There is strong evidence that the immune system plays a role in tumor growth and progression [4,5]. An effective immune response may lead to recognition of tumor cells, resulting in their eradication. However, due to their genetic unstable nature, tumor cells may arise which display properties that enables them to escape from immune recognition [4,5]. Indeed, downregulation or loss of proteins that are crucial for immune responses, like classical human leukocyte antigens (HLA) class I, or upregulation of proteins that confer resistance to immune recognition, like non-classical HLA class I, are frequently found in various types of tumors [6-10].

The activating receptor natural killer cell lec-tin-like receptor gene 2D (NKG2D) is a stimulatory immune receptor that is expressed on natural killer (NK) cells, NKT cells, γδ⁺ T cells and CD8+ T cells [11]. Ligands which bind NKG2D receptors comprise major histocompatibility complex class I chain-related proteins A and B (MIC-AB) and unique long 16 (UL16) binding proteins 1-6 (ULBP1-6) [12,13]. Expression of these ligands may be induced upon infection and other inducers of cellular stress and is unusual in normal cells [14]. By binding to the NKG2D receptors on NK and T cells, the NKG2D ligands may initiate an immune response against cells expressing these ligands. Overexpression and shedding of NKG2D ligands have been reported [14]. It is, however, unclear whether these features also results in activation of an immune response or lead to overstimulation and downregulation of NKG2D on immune cells [11].

Malignant transformation of cells may be among stimuli inducing expression of NKG2D ligands as such expression has been found in various tumor types [8-10,15-18]. This may be a mechanism for preventing tumor growth by advancing an anti-tumor immune response. Convincing evidence has been found in in vivo studies, which have shown that in mouse models transfection with NKG2D ligands resulted in a NKG2D-mediated tumor rejection [19,20]. Other studies showed that downregulation or complete knockout of NKG2D in mice resulted in an impaired immune response against tumor cells, higher expression levels of NKG2D ligands, and an increased incidence of certain tumors [21,22].

A few studies have investigated tumor expression of NKG2D ligands and associations with clinical outcome in human breast, colorectal, and ovarian cancer [8-10,15,16]. Expression of MIC-A was frequently found in all tumors studied and resulted in a statistically significant favorable patient's prognosis in colorectal cancer, while it was not statistically significantly associated with outcome in breast cancer and ovarian cancer [8-10,16]. ULBP1-5 expression was also found to be expressed in many tumor samples of colorectal and ovarian cancer [9,10,15]. In colorectal cancer expression of ULBP5 was an independent prognostic factor for a favorable clinical outcome [9]. In contrast to these results, expression of ULBP2 and ULBP4 were found to be independent prognostic factors for a worse outcome of ovarian cancer patients [10,15]. Taken together, several studies suggest that evasion of NKG2D-mediated immune regulation plays an important role in tumor progression, but some studies contradict this suggestion. Contradictory results may be explained by assuming functional differences in immune regulation of the different ligands. Moreover, expression of NKG2D ligands may behave different among different tumor types [9]. It is known that overexpression or shedding of these ligands leads to overstimulation and downregulation of NKG2D on immune cells [10,15], thereby evading an immune response.

NKG2D ligands are expressed on the cell surface in response to stress or malignant transformation [11]. Our study confirms that breast cancer tumor cells show frequent and high expression of NKG2D ligands, as has been found in other studies for various types of tumors such as ovarian cancer, colorectal cancer and breast cancer [8-10,15,16]. Though all studies show consistently frequent expression of NKG2D ligands, very diverse prognostic effects have been described for these types of ligands in different cancer types [8-10,15,16]. This may be explained by functional differences in immune regulation for varying expression levels of different ligands in different environments. Expression of NKG2D ligands may induce an immune response through binding to the NKG2D receptor, present on NK cells and a subset of T cells [11]. Therefore, selective outgrowth of malignant cells that do not express these NKG2D ligands may be a mechanism of tumor immune escape. On the other hand, overexpression of NKG2D ligands could lead to overstimulation and thereby insensibility or anergy of immune cells, which would result in evasion of immune attack by tumors overexpressing NKG2D ligands [11]. Adding to this hypothesis, it has been reported that NKG2D ligands on the cell membrane may be cleaved and produce soluble molecules. This shedding of NKG2D ligands could systemically downregulate NKG2D receptor expression and thereby result in an impaired anti-tumor reactivity of NK and T cells [11,27]. Taken together, the mechanisms by which NKG2D ligands mediate immune function or dysfunction may be diverse in different tumors and differ according to circumstances. The contradictory results on the prognostic effect of NKG2D ligands found between different studies on different tumors may be reflected by the functional and mechanistic implications of interaction between NKG2D and its ligands. In ovarian cancer expression of NKG2D ligands resulted in a worse patient outcome, probably due to chronic overexpression and shedding of these ligands, leading to overstimulation and downregulation of the NKG2D receptor of NK and T cells and, therefore, an impaired immune response [10,15]. Supporting the hypothesis that elevated expression of NKG2D ligands results in immune escape in ovarian cancer, one study found elevated levels of MIC-AB and ULBP-2 to be positively correlated to less intra-tumor epithelial CD57+ cells. The results found in breast cancer in the present study are contradictory to the results found in ovarian cancer, but similar to those found in colorectal cancer [9,16]. The results in our study and colorectal cancer are supported by the theory that expression of NKG2D ligands results in activation of immune cells which is reflected in a patient beneficial outcome for high ligand expression [9,16]. We found frequent and high expression of ligands in our study and statistically significant associations between expression levels of these ligands, indicating their cooperation with each other. Adding to the hypothesis that low expression of these ligands is a result of selective pressure by the immune system that results in cancer immune evasion or immunoediting, low expression of MIC-AB and ULBP-2 were prognostic factors for an unfavorable RFP of patients. When expression of MIC-AB and ULBP-2 were combined they showed to add to each others prognostic effect which is in line with the results found in previous studies [9,10] and suggests that NKG2D ligands operate together and in a similar manner.