Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling

Significance This manuscript describes the identification and characterization of two previously unidentified cancer genes, ribosomal protein L39 and myeloid leukemia factor 2, that play an important role in tumor initiation and metastasis. Knockdown of these genes in triple negative breast cancer (TNBC) models significantly reduces primary-tumor growth, as well as metastasis. Mutations in these genes are associated with worse survival in breast-cancer patients. Both genes are regulated by the nitric oxide signaling pathway. Identification of these two genes represents a significant breakthrough in our understanding of treatment resistance in TNBC. Targeting these genes could alter clinical practice for tumor metastasis in future and improve outcomes of patients with breast cancer. We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.