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.
Highlights
Metastatic prostate cancer (PCa) patients are treated with androgen deprivation therapies (ADT) to deplete systemic androgen levels and inhibit androgen binding to the androgen receptor (AR) protein in prostate cancer (PCa) cells [1]
CRPC is thought to be the consequence of dysregulated androgen signaling in PCa cells that develops subsequent to chronic ADT
We developed a robust procedure for the identification of new co-regulators of AR that may participate in progression to CRPC
Summary
Metastatic prostate cancer (PCa) patients are treated with androgen deprivation therapies (ADT) to deplete systemic androgen levels and inhibit androgen binding to the androgen receptor (AR) protein in prostate cancer (PCa) cells [1]. ADT maintains systemic castrate levels of androgen, studies of CRPC tissues and experimental models of CRPC support the notion that CRPC cells remain addicted to AR-driven signaling. This is reinforced by the clinical www.impactjournals.com/oncotarget success of new anti-androgens such as enzalutamide that significantly increase survival of CRPC patients. We tested siRNAs against gene targets that were enriched following initial selection for the ability to increase the expression of an AR-responsive reporter in androgen-depleted medium. SiRNAs that passed this validation were tested for their ability to enable androgen-independent growth of the cells Using this protocol, we have identified both known and novel effectors of androgen signaling in androgen-deprived PCa cells. Two of the AR signaling modulators identified in our screening, IGSF8 and CD9, appear to interact with each other in PCa progression
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