Immunohistochemical determination of the miR-1290 target arylamine N-acetyltransferase 1 (NAT1) as a prognostic biomarker in breast cancer

Abstract

Background: There are many molecular differences between estrogen receptor α (ERα)-positive and ER-negative breast cancers. Recent analyses have shown that the former can be divided into two subtypes, luminal A and luminal B. These differ in response to endocrine therapy and chemotherapy, and in prognosis. In a previous study, we found that microRNA (miR)-1290 that was significantly down-regulated in luminal A tumors and its potential target arylamine N-acetyltransferase 1 (NAT1). The aim of the present study was to determine whether NAT1 is a bona fide target of miR-1290, and to investigate the impact of NAT1 on breast cancer prognosis.

Methods: Luciferase reporter assays were employed to validate NAT1 as a putative miR-1290 target gene. Expression of NAT1, ERα, progesterone receptor (PgR) and HER2 was analyzed in 394 breast cancer samples by immunohistochemistry.

Results: NAT1 was confirmed to be a direct target of miR-1290. Levels of expression of NAT1 were positively correlated with those of ERα (P < 0.0001) and PgR (P < 0.0001), but negatively correlated with both tumor grade and size (P < 0.0001). Kaplan-Meier analysis showed that the presence of NAT1 was significantly associated with increased overall survival (OS) (P = 0.0416) in these patients. Similarly, significant associations of NAT1 with disease-free survival (DFS) (P = 0.0048) and OS (P = 0.0055) in those patients who received adjuvant endocrine therapy with tamoxifen (n = 176) were found. Moreover, NAT1 was also significantly associated with increased DFS (P = 0.0025) and OS (P = 0.0007) in the subset of lymph node-positive patients (n = 147). Univariate and multivariate analyses showed significant associations between levels of NAT1 and DFS (P = 0.0005 and 0.019, respectively).

Conclusions: We report that miR-1290 directly targets the NAT1 3'-UTR and that NAT1 protein expression is correlated with improved OS of breast cancer patients. NAT1 is a possible prognostic biomarker for lymph node-positive breast cancer. Thus, miR-1290 and its target NAT1 are associated with important characteristics of breast cancer.

Figure 1

Identification of miR-1290 target sites in the NAT1 3′-UTR. A, Schematic of the 3′-UTR-containing reporter constructs for potential miR-1290 target sites in NAT1. The 3′-UTR of the NAT1 gene was inserted just downstream of the firefly luciferase gene in the pMIR-report luciferase plasmid (NAT1-wt). Next, the mutated derivatives (Nat1-mut1, -mut2 and –mut1 + 2) of NAT1-wt were generated by inserting mutations into two putative binding sites corresponding to the seed-sequence of miR-1290. B-E, Cells were transfected with either miR-1290 or nonspecific control miRNA (NC). Luciferase activity was assayed 24 hr later. The data are shown as luciferase activity relative to the vehicle (pGL4.74).

Figure 2

Kaplan-Meier survival analyses of the 394 breast cancer patients. Disease-free survival (A) and overall survival (B) of the 394 breast cancer patients stratified according to the presence or absence of NAT1 protein.

Figure 3

Kaplan-Meier survival analyses of the patients who received adjuvant endocrine therapy with Tamoxifen. Disease-free survival (A) and overall survival (B) of the 176 patients who received adjuvant endocrine therapy with tamoxifen stratified according to expression of NAT1.

Figure 4

Kaplan-Meier survival analyses of the lymph node-positive breast cancer patients. Disease-free survival (A) and overall survival (B) of the 147 lymph node-positive breast cancer patients stratified according to expression of NAT1.