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.

中文翻译：

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PFKP在肺癌中高表达，并调节葡萄糖代谢。

目的

尽管已经报道磷酸果糖激酶（PFK）表达的上调是恶性肿瘤的主要特征，但是人们对血小板型PFK（PFKP）在肿瘤发生和发展中的作用还知之甚少。这项研究的目的是评估肺癌中PFKP的表达及其对糖酵解的影响，并探讨PFKP的表达水平与临床肺癌患者特征之间的相关性。

方法

使用癌症基因组图谱（TCGA）数据库比较了癌组织和邻近正常组织中PFKP mRNA的表达水平。新鲜的肺癌组织和细胞系中的PFKP mRNA和蛋白表达水平使用实时定量PCR和Western印迹法进行了评估。免疫组织化学（IHC）用于评估150份档案肺腺癌样品中的PFKP表达，然后评估随访数据及其与临床特征和患者预后的相关性。使用逆转录病毒shRNA介导的方法构建表达低水平PFKP的稳定细胞系。分别使用酶促，分光光度法和酶联免疫吸附（ELISA）测定法测定细胞培养上清液中的葡萄糖，乳酸和三磷酸腺苷浓度。分别使用集落形成，MTT和流式细胞术测定来评估PFKP表达对肺癌细胞增殖的影响。最后，从TCGA数据库中下载了533例肺腺癌患者和502例肺鳞癌患者的组织样本数据，然后使用基因集富集分析检索了途径和基因相关信息。

结果

我们发现PFKP在肺癌组织和细胞系中高表达。使用IHC，我们发现PFKP在原发性肺腺癌组织中高表达，并且高表达与不良预后相关。临床数据分析表明，PFKP表达水平与肿瘤大小和患者生存率相关。PFKP表达水平降低的肺癌细胞系显示葡萄糖摄取率，乳酸水平和三磷酸腺苷浓度显着降低。它们还表现出显着降低的增殖速率，集落形成能力和增加的G2 / M细胞周期相百分比。基因集富集分析显示，多种途径（包括糖酵解途径）可能参与了PFKP的调控。

结论

我们的数据表明，PFKP在肺癌组织和细胞系中高表达，并且与肿瘤大小和患者预后相关。因此，PFKP可以作为预后的生物标志物。我们还发现PFKP调节肺癌细胞中的糖酵解水平，并与肺癌细胞的增殖有关。这些数据可能有助于设计新的肺癌治疗方案。