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.

Leishmaniavirus-Dependent Metastatic Leishmaniasis Is Prevented by Blocking IL-17A

“…We subsequently tested whether digoxin (shown to inhibit IL-17A production [ 35 ] and possibly tumour metastasis [ 36 ]) and SR1001 (described to impede the differentiation and function of T H 17 cells [ 35 , 37 ]) could reduce LRV1 mediated disease severity in our murine model. Digoxin and SR1001 treatments were commenced at the onset of visible lesional swelling (2.5 weeks post infection).…”

Octreotide-periplocymarin conjugate prodrug for improving targetability and anti-tumor efficiency: synthesis, in vitro and in vivo evaluation

“…Recently, a number of publications have emphasized that apoptosis produced by cardiotonic glycosides in human tumor cells could be achieved at concentrations with no toxicity in humans, and therefore, these agents might be useful for cancer treatment [3, 4]. Over the last 10 years, interest in developing CS as anti-cancer agents has grown progressively.…”

Screening for Biologically Annotated Drugs That Trigger Triacylglycerol Accumulation in the Diatom Phaeodactylum

“…The screen was developed to select molecules that trigger the accumulation of TAG within P. tricornutum living cells. Theoretically, hit molecules from this repurpose collection should interact with P. tricornutum proteins that share some structural and/or functional features with the original drug targets (Blagosklonny, 2003;Jeong et al, 2015;Coleman et al, 2016). Our objective was then to identify biological processes or pathways affected by the active molecules and propose lines of research for future P. tricornutum strain developments.…”