Cancer Letters

Cancer Letters

Volume 522, 1 December 2021, Pages 105-118
Cancer Letters

AKT1/FOXP3 axis-mediated expression of CerS6 promotes p53 mutant pancreatic tumorigenesis

https://doi.org/10.1016/j.canlet.2021.06.024Get rights and content

Highlights

  • AKT phosphorylates FoxP3 at S418, which impairs FoxP3 transcriptional activity.

  • Phosphorylation of FoxP3 at S418 alleviates its ability of suppressing CerS6 expression.

  • CerS6 produces C16-ceramide, which binds and stabilizes WT or mutant p53, thus, presents opposite effect on tumor cells.

Abstract

Ceramide synthases (CerSs) catalyze the formation of ceramides from sphingoid bases and acyl-CoA substrates. Increasing evidence suggests that cancer cells generally exhibit altered sphingolipid metabolism in the tumorigenesis of multiple cancers. However, there is no evidence that CerSs are associated with pancreatic ductal carcinoma (PDAC). In the present study, we examined CerS expression in clinical tissue and conducted data mining to investigate the clinical significance of CerSs in the TCGA-PAAD database. We found that high CerS6 expression positively correlated with progression and predicted worse prognosis in PDAC patients, establishing CerS6 as a potential biomarker for PDAC. Furthermore, CerS6 promoted cell proliferation, colony formation and invasion by producing C16-ceramide and was required for tumor formation. Mechanistically, AKT1 interacted with and phosphorylated FOXP3 at S418, which decreased the binding of FOXP3 to the CERS6 promoter and in turn induced CerS6 expression by reconstituting an activated state on the CERS6 promoter. The AKT1/FOXP3 axis mediated the CerS6 expression and promoted p53 mutant pancreatic tumorigenesis by producing excessive C16-ceramide, which induced the accumulation of mutant p53. Thus, our study explores the relationship between PI3K/AKT signaling and sphingolipid metabolism, revealing an oncogenic role for CerS6, which may represent a potential target for PDAC treatment.

Introduction

Sphingolipid metabolism has emerged as an important mediator regulating cell growth, death, survival and many other aspects of biology [1,2]. Ceramide synthases, a group of enzymes that catalyzes the formation of ceramides from sphingolipid bases and acyl-CoA substrates, occupy the center of sphingolipid metabolism. By controlling acyl chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology [3,4]. At present, six mammalian CerSs (CerS1–CerS6) have been identified in mammalian cells [5]. Because there are currently no reports on the function and mechanism of CerSs in pancreatic ductal carcinoma (PDAC), we attempted to identify whether CerSs exert their activities during pancreatic tumorigenesis. Through clinical data mining and functional experiments, we ascertained that CerS6 plays an aggressive role in mutant p53 PDAC. Previous reports proved that CerS6 exerts an antitumor function by inducing the accumulation of wild-type (WT) p53 because the product of CerS6, C16-ceramide, disrupts the p53-MDM2 complex and stabilizes p53 [6,7]. However, p53 is frequently mutated or silenced in PDAC patients, and mutant p53 conveys poor prognosis to pancreatectomy [8], drives PDAC metastasis [9,10] and even alters RNA splicing to activate RAS signaling in pancreatic cancer [11]. Thus, it is significant to explore the role of CerS6 in p53-mutant PDAC.

CerS6 shows distinct functions in different cancer cell contexts. First, CerS6 promotes either the survival or death of cancer cells in stressful environments. For example, CerS6 confers resistance to BCL-2 inhibition in T-cell acute lymphoblastic leukemia by binding to CD95/Fas [12] or sensitivity to cisplatin in oral squamous carcinoma by altering mitochondrial fission and autophagy [13]. Second, CerS6 drives cancer malignancy by mediating signal transduction. For instance, CerS6 functions as an oncoprotein and dysregulates the SOCS2/JAK2/STAT3 pathway in human gastric cancer [14]. In addition, CerS6 mRNA is stabilized by the long noncoding RNA CerS6-AS1/IGF2BP3 axis and promotes breast cancer malignancy [15]. Despite these findings, there are still few reports describing the suppressive role of CerS6 with respect to its induction of apoptosis in breast cancer and head and neck squamous cancer [16,17].

CerS6 exhibits different functions under conditions such as cancer or obesity through its metabolite C16-ceramide [18]. C16-ceramide, generated by CerS6, has been implicated in cancer cell proliferation, whereas CerS1-generated C18-ceramide mediates cell death [19]. Obesity induces the accumulation of C16-ceramide produced by CerS6, promoting weight gain and glucose intolerance [20]. CerS6 promotes allogeneic T cell responses through C16-ceramide's effect on T cell proliferation and cytokine expression [21]. C16-ceramide directly interacts with mitochondrial fission factor (Mff) and promotes mitochondrial fragmentation in obesity [22]. Hence, the role of C16:0 ceramide in cancers and obesity in prompting CerS6 inhibition may represent a novel therapeutic approach [18].

Although CerS6 has demonstrated important functions from multiple biological aspects, the expression mechanism of CerS6 has seldom been examined. In this study, we screened signaling pathways required for CerS6 expression and observed that the PI3K/AKT axis greatly contributes to maintaining CerS6 mRNA levels. AKT, also known as protein kinase B (PKB), plays a central role in many cancer types and was validated as a therapeutic target nearly two decades ago [23]. Targeting AKT is viewed as a promising strategy to inhibit tumor growth [24,25]. To date, no AKT inhibitors have been clinically applied for cancer treatment due to their severe side effects and low efficacy [26,27]. Hence, identifying key substrates of AKT in PDAC represents an alternative and better strategy for identifying therapeutics.

In this study, we discovered a novel AKT1/FOXP3 axis that is involved in upregulating CerS6 expression. In detail, AKT1 phosphorylates FOXP3 at S418 and attenuates FOXP3 DNA binding activity, which results in the upregulation of CerS6 by reconstituting an activated state on its genetic promoter. The function of FOXP3 has been primarily characterized with respect to T cell regulation. FOXP3 is a transcription factor that is both necessary and sufficient for the induction of the immunosuppressive functions of regulatory T cells [[28], [29], [30]], and its expression was first considered specific for this cell type. However, abundant FOXP3 has also been detected in cancer cells [31]. The molecular mechanism by which FOXP3 controls gene expression in regulatory T cells involves interactions of FOXP3 with other chromatin proteins and regulates the repressive or active state of targeted gene promoters. For example, FOXP3 interacts with nuclear factor of activated T (NFAT), preventing its binding to AP-1, and then, FOXP3/NFATs act together to activate CTLA4 and IL2RA expression [32]. Binding of FOXP3 to the IL2 and IFNγ gene promoters induces the deacetylation of histone H3, a process that inhibits chromatin remodeling and hampers gene transcription [33]. In contrast, binding of FOXP3 to the CTLA4 and IL2RA gene promoters increases histone acetylation [34]. In this study, attenuation of FOXP3 through the CERS6 gene promoter resulted in increased CerS6 expression.

Enhanced CERS6 expression promoted the accumulation of its product, and we accordingly observed that supplementation with cell-permeable C16-ceramide also rescued the phenotypes of p53 mutant PDAC cells lacking CerS6, indicating that C16-ceramide may represent a potential target in PDAC patients expressing mutant p53.

Section snippets

Patient specimens

Tumors and paired normal pancreatic tissues were obtained from patients with PDAC who underwent surgery at Shanghai Fourth People's Hospital. This study protocol was approved by the ethics committee of Shanghai Fourth People's Hospital. Written informed consent was obtained from all participants in this study. All research was performed in accordance with the provisions of the Declaration of Helsinki of 1975.

Antibodies and reagents

Rabbit polyclonal or monoclonal antibodies against CerS6 (PA5-20648, 1:200 for IHC,

CerS6 expression is associated with PDAC diagnosis and prognosis

To ensure the significance of our study, we first assessed the mRNA expression of CerS1-6 genes in 12 paired pancreatic normal and tumor tissues. As shown in Fig. 1A, CerS1-2 expression displayed no significant differences between normal and tumor tissues, while CERS3-5 gene expression was higher in normal tissues than in tumor tissues. Only CERS6 was highly expressed in pancreatic tumor tissues (Fig. 1A). Using IB to analyze the protein levels of CerS6 in the 12 patient samples, we obtained

Discussion

To clarify the role of CerSs in pancreatic tumorigenesis, we examined CerS expression in clinical samples and revealed the clinical significance of CerS6. Next, we performed functional experiments and cell proliferation, colony formation, invasion and xenograft studies to confirm the oncogenic role of CerS6 by producing ceramide C16 in PDAC cells. Furthermore, we explored the mechanism by which CerS6 is upregulated by the AKT/FOXP3 axis. Thus, our study expands the relationship between PI3K/AKT

Conclusions

We conducted TCGA-PAAD data mining and found that high CerS6 expression predicted worse prognosis of PDAC patients. In vitro and in vivo experiments further explored the oncogenic role of CerS6. Mechanistically, AKT1 phosphorylates FOXP3 at S418 and suppresses the repressive effect on the CERS6 promoter; hence, AKT signaling drives CerS6 expression and enhances the accumulation of C16 ceramide, which induces mutant p53 stabilization in PDAC. Thus, our study explores the relationship between AKT

Author contribution statement

QD, ZZ and YH designed the experiments of this study. SX and YH performed most of the experiments in this study. XC, YH and CC conducted the experiments, data analysis, and critical discussions of the results. QD and YH performed the investigation and organized methodology. YW and SP collected clinical samples and recorded, organized the related information. QD and ZZ wrote and edited of the manuscript and approved the final draft of the manuscript.

Declaration of competing interest

The authors declare no competing interests.

Acknowledgments

This work was supported by Shanghai (China) Fourth People's Hospital Research Startup Fund.

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