Intratumoural mRNA expression of genes from the oestradiol metabolic pathway and clinical and histopathological parameters of breast cancer

Abstract

Introduction: The expression of the oestrogen receptor (ER) is one of the more important clinical parameters of breast cancer. However, the relationship between the ER and its ligand, oestradiol, and the enzymes that synthesise it are not well understood. The expression of mRNA transcripts of members of the oestradiol metabolic and signalling pathways including the ER was studied in detail.

Method: mRNA transcripts for aromatase (CYP19), 17-beta-hydroxysteroid dehydrogenase I, 17-beta-hydroxysteroid dehydrogenase II, ERalpha, ERbeta, steroid sulfatase (STS), oestradiol sulfotransferase (EST), cyclin D1 (CYCLD1) and ERBB2 were fluorometrically quantified by competitive RT-PCR using an internal standard in 155 breast carcinomas. In addition, the transcripts of CYP19 were analysed for alternative splicing/usage of exon 1 and an alternative poly A tail.

Results: A great variability of expression was observed, ranging from 0 to 2376 amol/mg RNA. The highest levels were observed for STS and EST, and the lowest levels (close to zero) were observed for the 17-beta-hydroxysteroid dehydrogenase isoenzymes. The levels of mRNA expression were analysed with respect to clinical and histopathological parameters as well as for disease-free survival. High correlation of the mRNA expression of STS, EST and 17-beta-hydroxysteroid dehydrogenase in the tumours suggested a common regulation, possibly by their common metabolite (oestradiol). Hierarchical clustering analysis in the 155 patients resulted in two main clusters, representing the ERalpha-negative and ERalpha-positive breast cancer cases. The mRNA expression of the oestradiol metabolising enzymes did not follow the expression of the ERalpha in all cases, leading to the formation of several subclasses of tumours. Patients with no expression of CYP19 and patients with high levels of expression of STS had significantly shorter disease-free survival time (P > 0.0005 and P < 0.03, respectively). Expression of ERbeta mRNA was a better prognostic factor than that of ERalpha in this material.

Conclusion: Our results indicate the importance of CYP19 and the enzymes regulating the oestrone sulfate metabolism as factors of disease-free survival in breast cancer, in addition to the well-known factors ER and ERBB2.

Figure 1

Oestradiol synthesis pathway from cholesterol. The present study was focused on the right branch of this panel – the final metabolism of oestradiol from androsenedione. Thick arrows indicate the enzymes and activities coded by the mRNA transcripts studied in the present report.

Figure 2

Clustering analysis of levels of mRNA relative to the median. Two distinct clusters of the oestrogen receptor (ER)-positive and ER-negative samples were observed. The mRNA expression of the oestradiol metabolising enzymes, however, did not follow the expression of the ER in all cases, leading to the formation of several subclasses of tumours: grey, ERα⁺/CYP19^-; dark green, ERα⁺/CYP19⁺; light green, ERα⁺/CYP19^-/ERβ^-; yellow, ERα^-/CYP19⁺/ERβ^-; pink, ERα^-/CYP19⁺/ERβ⁺; blue, ERα^-/CYP19^-/ERβ^-.

Figure 3

Kaplan–Meier analysis of disease-free survival of breast cancer patients dependent on the expression of mRNA coding for oestradiol metabolising enzymes. (a) CYP19 (aromatase) using the conventional, adipose tissue-specific first exon (curve 1), abnormally using the ovarian-specific first exon (curve 2), and with the 0-null expression (curve 0). (b) Steroid sulfatase with expression below (curve 1) and above (curve 2) the median. (c) Oestrogen receptor (ER)α below (curve 1) and above (curve 2) the median. (d) ERβ below (curve 2) and above (curve 1) the median.

Figure 4

Disease-free survival of breast cancer patients as a function of mRNA expression of ERBB2 below (curve 1) and above (curve 2) the median. P = 0.011.