Bad to the bone: An RNA-sequencing study identifying a role for IKK alpha inosteosarcoma cell division and progression

Introduction
Osteosarcoma (OS) is the most prevalent primary malignant bone tumour generally made up of osteoblasts and is diagnosed in one to three individuals per million people every year [1]. Despite research efforts, there has been little improvement to OS treatment and prognosis in the last30 years [2]. Given these marginal improvements, research at a molecular level is imperative to enable discovery of therapeutic drug targets for OS.

Methods
Our present study utilises short-read RNA-sequencing techniques (Illumina) to a human OS cell line, U2OS, with and without IKKα deletion byCRISPR-Cas9 knockdown, and in the absence or presence of IL-1β stimulation for 8 or 24 h (n = 3). Following RNA-sequencing analysis, we further validated gene expression of genes of interest by RT-qPCR and at protein level by utilising immunofluorescence techniques and SDS-PAGE and western blotting.

Results
Our findings highlight that IKKα knockdown significantly decreases expression of cell division genes associated with OS progression, including AURKA, AURKB, TPX2, BIRC5, GTSE1, E2F2, FOXM1 and SPC24 (****P < 0.0001). Interestingly, IKKα knockdown increased osteoclast-associated receptor (OSCAR) gene expression (****P < 0.0001), which is a central receptor in bone degradation processes. Osteoclast activity is associated with decreased OS-derived metastasis, and hence this implies a role for IKKα in promoting metastasis. Additionally, with IL-1β stimulation for 8 h, we identified that there are several IL-1β-dependent, IKKα-dependent genes associated with OS tumour progression including CXCL5 and GAS7 [3]. These results were validated in the laboratory by RT-qPCR. This reveals an important role for IKKα in osteogenesis mediated by the CXCR2 axis and GAS7 (Figure 1).

Conclusions
Overall, IKKα appears to play a role in osteogenesis, a key feature in OS cell proliferation and a role in down-regulating bone degradation. How-ever, validating these results in OS patient samples would be required before considering IKKα as a viable therapeutic drug target in OS.

References
1. Huang X, Wang L, Guo H, Zhang W. Single-cell RNA sequencing reveals SERPINE1-expressing CAFs remodelling tumour microenvironment in recurrent osteosarcoma. Clin Transl Med. 2024;14(1):e1527.
2. Gianferante DM, Mirabello L, Savage SA. Germline and somatic genetics of osteosarcoma—Connecting aetiology, biology and therapy. Nature Reviews Endocrinology. 2017;13(8):480-91.SUPPLEMENT ABSTRACT702