Metrics & MoreArticle Recommendations * sı Supporting Information ABSTRACT: (+)-Digoxin ( 1) is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides (2−5) and two new analogues, (+)-8(9)-β-anhydrodigoxigenin ( 6) and (+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7), were synthesized from 1 and evaluated for their cytotoxicity toward a small panel of human cancer cell lines. A preliminary structure−activity relationship investigation conducted indicated that the C-12 and C-14 hydroxy groups and the C-17 unsaturated lactone unit are important for 1 to mediate its cytotoxicity toward human cancer cells, but the C-3 glycosyl residue seems to be less critical for such an effect. Molecular docking profiles showed that the cytotoxic 1 and the noncytotoxic derivative 7 bind differentially to Na + /K + -ATPase. The HO-12β, HO-14β, and HO-3′aα hydroxy groups of (+)-digoxin (1) may form hydrogen bonds with the side-chains of Asp121 and Asn122, Thr797, and Arg880 of Na + /K + -ATPase, respectively, but the altered lactone unit of 7 results in a rotation of its steroid core, which depotentiates the binding between this compound and Na + /K + -ATPase. Thus, 1 was found to inhibit Na + /K + -ATPase, but 7 did not. In addition, the cytotoxic 1 did not affect glucose uptake in human cancer cells, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na + /K + -ATPase but not by interacting with glucose transporters.

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer deaths among women yet effective targeted therapies against this disease are limited. The heterogeneity of HGSOC, including few shared oncogenic drivers and origination from both the fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE), has hampered development of targeted drug therapies. PAX8 is a lineage-specific transcription factor expressed in the FTE that is also ubiquitously expressed in HGSOC where it is an important driver of proliferation, migration, and cell survival. PAX8 is not normally expressed in the OSE, but it is turned on after malignant transformation. In this study, we use proteomic and transcriptomic analysis to examine the role of PAX8 leading to increased migratory capabilities in a human ovarian cancer model, as well as in tumor models derived from the OSE and FTE. We find that PAX8 is a master regulator of migration with unique downstream transcriptional targets that are dependent on the cell’s site of origin. Importantly, we show that targeting PAX8, either through CRISPR genomic alteration or through drug treatment with micelle encapsulated thiostrepton, leads to a reduction in tumor burden. These findings suggest PAX8 is a unifying protein driving metastasis in ovarian tumors that could be developed as an effective drug target to treat HGSOC derived from both the OSE and FTE.

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High grade serous ovarian cancer (HGSC), the most common and lethal form of ovarian cancer, is a highly heterogeneous disease. HGSC is rarely detected early, and likely arises from the fimbriated end of the fallopian tube epithelium (FTE), and in some cases, the ovarian surface epithelium (OSE). PAX8 is a commonly used biomarker for ovarian serous tumors and is expressed in ~90% of HGSC. Although the OSE does not express PAX8, murine models of HGSC derived from the OSE acquire PAX8, suggesting that it is not only a marker of Müllerian origin, but also an essential part of cancer progression, potentially from both the OSE and FTE. Our data shows that PAX8 loss by CRISPR and shRNA in HGSC cell lines causes tumor cell death and reduces cell migration and invasion. Additionally, loss of PAX8 significantly reduced tumor burden in a xenograft model of HGSC. Herein, secretome analysis was performed on PAX8 deleted cells, and we identified a reduction of the extracellular matrix (ECM) components, collagen and fibronectin. Immunoblotting and immunofluorescence in PAX8 deleted OVCAR8 HGSC cells further validated the results from the secretome analysis. PAX8 loss reduced the amount of secreted TGFbeta, a cytokine that plays a crucial role in remodeling of the tumor microenvironment. Furthermore, PAX8 loss reduced the integrity of 3D spheroids and caused a reduction of ECM proteins in 3D cultures: fibronectin and collagen. Due to the ubiquitous expression of PAX8 in HGSC, regardless of cell origin, and evidence that reducing PAX8 protein levels inhibits tumor growth, a PAX8 inhibitor could be a promising drug lead against HGSC. To accomplish this, we generated a murine oviductal epithelial (MOE) cell line stably expressing the PAX8 promoter driving luciferase reporter protein. Using this cell line, we performed a screening assay with a library of FDA-approved drugs (Prestwick Library) and quantitatively assessed these compounds for their inhibition of PAX8-luciferase. We identified two hits: losartan and captropril, both inhibitors of the renin-angiotensin pathway that inhibit PAX8 expression and function. We are currently working to monitor if these compounds reduce tumor burden via PAX8 reduction. Further, if PAX8 reduction in vivo diminishes collagen and fibronectin, this may impact immune cell infiltration via changes in the tumor microenvironment. Overall, this study validates PAX8 as a regulator of ECM deposition in the tumor microenvironment. Citation Format: Amrita Salvi, Laura R. Hardy, Kimberly N. Heath, Samantha Watry, Melissa R. Pergande, Stephanie M. Cologna, Joanna E. Burdette. PAX8 modulates the tumor microenvironment of high grade serous ovarian cancer through changes in the secretome [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B030.