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<jats:title>Abstract</jats:title>
<jats:p>With three-dimensional growth conditions, multicellular tumor spheroids reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients, characteristic of poorly vascularized tumor regions. 3D high content screening (HCS) identified compounds that selectively kill tumor cells in the inner core of tumor cell spheroids by targeting the Stearoyl CoA Desaturase 1 (SCD1) pathway. SCD1 catalyzes the rate-limiting step in the production of mono-unsaturated fatty acids (MUFAs). Cancer cells are dependent on higher levels of MUFAs compared to normal cells and SCD1 is highly expressed in multiple tumor types. Changes in the MUFA / SFA (saturated fatty acid) ratio alters lipid biosynthesis and thus triggers cellular (ER) stress and induces the Unfolded Protein Response. Although the lead compound was very effective in vitro, it had unfavorable PK and physical chemistry properties, including low permeability and solubility and very high lipophilicity. This led to insufficient oral bioavailability, which could be overcome by optimization of PK and physical chemistry properties. Here, we report on the in vitro/in vivo effects of our 3D HCS compounds which showed high potency in the 3D spheroid inner core death assay with T47D breast cancer cells. In this in vitro model compound-induced inner core cell death is enhanced by SCD1 substrates palmitic or stearic acid and rescued by the SCD1 products palmitoleic or oleic acid. Furthermore, the effects can be reproduced in 2D cultures, which become increasingly sensitive to inhibition by our 3D HCS compounds with decreasing FBS concentration in the culture medium and this effect can also be rescued by addition of MUFAs but not of palmitic acid. Mode of action analysis showed that our compounds reduced palmitoleoyl- or oleoyl-CoA levels and simultaneously increased saturated fatty acyl-CoAs of palmitate or stearate in several cell lines as well as in vivo. In the sensitive T47D cells, the compounds induced expression of stress response genes and genes related to lipid metabolism. While these results support the SCD1 pathway as target for our 3D HCS compounds, we also observed striking differences to published SCD1 inhibitors suggesting a new cancer target beyond SCD1. Thus, further validation of our inhibitors in vitro and in vivo will be required, but these results suggest that 3D spheroid cultures may be a valuable tool for elucidation of new drug targets for cancer therapy.</jats:p>
<jats:p>Citation Format: Sylvia Gruenewald, Carolyn Sperl, Patrick Steigemann, Alexander Walter, Sylvia Zacharias, Uwe Eberspaecher, Roland Neuhaus, Ludwig Zorn, Wolfgang Schwede, Kai Thede, Sven Christian. 3D spheroid screen yields SCD1 pathway inhibitors for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4989. doi:10.1158/1538-7445.AM2017-4989</jats:p>