Identification of novel genes that regulate androgen receptor signaling and growth of androgen-deprived prostate cancer cells

Abstract

Prostate cancer progression to castration refractory disease is associated with anomalous transcriptional activity of the androgen receptor (AR) in an androgen-depleted milieu. To identify novel gene products whose downregulation transactivates AR in prostate cancer cells, we performed a screen of enzymatically-generated shRNA lenti-libraries selecting for transduced LNCaP cells with elevated expression of a fluorescent reporter gene under the control of an AR-responsive promoter. The shRNAs present in selected populations were analyzed using high-throughput sequencing to identify target genes. Highly enriched gene targets were then validated with siRNAs against selected genes, testing first for increased expression of luciferase from an AR-responsive promoter and then for altered expression of endogenous androgen-regulated genes in LNCaP cells. We identified 20 human genes whose silencing affected the expression of exogenous and endogenous androgen-responsive genes in prostate cancer cells grown in androgen-depleted medium. Knockdown of four of these genes upregulated the expression of endogenous AR targets and siRNAs targeting two of these genes (IGSF8 and RTN1) enabled androgen-independent proliferation of androgen-dependent cells. The effects of IGSF8 appear to be mediated through its interaction with a tetraspanin protein, CD9, previously implicated in prostate cancer progression. Remarkably, homozygous deletions of IGSF8 are found almost exclusively in prostate cancers but not in other cancer types. Our study shows that androgen independence can be achieved through the inhibition of specific genes and reveals a novel set of genes that regulate AR signaling in prostate cancers.

Keywords: IGSF8; androgen receptor; prostate cancer; shRNA; tumor progression.

Figure 1. High throughput screening for shRNAs stimulating AR activity in low androgen environment

A. AR-driven reporter construct containing Red fluorescent protein (DsRed II) under control of modified rat probasin promoter. B. FACS analysis of Prb-DsRed-LNCaP reporter cells. Cells were cultured in a hormone-free medium for 8 days (left). Cells were cultured in a hormone-free medium for 6 days followed by 48 h treatment with 100pm R881 (right). C. DsRed fluorescence is increased in Prb-DsRed-LNCaP reporter cells infected with AR-expressing (right) or a control lentiviral vector (left) and cultured for 8 days in hormone-free conditions. DIC (top) and fluorescence (bottom) microscopy, 20x magnification. D. Scheme of the selection and analysis of the shRNAs stimulating rat probasin promoter under hormone-free conditions.

Figure 2. Validation of shRNA library selection procedure

A. Scheme of library screening and validation procedure. B. The AR-driven reporter construct containing Firefly Luciferase (FFLuc) under control of modified rat probasin promoter (validation, step 1) C. Androgen dose-dependent activity of the integrated luciferase reporter in Prb-Luc-LNCaP cells D. Localization of Androgen Responsive Elements (AREs) in the endogenous genes selected for QPCR based validation. Grey boxes represent canonical AREs, black box represents a non-canonical ARE.

Figure 3. Effects of knockdown of proteasome complex subunits

A. Effects of siRNAs against the indicated proteasome components (4 siRNA per gene, sets A–D) on Prb-promoter activity measured by luciferase expression in Prb-Luc-LNCaP cells. Genes enriched in shRNA library selection are highlighted. Cells were transfected with siRNAs (5 nM) in duplicates, cultured in hormone-free medium for 6 days, followed by measurement of luciferase activity. B–C. Reporter Prb-Luc-LNCaP cells were transfected with siRNA against 19S proteosomal component PSMC4 (5 nM), cultured in hormone-free medium for 6 days, followed by QPCR analysis of expression of firefly luciferase B. or endogenous androgen responsive genes C. Results represent mean of 3 independent experiments +/– SD.

Figure 4. New regulators of androgen-responsive genes identified in the functional screening

A. Two groups of siRNA regulators were discovered: androgen independent activators and inhibitors of androgen responsive genes. The expression of 4 known androgen-responsive genes (KLF3 (PSA), KLK2, FKBP5 and TMPRSS2) was measured following transfection of selected siRNAs (at 5 nm) into LNCaP cells in triplicates (see Figure 2). Cells were cultured in androgen-free conditions for 6 days. mRNA was purified and subjected to QPCR analysis. Table represents the levels of gene expression as percentages compared to control (the description of genes is presented in Supplementary Table S5). First column represents the effect of selected siRNAs on expression of luciferase in Prb- Luc-LNCaP cells (in triplicate) B. Three GO categories enriched in the validated group of genes. C. Screen-selected siRNAs enable proliferation of LNCaP cells under hormone-free conditions. LNCaP cells were transfected with siRNAs as shown and cultured under androgen-free conditions. The number of viable cells was determined using spectrophotometric quantification of cell proliferation by WST-1 assay. The plot represents the mean of six independent transfections +/– SD.

Figure 5. siRNA-mediated depletion of IGSF8 activates the expression of androgen responsive genes

A. Two different siRNAs efficiently inhibit expression of IGSF8 gene (QPCR analysis). B. LNCaP cells were transfected with IGSF8-targeting or control siRNAs (5 nM) and cultured in hormone-free conditions for 6 days. Expression of androgen responsive genes was analyzed by QPCR. The results represents mean of 3 independent experiments +/– SD. C. Effect of the IGSF8 knockdown is AR-dependent. LNCaP cells were transfected with control, IGSF8 or AR siRNAs and cultured in hormone-free conditions for 6 days. Expression of androgen responsive genes was analyzed by QPCR. D. Effects of the IGSF8 knockdown depend on the expression of CD9. LNCaP cells were transfected with control, IGSF8 or CD9 siRNAs and cultured in hormone-free conditions for 6 days. Expression of androgen responsive genes was analyzed by QPCR. All QPCR results represent mean of 3 independent experiments +/– SD.

Figure 6. IGSF8 knockdown effects is dependent of AR but does not change AR expression

A. LNCaP cells were transfected with control or IGSF8-targeting siRNAs (5 nM) and cultured in hormone-free conditions for 6 days. Expression of AR and PSA was analyzed by immunoblotting. Quantification of AR signal intensity normalized to GAPDH signal is shown on the right, relative to AR level of untransfected LNCaP cells (mean of 3 independent experiments +/– SD). B. LNCaP were transfected as above, followed by fixation and immunostaining with DAPI and anti-AR antibodies. Images were taken and nuclear staining was quantified. The results represent average quantification of the AR to DAPI ratio of fluorescence intensity (30 nuclei per siRNA) +/– SD. C. LNCaP cells were transfected as in A and cultured in hormone-free conditions for 6 days in the presence of 10 μM of Enzalutamide or DMSO. Gene expression was analyzed by QPCR. The results represent mean of 3 independent experiments +/– SD.

Figure 7. Comparison of gene expression affected by IGSF8 knockdown or androgen stimulation

Affymetrix array analysis of gene expression profiles of cells with siRNA knockdown of IGSF8 or treated with R1881. Diagrams represent changes in gene expression > 1.4-fold (P < 0.05). A. Genes co-regulated by ISF8 knockdown and R1881 treatment. B. Genes differentially regulated by IGSF8 siRNA and R1881. C. Genetic alterations of IGSF8 in human cancers analyzed through cBioPortal tool (TCGA provisional data set).