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 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

STK17A protein is overexpressed in GBM cells lines compared to NT2/D1 and HCT116 cells.

<p><b>A</b>, <b>B</b>, Western analysis comparing STK17A expression in U87, U251, U118 and U563 GBM cells compared to NT2/D1 human embryonal carcinoma and HCT116 colon cancer cells. Arrow indicates STK17A specific band identified by the diminished signal in U87 cells stably expressing STK17A shRNA. </p

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 knockdown results in decreased GBM cell proliferation.

<p>U87, SF268 and U118 GBM cells stably expressing STK17A shRNA or a control shRNA were assessed for changes in cell proliferation by cell counting 4.5 days after plating. Cell counts were normalized to cell counts after 0.5 days of plating to control for plating errors and differences in cell adherence. Bars are the average of three biological replicates and error bars are SD. *, p < 0.05. Data are representative of at least two independent experiments. To the right of each graph the extent of STK17A knockdown was assessed by Western analysis. </p

Knockdown or overexpression of STK17A alters cell morphology of U87 cells.

<p>Representative micrographs of parental U87 cells (<b>A</b>), U87 cells stably expressing control shRNA (<b>B</b>), two distinct STK17A shRNAs (<b>C</b>, <b>D</b>), or U87 cells stably overexpressing STK17A (<b>E</b>). Cells become large, flatter and more rounded upon STK17A knockdown while cells take on an elongated, needle-shaped appearance upon STK17A overexpression. Representative of three independent derivations of each cell line. Pictures were taken at 10X magnification on a NIKON ELWD microscope. <b>F</b>, Western analysis of STK17A knockdown and overexpression. Efficiency of stable knockdown is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#pone-0081803-g003" target="_blank">Figure 3</a>.</p

STK17A knockdown decreases oncogenic properties of GBM cells.

<p><b>A</b>, STK17A knockdown decreases soft agar colony formation. U87 control or U87 STK17A knockdown cells were suspended in soft agar and cells were stained with MTT reagent after 2 weeks of culture. Representative of two independent experiments. <b>B</b>, STK17A knockdown decreases clonogenicity of GBM cells. U87 control or STK17A U87 knockdown cells were plated and stained with Giemsa after 10 days of cell culture. Representative of three independent experiments. <b>C</b>, STK17A knockdown sensitizes GBM cells to cisplatin and temozolomide. Left, Dose response after 3 days of cisplatin treatment of U87 or U251 control versus STK17A knockdown cells. Cell proliferation and survival was measured with Cell-Titer Glo reagent. Data points are the average of biological triplicates and error bars are SD. *, p < 0.05. Representative of three independent experiments. Right, Dose response after 3 days of temozolomide treatment of U87 control versus U87 STK17A knockdown cells. Cell proliferation and survival was measured with Cell-Titer Glo reagent. Data points are the average of biological triplicates and error bars are SD. *, p< 0.05. Representative of two independent experiments. </p

STK17A knockdown results in the formation of actin stress fibers and inhibition of cell motility and invasion.

<p><b>A</b>, Representative fluorescent images of U87 cells stably expressing control shRNA and two distinct STK17A shRNAs. Actin is stained green and nuclei are stained blue. Note the larger size of STK17A knockdown cells and the presence of actin stress fibers. Extent of knockdown is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081803#pone-0081803-g003" target="_blank">Figure 3</a>. This morphologic phenotype was apparent in independent derivations of the cell lines. Pictures were taken at 20X magnification on a NIKON ELWD fluorescent microscope. <b>B</b>, STK17A knockdown decreases cell migration and invasion of U87 cells. Cell migration and invasion was assessed as described in Methods. Bars are the average of biological triplicates and error bars are SD. *, p < 0.05. Representative of two independent experiments.</p