Lack of functional and expression homology between human and mouse aldo-keto reductase 1C enzymes: implications for modelling human cancers

Abstract

Background: Over recent years, enzymes of the aldo-keto reductase (AKR) 1C subfamily have been implicated in the progression of prostate, breast, endometrial and leukemic cancers. This is due to the ability of AKR1C enzymes to modify androgens, estrogens, progesterone and prostaglandins (PGs) in a tissue-specific manner, regulating the activity of nuclear receptors and other downstream effects. Evidence supporting a role for AKR1C enzymes in cancer derives mostly from studies with isolated primary cells from patients or immortalized cell lines. Mice are ideal organisms for in vivo studies, using knock-out or over-expression strains. However, the functional conservation of AKR1C enzymes between human and mice has yet to be described.

Results: In this study, we have characterized and compared the four human (AKR1C1,-1C2, -1C3 and -1C4) and the eight murine (AKR1C6, -1C12, -1C13, -1C14, -1C18, -1C19, -1C20 and -1C21) isoforms in their phylogeny, substrate preference and tissue distribution. We have found divergent evolution between human and murine AKR1C enzymes that was reflected by differing substrate preference. Murine enzymes did not perform the 11beta-ketoreduction of prostaglandin (PG) D2, an activity specific to human AKR1C3 and important in promoting leukemic cell survival. Instead, murine AKR1C6 was able to perform the 9-ketoreduction of PGE2, an activity absent amongst human isoforms. Nevertheless, reduction of the key steroids androstenedione, 5alpha-dihydrotestosterone, progesterone and estrone was found in murine isoforms. However, unlike humans, no AKR1C isoforms were detected in murine prostate, testes, uterus and haemopoietic progenitors.

Conclusions: This study exposes significant lack of phylogenetic and functional homology between human and murine AKR1C enzymes. Therefore, we conclude that mice are not suitable to model the role of AKR1C in human cancers and leukemia.

Figure 1

Proposed roles of AKR1C enzymes in prostate cancer, breast cancer and myeloid leukaemia. AR, androgen receptor. ER, estrogen receptor. CYP19arom, CYP19 aromatase. PG, prostaglandin. 15d-PGJ₂, 15-deoxy-Δ^12,14-PGJ₂. ROS, reactive oxygen species. PPARγ, peroxisome proliferator-activated receptor γ. NF-κB, nuclear factor κB.

Figure 2

Phylogeny of the AKR1C subfamily. A. phylogenetic tree showing the relationship between the 23 known AKR1C proteins. Protein sequences available at the AKR homepage http://www.med.upenn.edu/akr/ were aligned using ClustalW and a tree generated using TreeView X. Human AKR1C isoforms cluster closely together suggesting an early common ancestor while murine isoforms have higher diversity. B. Position of AKR1C genes in chromosome reflects phylogenetic relationships. To scale representation of murine (chromosome 13) and human (chromosome 10) AKR1C gene clusters. Grey boxes represent individual genes.

Figure 3

SDS-PAGE of purified recombinant AKR1C proteins. SDS-PAGE showing the purity of recombinant human and murine AKR1C enzymes. AKR proteins bearing a C-terminal 6× His tag were over-expressed in E. coli and purified in affinity column followed by FPLC. To determine purity 3 μg of protein were separated in SDS-PAGE and the gel stained with Coomassie Blue.

Figure 4

Reduction of 5α-DHT to 3α/β-adiol by human and murine AKR1C enzymes. Human and murine recombinant AKR1C enzymes were incubated with 25 μM 5α-DHT mixed with 1 μCi [³H]-5α-DHT and NADPH. At 30 and 90 minutes reactions were stopped and the steroids extracted and separated in TLC along with known standards. The radioactive traces were scanned and the percentage of 5α-DHT reduced to 3α- or 3β-adiol calculated. Bars represent average expression of three replicates and error bars represent standard deviation. * - Incubation with AKR1C21 resulted in efficient conversion to a product other than 3α- or 3β-adiol.

Figure 5

Tissue expression of the eight murine AKR1C enzymes. RNA was extracted from several murine tissues (in triplicate), converted to cDNA and gene expression quantified by Taqman QRT-PCR. Expression levels were normalized to 18S expression using the following formula: (2^{-(CTgene-CT18S)})x10⁶ and Gapdh expression was measured as control of cDNA quality. P - extracted from pregnant mice. Bars represent average expression of three biological replicates and error bars represent standard deviation.