High‐throughput Screening for Identification of Inhibitors of EpCAM‐Dependent Growth of Hepatocellular Carcinoma Cells

Abstract

The cancer stem cell marker, EpCAM, is an important indicator of wnt-β-catenin signaling activation and a functional component of hepatocellular tumor initiating cells. A high-throughput screening assay was developed to identify inhibitors of EpCAM-dependent growth of hepatocellular carcinoma cells. EpCAM(+) and EpCAM(−) HCC cell lines were assessed for differential sensitivity to a wnt-β-catenin pathway inhibitor. Libraries comprising 22,668 pure compounds and 107,741 crude or partially purified natural product extracts were tested and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express EpCAM), but resulted in generation of cell populations expressing lower levels of EpCAM. Sublethal concentrations (~IC50) reduced median EpCAM expression to 28% of control after 1 d and 19% of control after 2 d. Reduction in EpCAM expression preceded growth inhibition suggesting that a threshold of EpCAM expression may be required for growth of EpCAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth.

Three-dimensional Organotypic Culture Models of Human Hepatocellular Carcinoma

“…EpCAM + HCC cells, particularly Huh1 cells, can efficiently form organoid-like spheroids with features of glandular epithelium in vitro , which include acinar morphogenesis, but are chemoresistant and have elevated metastases in orthotopic liver cancer models. We also found that EpCAM + HCC cell organoids rely on the presence of EpCAM and β-catenin signaling since RNAi-mediated silencing of EpCAM or small molecular inhibitors of wnt-β-catenin signaling, identified from a high-throughput screen of small molecule libraries from the Developmental Therapeutics Program of NCI 25 , can effectively inhibit organoid formation. Further characterization of these wnt-β-catenin inhibitors, including examining a combination of these inhibitors with conventional chemotherapeutics, using this organotypic culture model is an important future endeavor.…”

“…3 ). Because EpCAM is a direct transcriptional target of Wnt/β-catenin signaling 17 , we also examined whether β-catenin inhibitors have an effect on Huh1 spheroid formation using small molecular inhibitors of wnt/β-catenin signaling that were previously identified via a high throughput screen (i.e., AV606 and fiduxosin 25 ). The optimum concentration of each drug was determined by adopting a dose in which the number of Huh1 spheroids formed (Wnt/β-catenin active) is at least 1.5-fold less than the number of MHCC97 spheroids formed (Wnt/β-catenin negative) ( Suppl Fig.…”

Epithelial‐to‐Mesenchymal Transition Induced by TGF‐β1 Is Mediated by AP1‐Dependent EpCAM Expression in MCF‐7 Cells

“…Recent studies have shown a role for EpCAM in cell signaling, carcinogenesis, and stem cell biology (Gadalla et al, ; Henrich et al, ; Ni et al, ). The observation that there is increased expression of EpCAM in breast cancer implies that increased expression of EpCAM may confer anti‐apoptostic property to further promote tumorigenesis and progression (Martowicz et al, ,b; Saadatmand et al, ).…”

Inhibition of wnt/β-catenin Signaling in Hepatocellular Carcinoma by an Antipsychotic Drug Pimozide

“…The repositioning of non-cancer drugs by screening for novel anti-cancer activity provides a powerful strategy to rapidly identify new cancer therapeutics from the existing pool of small molecules that have already undergone rigorous testing via clinical trials for human safety, thus potentially fast-tracking the development and approval process, while reducing risk 13 . Recently, in a high-throughput drug screen designed to identify inhibitors of EpCAM dependent growth, our laboratory discovered that an FDA-approved drug pimozide (PMZ), a psychotropic dopamine receptor antagonist 14 , preferentially inhibited proliferation in several EpCAM expressing HCC cell lines, compared to EpCAM negative HCC cell lines 15 .…”