Transcriptional regulation of aldo-keto reductase 1C1 in HT29 human colon cancer cells resistant to methotrexate: Role in the cell cycle and apoptosis

Abstract

While studying differentially expressed genes between sensitive and 10⁻⁵ M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 1.1.1.213), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX.

Introduction

Dihydrofolate reductase (DHFR), a key enzyme of the folate cycle and the one carbon unit metabolism [1], [2], [3], catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tertrahydrofolate (THF) [4]. DHFR enzymatic activity is necessary for the biosynthesis of purines and thymidylate that are needed for cell proliferation. Methotrexate (MTX) is a 4-amino 10-methyl analog of folic acid that inhibits DHFR activity by competing with DHF for the active site. MTX was one of the first antimetabolite drugs developed and nowadays continues to play an important role in the chemotherapy of human malignancies such as acute lymphoblastic leukemia, lymphoma, osteosarcoma, breast cancer, and head and neck cancer [5], [6]. Unfortunately, the efficacy of this chemotherapeutic agent is often compromised by the development of resistance in cancer cells.

The identification of suitable genes to target in combination with MTX could be a strategy to minimize the development of resistance. To this end, we studied the gene expression profile in MTX resistance using the HG U133A 2.0 cDNA microarrays from Affymetix containing 22.300 transcripts. The human colon adenocarcinoma cell line HT29 was chosen for this study because it can be adapted to grow in high concentrations of MTX [7] and concomitantly develop amplification of the dhfr gene [8]. Among the genes whose expression is changed in cells with acquired resistance to 10⁻⁵ M MTX, we noted some members of the aldo-keto reductase (AKR) superfamily.

Members of the AKR superfamily are monomeric cytoplasmic proteins of about 320 amino acid residues, which have related structures and common evolutionary origins [9]. These enzymes are present from prokaryotes to eukaryotes and they share similar sequences and properties [10], [11], [12]. The AKRs have been proposed to be involved in detoxification processes [11], [13], [14], [15], as they can catalyze the NAD(P)H-dependent oxido-reduction of a wide range of substrates [16]. Substrate specificity is dictated by the loops at the back of the structure. Fourteen families of AKRs exist, and the AKR1 family contains many of the human isoforms.

AKRs have previously been related to cancer. Hsu et al. [17] showed that AKR1C1, also known as Dihydrodiol dehydrogenase (DDH), is highly overexpressed in NSCLC patients and its high expression correlates with a poor prognostic outcome. Overexpression of AKR1C1 has also been found in esophageal cancer, and has been associated with disease progression [18]. This gene is upregulated in HER-2/neu-positive breast tumors, which could suggest an enhanced activation of the cellular detoxification processes within the breast tumor microenvironment [19]. In addition, AKRs contribute to polycyclic aromatic hydrocarbons (PAH)-induced oral carcinogenesis [20], as induced AKR1C isozymes can convert PAH trans-dihydrodiols to deleterious O-quinones that can cause oxidative DNA damage as well as change-in-function mutations in the p53 tumor suppressor gene [21].

AKR1C1 overexpression has also been related to drug-resistance in a variety of cancers. It has been proposed that the high similarity between the chemical structures of anticancer drugs and some compounds that can be metabolized by AKR1C1 could indicate that these drugs may be subject to this enzyme activity [17]. Several reports are on accordance with this hypothesis. On one hand, Ax et al. detected AKR1C1 overexpression in daunorubicin-resistant human stomach cancer cells and suggested an association of this enzyme to drug-resistance, which they postulated to be mediated through drug detoxification in these cancer cells [22], as it had been also proposed for NSCLC [17]. On the other hand, a study on human ovarian cancer cell lines indicated that overexpression of AKR1C1 was closely associated with resistance to cisplatin and probably to disease progression [23]. The same investigators suggested that an increase in AKR1C1 activity would be sufficient to detoxify ROS, induced by cisplatin, and could lead to apoptosis-related development of drug-resistance [24]. Chen et al. [25] also correlated AKR1C1 expression with cisplatin-based chemotherapy resistance using epithelial ovarian cancer patient samples. In addition, Hung et al. [26] concluded that resistance to cisplatin, adriamycin and radiotherapy in lung adenocarcinoma cells was closely associated with AKR activity. Furthermore, increased expression of AKR1C1 in ethacrynic acid-induced drug-resistant human colon cancer cells has been also identified [27], [28], and it has been proposed that this overexpression may give rise to an enhanced capacity to metabolize exogenous and endogenous substrates, thereby contributing to the drug-resistant phenotype.

In this work we detected an overexpression of AKR1C1 in HT29 MTX-resistant cells and proceeded with the study of the mechanism of action for this effect. We found that there is a transcriptional regulation of AKR1C1 in the resistant cells, which is mainly dependent on the transcription factor Sp1, and that AKR1C1 overexpression counteracts the effects of MTX both at the level of S-phase cell arrest and apoptosis.