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RAI2 has initially been identified as a metastasis-associated gene in breast cancer. Recently, we found that increased RAI2 protein in primary tumors predicts early biochemical relapse of prostate cancer patients. On the molecular level, RAI2 interacts with CtBP1 as transcriptional co-repressors via a non-consensus tandem ALDLS-motif in hormone-dependent cancer cells. In this study, we aim to investigate the cell-biological relevance of this molecular interaction in prostate cancer cells using the CDKN1A gene as an example of a transcriptional target of CtBP-mediated gene regulation. To analyze a molecular relation between the RAI2/CtBP1 interaction and repressor of the CDKN1A gene, we first applied a transactivation assay in 293T cells, transiently transfected with CtBP1 and different RAI2 protein variants. Next, we depleted the RAI2 protein in VCaP prostate cancer cells. We used this cell line model to analyze gene expression and protein concertation of RAI2, CtBPs and p21 under different conditions. We studied the subcellular localization and chromatin binding of the proteins of interest by confocal laser-scanning microscopy and ChIP analysis. The gene reporter assay revealed that combined overexpression of RAI2 and CtBP1 reliefs the repression of the proximal CDKN1A-promotor and that RAI2 with an intact ALDLS tandem motif is required for this process. RAI2 depletion in VCaP cells resulted in a significant reduction of both CtBP1 and CtBP2 and almost complete abolishment of p21 protein levels, which is accompanied by reduced interaction of RAI2 with CtBP1 in nuclei. In parental VCaP cells, genotoxic stress significantly induced CDKNA1 gene expression and p21 protein concentration. In contrast, in RAI2-depleted VCaP cells we did not observe any of these effects, demonstrating the requirement of RAI2 to cause the relief of CDKNA1 repression. RAI2 together with CtBP1 appeared as definite foci in the nuclei of VCaP cells that are co-localized with key factors of polycomb 1 and 2. The ChIP analysis revealed that RAI2-depletion significantly induced binding of CtBPs to the CDKN1A promoter. In contrast, the binding of the polycomb protein LCoR to chromatin is significantly reduced in RAI2-depleted cells. Irrespective of the RAI2 status, the EZH2 binding to the chromatin remained unaltered, whereas a significant increase in H3K27me3 in RAI2-depleted cells was observed. In summary, we found that interruption of the interaction of RAI2 with CtBP1 is leading to increased chromatin binding of CtBP1 and trimethylation of H3K27 of the CDKN1A promotor, which is associated with repression of CDKN1A gene expression. We conclude that the molecular interaction of RAI2 with CtBP1 is a new molecular mechanism of corepression by modulating the histone-modifications of the target gene. Citation Format: Sarah Greimeier, Bettina Steinbach, Simon Sander, Lina Merkens, Nishit Goradia, Matthias Wilmanns, Eric Metzger, Klaus Pantel, Stefan Werner. RAI2 controls polycomb-mediated repression of CDKN1A by its interaction with CtBP1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3724.

Previously, we found that increased Retinoic acid-induced 2 (RAI2) protein concentration in primary tumors predicts early biochemical relapse of prostate cancer patients and described a co-repressor function of RAI2 related to androgen receptor activity (1). The presence of a non-canonical tandem ALDLS motif on RAI2 protein prompted the present investigation on a possible role of a dual interaction of RAI2 with C-terminal binding proteins (CtBPs) in relation to prostate cancer progression. We analyzed RAI2 gene expression as well as CtBP1 protein localization and constitution in circulating tumors cells (CTCs) isolated from metastatic prostate cancer patients by semi-quantitative PCR analysis and immune-fluorescence staining. We employed an integrated structural biology approach combining biophysical techniques like isothermal calorimetry, circular dichroism, size exclusion chromatography with high-resolution structural biology techniques including X-ray crystallography and single particle cryo-electron microscopy. We established RAI2-depleted VCaP cells and used this model for validation of the obtained results. RAI2 gene expression is significantly increased in CTCs isolated from patients with castration-resistant disease in comparison to CTCs isolated from patients with hormone-sensitive, aggressive or neuroendocrine variant prostate cancer. We detected CtBP1 protein foci in the nuclei of individual CTCs. We show that RAI2 induces CtBP polymerization. Using single particle cryo-electron microscopy, we obtained crucial insights into the structural organization of this filament. RAI2 inactivation in VCaP cells caused a significant reduction of both CtBP proteins, which is accompanied by the disappearance of CtBP protein aggregates in nuclei. In addition, we observed the induction of neuroendocrine features, marked by the induction of Synaptophysin and Chromogranin A, a higher nucleus to cytoplasm ratio and smaller cell size of RAI2-depleted cells. In summary, we report tandem motifs-induced polymerization of CtBP’s for the first time. We found that elevated RAI2 gene expression is a characteristic of castration-resistant prostate cancer. Because RAI2 depletion resulted in loss of CtBP aggregates and the induction of neuroendocrine traits, we conclude that RAI2-driven CtBP polymerization sustains epithelial differentiation in castration-resistant prostate cancer. These findings holds potential for the diagnosis of castration-resistant disease and inhibiting the progression from castration-resistant to neuroendocrine prostate cancer. 1. Besler K, Weglarz A, Keller L, von Amsberg G, Bednarz-Knoll N, Offermann A, et al. Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer. Clin Chem 2022;68:973-83 Citation Format: Stefan Werner, Nishit Goradia, Edukondalu Mullapudi, Lina Merkens, Sarah Greimeier, Aleksandra Węglarz, Gunhild von Amsberg, Harriet Wikman, Klaus Pantel, Matthias Wilmanns. Retinoic acid-induced 2 driven polymerization of c-terminal binding proteins sustains epithelial differentiation in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1447.

Aggressive variants of prostate cancer (AVPC) emerge from prostate adenocarcinoma following enduring therapeutic pressure. They grow independent of the androgen receptor, progress rapidly and can show traits of neuroendocrine (NE) differentiation. As progression to AVPC is not well detected by prostate specific antigen monitoring, new biomarkers are urgently needed. The aim of this study is to evaluate the detection of circulating tumor cells (CTCs) in combination with gene expression analysis of NE genes to identify AVPC patients with neuroendocrine traits. Blood samples were collected from patients suffering from AVPC (n=78, including 25 patients with histological evidence of NEPC) as well as from patients with hormone-sensitive prostate cancer (HSPC) as controls (n=12) at the University Medical Center Hamburg-Eppendorf. CTC counts were determined by CellSearch analysis. In parallel, CTCs were immuno-magnetically enriched using the AdnaTest and bulk gene expression of prostate-specific and NE genes was analyzed by semi-quantitative PCR. From a subset of patients, CTCs were additionally enriched by Parsortix to compare gene expression of CTCs with EpCAM-based or size-based CTC enrichment. Using CellSearch analysis, CTCs were found in 88.9 % of AVPC patients. CTC counts ranged between 0 – 20,000 CTCs/7.5 ml of blood with a median of 32 CTCs/7.5 ml. Similarly, 81.8 % of NEPC patients were positive for CTCs with a median count of 35 CTCs/7.5 ml (range 0-13,000). In contrast, CTCs were found in only 50 % of HSPC patients at lower concentrations (median count of 0.5 CTCs/7.5 ml, range 0-34). CTC counts were significantly elevated in AVPC and NEPC patients compared to HSPC (p = 0.002, p = 0.012), but no significant differences were found between AVPC and NEPC samples.In 93.8 % of AVPC samples, CTCs expressed prostate-specific genes such as KLK3 or PSMA. At least one of the NE transcripts was detected in 37.5 % of AVPC patients. In CTCs from NEPC patients, the positivity for prostate markers was significantly reduced to 66.6 % (p = 0.01) and the positivity for NE markers was increased to 61.1 %. A random Forrest model trained on all analyzed transcripts allowed for a distinction of HSPC and NEPC with an AUC of 79.5 %. Comparison of CTC enrichment strategies in a subset of 13 patients showed similar NE marker detection by size-based enrichment (53.8 % NE-positive) compared to the AdnaTest (61.5 % NE-positive). AVPC and NEPC patients show a high CTC burden that will allow subsequent molecular analyses, and CTC counts allowed a distinction between blood samples from HSPC and NEPC patients. Gene expression analysis revealed a high degree of inter-patient heterogeneity for neuroendocrine-specific transcripts with a reduction of prostate markers in NEPC patients. Future molecular characterization and longitudinal monitoring of CTCs will shed more light on the evolution of AVPC and NEPC. Citation Format: Lina Merkens, Sarah Greimeier, Sabine Riethdorf, Klaus Pantel, Gunhild von Amsberg, Stefan Werner. Liquid biopsy approaches to determine tumor cell heterogeneity in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3370.