Blagodatski A et al. (2017), High-throughput targeted screening in triple-ne...

ECB-ART-45768

Sci Rep 2017 Sep 20;71:11964. doi: 10.1038/s41598-017-12232-7.

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High-throughput targeted screening in triple-negative breast cancer cells identifies Wnt-inhibiting activities in Pacific brittle stars.

Blagodatski A , Cherepanov V , Koval A , Kharlamenko VI , Khotimchenko YS , Katanaev VL .

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Pro-proliferative oncogenic signaling is one of the hallmarks of cancer. Specific targeting of such signaling pathways is one of the main approaches to modern anti-cancer drug discovery, as opposed to more traditional search for general cytotoxic agents. Natural products, especially from marine sources, represent a largely untapped source of chemical diversity, which so far have mostly been screened for cytotoxicity. Here we present a pioneering pipeline of high-throughput screening of marine-based activities targeted against the Wnt signaling pathway, which is one of the key factors in oncogenic transformation, growth and metastasis in different cancers, including the devastating triple-negative breast cancer (TNBC) currently lacking any targeted therapies. This pipeline consisted of collection and characterization of numerous invertebrates during the SokhoBio expedition to the Kuril Basin in North Pacific, preparation of extracts from these specimen, and their screening in dedicated assays monitoring Wnt signaling in TNBC cells. This approach yielded a number of promising hits, including highly specific anti-Wnt activities targeting multiple levels within the Wnt pathway from Ophiura irrorata and other Pacific brittle stars.

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Genes referenced: adcy10 LOC100887844

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	Figure 1. Map of the SokhoBio expedition of the research vessel “Akademik M.A. Lavrentiev”, departing from and returning to Vladivostok, from 5th of June to 6th of August 2015 in the Kuril Basin of the Sea of Okhotsk, northwestern Pacific Ocean. 1 to 11 illustrates the eleven stations where specimens were collected (see Supplementary Table S1). Sea depths are indicated. Insert in the upper right corner places the expedition region into a bigger map of the North Pacific area. The map was drawn using the QGIS 2.16.1-Nødebo - Quantum GIS Geographic Information System, Open Source Geospatial Foundation Project by the “Quantum GIS Development Team” (2016) - a free open source geoinformation system software available under following web address: http://www.qgis.org/eng/site.
	Figure 2. Photographs (not in scale) of representatives of the ca. 80 invertebrate specimens analyzed in this study. Specimen number(s) is provided below each image. Color coding refers to each of the eleven stations, where the specimens were collected (see Supplementary Table S1 and Fig. 1).
	Figure 3. Extracts showing specific Wnt pathway-modulating activities in TNBC cells, across multiple representatives of same invertebrate groups collected at different sites. (a) Extracts from holothurians can robustly and specifically inhibit the Wnt pathway. Extracts from Peniagone sp. (specimens 59 and 94) are shown in red, and from the skin and muscular sac of Molpadia musculus (specimens 4, 37, 54, 86) in blue. The X-axis reflects the dilution of the extract (i.e. the final part of the extract in the culture media, such that dilution of 0.01 corresponds to the 1:100 dilution). The Y-axis shows the activity of the Wnt-dependent luminescence, normalized to the Wnt-independent Renilla luminescence, setting to 100% the normalized luminescence activity of cells treated with the Wnt3a-conditioned medium without any extract added. Curves are fit using the equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y={ax}\,-\frac{{bx}}{(c+x)}$$\end{document}y=ax−bx(c+x), providing c as the IC50 of the extract-induced inhibition, which is 0.0019 (1:526 dilution) for Peniagone sp. and 0.0121 (1:83 dilution) for the skin and muscular sac of Molpadia musculus. (b) Co-activation of the Wnt pathway can be observed for extracts (all shown for the 1:100 dilution) from several invertebrate groups: polychaete Travisia sp. (specimens 7, 17, 53, 64, 65), various Actinias (specimens 16, 44, 55, 78), and gonads of M.musculus (specimens 9, 60, 90). Data are presented as mean ± sem. “” signs highlight experimental points with statistically significant pairwise difference from control (no extract added) as determined by the two-way ANOVA (a) and the Student t-test (b); p value < 0.05 is indicated as “”, <0.01 as “”, <0.001 as “”, and <0.0001 as “***”. Photographs of the specimens are provided on each panel. “n” stands for the number of experiments.
	Figure 4. Different ophiuras possess strong and specific anti-Wnt activity in TNBC cells. (a,b) Extracts from brittle stars collected at station 11 (Ophiura irrorata, specimens 22 and 47), 8 (Ophiura sp. #1, specimens 38 and 98), and station 9 (Ophiura sp. #2, specimen 76), all strongly inhibit the specific Wnt-dependent transcription in a concentration-dependent manner. Curves in (a) display representative experiments performed with the indicated specimens. Curve fitting in (b) is done as in Fig. 3, with the resulting IC50 of 0.0026 (extract dilution of 1:385). (c) Extract from Ophiura irrorata (#22) also inhibits, in a concentration-dependent manner, Wnt pathway activation induced by 15 mM LiCl. Due to incomplete inhibition, IC50 of the inhibition of the LiCl-induced response cannot be adequately calculated. Statistical analysis in (b,c) is performed with the 1-way ANOVA. Data presentation is as in Fig. 3. For each dataset in (c), n = 3.

References [+] :

Amini, Metastatic Inhibitory and Radical Scavenging Efficacies of Saponins Extracted from the Brittle Star (Ophiocoma erinaceus). 2015, Pubmed, Echinobase