Jianfei Shen1, Zixian Jin1, Haiyan Lv1, Ke Jin1, Kangberee Jonas2, Chengchu Zhu3, Baofu Chen4. 1. Department of Thoracic Surgery, Taizhou Hospital of Zhejiang Province, Whenzhou Medical University, Linhai, 317000, China. 2. Whenzhou Medical University, Whenzhou, 325000, China. 3. Department of Thoracic Surgery, Taizhou Hospital of Zhejiang Province, Whenzhou Medical University, Linhai, 317000, China. zhucc@enzemed.com. 4. Department of Thoracic Surgery, Taizhou Hospital of Zhejiang Province, Whenzhou Medical University, Linhai, 317000, China. chenbf@enzemed.com.
Abstract
PURPOSE: Although it has been reported that up-regulation of phosphofructokinase (PFK) expression is a major feature of malignant tumors, the role of platelet type PFK (PFKP) in tumor initiation and progression is as yet poorly understood. The objective of this study was to evaluate PFKP expression in lung cancer and its effect on glycolysis, and to explore correlations between PFKP expression levels and clinical lung cancer patient features. METHODS: PFKP mRNA expression levels in cancer tissues and adjacent normal tissues were compared using The Cancer Genome Atlas (TCGA) database. PFKP mRNA and protein expression levels in fresh lung cancer tissues and cell lines were assessed using quantitative real-time PCR and Western blotting. Immunohistochemistry (IHC) was used to assess PFKP expression in 150 archival lung adenocarcinoma samples, after which follow-up data and their correlations with clinical features and patient prognosis were evaluated. A retroviral shRNA-mediated method was used to construct stable cell lines expressing low levels of PFKP. Glucose, lactate and adenosine triphosphate concentrations in the cell culture supernatants were determined using enzymatic, spectrophotometric and enzyme-linked immunosorbent (ELISA) assays, respectively. The effect of PFKP expression on the proliferation of lung cancer cells was assessed using colony forming, MTT and flow cytometry assays, respectively. Finally, data from tissue samples of 533 patients with lung adenocarcinoma and 502 patients with lung squamous cell carcinoma were downloaded from the TCGA database, after which pathway and gene correlation information was retrieved using gene set enrichment analyses. RESULTS: We found that PFKP was highly expressed in lung cancer tissues and cell lines. Using IHC we found that PFKP was highly expressed in primary lung adenocarcinoma tissues and that a high expression was associated with a poor prognosis. Clinical data analysis revealed that the PFKP expression levels correlated with tumor size and patient survival. Lung cancer cell lines with decreased PFKP expression levels showed significant decreases in glucose uptake rates, lactate levels and adenosine triphosphate concentrations. They also exhibited significantly decreased proliferation rates, colony forming abilities and increased G2/M cell cycle phase percentages. Gene set enrichment analysis revealed that multiple pathways, including glycolytic pathways, may be involved in the regulation PFKP. CONCLUSIONS: Our data indicate that PFKP is highly expressed in lung cancer tissues and cell lines and is associated with tumor size and patient prognosis. As such, PFKP may serve as a prognostic biomarker. We also found that PFKP regulates the level of glycolysis in lung cancer cells and is associated with lung cancer cell proliferation. These data may be instrumental for the design of new lung cancer treatment options.
PURPOSE: Although it has been reported that up-regulation of phosphofructokinase (PFK) expression is a major feature of malignant tumors, the role of platelet type PFK (PFKP) in tumor initiation and progression is as yet poorly understood. The objective of this study was to evaluate PFKP expression in lung cancer and its effect on glycolysis, and to explore correlations between PFKP expression levels and clinical lung cancerpatient features. METHODS:PFKP mRNA expression levels in cancer tissues and adjacent normal tissues were compared using The Cancer Genome Atlas (TCGA) database. PFKP mRNA and protein expression levels in fresh lung cancer tissues and cell lines were assessed using quantitative real-time PCR and Western blotting. Immunohistochemistry (IHC) was used to assess PFKP expression in 150 archival lung adenocarcinoma samples, after which follow-up data and their correlations with clinical features and patient prognosis were evaluated. A retroviral shRNA-mediated method was used to construct stable cell lines expressing low levels of PFKP. Glucose, lactate and adenosine triphosphate concentrations in the cell culture supernatants were determined using enzymatic, spectrophotometric and enzyme-linked immunosorbent (ELISA) assays, respectively. The effect of PFKP expression on the proliferation of lung cancer cells was assessed using colony forming, MTT and flow cytometry assays, respectively. Finally, data from tissue samples of 533 patients with lung adenocarcinoma and 502 patients with lung squamous cell carcinoma were downloaded from the TCGA database, after which pathway and gene correlation information was retrieved using gene set enrichment analyses. RESULTS: We found that PFKP was highly expressed in lung cancer tissues and cell lines. Using IHC we found that PFKP was highly expressed in primary lung adenocarcinoma tissues and that a high expression was associated with a poor prognosis. Clinical data analysis revealed that the PFKP expression levels correlated with tumor size and patient survival. Lung cancer cell lines with decreased PFKP expression levels showed significant decreases in glucose uptake rates, lactate levels and adenosine triphosphate concentrations. They also exhibited significantly decreased proliferation rates, colony forming abilities and increased G2/M cell cycle phase percentages. Gene set enrichment analysis revealed that multiple pathways, including glycolytic pathways, may be involved in the regulation PFKP. CONCLUSIONS: Our data indicate that PFKP is highly expressed in lung cancer tissues and cell lines and is associated with tumor size and patient prognosis. As such, PFKP may serve as a prognostic biomarker. We also found that PFKP regulates the level of glycolysis in lung cancer cells and is associated with lung cancer cell proliferation. These data may be instrumental for the design of new lung cancer treatment options.