Ascites Tumor Research Articles

METTL3-mediated N6-methyladenosine modification and HDAC5/YY1 promote IFFO1 downregulation in tumor development and chemo-resistance

Ovarian cancer (OC) is a malignant tumor that seriously threatens women's health. Due to the difficulty of early diagnosis, most patients exhibit advanced disease or peritoneal metastasis at diagnosis. We discovered that IFFO1 is a novel tumor suppressor, but its role in tumorigenesis, development and chemoresistance is unknown. In this study, IFFO1 levels were downregulated across cancers, leading to the acceleration of tumor development, metastasis and/or cisplatin resistance. Overexpression of IFFO1 inhibited the translocation of β-catenin to the nucleus and decreased tumor metastasis and cisplatin resistance. Furthermore, we demonstrated that IFFO1 was regulated at both the transcriptional and posttranscriptional levels. At the transcriptional level, the recruitment of HDAC5 inhibited IFFO1 expression, which is mediated by the transcription factor YY1, and the METTL3/YTHDF2 axis regulated the mRNA stability of IFFO1 in an m6A-dependent manner. Mice injected with IFFO1-overexpressing cells had lower ascites volumes and tumor weights throughout the peritoneal cavity than those injected with parental cells expressing the vector control. In conclusion, we demonstrated that IFFO1 is a novel tumor suppressor that inhibits tumor metastasis and reverses drug resistance in ovarian cancer. IFFO1 was downregulated at both the transcriptional level and posttranscriptional level by histone deacetylase and RNA methylation, respectively, and the IFFO1 signaling pathway was identified as a potential therapeutic target for cancer.

A case of death of patient with ovarian fibroma combined with Meigs Syndrome and literature review

Ovarian fibroma is the most common benign pure stromal tumor. It has no specific clinical manifestation, most of which are pelvic or adnexal masses. 10-15% of cases with hydrothorax or ascites, after tumor resection, hydrothorax and ascites disappear, known as Meigs Syndrome. The elevated level of CA125 in a few patients was easily misdiagnosed as ovarian malignant tumor. A case of bilateral Ovarian fibroma associated with Meigs Syndrome is reported and the literature is reviewed in order to improve the understanding of the changes and avoid misdiagnosis.

Novel compound C150 inhibits pancreatic cancer through induction of ER stress and proteosome assembly.

Pancreatic cancer is a devastating disease with a dismal prognosis and poor treatment outcomes. Searching for new agents for pancreatic cancer treatment is of great significance. We previously identified a novel activity of compound C150 to inhibit pancreatic cancer epithelial-to-mesenchymal transition (EMT). Here, we further revealed its mechanism of action. C150 induced ER stress in pancreatic cancer cells and subsequently increased proteasome activity by enhancing proteasome assembly, which subsequently enhanced the degradation of critical EMT transcription factors (EMT-TFs). In addition, as cellular responses to ER stress, autophagy was elevated, and general protein synthesis was inhibited in pancreatic cancer cells. Besides EMT inhibition, the C150-induced ER stress resulted in G2/M cell cycle arrest, which halted cell proliferation and led to cellular senescence. In an orthotopic syngeneic mouse model, an oral dose of C150 at 150 mg/kg 3× weekly significantly increased survival of mice bearing pancreatic tumors, and reduced tumor growth and ascites occurrence. These results suggested that compound C150 holds promises in comprehensively inhibiting pancreatic cancer progression.

Utility of ascitic tumor markers and adenosine deaminase for differential diagnosis of tuberculous peritonitis and peritoneal carcinomatosis

BackgroundDifferential diagnosis between tuberculous peritonitis and peritoneal carcinomatosis remains challenging in clinical practice; thus, in-patients diagnosed with tuberculous peritonitis or peritoneal carcinomatosis were retrospectively enrolled, and diagnostic values of ascitic tumor markers and adenosine deaminase were determined.MethodsConsecutive patients diagnosed with tuberculous peritonitis or peritoneal carcinomatosis were retrospectively enrolled. The pertinent data of 169 patients enrolled were collected.ResultsA panel of ascitic tumor makers (CEA, CA15-3, CA19-9) had high specificity (96.83%) and accuracy (94.67%) in the differentiation of peritoneal carcinomatosis from tuberculous peritonitis; and ascitic ADA was a good discriminator between these patients, with an accuracy of 91.72%. Combined use of ascitic tumor makers and ADA (ascitic ADA < 22.5 IU/L or ascitic CEA > 3.65 ng/mL or CA15-3 > 42.70 U/mL or CA19-9 > 25.10 U/mL) performed high sensitivity (99.06%) and accuracy (94.08%) for the diagnosis of peritoneal carcinomatosis. In addition, combined ascitic ADA and tumor marker (positive ascitic tumor makers and ADA < 22.50 IU/L) had 100% of the specificity in diagnosing peritoneal carcinomatosis.ConclusionsCombined use of ascitic tumor markers and adenosine deaminase showed excellent efficiency in the differential diagnosis between tuberculous peritonitis and peritoneal carcinomatosis, thus these two simple and cost‐effective parameters should be determined when tuberculous peritonitis or peritoneal carcinomatosis was suspected in clinic practice.

Engineering Alendronate-Composed Iron Nanochelator for Efficient Peritoneal Carcinomatosis Treatment.

Iron is an essential element for various cellular metabolism. Cancer cells also have high requirement of iron in their proliferation, invasion, and metastasis processes. Alendronate (ALN), a kind of FDA‐approved bisphosphonates with metal‐chelating capability, is initially certified to selectively bind to intracellular Fe3+ theoretically and experimentally in this study. Hence, CaALN iron nanochelator is rationally designed to kill cancer cells by synergism of Fe‐depletion and calcium accumulation. In vitro experiments and RNA sequencing analysis indicate that CaALN nanomedicine inhibits the proliferation of cancer cells by depleting Fe, interfering with DNA replication, and triggering intracellular reactive oxygen species (ROS). Meanwhile, released Ca2+ and ROS mutually promote and induce damage of cellular macromolecules, which leads to mitochondrial apoptosis of cancer cells. In an intraperitoneal disseminated mouse model with the human ovarian cancer cells SKOV3, CaALN nanoparticles selectively accumulate in tumor tissues and result in significant retardation of tumor growth and ascites formation. The mean survival time of SKOV3‐bearing mice in treatment group is prolonged from 33 to 90 d. These results indicate that the alendronate‐originated iron chelator can serve as an efficient strategy for the treatment of peritoneal carcinomatosis.

Abstract PO024: Combination therapy with PXS-5505 improves chemotherapeutic efficacy and reduces myeloid immune suppression in murine cholangiocarcinoma – a novel therapeutic strategy for clinical translation

Abstract Introduction: Despite growing prevalence, 5-year survival for cholangiocarcinoma remains poor due to an immunosuppressive fibroinflammatory stroma, featuring abundant collagen and cancer-associated fibroblasts. Lysyl oxidases (LOX) consist of 5 secreted enzymes that catalyze collagen cross-linking, resulting in increased tumor stroma density and facilitate immune evasion. Here, we present our findings of pan-LOX inhibition with a novel orally dosed agent (PXS-5505) in a murine model of CCA. Methods: Immunohistochemistry (IHC) analysis was performed on a human tissue macro-array of CCA. Mice that develop spontaneous CCA (KPPC) or orthotopically injected with a syngeneic cell line derived from a KPPC tumor were screened for disease onset and progression with ultrasonography (U/S). Groups of KPPC mice or mice bearing orthotopic tumors were enrolled into treatment cohorts of chemotherapy with or without PXS-5505 to evaluate survival and to perform fixed time point studies with treated tumors including assessment of intratumoral interstitial fluid pressure (IFP), IHC analysis of tumor markers, chemotherapy drug perfusion into tumors by mass spectrometry, and flow cytometry analysis of tumor single cell suspensions for stromal and immune cell populations. Results: IHC analysis demonstrated significantly elevated LOX expression in both human and KPPC CCA (p&amp;lt;0.0001 and p=0.0025, respectively) compared to normal liver controls. High LOX expression was associated with worse disease-free (p=0.02) and overall (p=0.03) survival in resected human CCA specimens. KPPC mice treated with the combination of chemotherapy plus PXS-5505 demonstrated delayed tumor growth (p&amp;lt;0.0001) and ascites onset (p=0.005) with improved survival (median 77 vs. 59 days, p=0.001) compared to chemotherapy alone. In addition, combination therapy reduced IFP (p=0.002) and was associated with increased mean blood vessel area (p&amp;lt;0.0001) and increased concentration of intratumoral chemotherapy levels (p=0.02) compared to chemotherapy alone. Subsequent IHC revealed increased cell death through cleaved caspase-3 (p=0.0003) and damage-associated molecular pattern (DAMP) release through HMGB1 (p=0.0004). Flow cytometry analysis of tumors treated with combination therapy demonstrated reduced prevalence of tumor-infiltrating monocytic and granulocytic myeloid-derived suppressor cell populations (p=0.002 and p=0.01, respectively) and tumor associated macrophages (TAMs; p=0.02). There were increased CD4+ (p=0.01) T-cells with a reduced proportion of Treg+CD4+ (p&amp;lt;0.0001) T-cells. The ratio of CD8:Treg was significantly elevated in the combination therapy cohort (p=0.001) and depletion of T-cells in orthotopic tumors abrogated the improved survival with combination therapy. Conclusions: LOX is elevated in human CCA and its expression correlates with prognosis following resection. Combination therapy with PXS-5505 delays tumor progression and improves survival in murine CCA models. Thus, chemotherapy combined with PXS-5505 represents a novel therapeutic strategy for treating CCA. Citation Format: Paul R Burchard, Luis I Ruffolo, Nicholas A Ullman, Yatee Dave, Benjamin Dale, Mary Georger, Rachel Jewell, Nabeel Badri, Brian A Belt, David C Linehan, Roberto Hernandez-Alejandro. Combination therapy with PXS-5505 improves chemotherapeutic efficacy and reduces myeloid immune suppression in murine cholangiocarcinoma – a novel therapeutic strategy for clinical translation [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO024.

Effect of a novel thiazole derivative and its complexes with polymeric carriers on the processes of lipid peroxidation in lymphoma cells

Background. Many types of tumors are sensitive to changes in prooxidant-antioxidant balance. Thus, further studies on reactive oxygen species inducing antitumor drugs that generate oxidative stress-dependent cytotoxic effects are promising. Our previous works showed that thiazole derivatives in combination with polymeric carriers have a pronounced cytotoxic effect on tumor, while not being cytotoxic against pseudo-normal cells in vitro. It was found that thiazole derivatives in complex with PEG-based polymeric carriers affected the antioxidant system of lymphoma cells in vitro. The aim of this work was to study the in vitro effect of the complex of thiazole derivative N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (BF1) in combination with polymeric carriers poly(VEP-co-GMA)-graft-mPEG (Th1), poly(PEGMA) (Th3) and poly(PEGMA-co-DMM) (Th5) on the level of lipid peroxidation products in NK/Ly cells. Materials and Methods. The experiments were conducted on white wild-type male mice with a grafted NK/Ly lymphoma. Ascites tumor cells were inoculated into mice intraperitoneally. Abdominal drainage with ascites of anesthetized mice was performed with a sterile syringe on the 7th–10th days after inoculation. Investigated compounds BF1, polymeric carriers Th1, Th3, Th5 and combination of BF1 + Th1 (Th2), BF1 + Th3 (Th4) and BF1 + Th5 (Th6) at a final concentration of 10 μM were added to the lymphoma samples and incubated for 10 minutes. The level of lipid peroxidation products, such as lipid hydroperoxides and thiobarbituric acid-positive products) were determined according to the techniques described below. Results. All applied complexes based on thiazole derivative BF1 and PEG-based polymeric carriers at a concentration of 10 μM increased the activity of lipid hydroperoxi­des in the lymphoma cells by 29–36% compared to control. Complexes Th2 and Th6 increased the significance of BF1 influence on lymphoma cells from P &lt;0.05 to P &lt;0.01. Among all of the studied complexes, Th4 and Th6 significantly increased the level of TBA-positive products, while Th2 and BF1 did not change the content of the secondary products of lipid peroxidation. None of the unconjugated polymeric carriers affected the level of lipid peroxidation products. Conclusions. Thus, based on the results of this work, thiazole derivative BF1 in complex with polymeric carriers increases the level of primary and secondary products of lipid peroxidation in lymphoma cells. Polymeric carriers enhanced the effect of thiazole derivative on the studied parameters, so complexes of thiazole derivatives and PEG-containing polymeric carriers should be taken into consideration and further investigated as potential antitumor agents.

Mutation analysis of circulating tumor DNA and paired ascites and tumor tissues in ovarian cancer

Circulating tumor DNA (ctDNA) is one conventional type of liquid biopsy that can be collected to dynamically monitor disease status. However, its potential clinical value and concordance with ascites samples or tumor biopsy needs to be evaluated further for patients with ovarian cancer. Therefore, the present study compared the mutation profiles among ctDNA, paired tumor tissue and ascites samples to explore their possible clinical value in ovarian cancer. Targeted next-generation sequencing was used to screen for mutations in 18 peripheral blood samples, six paired ascites samples and eight paired tumor tissues collected from patients with ovarian cancer. Functional analyses were performed using public databases. WebGestalt was used to perform Gene Ontology and pathway enrichment analyses. The cBioPortal for Cancer Genomics was used to assess therapeutic targets. Chilibot and Search Tool for the Retrieval of Interacting Genes/Proteins were used to obtain key genes and their functional interactions. Comparative analysis was performed among the three types of samples using Venn diagram. A total of 104 cancer-associated mutant genes in ctDNA samples, 95 genes in tumor tissues and 44 genes in ascites samples were found. A cluster covering 10 genes, namely NOTCH2, NOTCH3, lysine methyltransferase 2A, PTEN, androgen receptor, DNA-activated protein kinase catalytic subunit, hepatocyte nuclear factor 1 homeobox A, SRC, insulin receptor substrate 2 and SRY-box transcription factor 10, was obtained by Chilibot analysis. This gene panel may have the potential to monitor metastasis and identify therapeutic targets in ovarian cancer. Taken together, the present study focused on the mutant genes in ctDNA, ascites and tumor tissues, and suggested that the integrated information of different samples could be examined to comprehensively reflect the mutational landscape in ovarian cancer. However, procedures and protocols to interpret and utilize the integrated information obtained from various forms of liquid biopsies will require optimization prior to their use for future clinical applications.

Switching indication of PEGylated lipid nanocapsules-loaded with rolapitant and deferasirox against breast cancer: Enhanced in-vitro and in-vivo cytotoxicity

Breast cancer (BC) is considered the leading cause of mortality and morbidity among adult women worldwide, and it is associated with many genetic or hormonal factors. Despite the advanced therapeutic and theranostic strategies for BC treatment, cancer metastasis and relapse are often observed among patients which lead to therapeutic failure. Accordingly, among the repositioned medication against BC proliferation is neurokinin receptor antagonists and iron chelating agents especially rolapitant HCl (RP) and deferasirox (DFO), respectively. However, RP and DFO are classified as class II with low aqueous solubility. Both drugs were nanoformulated into PEGylated lipid nanocapsules (LNCs) for enhancing their aqueous solubility and augmenting their efficacy. RP-LNCs, DFO-LNCs and their combinations were evaluated according to particle size (PS), zeta potential, polydispersity index (PDI) and surface morphology. Importantly, the antitumor effect of these novel molecules and their nanoforms was evaluated against the suppression of Ehrlich Ascites tumor model using female mice. Results revealed that RP-LNCs, DFO-LNCs and RP/DFO-LNCs exerted PS from 45.23 ± 3.54 to 60.1 ± 3.32 nm with PDI around 0.20 which indicates homogenous particles distribution. Also, RP-LNCs, DFO-LNCs and RP/DFO-LNCs displayed surface charges of +16.6 ± 6.9, −13.3 ± 5.82 and − 20.2 ± 5.40 mV, respectively. The obtained LNCs conferred a high potent cytotoxic effect against MCF7 cancer cells as compared to parent drugs, with IC50 of 10.86 ± 0.89, 3.34 ± 0.99 and 2.24 ± 0.97 μg/mL for RP-LNCs, DFO-LNCs and RP/DFO-LNCs, respectively. The in-vivo pharmacodynamics effect of the developed nano-formulations showed superior antitumor effect for the individual drugs rather than their combinations as compared to the control group. The current study confirmed the potential of RP and DFO nanoforms as promising therapeutic agents for BC treatment.

Green self-assembled lactoferrin carboxymethyl cellulose nanogels for synergistic chemo/herbal breast cancer therapy

The current treatment protocols for breast cancer have shifted from single agent therapies to combinatorial approaches that offer synergistic efficacies and reduced side effects. Self-assembled nanogels comprising natural polysaccharides and functional proteins provide an intelligent platform for the targeted co-delivery of therapeutic molecules. Herein, we report the fabrication of self-assembled nanogels utilizing hydrophilic biocompatible proteins, lactoferrin (Lf), and polysaccharide carboxy methyl cellulose (CMC), for the combined delivery of the antimetabolite pemetrexed (PMT) and the herbal polyphenol honokiol (HK). PMT was conjugated to LF via an amide bond. The conjugate was then electrostatically assembled into CMC under optimized conditions to form nanogels (Lf-CMC NGs). An inclusion complex of HK with hydroxypropyl-β-cyclodextrin was then encapsulated in the prepared Lf-CMC NGs with an entrapment efficiency of 66.67%. The dual drug-loaded cross-linked Lf-CMC NGs exhibited a particle size of 193.4 nm and zeta potential of − 34.5 mV and showed a sustained release profile for both drugs. PMT/HK-loaded Lf-CMC NGs were successfully taken up by MDA-MB-231 breast cancer cells and demonstrated superior in vitro cytotoxicity, as elucidated by a low combination index value (CI=0.17) and a higher dose reduction index (DRI) compared to those of the free drugs. An in vivo antitumor study using an Ehrlich ascites tumor (EAT) mouse model revealed the robust efficacy of PMT/HK-loaded Lf-CMC NGs in inhibiting tumor growth, which was ascribed to the reduced expression level of VEGF-1, elevated protein expression level of caspase-3, and suppressed Ki-67 protein level in the tumor tissue (P ˂0.05). In conclusion, our green fabricated self-assembled dual-loaded nanogels offer a promising biocompatible strategy for targeted combinatorial breast cancer therapy.

Abstract 6040: Orthotopic patient-derived xenograft models of appendiceal adenocarcinoma identify intraperitoneal paclitaxel treatment as an active therapeutic agent

Abstract Background Appendiceal adenocarcinomas (AAs) are a rare and heterogeneous mix of tumors for which only few preclinical models exist for drug discovery. These tumors are commonly treated with chemotherapy like those for colorectal cancer despite clear evidence that the two cancers are distinctly different in their clinical behavior and tumor molecular profiles. In this study, we created PDX models of appendix cancer and tested the efficacy of IP paclitaxel treatment. Methods After obtaining informed consent, fresh tumor samples were obtained from 9 patients with AA cancer undergoing surgical resection at MD Anderson Cancer Center. Tumors were implanted in both the flank and peritoneum of NSG mice; from each patient 5 mice were implanted with approx. 0.5 cm3 tumor. Mice were evaluated after 6 months to assess for tumor formation. Additionally, 3 AA PDX models were obtained from collaborators and implanted. A PDX model was treated with paclitaxel (25 mg/kg, IP, 3 weekly treatments and 1 week off, for two cycles). Tumor size in MRI was evaluated by mucinous peritoneal Response Evaluation Criteria In Solid Tumors (mpRECIST) method. Results None of 5 well differentiated (grade 1) tumors grew in either flank and peritoneum (0 of 10 mice and 0 of 15 mice). Two of 3 moderately differentiated (grade 2) tumors grew in the peritoneum [8 of 9 mice (89%) and 8 of 8 mice (100%)], while only 1 grew in the flank [2 of 4 mice (50%)]. Three poorly differentiated (grade 3) tumors grew in the peritoneum [13 of 13 mice (100%), 8 of 8 mice (100%), and 2 of 5 mice (40%)]. Two of these tumors also grew in the flank but with lower rate of formation [8 of 8 mice (100%), 2 of 4 mice (50%), and 0 of 1 mouse (0%)]. Overall, the tumor formation rate was greater in peritoneum vs flank (30.8% vs 16.7%). Although low-grade tumor didn’t grow, the formation rates of grade 2 and 3 tumors were 56% and 67% in the peritoneum. PMP-2 and PMCA-3 models grew as a mucinous ascites, MAd20-001 grew as solid tumors, while TM00351 formed a mix of solid tumor and mucinous ascites. Histologically, in MAd20-001, neoplastic cells were poorly differentiated and arranged in sheets with frequent mitoses. PMP-2 and PMCA-3, both with GNASR201C mutation, had a mucinous phenotype similar to GNAS mutant appendiceal tumors in humans. IP paclitaxel treatment of the TM00351 showed 77.9% reduction of tumor growth relative to no treatment control mice at 12 weeks. No significant side effect of the treatment was seen in any mice. IP paclitaxel treatment of additional models is ongoing. Conclusion Interaction with the peritoneal microenvironment is important to promote growth of appendiceal tumors. Orthotopic PDX models recapitulate clinical, histologic, and molecular features of AA, including the indolent growth of low-grade and aggressive growth of high-grade tumors. Using this system we identify IP paclitaxel as an active agent for high-grade AA. Citation Format: Ichiaki Ito, Michael G. White, Abdelrahman M. Yousef, Princess N. Dickson, Yue Gu, Keith F. Fournier, John P. Shen. Orthotopic patient-derived xenograft models of appendiceal adenocarcinoma identify intraperitoneal paclitaxel treatment as an active therapeutic agent [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 6040.

Abstract 128: SIRT3 supports anchorage-independent survival of ovarian cancer cells

Abstract Metabolic changes during tumor progression have numerous consequences on cancer cells, including an increased risk of cell death due to production of reactive oxygen species (ROS). Antioxidant defense adaptations thus accompany metabolic alterations as essential survival mechanisms during metastasis. We observe that oxidative phosphorylation is increased when ovarian cancer (OC) cells transition to anchorage independence (a-i), an important step during metastasis in the peritoneal cavity. To combat the rise in oxidative stress resulting from this metabolic shift, a rapid increase in the sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) axis occurs to reduce the mitochondrial superoxide anion surge and to maintain cellular viability. We demonstrate that this upregulation occurs in transcription, translation, and SIRT3-mediated regulation of SOD2 activity. However, the fate of the hydrogen peroxide (H2O2) resulting from superoxide dismutation through SIRT3/SOD2 remains unknown. RNAseq analysis of attached versus a-i conditions revealed a significant downregulation of peroxiredoxin 3, a major scavenger of H2O2 in the mitochondria, suggesting that OCs have a net increase in mitochondrial H2O2 in a-i. At sub-lethal levels, H2O2 serves as a second messenger of redox signaling, primarily via protein cysteine oxidation, which has been implicated with metastasis. We thus hypothesize that increases in SIRT3/SOD2/H2O2 play a role as mediators of redox signaling during OC anchorage-independent survival, spheroid formation and metastasis. Using the HyPer7 redox probe and manipulation of SIRT3 and SOD2 expression, H2O2 generation and localization in a-i are being assessed using 3D fluorescence microscopy. Moreover, siRNA mediated knock-down of SIRT3 in a-i leads to the downregulation of genes involved in specific signaling pathways, including NF-kB signaling, the inflammatory response, and metabolism. Mass-spec studies are underway to identify specific SIRT3 target proteins upstream of these transcriptional changes, by assessing global cysteine oxidation and the cellular acetylome to determine the role of H2O2-dependent redox signaling and SIRT3-dependent protein de-acetylation, respectively. Given the increase in SIRT3-dependent inflammatory signaling in a-i, we are exploring how the SIRT3/SOD2/H2O2 axis influences OC interaction with tumor associated macrophages and mesenchymal stem cells, commonly found in the ascites tumor environment. This work demonstrates a multi-faceted role for the SIRT3/SOD2/H2O2 axis in a-i survival, a key step during OC metastasis. Citation Format: Priscilla Tang, Yeon Soo Kim, Piyushi Gupta-Vallur, Amal T. Elhaw, Zaineb Javed, Weihua Pan, Nadine Hempel. SIRT3 supports anchorage-independent survival of ovarian cancer cells [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 128.

Abstract 107: Reciprocal Sox2 regulation by SMAD1 and SMAD3 is critical for anoikis resistance and metastasis in ovarian cancer

Abstract The tumor ascites environment is enriched with cytokines and growth factors especially members of the Transforming Growth Factor-β (TGF-β) family, that play crucial roles in modulating anoikis sensitivity and consequently metastatic progression in ovarian cancer. Here, we demonstrated for the first-time significant broad dichotomy between these TGF-β members: BMPs and TGF-β/activin and their regulated SMADs in this process. We previously reported BMP9/GDF2 to be in low to undetected levels in ovarian cancer patient-derived ascites and promoted anoikis resistance in both breast and ovarian cancer cell lines. We have now identified additional BMPs -BMP2, BMP4 and BMP9 as promoting anoikis sensitivity in a spectrum of ovarian cancer cells. Conversely, we found TGF-βs (1 and 2) to be in much higher expression and demonstrated that TGF-β and activin promoted anoikis resistance in ovarian cancer. Hence, in an attempt to identify genes downstream of BMPs that may provide anoikis resistance, transcriptomics was performed leading to the identification of Sox2, a developmental gene with prior established roles in OVCA, as being significantly downregulated in response to BMP9. We further uncovered Sox2 to be reciprocally regulated by anoikis-promoting BMPs (2, 4 and 9) and anoikis suppressing TGF-β and activin A. Our findings highlight a novel contrasting SMAD dependent regulation of Sox2 as a central node for controlling tumor cell survival in ovarian cancers demonstrating a subset of BMPs as a therapeutic strategy in cancer. The studies presented here begin to elucidate the mechanisms of reciprocal epigenetic regulation of Sox2 by BMP and TGF-β/activin in the context of ovarian cancer, which is the most lethal gynecologic malignancy in women due to peritoneal metastatic spread. Citation Format: Zainab M. Shonibare, Mehri Monavarian, Kathleen O'Connell, Diego Altamore, Abigail Shelton, Shubham Mehta, Renata Jaskula-Sztul, Rebecca Phaeton, Andrew Berchuck, Andrew B. Nixon, Rebecca Arend, Nam Y. Lee, Ryan Miller, Nadine Hempel, Karthikeyan Mythreye. Reciprocal Sox2 regulation by SMAD1 and SMAD3 is critical for anoikis resistance and metastasis in ovarian cancer [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 107.

Abstract 3264: Ex vivo treatment in high grade serous ovarian cancer demonstrates the benefit of EZH2 inhibition in combination with standard therapy

Abstract While high-grade serous ovarian cancers (HGSOC) are initially sensitive to platinum-based therapy, resistance to chemotherapy remains a major challenge. Recently, the role of EZH2 in establishing a chemo-resistant state has been described in small cell lung cancer with a potential mechanism thought to involve the silencing of SLFN111. Preclinical studies showed that EZH2 inhibition results in re-expression of SLFN11 protein and restores sensitivity to DNA damaging agents. A study in ovarian cancer2 emphasized the need to investigate the effect further. Here we describe the response of platinum-resistant HGSOC patient-derived xenograft (PDX) tumors to CPI-0209, an investigational new drug that is currently being explored for its potential as a reversible second generation EZH2 inhibitor as monotherapy and in combination with carboplatin or PARP inhibition. Four luciferized PDXs (DF101, DF149, DF181, and DF216) were selected based on known platinum sensitivity and BRCA mutation status. PDX tumor ascites were harvested from mice at the IVIS signal intensity of 1011. Xenograft-derived organotypic tumor spheroids (xDOTS3) were loaded into 3D devices (AIM Biotech), treated with either CPI-0209, carboplatin, or olaparib alone, or with CPI-0209 for 7 days prior to adding carboplatin or olaparib for an additional 5-11 days. H3K27me3 was assessed by immunofluorescence. Expression of SLFN11 following treatment was assessed by western blot. Activity was measured at endpoint by dual-labeling fluorescence microscopy. EZH2-dependent target engagement via demethylation of H3K27me3 was seen in all models. While DF181 (BRIP1m) showed resistance to all treatments, DF216 (BRCAwt) showed a consistent trend in reduction in live area in the combination of carboplatin or olaparib with CPI-0209 vs single agent, and significant reduction (&amp;gt;20%; P&amp;lt;0.05) vs control. DF101 (BRCAm) and DF149 (BRCAwt), which showed no evidence of sensitivity to carboplatin and CPI-0209 monotherapy, showed sensitivity to olaparib which was not enhanced by the addition of CPI-0209. Further assessment of DF216 via western blot analysis showed elevated expression of SLFN11 after 7 days of EZH2i treatment. Since PDX in vivo studies are time-consuming and costly, it is impractical to use them for large drug screens. Here we show that our short-term and efficient xDOTS platform allows for rapid evaluation of multiple targeted therapies or therapeutic combinations. The results suggested that CPI-0209 directed combination therapy could enhance activity over standard chemotherapy alone in a platinum-resistant model of ovarian cancer ex vivo. Increased SLFN11 protein level supported the importance of EZH2-SLFN11 axis. Further exploration of CPI-0209 combination in ovarian cancer in known genomic contexts is warranted. Citation Format: Ha V. Vo, Qing Zeng, David A. Barbie, Prafulla C. Gokhale, Elizabeth Adams, Cloud P. Paweletz, Elena Ivanova. Ex vivo treatment in high grade serous ovarian cancer demonstrates the benefit of EZH2 inhibition in combination with standard therapy [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 3264.

Flaxseed Ethanol Extracts' Antitumor, Antioxidant, and Anti-Inflammatory Potential.

The antitumoral, antioxidant, and anti-inflammatory effects of flaxseed ethanol extract was screened. Phytochemical analysis was performed by measuring the total phenolic content and by HPLC-DAD-ESI MS. In vitro antiproliferative activity was appreciated by MMT test of four adenocarcinomas and two normal cell lines. In vitro, antioxidant activity was evaluated by DPPH, FRAP, H2O2, and NO scavenging tests. The in vivo growth inhibitory activity against Ehrlich ascites carcinoma (EAC) in female BALB/c mice was determined using the trypan blue test. In EAC mice serum and ascites total oxidative status, total antioxidant reactivity, oxidative stress index, malondialdehyde, total thiols, total nitrites, 3-nitrotyrosine, and NFkB were measured. The phytochemical analysis found an significant content of phenols, with lignans having the highest concentration. The extract had an significant in vitro antioxidant effect and different inhibitory effects on different cell lines. After treatment of EAC mice with flaxseeds extract, body weight, ascites volume and viable tumour cell count, serum and ascites oxidative stress, and inflammatory markers decreased significantly. The ethanol flaxseeds extract has potential antiproliferative activity against some ovary and endometrial malignant cells and EAC. This effect can be attributed to the phenols content, and its antioxidant and anti-inflammatory activity.

DNA Repair Protein HELQ and XAB2 as Chemoresponse and Prognosis Biomarkers in Ascites Tumor Cells of High-Grade Serous Ovarian Cancer.

Nucleotide excision repair (NER) is an important mediator for responsiveness of platinum-based chemotherapy. Our study is aimed at investigating the NER-related genes expression in ascites tumor cells and its application in the prediction of chemoresponse in high-grade serous ovarian cancer (HGSC) patients. The relationship between 16 NER-related genes and the prognosis of ovarian cancer was analyzed in the TCGA database. NER-related genes including HELQ and XAB2 expressions were determined via immunocytochemistry in ascites cell samples from 92 ovarian cancer patients prior to primary cytoreduction surgery. Kaplan-Meier analysis and Cox model were used to investigate the association between NER-related gene expression and prognosis/chemotherapeutic response. Predicting models were constructed using a training cohort of 60 patients and validated in a validation cohort of 32 patients. We found that high expression of HELQ and XAB2 in the training cohort was associated with poor prognosis (for HELQ, P = 0.001, HR = 2.83, 95% CI: 1.46-5.49; for XAB2, P = 0.008, HR = 2.38, 95% CI: 1.23-4.63) and platinum resistance (for HELQ, P < 0.001; for XAB2, P = 0.006). In the validation cohort, the combination of HELQ and XAB2 (AUC = 0.863) showed the highest AUC. The expression levels of HELQ (RR 5.7, 95% CI 1.7-19.2) and XAB2 (RR 3.2, 95% CI 0.9-10.8) in ascites tumor cells were positively correlated to the risk of platinum resistance. In summary, we revealed that the expression levels of HELQ and XAB2 are candidate predictors for primary chemotherapy responsiveness and prognosis in HGSC. Ascites cytology is applicable as a promising method for chemosensitivity prediction in HGSC.

Usefulness of Tumor Markers in Ascitic Fluid for the Diagnosis of Gastric Cancer-associated Malignant Ascites

See the article "Diagnostic Value of Ascitic Tumor Markers for Gastric Cancer-associated Malignant Ascites" on page 38.

Diagnostic Value of Ascitic Tumor Markers for Gastric Cancer-associated Malignant Ascites

Background/Aims: Peritoneal carcinomatosis with malignant ascites is common in patients with advanced gastric cancer (GC). The detection of tumor cells is the gold standard for the diagnosis of malignant ascites; however, it often requires complementary tests because of its low sensitivity. Herein, we measured the levels of tumor markers in the malignant ascites of GC patients (GC-ascites) and benign ascites of liver cirrhosis patients (LC-ascites) to elucidate the diagnostic value of tumor markers in GC-ascites.Materials and Methods: The levels of CEA, cancer antigen 72-4 (CA 72-4), CA 19-9, and CA 125 were measured in 138 GC-ascites and 64 LC-ascites samples obtained from the National Biobank of Korea. We performed receiver operating characteristic curve analysis to determine the optimal cutoff value for each tumor marker.Results: CEA, CA 72-4, and CA 19-9 levels were significantly higher in GC-ascites than in LC-ascites. There was no difference in tumor marker levels between GC-ascites samples irrespective of cytology. CEA, CA 72-4, and CA 19-9 had sensitivities of 85.5%, 79.0%, and 61.6%, respectively, and specificities of 96.8%, 100.0%, and 89.1%, respectively, for distinguishing GC-ascites samples from LC-ascites samples. The diagnostic accuracy was improved by combining two or more tumor markers. The combination of CEA and CA 72-4 showed the highest sensitivity (86.2%) and specificity (100%).Conclusions: Measurement of tumor markers, such as CEA, CA 72-4, and CA 19-9, in ascites samples could help diagnose GC-ascites, and combining two or more tumor markers could further increase the diagnostic yield, even in cytology-negative patients.

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Beatrice Mintz (1921-2022): an innovator in embryo research and cancer biology

Long-term results of the enzyme glutation system activity in experimental ovarian cancer

The article presents the experimental results of the investigation the glutathione system activity (glutathione peroxidase and glutathione reductase) in rats simulated ovarian ascites tumor. In the experimental carcinogenesis of the ovaries, the activity of glutathione reductase and glutathione peroxidase was inhibited at all intervals of the study during long-term observation. After the presence of carcinogenesis in the body, the number of reactions that do not lead to complete utilization of metabolites. Glutathione reductase activity increases between 4 and 6 months. And the activation of glutathione peroxidase for the entire period only decreases. The experiment showed that gammaglobulin with long-term treatment can increase the activity of the glutathione system.