Elevated expression of LSD1 (Lysine-specific demethylase 1) during tumour progression from pre-invasive to invasive ductal carcinoma of the breast

LSD1 (Lysine-specific demethylase 1) is a key enzyme in posttranslational histone modification [1]. Histone modifications including acetylation, methylation, phosphorylation and ubiquitination play an important role in structural changes of chromatin [2, 3]. Chromatin modifying activities are dynamically regulated processes and form epigenetic marks on the histone substrate. Thus, enzymes and especially LSD1 are involved in epigenetic gene deregulation and initiate tumourgenesis [3–5]. So far little is known on the in vivo biochemical mechanism by which LSD1 overexpression is linked to neoplastic cell proliferation. Discovered by in vitro data it was shown that LSD1 interfers to G2-M phase cell-cycle and promotes cell proliferation [6]. LSD1 is localised in the nucleus and characterised by a C-terminal amino oxidase domain (AOD) and by an N-terminal domain. The catalytic centre of LSD1 is the Flavin Adenin Dinucleotide-dependent AOD region. This highly specific catalytic domain leads to demethylation of mono- or dimethylated lysine (Lys4) of histone 3 (H3) [5, 7].

LSD1 has been analysed in several human tumour entities. It has been shown to be overproduced in prostate cancer [8, 9], neuroblastoma [10], lung, colorectal and bladder cancer [11, 12]. Furthermore, LSD1 expression has also been investigated in invasive breast carcinoma. Lim et al. [13] showed a significant positive correlation between overexpression of LSD1 and negative oestrogen receptor status in breast carcinoma. Also there was an inverse correlation between high LSD1 expression levels and low progesterone receptor status. Other histopathological data like tumour size (pT1-pT4), nodal status and human epidermal growth factor receptor (HER 2) status reached no significant correlation with LSD1 expression. However in consideration of the fact that breast carcinoma with positive steroid hormone receptor status responds to endocrine treatment [14] and therefore reveals a better prognosis, Lim et al. supposed that upregulation of LSD1 and its correlation with negative oestrogen receptor status could be a biomarker for aggressive tumour biology in breast cancer.

Consistent with its tumour promoting role, the specificity of posttranslational modification conferred by LSD1 has been investigated by Bradley et al. [15]. They analysed human mammary epithelial cell lines after carcinogen exposure and examined equal levels of histone H3 total protein and histone H4, but significantly decreased levels of mono-methyl histone H3 (Lys4) compared with control groups which were not treated. They pointed out that downregulation of histone H3 (Lys4) was related to LSD1 upregulation after carcinogen treatment. In addition accumulated levels of carcinogen exposure were related to accumulated levels of LSD1 expression compared to control human mammary epithelial cell lines. Thus increased LSD1 expression was assumed to be an early step in breast cancer development.

Surprisingly, LSD1 has not been studied in pre-invasive breast cancer lesions, so far. To the best of our knowledge, this is the first study to analyse LSD1 protein expression in pre-invasive breast neoplasias. We systematically examined LSD1 expression in low, intermediate and high grade ductal carcinoma in situ in comparison to invasive ductal carcinoma.

LSD 1 is a member of monoaminoxidase enzymes with an important role in controlling gene expression by histone modification [5]. In accordance with the perceived role of LSD1 in cell proliferation, significant expression of LSD1 has been reported in diverse human tumour entities, including breast cancer. Until now, LSD1 expression has not been analysed in pre-invasive breast cancer lesions, so far. In this study we developed a first systematic expression analysis of LSD1 in pre-invasive (n = 88) and invasive (n = 32) breast carcinoma.

Using immunohistochemistry we here show that LSD1 is also expressed in pre-invasive ductal neoplasias of the breast. Additionally, we detected a significant gradual increase of LSD1 expression during tumour progression from low, intermediate and high grade ductal carcinoma in situ to invasive ductal breast carcinoma (p <0.05). Our results implicate that LSD1 could play a key role in breast cancer development. So upregulation of LSD1 may be an early tumour promoting event in breast carcinoma. This is supported by the work of Bradley et al. [15] showing the dynamic movement of LSD1 from the nuclear periphery into the nucleus after carcinogen treatment in human mammary epithelial cell lines. Furthermore increased LSD1 expression levels were found in increasing carcinogen treated human mammary epithelial cell lines compared to non-treated controls. They concluded that upregulation of LSD1 and localisation into the nucleus are mechanisms that are responsible for demethylation of histone H3 (Lys4) by LSD1 affecting genes like p57^kip2, a cyclin-dependent kinase inhibitor, which is known to be essential in tumourgenesis of breast cancer cells. It is supposed that LSD1 forms complexes with different co-factors and depending on the promoter context it has a gene activating or repression function [1]. A recent study by Scoumanne et al. [6] analysed the role of LSD1 in the human malignant breast cancer cell line MCF7. They found out that LSD1 downregulation decreased the number of proliferating breast cancer cells. Even though the concrete mechanism in which LSD1 is linked to cancer development has not been fully examined, it has been shown that high LSD1 expression is a characteristic feature of cancer cells.

Consistent with these data, we detected high expression levels of LSD1 in invasive ductal breast carcinoma which supports the results of a study by Lim et al. [13] showing abundant LSD1 expression in invasive breast cancer, as well. In conclusion our data imply a positive association between LSD1 overexpression and progression, proliferation as well as increasing invasiveness of breast cancer cells.

However, in our collective of invasive ductal breast carcinoma the inverse correlation of LSD1 expression with lymph node status, histological grade and oestrogen receptor status may be due to a inhomogeneous and relatively small group size of invasive breast carcinoma specimens because this study was not constructed to validate LSD1 expression in invasive breast carcinoma as it was already analysed in a previous work by Lim et al. [13]. Furthermore, our collective of invasive ductal breast carcinomas consists mainly of tumours with small tumour size accordingly pT1 tumours whereas Lim et al. [13] analysed more tumours of a higher stage respectively pT2 to pT4 invasive breast carcinomas. Nevertheless, the association between LSD1 expression and oestrogen receptor status has to be further investigated and validated in greater cohorts of invasive breast carcinomas.

In addition LSD1 could be an interesting target molecule in the treatment of breast carcinoma. Usually, DCIS treatment involves breast conserving surgery by local tumour excision or mastectomy depending on free margins to reduce the risk of ipsilateral recidive. Supplemental radiation is part of adjuvant therapeutic regime [18]. Until now, there has been an absence of common guidelines for the use of hormone therapy or Trastuzumab, a monoclonal antibody, for patients with (human epidermal growth factor) HER 2 positive DCIS [19–22]. Boughey et al. [19] suggested that anti-hormone therapy should be part of adjuvant therapy regime in oestrogen receptor positive DCIS and the use of Trastuzumab for DCIS was seen as an option which needs to be more specified in future. Nonetheless, it was shown by clinical evidence that only patients with positive oestrogen receptor status responded to anti-hormone treatment and a subgroup of patients developed resistance after extended treatment.

Therefore there is a need for alternative drug treatment in case of resistance to anti-oestrogens. With regard to offering other suitable options of breast cancer therapy in connection with other eligible targets, LSD1 may be such a target mark as LSD1 inhibitors were discussed as novel breast cancer therapeutics [13].

Because of its structural analogy with monoaminoxidases, LSD1 was shown to be inhibited by nonspecific monoaminoxidase inhibitors like tranylcypromine [23]. In a previous work of Lee et al. [23] tranylcypromine was found to inhibit embryonal carcinoma cells. Therefore tranylcypromine was discussed as a possible novel anti-cancer target referring to breast cancer, as well. In concordance with this, Lim et al. [13] showed inhibition of LSD1 in breast cancer cells in vitro by tranylcypromine and clorgyline, a selecitve monoaminoxidase inhibitor, leading to inhibitory effect on LSD1 and reduced growth of the breast cancer cell lines MCF7, T47D, MDA-MB 453 and MDA-MB 231.

In the future LSD1 detection may be an early identification marker of breast cancer and a potential target for early therapeutic strategies. However, in our collective of invasive ductal breast carcinoma the inverse correlation of LSD1 expression with oestrogen receptor status may be due to a inhomogeneous and relatively small group size of invasive breast carcinoma specimens mainly consisting of pT1 tumours [24]. Further studies are underway to analyse the expression and function of LSD1 in a larger cohort of breast cancer specimens including patients` survival data to evaluate the prognostic relevance of LSD1 in breast carcinoma.

The study was supported by a grant (BONFOR program) of the Faculty of Medicine, University of Bonn, to Dr. Nuran Serce.

The study was approved by the ethics committee of the Faculty of Medicine, University of Bonn.