A p53-inducible microRNA-34a downregulates Ras signaling by targeting IMPDH

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Abstract

p53 is a well-known transcription factor that controls cell cycle arrest and cell death in response to a wide range of stresses. Moreover, p53 regulates glucose metabolism and its mutation results in the metabolic switch to the Warburg effect found in cancer cells. Nucleotide biosynthesis is also critical for cell proliferation and the cell division cycle. Nonetheless, little is known about whether p53 regulates nucleotide biosynthesis. Here we demonstrated that p53-inducible microRNA-34a (miR-34a) repressed inosine 5′-monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo GTP biosynthesis. Treatment with anti-miR-34a inhibitor relieved the expression of IMPDH upon DNA damage. Ultimately, miR-34a-mediated inhibition of IMPDH resulted in repressed activation of the GTP-dependent Ras signaling pathway. In summary, we suggest that p53 has a novel function in regulating purine biosynthesis, aided by miR-34a-dependent IMPDH repression.

Highlights

► p53 downregulates IMPDH. ► p53-dependent miR-34a transactivation inhibits IMPDH transcription. ► miR-34a-mediated inhibition of IMPDH downregulates GTP-dependent Ras signal.

Introduction

The tumor suppressor p53 plays diverse roles in the response to cellular stress [1], [2]. Besides the well-known functions of p53 in the cell cycle, apoptosis and aging, recently described functions of p53 as metabolic regulator suggest roles in normal cellular homeostasis as well as in cancer cell homeostasis [3]. For example, p53 can modulate glycolysis, mitochondrial respiration, glutaminolysis and fatty acid oxidation [4], [5], [6], [7].

p53 has been known to transactivate the miR-34 family, which targets broad spectrum genes involved in cell cycle, apoptosis, and DNA damage [8], [9], [10], [11]. However, there is no direct evidence that p53-inducible miR-34 regulates cellular metabolism.

Meanwhile, IMPDH is a rate-limiting enzyme in the de novo synthesis of purine nucleotides [12]. Due to its role in GTP biosynthesis, IMPDH has been implicated in the oncogenic functions of various cancers. Numerous studies have demonstrated a role for IMPDH in cancer, with strong correlation between IMPDH activity and proliferating properties [13].

Until now, two isoforms of IMPDH (IMPDH1 and IMPDH2) have been identified, each consisting of 514 amino acids with 84% sequence identity [14]. Whereas IMPDH1 is constitutively expressed in normal cells, IMPDH2 is selectively up-regulated in proliferating cells, especially in leukocytes and tumor cells [15], [16]. Moreover, accumulation of IMPDH2 is reported in human ovarian cancer, leukemic cells from patients with chronic granulocytic/lymphocytic and acute myeloid leukemias [17], [18]. According to these clinical results, imbalance of purine metabolism occurs in cancer cells. For instance, up-regulation of purine metabolic enzymes like IMPDH, GMP synthase (GMPS), adenylosuccinate synthetase (ADSS), adenylosuccinase (ADSL), AMP deaminase (AMPD) and amidophosphoribosyltransferase (ATase) was identified in transplantable hepatomas in rat [19]. Interestingly, overall reduction of guanine nucleotide pools by IMPDH inhibition influences the interruption of DNA and RNA synthesis, cell cycle arrest, differentiation and cell death [20]. Therefore IMPDH is an attractive target for anticancer drugs, antiviral drugs, and immunosuppressive chemotherapy [21], [22].

Considering that p53 is associated with various metabolic pathways and abnormal metabolism is a distinct feature in tumor cells, we speculate that p53 has a regulatory function in the purine metabolic pathway. Although IMPDH has been suggested as a rate-determining factor for p53-dependent growth regulation and for p53-dependent asymmetric self-renewal [23], [24], the precise molecular mechanism for direct regulation of the purine metabolic pathway by p53 has not been fully elucidated yet. Thus, we investigated the mechanism for p53-dependent purine metabolism regulation.

In this study, we showed that p53-inducible microRNA, miR-34a, directly targets both IMPDH isoforms under DNA-damaging conditions, ultimately leading to reductions in the GTP-dependent Ras signaling pathway.

Section snippets

Cells

HEK293, H1299 and HCT116 cells were obtained from ATCC. H1299/RasG12V stable cells were selected using G418 (1 mg/ml) in H1299 cells transiently transfected with pcDNA3-RasG12V plasmid. Cells were cultured in DMEM containing 10% (v/v) fetal bovine serum and 50U /ml of streptomycin and penicillin.

Plasmids and antibodies

The expression vector for full-length IMPDH2 was generated by inserting PCR fragments into pCMV-Tag2B-Flag. The antibodies used in this study were as follows: anti-Flag (M2) and anti-β-actin monoclonal

p53 downregulates IMPDH

To examine the role of p53 in purine metabolism, we first measured the mRNA level of genes involved in the purine metabolic pathway (Fig. 1A). With adenoviral expression of p53 in p53-null H1299 cells, successful p53-mediated transactivation was confirmed by increasing amounts of mRNA of well-known p53 target genes (p21 and TIGAR) (Fig. 1B) [4], [31]. Interestingly, mRNA levels of IMPDH1 and IMPDH2 specifically decreased with p53 overexpression, compared to other metabolic enzymes in the purine

Discussion

The altered metabolism in cancer provides clues as to how a normal cell can acquire pathogenesis and progress to cancer cells. For example, altered glucose metabolism in cancer cells contributes to tumorigenesis by supporting rapid proliferation [38]. Interestingly, the latest cancer research has focused on unveiling novel metabolic functions of classical tumor suppressors such as p53 [3]. From this point of view, metabolic change can occur in a cancer cell by uncontrolled p53 regulation. When

Acknowledgments

We thank Dr. VN Kim (Seoul National University) for invaluable materials. This work was supported by grants from the Korea Healthcare Technology R&D Project (A090281) to H.D.Y.

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