Adipose microenvironment promotes hypersialylation of ovarian cancer cells.

Sialylation, the addition of negatively charged sialic acid sugars to terminal ends of glycans, is upregulated in most cancers. Hypersialylation supports multiple pro-tumor mechanisms such as enhanced migration and invasion, resistance to apoptosis and immune evasion. A current gap in knowledge is the lack of understanding on how the tumor microenvironment regulates cancer cell sialylation. The adipose niche is a main component of most peritoneal cancers' microenvironment. This includes ovarian cancer (OC), which causes most deaths from all gynecologic cancers. In this report, we demonstrate that the adipose microenvironment is a critical regulator of OC cell sialylation. In vitro adipose conditioning led to an increase in both ⍺2,3- and ⍺2,6-linked cell surface sialic acids in both human and mouse models of OC. Adipose-induced sialylation reprogramming was also observed in vivo from intra-peritoneal OC tumors seeded in the adipose-rich omentum. Mechanistically, we observed upregulation of at least three sialyltransferases, ST3GAL1, ST6GAL1 and ST3GALNAC3. Hypersialylated OC cells consistently formed intra-peritoneal tumors in both immune-competent mice and immune-compromised athymic nude mice. In contrast, hyposiaylated OC cells persistently formed tumors only in athymic nude mice demonstrating that sialylation impacts OC tumor formation in an immune dependent manner. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancers.

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Adipose-derived exosomal miR-421 targets CBX7 and promotes metastatic potential in ovarian cancer cells.

BACKGROUND: Chromobox protein homolog 7 (CBX7), a member of the Polycomb repressor complex, is a potent epigenetic regulator and gene silencer. Our group has previously reported that CBX7 functions as a tumor suppressor in ovarian cancer cells and its loss accelerated formation of carcinomatosis and drove tumor progression in an ovarian cancer mouse model. The goal of this study is to identify specific signaling pathways in the ovarian tumor microenvironment that down-regulate CBX7. Given that adipocytes are an integral component of the peritoneal cavity and the ovarian tumor microenvironment, we hypothesize that the adipose microenvironment is an important regulator of CBX7 expression. RESULTS: Using conditioned media from human omental explants, we found that adipose-derived exosomes mediate CBX7 downregulation and enhance migratory potential of human ovarian cancer cells. Further, we identified adipose-derived exosomal miR-421 as a novel regulator of CBX7 expression and the main effector that downregulates CBX7. CONCLUSION: In this study, we identified miR-421 as a specific signaling pathway in the ovarian tumor microenvironment that can downregulate CBX7 to induce epigenetic change in OC cells, which can drive disease progression. These findings suggest that targeting exosomal miR-421 may curtail ovarian cancer progression.

Adipose-derived exosomal miR-421 targets CBX7 and promotes metastatic potential in ovarian cancer cells.

Background: Chromobox protein homolog 7 (CBX7), a member of the Polycomb repressor complex, is a potent epigenetic regulator and gene silencer. Our group has previously reported that CBX7 functions as a tumor suppressor in ovarian cancer cells and its loss accelerated formation of carcinomatosis and drove tumor progression in an ovarian cancer mouse model. The goal of this study is to identify specific signaling pathways in the ovarian tumor microenvironment that down-regulate CBX7. Given that adipocytes are an integral component of the peritoneal cavity and the ovarian tumor microenvironment, we hypothesize that the adipose microenvironment is an important regulator of CBX7 expression. Results: Using conditioned media from human omental explants, we found that adipose-derived exosomes mediate CBX7 downregulation and enhance migratory potential of human ovarian cancer cells. Further, we identified adipose-derived exosomal miR-421 as a novel regulator of CBX7 expression and the main effector that downregulates CBX7. Conclusion: In this study, we identified miR-421 as a specific signaling pathway in the ovarian tumor microenvironment that can downregulate CBX7 to induce epigenetic change in OC cells, which can drive disease progression. These findings suggest that targeting exosomal miR-421 may curtail ovarian cancer progression.

A Novel Role of Connective Tissue Growth Factor in the Regulation of the Epithelial Phenotype.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells lose their adhesive properties and gain invasive, metastatic, and mesenchymal properties. Maintaining the balance between the epithelial and mesenchymal stage is essential for tissue homeostasis. Many of the genes promoting mesenchymal transformation have been identified; however, our understanding of the genes responsible for maintaining the epithelial phenotype is limited. Our objective was to identify the genes responsible for maintaining the epithelial phenotype and inhibiting EMT. METHODS: RNA seq was performed using an vitro model of EMT. CTGF expression was determined via qPCR and Western blot analysis. The knockout of CTGF was completed using the CTGF sgRNA CRISPR/CAS9. The tumorigenic potential was determined using NCG mice. RESULTS: The knockout of CTGF in epithelial ovarian cancer cells leads to the acquisition of functional characteristics associated with the mesenchymal phenotype such as anoikis resistance, cytoskeleton remodeling, increased cell stiffness, and the acquisition of invasion and tumorigenic capacity. CONCLUSIONS: We identified CTGF is an important regulator of the epithelial phenotype, and its loss is associated with the early cellular modifications required for EMT. We describe a novel role for CTGF, regulating cytoskeleton and the extracellular matrix interactions necessary for the conservation of epithelial structure and function. These findings provide a new window into understanding the early stages of mesenchymal transformation.

Immunological modifications following chemotherapy are associated with delayed recurrence of ovarian cancer.

Introduction: Ovarian cancer recurs in most High Grade Serous Ovarian Cancer (HGSOC) patients, including initial responders, after standard of care. To improve patient survival, we need to identify and understand the factors contributing to early or late recurrence and therapeutically target these mechanisms. We hypothesized that in HGSOC, the response to chemotherapy is associated with a specific gene expression signature determined by the tumor microenvironment. In this study, we sought to determine the differences in gene expression and the tumor immune microenvironment between patients who show early recurrence (within 6 months) compared to those who show late recurrence following chemotherapy. Methods: Paired tumor samples were obtained before and after Carboplatin and Taxol chemotherapy from 24 patients with HGSOC. Bioinformatic transcriptomic analysis was performed on the tumor samples to determine the gene expression signature associated with differences in recurrence pattern. Gene Ontology and Pathway analysis was performed using AdvaitaBio's iPathwayGuide software. Tumor immune cell fractions were imputed using CIBERSORTx. Results were compared between late recurrence and early recurrence patients, and between paired pre-chemotherapy and post-chemotherapy samples. Results: There was no statistically significant difference between early recurrence or late recurrence ovarian tumors pre-chemotherapy. However, chemotherapy induced significant immunological changes in tumors from late recurrence patients but had no impact on tumors from early recurrence patients. The key immunological change induced by chemotherapy in late recurrence patients was the reversal of pro-tumor immune signature. Discussion: We report for the first time, the association between immunological modifications in response to chemotherapy and the time of recurrence. Our findings provide novel opportunities to ultimately improve ovarian cancer patient survival.

Generation of Stable Epithelial-Mesenchymal Hybrid Cancer Cells with Tumorigenic Potential.

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.

MNRR1 is a driver of ovarian cancer progression.

Cancer progression requires the acquisition of mechanisms that support proliferative potential and metastatic capacity. MNRR1 (also CHCHD2, PARK22, AAG10) is a bi-organellar protein that in the mitochondria can bind to Bcl-xL to enhance its anti-apoptotic function, or to respiratory chain complex IV (COX IV) to increase mitochondrial respiration. In the nucleus, it can act as a transcription factor and promote the expression of genes involved in mitochondrial biogenesis, migration, and cellular stress response. Given that MNRR1 can regulate both apoptosis and mitochondrial respiration, as well as migration, we hypothesize that it can modulate metastatic spread. Using ovarian cancer models, we show heterogeneous protein expression levels of MNRR1 across samples tested and cell-dependent control of its stability and binding partners. In addition to its anti-apoptotic and bioenergetic functions, MNRR1 is both necessary and sufficient for a focal adhesion and ECM repertoire that can support spheroid formation. Its ectopic expression is sufficient to induce the adhesive glycoprotein THBS4 and the type 1 collagen, COL1A1. Conversely, its deletion leads to significant downregulation of these genes. Furthermore, loss of MNRR1 leads to delay in tumor growth, curtailed carcinomatosis, and improved survival in a syngeneic ovarian cancer mouse model. These results suggest targeting MNRR1 may improve survival in ovarian cancer patients.

Regulatory Role of the Adipose Microenvironment on Ovarian Cancer Progression.

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.

Detection of Anoikis Using Cell Viability Dye and Quantitation of Caspase Activity.

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.

Transimmunization restores immune surveillance and prevents recurrence in a syngeneic mouse model of ovarian cancer.

Ovarian cancer accounts for most deaths from gynecologic malignancies. Although more than 80% of patients respond to first-line standard of care, most of these responders present with recurrence and eventually succumb to carcinomatosis and chemotherapy-resistant disease. To improve patient survival, new modalities must, therefore, target or prevent recurrent disease. Here we describe for the first time a novel syngeneic mouse model of recurrent high-grade serous ovarian cancer (HGSOC), which allows immunotherapeutic interventions in a time course relevant to human carcinomatosis and disease course. Using this model, we demonstrate the efficacy of Transimmunization (TI), a dendritic cell (DC) vaccination strategy that uses autologous and physiologically derived DC loaded with autologous whole tumor antigens. TI has been proven successful in the treatment of human cutaneous T cell lymphoma and we report for the first time its in vivo efficacy against an intra-peritoneal solid tumor. Given as a single therapy, TI is able to elicit an effective anti-tumor immune response and inhibit immune-suppressive crosstalks with sufficient power to curtail tumor progression and establishment of carcinomatosis and recurrent disease. Specifically, TI is able to inhibit the expansion of tumor-associated macrophages as well as myeloid-derived suppressive cells consequently restoring T cell immune-surveillance. These results demonstrate the possible value of TI in the management of ovarian cancer and other intra-peritoneal tumors.

CBX7 binds the E-box to inhibit TWIST-1 function and inhibit tumorigenicity and metastatic potential.

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 reacquire 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 nonfunctional 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 subclassification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

The Role of Intra-Tumoral Heterogeneity and Its Clinical Relevance in Epithelial Ovarian Cancer Recurrence and Metastasis.

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.

Protein kinase Cα-mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it.

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.

p53-Pirh2 Complex Promotes Twist1 Degradation and Inhibits EMT.

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.

Effects of serum adsorption on cellular uptake profile and consequent impact of titanium dioxide nanoparticles on human lung cell lines.

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.