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. 2012 Mar 20;109(12):4609-14.
doi: 10.1073/pnas.1200305109. Epub 2012 Mar 6.

Versatile pathway-centric approach based on high-throughput sequencing to anticancer drug discovery

Hairi Li  1 , Hongyan Zhou, Dong Wang, Jinsong Qiu, Yu Zhou, Xiangqiang Li, Michael G Rosenfeld, Sheng Ding, Xiang-Dong Fu
Affiliations

Versatile pathway-centric approach based on high-throughput sequencing to anticancer drug discovery

Hairi Li et al. Proc Natl Acad Sci U S A. .

Abstract

The advent of powerful genomics technologies has uncovered many fundamental aspects of biology, including the mechanisms of cancer; however, it has not been appropriately matched by the development of global approaches to discover new medicines against human diseases. Here we describe a unique high-throughput screening strategy by high-throughput sequencing, referred to as HTS(2), to meet this challenge. This technology enables large-scale and quantitative analysis of gene matrices associated with specific disease phenotypes, therefore allowing screening for small molecules that can specifically intervene with disease-linked gene-expression events. By initially applying this multitarget strategy to the pressing problem of hormone-refractory prostate cancer, which tends to be accelerated by the current antiandrogen therapy, we identify Peruvoside, a cardiac glycoside, which can potently inhibit both androgen-sensitive and -resistant prostate cancer cells without triggering severe cytotoxicity. We further show that, despite transcriptional reprogramming in prostate cancer cells at different disease stages, the compound can effectively block androgen receptor-dependent gene expression by inducing rapid androgen receptor degradation via the proteasome pathway. These findings establish a genomics-based phenotypic screening approach capable of quickly connecting pathways of phenotypic response to the molecular mechanism of drug action, thus offering a unique pathway-centric strategy for drug discovery.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The scheme of the HTS2 technology. (A) The flow of the HTS2 technology. (B and C) Representative responses to androgen (DHT) and the effects of known antiandrogen compounds on a panel of DHT-responsive genes (B) and a set of housekeeping and cytotoxicity-related genes (C). Three vehicle-treated samples are averaged to serve as the baseline and variations from the baseline are color-coded: red for DHT-induced genes and blue for DHT-suppressed genes. The ES, SS, and SD are shown on the bottom of each panel for each compound.
Fig. 2.
Fig. 2.
Identification of unique anti-AR compounds. (A) Two-dimensional plot of screened compounds (plots of individual plates are shown in Fig. S3). Blue, full-effect mimics (no DHT treatment and no compound; pink, DHT treatment alone; red, CDX on DHT-treated cells; and green, compound on DHT-treated cells. (B) Clustering analysis of top candidate hits. Red represents the effect on suppressing DHT-induced genes; green shows the effect on restoring DHT-repressed genes. The compounds identified from published screenings as described in the text are highlighted in green, blue, and pink; cardiac glycosides are labeled in red. (C) Titration and deduced ESmax and IC50 for individual compounds. SDs are based on triplicate measurements. Compound structure is shown on top of each titration curve.
Fig. 3.
Fig. 3.
Effects of lead compounds on cell proliferation and apoptosis. (A) Effect of each compound (5 μM) on DHT-treated LNCaP cells and on androgen-independent LNCaP-abl, PC3, and PC3-AR cells. (B) Effect of each compound at different concentrations on androgen-resistant LNCaP-abl cells. (C) Apoptosis induced on LNCaP (Left) and LNCaP-abl (Right) cells by individual compounds with Nigericin as a positive control. (D) Competitive binding determined with 3H-labeled DHT in the presence of increasing concentrations of individual cold DHT, CDX, and Peruvoside.
Fig. 4.
Fig. 4.
Global analysis of cardiac glycosides in comparison with AR RNAi on LNCaP-abl cells. (A) LNCaP-abl cells treated with 5 μM of individual cardiac glycosides were compared with the effect of AR RNAi (the knockdown efficiency is shown next to the heatmap). Significant changes (>twofold; P < 0.01) were identified, which added up to a total of 2,056 genes that were either up-regulated (red) or down-regulated (blue) on at least one treatment condition. (B) Venn diagrams of overlapped changes between AR RNAi and individual cardiac glycosides. (C) Overlapped genes showed changes largely in the same directions.
Fig. 5.
Fig. 5.
Cardiac glycosides induce rapid AR degradation. (A and B) Effect on the expression of KLK3 (A) and AR (B) in LNCaP-abl cells. SDs are based on triplicate experiments. (C and D) Western blot of AR in LNCaP-abl cells treated with different concentrations of compound for 24 h (C) or with Digoxin for different periods of time (D). (E) Western blot of AR in PC3-AR cells treated with different concentrations of compound for 24 h. (F) Knockdown of the catalytic subunit (ATP1α1) of the Na+/K+ ATPase, showing no effect on AR protein in LNCaP-abl cells. (G) Prevention of induced AR degradation by the proteasome inhibitor MG132. Note that a truncated AR because of calpain-mediated AR cleavage (31) or alternative splicing (32) became detectable in MG132-treated LNCaP-abl cells.
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