Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer cases. It tends to be aggressive, high-grade and poorly differentiated tumour with poor clinical outcome. Lack of expression of oestrogen, progesterone receptors, and human epidermal growth factor receptor 2 make TNBC patients ineligible to hormonal therapy. For these reasons the identification of novel clinical targets still remains a priority. With the advances of multiomics, many of the genes transcriptionally upregulated in TNBC have been identified, but how they are disregulated is still unknown: the understanding of how this works at a transcriptional level could contribute to the development of a novel therapeutic approach.

We report here a new methodology to identify key transcription regulators that we applied to investigate the expression of highly expressed genes in TNBC: our approach combines RIME proteomics with CRISPR/Cas9 technology. In brief, we targeted putative regulatory regions of differentially regulated genes in TNBC compared to other subtypes of breast cancer using a catalytically dead version of the Cas9 protein (dCas9). In particular, we focused on transcription regulators like FOXC1, NFIB and NFE2L3. We then performed RIME proteomics to identify which proteins are in close proximity to dCas9 and thus potentially bound to these putative regulatory regions. In addition, we developed a novel, statistical approach to analyse these particular proteomic datasets based on the relative abundance of the protein of interest, and a powerful ranking method to identify biologically and therapeutic meaningful candidates.

Through this process, we identified three putative regulatory proteins, MTA2, CDK1 and CDK6, bound to all three loci. In here, we reported how their knockdown, performed by shRNA, directly affects the expression of the investigated genes in a panel of TNBC cell lines, and their oncogenic capacity in vitro. These results demonstrate the importance of these transcription regulators for TNBC biology, and they highlight the necessity of a deeper understanding of their roles in gene expression regulation.