title: Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation.
abstract: The tumor microenvironment, primarily composed of myofibroblasts, directly influences the progression of solid tumors. Through secretion of growth factors, extracellular matrix deposition, and contractile mechanotransduction, myofibroblasts, or cancer-associated fibroblasts (CAFs), support angiogenesis and cancer cell invasion and metastasis. The differentiation of fibroblasts to CAFs is primarily induced by TGF-β from cancer cells. To discover agents capable of blocking CAF differentiation, we developed a high content immunofluorescence-based assay to screen repurposed chemical libraries utilizing fibronectin expression as an initial CAF marker. Screening of the Prestwick chemical library and NIH Clinical Collection repurposed drug library, totaling over 1700 compounds, identified cardiac glycosides as particularly potent CAF blocking agents. Cardiac glycosides are traditionally used to regulate intracellular calcium by inhibiting the Na+/K+ ATPase to control cardiac contractility. Herein, we report that multiple cardiac glycoside compounds, including digoxin, are able to inhibit TGF-β-induced fibronectin expression at low nanomolar concentrations without undesirable cell toxicity. We found this inhibition to hold true for multiple fibroblast cell lines. Using real-time qPCR, we determined that digoxin prevented induction of multiple CAF markers. Furthermore, we report that digoxin is able to prevent TGF-β-induced fibroblast contraction of extracellular matrix, a major phenotypic consequence of CAF differentiation. Assessing the mechanism of inhibition, we found digoxin reduced SMAD promoter activity downstream of TGF-β, and we provide data that the effect is through inhibition of its known target, the Na+/K+ ATPase. These findings support a critical role for calcium signaling during CAF differentiation and highlight a novel, repurposable modality for cancer therapy.
authors.affiliation: Louisiana State University Health Sciences Center, Feist-Weiller Cancer Center, Shreveport, LA, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Coleman
authors.affiliation: Louisiana State University Health Sciences Center, Feist-Weiller Cancer Center, Shreveport, LA, USA.
authors.forename: Alana L
authors.initials: AL
authors.lastname: Gray
authors.affiliation: Louisiana State University Health Sciences Center, Feist-Weiller Cancer Center, Shreveport, LA, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Stephens
authors.affiliation: Louisiana State University Health Sciences Center, Feist-Weiller Cancer Center, Shreveport, LA, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Scott
authors.affiliation: Louisiana State University Health Sciences Center, Feist-Weiller Cancer Center, Shreveport, LA, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Cardelli
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D020013:Calcium Signaling / Q000187:drug effects; D000072645:Cancer-Associated Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002301:Cardiac Glycosides / Q000494:pharmacology* / Q000633:toxicity; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D058492:Drug Repositioning; D005353:Fibronectins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D058628:Myofibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D059016:Tumor Microenvironment
chemical_list: D000970:Antineoplastic Agents; D002301:Cardiac Glycosides; D005353:Fibronectins; D051785:Smad Proteins; D054852:Small Molecule Libraries; D016212:Transforming Growth Factor beta; D000254:Sodium-Potassium-Exchanging ATPase
keywords: cancer-associated fibroblast; cardiac glycosides; digoxin; drug screen; tumor microenvironment