IJBM eISSN 1724-6008
Int J Biol Markers 2016; 00(00): e000-e000 DOI: 10.5301/jbm.5000189
ORIGINAL ARTICLE
Genetic alteration in phosphofructokinase family promotes growth of muscle-invasive bladder cancer Chen-min Sun, Da-bo Xiong, Yang Yan, Jiang Geng, Min Liu, Xu-dong Yao Department of Urology, Shanghai Tenth People’s Hospital, Tongji University, Shanghai - China Chen-min Sun and Da-bo Xiong contributed equally to this work.
ABSTRACT Aims: Metabolic alterations in cancer, including bladder cancer, have been addressed in recent years. We aimed to study the role of phosphofructokinase (PFK) in muscle-invasive bladder cancer (MIBC). Method: By in silico analysis of the bladder cancer data from the Cancer Genome Atlas (TCGA) database using the cBioPortal platform, we studied genetic alteration of genes within the PFK family (PFKL, PFKM, PFKP, PFKFB1, PFKFB2, PFKFB3, and PFKFB4). In vitro studies were carried out using the PFK inhibitor 2,5-anhydro-D-glucitol6-phosphate. Results: Genetic alterations of PFK family genes were observed in ~44% of MIBC cases in TCGA. The main alterations were amplification and upregulation. Patients with altered PFK gene status were more likely to have a history of noninvasive bladder cancer. Altered PFK status was not associated with survival or disease relapse. Use of the PFK inhibitor significantly decreased the level of glycolysis and inhibited the growth and invasion of bladder cancer cells. Conclusions: PFKs were critical genes in charge of glycolysis and were upregulated in bladder cancer. Targeting this pathway could inhibit cell growth in bladder cancer. Keywords: PFK, MIBC, Phosphofructokinase family, Muscle-invasive bladder cancer
Introduction Bladder cancer, also known as urothelial carcinoma of the bladder, is a common malignancy in the genitourinary system and is characterized by multicentricity, high recurrence and potent invasiveness (1, 2). The necessity of lifelong surveillance has made bladder cancer one of the most costly diseases from diagnosis to death (3, 4). Despite the variety of current therapies for bladder tumors, including radical or palliative resection, chemotherapy, and radiotherapy, there is still an incorrigible rate of recurrence (5, 6). The recurrence rate of bladder carcinomas is up to 80%, of which 16%-25% face progression in grade and 10% become invasive (7-9). Cancer adopts various types of metabolic rewiring, and the glucose metabolism is one of the most common energy Received: Accepted: December 2, 2015 Published online: Corresponding author: Xu-dong Yao Department of Urology Shanghai Tenth People’s Hospital Tongji University 200072 Shanghai, China
[email protected]
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shifts for cells to survive in the presence of hypoxia and overgrowth (10). Examples of this reprogramming in cancer include the well-established Warburg effect, in which glycolysis occurs constantly even in the presence of an affluent energy supply (10). Phosphofructokinase (PFK) is a kinase enzyme with a fundamental role in glycolysis. The enzyme-catalyzed transfer of a phosphoryl group from adenosine triphosphate (ATP) is an important reaction that occurs in many biological processes (10). PFK is one of the main enzymes that utilize this reaction: it catalyzes the phosphorylation of fructose6-phosphate to fructose-1,6-bisphosphate, a key regulatory step in the glycolytic pathway. The clinical significance of PFK can be classified into 3 groups: a) a genetic mutation in the PFKM gene results in Tarui’s disease, a glycogen storage disease where the ability of certain cell types to utilize carbohydrates as a source of energy is impaired (11); b) some viruses affect cellular metabolic pathways such as glycolysis by a titer-dependent increase in the activity of PFK (12); for instance, herpes virus increases the PFK activity by phosphorylating the enzyme at the serine residues; and c) in order for cancer cells to meet their energy requirements due to rapid cell growth and division, the PFK1 enzyme is usually hyperactivated to cater for swift energy supply (13, 14). During rapid division in the cancer context, angiogenesis is always lagging, combined with even more slow maturation of the vessels. Such drastic changes in tumor mass induce hypoxia, which in turn leads to O-GlcNAcylation
PFK promotes MIBC
e2
at serine 529 of PFK, giving cancer cells a selective growth advantage. Given the critical role of PFK in glycolysis and cancer, and the lack of reports on PFK in bladder cancer, we conducted an in silico and in vitro study in an effort to depict the role of PFK family members in bladder cancer.
Materials and methods Analysis of public dataset The Cancer Genome Atlas (TCGA) database stores genomic and clinical data of a series of common cancers including bladder cancer; all data have been made public for analysis (15). The official TCGA report on bladder cancer was released and the database was analyzed on the cBioPortal platform (http://www.cbioportal.org/) (16). We used the provisional dataset for bladder cancer, which contained 126 MIBC samples whose somatic mutations, putative copy-number alterations, RNA-seq, and protein/phosphoprotein data detected with reverse-phase protein array (RPPA) were complete (17, 18). We input genes from the PFK family (PFKL, PFKM, PFKP, PFKFB1, PFKFB2, PFKFB3, and PFKFB4) and the cBioPortal automatically calculated the mutual exclusivity, frequencies of mutation and copy number variance (CNV), correlation between mRNA expression and CNV, coexpression analysis, enrichment analysis, survival data, and network analysis.
Colony formation assay The established protocol was reported previously (20, 22, 23). Roughly 1,000 cells were resuspended in complete medium mixed with 0.4% agarose. The mixture was layered on top of the solidified mixture of complete medium and 0.6% agarose. On top of both layers was 1 mL of complete medium. After 2 weeks of culture, plates were stained with 0.005% crystal violet for 1 hour. Colony tubercles were counted. Statistical analysis In silico statistical analysis of the TCGA data was automatically performed through the cBioPortal web interface [AUTHORS: Change OK?]. All in vitro assays were run in triplicate in 3 independent experiments. Student’s t-test was used to evaluate means between 2 groups. A p value of 10
0.0209
0.609
Tendency towards co-occurrence
MST1
3p21
5.45%
0.00%
>10
0.0209
0.609
Tendency towards co-occurrence
TABLE IV - Top 5 changes in mRNA level detected by RNA-seq between PKF-altered and PKF-unaltered cases Gene
Cytoband
Mean of alteration PFK altered
SD of alteration
PFK unaltered
PFK altered
PFK unaltered
P value
Q value
0.979
MLLT10
10p12
9.49
9.22
0.54
0.43
3.040E-03
MLST8
16p13.3
9.58
9.8
0.37
0.45
4.628E-03
TRAF7
16p13.3
11.16
11.36
0.4
0.4
7.121E-03
0.979
HOXB13
17q21.2
5.73
4.45
2.66
2.74
9.677E-03
0.979
AKAP9
7q21-q22
10.41
10.13
0.5
0.69
9.913E-03
0.979
TABLE V - Top 5 changes in phosphoprotein level detected by RPPA between PKF-altered and PKF-unaltered cases Protein_residue
Mean of alteration
SD of alteration
P value
Q value
PFK altered
PFK unaltered
PFK altered
PFK unaltered
PRKCD_PS664
0.21
0.35
0.19
0.2
2.90E-04
0.0125
PRKCA_PS657
-0.01
0.28
0.46
0.56
3.52E-03
0.0756
EGFR_PY1173
1.13
1.03
0.26
0.13
0.0155
0.223
RPS6_PS235
-0.29
-0.01
0.62
0.67
0.0216
0.232
RPS6_PS240
-0.07
0.17
0.64
0.68
0.0551
0.452
metabolism in different tissues. It is thus intriguing that in our study all 3 isoforms were hyperactive in bladder cancer. We speculate that bladder cancer is in need of glycolysis to maintain its malignant potential. Also, the TCGA database included only MIBC, an aggressive subtype of bladder cancer. The increased glycolysis could contribute to its ability to evade and metastasize (25). As expected, our in vitro study showed that inhibiting PFK could combat against the cell motility of bladder cancer. We also found in the current study that patients with altered PFK gene status tended to have a history of noninvasive bladder cancer. Such a population is believed to have undergone several resections of the tumor before cystectomy. Therefore, one interpretation of the results is that PFK gene alterations could be more associated
with a progressive phenotype and less with a phenotype that is initially aggressive. PKF1 plays a key role in the glycolytic pathway as it curbs the step that is in charge of both energy release and the first irreversible glycolytic reaction (26). Such a metabolic switch mediates the precise control of the downstream elements of glycolysis. By contrast, glucose-6-phosphate can also join other elements of the glucose metabolism like the pentose phosphate pathway or glycogenesis upstream of the PFK1 switch. Activation of PFK1 activity is dependent in part on the ATP/ AMP (adenosine monophosphate) ratio, as a high ATP level inhibits PFK1. This accounts for the initiation of glycolysis in case of energy shortage. In the current study, we have used 2,5-anhydro-D-glucitol-6-phosphate as the inhibitor of PFK © 2016 Wichtig Publishing
Sun et al
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3.
4. 5. 6.
7.
Fig. 5 - Reproduced from the TCGA bladder cancer database using the cBioPortal platform. The Network function demonstrated cross talk between PFK family genes.
8. 9.
activity. The compound exhibited potent inhibition of PFK in our in vitro assays. However, clinical application of the inhibitor still faces tremendous challenges and requires accurate validation, as broad inhibition of PFK could lead to serious adverse events. Metabolic reprogramming is one of the characteristics of cancer. In particular, the major alterations in cancer cell metabolism are not focused on maximizing ATP production but on providing the ingredients needed to support macromolecular synthesis. In renal cell carcinoma, HIF1A increases the glucose uptake and controls the glucose flux through the glycolytic and pentose phosphate pathways (27). In our study, network analysis also designated HIF1A and MYC at the hub of the cross talk of PFK, both of which were key regulators between metabolism and multiple oncogenic pathways (Fig. 5). In conclusion, our findings provide evidence that PFK family genes exert important functions in bladder cancer. Because novel treatment modalities for bladder cancer are urgently needed, targeting glycolysis via PFK could be a promising approach. However, the current targeted therapies aiming at metabolic shifts in cancer face an important challenge as resistance develops quickly in response to energy depletion. Our findings may serve to suggest a supplementary approach to the standard treatment modalities.
Disclosures Financial support: None. Conflict of interest: The authors declare there is no conflict of interest.
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