Elucidating the Role of NPM1 in AKT/mTOR/S6K1 Signaling and Metastatic Progression of Triple-Negative Breast Cancer

Abstract

Background: Metastasis remains the leading cause of cancer-related mortality. It is governed by a complex cascade that includes tumor growth, angiogenesis, epithelial-to mesenchymal transition (EMT), invasion, immune evasion, and organ-specific colonization. Breast cancer (BC) classifies among the leading cancers worldwide and shows rising mortality-to-incidence ratios in the Middle East. Triple-negative breast cancer (TNBC), an aggressive BC subtype, carries a particularly poor prognosis due to its high metastatic potential and lack of effective targeted therapies. In TNBC, the pleiotropic chaperone protein Nucleophosmin-1 (NPM1) which regulates processes such as ribosomal biogenesis, is overexpressed and associated with poor clinical outcomes. The ribosomal protein S6 kinase B1 (S6K1), a key mediator of ribosomal biogenesis and EMT, is activated downstream of the hyperactivated AKT/mTOR pathway in BC, likely contributing to tumor progression and metastasis. Methods: Using short hairpin RNA, we evaluated the impact of knocking-down NPM1 on the proliferation of TNBC cell lines and on key molecular drivers of metastasis, including AKT/mTOR signaling, S6K1 activation, and EMT markers. RNA sequencing was performed to assess the global transcriptomic changes following NPM1 modulation. Laser-dissection microscopy was performed on paraffin-embedded blocks from primary TNBC tumors or their metastatic counterparts to different organs. The in vivo capacity of NPM1-modulated TNBC cells to metastasize to bone, liver, lung, and brain was also tested. Finally, the effect of NPM1 modulation on AKT/mTOR pathway and the pharmacological efficacy of a combinatorial therapy targeting this pathway were elucidated. Results: We showed that knockdown of NPM1 in TNBC cells reduced cell proliferation, promoted mesenchymal-to-epithelial transition (MET), and significantly reduced tumor growth, metastasis, and mortality in vivo. Patient RNA-seq analysis demonstrated higher NPM1 expression in metastatic versus primary sites. Pharmacological inhibition of the NPM1/AKT/mTOR pathway effectively inhibited the proliferation of TNBC cells and inactivated AKT/mTOR/S6K1 signaling in vitro. Conclusion: Integrating transcriptomics on modulated TNBC cells, patient-derived samples, and in-vivo models, we defined a role for NPM1 in driving metastatic progression in TNBC and investigated NPM1-targeted therapies as an avenue to impede TNBC metastasis.