The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex

Abstract

Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority. The molecular basis for this clinical heterogeneity remains incompletely understood. Here we characterize a long noncoding RNA termed SChLAP1 (second chromosome locus associated with prostate-1; also called LINC00913) that is overexpressed in a subset of prostate cancers. SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo gain-of-function and loss-of-function experiments indicate that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. These results suggest that SChLAP1 contributes to the development of lethal cancer at least in part by antagonizing the tumor-suppressive functions of the SWI/SNF complex.

Figure 1. Discovery of SChLAP1 as a prostate cancer lncRNA

(a) Cancer outlier profile analysis (COPA) for intergenic lncRNAs. (b) A representation of the SChLAP1 gene and its annotations in current databases. An aggregated representation of current gene annotations for Ensembl, ENCODE, UCSC, Ref-Seq, and Vega shows no annotation for SChLAP1. ChIP-Seq data for H3K4me3 and H3K36me3 show enrichment at the SChLAP1 gene. Also, RNA-Seq data showing an outlier sample for SChLAP1 illustrates its expression. (c) qPCR for SChLAP1 on a panel of benign prostate (n=33), localized prostate cancer (n=82), and metastatic prostate cancer (n=33) samples. qPCR data is normalized to the average of (GAPDH + HMBS) and represented as standardized expression values. (d) Fractionation of prostate cell lysates demonstrates nuclear expression of SChLAP1. U1 is a positive control for nuclear gene expression. (e)In situ hybridization of SChLAP1 in human prostate cancer. SChLAP1 staining is shown for both localized and metastatic tissues.

Figure 2. SChLAP1 expression characterizes aggressive prostate cancer

(a) Network representation of genes positively or negatively correlated with SChLAP1 in localized prostate cancers using Oncomine concepts analysis and visualized with the Force Directed Layout algorithm in the Cytoscape tool. Node sizes reflect the number of genes that comprise each molecular concept and node names are labeled according to the author of the primary study as detailed in Supplementary Table 3b. The nodes are colored according to the concept categories indicated in the figure legend. Edges are drawn between nodes with statistically significant enrichment (p-value < 1e-6, odds ratio > 3.0) and darker edge shading implies higher odds ratio. (b) Heatmap representation of comparisons between co-expression gene signatures and molecular concepts. Comparisons to positively (top portion) and negatively correlated (bottom portion) gene signatures are shown separately. Comparisons that do not reach statistical significance (q > 0.01 or odds ratio < 2) are shown in grey. Associations with over-expression concepts are colored red, and under-expression concepts blue. (c-e) Kaplan-Meier analyses of prostate cancer outcomes in the Mayo Clinic cohort. SChLAP1 expression was measured using Affymetrix exon arrays and patients were stratified according to their SChLAP1 expression. Patient outcomes were analyzed for biochemical recurrence (c), clinical progression to systemic disease (d), and prostate cancer-specific mortality (e). The shaded regions represent the 95% confidence interval.

Figure 3. SChLAP1 coordinates cancer cell invasion in vitro and metastatic seeding in vivo

(a) siRNA knockdown of SChLAP1 in vitro in three prostate cell lines (LNCaP, 22Rv1, Du145) impairs cellular invasion through Matrigel in a Boyden chamber assay. EZH2 siRNA serves as a positive control. (b) Overexpression of SChLAP1 in RWPE cells results in increased cellular invasion through Matrigel in Boyden chamber assays. (c) Intracardiac injection of 22Rv1 cells with stable SChLAP1 knockdown in severe combined immunodeficient (SCID) mice. Example luciferase bioluminescence images from 22Rv1 shNT, shSChLAP1 #1, and shSChLAP1 #2 mice five weeks following intracardiac injection. Mouse IDs are given above each image. (d) The relative intensity of whole-mouse luciferase signal is plotted for 22Rv1 shNT (n=9), shSChLAP1 #1 (n=14) and shSChLAP1 #2 (n=14) intracardiac injection experiments. (e) The number of gross metastatic sites observed by luciferase signal in 22Rv1 shSChLAP1 cells or shNT controls. Independent foci of luciferase signal were averaged for shNT (n=9), shSChLAP1 #1 (n=14) and shSChLAP1 #2 (n=14) mice. (f) Invasion of 22Rv1-shNT and 22Rv1 shSChLAP1 cells in the chick chorioallantoic membrane (CAM) assay. 22Rv1 cells are labeled with GFP. The image is counterstained with chicken collagen IV for vasculature (RFP) and DAPI for nuclei. (g-i) Using the CAM assay, 22Rv1 shSChLAP1 cells demonstrate decreased intravasation (g), metastatic spread to the liver and lungs (h), and reduced tumor weight (i). (j) Quantification of intravasation of RWPE-LacZ and RWPE-SChLAP1 cells in the CAM assay. All data in this figure are represented as mean +/- S.E.M. Statistical significance was determined by a two-tailed Student's t-test. An asterisk (*) indicates a p-value < 0.05.

Figure 4. SChLAP1 antagonizes SNF5 function and attenuates SNF5 genome-wide localization

(a) Heatmap results for SChLAP1 or SNF5 knockdown in LNCaP and 22Rv1 cells. (b) RNA immunoprecipitation (RIP) of SNF5 or AR in SNF5 in 22Rv1 and LNCaP cells. Inset Western blots demonstrate pulldown efficiency. (c) RIP analysis of SNF5 in RWPE cells overexpressing LacZ, SChLAP1 isoform #1, or SChLAP1 isoform #2. Inset Western blots demonstrate pulldown efficiency. (d) Pulldown of SChLAP1 RNA using Chromatin Isolation by RNA Purification (ChIRP) recovers SNF5 protein in RWPE-SChLAP1 isoform 1 cells. LacZ and TERC serve as controls. (e) A global representation of SNF5 genomic binding over ±2kb window surrounding each SNF5 ChIP-Seq peak in RWPE-LacZ, RWPE-SChLAP1 isoform 1, and RWPE-SChLAP1 isoform 2 cells. (f) A heatmap representation of SNF genomic binding at target sites in RWPE-LacZ, RWPE-SChLAP1 isoform 1, and RWPE-SChLAP1 isoform 2 cells. A ±1kb interval surrounding the called SNF5 peak is shown. (g) Gene set enrichment analysis results showing significant enrichment of ChIP-Seq promoter peaks with >2-fold loss of SNF5 binding for underexpressed genes in RWPE-SChLAP1 cells.