Abstract P5-05-01: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive aggressive metastatic breast cancer
Authors: Dasgupta S , Anand V , John H , Sawant Dessai A , Katsuta E , Takabe K , O'Malley B 
Published: May 2019

Background: Metabolic rewiring is one of the central hallmarks of cancer progression and survival to support anabolic and energetic demands. Tumor cells constantly alter their metabolic state in response to oncogenic stimuli, nutrient availability, and interaction with immune cells however the precise regulation that precedes the metabolic alteration is poorly understood. Here we report a direct interaction of glycolytic enzyme PFKFB4 with transcriptional coregulator SRC-3. PFKFB4 functions as a critical regulator of Warburg effect and our study reveals that upon glucose stimulation PFKFB4 activates SRC-3 driving an invasive-metastatic breast cancer.

Methods: Molecular experiments were performed to understand the transcriptional activation of SRC-3 by PFKFB4 enzyme. Chromatin immunoprecipitation and gene expression studies were performed to investigate the functions of PFKFB4/SRC-3 crosstalk on transcriptional regulation. Metabolomics and isotope tracing studies were performed to identify the metabolic adaptations regulated by PFKFB4/SRC-3 in breast tumors. PFKFB4-knockout was established using CRISPR-Cas9 system and functional studies were carried out to define its role in tumor cell proliferation, invasion-migration, and breast to lung metastasis. Human breast tumor samples were evaluated to identify the clinical importance of PFKFB4/SRC-3 crosstalk in patients.

Results:Molecular studies revealed that PFKFB4 enzyme phosphorylates SRC-3 at serine 857 (S857) enhancing its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient SRC-3 mutant S857A (SRC-3S857A) significantly abolished SRC-3-mediated transcriptional output (p&lt;0.000001). Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway enabling purine synthesis by transcriptionally upregulating the expression of enzyme transketolase (TKT). Deletion of PFKFB4 by CRISPR-Cas9 system resulted in significantly reduced proliferation (p&lt;0.05) and migration-invasion (p&lt;0.001) compared to wildtype breast tumor cells. Ablation of SRC-3 or PFKFB4 suppressed in vivo breast tumor growth and prevents metastasis to the lung from an orthotopic setting (p&lt;0.0001). PFKFB4 and phosphorylated SRC-3 levels are significantly increased in breast tumors (p=0.02), whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of TNBC patients (p=0.03).

Conclusion:Our data suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumors. It also provides first evidence how Warburg pathway drives aggressive breast tumorigenesis by directly activating powerful oncogene SRC-3. Our work suggests that targeting the PFKFB4–SRC-3 axis may be therapeutically valuable in breast tumors that are notably dependent on glucose metabolism.

(This work is funded by grants from Susan G. Komen and NCI to S.D.)

Citation Format: Dasgupta S, Anand V, John H, Sawant Dessai A, Katsuta E, Takabe K, O'Malley B. Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive aggressive metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-01.