hNaa10p contributes to tumorigenesis by facilitating DNMT1-mediated tumor suppressor gene silencing

Abstract

Hypermethylation-mediated tumor suppressor gene silencing plays a crucial role in tumorigenesis. Understanding its underlying mechanism is essential for cancer treatment. Previous studies on human N-alpha-acetyltransferase 10, NatA catalytic subunit (hNaa10p; also known as human arrest-defective 1 [hARD1]), have generated conflicting results with regard to its role in tumorigenesis. Here we provide multiple lines of evidence indicating that it is oncogenic. We have shown that hNaa10p overexpression correlated with poor survival of human lung cancer patients. In vitro, enforced expression of hNaa10p was sufficient to cause cellular transformation, and siRNA-mediated depletion of hNaa10p impaired cancer cell proliferation in colony assays and xenograft studies. The oncogenic potential of hNaa10p depended on its interaction with DNA methyltransferase 1 (DNMT1). Mechanistically, hNaa10p positively regulated DNMT1 enzymatic activity by facilitating its binding to DNA in vitro and its recruitment to promoters of tumor suppressor genes, such as E-cadherin, in vivo. Consistent with this, interaction between hNaa10p and DNMT1 was required for E-cadherin silencing through promoter CpG methylation, and E-cadherin repression contributed to the oncogenic effects of hNaa10p. Together, our data not only establish hNaa10p as an oncoprotein, but also reveal that it contributes to oncogenesis through modulation of DNMT1 function.

Figure 1. hNaa10p overexpression in human lung cancer tissues.

(A) Relative mRNA level of hNAA10 in lung cancerous specimens or surrounding non-neoplastic stroma tissues after normalization to actin. Data are mean ± SD from 3 independent assays. (B) IHC detection of hNaa10p protein in normal and cancerous human lung tissues. Shown are 1 example of normal adjacent tissue and 3 examples of tumor sections. Original magnification, ×100 (left panels); ×200 (right panels). (C) IHC analysis of 90 lung adenocarcinoma patients indicated that 48 patients with high hNaa10p protein level in tumor tissues showed poor survival compared with 42 patients with hNaa10p expression below the detection limit, as in normal adjacent tissues. P = 0.0089.

Figure 2. hNaa10p contributes to clonogenesis and xenograft tumor formation in lung cancer cells.

(A) hNaa10p overexpression enhances the colony formation ability of NIH3T3 cells on soft agar. hNaa10p-V5, hNaa10p with C-terminal V5 tag (containing 14 amino acids, GKPIPNPLLGLDST, derived from the P and V proteins of the paramyxovirus of simian virus 5). Representative microscopic images of the colonies are shown. Scale bars: 100 μm. (B) Western blots showing successful knockdown of hNaa10p protein level by si-hNAA10-1 and si-hNAA10-2. si-scramble and si-Tax were used as negative controls. (C) Lung cancer H1299 cells transfected with si-hNAA10-1 exhibited poor proliferation rate compared with cells mock transfected or transfected with si-Tax. (D) Transfection with si-hNAA10-1 or si-hNAA10-2 impaired the ability of H1299 cells to form colonies on soft agar, compared with cells mock transfected or transfected with si-scramble. Photographs of representative soft agar plates are shown. (E) H1299 cells transfected with si-hNAA10-1 or si-hNAA10-2 generated smaller tumors in NOD-SCID mice than did cells mock transfected or transfected with si-scramble. The volume of the derived tumors was measured at the indicated days after injection. Representative tumor masses are shown. A total of 5–6 mice was analyzed in each group. Data in A, C, D, and E are mean ± SD from 3 independent assays.

Figure 3. hNaa10p associates with DNMT1 both in vivo and in vitro.

(A) Co-IP showed that anti-hNaa10p pulled down hNaa10p and DNMT1, but not DNMT3a or DNMT3b, from H1299 cell exacts. (B) Co-IP showed that anti-hNaa10p pulled down hNaa10p and all DNMT1 fragments (V5-tagged) containing aa 291–570 exogenously expressed in H1299 cells, as revealed by Western blot using hNaa10p or V5 Ab. The schematic illustration of DNMT1 fragments used here, and the results of interaction, are summarized above. DMAP, DNMT1-associated protein; PCNA, proliferating cell nuclear antigen; NLS, nuclear localization signal; FTR, replication foci targeting sequence. (C and D) GST pulldown assays showed that the recombinant GST-fused DNMT1 fragments containing aa 291–570 precipitated hNaa10p-V5 in vitro translated from rabbit reticular lysate (C) or His-Xpress-hNaa10p expressed in and purified from E. coli (D), as revealed by Western blot using the respective Abs. In D, lanes 2–4 and 5–7 show results using NP40 and PBS-T, respectively, as incubation/wash buffer. (E) GST-DNMT1 aa 291–570 was used to pull down the in vitro translated V5-tagged hNaa10p mutant lacking aa 102–122 or aa 182–201, or GFP-fused hNaa10p fragments containing aa 1–21, aa 62–81, aa 102–122, or aa 182–201, followed by Western blotting with V5 or GFP Ab. Lanes were run on the same gel but were noncontiguous (white lines).

Figure 4. The oncogenic potential of hNaa10p depends on its binding to DNMT1.

(A and B) si-hNAA10-1–treated H1299 cells expressing the resistant V5-tagged WT or mutant hNaa10p lacking aa 182–201 or aa 102–122 were subjected to soft agar assay and Western blot (A) as well as cell proliferation study (B). Data are mean ± SD from 3 independent assays. Lanes in A were run on the same gel but were noncontiguous (white lines).

Figure 5. hNaa10p maintains and stimulates DNMT1 activity by increasing DNMT1 binding to DNA.

(A) H1299 cells transfected with si-hNAA10-1 or si-DNMT1 or treated with 5'-AzadC had reduced DNMT activity compared with cells mock transfected or transfected with si-Tax. Western blot shows expression of corresponding proteins. (B) Overexpressing WT hNaa10p, acetylase-dead mutant hNaa10p R82A, or DNMT1-V5 increased DNMT activity of 293T cells. (C) hNaa10p stimulated DNMT1 activity independently of acetyl-CoA. Recombinantly purified DNMT1 and His-hNaa10p, alone or in combination, were mixed with or without acetyl-CoA, followed by DNMT assay. (D) hNaa10p facilitated DNMT1 binding to DNA in vitro. EMSA assays were applied with the biotin-labeled hemimethylated DNA probe alone or with the recombinantly purified DNMT1 in the absence or presence of increasing amounts of E. coli–expressed and purified His-hNaa10p. (E) ChIP assay showed that enforced expression of vector or hNaa10p in H1299 cells enhanced DNMT1 binding to promoter of E-cadherin, but not p21. (F) ChIP showed that depleting hNaa10p from H1299 cells by si-hNAA10-2 abolished DNMT1 binding to the E-cadherin promoter, whereas DNMT1 loss of function did not impair hNaa10p binding to the same promoter. Lanes in A and B were run on the same gel but were noncontiguous (white lines).

Figure 6. hNaa10p and E-cadherin promoter methylation.

(A) The sequence of the proximal promoter region –202 to +31 bp of the E-cadherin gene containing 17 CpGs (shown by bold and underline). (B and C) Bisulfite sequencing analysis. After bisulfite treatment, 10 independent clones of the indicated groups were sequenced. Black circles denote methylated cytosine in specific CpG; white circles denote nonmethylated cytosine in specific CpG. (B) Depleting hNaa10p or DNMT1 from H1299 cells reduced E-cadherin promoter methylation. (C) Overexpression of siRNA-resistant hNaa10p restored E-cadherin promoter methylation.

Figure 7. DNMT1-dependent repression of E-cadherin by hNaa10p contributes to hNaa10p oncogenic ability.

(A) hNaa10p inhibition of E-cadherin promoter activity was dependent on DNMT1. H1299 cells with lentivirus infection of si-scramble or si-DNMT1 were transfected with the luciferase reporter driven by the E-cadherin promoter, together with vector or hNaa10p-V5 expression plasmid, followed by luciferase assay. (B) E-cadherin promoter activity was only inhibited by transfected WT hNaa10p or hNaa10p mutant lacking aa 182–201, not by the mutant lacking DNMT1 binding region (aa 102 to 122). (C and D) Depletion of hNaa10p from H1299 cells increased mRNA (C) and protein (D) levels of E-cadherin gene. Quantified E-cadherin protein levels (relative to β-tubulin) are shown below the lane numbers. (E) Western blot showed that hNaa10p lacking DNMT1 interaction domain (aa 102–122) failed to repress E-cadherin protein level. (F) Knocking down E-cadherin partly rescued the colony formation ability inhibited by depleting hNaa10p. The indicated siRNAs were cotransfected into H1299 cells, followed by colony formation assay. All data in A, B, C, and F are mean ± SD from 3 independent experiments. *P < 0.05.