Transforming growth factor ß1 upregulates 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-4 expression in A549 and MCF-10A cells.

Transforming growth factor ß1 (TGFß1) induces a cellular process known as epithelial-mesenchymal transition (EMT) associated with metabolic reprogramming, including enhanced glycolysis. Given the involvement of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB) enzymes in glycolysis, we aimed to investigate whether TGFß1 regulates expressions of PFKFB genes and if PFKFBs are required for TGFß1-driven phenotypes. A549 and MCF-10A cell lines were used as TGFß1-driven EMT models. Messenger RNA expressions of PFKFB and EMT genes were determined by real-time quantitative polymerase chain reaction. A small interfering RNA approach was used to deplete PFKFB4 expression. A Matrigel invasion assay was conducted to assess the effect of PFKFB4 silencing on the TGFß1-enhanced invasion of A549 cells. F2,6BP levels were analyzed using an enzyme-coupled assay. Glucose and lactate concentrations were determined using colorimetric assays. TGFß1 robustly induced expression of the fourth isoform of PFKFBs, PFKFB4, in both cell lines. PFKFB4 depletion partially inhibits mesenchymal transdifferentiation caused by TGFß1 in A549 cells, as assessed by microscopy. Inductions of Snail in MCF-10A cells and Fibronectin in A549 cells and repressions of E-cadherin in both cell lines by TGFß1 are attenuated by PFKFB4 silencing. PFKFB4 silencing reduces F2,6BP and glycolytic activity, although TGFß1 alone does not affect these parameters. Finally, PFKFB4 depletion suppresses the TGFß1-driven invasion of A549 cells through Matrigel. Presented data suggest that TGFß1 induces the expression of PFKFB4 in A549 and MCF-10 cells, and PFKFB4 may be required for TGFß1-driven phenotypes such as EMT and invasion in these models.

Identification of genomic determinants contributing to cytokine release in immunotherapies and human diseases.

BACKGROUND: Cytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS. METHODS: Forty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes. RESULTS: We found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE). CONCLUSION: Our data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.

KDM3A-mediated SP1 activates PFKFB4 transcription to promote aerobic glycolysis in osteosarcoma and augment tumor development.

BACKGROUND: Lysine-specific histone demethylase 3A (KDM3A) is a potent histone modifier that is frequently implicated in the progression of several malignancies. However, its role in aerobic glycolysis of osteosarcoma (OS) remains unclear. METHODS: KDM3A expression in OS tissues was determined by immunohistochemistry, and that in acquired OS cells was determined by RT-qPCR and western blot assays. KDM3A was silenced in OS cells to examine cellular behaviors and the aerobic glycolysis. Stably transfected cells were injected into nude mice for in vivo experiments. The downstream targets of KDM3A were predicted by bioinformatics systems and validated by ChIP-qPCR. Rescue experiments of SP1 and PFKFB4 were performed to examine their roles in the KDM3A-mediated events. RESULTS: KDM3A was highly expressed in OS tissues and cells. Knockdown of KDM3A weakened OS cell growth and metastasis in vivo and in vitro, and it suppressed the aerobic glycolysis in OS cells. KDM3A enhanced the transcription of SP1 by demethylating H3K9me2 on its promoter. Restoration of SP1 rescued growth and metastasis of OS cells and recovered the glycolytic flux in cells suppressed by knockdown of KDM3A. SP1 bound to the PFKFB4 promoter to activate its transcription and expression. PFKFB4 expression in OS cells was suppressed by KDM3A silencing but increased after SP1 restoration. Overexpression of PFKFB4 significantly promoted OS cell growth and metastasis as well as the glycolytic flux in cells. CONCLUSION: This paper elucidates that upregulation of PFKFB4 mediated by the KDM3A-SP1 axis promotes aerobic glycolysis in OS and augments tumor development.

PFKFB4 modulated by miR-195-5p can boost the malignant progression of cervical cancer cells.

PFKFB4 is dysregulated in varying tumors and has the biological function of regulating tumor progression. However, its biological function in cervical cancer is poorly understood. We obtained the upstream regulatory gene (miR-195-5p) of PFKFB4 through bioinformatics analysis. Then, experiments were introduced to measure expression and targeting relationship of miR-195-5p and PFKFB4 in cervical cancer cells, in order to evaluate their influence on proliferation, migration, invasion and angiogenesis of cervical cancer cells. As expressed in results, PFKFB4 was abnormally increased and boosted malignant progression of cervical cancer cells. Besides, miR-195-5p was markedly decreased and restrained PFKFB4 in cervical cancer. While tumor-suppressive effect of miR-195-5p was partially restored by overexpressing PFKFB4, indicating that miR-195-5p and PFKFB4 may be new therapeutic targets for cervical cancer patients.

Loss of PFKFB4 induces cell cycle arrest and glucose metabolism inhibition by inactivating MEK/ERK/c-Myc pathway in cervical cancer cells.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) was reported to be necessary for tumour growth in several cancers. However, the function of PFKFB4 in cervical cancer has not been clearly elucidated. Bioinformatics analysis was applied to detect the expression of PFKFB4 in cervical cancer and the association with survival prognosis. The effect of PFKFB4 on cervical cancer cells growth, cycle, invasion, migration and glucose metabolism was investigated by loss-of-function approaches in vitro. The association between PFKFB4 and MEK/ERK/c-Myc pathway was identified by western blot assay. We found that PFKFB4 was highly expressed in cervical cancer samples and its overexpression led to a poor prognosis of cervical cancer patients. Knock down of PFKFB4 reduced cell growth, blocked cell cycle, inhibited cell invasion and migration, and blocked glucose metabolism in cervical cancer cells. Our findings afforded a theoretical basis for further research on the treatment of cervical cancer based on the control of PFKFB4 expression. Impact StatementWhat is already known on this subject? PFKFB4 was overexpressed in several kinds of cancers and its requirement for tumour growth has been confirmed in cancers such as glioma and breast cancer. However, the function of PFKFB4 in cervical cancer cells has not been clearly elucidated. A bioinformatics study showed that PFKFB4 was a member of a six-gene signature associated with glycolysis to predict the prognosis of patients with cervical cancer. However, the relationship between PFKFB4 and glucose metabolism in cervical cancer has not been revealed.What do the results of this study add? Our results showed that PFKFB4 was highly expressed in cervical cancer samples and its overexpression led to a poor prognosis of cervical cancer patients. Moreover, the administration of si-PFKFB4 significantly reduced cell growth ability, blocked cell cycle, restrained the mobility and suppressed the glucose metabolism in cervical cancer cells partially by inactivating MEK/ERK/c-Myc pathway.What are the implications of these findings for clinical practice and/or further research? Our findings afforded a theoretical basis for further research on the treatment of cervical cancer based on the control of PFKFB4 expression.

Bruceine A induces cell growth inhibition and apoptosis through PFKFB4/GSK3ß signaling in pancreatic cancer.

Pancreatic cancer is one of the most aggressive cancers with a poor prognosis and 5-year low survival rate. In the present study, we report that bruceine A, a quassinoid found in Brucea javanica (L.) Merr. has a strong antitumor activity against human pancreatic cancer cells both in vitro and in vivo. Human proteome microarray reveals that 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is the candidate target of bruceine A and both fluorescence measurement and microscale thermophoresis suggest bruceine A binds to PFKFB4. Bruceine A suppresses glycolysis by inhibiting PFKFB4, leading to cell cycle arrest and apoptosis in MIA PaCa-2 cells. Furthermore, glycogen synthase kinase-3 ß (GSK3ß) is identified as a downstream target of PFKFB4 and an PFKFB4-interacting protein. Moreover, bruceine A induces cell growth inhibition and apoptosis through GSK3ß, which is dysregulated in pancreatic cancer and closely related to the prognosis. In all, these findings suggest that bruceine A inhibits human pancreatic cancer cell growth via PFKFB4/GSK3ß-mediated glycolysis, and it may serve as a potent agent for curing human pancreatic cancer.

PFKFB4 promotes lung adenocarcinoma progression via phosphorylating and activating transcriptional coactivator SRC-2.

BACKGROUND: To investigate the role and its potential mechanism of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) in lung adenocarcinoma. METHODS: Co-immunoprecipitation was performed to analyze the interaction between PFKFB4 and SRC-2. Western blot was used to investigate the phosphorylation of steroid receptor coactivator-2 (SRC-2) on the condition that PFKFB4 was knockdown. Transcriptome sequencing was performed to find the downstream target of SRC-2. Cell Counting Kit-8 (CCK-8) assay, transwell assay and transwell-matrigel assay were used to examine the proliferation, migration and invasion abilities in A549 and NCI-H1975 cells with different treatment. RESULTS: In our study we found that PFKFB4 was overexpressed in lung adenocarcinoma associated with SRC family protein and had an interaction with SRC-2. PFKFB4 could phosphorylate SRC-2 at Ser487, which altered SRC-2 transcriptional activity. Functionally, PFKFB4 promoted lung adenocarcinoma cells proliferation, migration and invasion by phosphorylating SRC-2. Furthermore, we identified that CARM1 was transcriptionally regulated by SRC-2 and involved in PFKFB4-SRC-2 axis on lung adenocarcinoma progression. CONCLUSIONS: Our research reveal that PFKFB4 promotes lung adenocarcinoma cells proliferation, migration and invasion via enhancing phosphorylated SRC-2-mediated CARM1 expression.

Expression of Alternative Splice Variants of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-4 in Normoxic and Hypoxic Melanoma Cells.

Cancer-specific isoenzyme of phosphofructokinase II (PFKFB4), as our previous research has shown, may be one of the most important enzymes contributing to the intensification of glycolysis in hypoxic malignant melanoma cells. Although the PFKFB4 gene seems to play a crucial role in the progression of melanoma, so far there are no complete data on the expression of PFKFB4 at the isoform level and the influence of hypoxia on alternative splicing. Using RT-qPCR and semi-quantitative RT-PCR, we presented the PFKFB4 gene expression profile at the level of six isoforms described in the OMIM NCBI database in normoxic and hypoxic melanoma cells. Additionally, using VMD software, we analyzed the structure of isoforms at the protein level, concluding about the catalytic activity of individual isoforms. Our research has shown that five isoforms of PFKFB4 are expressed in melanoma cells, of which the D and F isoforms are highly constitutive, while the canonical B isoform seems to be the main isoform induced in hypoxia. Our results also indicate that the expression profile at the level of the PFKFB4 gene does not reflect the expression at the level of individual isoforms. Our work clearly indicates that the PFKFB4 gene expression profile should be definitely analyzed at the level of individual isoforms. Moreover, the analysis at the protein level allowed the selection of those isoforms whose functional validation should be performed to fully understand the importance of PFKFB4 expression in the metabolic adaptation of malignant melanoma cells.

PFKFB4 is critical for the survival of acute monocytic leukemia cells.

Acute myeloid leukemia (AML), which is characterized by an overproliferation of blood cells, is divided into several subtypes in adults and children. Of those subtypes, acute monocytic leukemia (M4/M5, AMoL) is reported to be associated with abnormal gene fusions that result in monocytic cell differentiation being blocked. However, few studies have shown a relationship between cellular metabolism and the initiation of AMoL. Here, we use the open-access database TCGA to analyze the expression of enzymes in the metabolic cycle and find that PFKFB4 is highly expressed in AMoL. Subsequently, knocking down PFKFB4 in THP-1 and U937 cells significantly inhibits cell growth and increases the sensitivity of cells to chemical drug-induced apoptosis. In line with the gene-editing alterations, treatment with a PFKFB4 inhibitor exhibits similar effects on THP-1 and U937 proliferation and apoptosis. In addition, we find that PFKFB4 functions as a reliable target of the epigenetic regulator MLL, which is a well-known modulator in AMoL. Mechanistically, MLL promotes PFKFB4 expression at the transcriptional level through the putative E2F6 binding site in the promoter of the pfkfb4 gene. Taken together, our results suggest PFKFB4 serves as a downstream target of MLL and functions as a potent therapeutic target in AMoL.

The metabolic role of PFKFB4 in androgen-independent growth in vitro and PFKFB4 expression in human prostate cancer tissue.

BACKGROUND: It is well known that androgen-deprivation therapy (ADT) can inevitably drive prostate cancer (PCa) cells into a castration-resistant state. According to the "Warburg effect", the metabolism of aggressive tumor cells increases significantly. The growth of cancer cells depends on glycolysis, which may be a potential target for cancer control. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) plays key roles in the proliferation and metastasis of PCa cells. However, there is very limited knowledge on the role of PFKFB4 in the conversion to castration resistance. The present study aimed to determine the changes in glucose consumption and PFKFB4 expression in LNCaP cells and androgen-independent LNCaP (LNCaP-AI) cells during the whole process of androgen-independent growth. Additionally, PFKFB4 expression in human PCa tissues was evaluated. METHODS: We established an androgen-independent LNCaP-AI cell line derived from LNCaP cells to mimic the traits of castration resistance in vitro. LNCaP-AI and LNCaP cells were cultured in the corresponding medium containing the same amount of glucose. At the end of experiments, the medium supernatant and blank medium were collected, and absorbance was measured. LNCaP-AI and LNCaP cells were harvested to detect PFKFB4 expression by Western blotting. Prostate tissue samples including PCa tissue, carcinoma-adjacent tissue and benign prostatic hyperplasia (BPH) tissue specimens were evaluated for PFKFB4 expression using immunohistochemistry. RESULTS: In 18 h supernatant samples, the glucose consumption and lactate secretion of LNCaP-AI cells were higher than those of LNCaP cells. The Western blot results indicated that PFKFB4 expression was increased in LNCaP-AI cells compared with LNCaP cells. Immunohistochemistry revealed that the expression of PFKFB4 in PCa tissue specimens was higher than that in BPH and adjacent tissue specimens. However, the differences in PCa tissue before and after ADT were not statistically significant. CONCLUSION: PFKFB4 may be associated with enhanced glycolysis during the androgen-independent growth of PCa cells in vitro. PFKFB4 may be a marker of PCa progression. Our results provide a rationale for further clinical investigation of PCa treatment focused on controlling PFKFB4 expression.

High expression of metabolic enzyme PFKFB4 is associated with poor prognosis of operable breast cancer.

BACKGROUND: Enhanced glycolysis in tumors, known as the Warburg effect, provides the metabolic basis of enhanced cancer cell proliferation and metastasis. The Warburg pathway enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is a newly identified key kinase that regulates transcriptional reprogramming and cell proliferation. Here we show the prognostic value of PFKFB4 expression in patients with operable breast cancer. METHODS: PFKFB4 expression was evaluated by immunohistochemistry in surgical specimens retrospectively collected from 200 patients with histologically proven invasive ductal breast cancer. Kaplan-Meier survival analysis and Cox regression analysis were performed to assess the prognostic significance of PFKFB4 expression. RESULTS: Kaplan-Meier survival analysis revealed that breast cancer patients with high PFKFB4 expression demonstrated unfavorable disease-free survival (p = 0.008) and overall survival (p = 0.002). PFKFB4 had an hazard ratio (HR) of 7.38 (95% CI 1.69-32.3; p = 0.008) in univariate Cox analysis and retained prognostic power (HR 7.44, 95% CI 1.67-33.2; p = 0.009) when adjusted by tumor size, lymph node status, grade, estrogen receptor status, human epidermal growth factor receptor 2 status and subtype, which indicated PFKFB4 was an independent prognostic factor in breast cancer. CONCLUSIONS: Together, our findings establish the prognostic value of metabolic enzyme PFKFB4 in patients with operable breast cancer.

The influence of PFK-II overexpression on neuroblastoma patients' survival may be dependent on the particular isoenzyme expressed, PFKFB3 or PFKFB4.

BACKGROUND/AIM: During cancer progression metabolic reprogramming is observed in parallel to the alternation in transcriptional profiles of malignant cells. Recent studies suggest that metabolic isoenzymes of phosphofructokinase II (PFK-II) - PFKFB3 and PFKFB4, often induced in hypoxic environment, significantly contribute to enhancement of glucose metabolism and in consequence cancer progression. MATERIALS AND METHODS: Using the publicly available data deposited in the R2 data base we performed a Kaplan-Meyer analysis for cancer patients divided into groups with high and low expression levels of PFKFB3/4, determined based on the median. RESULTS: Our data showed that high PFKFB3/4 expression significantly correlates with shorter overall survival in several cancers. Moreover, we found that neuroblastoma patients with poor overall survival and evidence free survival are characterized by high PFKFB3 and at the same time low PFKFB4 expression, whereas patients with high PFKFB4 expressions are characterized by significantly better overall survival/evidence free survival rates. CONCLUSION: Our analysis clearly indicates that expression of PFKFB3/4 isoenzymes may have a key prognostic value for several cancers. What's more, it seems that in neuroblastoma the prognostic value of PFK-II may be dependent on the relation between PFKFB3 and PFKFB4 isoenzyme expression, indicating that further studies analyzing the role of both cancer specific PFK-II isoenzymes are highly desired.

PFKFB4 Promotes Breast Cancer Metastasis via Induction of Hyaluronan Production in a p38-Dependent Manner.

BACKGROUND/AIMS: The bi-functional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase-4 (PFKFB4) is highly expressed in many types of cancer and its requirement for tumor survival has been demonstrated in glioma, lung, and prostate cancers. However, whether PFKFB4 plays a role in the tumor metastasis remains uncertain. This study explores the role of PFKFB4 in tumor metastasis and its underlying mechanisms in breast cancer cells. METHODS: The expression of PFKFB4 was first analyzed using the Cancer Genome Atlas (TCGA) dataset, and confirmed by immunohistochemical staining of tissue microarray and breast cancer tissues from patient samples. Gain- and loss-of- function approaches were used to investigate the effects of PFKFB4 on breast cancer cell migration in vitro. Orthotopic xenograft model and experimental metastasis model were used to assess the effects of PFKFB4 on breast cancer cell metastasis in vivo. ELISA and immunofluorescence staining were used to examine HA production. Quantitative RT-PCR and western blotting were used to explore the mRNA and protein levels of HAS2, respectively. RESULTS: We found that PFKFB4 enhances the migration/invasiveness of breast cancer cells in vitro as well as in vivo. Notably, the effects of PFKFB4 on migration are mediated by induction of HAS2 expression and HA production. Moreover, PFKFB4-induced HAS2 up-regulation depends upon the activation of p38 signaling. CONCLUSION: PFKFB4 promotes the metastasis of breast cancer cells via induction of HAS2 expression and HA production in a p38-dependent manner. Therefore, the PFKFB4/p38/HAS2 signaling pathway may serve as a potential therapeutic target for metastatic breast cancer.

HIF-1α activates hypoxia-induced PFKFB4 expression in human bladder cancer cells.

PFKFB4 is reported to regulate glycolysis by synthesizing fructose-2, 6-bisphosphate (F2,6BP) and has proved to be associated with most malignancies. However, the underlying mechanism for increased PFKFB4 expression in bladder cancer remains unclear. The present study demonstrated that PFKFB4 was overexpressed in bladder cancer tissues. In addition, the expression of PFKFB4 elevated in bladder cancer cells in the hypoxic condition, while in nomoxic condition, the expression of PFKFB4 still very low. Furthermore, we identified the hypoxia-responsive elements (HRE)-D from five putative HREs in the promoter region of PFKFB4 and demonstrated that the HRE-D was transactivated by the HIF-1α in bladder cancer cells. By using the Double-immunofluorescence co-localization assay, we revealed that the HIF-1α expression was associated with PFKFB4 expression in human bladder cancer specimens. Altogether, our study for the first time identified the pivotal role of HIF-1α in the connection between PFKFB4 and hypoxia in bladder cancer, which may prove to be a potential target for the treatment of bladder cancer.

Fibrillarin reprograms glucose metabolism by driving the enhancer-mediated transcription of PFKFB4 in liver cancer.

DNA- and RNA-binding proteins (DRBPs) are versatile proteins capable of binding to both DNA and RNA molecules. In this study, we identified fibrillarin (FBL) as a key DRBP that is upregulated in liver cancer tissues vs. normal tissues and is correlated with patient prognosis. FBL promotes the proliferation of liver cancer cells both in vitro and in vivo. Mechanistically, FBL interacts with the transcription factor KHSRP, thereby regulating the expression of genes involved in glucose metabolism and leading to the reprogramming of glucose metabolism. Specifically, FBL and KHSRP work together to transcriptionally activate the glycolytic enzyme PFKFB4 by co-occupying enhancer and promoter elements, thereby further promoting liver cancer growth. Collectively, these findings provide compelling evidence highlighting the role of FBL as a transcriptional regulator in liver cancer cells, working in conjunction with KHSRP. The FBL/KHSRP-PFKFB4 regulatory axis holds potential as both a prognostic indicator and a therapeutic target for liver cancer. SIGNIFICANCE: A novel role of FBL in the transcriptional activation of PFKFB4, leading to glucose metabolism reprogramming in liver cancer.

Multi-omics analysis reveals that Cas13d contributes to PI3K-AKT signaling and facilitates cell proliferation via PFKFB4 upregulation.

The CRISPR-Cas system is a powerful gene editing technology, the clinical application of which is currently constrained due to safety concerns. A substantial body of safety research concerning Cas9 exists; however, scant attention has been directed toward investigating the safety profile of the emergent Cas13 system, which confers RNA editing capabilities. In particular, uncertainties persist regarding the potential cellular impacts of Cas13d in the absence of reliance on a cleavage effect. In this study, we conducted an initial exploration of the effects of Cas13d on HeLa cells. Total RNA and protein samples were extracted after transfection with a Cas13d-expressing plasmid construct, followed by transcriptomic and proteomic sequencing. Differential gene expression analysis identified 94 upregulated and 847 downregulated genes, while differential protein expression analysis identified 185 upregulated and 231 downregulated proteins. Subsequently, enrichment analysis was conducted on the transcriptome and proteome sequencing data, revealing that the PI3K-Akt signaling pathway is a common term. After intersecting the differentially expressed genes enriched in the PI3K-Akt signaling pathway with all the differentially expressed proteins, it was found that the expression of the related regulatory gene PFKFB4 was upregulated. Moreover, western blot analysis demonstrated that Cas13d can mediate PI3K-Akt signaling upregulation through overexpression of PFKFB4. CCK-8 assay, colony formation, and EdU experiments showed that Cas13d can promote cell proliferation. Our data demonstrate, for the first time, that Cas13d significantly impacts the transcriptomic and proteomic profiles, and proliferation phenotype, of HeLa cells, thus offering novel insights into safety considerations regarding gene editing systems.

Overexpression of SLC2A1, ALDOC, and PFKFB4 in the glycolysis pathway drives strong drug resistance in 3D HeLa tumor cell spheroids.

The 3D multicellular tumor spheroid (MTS) model exhibits enhanced fidelity in replicating the tumor microenvironment and demonstrates exceptional resistance to clinical drugs compared to the 2D monolayer model. In this study, we used multiomics (transcriptome, proteomics, and metabolomics) tools to explore the molecular mechanisms and metabolic differences of the two culture models. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways revealed that the differentially expressed genes between the two culture models were mainly enriched in cellular components and biological processes associated with extracellular matrix, extracellular structural organization, and mitochondrial function. An integrated analysis of three omics data revealed 11 possible drug resistance targets. Among these targets, seven genes, AKR1B1, ALDOC, GFPT2, GYS1, LAMB2, PFKFB4, and SLC2A1, exhibited significant upregulation. Conversely, four genes, COA7, DLD, IFNGR1, and QRSL1, were significantly downregulated. Clinical prognostic analysis using the TCGA survival database indicated that high-expression groups of SLC2A1, ALDOC, and PFKFB4 exhibited a significant negative correlation with patient survival. We further validated their involvement in chemotherapy drug resistance, indicating their potential significance in improving prognosis and chemotherapy outcomes. These results provide valuable insights into potential therapeutic targets that can potentially enhance treatment efficacy and patient outcomes.

Reprogramming of glucose metabolism via PFKFB4 is critical in FGF16-driven invasion of breast cancer cells.

Fibroblast growth factors (FGFs) are expressed in both developing and adult tissues and play important roles in embryogenesis, tissue homeostasis, angiogenesis, and neoplastic transformation. Here, we report the elevated expression of FGF16 in human breast tumor and investigate its potential involvement in breast cancer progression. The onset of epithelial-mesenchymal transition (EMT), a prerequisite for cancer metastasis, was observed in human mammary epithelial cell-line MCF10A by FGF16. Further study unveiled that FGF16 alters mRNA expression of a set of extracellular matrix genes to promote cellular invasion. Cancer cells undergoing EMT often show metabolic alteration to sustain their continuous proliferation and energy-intensive migration. Similarly, FGF16 induced a significant metabolic shift toward aerobic glycolysis. At the molecular level, FGF16 enhanced GLUT3 expression to facilitate glucose transport into cells, which through aerobic glycolysis generates lactate. The bi-functional protein, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 4 (PFKFB4) was found to be a mediator in FGF16-driven glycolysis and subsequent invasion. Furthermore, PFKFB4 was found to play a critical role in promoting lactate-induced cell invasion since silencing PFKFB4 decreased lactate level and rendered the cells less invasive. These findings support potential clinical intervention of any of the members of FGF16-GLUT3-PFKFB4 axis to control the invasion of breast cancer cells.

Effect of PFKFB4 on the Prognosis and Immune Regulation of NSCLC and Its Mechanism.

Background: NSCLC (non-small cell lung cancer) has become the malignancy with the highest incidence and mortality rate worldwide. Fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is a key regulator of glycolysis with both kinase and phosphatase activities. The Warburg effect, or increased glycolysis in tumors, provides the metabolic basis for cancer cell proliferation and metastasis, and the Warburg pathway enzyme PFKFB4 is a newly identified important kinase. This study aimed to elucidate the poor prognostic relevance of PFKFB4 in non-small cell lung cancer tissues and its relationship with immune cell infiltration, immune cell biomarkers, and immune checkpoints. Methods: In this study, immunohistochemical methods were used to assess PFKFB4 expression levels in 140 surgical specimens from patients with histologically confirmed non-small cell lung cancer and to investigate the relationship between PFKFB4 expression levels and the patients' clinicopathological characteristics. The impact of PFKFB4 expression on prognosis was evaluated using Kaplan-Meier survival analysis and Cox regression analysis. Results: When compared to normal paracrine tissues, PFKFB4 expression was enhanced in lung cancer tissues, and Kaplan-Meier survival analysis revealed that patients with high PFKFB4 expression had a worse prognosis. In NSCLC, PFKFB4 was found to be associated with immune cell infiltration and immunological checkpoints. Conclusion: PFKFB4 expression may be upregulated as a sign of poor prognosis in NSCLC, and PFKFB4 may be implicated not only in the genesis and progression of NSCLC but also in its immunological control.

Iron controls T helper cell pathogenicity by promoting glucose metabolism in autoimmune myopathy.

BACKGROUND: T helper cells in patients with autoimmune disease of idiopathic inflammatory myopathies (IIM) are characterized with the proinflammatory phenotypes. The underlying mechanisms remain unknown. METHODS: RNA sequencing was performed for differential expression genes. Gene expression in CD4+ T-cells was confirmed by quantitative real-time PCR. CD4+ T-cells from IIM patients or healthy controls were evaluated for metabolic activities by Seahorse assay. Glucose uptake, T-cell proliferation and differentiation were evaluated and measured by flow cytometry. Human CD4+ T-cells treated with iron chelators or Pfkfb4 siRNA were measured for glucose metabolism, proliferation and differentiation. Signalling pathway activation was evaluated by western blot and flow cytometry. Mouse model of experimental autoimmune myositis (EAM) were induced and treated with iron chelator or rapamycin. CD4+ T-cell differentiation and muscle inflammation in the EAM mice were evaluated. RESULTS: RNA-sequencing analysis revealed that iron was involved with glucose metabolism and CD4+ T-cell differentiation. IIM patient-derived CD4+ T-cells showed enhanced glycolysis and mitochondrial respiration, which was inhibited by iron chelation. CD4+ T-cells from patients with IIM was proinflammatory and iron chelation suppressed the differentiation of interferon gamma (IFNγ)- and interleukin (IL)-17A-producing CD4+ T-cells, which resulted in an increased percentage of regulatory T (Treg) cells. Mechanistically, iron promoted glucose metabolism by an upregulation of PFKFB4 through AKT-mTOR signalling pathway. Notably, the knockdown of Pfkfb4 decreased glucose influx and thus suppressed the differentiation of IFNγ- and IL-17A-producing CD4+ T-cells. In vivo, iron chelation inhibited mTOR signalling pathway and reduced PFKFB4 expression in CD4+ T-cells, resulting in reduced proinflammatory IFNγ- and IL-17A-producing CD4+ T-cells and increased Foxp3+ Treg cells, leading to ameliorated muscle inflammation. CONCLUSIONS: Iron directs CD4+ T-cells into a proinflammatory phenotype by enhancing glucose metabolism. Therapeutic targeting of iron metabolism should have the potential to normalize glucose metabolism in CD4+ T-cells and reverse their proinflammatory phenotype in IIM.