMT by inhibiting the active degradation of Twist1 is critical for prevention of carcinomatosis. In this study, we report the identification of PKCα as a central regulator of Twist1. We identified 11 possible PKCα phosphorylation sites on Twist1. We show that Twist1 is a novel substrate of the kinase PKCα and that PKCα-induced Twist1 phosphorylation abrogates Twist1 ubiquitination leading to its stabilization and consequently EMT. METHODS: The following cell lines were used: (1) ovarian cancer cells lines OVCAR3 and OVCA432; (2) in-house developed cultures of ovarian cancer cells, R182 and R2615; and (3) HEK293T cells. The effect of constitutively active (PKCαCAT), dominant negative (PKCαDN), and wild-type (wt-PKCα) PKCα on levels of Twist1 were determined by transient transfection using qRT-PCT and western blot analysis. Levels of phosphorylated Twist1 were measured in Twist1 immunoprecipitate-complex using anti-phospho-serine/threonine/tyrosine antibody. The phospho-deficient mutant Twist1 S144A was constructed using QuickChange Site-directed Mutagenesis Kit. PKCα was knocked-out in ovarian cancer cells using CRISPR/Cas9. EMT was induced by treatment with 10 ng/ml TGFβ1 and confirmed molecularly (i.e. loss of epithelial markers E-cadherin, Ck18, Claudin 3 and gain of mesenchymal markers Twist1, N-cadherin, and vimentin). Intra-peritoneal tumors were established by injecting 10 million ovarian cancer cells in athymic nude mice. RESULTS: Transfection with PKCαCAT(active) resulted in a significant increase in Twist1 protein levels compared to PKCαDN(inactive) or empty vector control in EOC and HEK293T cells. This was not a transcriptional effect (no increase in Twist1 mRNA) but due to increase in the levels of phospho-serine/threonine/tyrosine on Twist1. We identified S144 as the putative PKCα phosphorylation site on Twist1 protein. Whereas wt-Twist1 was readily ubiquitinated when transfected in ovarian cancer cells and HEK293T, the phosphomimic S144D Twist1 demonstrated significantly less ubiquitination. Furthermore, we identified TGFβ1 as an activator of the endogenous PKCα-Twist1 axis in ovarian cancer cells. TGFβ1 is able to: (1) activate PKCα; (2) increase Twist1 protein levels; (3) and induce EMT (spheroid formation). Knock-out of PKCα in ovarian cancer cells abrogated TGFβ1-induced EMT in vitro and inhibit carcinomatosis in vivo in athymic nude mice model. CONCLUSION: We demonstrate for the first time a TGFβ1-PKCα-Twist1 signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization. This is a non-classical pathway of TGFβ1 induced EMT. Moreover, we identify S144 on Twist1 as novel and direct PKCα-phosphorylation site that can control Twist1 stability. Given the pleiotropic nature of TGFβ1 signaling, the identification of PKCα as a novel target may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation in ovarian cancer. Citation Format: Roslyn Tedja, Cai Roberts, Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Yang Yang-Hartwich, Carlotta Glackin and Gil G. Mor. PKCα INDUCES TWIST1 PHOSPHORYLATION AT SERINE 144 AND PROMOTES EMT IN OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-084.

Increasing use of titanium dioxide (TiO2) nanoparticles in many commercial applications has led to emerging concerns regarding the safety and environmental impact of these materials. In this study, we have investigated the biological impact of nano-TiO2 (with particle primary size of 20 nm Aeroxide P25) on human lung cell lines in vitro and also the effect of particle size distribution on the particle uptake and apparent toxicity. The biological impact of nano-TiO2 is shown to be influenced by the concentration and particle size distribution of the TiO2 and the impact was shown to differ between the two cell lines (A549 and H1299) investigated herein. A549 cell line was shown to be relatively resistant to the total amount of TiO2 particles uptaken, as measured by cell viability and metabolic assays, while H1299 had a much higher capacity to ingest TiO2 particles and aggregates, with consequent evidence of impact at concentrations as low as 30–150 μg/mL TiO2. Evidence gathered from this study suggests that both viability and metabolic assays (measuring metabolic and mitochondrial activities and also cellular ATP level) should be carried out collectively to gain a true assessment of the impact of exposure to TiO2 particles.

Background: Epithelial - mesenchymal transition (EMT) is a pre-requisite for cancer metastasis. Twist-1 is a transcription factor with a central role in the process of EMT and therefore metastasis formation. Using ovarian cancer models, our group has previously shown that although the stationary epithelial phenotype express Twist1 mRNA, this phenotype is enforced by mechanisms that constitutively ubiquitinates and degrades the Twist1 protein. The objective of this study is to identify molecular mechanisms that promote Twist1 protein stabilization and therefore confer a mesenchymal and mobile phenotype. Materials and methods: Novel phosphorylation sites on Twist1 protein, which are adjacent to ubiquitination sites, were identified using NetPhosK and Ubpred. Further analysis demonstrates that most of these sites contain the consensus sequence for PKCα. Constitutively active PKCα (PKCαcat) was ectopically expressed in HEK293T cells and patient-derived epithelial ovarian cancer cell lines. Protein expression and phosphorylation status were determined using western blot analysis using either whole cell lysates or cellular fractions. Protein-protein interaction was determined by immunoprecipiation. The specific Twist1 domain required for PKCα interaction was determined by co-transfection with Twist1 deletion mutants. Results: Ectopic expression of PKCαcat in HEK293T cells and ovarian cancer cells resulted in increased levels of Twist1 protein compared to empty vector control. In ovarian cancer cells, PKCcat-induced upregulation of Twist1 protein is not associated with an increase in Twist1 mRNA but instead is associated with Twist1 phosphorylation, decreased ubiquitination, and enhanced stabilization. We identify the twist box (WR) domain of Twist1 as a pre-requisite for PKCα binding and PKCαcat-induced Twist1 stabilization. Moreover, we identify TGFβ1 as a potent activator of endogenous PKCα in epithelial ovarian cancer cells. TGFβ1 (1 ng/ml) is able to: promote the activation and membranal translocation of PKCα; increase Twist1 protein levels; and induce spheroid formation and EMT in epithelial ovarian cancer cells. Interestingly, TGFβ1 had no effect on Twist1 mRNA suggesting that this mechanism is independent of the classical TGFβ-Smad pathway. Conclusions: We demonstrate for the first time a novel TGFβ-PKCα signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization resulting in EMT. This mechanism is independent of the classical TGFβ-Smad pathway that controls EMT via transcriptional regulation. Since TGFβ is a pleiotropic cytokine that can affect multiple cell types, the identification of PKCα as a novel target in ovarian cancer cells may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation. Citation Format: Roslyn Tedja, Ayesha B. Alvero, Carlos Cardenas, Mary Pitruzzello, Gang Yin, Yang Yang-Hartwich, Cai Roberts, Carlotta Glackin, Gil G. Mor. PKCα-induced Twist1 phosphorylation is a novel regulator of Twist1 stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1969. doi:10.1158/1538-7445.AM2017-1969

Background: Ovarian cancer is the most lethal of all gynecologic cancers. It is characterized by the presence of carcinomatosis at time of presentation and chemoresistant micrometastatic seedings at the time of recurrence. The omentum, an adipocyte-rich organ, is a frequent site of ovarian cancer metastasis in patients and the most common site of post-chemotherapy residual disease in animal models. Whereas previous studies have shown how the adipocyte microenvironment can induce metabolic re-programming in cancer cells, it is not clear if it can regulate pathways that affect chemoresponse. The objective of this study is to determine the effect of the adipocyte microenvironment on pathways that control cell cycle and apoptosis. Methods: In vitro: Patient-derived epithelial ovarian cancer cell lines were cultured in the presence of absence of omentum conditioned-media. Cell growth was determined by measuring culture confluence using IncucyteTM. Protein expression was determined by western blot analysis, and flow cytometry. In vivo: Human ovarian cancer xenografts were established intra-peritoneally in nude mice. Tumor implants from adipocyte-rich organs such as the omentum, pelvic fat, and mesentery and tumor implants from non-adipocyte-rich organs such as liver, ovaries, and GI tract were collected and analyzed independently. Results: Compared to cells cultured in growth media, epithelial ovarian cancer cells cultured in omentum CM demonstrated a more fibroblastic morphology characterized by elongated shape and bipolarity, significantly slower growth kinetics (p < 0.0001) accompanied by upregulation of the cell cycle inhibitor, p27, and enhanced secretion of the pro-angiogenic factor, VEGF. More importantly, these cells express higher levels of the anti-apoptotic proteins Bclxl and Mcl-1. Consequently, ovarian cancer cells in growth media treated with Carboplatin had a IC50 of 14.5 μg/ml. In contrast, cells cultured in omentum CM demonstrated enhanced resistance to carboplatin with IC50 of > 50 μg/ml. Analysis of tumor implants in vivo showed similar results as observed in vitro. Thus, tumor implants isolated from adipocyte-rich organs express higher levels of p27, Bclxl, and Mcl-1. Conclusion: We demonstrate in this study that the adipocyte microenvironment induces major changes in the phenotype of ovarian cancer cells characterized by morphological changes chemoresistance and growth rate. These findings highlight the importance of the adipocyte microenvironment in the progression of ovarian cancer. Further studies that can identify specific therapeutic targets in the adipocyte-educated chemoresistant ovarian cancer cells may aid in the development of novel therapies and improve patient survival. Citation Format: Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Roslyn Tedja, Gil G. Mor. The omentum promotes ovarian cancer cell survival by increasing cell cycle duration and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2017-1995

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.

A successful modification of titanium dioxide (TiO2) nanoparticles surfaces by a grafting-to polymer technique combining catalytic chain transfer and thiol–ene click chemistry is reported. Vinylic end functional polymers were first prepared by catalytic chain transfer polymerization (CCTP) using oligo(ethylene glycol) methacrylate as a monomer. The presence of vinylic end groups was then exploited to attach the polymers to thiol functionalized TiO2 nanoparticles via thiol–ene Michael nucleophilic reactions. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-infrared (ATR-IR), dynamic light scattering (DLS), and thermogravimetric analyses (TGA) were used to verify the successful modification of the TiO2 surface. The modified TiO2 nanoparticles were stable in cell culture media and formed smaller aggregates when compared to non-surface modified nanoparticles. Cellular toxicity of the hybrid TiO2–polymer particles towards human lung cell lines A549 and H1299 in vitro was evaluated. Results from one-dimensional gel electrophoresis show the presence of polymer layers around the particles affects the adsorption of protein onto the TiO2 surface. The reduction in particle aggregate size and changes to the particle surface chemistry, following polymer grafting, was found to reduce cellular uptake and diminish cytotoxicity for both human lung cell lines tested.