SAMHD1 is down regulated in lung cancer by methylation and inhibits tumor cell proliferation

doi:10.1016/j.bbrc.2014.10.153

Biochemical and Biophysical Research Communications

Volume 455, Issues 3–4, 12 December 2014, Pages 229-233

https://doi.org/10.1016/j.bbrc.2014.10.153 Get rights and content

Highlights

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SAMHD1 expression level is down regulated in lung adenocarcinoma.
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The promoter of SAMHD1 is methylated in lung adenocarcinoma.
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Over expression of SAMHD1 inhibits the proliferation of lung cancer cells.

Abstract

The function of dNTP hydrolase SAMHD1 as a viral restriction factor to inhibit the replication of several viruses in human immune cells was well established. However, its regulation and function in lung cancer have been elusive. Here, we report that SAMHD1 is down regulated both on protein and mRNA levels in lung adenocarcinoma compared to adjacent normal tissue. We also found that SAMHD1 promoter is highly methylated in lung adenocarcinoma, which may inhibit its gene expression. Furthermore, over expression of the SAMHD1 reduces dNTP level and inhibits the proliferation of lung tumor cells. These results reveal the regulation and function of SAMHD1 in lung cancer, which is important for the proliferation of lung tumor cells.

Introduction

SAMHD1. (sterile alpha motif and histidine-aspartic domain containing protein 1) is a deoxynucleoside triphosphate phosphohydrolase which cleaves deoxynucleoside triphosphates (dNTP) into deoxynucleosides (dN) and inorganic triphosphates, thus depleting the cellular dNTP pool required for cellular DNA polymerase [1], [2]. It can efficiently prevent HIV-1 replication by degrading the dNTP pool, thus, inhibits the early steps of reverse transcription. Blockade at this step prevents the synthesis of full-length double-strand DNA and disrupts later stages of the viral life cycle, including nuclear translocation and integration of proviral DNA [3], [4], [5]. This function of SAMHD1 has been recognized predominantly in non-cycling cell types including monocytes [6], macrophages [7], dendritic cells [8], [9] and resting CD4+ T cells [10].

The function of SAMHD1 in human immune cells was well established. It can be regulated by a number of factors, including phosphorylation state, the production of type 1 IFN and combination of IL-12 and IL-18. Recently, three studies demonstrate that the antiretroviral activity of SAMHD1 is regulated by phosphorylation in a cell-cycle-dependent manner [11], [12], [13]. IFN-α can up regulate SAMHD1 expression in primary monocytes [6] and a combination of IL-12 and IL-18 can lead to an increase in SAMHD1 expression in monocyte-derived macrophage (MDM), resulting in the generation of cells that are more resistant to HIV-1 infection [14]. However, its regulation and function in cancer have been elusive. Lately, one group found that SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and the acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed and refractory CLL patients [15], indicating it may play a role in tumorigenesis and development.

In this study, we studied the regulation and function of SAMHD1 in lung cancer and found that SAMHD1 is down regulated both on protein and mRNA levels in lung adenocarcinoma compared to adjacent normal tissue, which may be regulated by methylation on its promoter. Furthermore, over expression of the SAMHD1 reduces dNTP level and inhibits the proliferation of lung tumor cells. These results reveal the regulation and function of SAMHD1 in lung cancer, which is important for the proliferation of lung tumor cells.

Section snippets

Cell lysis and immunological procedures

Cells were lysed in an NP40 buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM Na₃VO₄ and 1 mM PMSF. Western blot analysis was performed according to standard methods. Antibodies specific to SAMHD1 (Abcam) and GAPDH (Santa Cruz) were purchased commercially.

Preparation and analysis of lung adenocarcinoma

Lung adenocarcinoma samples were acquired from Huadong Hospital affiliated to Fudan University. An informed consent was obtained from the patients. The procedures

SAMHD1 expression level is down regulated in lung adenocarcinoma

SAMHD1 is the first identified mammalian dNTP triphosphohydrolase, its regulation and function in cancer have been unclear. A recent paper reported that acquired SAMHD1 mutations are associated with reduced SAMHD1 expression and occur in 11% of relapsed and refractory chronic lymphocytic leukemia patients compared to a mutation frequency of 3% in the pretreatment group, suggesting a potential role of SAMHD1 in tumor. So we compared SAMHD1 protein level between five pairs of lung adenocarcinoma

Conflict of interest

The authors declare that they have no conflict of interest.

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Whole-exome sequencing in 415,422 individuals identifies rare variants associated with mitochondrial DNA copy number
2023, Human Genetics and Genomics Advances
Inter-individual variation in the number of copies of the mitochondrial genome, called mitochondrial DNA copy number (mtDNA-CN), reflects mitochondrial function and has been associated with various aging-related diseases. We examined 415,422 exomes of self-reported White ancestry individuals from the UK Biobank and tested the impact of rare variants, at the level of single variants and through aggregate variant-set tests, on mtDNA-CN. A survey across nine variant sets tested enrichment of putatively causal variants and identified 14 genes at experiment-wide significance and three genes at marginal significance. These included associations at known mtDNA depletion syndrome genes (mtDNA helicase TWNK, p = 1.1 × 10⁻³⁰; mitochondrial transcription factor TFAM, p = 4.3 × 10⁻¹⁵; mtDNA maintenance exonuclease MGME1, p = 2.0 × 10⁻⁶) and the V617F dominant gain-of-function mutation in the tyrosine kinase JAK2 (p = 2.7 × 10⁻¹⁷), associated with myeloproliferative disease. Novel genes included the ATP-dependent protease CLPX (p = 8.4 × 10⁻⁹), involved in mitochondrial proteome quality, and the mitochondrial adenylate kinase AK2 (p = 4.7 × 10⁻⁸), involved in hematopoiesis. The most significant association was a missense variant in SAMHD1 (p = 4.2 × 10⁻²⁸), found on a rare, 1.2-Mb shared ancestral haplotype on chromosome 20. SAMHD1 encodes a cytoplasmic host restriction factor involved in viral defense response and the mitochondrial nucleotide salvage pathway, and is associated with Aicardi-Goutières syndrome 5, a childhood encephalopathy and chronic inflammatory response disorder. Rare variants were enriched in Mendelian mtDNA depletion syndrome loci, and these variants implicated core processes in mtDNA replication, nucleoid structure formation, and maintenance. These data indicate that strong-effect mutations from the nuclear genome contribute to the genetic architecture of mtDNA-CN.
Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins
2021, Journal of Biological Chemistry
Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine–aspartate domain–containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.
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¹: These authors should be regarded as joint first authors.

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SAMHD1 is down regulated in lung cancer by methylation and inhibits tumor cell proliferation

Highlights

Abstract

Introduction

Section snippets

Cell lysis and immunological procedures

Preparation and analysis of lung adenocarcinoma

SAMHD1 expression level is down regulated in lung adenocarcinoma

Conflict of interest

J. Biol. Chem.

Cell Rep.

Cell Host Microbe

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Mutat. Res.

HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase

Nature

SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells

Nat. Med.

Evidence for IFNalpha-induced, SAMHD1-independent inhibitors of early HIV-1 infection

Retrovirology