Specific activation of tumor suppressors repressed by oncogenes to treat cancer
Biochemistry
Final Report Abstract
As therapeutic options for hepatocellular carcinoma (HCC) patients are currently limited, there is an urgent need to develop targeted treatments. The transcription factor Sal-like protein 4 (SALL4) is a promising gene target, expressed in a significant number of solid cancers, including HCC, but not in healthy adult tissues. SALL4 binds to the retinoblastoma-binding protein 4 (RBBp4) of the nucleosome remodeling and deacetylase (NuRD) complex, thereby repressing various genes, including tumor suppressors, which can ultimately lead to cancer. The funded project aimed at disrupting the SALL4/NuRD complex and activating tumor suppressor genes in HCC cells. In preliminary studies, a set of promoters was identified where the SALL4/NuRD complex binds directly and suppresses gene expression. Notably, this repression involves tumor suppressors such as SPEN, MADCAM1, and KDM6B, whose significant fold changes and antitumorigenic properties in HCC, confirmed by RNA-seq, led to their selection for further experiments. To study the effects of disrupting SALL4/NuRD complex activity at the promoters of these tumor suppressors, the CRISPR/dCas9 approach was employed. Specific guide RNAs (gRNAs) targeting the promoters of SPEN, MADCAM1, and KDM6B were designed, and the corresponding plasmids were successfully cloned. These plasmids were then used in subsequent experiments to direct the Cas9-based system to the target promoters. To interfere with the SALL4/NuRD complex, a site-specific dCas9-FFW construct was created. The dCas9 protein, lacking endonuclease activity, was fused to FFW peptides either directly or through linkers. The plasmid was successfully modified through site-directed mutagenesis, ensuring proper insertion of the FFW peptide sequences. The experimental setup involved transfecting the HCC cell line SNU398 with plasmids containing dCas9-FFW constructs and gRNAs. Despite confirming the expression of the dCas9-FFW constructs via mass spectrometry, the results showed no activation of SPEN, MADCAM1, or KDM6B at the RNA level, suggesting that the SALL4 suppression on these genes was not disrupted. Due to the lack of success with transient transfections, stable SNU398 cell lines expressing dCas9-FFW and various gRNAs were generated. However, even with stable expression, no upregulation of the target genes was detected. To understand the underlying reason for the lack of gene activation, co-immunoprecipitation (Co-IP) experiments were conducted to analyze the binding of SALL4 to the NuRD complex. The results revealed no significant change in the binding of SALL4 to the RBBp4 subunit, indicating that the SALL4-NuRD complex remained intact, and the expected gene repression was still occurring. Despite the successful design of gRNAs, generation of dCas9-FFW plasmids, and creation of stable cell lines, the disruption of SALL4-mediated gene repression on SPEN, MADCAM1, and KDM6B could not be demonstrated. The failure to activate these tumor suppressor genes in both transient and stable cell lines, combined with the Co-IP results, suggests that the SALL4/NuRD complex may not have been effectively disrupted in these experimental settings. Therefore, alternative strategies may be required to study the role of these tumor suppressors in HCC.
Publications
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Molecular Mechanisms to Target Cellular Senescence in Hepatocellular Carcinoma. Cells, 9(12), 2540.
Mittermeier, Constanze; Konopa, Andreas & Muehlich, Susanne
