3PO inhibits glycolysis but does not bind to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3)

Veseli, Besa Emini; Perrotta, Paola; Abdali, Anahita; Wielendaele, Pieter Van; Monaco, Giovanni; Gelmi, Maria Luisa; Bultynck, Geert; Martinet, Wim; Meyer, Guido De

doi:10.1016/j.atherosclerosis.2020.10.268

Cited by 4 publications

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“…Research studies have demonstrated that 3PO does not bind to PFKFB3, and is inactive in a PFKFB3 kinase assay. 22,23 However, 3PO analog PFK15 (used in the present study) has been shown to reduce F-2,6-P 2 levels and lactate production in cancer cell lines. 21…”

Section: Introductionmentioning

confidence: 60%

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK‐1 activity of triple‐negative breast cancers by reducing the glycolysis

Kashyap

Umar

et al. 2023

J of Cellular Biochemistry

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Among all the subtypes of breast cancer, triple‐negative breast cancer (TNBC) has been associated with the worst prognosis. Recently, for many solid tumors (including breast cancer) metabolic reprogramming has appeared as a cancer cell hallmark, and the elevated glycolytic pathway has been linked to their aggressive phenotype. In the present study, we evaluated the prognostic and therapeutic relevance of PFKFB3 (6‐phosphofructo‐2‐ kinase/fructose‐2,6‐bisphosphatase) in TNBCs. Prognostic significance of PFKFB3 expression was evaluated in overall breast cancers as well as in TNBCs. PFKFB3 inhibitor (3PO potent analogue i.e., PFK15) cytotoxicity in TNBC cell lines (MDA‐MB‐231 and MDA‐MB‐468) was analyzed using an MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. Cancer cell physiological characteristics like clonogenicity and migration were also investigated after PFK15 treatment. As fructose‐2,6‐bisphosphate (F‐2,6‐BP), has been associated with increased PFK‐1 activity, the effect of PFKFB3 inhibition by PFK15 was investigated on two major isoforms of phosphofructokinase‐1 (PFK‐1) in breast cancer, that is, phosphofructokinase‐platelet type (PFKP) and phosphofructokinase‐liver type (PFKL) (relevant to breast cancer). For PFKL inhibition, the siRNA approach was used. PFKFB3 expression was significantly correlated with inferior overall survival in breast cancer patients including TNBCs. PFK15 treatment in TNBC cells (i.e., MDA‐MB‐231 and MDA‐MB‐468) resulted in a decreased PFKP expression, thereby leading to reduced colony formation ability, migration rate, and extracellular lactate levels. However, to our surprise PFK15 treatment in both TNBC cells also resulted in elevated PFKL levels. Our results demonstrated that the combinatorial inhibition of PFK15 with siPFKL was more effective in TNBC cells, as it led to a decrease in colony formation ability, migration rate, extracellular lactate levels, and PFK‐1 activity when compared with individual treatments. Using bona fide PFKFB3 inhibitor, that is, AZ67, we further show that AZ67 treatment to TNBC cells has no effect either on the expression of PFKP and PFKL, or on the lactate production. In summary, our present in vitro study demonstrated that 3PO derived PFK15 mechanism of action is totally different from AZ67 in TNBC cells. However, we advocate that the PFK15–mediated inhibition (along with PFKL) on the TNBCs migration, colony formation, and PFK‐1 activity can be further explored for the therapeutic advantage of TNBC patients.

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Section: Introductionmentioning

confidence: 60%

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK‐1 activity of triple‐negative breast cancers by reducing the glycolysis

Kashyap

Umar

et al. 2023

J of Cellular Biochemistry

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“…We therefore used AZ PFKFB3 67, a PFKFB3 inhibitor [ 25 ]. We exposed the cells to 25 µM AZ PFKFB3 67, a concentration limiting PFKFB3 activity in cells [ 33 ]. Similarly to Torin1 and BAPTA i , AZ PFKFB3 67 decreased the phospho-p70S6K/total p70S6K ratio and reduced MCL-1-protein levels in both SU-DHL-4 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells

Sneyers,

Kerkhofs,

Speelman-Rooms

et al. 2023

Cell Death Dis

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Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. In this study, we discovered that BAPTAi alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTAi provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTAi diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTAi. Notably, these effects were also induced by a BAPTAi analog with low affinity for Ca2+. Consequently, our findings uncover PFKFB3 inhibition as an Ca2+-independent mechanism through which BAPTAi impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

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Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing Mcl-1-addicted cancer cells

Sneyers¹,

Kerkhofs²,

Welkenhuyzen³

et al. 2022

Preprint

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Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). We previously demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a Bcl-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). These findings implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic Bcl-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. We observed that BAPTAi induced apoptosis in lymphoma cell models that were highly sensitive to S63845, an Mcl-1 antagonist. BAPTAi provoked a rapid decline in Mcl-1-protein levels by inhibiting mTORC1-driven MCL-1 translation. Overexpression of nondegradable Mcl-1 rescued BAPTAi-induced cell death. We further examined how BAPTAi diminished mTORC1 activityand found that BAPTAi impaired glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, an up to now unappreciated effect of BAPTAi. All aforementioned effects of BAPTAi were also elicited by a BAPTAi analog with low affinity for Ca2+. Thus, our work reveals PFKFB3 inhibition as an unappreciated Ca2+-independent mechanism by which BAPTAi impairs cellular metabolism and ultimately the survival of Mcl-1-dependent cancer cells. Our work has two important implications. First, direct inhibition of PFKFB3 emerged as a promising target in cancer treatment. Second, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi.

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3PO inhibits glycolysis but does not bind to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3)

Cited by 4 publications

References 0 publications

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK‐1 activity of triple‐negative breast cancers by reducing the glycolysis

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK‐1 activity of triple‐negative breast cancers by reducing the glycolysis

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing MCL-1-addicted cancer cells

Intracellular BAPTA directly inhibits PFKFB3, thereby impeding mTORC1-driven Mcl-1 translation and killing Mcl-1-addicted cancer cells

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