PFKFB3 Regulates Epithelial-To-Mesenchymal Transition in Tumor Cells

Year 2023, Volume: 2 Issue: 1, 15 - 27, 31.03.2023

Tuğba Hazal Altunok , Abdullah Yalçın

Abstract

Background and aim: Reprogramming of energy metabolism in tumor cells is suggested to play a key role in promotion of the epithelial-to-mesenchymal transition (EMT) program that is associated with malignant features including metastasis and chemoresistance. Given the role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 (PFKFB3) in regulation of glycolytic activity, we sought to determine whether PFFKB3 is required to maintain the steady-state expression levels of EMT genes in tumor cell lines.
Materials and methods: HCT116 (colon adenocarcinoma), S2VP10 (a metastatic variant of SUIT-2, pancreatic adenocarcinoma), MCF-7 (breast carcinoma), MDA-MB-231 (breast carcinoma), and HeLa (cervix carcinoma) cell lines were used as in vitro models. Endogenous PFKFB3 expression was silenced by transfecting cells with a combination of two different siRNA molecules specific for the coding region of the PFKFB3 mRNA. mRNA expression and protein levels were measured using real-time quantitative PCR and Western blot, respectively. Glucose consumption and lactate production were determined spectrophotometrically using commercial kits. HCT116 cells were stably transfected with an expression vector containing PFKFB3 cDNA for oncosphere formation assays. The Cancer Genom Atlas (TCGA) datasets were analyzed using cBioportal tool to study the correlation between PFKFB3 and EMT gene expressions.
Results and conclusions: We demonstrated that silencing of PFKFB3 resulted in changes in expressions of EMT genes, including E-cadherin, Vimentin and Snail in various tumor cell lines and that PFKFB3 mRNA expression correlates with mRNA levels of mesenchymal genes in colorectal adenocarcinoma patients. We further show that ectopic expression of PFKFB3 increases the ability of HCT116 cells to form oncospheres. Manipulation of PFKFB3 activity may be considered a viable approach to target malignant traits such as metastasis and chemoresistance that is associated with EMT.

Keywords

Epithelial-to-mesenchymal transition, Glycolysis, PFKFB3

PFKFB3 Tümör Hücrelerinde Epitelyal-Mezenkimal Geçişi Düzenler

Abstract

Amaç: Tümör hücrelerinde enerji metabolizmasının yeniden programlanmasının, metastaz ve kemorezistans dahil olmak üzere malign özelliklerle ilişkili epitelyal-mezenkimal geçiş (EMT) programının desteklenmesinde önemli bir rol oynadığı bilinmektedir. 6-fosfofrukto-2-kinaz/fruktoz 2,6-bifosfataz-3 (PFKFB3)'ün glikolitik aktivitenin düzenlenmesindeki rolü nedeniyle bu çalışmada, PFFKB3'ün EMT genlerinin ekspresyonundaki olası rolünü belirlemeyi amaçladık.
Yöntem: HCT116 (kolon adenokarsinomu), S2VP10 (SUIT-2'nin metastatik bir varyantı, pankreatik adenokarsinom), MCF-7 (meme karsinomu), MDA-MB-231 (meme karsinomu) ve HeLa (serviks karsinomu) hücre hatları in vitro modeller olarak kullanıldı. Endojen PFKFB3 ekspresyonu, hücrelerin PFKFB3 mRNA'nın kodlama bölgesine özgü iki farklı siRNA molekülünün kombinasyonu ile transfekte edilmesiyle susturuldu. mRNA ve protein ekspresyon seviyeleri, sırasıyla gerçek-zamanlı kantitatif PCR ve Western blot kullanılarak ölçüldü. Glikoz tüketimi ve laktat üretimi, ticari kitler kullanılarak spektrofotometrik olarak belirlendi. HCT116 hücreleri, onkoküre oluşum analizleri için PFKFB3 cDNA içeren bir ekspresyon vektörü ile stabil bir şekilde transfekte edildi. Kanser Genom Atlası (TCGA) veri kümeleri, PFKFB3 ve EMT gen ifadeleri arasındaki korelasyonu incelemek için cBioPortal aracı kullanılarak analiz edildi.
Bulgular ve Sonuç: PFKFB3'ün susturulmasının çeşitli tümör hücre hatlarında E-cadherin, Vimentin ve Snail adlı EMT genlerinin ifadelerinde değişikliklere yol açtığını ve PFKFB3 mRNA ifadesinin kolorektal adenokarsinom hastalarında mezenkimal genlerin mRNA seviyeleri ile korelasyon gösterdiğini gözlemledik. Ayrıca, PFKFB3'ün ektopik ifadesinin, HCT116 hücrelerinin onkoküre oluşturma yeteneğini arttırdığını raporladık. PFKFB3 aktivitesinin manipülasyonu, metastaz ve EMT ile ilişkili kemorezistans gibi habis özellikleri hedeflemek için geçerli bir yaklaşım olarak kabul edilebilir.

Keywords

Epitelyal-mezenkimal geçiş, glikoliz, PFKFB3

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