Skip to main content

Comparative clinical significance and biological roles of PFKFB family members in oral squamous cell carcinoma

Abstract

Background

Cancer cells promote glycolysis, which supports rapid cell growth and proliferation. Phosphofructokinase-fructose bisphosphatases (PFKFBs), a family of bidirectional glycolytic enzymes, play key roles in the regulation of glycolysis in many types of cancer. However, their roles in oral squamous cell carcinoma (OSCC), the most common type of oral cancer, are still unknown.

Methods

We compared the gene expression levels of PFKFB family members and analyzed their clinical significance in oral cancer patients, whose clinical data were obtained the Cancer Genome Atlas database. Moreover, real-time quantitative polymerase chain reaction, western blotting, assays for cell viability, cell cycle, cell migration and viability of cell spheroid were performed in scramble and PFKFB-silenced cells.

Results

We discovered that PFKFB3 expression in tumor tissues was slightly higher than that in tumor adjacent normal tissues but that PFKFB4 expression was significantly higher in the tumor tissues of oral cancer patients. High PFKFB3 and PFKFB4 expression had different effects on the prognosis of oral cancer patients with different clinicopathological outcomes. Our data showed that PFKFB3 and PFKFB4 play different roles; PFKFB3 is involved in cell viability, G2/M cell cycle progression, invasion, and migration, whereas PFKFB4 is involved in the drug resistance and cancer stemness of OSCC cells. Furthermore, oral cancer patients with co-expressions of PFKFB3/cell cycle or EMT markers and PFKFB4/stemness markers had poor prognosis.

Conclusions

PFKFB3 and PFKFB4 play different biological roles in OSCC cells, which implying that they might be potential prognostic biomarkers for OSCC patients with certain clinicopathological outcomes.

Introduction

Oral squamous cell carcinoma (OSCC), which constitutes more than 90% of oral cancers, originates in areas of the oral cavity, including the lip, tongue, and cheek [1]. The incidence of OSCC is increasing in many countries, especially in individuals aged under 45 years [2]. Although various treatments for OSCC are available, including surgery, chemotherapy, and chemoradiation, low public awareness and insufficient screening methods have resulted in a low 5-year survival rate and poor prognosis for OSCC patients [3]. Accurate diagnostic and prognostic biomarkers are thus urgently required.

Cancer cells alter their glycolytic metabolism under aerobic conditions to maintain the high energy levels required for their growth and proliferation [4]. Aerobic glycolysis (or Warburg effect) regulates the tumorigenesis and prognosis of OSCC [5]. Several anticancer drugs targeting to glucose metabolism enzyme such as glucose transferase, hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase have been developed [6]. Thus, elucidating more precise metabolic enzymes regarding to glycolytic metabolism in OSCC could provide new biomarkers or therapeutic targets for OSCC patients.

Phosphofructokinase-fructose bisphosphatases (PFKFBs), a family of bidirectional glycolytic enzymes, modulate the formation and degradation of fructose-2,6-bisphosphate (F-2,6-BP), thereby regulating glycolysis [7]. PFKFBs is encoded by four genes (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) in humans [8]. PFKFB1 is found in the liver and skeletal muscles, PFKFB2 is found in cardiac muscles, PFKFB3 is ubiquitously expressed, and PFKFB4 occurs mainly in the testes [8, 9]. PFKFB1 expression has not been detected in any cancers. The expression of PFKFB2, PFKFB3, and PFKFB4 has been observed in several types of cancers. For example, PFKFB2 has been highly expressed in lung cancer [10], gastric cancer [11], retinoblastoma [12], osteosarcoma [13], and breast cancer [14]. The overexpression of PFKFB3 was observed in breast cancer [15], colon cancer [16], non-small cell lung cancer (NSCLC) [17], and hepatocellular carcinoma (HCC) [18]. The overexpression of PFKFB4 was found in breast cancer [19], triple-negative breast cancer (TNBC) [20], osteosarcoma [21], cervical cancer [22], clear-cell renal cell carcinoma [23], melanoma[24], HCC [18], glioblastoma [25], bladder cancer [26], gastric cancer [27], pancreatic cancer [28], and prostate cancer [29]. PFKFB2 is related to the cell proliferation, invasion, and migration of lung cancer [10]. PFKFB3 has emerged as a key oncogene in several types of cancer; it plays a considerable role in the regulation of glycolysis in cancer cells and in the proliferation and survival of cancer cells [30]. PFKFB4 promotes chemoresistance in clear-cell renal cell carcinoma [23]. The distinct activity, synthesis, distribution, and function of PFKB1-4 were determined by different conditions or response to different physiological or pathological stimuli [8]. However, most studies only focused on investigating the role of a member of PFKFB family, the roles of a set of PFKFB family members in cancer, especially in OSCC, remain unknown.

In the study, we performed a comprehensive analysis of the expression levels and prognostic value of a set of PFKFB family members in oral cancer patients. We found that oral cancer patients with high expression of PFKFB3 and PFKFB4 had poor prognosis. We also investigated their biological roles in OSCC cells, which PFKFB3 is linked to critical aspects such as cell survival, G2/M cell cycle progression, invasion, and migration, while PFKFB4 is strongly associated with drug resistance and the acquisition of cancer stemness characteristics. The study first reports the clinical significance and biological roles of PFKFB3 and PFKFB4, which could provide potential and specific biomarkers or therapeutic targets for OSCC patients.

Materials and methods

Cell culture

Two OSCC cell lines, namely SAS and TW2.6 cells, were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco™, Carlsbad, CA, USA), to which 10% heat-inactivated fetal bovine serum (Biological Industries, Cromwell, CT, USA), 1% minimum essential medium nonessential amino acids, 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA) were added, then stored at 37 °C in a 5% CO2 atmosphere.

Transient transfection

The cells (2 × 105 cells/well, 6 wells) were transfected with 10 nM scramble siRNA or siRNA against PFKFB3 or PFKFB4 (Ambion, Austin, TX, USA) for 72 h by using an RNAiMAX transfection kit (Invitrogen Life Technologies, Carlsbad, CA, USA).

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from cells by using a TRIzol reagent and then reverse transcribed using SuperScriptIII RNase Reverse Transcriptase in accordance with the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). The expression levels of the genes were analyzed using SYBR Green Master Mix and QuantStudio real-time polymerase chain reaction systems (Applied Biosystems, Foster City, CA, USA). PFKFB3 primer (Forward 5′-GGGACCGACGACACGC-3′; Reverse 5′-ATCTTCTGCACTCGGCTCTG-3′), PFKFB4 primer (Forward 5′-TCCCCACGGGAATTGACAC-3’; Reverse 5′-GGGCACACCAATCCAGTTCA-3′) Slug primer (Forward 5′-TGTGACAAGGAATATGTGAGCC-3’; Reverse 5′- TGAGCCCTCAGATTTGACCTG-3′), E-cadherin primer (Forward 5′-ATTTTTCCCTCGACACCCGAT-3′; Reverse 5′-TCCCAGGCGTAGACCAAGA-3′), CD166 primer (Forward 5′-ACTTGACGTACCTCAGAATCTCA -3′; Reverse 5’-CATCGTCGTACTGCACACTTT -3′), CD44 primer (Forward 5′-CTGCCGCTTTGCAGGTGTA -3′; Reverse 5′-CATTGTGGGCAAGGTGCTATT-3’), ABCG2 primer (Forward 5′-TGAGCCTACAACTGGCTTAGA-3′; Reverse 5’-CCCTGCTTAGACATCCTTTTCAG-3′), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) primer (Forward 5’-CCGTGGCGTACTATGGATGC-3′; Reverse 5′-GCAGCAGACGATCTCTTTCGAT-3′), aldehyde dehydrogenase 1 family, member A2 (ALDH1A2) primer (Forward 5’-GGGTGTGTTTTGATGCAGCCT-3′; Reverse 5′-TGGTGGGGTCAAAGGGACT-3′), and EpCAM (Forward 5′-AATCGTCAATGCCAGTGTACTT-3′; Reverse 5′- TCTCATCGCAGTCAGGATCATAA-3′) primer were used for mRNA amplification. The internal control GAPDH gene was used for normalization.

Western blotting

Following electrophoretic separation, proteins were transferred from a polyacrylamide gel onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). Blocked membranes with 5% skim milk were incubated with PFKFB3 (ab181861, Abcam, Trumpington, Cambridge, UK) or PFKFB4 (ab137785, Abcam, Abcam, Trumpington, Cambridge, UK) antibody overnight at 4 °C then with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The ECL reagent was used for chemiluminescent detection using the a Syngene GeneGnome XRQ chemiluminescence imaging system (GeneGnome XRQ, SYNGENE, Cambridge, UK).

Cell viability

Cell viability (6 × 105 cells/mL, 96 wells) was analyzed using a CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA) in accordance with the method used in our previous study [31].

Cell cycle assay

The cells fixed by ice-cold 75% ethanol were stained with propidium iodide (50 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) then analyzed with the FACScan analyzer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The percentages of cell cycle distribution were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Wound-healing assay

IBIDI Culture-Inserts (IBIDI, Inc., Planegg, Bavaria, Germany) was used to analyze cell migration. The procedure for the wound-healing assay is described in our previous study [32].

Sensitivity of cell spheroids to drug treatment

The OSCC cells (5 × 103/mL) were seeded into a 96-well, round-bottom, ultra-low-attachment microplate (Corning Costar, Cambridge, MA, USA) for cell spheroid formation. The viability of cell spheroids treated or untreated with 50 μM of cisplatin (CIS; Sigma-Aldrich Corporation, St. Louis, Missouri, USA) or 25–200 nM of paclitaxel (PTX; Selleckchem, Houston, TX, USA) for 24 h was analyzed using the CellTiterGlo 3D assay (Promega, Madison, WI, USA).

Statistical analysis

Transcriptome data on 30 tumor-adjacent normal tissues and 315 tumor tissues from oral cancer patients were downloaded from the public Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov). All gene expression levels and survival rate were analyzed using SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). Student’s t test was used to compare PFKFBs between tumor-adjacent normal tissues and tumor tissues. Univariate and multivariate Cox proportional hazards models were used to analyze survival; overall survival (OS), progression-free interval survival (PFI), disease-specific survival (DSS), and disease-free interval survival (DFI) were defined using the time intervals from the TCGA database. Cumulative survival curves were estimated using the Kaplan–Meier method. A receiver operating characteristic curve was used to determine high and low expression levels of PFKFB family members.

Results

Comparison of the expression of PFKFB family members between normal tissues and tumor tissues in oral cancer patients

PFKFB family members differentially express in many cancer patients [8]. However, their expression levels in oral cancer patients are still unknown. After analyzing transcriptome data of oral cancer patients from TCGA database, we found that PFKFB1 expression was lower in the tumor tissues than in the tumor-adjacent normal tissues (p = 0.001, Table 1, Fig. 1A), but PFKFB2 expression did not differ significantly (p = 0.322; Table 1, Fig. 1B). PFKFB3 expression was slightly higher in the tumor tissues than in the tumor-adjacent normal tissues (p = 0.098, Table 1, Fig. 1C). PFKFB4 expression in tumor tissues was significantly higher than that in normal tissues (p < 0.001, Table 1, Fig. 1D). Our results indicate that the expression levels of PFKFB family members differs between patients with and without oral cancer.

Table 1 The comparison of gene expressions of PFKFB family members between tumor adjacent normal and tumor tissues in oral cancer patients from TCGA database
Fig. 1
figure 1

Expression and prognostic roles of PFKFB family members in oral cancer patients. Comparison of A PFKFB1 B PFKFB2 C PFKFB3 D PFKFB4 expression between 30 tumor- adjacent normal and 315 tumor tissues of oral cancer patients. The association of high and low levels of PFKFB3 with E overall survival (OS), F progression-free interval survival (PFI), G disease-free interval survival (DFI) and H disease-specificl survival (DSS)

Association between the expression of PFKFB family members and the prognosis of oral cancer patients

High expression of PFKFBs were associated with prognosis in many cancer patients [8]. However, their prognostic roles in oral cancer patients are still unknown. Next, we analyzed the association between the expression of PFKFB family members and various measures of survival, namely OS, PFI, DFI, and DSS. As data shown, high PFKFB3 expression was associated with poor OS [crude hazard ratio [25] = 2.77 (1.02–7.51), p = 0.046, Table 2; p = 0.0037, Fig. 1E], poor PFI [adjusted hazard ratio (AHR) = 1.85 (1.08–3.19), p = 0.025, Table 2; p = 0.004, Fig. 1F]. Moreover, high PFKFB3 expression was not related to DFI [1.82 (0.64–5.21), p = 0.264, Table 2; p = 0.257, Fig. 1G] but to DSS [AHR = 2.43 (1.36–4.37), p = 0.003, Table 2; p < 0.001, Fig. 1H] in patients with oral cancer. PFKFB1 and PFKFB2 expression was not associated with OS, PFI, DFI, or DSS in patients with oral cancer when their data were stratified by clinicopathological outcome (Table 2, Additional file 1: Table S1 and Table S2). However, high PFKFB3 expression was associated with a short PFI in patients with larger tumor size (T classification, III and IV, AHR = 1.86, p = 0.046, Table 3; p = 0.037, Fig. 2A]. High PFKFB3 expression was associated with a short DFI in patients with lymph node metastasis [N classification, N1, N2 and N3, AHR = 7.88, p = 0.042, Table 3; p = 0.007, Fig. 2B]. High PFKFB3 expression was associated with poor DSS in patients with moderate or poor cell differentiation [AHR = 2.22, p = 0.009, Table 3; p = 0.002, Fig. 2C], lymph node metastasis [N1, N2, and N3, AHR = 2.12, p = 0.04, Table 3; p = 0.007, Fig. 2D], larger tumors [T3 and T4, AHR = 2.38, p = 0.006, Table 3; p = 0.004, Fig. 2E] and advanced pathological stages [III or IV; AHR = 2.35, p = 0.006, Table 3; p = 0.002, Fig. 2F],

Table 2 The association of gene expression of PFKFB family members with survival in oral cancer patients from TCGA database
Table 3 The association of PFKFB3 expression with prognosis in oral cancer patients stratified by different clinicopathological features
Fig. 2
figure 2

Different prognostic roles of oral cancer patients stratified by different clinicopathological outcomes depending on levels of PFKFB3 and PFKFB4. AF The association of high and low levels of PFKFB3 with PFI, DFI, DSS in oral cancer patients stratified by cell differentiation, N-classification, T-classification, and pathological stage. GI The association of high and low levels of PFKFB4 with PFI and DSS in oral cancer patients stratified by pathological stage and N-classification

Moreover, high PFKFB4 expression was associated with a short PFI in patients with lymph node metastasis [N1, N2, and N3, AHR = 2.00, p = 0.009, Table 4; p = 0.003, Fig. 2G] and late pathological stages [III and IV; AHR = 1.63, p = 0.03, Table 4; p = 0.025, Fig. 2H]. High PFKFB4 expression was also associated with poor DSS in patients with lymph node metastasis [N1, N2, and N3, AHR = 1.88, p = 0.041, Table 4; p = 0.011, Fig. 2I]. Our results indicate that PFKFB3 and PFKFB4 expression levels have different effects on the prognosis of oral cancer patients with different clinicopathological outcomes.

Table 4 The association of PFKFB4 expression with prognosis in oral cancer patients stratified by different clinicopathological features

Effects of PFKFB3 and PFKFB4 on the viability of OSCC cells

PFKFBs play different roles in many cancers, such as cell viability and migration [8]. However, their biological roles in oral cancer are still unclear. To investigate if PFKFB3 and PFKFB4 play role in cell viability of oral cancer cells, OSCC cells were knocked down using siRNA against PFKFB3 or PFKFB4. After knockdown, the gene (Fig. 3A) and protein (Fig. 3B) levels of PFKFB3 or PFKFB4 were decreased. Moreover, the cell viability of PFKFB3-silenced OSCC cells was significantly lower, whereas the cell viability did not differ between scramble and PFKFB4-silenced OSCC cells (Fig. 3C). Also, the PFKFB3- or PFKFB4-silenced OSCC cells showed G2/M arrest (Fig. 3D), lower level of cell cycle regulator (cyclin B) but higher level of two cell cycle inhibitors (p21 and p27) (Fig. 3E). Moreover, high PFKFB3 expression was associated with poor PFI and DSS in OSCC patients with larger tumors (Table 3). Our results indicate that PFKFB3 (but not PFKFB4) might be involved in tumor growth through regulating G2/M cell cycle progression in OSCC.

Fig. 3
figure 3

Cell viability and cell cycle progression of PFKFB3-or PFKFB4-silenced SAS and TW2.6 cells. The mRNA and protein levels of PFKFB3 or PFKFB4 were evaluated by A RT-qPCR and B Western blotting in PFKFB3- or PFKFB4-silenced cells. C Cell viability of PFKFB3-or PFKFB4-knockeddowned cells was analyzed by CellTiter-Glo assay. D Cell cycle progression of PFKFB3-silenced cells was analyzed by flow cytometry. E Cell cycle-related proteins in PFKFB3-silenced cells were analyzed by Western blotting. The 10 nM scrambled siRNA (siCtrl) or siRNA against PFKFB3 or PFKFB4 (siPFKFB3 or siPFKFB4) were transfected into cells for 72 h. All data were represented as the average ± SD from 3 independent experiments. The significant differences between the scrambled control and knocked down cells were indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001

Effects of PFKFB3 and PFKFB4 on the migration of OSCC cells

We further investigated if PFKFB3 and PFKFB4 involve in migration of OSCC cells. SAS and TW2.6 cells were transiently knocked down with scramble siRNA and siRNA against PFKFB3 or PFKFB4. The migration ability of PFKFB3-silenced cells was significantly weaker than that of the control cells (Fig. 4A). However, the migration ability of PFKFB4-silenced OSCC cells did not differ from that of the scramble cells (Fig. 4B). In addition, the expression of epithelial–mesenchymal transition (EMT) markers such as Slug was significantly lower, but that of E-cadherin was higher in the PFKFB3–knocked down OSCC cells (Fig. 4C). Moreover, high PFKFB3 expression was associated with poor DFI and DSS in patients with OSCC with lymph node metastasis (Table 3). Our results indicate that PFKFB3 might be involved in metastasis through EMT regulation in OSCC.

Fig. 4
figure 4

Cell migration and expression of EMT-related markers in PFKFB3- or PFKFB4-silenced SAS and TW2.6 cells. The cell migration of A PFKFB3-silenced and B PFKFB4-silenced cells was measured by the wound-healing assay. C Expression of EMT markers (Slug and E-cad) in PFKFB3-silenced cells were measured by qRT-PCR. The 10 nM scrambled siRNA (siCtrl) or siRNA against PFKFB3 or PFKFB4 (siPFKFB3 or siPFKFB4) were transfected into cells for 72 h. All data were represented as the average ± SD from 3 independent experiments. The significant differences between the scrambled control and knocked down cells were indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001

Effects of PFKFB3 and PFKFB4 on the chemoresistance and cancer stemness of OSCC cells

It is known that PFKFBs play roles in chemoresistance and cancer stemness [33]. We further explored the effects of PFKFB3 and PFKFB4 on chemoresistance and cancer stemness in OSCC cells. After knockdown, we observed no difference in viability of cell spheroids between PFKFB3-silenced SAS and TW2.6 cells untreated or treated with 0.025–0.2 μM of PTX or 50 μM of CIS (Fig. 5A). However, the PFKFB4-silenced SAS and TW2.6 cells exhibited significantly lower viability of cell spheroids in the presence of PTX and CIS compared with the scramble cells (Fig. 5B). To further confirm the role of PFKFB4 in cancer stemness-related chemoresistance, we investigated the expression of several cancer stemness markers, namely CD44, CD166, ABCG2ALDH1A1, ALDH1A2, and EpCAM and found that their expressions were lower in PFKFB4–knocked down SAS and TW2.6 cells than scramble cells (Fig. 5C). Taken together, our results indicate that PFKFB4 might be involved in the chemoresistance and cancer stemness of OSCC.

Fig. 5
figure 5

Sensitivity of cancer cell sphorids to cancer drugs in PFKFB3-or PFKFB4-silenced SAS and TW2.6 cells. The cell viability of spheroid cells silenced with scrambled siRNA or siRNA against A PFKFB3 or B PFKFB4 for 3 days then in the absence or presence of cisplatin (CIS, 50 µM) or paclitaxel (PTX, 0.025 and 2 µM) for 24 h was measured using the CellTiterGlo 3D assay. C mRNA levels of cancer stemness markers (CD166, CD44, ABCG2, ALDH1A1, ALDH1A2 and EpCAM) in PFKFB4-silenced SAS cells were assessed by RT-qPCR. The 10 nM scrambled siRNA (siCtrl) or siRNA against PFKFB3 or PFKFB4 (siPFKFB3 or siPFKFB4) were transfected into SAS cells for 72 h. All data were represented as the average ± SD from 3 independent experiments. The significant differences between scramble control and knocked down cells were indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001

The co-expressions of PFKFB3/cell cycle or EMT markers and PFKFB4/stemness markers in prognosis of oral cancer patients

Previous studies indicate that PFKFBs expression was significantly correlated with EMT-related [34] or stemness markers [33]. Moreover, our results indicated that PFKFB3 promoted tumor growth through regulating G2/M cell cycle and involved in metastasis through EMT regulation in OSCC cells. In oral cancer patients, we found that co-expressions of high PFKFB3/low p27 or high PFKFB3/high cyclin B1 or Slug were associated with poor DSS (Fig. 6A). Moreover, co-expressions of high PFKFB4/ high stemness markers such as ABCG2, ALDH1A1 and EpCAM were also associated with poor PFI (Fig. 6B). Our analyzed data confirmed the possible effect of PFKFB3 in cell cycle progression and migration as well as  the effect of PFKFB4 in cancer stemness in OSCC.

Fig. 6
figure 6

The association of co-expression of PFKFB3/p27, PFKFB3/cyclin B1, PFKFB3/slug and PFKFB4/stemness markers (ABCG2, ALDH1A1 and EpCAM) with prognosis of oral cancer patients. A Co-expressions of PFKFB3/p27, PFKFB3/cyclin B1, PFKFB3/slug in DSS. B Co-expressions of PFKFB4/stemness markers (ABCG2, ALDH1A1 and EpCAM) in PFI

Discussion

PFKFBs are bidirectional glycolytic enzymes that control the steady-state cytoplasmic levels of F-2,6-BP, and increased F-2,6-BP concentration is a marker of glycolysis in many cancer cells [35]. PFKFBs have been reported to be involved in tumor progression and are considered potential biomarkers of various types of cancer [8]. However, their roles in oral cancer have not been reported. In the present study, we revealed that the expression of PFKFB4 was higher in the tumor tissues of oral cancer patients than in tumor-adjacent normal tissues. In addition, high PFKFB3 expression was associated with a shorter survival  in oral cancer patients with poor cell differentiation, large tumors, and larger tumor sizes. High PFKFB4 expression was associated with a shorter survival  in oral cancer patients with advanced lymph node metastasis and clinicopathological stages. Furthermore, PFKFB3 is involved in the growth and metastasis of OSCC cells, but PFKFB4 is involved in chemoresistance and the cancer stemness of OSCC cells. The co-expressions of PFKFB3/cell cycle or EMT markers and PFKFB4/stemness markers were associated with poor prognosis in oral cancer patients. These findings suggest that PFKFB family members have different biological roles and clinical significance in oral cancer patients.

PFKFB1 was originally identified in tissues of the liver, muscle tissues, and fetal tissues but was not observed in cancer cells [36]. However, we discovered that PFKFB1 had lower expression in tumor tissues than in normal tissues of patients with oral cancer. In addition, PFKFB1 expression was not significantly associated with survival in patients with oral cancer. PFKFB2 is expressed in the heart and kidney. PFKFB2 is highly expressed in lung cancer [10], gastric cancer [11], melanoma [37], retinoblastoma [12], osteosarcoma [38], HCC [39] breast cancer [14], and prostate cancer [40]. Our results indicate that PFKFB2 expression is not significantly different between tumor-adjacent normal and tumor tissues in oral cancer patients. In addition, PFKFB2 expression was not associated with prognosis for oral cancer patients.

PFKFB3 is frequently observed in pancreatic cancer, gastric cancer, nasopharyngeal carcinoma, and many other neoplasms [8]. PFKFB3 is overexpressed in breast cancer [15], colon cancer [16], NSCLC [17], and HCC [18]. Moreover, high PFKFB3 expression is linked to poor survival in brain tumors [9], indicating that PFKFB3 might be a therapeutic target for various types of cancer. It is known that PFKFB3 promotes the proliferation, invasion, and migration of breast cancer cells [41]. The blockage of PFKFB3 decreases tumor growth and metastasis in head and neck squamous cell carcinoma (HNSCC) [42]. Our results indicate that PFKFB3 was highly expressed in OSCC tissues and associated with a poor OS in oral cancer patients. We also discovered that the knockdown of PFKFB3 significantly suppressed cell growth and migration of OSCC cells.

PFKFB3–knocked down HeLa cells have exhibited G1/S arrest [43]. PFKFB3 expression has been induced during the G1/S transition [44]. The 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (a kind of PFKFB3 inhibitor) can induce G0/G1 arrest in A375 human melanoma cells [44] and G2/M arrest in Jurkat cells [45]. PFKFB3 knockdown results in the G2/M arrest of HCC cells. PFKFB3 upregulates some cyclin-dependent kinases (including Cdk-1, Cdc25C, and cyclin D3) and downregulates the p27 protein for G1/S transition and cell proliferation [46]. Our results indicated that PFKFB3 might control G2/M cell-cycle progression. On the other hand, PFKFB3 modulates cell proliferation with the concomitant activation of NF-kB signaling in gastric cancer [47]. However, if PFKFB3 is involved in cell growth through the regulation of NF-kB signaling in oral cancer, this topic will require further study.

Many studies have indicated that PFKFB3 promotes metastasis by regulating EMT. PFKFB3 knockdown inhibits invasiveness by upregulating E-cadherin and downregulating vimentin and N-cadherin levels in CNE2 human nasopharyngeal carcinoma cells [48]. The knockdown of PFKFB3 reduces Snail expression and upregulates E-cadherin levels in pancreatic cancer cells [49]. Our results indicate that PFKFB3 also regulates EMT-related Slug and E-cad, indicating that the upregulation of glycolysis promotes the EMT [50]. PFKFB3-modulating glycolysis is essential for lymphotoxin α–promoted tumor angiogenesis in HNSCC [51]. Many studies have indicated that PFKFB3 is involved in the angiogenesis of OSCC [52], especially for lymphangiogenesis [53]. Our results suggest that oral cancer patients with higher PFKFB3 expression exhibits lymph node metastasis (Table 4), implying that PFKFB3 might promote lymphangiogenesis for lymph node metastasis.

Dysfunctional glycolysis results in drug resistance in clinical tumor therapy [27]. The knockdown of PFKFB3 inhibits the expression of cancer stemness markers such as CD133, ALDH1A1, CD44, Sox2, and ABCG2, which are also associated with chemotherapy resistance [14]. Our results demonstrate that PFKFB3 is involved in cell growth by regulating G2/M cell cycle progression and migration but not in chemoresistance and cancer stemness in OSCC cells.

We found that high PFKFB3 expression was associated with a short PFI in patients with larger tumor size (T3 and T4) and was associated with a short DFI  in oral cancer patients with lymph node metastasis (N1, N2 and N3). In addition, high PFKFB3 expression was also associated with poor DSS in oral cancer patients with moderate or poor cell differentiation, lymph node metastasis, and larger tumors.. Moreover, we found that PFKFB3 is associated with cell growth and migration in OSCC cells. These results indicate that effects of PFKFB3 on cell growth and metastasis are associated with tumor growth and metastasis in oral cancer patients.

Increasing PFKFB4 expression contributes to the proliferation of HCC cells [54]. PFKFB4 increases proliferative action in breast cancer cells [55]. PFKFB4 mediates CD44-driven proliferation in prostate cancer cells [56]. PFKFB4 is key to the survival of glioma stem-like cells [57]. PFKFB4 is involved in androgen-independent growth in human prostate cancer tissues [29]. It was reported that PFKFB4 seems to contribute to tumor growth by regulating G1/0 phase progression for cell proliferation [8]. For example, the loss of PFKFB4 induces cell cycle arrest in cervical cancer cells [58]. PFKFB4 promotes G1/S transition for the cell proliferation of TNBC [20]. On the other hand, the knockdown of PFKFB4 inhibits invasiveness through the upregulation of histone acetyltransferase GCN5 in IHH-4 thyroid cancer cells [59]. PFKFB4 plays a role in the motility of cervical cancer cells [60]. PFKFB4 activates cell migration in melanoma [24]. However, our study indicates that PFKFB4 was not involved in cell growth and migration in OSCC cells.

PFKFB4 enhances cancer stemness and contributes to chemoresistance to palbociclib in estrogen receptor–positive breast cancer [7]. PFKFB4 is involved in chemoresistance to sunitinib in clear-cell renal cell carcinoma [23]. In addition, PFKFB4 is involved in chemoresistance of HCC [18]. Previous studies showed that CD44 may be a therapeutic target for glycolytic cancer cells that exhibit drug resistance [61]. Cancer cells with high glycolysis can release a large number of exosomes containing cancer stemness markers, including ABCG2, ALDH1A1, and EpCAM [62]. Our data also indicate that PFKFB4 is involved in the chemoresistance of OSCC, which the inhibition of PFKFB4 decreased the expression levels of CD44, CD166ABCG2, ALDH1A1, ALDH1A2, and EpCAM. These data indicate that glucose metabolic reprogramming was involved in chemoresistance [62], which will need to be further verified.

We found that PFKFB4 expression in tumor tissues was significantly higher than that in normal tissues and high PFKFB4 expression was associated with a short PFI in oral cancer patients with lymph node metastasis and late pathological stages. High PFKFB4 expression was also associated with poor DSS in oral cancer patients with lymph node metastasis. Moreover, we found that PFKFB4 is associated with drug resistance and cancer stemness in OSCC cells. Since cancer stemness and drug resistance confer to survival of cancer patients, our results suggest that elevated PFKFB4 might modulate signaling pathway required for drug resistance and cancer stemness, which in turn to contribute worse survival of oral cancer patients.

Our results showed that PFKFB3 contributes to cellular proliferation and migration of OSCC. Previous study indicated that PFKFB3 regulate both of proliferation and migration of ovarian cancer by regulating cytosolic protein tyrosine kinase 2 (focal adhesion kinase) [63]. Moreover, PFKFB3 involves in the Ras signaling pathway, which is considered regulators of both proliferation and migration [64]. On the other hand, we revealed that PFKFB4 involves in chemoresistance and cancer stemness of OSCC. It is reported that PFKFB4-mediated glycolysis pathway is associated with stemness features of breast cancer [65]. Moreover, PFKFB4 modulates the chemoresistance of small-cell lung cancer by regulating autophagy [66]. According to above findings, PFKFB3 and PFKFB4 could contribute to many facets of oral cancer progression including controlling cell cycle progression, metastasis, and chemoresistance, which might not only act as regulators of glucose metabolism, but also act in a non-glycolysis-dependent manner (such as cell cycle regulation, autophagy, and transcriptional regulation) in OSCC [67]. Thus, the therapeutic implications of targeting PFKFB3 and PFKFB4 could disrupt glycolysis or Warburg effect and eliminate other signaling mechanisms for cancer progression.

Several studies revealed that targeting PFKFB3 and PFKFB4 could inhibit glycolysis in cancer cells [68]. Although PFKFB3 inhibitors such as 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) [69] or PFKFB4 inhibitor such as 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN) have been reported, their problems in low specificity and off targets are difficult to avoid [67]. Therefore, identifying more effective small molecule by computational approach involving virtual screening, drug-likeness, ADEM (absorption, distribution, metabolism, and excretion), molecular docking simulation, thermodynamic free energy calculations, per residue binding free energy contribution[70] and silico approach [71] or identifying plant extracts for inhibition of PFKFB3 and PFKFB4 enzymes for OSCC therapy is essential [72].

Our current study supports the clinical relevance and biological functions of PFKFB3 and PFKFB4 in oral cancer. Nevertheless, some of the detailed effects are still inconclusive due to the limitations of this study: (1) the cohort in TCGA database that we use to analyze the clinical significance of PFKFBs in most oral cancer patients is obtained from western countries, which needs more cohorts from other countries to further verify its importance in oral cancer; (2) the biological roles of PFKFBs was evaluated with oral cancer cell lines in this study, which needs the animal model to elucidate complex biological mechanisms that occur in OSCC patients; (3) The relation between PFKFB3 and PFKFB4 expression determine their prognostic value in several cancers [68], which needs further studies to analyze the relationship between both enzymes; (4) Previous study has shown that phosphorylation of PFKFB3 [73] and PFKFB4 [74] isoenzyme increases their kinase activity. Thus, additional studies are needed to explore the protein levels of phosphorylated PFKFB3 and PFKFB4 in tumor tissues of OSCC patients.

Conclusion

Our study first investigates roles of the PFKFB family in oral cancer patients. The overexpression of PFKFB3 and PFKFB4 was associated with low survival in oral cancer patients and was involved in cell growth/migration and chemoresistance/cancer stemness in OSCC cells, respectively. The co-expressions of PFKFB3/cell cycle or EMT markers and PFKFB4/stemness markers in oral cancer patients were also related to poor prognosis. Thus, we speculate that PFKFB3 and PFKFB4 might be potential prognostic biomarkers and therapeutic targets for OSCC patients.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

PFKFBs:

Phosphofructokinase-fructose bisphosphatases

OSCC:

Oral squamous cell carcinoma

NSCLC:

Non-small cell lung cancer

HNSCC:

Head and neck squamous cell carcinoma

HCC:

Hepatocellular carcinoma

TNBC:

Triple-negative breast cancer

DMEM:

Dulbecco’s modified Eagle’s medium

OS:

Overall survival

PFI:

Progression-free interval survival

DSS:

Disease-specific survival

DFI:

Disease-free interval survival

CHR:

Crude hazard ratio

AHR:

Adjusted hazard ratio

EMT:

Epithelial–mesenchymal transition

References

  1. Zhang C, Cai Q, Ke J. Poor prognosis of oral squamous cell carcinoma correlates With ITGA6. Int Dent J. 2023;73:178–85.

    Article  PubMed  Google Scholar 

  2. Al-Jamaei AAH, van Dijk BAC, Helder MN, Forouzanfar T, Leemans CR, de Visscher J. A population-based study of the epidemiology of oral squamous cell carcinoma in the Netherlands 1989–2018, with emphasis on young adults. Int J Oral Maxillofac Surg. 2022;51:18–26.

    Article  CAS  PubMed  Google Scholar 

  3. Wang S, Yang M, Li R, Bai J. Current advances in noninvasive methods for the diagnosis of oral squamous cell carcinoma: a review. Eur J Med Res. 2023;28:53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Vaupel P, Multhoff G. Revisiting the Warburg effect: historical dogma versus current understanding. J Physiol. 2021;599:1745–57.

    Article  CAS  PubMed  Google Scholar 

  5. Zhou X, Xue D, Qiu J. Identification of biomarkers related to glycolysis with weighted gene co-expression network analysis in oral squamous cell carcinoma. Head Neck. 2022;44:89–103.

    Article  PubMed  Google Scholar 

  6. Zhang Y, Li Q, Huang Z, Li B, Nice EC, Huang C, Wei L, Zou B. Targeting glucose metabolism enzymes in cancer treatment: current and emerging strategies. Cancers. 2022. https://doi.org/10.3390/cancers14194568.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Wang S, Bei Y, Tian Q, He J, Wang R, Wang Q, Sun L, Ke J, Xie C, Shen P. PFKFB4 facilitates palbociclib resistance in oestrogen receptor-positive breast cancer by enhancing stemness. Cell Prolif. 2023;56: e13337.

    Article  CAS  PubMed  Google Scholar 

  8. Kotowski K, Rosik J, Machaj F, Supplitt S, Wiczew D, Jablonska K, Wiechec E, Ghavami S, Dziegiel P. Role of PFKFB3 and PFKFB4 in cancer: genetic basis, impact on disease development/progression, and potential as therapeutic targets. Cancers. 2021. https://doi.org/10.3390/cancers13040909.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Alvarez R, Mandal D, Chittiboina P. Canonical and non-canonical roles of PFKFB3 in brain tumors. Cells. 2021. https://doi.org/10.3390/cells10112913.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Sha L, Lv Z, Liu Y, Zhang Y, Sui X, Wang T, Zhang H. Shikonin inhibits the Warburg effect, cell proliferation, invasion and migration by downregulating PFKFB2 expression in lung cancer. Mol Med Rep. 2021. https://doi.org/10.3892/mmr.2021.12199.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Shen X, Zhu X, Hu P, Ji T, Qin Y, Zhu J. Knockdown circZNF131 inhibits cell progression and glycolysis in gastric cancer through miR-186-5p/PFKFB2 Axis. Biochem Genet. 2022;60:1567–84.

    Article  CAS  PubMed  Google Scholar 

  12. Ji F, Dai C, Xin M, Zhang J, Zhang Y, Liu S. Long intergenic non-protein coding RNA 115 (LINC00115) aggravates retinoblastoma progression by targeting microRNA miR-489-3p that downregulates 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2). Bioengineered. 2022;13:5330–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pan X, Li H, Tan J, Weng X, Zhou L, Weng Y, Cao X. miR-1297 suppresses osteosarcoma proliferation and aerobic glycolysis by regulating PFKFB2. Onco Targets Ther. 2020;13:11265–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liu Y, Ma L, Hua F, Min Z, Zhan Y, Zhang W, Yao J. Exosomal circCARM1 from spheroids reprograms cell metabolism by regulating PFKFB2 in breast cancer. Oncogene. 2022;41:2012–25.

    Article  CAS  PubMed  Google Scholar 

  15. Cheng X, Jia X, Wang C, Zhou S, Chen J, Chen L, Chen J. Hyperglycemia induces PFKFB3 overexpression and promotes malignant phenotype of breast cancer through RAS/MAPK activation. World J Surg Oncol. 2023;21:112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Larionova I, Patysheva M, Iamshchikov P, Kazakova E, Kazakova A, Rakina M, Grigoryeva E, Tarasova A, Afanasiev S, Bezgodova N, et al. PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse. Front Immunol. 2022;13:1080501.

    Article  CAS  PubMed  Google Scholar 

  17. Wan K, Shao J, Liu X, Cai Y, Xu Y, Li L, Xiong L, Liang S. HOXD9 contributes to the Warburg effect and tumor metastasis in non-small cell lung cancer via transcriptional activation of PFKFB3. Exp Cell Res. 2023;427: 113583.

    Article  CAS  PubMed  Google Scholar 

  18. Dou Q, Grant AK, Callahan C, Coutinho de Souza P, Mwin D, Booth AL, Nasser I, Moussa M, Ahmed M, Tsai LL. PFKFB3-mediated pro-glycolytic shift in hepatocellular carcinoma proliferation. Cell Mol Gastroenterol Hepatol. 2023;15:61–75.

    Article  CAS  PubMed  Google Scholar 

  19. Li D, Tang J, Gao R, Lan J, Shen W, Liu Y, Chen Y, Sun H, Yan J, Nie Y, et al. PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer. J Cancer. 2022;13:212–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cai YC, Yang H, Shan HB, Su HF, Jiang WQ, Shi YX. PFKFB4 overexpression facilitates proliferation by promoting the G1/S transition and is associated with a poor prognosis in triple-negative breast cancer. Dis Markers. 2021;2021:8824589.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Wang W, Wang B. KDM3A-mediated SP1 activates PFKFB4 transcription to promote aerobic glycolysis in osteosarcoma and augment tumor development. BMC Cancer. 2022;22:562.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Sun J, Jin R. PFKFB4 modulated by miR-195-5p can boost the malignant progression of cervical cancer cells. Bioorg Med Chem Lett. 2022;73: 128916.

    Article  CAS  PubMed  Google Scholar 

  23. Feng C, Li Y, Li K, Lyu Y, Zhu W, Jiang H, Wen H. PFKFB4 is overexpressed in clear-cell renal cell carcinoma promoting pentose phosphate pathway that mediates Sunitinib resistance. J Exp Clin Cancer Res. 2021;40:308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sittewelle M, Kappes V, Zhou C, Lecuyer D, Monsoro-Burq AH. PFKFB4 interacts with ICMT and activates RAS/AKT signaling-dependent cell migration in melanoma. Life Sci Alliance. 2022. https://doi.org/10.26508/lsa.202201377.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Phillips E, Balss J, Bethke F, Pusch S, Christen S, Hielscher T, Schnolzer M, Fletcher MNC, Habel A, Tessmer C, et al. PFKFB4 interacts with FBXO28 to promote HIF-1alpha signaling in glioblastoma. Oncogenesis. 2022;11:57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang H, Lu C, Fang M, Yan W, Chen M, Ji Y, He S, Liu T, Chen T, Xiao J. HIF-1alpha activates hypoxia-induced PFKFB4 expression in human bladder cancer cells. Biochem Biophys Res Commun. 2016;476:146–52.

    Article  CAS  PubMed  Google Scholar 

  27. Peng J, Cui Y, Xu S, Wu X, Huang Y, Zhou W, Wang S, Fu Z, Xie H. Altered glycolysis results in drug-resistant in clinical tumor therapy. Oncol Lett. 2021;21:369.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang P, Tao W, Lu C, Fan L, Jiang Q, Yang C, Shang E, Cheng H, Che C, Duan J, et al. Bruceine A induces cell growth inhibition and apoptosis through PFKFB4/GSK3beta signaling in pancreatic cancer. Pharmacol Res. 2021;169: 105658.

    Article  CAS  PubMed  Google Scholar 

  29. Li X, Chen Z, Li Z, Huang G, Lin J, Wei Q, Liang J, Li W. The metabolic role of PFKFB4 in androgen-independent growth in vitro and PFKFB4 expression in human prostate cancer tissue. BMC Urol. 2020;20:61.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Shi L, Pan H, Liu Z, Xie J, Han W. Roles of PFKFB3 in cancer. Signal Transduct Target Ther. 2017;2:17044.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Cheng JS, Tsai WL, Liu PF, Goan YG, Lin CW, Tseng HH, Lee CH, Shu CW. The MAP3K7-mTOR axis promotes the proliferation and malignancy of hepatocellular carcinoma cells. Front Oncol. 2019;9:474.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kang BH, Shu CW, Chao JK, Lee CH, Fu TY, Liou HH, Ger LP, Liu PF. Author Correction: HSPD1 repressed E-cadherin expression to promote cell invasion and migration for poor prognosis in oral squamous cell carcinoma. Sci Rep. 2020;10:1829.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jiang YX, Siu MKY, Wang JJ, Leung THY, Chan DW, Cheung ANY, Ngan HYS, Chan KKL. PFKFB3 regulates chemoresistance, metastasis and stemness via IAP proteins and the NF-kappaB signaling pathway in ovarian cancer. Front Oncol. 2022;12: 748403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. He X, Cheng X, Ding J, Xiong M, Chen B, Cao G. Hyperglycemia induces miR-26-5p down-regulation to overexpress PFKFB3 and accelerate epithelial-mesenchymal transition in gastric cancer. Bioengineered. 2022;13:2902–17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ros S, Schulze A. Balancing glycolytic flux: the role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases in cancer metabolism. Cancer Metab. 2013;1:8.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Bartrons R, Simon-Molas H, Rodriguez-Garcia A, Castano E, Navarro-Sabate A, Manzano A, Martinez-Outschoorn UE. Fructose 2,6-bisphosphate in cancer cell metabolism. Front Oncol. 2018;8:331.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Houles T, Gravel SP, Lavoie G, Shin S, Savall M, Meant A, Grondin B, Gaboury L, Yoon SO, St-Pierre J, et al. RSK regulates PFK-2 activity to promote metabolic rewiring in melanoma. Cancer Res. 2018;78:2191–204.

    Article  CAS  PubMed  Google Scholar 

  38. Zhao SJ, Shen YF, Li Q, He YJ, Zhang YK, Hu LP, Jiang YQ, Xu NW, Wang YJ, Li J, et al. SLIT2/ROBO1 axis contributes to the Warburg effect in osteosarcoma through activation of SRC/ERK/c-MYC/PFKFB2 pathway. Cell Death Dis. 2018;9:390.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Ji D, Lu ZT, Li YQ, Liang ZY, Zhang PF, Li C, Zhang JL, Zheng X, Yao YM. MACC1 expression correlates with PFKFB2 and survival in hepatocellular carcinoma. Asian Pac J Cancer Prev. 2014;15:999–1003.

    Article  PubMed  Google Scholar 

  40. Moon JS, Jin WJ, Kwak JH, Kim HJ, Yun MJ, Kim JW, Park SW, Kim KS. Androgen stimulates glycolysis for de novo lipid synthesis by increasing the activities of hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 in prostate cancer cells. Biochem J. 2011;433:225–33.

    Article  CAS  PubMed  Google Scholar 

  41. Peng F, Li Q, Sun JY, Luo Y, Chen M, Bao Y. PFKFB3 is involved in breast cancer proliferation, migration, invasion and angiogenesis. Int J Oncol. 2018;52:945–54.

    CAS  PubMed  Google Scholar 

  42. Li HM, Yang JG, Liu ZJ, Wang WM, Yu ZL, Ren JG, Chen G, Zhang W, Jia J. Blockage of glycolysis by targeting PFKFB3 suppresses tumor growth and metastasis in head and neck squamous cell carcinoma. J Exp Clin Cancer Res. 2017;36:7.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Domenech E, Maestre C, Esteban-Martinez L, Partida D, Pascual R, Fernandez-Miranda G, Seco E, Campos-Olivas R, Perez M, Megias D, et al. AMPK and PFKFB3 mediate glycolysis and survival in response to mitophagy during mitotic arrest. Nat Cell Biol. 2015;17:1304–16.

    Article  CAS  PubMed  Google Scholar 

  44. Kotowski K, Supplitt S, Wiczew D, Przystupski D, Bartosik W, Saczko J, Rossowska J, Drag-Zalesinska M, Michel O, Kulbacka J. 3PO as a selective inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 in a375 human melanoma cells. Anticancer Res. 2020;40:2613–25.

    Article  PubMed  Google Scholar 

  45. Clem B, Telang S, Clem A, Yalcin A, Meier J, Simmons A, Rasku MA, Arumugam S, Dean WL, Eaton J, et al. Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth. Mol Cancer Ther. 2008;7:110–20.

    Article  CAS  PubMed  Google Scholar 

  46. Yalcin A, Clem BF, Simmons A, Lane A, Nelson K, Clem AL, Brock E, Siow D, Wattenberg B, Telang S, et al. Nuclear targeting of 6-phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases. J Biol Chem. 2009;284:24223–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lei L, Hong LL, Ling ZN, Zhong Y, Hu XY, Li P, Ling ZQ. A potential oncogenic role for PFKFB3 overexpression in gastric cancer progression. Clin Transl Gastroenterol. 2021;12: e00377.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Gu M, Li L, Zhang Z, Chen J, Zhang W, Zhang J, Han L, Tang M, You B, Zhang Q, et al. PFKFB3 promotes proliferation, migration and angiogenesis in nasopharyngeal carcinoma. J Cancer. 2017;8:3887–96.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Yalcin A, Solakoglu TH, Ozcan SC, Guzel S, Peker S, Celikler S, Balaban BD, Sevinc E, Gurpinar Y, Chesney JA. 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for transforming growth factor beta1-enhanced invasion of Panc1 cells in vitro. Biochem Biophys Res Commun. 2017;484:687–93.

    Article  CAS  PubMed  Google Scholar 

  50. Marcucci F, Rumio C. Tumor cell glycolysis-at the crossroad of epithelial-mesenchymal transition and autophagy. Cells. 2022. https://doi.org/10.3390/cells11061041.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Yang JG, Wang WM, Xia HF, Yu ZL, Li HM, Ren JG, Chen G, Wang BK, Jia J, Zhang W, et al. Lymphotoxin-alpha promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner. Int J Cancer. 2019;145:1358–70.

    Article  CAS  PubMed  Google Scholar 

  52. Li X, Jiang E, Zhao H, Chen Y, Xu Y, Feng C, Li J, Shang Z. Glycometabolic reprogramming-mediated proangiogenic phenotype enhancement of cancer-associated fibroblasts in oral squamous cell carcinoma: role of PGC-1alpha/PFKFB3 axis. Br J Cancer. 2022;127:449–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Li J, Tang X. Increased expression of PFKFB3 in oral squamous cell carcinoma and its association with lymphangiogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol. 2021;132:57–65.

    Article  PubMed  Google Scholar 

  54. Shen C, Ding L, Mo H, Liu R, Xu Q, Tu K. Long noncoding RNA FIRRE contributes to the proliferation and glycolysis of hepatocellular carcinoma cells by enhancing PFKFB4 expression. J Cancer. 2021;12:4099–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Dasgupta S, Rajapakshe K, Zhu B, Nikolai BC, Yi P, Putluri N, Choi JM, Jung SY, Coarfa C, Westbrook TF, et al. Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer. Nature. 2018;556:249–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Li W, Qian L, Lin J, Huang G, Hao N, Wei X, Wang W, Liang J. CD44 regulates prostate cancer proliferation, invasion and migration via PDK1 and PFKFB4. Oncotarget. 2017;8:65143–51.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Goidts V, Bageritz J, Puccio L, Nakata S, Zapatka M, Barbus S, Toedt G, Campos B, Korshunov A, Momma S, et al. RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival. Oncogene. 2012;31:3235–43.

    Article  CAS  PubMed  Google Scholar 

  58. Wu Y, Zhang L, Bao Y, Wan B, Shu D, Luo T, He Z. Loss of PFKFB4 induces cell cycle arrest and glucose metabolism inhibition by inactivating MEK/ERK/c-Myc pathway in cervical cancer cells. J Obstet Gynaecol. 2022;42:2399–405.

    Article  CAS  PubMed  Google Scholar 

  59. Lu H, Chen S, You Z, Xie C, Huang S, Hu X. PFKFB4 negatively regulated the expression of histone acetyltransferase GCN5 to mediate the tumorigenesis of thyroid cancer. Dev Growth Differ. 2020;62:129–38.

    Article  PubMed  Google Scholar 

  60. Hsin MC, Hsieh YH, Hsiao YH, Chen PN, Wang PH, Yang SF. Carbonic anhydrase IX promotes human cervical cancer cell motility by regulating PFKFB4 expression. Cancers. 2021. https://doi.org/10.3390/cancers13051174.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Tamada M, Nagano O, Tateyama S, Ohmura M, Yae T, Ishimoto T, Sugihara E, Onishi N, Yamamoto T, Yanagawa H, et al. Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells. Cancer Res. 2012;72:1438–48.

    Article  CAS  PubMed  Google Scholar 

  62. Lin J, Xia L, Liang J, Han Y, Wang H, Oyang L, Tan S, Tian Y, Rao S, Chen X, et al. The roles of glucose metabolic reprogramming in chemo- and radio-resistance. J Exp Clin Cancer Res. 2019;38:218.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Boscaro C, Baggio C, Carotti M, Sandona D, Trevisi L, Cignarella A, Bolego C. Targeting of PFKFB3 with miR-206 but not mir-26b inhibits ovarian cancer cell proliferation and migration involving FAK downregulation. FASEB J. 2022;36: e22140.

    Article  CAS  PubMed  Google Scholar 

  64. Li J, Zhang S, Liao D, Zhang Q, Chen C, Yang X, Jiang D, Pang J. Overexpression of PFKFB3 promotes cell glycolysis and proliferation in renal cell carcinoma. BMC Cancer. 2022;22:83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Gao R, Li D, Xun J, Zhou W, Li J, Wang J, Liu C, Li X, Shen W, Qiao H, et al. CD44ICD promotes breast cancer stemness via PFKFB4-mediated glucose metabolism. Theranostics. 2018;8:6248–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang Q, Zeng F, Sun Y, Qiu Q, Zhang J, Huang W, Huang J, Huang X, Guo L. Etk interaction with PFKFB4 modulates chemoresistance of small-cell lung cancer by regulating autophagy. Clin Cancer Res. 2018;24:950–62.

    Article  CAS  PubMed  Google Scholar 

  67. Yi M, Ban Y, Tan Y, Xiong W, Li G, Xiang B. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4: a pair of valves for fine-tuning of glucose metabolism in human cancer. Mol Metab. 2019;20:1–13.

    Article  PubMed  Google Scholar 

  68. Trojan SE, Markiewicz MJ, Leskiewicz K, Kocemba-Pilarczyk KA. The influence of PFK-II overexpression on neuroblastoma patients’ survival may be dependent on the particular isoenzyme expressed, PFKFB3 or PFKFB4. Cancer Cell Int. 2019;19:292.

    Article  PubMed  PubMed Central  Google Scholar 

  69. Emini Veseli B, Van Wielendaele P, Delibegovic M, Martinet W, De Meyer GRY. The PFKFB3 inhibitor AZ67 inhibits Angiogenesis Independently of Glycolysis Inhibition. Int J Mol Sci. 2021. https://doi.org/10.3390/ijms22115970.

    Article  PubMed  PubMed Central  Google Scholar 

  70. Gupta D, Kumar M, Singh M, Salman M, Das U, Kaur P. Identification of polypharmacological anticancerous molecules against Aurora kinase family of proteins. J Cell Biochem. 2022;123:719–35.

    Article  CAS  PubMed  Google Scholar 

  71. Kumar M, Dubey R, Kumar Shukla P, Dayal D, Kumar Chaubey K, Tsai LW, Kumar S. Identification of small molecule inhibitors of RAD52 for breast cancer therapy: in silico approach. J Biomol Struct Dyn. 2023. https://doi.org/10.1080/07391102.2023.2220822.

    Article  PubMed  Google Scholar 

  72. Naik S, Rawat RS, Khandai S, Kumar M, Jena SS, Vijayalakshmi MA, Kumar S. Biochemical characterisation of lectin from Indian hyacinth plant bulbs with potential inhibitory action against human cancer cells. Int J Biol Macromol. 2017;105:1349–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Lu C, Qiao P, Sun Y, Ren C, Yu Z. Positive regulation of PFKFB3 by PIM2 promotes glycolysis and paclitaxel resistance in breast cancer. Clin Transl Med. 2021;11: e400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Lu C, Qiao P, Fu R, Wang Y, Lu J, Ling X, Liu L, Sun Y, Ren C, Yu Z. Phosphorylation of PFKFB4 by PIM2 promotes anaerobic glycolysis and cell proliferation in endometriosis. Cell Death Dis. 2022;13:790.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

We acknowledge the support from the National Science and Technology Council (MOST 109-2320-B-037-015-MY3; NSTC 111-2320-B-110-006, NSTC 112-2320-B-037-026), the Kaohsiung Medical University Research Center Grant (KMU-TC108A04), the Kaohsiung Medical University Hospital (KMUH110-0M68), the National Sun Yat-sen University-KMU Joint Research Project (NSYSU-KMU-112-P02; NSYSU-KMU-112-P06), the joint grant of National Sun Yat-sen University with Kaohsiung Veterans General Hospital (KSVNSU112-006) and the NSYSU-KCGMH Joint Research Project (111-05).

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, K-FH, C-WS and P-FL; resources, C-WS; methodology, C-WS; software, Y-HC; formal analysis, K.-FH and C-HL; validation, K-FH and C-HL; data curation, K-FH and C-HL; supervision, C-JT and P-FL; writing—original draft, K-FH and P-FL; writing—review and editing, P-FL.

Corresponding author

Correspondence to Pei-Feng Liu.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

. The association of PFKFB1 expression with prognosis in oral cancer patients stratified by different clinicopathological features. Table S2.. The association of PFKFB2 expression with prognosis in oral cancer patients stratified by different clinicopathological features.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, KF., Shu, CW., Lee, CH. et al. Comparative clinical significance and biological roles of PFKFB family members in oral squamous cell carcinoma. Cancer Cell Int 23, 257 (2023). https://doi.org/10.1186/s12935-023-03110-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12935-023-03110-6

Keywords