ARTIK-52 induces replication-dependent DNA damage and p53 activation exclusively in cells of prostate and breast cancer origin

<p>The realization, that the androgen receptor (AR) is essential for prostate cancer (PC) even after relapse following androgen deprivation therapy motivated the search for novel types of AR inhibitors. We proposed that targeting AR expression versus its function would work in cells having either wild type or mutant AR as well as be independent of androgen synthesis pathways. Previously, using a phenotypic screen in androgen-independent PC cells we identified a small molecule inhibitor of AR, ARTIK-52. Treatment with ARTIK-52 caused the loss of AR protein and death of AR-positive, but not AR-negative, PC cells. Here we present data that ARTIK-52 induces degradation of AR mRNA through a mechanism that we were unable to establish. However, we found that ARTIK-52 is toxic to breast cancer (BC) cells expressing AR, although they were not sensitive to AR knockdown, suggesting an AR-independent mechanism of toxicity. Using different approaches we detected that ARTIK-52 induces replication-dependent double strand DNA breaks exclusively in cancer cells of prostate and breast origin, while not causing DNA damage, or any toxicity, in normal cells, as well as in non-PC and non-BC tumor cells, independent of their proliferation status. This amazing specificity, combined with such a basic mechanism of toxicity, makes ARTIK-52 a potentially useful tool to discover novel attractive targets for the treatment of BC and PC. Thus, phenotypic screening allowed us to identify a compound, whose properties cannot be predicted based on existing knowledge and moreover, uncover a barely known link between AR and DNA damage response in PC and BC epithelial cells.</p

Patient #2 targeted resequencing.

<p>A) The <i>ESR1</i> mutation was sequenced to greater depth on the Ion Torrent PGM. B) Comparison of allele frequencies between pre- and during-TDM1 treatment cfDNA samples for eight mutations present in the pre-treatment sample.</p

Sequencing Statistics.

<p>Summary of sequencing info for all ten sequencing runs. All reads are listed in millions. Accession numbers for.bam files uploaded to European Nucleotide Archive provided, for sarcoma patient all 3 cfDNA runs were combined in a single.bam file separated by read group.</p

Variant allele percentage correlation.

<p>A) cfDNA variant allele percentage is poorly correlated with Primary tumor variant allele percentage.</p

Overview of metastatic sarcoma patient treatment history.

<p>A) Patient diagnosed with intimal spindle cell sarcoma of the pulmonary artery. Treatments and sample collection indicated in months.</p

Patient #1 mutation calls and validation.

<p>A) Using a cutoff of 1.5% variant allele percentage, 46 of the 47 mutations present in the tumor were identified in the cfDNA. Estimating from the average variant allele percentage of 3.8%, 7.5% of the cfDNA was derived from the tumor. B) Fifteen additional mutations were called in the cfDNA which were not called in the tumor sample. Four of these are present in the tumor, but below our calling cutoff of 10% for the tumor. Genes highlighted in red were successfully validated via sequencing on the Ion Torrent PGM. Approximately 4,000 genomes of cfDNA were used as input to the validations, giving us a lower sensitivity bound of 0.025–0.5% depending on the site-specific background error rate.</p

ATM Suppresses SATB1-Induced Malignant Progression in Breast Epithelial Cells

<div><p>SATB1 drives metastasis when expressed in breast tumor cells by radically reprogramming gene expression. Here, we show that SATB1 also has an oncogenic activity to transform certain non-malignant breast epithelial cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature. We obtained aliquots from two different sources (here we refer to them as MCF10A-1 and MCF10A-2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in three-dimensional cultures, led to tumor formation in immunocompromised mice, and when injected into tail veins, led to lung metastasis. The number of metastases correlated positively with the level of SATB1 expression. In contrast, SATB1 expression in MCF10A-2 did not lead to any of these outcomes. Yet DNA copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2. However, gene expression profiling analysis revealed that these cell lines have significantly divergent signatures for the expression of genes involved in oncogenesis, including cell cycle regulation and signal transduction. Above all, the early DNA damage-response kinase, ATM, was greatly reduced in MCF10A-1 cells compared to MCF10A-2 cells. We found the reason for reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled SATB1 to induce malignant phenotypes similar to that observed for MCF10A-1. These data indicate a novel role for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, but also raise a cautionary note that phenotypic drift could lead to dramatically different functional outcomes.</p> </div

cfDNA and liver metastasis DNA are well correlated.

<p>A) cfDNA variant allele percentage is correlated with liver metastasis variant allele percentage B) Maximum parsimony tree showing relatedness of samples, branch length are number of somatic, nonsynonymous mutations C) Seventeen additional mutations were identified uniquely in cfDNA, 9 of which have reads supporting them in the primary and/or met, but where not called due to insufficient sequencing depth or variant allele percentage. Genes in red were successfully validated on the Ion Torrent PGM, genes in blue failed to validate, genes were black were not validated.</p

Patient #2 diagnosed with ER+/PR+/HER2+/Node+ breast carcinoma.

<p>A) Treatments and sample collection indicated in months. B) 48 total somatic mutations were called in the primary breast tumor and/or liver metastasis. 38 mutations were called in the cfDNA using a variant allele percentage cutoff of 1.5%. Genes in red were successfully validated on the Ion Torrent PGM, genes in blue failed to validate, genes were black were not validated.</p

SATB1 is endogenously expressed in aggressive breast cancer cell lines and its ectopic expression induces malignant phenotype in non-malignant MCF10A-1, but not in MCF10A-2 cells.

<p><b>A</b>) (Top) Quantitative RT-PCR analysis for SATB1 expression relative to GAPDH in MCF10A cell lines from two different sources (MCF10A-1 and MCF10A-2), 184A1, 184B5 and MCF10A progression series (MCF10A-neoT and CA1d), BT549 and MDA-MB-231. (Bottom) Immunoblot for SATB1 expression using the same cell lines as in quantitative RT-PCR analysis. α-tubulin was used as an internal loading control. <b>B</b>) Immunoblot showing the expression level of SATB1 before and after SATB1 overexpression in MCF10A-1 and MCF10A-2 cells. ß-actin was used as an internal loading control. <b>C</b>) Colony morphologies of MCF10A-1 and MCF10A-2 control and SATB1 overexpressing cells (pLXSN-SATB1: pooled populations), and MDA-MB-231 control and SATB1 depleted (SATB1 shRNA) cells at six days of 3D matrix on-top culturing. Images were captured with Phase 1 at 20X magnification. Scale bars, 100 µm. Note that SATB1 overexpression caused MCF10A-1 to form a mixture of large spheroid and spindle structures, indicated by white and blue arrows, respectively, while it caused MCF10A-2 cells to form larger spheroid structures. The aggressive breast cancer cell line, MDA-MB-231, formed network-like spindle structures, which was inhibited by knockdown of SATB1. <b>D</b>) Invasion assay of MCF10A-1, MCF10A-2, MCF10A-neoT and CA1d cell lines before and after SATB1 overexpression. Parental cells lines are shown in blue; SATB1 overexpressing cells are shown in pink. Error bars indicate s.e.m., n = 3 experiments. <b>E</b>) Vector control (top row) and SATB1-expressing MCF10A-1 (pLXN-SATB1) (bottom two rows) grown on Matrigel were stained for F-actin (red), ß-catenin (green), integrin α6 (green), SATB1 (green) and DAPI (blue). Note that control cells showed the typical acinar structure whereas SATB1-expressing cells showed large spheroid (i) and spindle (ii) structures. No acinar structures were detected in SATB1-expressing MCF10A-1. Scale bars, 15 µm.</p