Abstract
Purpose :
Retinoblastoma (RB) is an intraocular malignancy caused by biallelic inactivation of the RB1 tumor suppressor gene. In addition to RB1 mutations, the amplification/overexpression of MYCN oncogene has been linked to tumor pathogenesis. MYCN has previously been shown to regulate multiple tumor hallmarks in RB, including alterations in metabolic pathways. We discovered that 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a major regulator of glucose metabolism, is overexpressed in RB. Here, we investigated the functional role of PFKFB3 in RB.
Methods :
Immunoblotting and immunohistochemistry were performed to determine PFKFB3 protein expression in RB cell lines and patient specimens. shRNA knockdown/pharmacological inhibition with PFK15 and YZ9 were employed to study the role of PFKFB3 and assays for cell viability, cell cycle, and apoptosis were carried out. To understand PFKFB3 regulation by MYCN, protein levels of PFKFB3 were determined in RB cells with MYCN knockdown. Lactate production and glucose uptake were measured. Wound healing assay was performed, and mRNA expression of extracellular matrix (ECM) genes was measured using qRT-PCR to determine migration ability of cells with PFKFB3 inhibition. Combination studies were carried out with PFKFB3 inhibition and the chemotherapeutic agent melphalan.
Results :
PFKFB3 expression was found to be elevated in 90% of the RB specimens. Pharmacological inhibition and shRNA-mediated knockdown of PFKFB3 resulted in reduced cell survival, enhanced apoptosis, and cell cycle arrest. Immunoblotting revealed a reduction in PFKFB3 levels in response to MYCN knockdown. Inhibiting PFKFB3 led to decrease in glucose uptake, lactate production, cell migration and a concurrent downregulation in mRNA expression of ECM genes. Combination studies show that melphalan and PFK15 have a synergistic effect on RB cell viability.
Conclusions :
PFKFB3 overexpression alters metabolic parameters, and promotes cell growth and migration of RB cells. Overall, our findings suggest that PFKFB3 is induced in RB, and could be specifically inhibited, either alone or in conjunction with melphalan, to control tumor cell growth and migration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.