Role of PFKFB3 and CD163 in Oral Squamous Cell Carcinoma Angiogenesis

6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), an enzyme producing fructose 2, 6-bisphosphate (F-2, 6-BP), serves as a switch to activate phosphofructokinase-1, and is a critical enzyme for endothelial glycolysis, mediating circadian control of carcinogenesis. Also, tumor-associated macrophages (TAMs) play an important role in the progression and prognosis of numerous cancers. However, the role and clinical significance of PFKFB3 and TAMs in oral squamous cell carcinoma (OSCC) have not been elucidated. The present study was designed to investigate the correlation between PFKFB3 expression, CD 163+ TAMs infiltration and tumor angiogenesis in OSCC by tissue microarray. Tissue microarrays containing 117 OSCC specimens and 56 matched paracarcinoma tissues were studied by immunohistochemistry. The expression levels of PFKFB3, CD163 and CD31 were significantly increased in OSCC specimens as compared with normal oral mucosa (P<0.05), and PFKFB was significantly correlated with tumor differentiation and tumor size (P<0.05), and CD 163 was significantly correlated with areca nut chewing habit among OSCC tissues (P<0.05). Furthermore, Pearson's correlation analysis revealed that PFKFB3 was significantly correlated with both CD 163 and CD31 (P<0.05), meanwhile CD 163 was significantly correlated with CD31 (P<0.001), suggesting PFKFB3 may promote angiogenesis in tumor progression and metastases by regulating CD 163+ TAMs infiltration in OSCC.

Mechanisms of regulation of PFKFB expression in pancreatic and gastric cancer cells

Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expression of these mRNAs is increased in malignant tumors and strongly induced in different cancer cell lines by hypoxia inducible factor (HIF) through active HIF binding sites in promoter region of PFKFB-4 and PFKFB-3 genes. Moreover, the expression and hypoxia responsibility of PFKFB-4 and PFKFB-3 was also shown for pancreatic (Panc1, PSN-1, and MIA PaCa-2) as well as gastric (MKN45 and NUGC3) cancer cells. At the same time, their basal expression level and hypoxia responsiveness vary in the different cells studied: the highest level of PFKFB-4 protein expression was found in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with a stronger response to hypoxia in the pancreatic cancer cell line. Overexpression of different PFKFB in pancreatic and gastric cancer cells under hypoxic condition is correlated with enhanced expression of vascular endothelial growth factor (VEGF) and Glut1 mRNA as well as with increased level of HIF-1&#x003b1; protein. Increased expression of different PFKFB genes was also demonstrated in gastric, lung, breast, and colon cancers as compared to corresponding non-malignant tissue counterparts from the same patients, being more robust in the breast and lung tumors. Moreover, induction of PFKFB-4 mRNA expression in the breast and lung cancers is stronger than PFKFB-3 mRNA. The levels of both PFKFB-4 and PFKFB-3 proteins in non-malignant gastric and colon tissues were more pronounced than in the non-malignant breast and lung tissues. It is interesting to note that Panc1 and PSN-1 cells transfected with dominant/negative PFKFB-3 (dnPFKFB-3) showed a lower level of endogenous PFKFB-3, PFKFB-4, and VEGF mRNA expressions as well as a decreased proliferation rate of these cells. Moreover, a similar effect had dnPFKFB-4. In conclusion, there is strong evidence that PFKFB-4 and PFKFB-3 isoenzymes are induced under hypoxia in pancreatic and other cancer cell lines, are overexpressed in gastric, colon, lung, and breast malignant tumors and undergo changes in their metabolism that contribute to the proliferation and survival of cancer cells. Thus, targeting these PFKFB may therefore present new therapeutic opportunities.

Adoptive transfer of Pfkfb3-disrupted hematopoietic cells to wild-type mice exacerbates diet-induced hepatic steatosis and inflammation

Background and objectives:Hepatic steatosis and inflammation are key characteristics of non-alcoholic fatty liver disease(NAFLD).However,whether and how hepatic steatosis and liver inflammation are differentially regulated remains to be elucidated.Considering that disruption of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(Pfkfb3/iPfk2)dissociates fat deposition and inflammation,the present study examined a role for Pfkfb3/iPfk2 in hematopoietic cells in regulating hepatic steatosis and inflammation in mice.Methods:Pfkfb3-disrupted(Pfkfb3^(+/-))mice and wild-type(WT)littermates were fed a high-fat diet(HFD)and examined for NAFLD phenotype.Also,bone marrow cells isolated from Pfkfb3^(+/-)mice andWT mice were differentiated into macrophages for analysis of macrophage activation status and for bone marrow transplantation(BMT)to generate chimeric(WT/BMT-Pfkfb3^(+/-))mice in which Pfkfb3 was disrupted only in hematopoietic cells and control chimeric(WT/BMT-WT)mice.The latter were also fed an HFD and examined for NAFLD phenotype.In vitro,hepatocytes were co-cultured with bone marrowderived macrophages and examined for hepatocyte fat deposition and proinflammatory responses.Results:After the feeding period,HFD-fed Pfkfb3^(+/-)mice displayed increased severity of liver inflammation in the absence of hepatic steatosis compared with HFD-fed WT mice.When inflammatory activation was analyzed,Pfkfb3^(+/-)macrophages revealed increased proinflammatory activation and decreased anti-proinflammatory activation.When NAFLD phenotype was analyzed in the chimeric mice,WT/BMT-Pfkfb3^(+/-) mice displayed increases in the severity of HFD-induced hepatic steatosis and inflammation compared with WT/BMT-WT mice.At the cellular level,hepatocytes co-cultured with Pfkfb3^(+/-) macrophages revealed increased fat deposition and proinflammatory responses compared with hepatocytes co-cultured with WT macrophages.Conclusions:Pfkfb3 disruption only in hematopoietic cells exacerbates HFD-induced hepatic steatosis and inflammation whereas the Pfkfb3/iPfk2 in nonhematopoietic cells appeared to be needed for HFD feeding to induce hepatic steatosis.As such,the Pfkfb3/iPfk2 plays a unique role in regulating NAFLD pathophysiology.