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.

Adipocyte microenvironment promotes Bclxl expression and confers chemoresistance in ovarian cancer cells.

Resistance to mitochondria-initiated apoptosis is a hallmark of chemoresistant cancer stem cells including CD44+/MyD88+ epithelial ovarian cancer (EOC) stem cells. This is controlled by members of the Bcl2 family of proteins, which function as rheostats of mitochondrial stability. We observed a differential expression profile of Bcl2 family members comparing the chemoresistant EOC stem cells and the chemosensitive CD44-/MyD88- EOC cells. Chemoresistant EOC stem cells surprisingly express higher levels of the pro-apoptotic members Bak and Bax compared to the chemosensitive EOC cells. In addition, whereas chemosensitive EOC cells preferentially express Bcl2, chemoresistant EOC stem cells preferentially express Bclxl. In the EOC stem cells, 40% knock-down of Bclxl expression was sufficient to induce the full activation of caspases and this can be reversed by concurrent knock-down of Puma. More importantly, we demonstrate that Bclxl expression levels in EOC cells is dynamic and can be regulated by microenvironments that are enriched with the pro-inflammatory cytokine IL-6 such as the cancer stem cell and adipocyte niches. Adipocyte-induced upregulation of Bclxl correlated with acquisition of chemoresistance and thus demonstrates how a specific microenvironment can regulate the expression of apoptotic proteins and confer chemoresistance.

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 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.

Immune Modulation of Innate and Adaptive Responses Restores Immune Surveillance and Establishes Antitumor Immunologic Memory.

Current immunotherapies have proven effective in strengthening antitumor immune responses, but constant opposing signals from tumor cells and the surrounding microenvironment eventually lead to immune escape. We hypothesized that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system would provide a robust and long-term antitumor effect by creating immunologic memory against tumors. To achieve this, we developed CARG-2020, a genetically modified virus-like vesicle (VLV) that is a self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three immune modulators: (i) the pleiotropic antitumor cytokine IL12, in which the subunits (p35 and p40) are tethered together; (ii) the extracellular domain (ECD) of the protumor IL17RA, which serves as a dominant-negative antagonist; and (iii) a shRNA targeting PD-L1. Using a mouse model of ovarian cancer, we demonstrated the oncolytic effect and immune-modulatory capacities of CARG-2020. By enhancing IL12 and blocking IL17 and PD-L1, CARG-2020 successfully reactivated immune surveillance by promoting M1, instead of M2, macrophage differentiation, inhibiting MDSC expansion and establishing a potent CD8+ T cell-mediated antitumoral response. Furthermore, we demonstrated that this therapeutic approach provided tumor-specific and long-term protection against the establishment of new tumors. Our results provide a rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance antitumor responses and prevent a recurrence.

BRCA status dictates Wnt responsiveness in epithelial ovarian cancer.

The association of BRCA1 and BRCA2 mutations with increased risk for developing epithelial ovarian cancer is well established. However, the observed clinical differences, particularly the improved therapy response and patient survival in BRCA2 mutant (BRCA2mt) patients are unexplained. Our objective is to identify molecular pathways that are differentially regulated upon the loss of BRCA1 and BRCA2 function in ovarian cancer. Transcriptomic and pathway analysis comparing BRCA1 mutant (BRCA1mt), BRCA2mt and homologous recombination wild-type (HRwt) ovarian tumors showed differential regulation of the Wnt/ß-catenin pathway. Using Wnt3A-treated BRCA1/2 wild-type (BRCAwt), BRCA1null and BRCA2null mouse ovarian cancer cells, we observed preferential activation of the canonical Wnt/ß-catenin signaling in BRCAwt ovarian cancer cells while the non-canonical Wnt/ß-catenin signaling was preferentially activated in the BRCA1null cells. Interestingly, BRCA2null mouse ovarian cancer cells, demonstrated a unique response to Wnt3A with the preferential upregulation of the Wnt signaling inhibitor, Axin2. In addition, decreased phosphorylation and enhanced stability of ß-catenin were observed in BRCA2null mouse ovarian cancer cells, which correlated with increased inhibitory phosphorylation on GSK3ß. These findings open venues for the translation of these molecular observations into modalities that can impact 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.

Immune modulation of innate and adaptive responses restores immune surveillance and establishes anti-tumor immunological memory.

Current immunotherapies have proven effective in strengthening anti-tumor immune responses but constant opposing signals from tumor cells and surrounding microenvironment eventually lead to immune escape. We hypothesize that in situ release of antigens and regulation of both the innate and adaptive arms of the immune system will provide a robust and long-term anti-tumor effect by creating immunological memory against the tumor. To achieve this, we developed CARG-2020, a virus-like-vesicle (VLV). It is a genetically modified and self-amplifying RNA with oncolytic capacity and encodes immune regulatory genes. CARG-2020 carries three transgenes: 1 ) the pleiotropic antitumor cytokine IL-12 in which the subunits (p35 and p40) are tethered together; 2) the extracellular domain (ECD) of the pro- tumor IL-17RA, which can serve as a dominant negative antagonist; and 3) shRNA for PD-L1. Using a mouse model of ovarian cancer, we demonstrate the oncolytic effect and immune modulatory capacities of CARG-2020. By enhancing IL-12 and blocking IL-17 and PD-L1, CARG-2020 successfully reactivates immune surveillance by promoting M1 instead of M2 macrophage differentiation, inhibiting MDSC expansion, and establishing a potent CD8+ T cell mediated anti-tumoral response. Furthermore, we demonstrate that this therapeutic approach provides tumor-specific and long-term protection preventing the establishment of new tumors. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients to enhance the anti-tumor response and to prevent recurrence.

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.

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.

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.

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.

TWIST1 induces proteasomal degradation of ß-catenin during the differentiation of ovarian cancer stem-like cells.

Ovarian cancer (OC) is one of the leading gynecologic cancers worldwide. Cancer stem-like cells are correlated with relapse and resistance to chemotherapy. Twist1, which is involved in ovarian cancer stem-like cell differentiation, is positively correlated with CTNNB1 in different differentiation stages of ovarian cancer cells: primary epithelial ovarian cancer cells (primary EOC cells), mesenchymal spheroid-forming cells (MSFCs) and secondary epithelial ovarian cancer cells (sEOC cells). However, the expression of ß-catenin is inversed compared to CTNNB1 in these 3 cell states. We further demonstrated that ß-catenin is regulated by the protein degradation system in MSFCs and secondary EOC but not in primary EOC cells. The differentiation process from primary EOC cells to MSFCs and sEOC cells might be due to the downregulation of ß-catenin protein levels. Finally, we found that TWIST1 can enhance ß-catenin degradation by upregulating Axin2.

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.

Triphendiol (NV-196), development of a novel therapy for pancreatic cancer.

Despite incremental progress in the treatment of pancreatic adenocarcinoma, the prognosis of patients remains poor. Here, we report the preclinical studies in pancreatic cancer cells that demonstrate the efficacy of triphendiol (NV-196, a synthetic isoflavene) both as a monotherapy and as a gemcitabine sensitizer. The in-vitro effects of triphendiol on the pancreatic cancer cell lines HPAC and MIAPaCa-2 were determined using cell proliferation, flow cytometry, and western blot analysis. The antiproliferative activity of triphendiol was also investigated in two xenograft models of pancreatic cancer (HPAC and MIAPaCa-2). As a monotherapy, triphendiol-inhibited cell proliferation-induced p53-independent G2/M cell cycle arrest and activation of the intrinsic (mitochondrial) apoptosis pathway. Triphendiol-induced apoptosis was caspase independent and death receptor independent, whereas cell necrosis was caspase mediated. Using combination index analysis, we have shown that pretreatment of pancreatic cancer cells with triphendiol enhanced the cytotoxic effect of gemcitabine, the standard of care used to treat advanced pancreatic cancer. In xenograft models of pancreatic cancer, the rate of tumor proliferation on mice coadministered with triphendiol and gemcitabine was significantly reduced when compared with the corresponding tumor proliferation rates from the respective monotherapy-control and vehicle-control groups. Triphendiol was recently granted Investigational New Drug status by the US Food and Drug Administration. These data justify the commencement of clinical studies investigating the utility of combining triphendiol and gemcitabine in patients with early-stage and late-stage pancreatic cancer.

Detection of Unfolded Protein Response by Polymerase Chain Reaction.

The unfolded protein response is a cellular adaptive mechanism localized in the endoplasmic reticulum. It involves three phases: the detection of increased presence of unfolded proteins as a result of cellular stressors; the execution of an adaptive cascade of events aimed at the enhancement of proper protein folding and degradation of improperly folded proteins; and finally, when stress is not alleviated, the execution of programmed cell death. The main effectors of the UPR are transcription factors involved in the upregulation of either chaperone proteins or proapoptotic proteins. Two of these transcription factors are CHOP and the spliced variant of XBP-1 (XBP1s). In this chapter, we describe a quantitative PCR method to detect the upregulation of CHOP and XBP1s mRNA during Tunicamycin-induced UPR.

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.

Determination of Caspase Activation by Western Blot.

Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Apoptosis is executed by a class of cysteine proteases called caspases. Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. The described methods can be applied to any particular protein of interest.

Subcellular Fractionation to Demonstrate Activation of Intrinsic Apoptotic Pathway.

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.