Promoter methylation and mRNA expression of HLA-G in relation to HLA-G protein expression in colorectal cancer
Introduction
Evasion of immune surveillance is considered one of the emerging hallmarks of cancer [1], [2]. Understanding the complex mechanisms used by tumor cells to differentiate towards cells with reduced immunogenicity is a major challenge. It is suggested that tumor cells can escape immune recognition and destruction by expression of human leukocyte antigen-G (HLA-G) and it provides to an explanation why expression of the HLA-G molecule is associated with poor patient prognosis [3], [4], [5]. HLA-G is a non-classical HLA class I (class Ib) molecule, expressed at immune-privileged sites such as the placenta, in extravillous trophoblast cells [6], [7], [8]. Alternative splicing of the primary transcript has been reported to result in seven different HLA-G protein isoforms; four membrane-bound (HLA-G1, G2, G3, G4) and three soluble (HLA-G5, G6, G7) isoforms [9]. Advances in the field have made it evident that HLA-G is also expressed in different types of malignancies in a de novo manner [10]. This tumor driven expression of HLA-G inhibits the function of several types of immune cells, among which T cells and natural killer (NK) cells, inhibits proliferation of immune cells, and can additionally induce expansion of an immunosuppressive T cell subset [11]. Therefore HLA-G expression in tumors was recently described as an immune checkpoint molecule [12]. Expression of HLA-G appears as a promising clinical prognostic factor in several types of cancer, including colorectal cancer (CRC) [13], [14], [15], [16]. However, among and within different tumors types variances in HLA-G expression were observed [17]. For example, HLA-G expression has been reported by Zeestraten et al. in 20% of colon tumors, whereas Guo et al. reported levels up to 72% in CRC [15], [18]. Immunohistochemistry (IHC) is a widely accepted technique to detect HLA-G expression, although the obtained results are still controversial [17], [19]. It is important to note that most studies used a single type of monoclonal antibody (mAb), usually 4H84. However, in CRC we showed previously discrepant expression profiles among various types anti-HLA-G mAbs [20]. For that reason and because of the known cross reactivity of mAb 4H84, it is recommended to use multiple HLA-G specific mAbs [17], [19]. Furthermore, additional molecular biological and biochemical analyses will be required to evaluate HLA-G expression in cancer and to firmly validate HLA-G expression patterns.
Previously it has been shown that HLA-G transcription is regulated by epigenetic mechanisms, including by DNA methylation [21], [22]. Nevertheless, many of the established cell lines utilized in research have been in culture for decades and may present aberrant genetic and epigenetic characteristics. In the current study we therefore investigated HLA-G DNA methylation level in 21 recently established CRC cell lines never investigated before for HLA-G expression. Furthermore, the presence of HLA-G mRNA was measured. Membrane expression of the HLA-G protein was evaluated by flow cytometry and IHC. We used three different anti-HLA-G mAbs for analyzing expression of HLA-G by IHC in the CRC cell lines and results were compared with paraffin-embedded tumor tissue of which the tumor cell lines were derived from.
Section snippets
Tumor cell lines
21 CRC cell lines were established from primary CRC tumors and colorectal liver metastasis at the Department of Pathology, LUMC (Table 1). The cell lines have been extensively characterized for several cancer related mutations by Boot et al. [23]. The CRC cell lines were cultured in RPMI-1640 (Gibco™,Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal calf serum (FCS). Dulbecco’s Modified Eagle medium (DMEM)/F12 (Gibco™) with 10% FCS was used for culturing JVE222 and JVE371. DMEM
HLA-G promoter methylation
Transcription of HLA-G may be controlled by epigenetic mechanisms including DNA methylation. The methylation levels of 10 CpG dinucleotides in the minimal promoter and 15 CpG dinucleotides in CpG island-3 of HLA-G were analyzed in the early CRC cell lines. The methylation status was compared with the HLA-G expressing cell line JEG-3. The calculated JEG-3 methylation levels were 72% and 85% in the investigated CpG dinucleotides in the promoter and CpG Island-3, respectively. Compared with JEG-3,
Discussion
HLA-G has been of interest for the last decades as a possible immune tolerogenic molecule in several malignancies such as CRC. As a consequence HLA-G is considered as a potential target for immunotherapy strategies [30]. However, the results regarding HLA-G expression in cancer are controversial. Therefore, careful evaluation of HLA-G expression is necessary. In the current literature, several factors have been suggested to be essential for HLA-G expression such as the epigenetic status,
Disclaimers
The authors declare that they have no competing interests related to the content of this manuscript.
Acknowledgements
The authors acknowledge colleagues of the Department Surgery, including, Geeske G. Dekker-Ensink, Marieke Prevoo and Rob Keyzer, and Hans Morreau and Jaap D.H. van Eendenburg of the Department of Pathology, for their valuable contribution to this study.
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Both authors contributed equally.