The bipolar androgen therapy (BAT) to treat prostate cancer (PCa) includes cycles of supraphysiological androgen levels (SAL) under androgen-deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and in ex vivo-treated patient PCa tumor samples. Here, we analyzed the underlying molecular pathway and reveal that SAL induces cellular senescence in both, castration-sensitive (CSPC) LNCaP and castration-resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15INK4b and increased phosphorylation of AKT. Treatment with the AKT inhibitor (AKTi) potently inhibited SAL-induced expression of p15INK4b and cellular senescence in both cell lines. Proximity-ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with AKT in the cytoplasm as well as in the nucleus detectable after three days. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as with AKTi-treated cell transcriptomes revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo upregulates lncRNASAT1. In PCa tumor tissues, lncRNASAT1 is downregulated compared with nontumor tissues of the same patients. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer-cell senescence as an upstream factor for pAKT and for p15INK4b. Further, knockdown of lncRNASAT1 enhances cell proliferation by SAL, suggesting that lncRNASAT1 serves as a tumor suppressor at SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR-interacting partner that regulates AR target-gene expression. Similarly, RNA-ChIP experiments revealed the interaction of AR with lncRNASAT1 on chromatin. Thus, we identified a novel AR-lncRNASAT1-AKT-p15INK4b signaling axis to mediate SAL-induced cellular senescence.

Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial–mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.

No abstract

No abstract

No abstract

No abstract

No abstract