G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer

<p>G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.</p

Low-dose 5-aza and DNMT3B knockdown alters genome-wide promoter demethylation in NT2/D1-R1 cells.

<p><b>A</b>, Summary of results from Illumina 27K beadchip DNA promoter methylation analysis of NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. The number of promoter methylation alterations was based on the average of 3 replicates with 1.3-fold or greater change and a p value<0.05. <b>B</b>, Venn diagrams depicting degree of overlap in genes altered in expression or DNA promoter methylation in NT2/D1-R1 cells due to low dose 5-aza or DNMT3B knockdown. <b>Top</b>, High degree of overlap in genes in NT2/D1-R1 cells demethylated with low dose 5-aza and demethylated with DNMT3B knockdown (left). Little overlap in genes in NT2/D1-R1 cells undergoing increased DNA methylation with low dose 5-aza and increased DNA methylation with DNMT3B knockdown (right). The numbers represent methylation changes of 1.3-fold or greater with p<0.05. <b>Middle</b>, A moderate degree of overlap in genes in NT2/D1-R1 cells that underwent decreased DNA methylation and increased gene expression with 5-aza (left) and little overlap in genes in NT2/D1-R1 cells that underwent increased DNA methylation and increased gene expression with 5-aza. The numbers represent methylation changes of 1.3-fold or greater with p<0.05 and expression changes of 1.5-fold or greater with p<0.01. <b>Bottom</b>, A large degree of overlap in genes demethylated after 5-aza treatment of NT2/D1 and NT2/D1-R1 cells. The six overlapping genes are shown. The numbers represent methylation changes of 1.3-fold or greater with p<0.05. <b>C</b>, Unsupervised hierarchical clustering of the promoter DNA methylation of the 4 treatment arm values among just the 388 genes changed 1.3-fold or greater, p value<0.05 with 5-aza in the control cells depicting the large degree of overlap in genes undergoing demethylation with 5-aza and DNMT3B knockdown. Increased methylation = red, decreased methylation = green.</p

Confirmation of promoter DNA demethylation with low-dose 5-aza in NT2/D1 cells.

<p><b>A</b>, Representative bisulfite pyrosequencing tracing from NT2/D1 cells treated with vehicle control or 10 nM 5-aza for 3 days in the promoter region of SOX15. Shaded areas are CpG sites. <b>B</b>, Decrease in DNA methylation of the RIN1, SOX15 and TLR4 promoter with 3 day 10 nM 5-aza treatment of NT2/D1 cells as determined by bisulfite pyrosequencing. Average of biological triplicate determinations. Error bars are standard deviation. ** = p<0.005. SOX15 values represent the average methylation value across two CpG sites. RIN1 and TRL4 values represent the average methylation across three CpG sites. <b>C</b>, Increased gene expression of RIN1, SOX15 and TLR4 with 3 day 10 nM 5-aza treatment of NT2/D1 cells as determined by real-time PCR. Average of biological triplicate determinations. Error bars are standard deviation. * = p<0.05; ** = p<0.005.</p

Knockdown of DNMT3B results in resistance to low-dose 5-aza downstream of DNA damage and p53 protein induction.

<p><b>A</b>, DNMT3B knockdown in NT2/D1-R1 cells leads to resistance to low-dose 5-aza. Indicated doses of 5-aza were added fresh each day for 3 days to exponentially growing cultures of NT2/D1-R1 lentiviral control cells and NT2/D1-R1 cells stably expressing a lentiviral shDNMT3B construct. Viable cell growth and survival were measured. Data was normalized to no drug treatment. Error bars (within symbols) are standard deviation. * p = <0.05 compared to untreated controls. <b>B</b>, Low dose 5-aza induces DNA damage in NT2/D1-R1 cells independent of apoptosis. Cells were pretreated with vehicle or 20 µM Z-VAD-FMK (z-VAD) and treated with 100 nM 5-aza or 2 µM cisplatin for 1 day prior to Western analysis. Accumulation of pH2AX is indicative of double strand breaks. The arrow labeled cPARP indicates cleaved PARP that is indicative of apoptosis. Densitometry of pH2AX normalized to actin is shown. <b>C</b>, DNMT3B knockdown in NT2/D1-R1 cells results in resistant to low-dose 5-aza induced cell death and G2 cell cycle arrest. Cells were treated with 10 nM 5-aza for 3 days and then assayed for cell cycle analysis. Substantial cells in subG1 are indicative of cell death. <b>D</b>, Knockdown of DNMT3B in NT2/D1-R1 cells inhibits low-dose 5-aza mediated cell death but not p53 protein induction or induction of DNA damage. NT2/D1-R1 cells with no lentivirus and cells treated with sh-control lentvirus and sh-DNMT3B lentivirus were treated with 10 nM 5-aza for 3 days, 1 µM cisplatin for 12 hours or the combination. Western analysis was performed for p53, PARP and pH2AX.</p

Low-dose 5-aza induces an early, robust, and unique reprogramming of gene expression in NT2/D1 cells.

<p><b>A</b>, Scatter plot of microarray gene expression differences in NT2/D1 cells. Each point represents the average of 3 biological replicates. Points above the diagonal line represent genes upregulated and points below the diagonal line repressed with 1 day 5-aza treatment (top), 3 day 5-aza treatment (middle), and cisplatin treatment (bottom), compared to control. The number of altered genes above the indicated thresholds were changed with p<0.05. <b>B</b>, Robust induction of gene expression occurs with low-dose 5-aza. Box-whisker plot of expression levels of genes (1296) changed among groups >1.2-fold and p<0.02 ANOVA, Benjamini Hochberg (BH) corrected indicated a upward shift in gene expression with 5-aza. <b>C</b>, Low dose 5-aza induced an early and unique program of gene expression compared to cisplatin. Unsupervised hierarchical cluster analysis of the expression profile of 5-aza and cisplatin altered genes was performed on the 898 genes (rows) altered between the samples (columns) more than 1.5-fold with p value<0.01 ANOVA, BH corrected. Upregulated gene are red, downregulated genes are green. Large overlap in 1 day and 3 day 5-aza treatments and distinct regulation compared to cisplatin is evident. Genes regulated in a similar manner by 5-aza and cisplatin are in brackets.</p

DNMT3B knockdown inhibits low-dose 5-aza mediated genome-wide activation of p53 target genes and repression of pluripotency genes.

<p><b>A</b>, Summary of results from expression microarray data of NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. Knockdown of DNMT3B greatly reduced the number of genes altered with 5-aza treatment while DNMT3B knockdown alone results in minimal expression changes. The number of genes altered was based on the average of 3 biological replicates with 1.5-fold or greater change and a p value<0.01. <b>B</b>, Unsupervised hierarchical cluster analysis of the expression data in (A) was performed on the 541 genes altered more than 1.8-fold with p value<0.01 ANOVA BH corrected, between NT2/D1-R1 sh-control cells (sh-ctrl) and NT2/D1-R1 sh-DNMT3B cells (sh3B) untreated or treated for 3 days with 10 nM 5-aza. Upregulated gene are red, downregulated genes are green. C, Partitioning around mediods (PAM) analysis of 1169 genes changed 1.5-fold or greater with BH corrected p-value of <0.01. Cluster 1 and Cluster 2 of 6 total clusters are shown. All clusters are provided in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone.0053003.s004" target="_blank">Fig S4</a>. The number of genes in the clusters and the mean silhouette width (MSW) value is indicated. Expression intensity values for representative genes in Cluster 1 (pluripotent genes) and Cluster 2 (p53 target genes) is provided on the right and additional prominent members are provided below each cluster. Error bars are S.E.M. * = p<0.05 compared to untreated control.</p

Low-dose 5-aza induces distinct genome-wide activation of p53 target genes and unique repression of pluripotency genes in NT2/D1 cells.

<p><b>A</b>, Venn diagrams of expression microarray data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone-0053003-g002" target="_blank">Figure 2</a> indicating a large overlap in the genes upregulated (left) and down regulated (middle) in NT2/D1 cells with 1 day 5-aza treatment (fold change >1.3) compared to 3 day 5-aza treatment (fold change >1.5). A Venn diagram (right) of microarray data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#pone-0053003-g002" target="_blank">Figure 2</a> showing a large degree of overlap in genes upregulated 1.5-fold or greater by both 3 day 5-aza and cisplatin treatments. Genes listed in each Venn diagram are altered with p<0.05. Of the overlap genes, 13 are known p53 target genes in NT2/D1 cells. <b>B</b>, Partitioning around mediods (PAM) analysis of 5-aza and cisplatin regulated genes in NT2/D1 cells. The 1130 genes changed 1.5-fold or greater with BH corrected p-value of <0.02 were subjected to PAM analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053003#s4" target="_blank">Methods</a>. The number of genes in each of the five clusters and the mean silhouette width (MSW) value for each cluster is indicated. Expression intensity values for representative genes in Cluster 2, Cluster 3, and Cluster 4 is provided on the left. Error bars are S.E.M. * = p<0.05; ** = p<0.005 compared to untreated controls.</p

Serine/Threonine Kinase 17A Is a Novel Candidate for Therapeutic Targeting in Glioblastoma

<div><p>STK17A is a relatively uncharacterized member of the death-associated protein family of serine/threonine kinases which have previously been associated with cell death and apoptosis. Our prior work established that STK17A is a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner. In this study we have uncovered an additional, unanticipated role for STK17A as a candidate promoter of cell proliferation and survival in glioblastoma (GBM). Unexpectedly, it was found that STK17A is highly overexpressed in a grade-dependent manner in gliomas compared to normal brain and other cancer cell types with the highest level of expression in GBM. Knockdown of STK17A in GBM cells results in a dramatic alteration in cell shape that is associated with decreased proliferation, clonogenicity, migration, invasion and anchorage independent colony formation. STK17A knockdown also sensitizes GBM cells to genotoxic stress. STK17A overexpression is associated with a significant survival disadvantage among patients with glioma which is independent of age, molecular phenotype, IDH1 mutation, PTEN loss, and alterations in the p53 pathway and partially independent of grade. In summary, we demonstrate that STK17A provides a proliferative and survival advantage to GBM cells and is a potential target to be exploited therapeutically in patients with glioma. </p> </div

STK17A is overexpressed in GBM.

<p><b>A</b>, Data from Sun Brain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#B19" target="_blank">19</a>], and TCGA Brain (<a href="https://tcga-data.nci.nih.gov/tcga/tcgahome2.jsp" target="_blank"><u>https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp</u></a>) comparing microarray-based STK17A expression from clinical nonmalignant brain specimens (Norm. Brain) and clinical GBM indicating high expression of STK17A in GBM. <b>B</b>, Real-time PCR analysis of STK17A expression in GBM cell lines compared to expression in other cancer types and normal brain. Expression is normalized to GAPDH. Bars are the average of triplicate or duplicate biological replicates except for normal brain RNA which is the average of technical duplicates. Error bars are SD. All cell lines are human. Non-GBM cell lines are as follows: NT2 (NT2/D1), embryonal carcinoma; U2OS, osteosarcoma; H23, A549, and Hop62, lung adenocarcinomas; MCF7, breast cancer. <b>C</b>, Real-time PCR analysis of STK17A expression in normal brain mRNA and four clinical GBM samples from Dartmouth Hitchcock Medical Center. Expression is normalized to GAPDH. The bars represent the averages of technical duplicate determinations. </p

STK17A expression in glioma is associated with high grade and decreased survival.

<p><b>A</b>, Expression data was downloaded from the Rembrandt and TCGA databases and data from Sun Brain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#B19" target="_blank">19</a>] was downloaded from Oncomine and grouped according to grade. Rembrandt has 21 normal, 99 grade II, 85 grade III and 130 grade IV samples. TCGA has 10 normal, 7 grade II, 20 grade III and 482 grade IV samples and Sun Brain has 26 normal, 45 grade II, 31 grade III and 81 grade IV samples. Error bars are SEM. *, p < 0.02. <b>B</b>, STK17A expression is associated with poor overall survival in gliomas. Kaplan-Meier log-rank tests were performed on data obtained from the TCGA database. In all cases high and low expressing groups were divided at the median. All glioma and low grade glioma expression was from RNA-seq data while GBM expression was from Affymetrix microarray data. </p