Quantitative high-throughput phenotypic screening for environmental estrogens using the E-Morph Screening Assay in combination with in silico predictions.

BACKGROUND: Exposure to environmental chemicals that interfere with normal estrogen function can lead to adverse health effects, including cancer. High-throughput screening (HTS) approaches facilitate the efficient identification and characterization of such substances. OBJECTIVES: We recently described the development of the E-Morph Assay, which measures changes at adherens junctions as a clinically-relevant phenotypic readout for estrogen receptor (ER) alpha signaling activity. Here, we describe its further development and application for automated robotic HTS. METHODS: Using the advanced E-Morph Screening Assay, we screened a substance library comprising 430 toxicologically-relevant industrial chemicals, biocides, and plant protection products to identify novel substances with estrogenic activities. Based on the primary screening data and the publicly available ToxCast dataset, we performed an insilico similarity search to identify further substances with potential estrogenic activity for follow-up hit expansion screening, and built seven insilico ER models using the conformal prediction (CP) framework to evaluate the HTS results. RESULTS: The primary and hit confirmation screens identified 27 'known' estrogenic substances with potencies correlating very well with the published ToxCast ER Agonist Score (r=+0.95). We additionally detected potential 'novel' estrogenic activities for 10 primary hit substances and for another nine out of 20 structurally similar substances from insilico predictions and follow-up hit expansion screening. The concordance of the E-Morph Screening Assay with the ToxCast ER reference data and the generated CP ER models was 71% and 73%, respectively, with a high predictivity for ER active substances of up to 87%, which is particularly important for regulatory purposes. DISCUSSION: These data provide a proof-of-concept for the combination of in vitro HTS approaches with insilico methods (similarity search, CP models) for efficient analysis of large substance libraries in order to prioritize substances with potential estrogenic activity for subsequent testing against higher tier human endpoints.

PDE5 inhibition eliminates cancer stem cells via induction of PKA signaling.

Cancer stem cells (CSCs) are involved in metastasis and resistance development, thus affecting anticancer therapy efficacy. The underlying pathways required for CSC maintenance and survival are not fully understood and only a limited number of treatment strategies to specifically target CSCs have been identified. To identify novel CSC targeting compounds, we here set-up an aldehyde dehydrogenase (ALDH)-based phenotypic screening system that allows for an automated and standardized identification of CSCs. By staining cancer cells for ALDH activity and applying high-content-based single-cell population analysis, the proportion of a potential CSC subpopulation with significantly higher ALDH activity (ALDHhigh) can be quantified in a heterogeneous cell population. We confirmed high ALDH activity as surrogate marker for the CSC subpopulation in vitro and validated Wnt signaling as an essential factor for the maintenance of CSCs in SUM149 breast cancer cells. In a small molecule screen, we identified phosphodiesterase type 5 (PDE5) inhibition as potential strategy to target CSC maintenance and survival in multiple cancer cell lines. CSC elimination by PDE5 inhibition was not dependent on PKG signaling, and we suggest a novel mechanism in which PDE5 inhibition leads to elevated cGMP levels that stimulate cAMP/PKA signaling to eliminate CSCs.

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death.

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.