PFKFB4 adenovirus

Alterations in cellular metabolic and transcriptional programs are hallmarks of cancers that sustain rapid proliferation and metastasis. However, the mechanisms that control the interplay between metabolic reprogramming and transcriptional regulation remain unclear. Here, researchers show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating oncogenic steroid receptor coactivator-3 (SRC-3). They used a kinome-wide RNA interference-based screening approach to identify potential kinases that modulate the intrinsic SRC-3 transcriptional response. PFKFB4, a regulatory enzyme that synthesizes a potent glycolytic stimulator, was found to be a potent stimulator of SRC-3 that co-regulates the estrogen receptor. PFKFB4 phosphorylates SRC-3 at serine 857 and enhances its transcriptional activity, whereas inhibition of PFKFB4 or ectopic expression of the phosphorylation-deficient Ser857Ala mutant SRC-3 abolishes SRC-3-mediated transcriptional output. Ablation of SRC-3 or PFKFB4 inhibits breast tumor growth in mice and prevents tumor metastasis from orthotopic to lung, as does the Ser857Ala mutant SRC-3. PFKFB4 and phosphorylated SRC-3 levels are elevated and correlated in estrogen receptor-positive tumors, and in basal subtype patients, PFKFB4 and SRC-3 drive a common protein signature that correlates with poor survival in breast cancer patients. These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular pivot that couples sugar metabolism to transcriptional activation by stimulating SRC-3, thereby promoting aggressive metastatic tumors.

As a starting point to identify kinases that regulate SRC-3 transcriptional activity, the researchers performed an unbiased RNA interference (RNAi) screening assay using a kinome library containing short interfering RNAs (siRNAs) targeting 636 human kinases (median 3 siRNAs per kinase) in the presence of SRC-3 fused to the GAL4-DNA binding domain (pBIND-SRC-3) and a GAL4 DNA binding site containing a luciferase reporter gene (pG5-luc) (Figure 1a). As a positive control, they used siRNA targeting PRKCZ1, a protein kinase known to activate SRC-313, and compared the inhibition of coregulatory activity after kinase knockdown to a non-targeting control green fluorescent protein (GFP) siRNA. The kinome-wide screen identified several kinases as regulators of SRC-3 activity (Figure 1b), as either stimulators or repressors compared to the controls.

Ten kinases were designated as reproducible and significant hits in the screen (Figure 1c), with the metabolic kinase PFKFB4 identified as the most potent positive regulator of SRC-3 activity. Silencing PFKFB4 using different short hairpin RNAs (shRNAs) and siRNAs reduced SRC-3 activity in several cancer lines, and PFKFB4 levels were reduced. In contrast, ectopic overexpression of PFKFB4 using adenoviral infection (ad-PFKFB4) enhanced SRC-3 activity (Figure 1d). Interestingly, SRC-3 protein levels were increased upon ectopic PFKFB4 expression (Figure 1e), but SRC-3 (also known as NCOA3) mRNA levels were unaffected. Proximity ligation assays supported a direct interaction between SRC-3 and PFKFB4, consistent with PFKFB4-dependent regulation of SRC-3 activity.

Figure 1. PFKFB4 is an essential activator of transcriptional coregulator SRC-3. (Dasgupta S, et al., 2018)