PNAS: Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling
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Abstract
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 inpatient lung metastases (n = 53) and were statistically associated with a 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.
Large-scale sequencing analyses of solid cancers have identified extensive tumor heterogeneity within individual primary cancers (1). Recent studies indicate that such tumoral heterogeneity is associated with heterogeneous protein function, which fosters tumor adaptation, treatment resistance, and failure through Darwinian selection (2⇓–4). Cancer stem cells are a subpopulation of cells within the primary tumor responsible for tumor initiation and metastases (5⇓⇓⇓–9). Three groups have recently independently provided functional evidence for the presence of cancer stem cells by lineage-tracing experiments (10⇓–12). These observations suggest that these subpopulations of cancer stem cells (CSCs) within the bulk primary tumor are resistant to conventional therapies through different adaptive mechanisms with the potential for self-renewal and metastases (7, 13, 14). However, few studies have determined the genetic profile of the cells that escape primary cancer and evolve in distant metastatic sites (1). Additionally, no large-scale sequencing studies of metastases have been conducted because the majority of patients are treated with systemic therapies and not surgery.
Tumor clonal heterogeneity within a primary tumor may in part be explained by hypoxic regions within the bulk tumor that has been correlated with invasiveness, therapeutic resistance, and metastasis (15⇓⇓–18). Cancer stem cells have been found to reside near hypoxic regions in some solid cancers (19⇓–21). We have previously published a 477-gene tumorigenic signature by isolating breast cancer stem cells (BCSCs) derived from patient biopsies (22). Here, we have identified two previously unidentified cancer genes, ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2), by selective shRNA knockdown of genes from this tumorigenic signature, that impact breast cancer stem cell self-renewal and lung metastases. Analysis of 53 patient lung metastases confirmed damaging mutations in RPL39 and MLF2 in a significant number of samples, which conferred a gain-of-function phenotype. These mutations were statistically associated with a shorter median time to distant relapse. We further describe a common mechanism of action through nitric oxide synthase signaling that is regulated by hypoxia.
Since 2004, UT Health San Antonio, Greehey Children’s Cancer Research Institute’s (Greehey CCRI) mission has been to advance scientific knowledge relevant to childhood cancer, contribute to understanding its causes, and accelerate the translation of knowledge into novel therapies. Greehey CCRI strives to have a national and global impact on childhood cancer by discovering, developing, and disseminating new scientific knowledge. Our mission consists of three key areas — research, clinical, and education.
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