Elevating PKM2 Expression Indicates a Biomarker of Poor Prognosis in Patients With Liver Cancer

M2 isoform of pyruvate kinase (PKM2) plays an important role in reprogramming of cell metabolism which is a hall-marker of tumorigenesis. PKM2 expression altering is closely related to cancer metabolism and tumor growth. In the present study, we analyzed the role of PKM2 expression in liver cancer in order to clarify its potential application value in the diagnosis and prognosis of liver cancer patients. In cancerous liver tissues, the PKM2 expression was signicantly higher than normal tissues. High PKM2 expressing was related to patient’s age, gender, histological type, grade, stage, T classication and poor survival. Patients with Higher PKM2 expression had a shorter OS (P = 0.0013) and RFS (P = 0.027). ROC and Multivariate Cox analysis showed that high PKM2expression was a risk factor for patients’ poor prognosis. GSEA identied mitotic spindle, PI3K/Akt/mTOR signaling, notch signaling, apoptosis, G2M checkpoint and Wnt/β- Cantenin signaling were enriched with high PKM2 expression phenotype. These ndings suggested PKM2 expression has potential as a predictive biomarker for the diagnosis and prognosis of patients with liver cancer.


Introduction
Liver cancer, a clinically common malignant tumor with limited e cacy and poor prognosis, stills a main cause of global cancer-related deaths [1] . Despite tremendous progress in the diagnosis and treatment of liver cancer, the overall prognosis of patients is still unsatisfactory [2,3] . Therefore, it is extremely valuable and urgent to nd more speci c and reliable prognostic evaluation indicators of liver cancer.
Metabolic abnormalities are an important hall-marker for tumorigenesis. Pyruvate-kinase (PKM) is the enzyme that catalyzes the nal step in glycolysis, generating pyruvate and ATP from phosphoenolpyruvate and ADP [4] .
This process consumes glucose, causing pyruvate to be converted to lactate [5] . In mammals, pyruvate kinase has four isoforms, including PKL, PKR, PKM1 and PKM2 [6] . The PKL and PKR show tissue-speci c expression in the liver tissue and red-blood cells, respectively [7] . They have different rst exons and are de ned by tissue-speci c promoters [8] . The PKM consists of 12 exons, in which exons 9 and 10 are alternately spliced in mutually exclusive ways to produce PKM1 and PKM2 isoforms, respectively [9] . PKM2 is mainly expressed in a range of cancer cells, as well as in fetal and undifferentiated adult tissues, whereas PKM1 is predominantly expressed in terminally differentiated tissues [10] . Previous researches have shown that tissue-speci c PKM1/L/R expression gradually diminishes and is replaced by PKM2 expression during tumorigenesis [11] .
As a key regulator of aerobic glycolysis, the expression of PKM2 is essential for metabolic regulation [12] . In recent years, accumulating studies have shown that PKM2, in addition to its involvement in metabolic regulation, can also directly participate in gene transcription as a transcription coactivator and protein kinase, regulating the proliferation and apoptosis of undifferentiated cells [13][14][15] . PKM2 has also been found to have a potential prognostic role in the treatment of several cancers, including lung cancer [16] , cervical cancer [17] and endometrial carcinoma [18][19] . This means that PKM2 may be used as a predictor in clinical diagnosis and prognosis evaluation of cancer patients.
In the present study, we investigated the relationship between the expression pattern of PKM2 in cancerous and non-cancerous liver tissues, as well as the association between high PKM2 expression and histological grade, stage, T/N/M classi cation, patient age, gender and survival status. The prognostic signi cance of PKM2 expression in liver cancer was also analyzed to clarify its potential application value in the treatment and prognosis of liver cancer patients.

Materials And Methods
Data mining and preprocessing from The Cancer Genome Atlas (TCGA) database The RNAseq data of PKM2 (gene ID: 5315) and clinical information of liver hepatocellular carcinoma (LIHC) patient were obtained from The Cancer Genome Atlas (TCGA) database. The RNAseq value is estimated as log2 (x + 1) converted RSEM normalized counts, and these data were processed using R software (version 4.0.1). 20

Gene Set Enrichment Analysis
To explore the distribution of prede ned genomes and determine the potential mechanism to in uence the effect of PKM2 expression on the prognosis of LIHC patients, we opted for GSEA (version 4.0.3). This analysis was performed through the "h.all.v7.2.symbols.gmt" gene set in the Molecular Signatures database. 21 Gene-sets with a normal P value <0.05 was regarded as signi cantly enriched.
Statistical Analysis R software package was used for statistical analysis. The ggpot2 package in R was performed for visualization.
Chi-square test was used to evaluate the correlation between PKM2 expression and clinical characteristics of liver cancer. ROC analysis was used to evaluate the diagnostic signi cance of PKM2 expression. Kaplan-meier survival curve was used to analyze the correlation between PKM2 expression and patients OS and RFS. Univariate and multivariate Cox regression analysis were used to verify the correlation between PKM2 expression and survival rate and other clinical features. Statistical signi cance was de ned as P <0.05.

Results
The patients' clinical characteristics and expression pattern of PKM2 in human liver cancer We obtained RNAseq of PKM2 expression and clinical information of 377 liver cancer patients from the TCGA database, including the patient age and gender; as well as histological type, histological grade, pathologic stage, T/N/M classi cation, radiation therapy, survival status and relapse (Table 1). Subsequently, we found that the expression of PKM2 in tumors was signi cantly higher than normal tissues (P < 0.0001, Fig. 1). Furthermore, PKM2 expression was also closely related to histological grade (P = 0.00011), histological stage (P = 0.00024), T classi cation (P < 0.0001), age (P = 0.0336), gender (P = 0.0136) and survival status (P = 0.00261).

Diagnostic signi cance of PKM2 expression and relationship with clinical characteristics in human liver cancer
To evaluate the role of PKM2 expression in diagnosis, ROC analysis was performed. We found that PKM2 expression had well diagnostic value (AUC = 0.744; Fig. 2A). In addition, we also analyzed the diagnostic value of PKM2 expression in different stages of the liver cancer, including AUC value of 0.698 in the rst stage, 0.768 in the second stage, 0.786 in the third stage and 0.860 in the fourth stage ( Fig. 2B-E). Subsequently, we analyzed the association between PKM2 expression and clinical features of liver cancer by divided patients into two groups (high or low PKM2 expression) according to median expression cutoff ( Table 2). We found that high PKM2 expression was related to patient age (P = 0.05), gender (P = 0.002), histological type (P = 0.041), histological grade (P = 0.003), pathologic stage (P = 0.001), T classi cation (P = 0.000) and poor survival (p = 0.020).
Effect of PKM2 expression on RFS among liver cancer patients Next, we used Kaplan-Meier curves to explore the correlation of PKM2 expression with patient RFS (Fig. 4). Survival analysis showed that high PKM2 expression was associated with poor RFS (P = 0.027). Additionally, subgroup analysis also showed that high PKM2 expression signi cantly affected the RFS for patients with N0/N1 (P = 0.039), and M0/M1 (P = 0.004). However, contrary to expectations, we did not observe that high PKM2 expression is a risk factor for RFS by Univariate Cox analyses (Table S2). Unexpectedly, we found that high PKM2expression is not a risk factor for poor RFS in both male and female patients with LIHC (Table S2, 3).
High PKM2 expression-related signaling pathway Identifying the activation of signaling pathways would facilitate a better understanding of molecular interactions, reactions and relationships, as well as disease process. 22 To determine the signaling pathways activated in LIHC, we used GSEA to analyze the high and low PKM2 expression datasets. The results showed that mitotic spindle, PI3K/Akt/mTOR signaling, notch signaling, apoptosis, G2M checkpoint and Wnt/β-Cantenin signaling were enriched to the high PKM2 expression phenotype (Table 5, Fig. 5).

Discussion
In the current study, we investigated the role of tumor PKM2 expression as a predictor in cancerous and healthy liver tissues. This observation clearly shows that the PKM2 level of tumors is signi cantly higher than healthy tissues, and elevating expression of PKM2 is associated with histological grade, stage and vital status. In addition, univariate and multivariate COX analysis indicated that high PKM2 expression was a predictor for decreased OS in the diagnosis and treatment of liver cancer patients.
Pyruvate Kinase Isotype M2 (PKM2) was a key enzyme involved in Warburg effect, and its activity was important for tumor metabolism and growth regulation. 23 In most cancer cells, high PKM2expression was able to promote cancer cell proliferation and increase the degree of tumor malignancy. 24 In addition, the necrosis and renewal of tumor cells can cause PKM2 to be released into surrounding tissues, peripheral blood, feces of patients with gastrointestinal malignancies, and pleural effusion of patients with chest tumors, which can be used as biomarkers to detect tumor metabolism and proliferation. 25,26 In early tumors, PKM2 expression showed heterogeneity, but the staining of metastatic tumor tissue was uniform and strong. 27 This also indicated that PKM2 may play an important role in the occurrence and development of tumors. Previous studies had shown that the re-expression of PKM2 in tumor cells not only has important signi cance in regulating tumor cell sugar metabolism, but also gives tumor cells the advantage of selective growth. 28 In this study, our results show that PKM2 expression also gradually increased as histologic grade increased from G1 to G4, as histologic stage increased from I to IV, and as T classi cation increased from T1 to T4. These results suggested that PKM2 expression was closely related with staging and grading of cancer, indicating that it can be used as a prognostic monitoring indicator in LIHC.
The phenotype of gene expression and genetic characteristics in tumors are related to signal activation occurrence and development of tumor. It had been reported that knockout of PKM2 in HCC cells inhibit cell proliferation and induce apoptosis in vivo and in vitro. 29 In addition, PKM2 knockout was able to be used as a chemotherapeutic sensitizer of docetaxel in non-small cell lung cancer cells, resulting in cell viability inhibition, G2/M cell arrest and increased apoptosis. 30 Moreover, PKM2 promotes cell migration and inhibits autophagy by mediating PI3K/AKT activation, and promotes the malignant development of gastric cancer. 31 In fact, our results indicate that PKM2 expression is related to liver cancer progression and malignant tumors, and the implicit mechanism may be linked to mitotic spindle, PI3K/Akt/mTOR signaling, notch signaling, apoptosis, G2M checkpoint and Wnt/β-Cantenin signaling as GSEA identi ed. These ndings, combined with previous reports, greatly enrich our comprehension and understanding of the physiological role of PKM2 in tumor development and deterioration.
Recent studies have shown that the PKM2 expression was also a potential histopathological marker for the differential diagnosis of malignant and precancerous endometrial lesions, and high PKM2 expression in endometrial cancer is conducive to poor prognosis. 32-34 C Papadaki et al. also indicated that PKM2 as a biomarker for chemosensitivity to front-line platinum-based chemotherapy in patients with metastatic non-smallcell lung cancer. 35 In this study, we explored the correlation between high PKM2 expression and clinical diagnosis. In addition, high PKM2 expression reduces OS in patients. Interestingly, the high PKM2 expression was signi cantly correlated with OS in male patients (P = 0.00011), but not in female (P = 0.91; Fig. 3). Additionally, although not signi cant at the 5% level, high PKM2 expression is a risk factor for RFS in liver cancer male patients (P = 0.071; Fig. 4). Univariate and multivariate Cox analysis suggested that high PKM2 was an independent risk factor for poor OS in male patients, not in female patients with LIHC (Table 4 and S1). Thus, we speculate that PKM2 may be more suitable for male patients as a potential biomarker for diagnosis and prognosis in liver cancer.
In conclusion, we provided a reliable and comprehensive analysis of PKM2 expression patterns, diagnosis value and prognosis signi cance. It has possible to be a valuable potential prognostic biomarker and therapeutic target for liver cancer. It will give us a comprehensive understanding of the biological functions of PKM2 and provide new reference value through its regulated network of signaling pathway. This will help formulate a better supervision, diagnosis and treatment strategies for malignant tumor in clinical applications.

Declarations
This work was supported by Sichuan Social Science Planning Project (SC18B080). It also wanted to thank Pro. Chaofeng Lv from Southwestern University of Finance and Economics for the providing assistance in the early stage of manuscript writing.

Authors' contributions
Chaoxiang Lv and Qiqi Zhang collected and analyzed the data. Yuanguo Li and Fangxu Li performed the data curation. Tian Qin performed visualization of data-set. Dandan Zhu, Tiecheng Wang and Chaoxiang Lv manuscript writing and revision. All authors read and approved the nal manuscript.

Con icts of Interest
The authors declared that there were no con icts of interest regarding the publication of this manuscript.

Data availability statement
The original data-set used in this manuscript have been deposited in the TCGA database (https://cancergenome.nih.gov/).