ESTRO 2024 - Abstract Book

S5385

Radiobiology - Tumour biology

ESTRO 2024

1 University Medical Center Hamburg-Eppendorf, Radiotherapy & Radiation Oncology, Hamburg, Germany. 2 University Medical Center Hamburg-Eppendorf, Clinical Chemistry & Laboratory Medicine, Hamburg, Germany. 3 University Medical Center Hamburg-Eppendorf, Pediatric Hematology & Oncology, Hamburg, Germany. 4 University Medical Center Hamburg-Eppendorf, Mildred-Scheel Cancer Careers Center, Hamburg, Germany. 5 University Medical Center Hamburg-Eppendorf, II. Department of Medicine, Hamburg, Germany

Purpose/Objective:

Speckle-type POZ protein (SPOP) - a tumor suppressor gene frequently mutated in prostate cancer – is an adaptor of the cullin 3-based ubiquitin ligase, which is responsible for the ubiquitin-mediated degradation of several proteins. SPOP is crucial for maintaining genome stability, and therefore, its mutation correlates with chromosomal aberrations during tumorigenesis. However, the exact mechanism underlying this function is still elusive. Here we aim to unveil the role of SPOP in maintaining genomic stability.

Material/Methods:

CRISPR-Cas9 technology was used to establish SPOP-knockout (KO) clones from LNCaP and DU145 prostate cancer cells. Western blot, immunofluorescence microscopy, plasmid reporter assay for homologous recombination (HR) and DNA fiber assay were used to investigate the DNA damage response, DNA double-strand break repair, co transcriptional R-loop formation and DNA replication. Transcription was investigated using EU incorporation assay. Proteomic changes and histone post-translational modifications (HPTM) were assessed using mass spectrometry. Chromatin accessibility was monitored by ATACseq and DNA methylation profiling was investigated using the Illumina Infinium HumanMethylation450 BeadChip (450 K) array.

Results:

SPOP-KO sublines were generated from LNCaP and DU145 cells using CRISPR-Cas9 technology and the lack of SPOP expression was validated using Western blotting. No difference was observed in cell growth or DNA damage response between SPOP-wt and SPOP-KO clones. Immunofluorescence analysis revealed accumulation of spontaneous DNA double-strand break foci in SPOP-KO DU145 and LNCaP cells in a cell cycle-unrelated manner. SPOP-KO cells (Du145 & LNCaP) showed a slower replication rate and increased stalled replication forks. In contrary to the so-far published data, we could not observe any signs for HR deficiency in SPOP-KO cells, as confirmed by (i) a plasmid HR reporter assay and (ii) RAD51 immunofluorescence microscopy. Instead, we observed a slower transcription rate in DU145 SPOP-KO cells which was associated with accumulation of higher numbers of the transcription intermediate ‘R-loops’. Further investigation suggested a slower transcription elongation rate in DU145 SPOP-KO cells as evidenced by (i) efficient Pol II chromatin enrichment, (ii) normal Pol II phosphorylation at S5 and (iii) reduced levels of S2 phorphorylated Pol II. Proteomics reported deregulation in several chromatin-structure-associated pathways in SPOP-KO cells, such as chromatin remodelers and histone-methyltransferases. This indicates that SPOP may control transcription by regulating chromatin structure and DNA accessibility. In line with this finding, ATACseq analysis revealed a less accessible chromatin structure in DU145 SPOP-KO clones. Mass spectrometry-based global HPTM analysis revealed more methylated histones in DU145 SPOP-KO sublines. In addition, SPOP-KO DU145 cells showed significantly higher global DNA methylation levels compared to their parental SPOP-wt counterparts. Intriguingly, in the hormone-sensitive LNCaP SPOP-KO cells, we observed upregulated androgen receptor signaling that distinctly increased the transcription rate, causing accumulation of R-loops, replication stress, DNA double-strand breaks and genomic instability.