Metastatic Ovarian Cancer Cells Research Articles

Adipocyte pyroptosis occurs in omental tumor microenvironment and is associated with chemoresistance of ovarian cancer

BackgroundOvarian carcinoma (OC) is a fatal malignancy, with most patients experiencing recurrence and resistance to chemotherapy. In contrast to hematogenous metastasizing tumors, ovarian cancer cells disseminate within the peritoneal cavity, especially the omentum. Previously, we reported omental crown-like structure (CLS) number is associated with poor prognosis of advanced-stage OC. CLS that have pathologic features of a dead or dying adipocyte was surrounded by several macrophages is well known a histologic hallmark for inflammatory adipose tissue. In this study, we attempted to clarify the interaction between metastatic ovarian cancer cells and omental CLS, and to formulate a therapeutic strategy for advanced-stage ovarian cancer.MethodsA three-cell (including OC cells, adipocytes and macrophages) coculture model was established to mimic the omental tumor microenvironment (TME) of ovarian cancer. Caspase-1 activity, ATP and free fatty acids (FFA) levels were detected by commercial kits. An adipocyte organoid model was established to assess macrophages migration and infiltration. In vitro and in vivo experiments were performed for functional assays and therapeutic effect evaluations. Clinical OC tissue samples were collected for immunochemistry stain and statistics analysis.ResultsIn three-cell coculture model, OC cells-derived IL-6 and IL-8 could induce the occurrence of pyroptosis in omental adipocytes. The pyroptotic adipocytes release ATP to increase macrophage infiltration, release FFA into TME, uptake by OC cells to increase chemoresistance. From OC tumor samples study, we demonstrated patients with high gasdermin D (GSDMD) expression in omental adipocytes is highly correlated with chemoresistance and poor outcome in advanced-stage OC. In animal model, by pyroptosis inhibitor, DSF, effectively retarded tumor growth and prolonged mice survival.ConclusionsOmental adipocyte pyroptosis may contribute the chemoresistance in advanced stage OC. Omental adipocytes could release FFA and ATP through the GSDMD-mediate pyroptosis to induce chemoresistance and macrophages infiltration resulting the poor prognosis in advanced-stage OC. Inhibition of adipocyte pyroptosis may be a potential therapeutic modality in advanced-stage OC with omentum metastasis.

Omentum-derived matrix enables the study of metastatic ovarian cancer and stromal cell functions in a physiologically relevant environment

High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and in vitro models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients.Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.

Suppression of Metastatic Ovarian Cancer Cells by Bepridil, a Calcium Channel Blocker.

Although surgery followed by platinum-based therapy is effective as a standard treatment in the early stages of ovarian cancer, the majority of cases are diagnosed at advanced stages, leading to poor prognosis. Thus, the identification of novel therapeutic drugs is needed. In this study, we assessed the effectiveness of bepridil-a calcium channel blocker-in ovarian cancer cells using two cell lines: SKOV-3, and SKOV-3-13 (a highly metastatic clone of SKOV-3). Treatment of these cell lines with bepridil significantly reduced cell viability, migration, and invasion. Notably, SKOV-3-13 was more sensitive to bepridil than SKOV-3. The TGF-β1-induced epithelial-mesenchymal transition (EMT)-like phenotype was reversed by treatment with bepridil in both cell lines. Consistently, expression levels of EMT-related markers, including vimentin, β-catenin, and Snail, were also substantially decreased by the treatment with bepridil. An in vivo mouse xenograft model was used to confirm these findings. Tumor growth was significantly reduced by bepridil treatment in SKOV-3-13-inoculated mice, and immunohistochemistry showed consistently decreased expression of EMT-related markers. Our findings are the first to report anticancer effects of bepridil in ovarian cancer, and they suggest that bepridil holds significant promise as an effective therapeutic agent for targeting metastatic ovarian cancer.

Purinergic Activation of Store-Operated Calcium Entry (SOCE) Regulates Cell Migration in Metastatic Ovarian Cancer Cells.

Store-operated calcium entry (SOCE) is an important process in calcium signaling. Its role in physiological and pathological events is well recognized. However, in cancerous systems, the importance of SOCE in relation to the degree of cancer aggressiveness, as well as its regulation by ligands such as purinergic molecules, are not well documented. This study aimed to characterize a differential effect of the P2Y2 receptor (promoted by UTP of 10 µM and inhibited by ARC118925XX of 1 µM) on intracellular calcium response between metastatic (SKOV-3) and non-metastatic (CAOV-3) ovarian cell lines in conditions of normal (1.5 mM) and zero extracellular calcium concentration. The sustained calcium influx observed exclusively in SKOV-3 cells was associated with the presence of SOCE (promoted by thapsigargin (74.81 ± 0.94 ΔF) and sensitive to 2-APB (20.60 ± 0.85 ΔF)), whereas its absence in CAOV-3 cells (26.2 ± 6.1 ΔF) was correlated with a low expression of ORAI1. The relevance of SOCE in metastatic SKOV-3 cells was further corroborated when 2-APB significantly inhibited (40.4 ± 2.8% of covered area) UTP-induced cell migration (54.6 ± 3.7% of covered area). In conclusion, our data suggest that SOCE activation elicited by the P2Y2 receptor is involved in the aggressiveness of ovarian cancer cells.

Abstract 5952: Pyroptotic adipocytes contribute to chemoresistance in advanced stage ovarian cancer

Abstract Background: Ovarian cancer is known the gynecologic malignancy with the highest case-to-fatality ratio. Once appear metastasis, the 5 years survival rate reduce to 30-40% in these patients. Different with hematogenously metastasizing tumors, ovarian cancer cells primarily disseminate within the peritoneal cavity. Omentum, a lipid-rich tissue, is the most common metastatic site of ovarian cancer. In addition, the adipocyte-released free fatty acid can be used by cancer cells to fuel its growth and proliferation. In this study, we attempt to clarify the regulation between metastatic ovarian cancer cell and omental adipocytes, and find a therapeutic strategy for advanced stage ovarian cancer. Materials and Methods: A three-cell (SKOV3, 3T3L1 and THP-1) co-culture model was established to mimic in vitro tumor microenvironment of ovarian cancer omental metastasis. Flow cytometry was performed for the adipocytes survival rate with Annexin V/PI double staining. Caspase-1 activity assay was used to detect the cleavage of substrate YVAD-AFC level in adipocytes and quantified by a fluorescence microliter plate reader. The free fatty acid assay kit was used to detect free fatty acid level in condition medium and ascites. Invasion assay and MTT assay were used to validate the invasive ability and cell viability of ovarian cancer cells. Immunohistochemistry was used to CD36 staining in clinical specimens, and correlation analysis through prism 8 software. Results: We observed the adipocyte pyroptosis in 3-cell co-culture model, and the free fatty acid increased in culture medium by flow cytometry analysis, caspase-1 activity assay kit and free fatty acid assay kit. In the meantime, the invasive ability and cisplatin resistance increased in ovarian cancer cells. The free fatty acid level in ascites positive correlated with CD36 expression (r2=0.2189, p=0.0375). Of note, combination treatment of cisplatin and sulfosuccinimidyl oleate (SSO) can reverse chemoresistance in 3-cell co-culture model. Overall, free fatty acid released form the pyroptotic adipocytes and increase invasive ability and chemoresistance in ovarian cancer cells. Block the interaction of free fatty acid and CD36 can reverse the chemoresistance in advanced ovarian cancer. Conclusion: In this study, we demonstrated adipocytes pyroptosis can increase the free fatty acid release to the microenvironment, and then promote ovarian cancer cell invasion and increase chemoresistance. For more important, inhibition of free fatty acid and CD36 interaction can reverse the chemoresistance in the in vitro experiments. Thus, reduce the adipocytes pyroptosis may be a novel therapeutic strategy for the omentum metastatic ovarian cancer. Citation Format: Chang-Ni Lin, Yu-Ling Liang, Keng-Fu Hsu, Yi-Ying Wu. Pyroptotic adipocytes contribute to chemoresistance in advanced stage ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5952.

Abstract 293: Non-metabolic regulatory functions of nuclear pyruvate kinase M2 induce a MAPK mediated phenotypic switch in invasive cancers

Abstract Accumulating evidence suggests that PKM2, the differentially spliced pyruvate kinase isoform that is expressed in most cancers and embryonic tissue, is linked with epithelial-mesenchymal transition (EMT). However, how PKM2 may drive this process of tumor invasiveness remains unclear. Here we demonstrate that in metastatic hormone-sensitive and ovarian cancer cells PKM2 undergoes modification by phosphorylation on its Serine 37 residue (pS37) and that this modified protein displays distinct localization patterns in response to pro-migratory cues emanating from an artificial nanopatterned substrate mimicking the tumor associated extracellular matrix. We find that in epithelial-mesenchymal (EM) hybrid cohorts, migrating cells undergo phenotypic switching from followers to leaders and the PKM2-pS37 signals correlate with different cell phenotypes (less/more invasive and leaders vs followers). This phenotypic switching occurs as a function of cell distance from the leading edge in an expanding epithelial sheet. We found that the putative novel non-metabolic functions of PKM2 are attributed to its autoregulation that results in not only a PKM2-pS37 upregulation but also an induction in PKM2 expression via ERK signaling. Cues emanating from either the extracellular matrix (mediated via Integrin/FAK) or through growth factor signaling (EGFR signaling), culminating in ERK activation induce PKM2 phosphorylation at S37. Thus, our data suggest that an increase in PKM2pS37 is accompanied by a switch to less enzymatically active molecular complexes which can translocate to the nucleus and increase PKM2 expression through an auto-positive feedback loop resulting in increased total PKM2 concentration and function in the leader cells. To further confirm the existence of this positive feedback loop we developed a mathematical model for PKM2 regulation. This model allows for the evaluation of inputs emerging from the extracellular matrix, such as the integrin/FAK signaling, and growth factor receptor signaling both of which regulate PKM2 expression through ERK activation in a switch-like fashion. The mathematical model includes non-linearity in the phosphorylation of S37 on PKM2 and the non-linearity in the nuclear localization and upregulation of PKM2. Further experimental analysis supports the functional role of the switch, as the observed PKM2 expression states correlate with the expression of characteristic EMT markers and invasion-promoting pseudo-hypoxic stress in the leader cells. Importantly, the small molecule activator of PKM2, TEPP 46, can restore a more epithelial-like state, decrease invasion, and increase glucose uptake in these EM hybrid ovarian cancer cells. Taken together, our results highlight a link between PKM2 and cancer aggressiveness driven by novel regulatory mechanisms that support both metabolic rewiring and invasive spread of tumor cells. Citation Format: Kiran Vanaja, Maria Apostolidi, Andre Levchenko, Jesse Rinehart. Non-metabolic regulatory functions of nuclear pyruvate kinase M2 induce a MAPK mediated phenotypic switch in invasive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 293.

Artificial Diets with Altered Levels of Sulfur Amino Acids Induce Anticancer Activity in Mice with Metastatic Colon Cancer, Ovarian Cancer and Renal Cell Carcinoma.

Sulfur-containing amino acids methionine (Met), cysteine (Cys) and taurine (Tau) are common dietary constituents with important cellular roles. Met restriction is already known to exert in vivo anticancer activity. However, since Met is a precursor of Cys and Cys produces Tau, the role of Cys and Tau in the anticancer activity of Met-restricted diets is poorly understood. In this work, we screened the in vivo anticancer activity of several Met-deficient artificial diets supplemented with Cys, Tau or both. Diet B1 (6% casein, 2.5% leucine, 0.2% Cys and 1% lipids) and diet B2B (6% casein, 5% glutamine, 2.5% leucine, 0.2% Tau and 1% lipids) showed the highest activity and were selected for further studies. Both diets induced marked anticancer activity in two animal models of metastatic colon cancer, which were established by injecting CT26.WT murine colon cancer cells in the tail vein or peritoneum of immunocompetent BALB/cAnNRj mice. Diets B1 and B2B also increased survival of mice with disseminated ovarian cancer (intraperitoneal ID8 Tp53-/- cells in C57BL/6JRj mice) and renal cell carcinoma (intraperitoneal Renca cells in BALB/cAnNRj mice). The high activity of diet B1 in mice with metastatic colon cancer may be useful in colon cancer therapy.

New Insights into Ferroptosis Initiating Therapies (FIT) by Targeting the Rewired Lipid Metabolism in Ovarian Cancer Peritoneal Metastases

Ovarian cancer is one of the most lethal gynecological cancers worldwide. The poor prognosis of this malignancy is substantially attributed to the inadequate symptomatic biomarkers for early diagnosis and effective remedies to cure the disease against chemoresistance and metastasis. Ovarian cancer metastasis is often relatively passive, and the single clusters of ovarian cancer cells detached from the primary ovarian tumor are transcoelomic spread by the peritoneal fluid throughout the peritoneum cavity and omentum. Our earlier studies revealed that lipid-enriched ascitic/omental microenvironment enforced metastatic ovarian cancer cells to undertake metabolic reprogramming and utilize free fatty acids as the main energy source for tumor progression and aggression. Intriguingly, cell susceptibility to ferroptosis has been tightly correlated with the dysregulated fatty acid metabolism (FAM), and enhanced iron uptake as the prominent features of ferroptosis are attributed to the strengthened lipid peroxidation and aberrant iron accumulation, suggesting that ferroptosis induction is a targetable vulnerability to prevent cancer metastasis. Therefore, the standpoints about tackling altered FAM in combination with ferroptosis initiation as a dual-targeted therapy against advanced ovarian cancer were highlighted herein. Furthermore, a discussion on the prospect and challenge of inducing ferroptosis as an innovative therapeutic approach for reversing remedial resistance in cancer interventions was included. It is hoped this proof-of-concept review will indicate appropriate directions for speeding up the translational application of ferroptosis-inducing compounds (FINs) to improve the efficacy of ovarian cancer treatment.

Abstract 2429: Tumor and stromal AXL expression regulate ovarian cancer metastasis

Abstract Objective: Over 75% of patients with ovarian cancer present with widely metastatic disease. We have previously published that AXL is highly expressed in primary ovarian cancer tumors. However, it is unknown whether AXL expression in metastatic tumors correlates with outcome. Additionally, the role of stromally expressed AXL in metastasis has not been fully evaluated. Thus, the objective of this study is to evaluate the role of tumor and stromal AXL in the key steps of tumor metastasis. Methods: Immunohistochemistry was used to measure AXL expression in a tissue microarray with primary and metastatic high grade ovarian tumors. Attachment assays as well as transwell and spheroid invasion assays were performed on established ovarian cancer cell lines. Mesothelial cell clearance assays utilizing human omentum-cultured mesothelial cells (HPMC) and additional invasion assays utilizing normal omental fibroblasts (NOF) were performed. Results: 214 tumor samples were included from 139 patients: 122 primary and 92 metastatic sites, contributing to 85 matched samples. Most had high-grade serous histology (79%) and stage III-IV disease (79%). High AXL expression in metastatic tumors was associated with decreased overall survival (HRadj 1.8, 95% CI 1.03-3.1), but expression in primary tumors was not (1.1, 0.61-2.0). High vs. low AXL expression in metastases was associated with a decreased platinum-free interval (5.92 vs 15.9 months, P=0.03) and decreased progression-free survival (11.6 vs 18.8 months, P=0.02).Tumor AXL expression correlated with stromal AXL expression (R2=0.50, P<0.001). Human ovarian tumor cells depleted of AXL had decreased attachment (OVCAR3-TPMES, 0.55-fold decrease P<0.001) and invasion in transwell (OVCAR5, 173 vs 262 invasive tumor cells/hpf, P<0.01) and spheroid (ES2, 87% vs 31% area invaded, P<0.0001) invasion assays. Human ovarian cancer cells plated on AXL-deficient HPMCs had less cell clearance than those plated on AXL-expressing HPMCs (ES2, 6.0 vs 3.9 normalized area cleared, P<0.001). Further, human ovarian cancer cells plated on AXL-deficient NOFs had less invasion than tumor cells plated on AXL expressing NOFs (OVCAR5, 173 vs 262 invasive tumor cells/hpf, P<0.01). Conclusions: High AXL expression in metastatic ovarian cancer cells correlates with worse survival and worse response to chemotherapy. Further, AXL expression in both tumor and stromal cells contributes to the steps of ovarian cancer metastasis. Citation Format: Mary Margaret Mullen, Lillian N. van Biljon, Jeanne Quinn, Molly Greenwade, Gregory Opara, Hollie Noia, Ian S. Hagemann, Andrea R. Hagemann, Lindsay M. Kuroki, Carolyn K. McCourt, Premal H. Thaker, Matthew A. Powell, David G. Mutch, Dineo Khabele, Katherine C. Fuh. Tumor and stromal AXL expression regulate ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2429.

G6PD inhibition sensitizes ovarian cancer cells to oxidative stress in the metastatic omental microenvironment.

SUMMARYOvarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of OC-related deaths. In particular, OC tumors preferentially metastasize to and proliferate rapidly in the omentum. Here, we show that metastatic OC cells experience increased oxidative stress in the omental microenvironment. Metabolic reprogramming, including upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, allows OC cells to compensate for this challenge. Inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting that this adaptive metabolic dependency is important for OC omental metastasis.

Half-Sandwich Arene Ruthenium(II) Thiosemicarbazone Complexes: Evaluation of Anticancer Effect on Primary and Metastatic Ovarian Cancer Cell Lines.

In this study, we describe the synthesis, characterization and antiproliferative activity of three organo-ruthenium(II) half-sandwich complexes [RuCl(η6-p-cym)(N,S-L)]Cl (I, II, and III). To form these complexes, three thiosemicarbazone ligands (TSCs) were synthesized; L = 5-nitro-2-carboxyaldehyde-thiophen-N-methyl-thiosemicarbazone, (L1); 2-acetyl-5-bromo-thiophen-N-methyl-thiosemicarbazone, (L2) and 2-acetyl-5-bromo-thiophen-N,N-dimethyl-thiosemicarbazone, (L3). The isolated compounds were analyzed using spectroscopic techniques such as elemental analysis, conductance measurements, FT-IR, 1H NMR spectroscopy, MALDI-TOF mass spectrometry, and single-crystal XRD. Our results demonstrated that the synthesized thiosemicarbazone ligands (TSCs) are bound to the metal ion as a bidentate ligand that coordinates through the thiocarbonyl sulfur and azomethine nitrogen atoms in all complexes (I, II, and III). The X-ray crystal structures of L1 and L2 revealed that both compounds are crystallized in the triclinic crystal system with space group P-1. The biological potency of newly synthesized TSC ligands (L1, L2, and L3) and their corresponding ruthenium complexes (I, II, and III) were investigated on human primary ovarian (A2780) and human metastatic ovarian (OVCAR-3) cell lines. To get detailed information respecting antitumor properties, cytotoxicity, DNA/BSA binding affinity, cellular uptake, DNA binding competition, and trans-epithelial resistance measurement assays were performed. Our results demonstrate that newly synthesized ruthenium(II) complexes possess potential biological activity. Moreover, we observe that the ruthenium complexes reported here show anticancer activity on primary (A2780) and metastatic (OVCAR-3) ovarian cancer cells.

Metabolic Reprogramming and Lipophagy Mediates Survival of Ascites Derived Metastatic Ovarian Cancer Cells.

Objective:The study was aimed at understanding the survival of metastatic ovarian cancer spheroids in the malignant ascites microenvironment. Methods:All the assays were performed using aseptically collected patient samples. The cells were characterized for the expression of ovarian and cancer stem cell markers using immunocytochemistry. The presence of lipid in the primary metastatic cancer spheroids were confirmed by neutral fat staining using Oil Red-O and transmission electron microscopy. The mRNA expression of autophagy and lipid metabolism genes was analyzed using RT-PCR. The lipid content was analyzed using lipidomics analysis. Etomoxir and chloroquine were used to study the effect of inhibition of autophagy in the metastatic cells. The data were analyzed using appropriate statistical tools and a p-value <0.05 was considered to be statistically significant.Results:Metastatic ovarian cancer spheroids exhibit cancer stem like properties and undergo a metabolic reprogramming when they disseminate from the primary tumor. We report here the accumulation of numerous cytoplasmic lipid droplets and lipophagic vesicles in the metastatic cells in contrast to their primary tumors. In addition we also report that these cells depend on lipophagy for the utilization of lipids rather than the conventional lipolytic pathway. The lipidomics analysis data reveals that the metastatic cells possess high levels of unsaturated fatty acids. We have also reported the occurrence of distinct accumulation of multiple nuclei in the patient derived metastatic cells. Inhibition of beta-oxidation and autophagic machinery using etomoxir and chloroquine resulted in cell death suggesting a potential mode to suppress metastatic cancer cells. Conclusion: Metabolic reprogramming is a characteristic feature of the metastatic ovarian cancer cells that are persisting in the malignant ascites. Targeting of the metastatic cells by gaining an insight into the various metabolic and molecular changes that occur in the metastatic niche provides a promising therapeutic approach in management of the disease.

Targeting LRRC15 Inhibits Metastatic Dissemination of Ovarian Cancer.

Dissemination of ovarian cancer (OC) cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in OC bowel metastases compared to matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of OC demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic OC cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived OC xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting OC metastasis and suggest a novel and promising therapy to target OC metastases.

Chromatin softening and nuclear envelope breaching potentiates killing of chemoresistant cancer cells

The nuclei of metastatic cancer cells are more pliable because of reduced lamin A/C expression and decreased chromatin condensation, facilitating effective invasion through tissue interstices. Taking advantage of the fact that nuclei are already softer in chemoresistant metastatic ovarian cancer cells, we strategize to further compromising the nucleus integrity by treating cancer cells with low-dose drug paclitaxel (PTX) and fostamatinib. PTX is a commonly used chemotherapeutic drug for many cancer types, and fostamatinib is a recently approved adjuvant by FDA to treat ovarian cancer.

Epigenetic Silencing of miR-33b Promotes Peritoneal Metastases of Ovarian Cancer by Modulating the TAK1/FASN/CPT1A/NF-κB Axis.

Simple SummaryOmental metastasis and peritoneal dissemination are frequently observed in ovarian cancer peritoneal metastases and are associated with high mortality and poor prognosis. The tumor microenvironment is known to influence cancer epigenomics, which plays an essential role in promoting tumor development and metastatic progression. Therefore, investigation of the epigenetic mechanisms underlying the growth of ovarian cancer cells in the omental metastatic microenvironment is of great importance. Here, we report that miR-33b is significantly silenced by DNA hypermethylation in metastatic ovarian cancer cells to adapt to a lipid-rich microenvironment. Restoration of miR-33b was shown to impair lipid metabolic activities and reduce the oncogenic properties of ovarian cancer cells by negatively regulating the TAK1/FASN/CPT1A/NF-κB pathway, indicating that targeting this signaling cascade may be a molecular therapeutic choice for ovarian cancer metastatic progression.Peritoneal metastases are frequently found in high-grade serous carcinoma (HGSOC) patients and are commonly associated with a poor prognosis. The tumor microenvironment (TME) is a complex milieu that plays a critical role in epigenetic alterations driving tumor development and metastatic progression. However, the impact of epigenetic alterations on metastatic ovarian cancer cells in the harsh peritoneal microenvironment remains incompletely understood. Here, we identified that miR-33b is frequently silenced by promoter hypermethylation in HGSOC cells derived from metastatic omental tumor tissues. Enforced expression of miR-33b abrogates the oncogenic properties of ovarian cancer cells cocultured in omental conditioned medium (OCM), which mimics the ascites microenvironment, and in vivo tumor growth. Of note, restoration of miR-33b inhibited OCM-upregulated de novo lipogenesis and fatty acid β-oxidation in ovarian cancer cells, indicating that miR-33b may play a novel tumor suppressor role in the lipid-mediated oncogenic properties of metastatic ovarian cancer cells found in the omentum. Mechanistic studies demonstrated that miR-33b directly targets transforming growth factor beta-activated kinase 1 (TAK1), thereby suppressing the activities of fatty acid synthase (FASN) and carnitine palmitoyltransferase 1A (CPT1A) in modulating lipid metabolic activities and simultaneously inhibiting the phosphorylation of NF-κB signaling to govern the oncogenic behaviors of ovarian cancer cells. Thus, our data suggest that a lipid-rich microenvironment may cause epigenetic silencing of miR-33b, which negatively modulates ovarian cancer peritoneal metastases, at least in part, by suppressing TAK1/FASN/CPT1A/NF-κB signaling.

The Stress-Inducible BCL2A1 Is Required for Ovarian Cancer Metastatic Progression in the Peritoneal Microenvironment.

Simple SummaryCancer metastasis is still the main cause of cancer-related mortality. Peritoneal metastases are the first presentation of advanced ovarian cancer, and metastatic cancer cells tend to disseminate trans-peritoneally in the peritoneal cavity. Hypoxia is a critical factor in governing transcoelomic metastases of ovarian cancer. Therefore, targeting hypoxia appears to be a promising approach to arrest cancer metastasis. In the present work, we identified that BCL2A1, a BCL2 family member, is significantly induced by hypoxia and other physiological stresses by NF-κB signaling and followed by a gradual degradation. The upregulated BLC2A1 has been shown to enhance ovarian cancer survival, tumor growth, and tumor dissemination by suppressing intrinsic cell apoptosis. These data indicate BCL2A1 is an early response factor in the stressed tumor microenvironment, and targeting BCL2A1 may be a potential therapeutic approach in eradicating peritoneal metastases of ovarian cancer.Emerging evidence indicates that hypoxia plays a critical role in governing the transcoelomic metastasis of ovarian cancer. Hence, targeting hypoxia may be a promising approach to prevent the metastasis of ovarian cancer. Here, we report that BCL2A1, a BCL2 family member, acts as a hypoxia-inducible gene for promoting tumor progression in ovarian cancer peritoneal metastases. We demonstrated that BCL2A1 was induced not only by hypoxia but also other physiological stresses through NF-κB signaling and then was gradually reduced by the ubiquitin-proteasome pathway in ascites-derived ovarian cancer cells. The upregulated BCL2A1 was frequently found in advanced metastatic ovarian cancer cells, suggesting its clinical relevance in ovarian cancer metastatic progression. Functionally, BCL2A1 enhanced the foci formation ability of ovarian cancer cells in a stress-conditioned medium, colony formation in an ex vivo omental tumor model, and tumor dissemination in vivo. Under stress conditions, BCL2A1 accumulated and colocalized with mitochondria to suppress intrinsic cell apoptosis by interacting with the BH3-only subfamily BCL2 members HRK/BAD/BID in ovarian cancer cells. These findings indicate that BCL2A1 is an early response factor that maintains the survival of ovarian cancer cells in the harsh tumor microenvironment.

Abstract 3188: β-catenin in metastatic ovarian cancer cells mediates polyploidy induced by tumor-associated macrophages

Abstract Tumor-associated macrophages (TAMs) closely associate with tumor progression and could promote metastatic spread. However, how TAMs interact with individual cancer cell in the heterogeneous cancer cell population is unknown. Here, using an isogenic cell line pair with different metastatic potentials, we showed that highly metastatic (HM) cells, but not non-metastatic (NM) cells, could skew the co-cultured macrophages to TAM-like phenotypes. Single-cell tracking by time lapse imaging revealed that during co-culture, macrophages could induce failed cytokinesis in a subset of HM cells. Subsequent analyses suggested that this induction could be facilitated by direct cell-cell contact. We further identified that the cell-surface metadherin expression induced by β-catenin in HM cells is involved in this induction, while surface CEACAM1 expressed on macrophages could be responsible for the interaction with metadherin. HM-macrophage interaction promotes macrophage secretion of the cytokine CCL3, a strong chemoattractant for monocytes, which may further amplify the interaction. In an orthotopic humanized mice model, knockdown of β-catenin in HM cells reduced tumor burden as well as the number of tumor-infiltrating CD163+ TAMs. These findings demonstrate a positive feedback loop between macrophages and a subpopulation of cancer cells mediated by β-catenin to enhance the metastatic cascade (This work is supported by RGC GRF grant 17141216). Citation Format: Sally Kit Yan To, Jue Shi, Alice Sze Tsai Wong. β-catenin in metastatic ovarian cancer cells mediates polyploidy induced by tumor-associated macrophages [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 3188.

A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis.

Transcoelomic spread of cancer cells across the peritoneal cavity occurs in most initially diagnosed ovarian cancer (OC) patients and accounts for most cancer-related death. However, how OC cells interact with peritoneal stromal cells to evade the immune surveillance remains largely unexplored. Here, through an in vivo genome-wide CRISPR/Cas9 screen, we identified IL20RA, which decreased dramatically in OC patients during peritoneal metastasis, as a key factor preventing the transcoelomic metastasis of OC. Reconstitution of IL20RA in highly metastatic OC cells greatly suppresses the transcoelomic metastasis. OC cells, when disseminate into the peritoneal cavity, greatly induce peritoneum mesothelial cells to express IL-20 and IL-24, which in turn activate the IL20RA downstream signaling in OC cells to produce mature IL-18, eventually resulting in the polarization of macrophages into the M1-like subtype to clear the cancer cells. Thus, we show an IL-20/IL20RA-mediated crosstalk between OC and mesothelial cells that supports a metastasis-repressing immune microenvironment.

Glutamine regulates ovarian cancer cell migration and invasion through ETS1

Cancer cells are dependent on glutamine for their metabolism and growth. Despite being the most abundant amino acid in the blood, glutamine deprivation occurs in the core of the tumor rendering less access to glutamine to the nearby tumor cells. Tumor cells mostly use the glutamine for mitochondrial oxidative phosphorylation (OXPHOS) to produce energy and the ingredients of the biomass required for the highly proliferating and metastatic ovarian cancer cells. But there is a lack of reports on the regulation of glutamine starvation on metastatic behavior and epithelial to mesenchymal transition (EMT) of ovarian cancer cells. We found that glutamine starvation reduced the migration and invasion properties of the ovarian cancer cells, PA1 and SKOV3. The expression of the invasion-inducing proteins, like matrix metalloproteinases (MMP2 and MMP9), were downregulated upon glutamine starvation. MMP genes are mostly regulated by the ETS1 oncogenic transcription factor in invasive tumor cells. Here we demonstrated the significant involvement of ETS1 on EMT and invasion in glutamine-deprived cells. We have further shown that the regulation of ETS1 expression and nuclear localization upon glutamine starvation is controlled in a cell type-specific manner. In PA1 cells, glutamine-induced ETS1 over-expression is HIF1α-dependent, while in SKOV3, its translocation to the nucleus is regulated through the mTOR pathway. Considering all, our study suggests that glutamine plays a very significant role in migration and invasion in ovarian cancer cells and ETS1 plays a key role in inducing such oncogenic parameters.

Metastatic ovarian cancer spreading into mammary ducts mimicking an in situ component of primary breast cancer: a case report

BackgroundAccurate diagnosis of metastatic tumors in the breast is crucial because the therapeutic approach is essentially different from primary tumors. A key morphological feature of metastatic tumors is their lack of an in situ carcinoma component. Here, we present a unique case of metastatic ovarian carcinoma spreading into mammary ducts and mimicked an in situ component of primary carcinoma. To our knowledge, this is the second case (and the first adult case) confirming the in situ-mimicking growth pattern of a metastatic tumor using immunohistochemistry.Case presentationA 69-year-old Japanese woman was found to have a breast mass with microcalcifications. She had a known history of ovarian mixed serous and endocervical-type mucinous (seromucinous) carcinoma. Needle biopsy specimen of the breast tumor revealed adenocarcinoma displaying an in situ-looking tubular architecture in addition to invasive micropapillary and papillary architectures with psammoma bodies. From these morphological features, metastatic serous carcinoma and invasive micropapillary carcinoma of breast origin were both suspected. In immunohistochemistry, the cancer cells were immunoreactive for WT1, PAX8, and CA125, and negative for GATA3, mammaglobin, and gross cystic disease fluid protein-15. Therefore, the breast tumor was diagnosed to be metastatic ovarian serous carcinoma. The in situ-looking architecture showed the same immunophenotype, but was surrounded by myoepithelium confirmed by immunohistochemistry (e.g. p63, cytokeratin 14, CD10). Thus, the histogenesis of the in situ-like tubular foci was could be explained by the spread of metastatic ovarian cancer cells into existing mammary ducts.ConclusionMetastatic tumors may spread into mammary duct units and mimic an in situ carcinoma component of primary breast cancer. This in situ-mimicking growth pattern can be a potential pitfall in establishing a correct diagnosis of metastasis to the breast. A panel of breast-related and extramammary organ/tumor-specific immunohistochemical markers may be helpful in distinguishing metastatic tumors from primary tumors.