Background
One of the most important hallmarks of cancer is aerobic glycolysis or the so-called Warburg effect. The Warburg effect was first described by Warburg over 90 years ago and states that cancer cells heavily rely on glycolysis for energy metabolism even under normal oxygen concentrations [
1]. Consequently, unlike most normal cells, cancer cells derive a substantial amount of their energy from aerobic glycolysis, converting most incoming glucose to lactate rather than metabolizing it in the mitochondria through oxidative phosphorylation [
2,
3]. Although the Warburg effect has been documented in many cancers, the underlying mechanisms driving and regulating aerobic glycolysis are not fully understood [
4]. Because cancer cells adapt in various ways that distinguish cancer cells from normal cells, this is a need to know how and why cancer cells adapt to the aerobic glycolysis, which is faster in total glucose utilization but is more wasteful for energy supply. It is well known that cancer-specific metabolism is largely responsible for the growth advantage of cancer cells. Thus, uncovering the mechanisms underlying aerobic glycolysis in cancer cells could be helpful for the development of new therapeutic targets of human cancers [
5].
The canonical Wnt signaling pathway plays a central role in normal development and tumorigenesis [
6,
7]. The canonical Wnt pathway involves activation of the key effector molecule, β-catenin, that functions as part of a bipartite transcription factor that activates WNT-target genes by interacting with the LEF1/TCF family of transcription factors. In the absence of Wnt stimulation, β-catenin is anchored by the Axin-APC complex, subsequently phosphorylated by casein kinase Iα (CKIα) and glycogen synthase kinase-3β (GSK3β), and then targeted for ubiquitin-mediated proteasomal degradation [
8]. Upon the stimulation by Wnt ligands, the Axin-APC destruction complex is inactivated through the recruitment of the intracellular signaling protein, disheveled (DVL), which prevents β-catenin degradation and allows nuclear translocation. In turn, the β-catenin–LEF1/TCF complex regulates the expression of downstream target genes involved in diverse cellular processes [
9,
10].
Chibby was identified as β-catenin antagonist in a protein-protein interaction screen using the bait of C-terminal region of β-catenin in 2003 [
11]. Chibby physically interacts with the C-terminal activation domain of β-catenin and represses β-catenin–mediated transcriptional activation by competing with Tcf/Lef factors for β-catenin binding [
11,
12]. Moreover, Chibby facilitates β-catenin export from the nucleus in conjunction with the proteins 14–3-3 and the nuclear export receptor chromosomal region maintenance 1 (CRM1) [
13,
14]. The regulatory effect of Chibby on the Wnt/β-catenin signaling pathway suggests the biological importance of Chibby as a potential tumor suppressor [
11]. Several studies have shown that the expression of Chibby was down-regulated only in thyroid cancer, pediatric ependymomas and colon carcinoma cell lines [
15‐
17]. Our previous study also indicated that the expression of Chibby is decreased in Laryngeal Squamous Cell Carcinoma (LSCC) [
18]. However, the biological function of Chibby in NPC and the underlying molecular mechanism has not yet been defined. In the present study, we have demonstrated that Chibby suppresses aerobic glycolysis and the proliferation of nasopharyngeal carcinoma and that the Wnt/β-catenin-Lin28/let7-PDK1 cascade mediates this activity. Our study reveals an association between Chibby expression and aerobic glycolysis in cancer, which highlights the importance of the Wnt/β-catenin pathway in regulating energy metabolism in nasopharyngeal carcinoma.
Methods
Patient tissue samples
Clinical Chibby, β-Catenin, PDK1 protein levels were detected from primary human nasopharyngeal cancer or normal tissue. All samples were obtained from the First Affiliated Hospital of Xiamen University with patient consent and institutional review board approval. These samples were subsequently de-identified to protect patient confidentiality.
Animals
Four-week-old female BALB/c nude mice were used. A total of 4 × 106 cells were injected subcutaneously into the dorsal thighs of mice. Tumor growth was monitored regularly for 6 weeks, then the tumor volume was calculated every week. All mice were kept under specific pathogen-free conditions at Xiamen University Laboratory Animal Center (Xiamen University, China) in accordance with institutional guidelines. This study was approved by the local Ethical Committee of Xiamen University.
Statistical analysis
Date were analyzed using GraphPad Prism software. Data are presented as the means ± standard error. The Student’s t-test (two-tailed), Fisher’s exact test, and Pearson’s r were used to compare data and to calculate their probability value (p). p < 0.05 was considered statistically significant.
Other procedures
Protocols for other procedures are described in the Additional file
1.
Discussion
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors and is reported as an endemic disease with high prevalence in Southeast Asia, particularly in South China [
22,
23]. The etiology and pathogenesis of NPC have not yet been completely defined. Emerging studies have suggested that environmental factors, genetic susceptibility, and Epstein-Barr virus may play crucial roles in its carcinogenesis. Although the 5-year survival rate of NPC has been greatly improved through comprehensive treatments such as radiotherapy and chemotherapy [
24], long-term prognosis remains unsatisfactory. The approaches that change or modify some important genes or their expression have become a research hotspot in the biological treatment of NPC. Therefore, there is an urgent need to further explore the molecular mechanism during carcinogenesis of NPC. Many signaling pathways have been reported to be involved in this process. However, there is very little knowledge regarding Wnt/β-catenin signaling cascade genes in NPC [
25]. Numerous studies have revealed the role of Wnt/β-catenin signaling in the carcinogenesis of many cancers; however, the regulation of this signaling process during carcinogenesis has not been completely defined. Moreover, since somatic mutations of Wnt/β-catenin signaling components are rare in NPC, regulators of Wnt/β-catenin signaling components primarily control the Wnt/β-catenin output level. Accumulating evidence has demonstrated that the inhibition of Wnt/β-catenin by ZNRF3 [
26], YPEL3 [
27], SFRP1 [
28], Wnt-C59 [
29], SOX1 [
30] and WIF-1 [
31] in NPC cells was significantly compromised, resulting in elevated Wnt/β-catenin output levels. Chibby is an interaction partner and negative regulator of β-catenin; however, its role in NPC has not been elucidated. To the best of our knowledge, this report is the first to link Chibby to NPC.
Wnt/β-catenin signaling has been implicated in the mediation of cancer cell metabolism via multiple mechanisms [
32]. Specifically, it was reported that PDK1 served as a direct downstream target gene of Wnt/β-catenin signaling in colon cancer cells and mediated aerobic glycolysis [
33]. And PDK1 would down-regulate pyruvate dehydrogenase (PDH) to shutting down pyruvate entry into the tricarboxylic acid cycle (TCA) [
34]. However, in the present study we did not observe changes in PDK1 mRNA levels upon Wnt/β-catenin activation in NPC cells. Instead, PDK1 was post-transcriptionally regulated by Wnt/β-catenin signaling via the Lin28-Let-7 pathway in NPC cells, which reflects the tissue specificity and cancer-type dependence. Moreover, we noticed that, compared to PDK1 mRNA levels, blocking Wnt/β-catenin activity in colon cancer cells resulted in a further reduction in PDK1 protein levels [
33], which suggests that post-transcriptional regulation of PDK1 by Wnt/β-catenin signaling at least partially contributes to the metabolism of colon cancer cells. Previous studies also identified other mechanisms downstream of Wnt/β-catenin signaling to regulate aerobic glycolysis. For example, the well-known Wnt/β-catenin target gene c-Myc plays an important role in cancer metabolism, driving both aerobic glycolysis and glutaminolysis [
35‐
37]. Moreover, c-Myc has been shown to enhance HIF-1a-mediated regulation of PDK1 [
38]. However, we found that c-Myc is not required in our system to mediate changes in metabolism as its levels are not altered in NPC cells upon Wnt/β-catenin activation (data not shown), which also suggests a context dependence.
Given the strong evidence for regulation of PDK1 protein expression by Chibby through Wnt/β-catenin signaling, we asked whether Chibby, Wnt/β-catenin signaling and PDK1 were correlated in primary human NPC specimens. We used 45 pairs of fresh NPC samples with normal tissues to detect the expression of Chibby, nuclear β-catenin, and PDK1 by immunostaining. Indeed, we observed a strong inverse correlation between Chibby and nuclear β-catenin or PDK1 levels and a strong correlation between nuclear β-catenin and PDK1, which supports the results observed in in vitro experiments.
Conclusions
Taken together, our findings identified Chibby as a negative regulator of proliferation that suppresses NPC aerobic glycolysis via the inhibition of Wnt/β-catenin signaling in vitro and in vivo. The modulation of this molecular process may be a method of inhibiting NPC cell growth by restoring Chibby expression to interfere with cell metabolism. In particular, the PDK1 may become a new target for further inhibitor design to interfere with Wnt/β-catenin dependent NPC progression.
Acknowledgments
We would like to thank Dr. Yuan-ji Xu for providing series cell lines of NPC; Prof. Bo-an Li for providing the series plasmids of β-catenin.
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