We generated four monoclonal antibodies against the human GPCR RAI3 and carried out a systematic analysis of RAI3 expression in human breast cancer. Members of the GPCR superfamily are known to be involved in the regulation of many physiological processes including cell growth and differentiation, and are therefore regarded by the pharmaceutical industry as interesting therapeutic targets [1
]. In terms of antibody generation, GPCRs in general are often considered as difficult antigens since the expression and purification of the recombinant protein is a challenging approach that limits the availability of the protein [10
]. We have overcome this challenge by combining recombinant protein expression in bacteria with a technique for reformulating the RAI3 protein in liposomes prior to immunisation, allowing the generation of monoclonal antibodies using standard hybridoma technology. Only polyclonal antibodies against RAI3 are commercially available, so our work represents an important step forward, i.e. the first monoclonal anti-RAI3 antibodies. Monoclonal antibodies are more suitable as therapeutics because nonspecific cross-reactions are less likely to occur. Furthermore, the quality of polyclonal antibodies may vary considerably between batches whereas monoclonal antibodies, which originate from a single cell, are more consistent. Among four independent monoclonal antibodies generated by hybridoma technology, we selected Mab 24 2.3 for further immunohistochemical experiments as it provided the clearest signals and was able to detect RAI3 in formaldehyde-fixed, paraffin-embedded tissues which makes it easily applicable in the clinicopathological routine.
To determine whether Mab 24 2.3 was useful for the characterisation of human breast carcinomas, we carried out a systematic analysis of breast cancer and normal breast tissue on a tissue microarray. Previous studies have shown that RAI3
mRNA is upregulated in several breast cancer tissues and cell lines [5
] but there has been no similar study at the protein level because of the lack of useful reagents and a immunohistochemical characterisation of the RAI3 protein in normal and breast cancer tissue has not been published. The systemic analysis of RAI3 protein expression in human breast cancer tissue is interesting because the protein has been proposed as membrane-bound [3
], making it a potential therapeutic target as well as a useful biomarker. The most prominent therapeutic monoclonal antibody is Herceptin®
(trastuzumab) which is indicated for breast cancers involving the upregulation of HER2, a receptor tyrosine kinase belonging to the epidermal growth factor receptor (EGFR)/HER family [21
]. Upregulation of HER2 is observed in 10–34% of invasive breast tumours and offers a poor prognosis. The HER2 status of a breast cancer patient is therefore a very important clinical indicator used to select a therapeutic strategy, and HER2-positive breast tumours are often treated with Herceptin®
. Nevertheless, most patients develop resistance to Herceptin during therapy [22
], so the discovery of additional biomarkers and potential therapeutic targets is of great interest, since this provides scope for novel or combined therapies in Herceptin-resistant tumours. RAI3 may be such a candidate molecule and monoclonal antibodies against RAI3 could be used in diagnosis and in the treatment of breast cancer. However, because the function of RAI3 in normal and malignant cells is poorly understood and controversial [5
], a thorough analysis of its expression in human breast cancer tissue is necessary prior to further development. Expression of RAI3 in human cancerous and normal breast tissue has not been analysed by immunohistochemistry, thus far. In the current study, a systematic characterisation of RAI3 expression in human cancerous and normal breast tissue, both at the cDNA and the protein level, is here provided for the first time.
In a previous study [6
mRNA levels were measured by quantitative RT-PCR in the normal mammary cell lines HMEC 50 and HMEC 21 as well as in the malignant breast epithelial cell lines MDA-MB-468, BT-20, BT-549, SK-BR-3, T47D, MCF7, ZR-75-1 and BT474. Upregulation of RAI3
mRNA (50%) was observed in the oestrogen receptor-negative breast cancer cell lines MDA-MB-468, BT-20, BT-549, SK-BR-3 compared to the normal mammary cell lines HMEC 50 and HMEC 21. However, we could not detect a significant correlation between RAI3 expression and the hormone receptor status in our analysis. Furthermore, similar results indicating RAI3 upregulation were obtained in a small set of lung, colon and pancreas cell lines, compared to one sample originating from normal tissue [6
]. The overexpression of RAI3 in human breast cancer was also shown by Nagahata et al.
]. Quantitative RT-PCR also showed that RAI3 levels were higher in 19 out of 25 primary breast cancers compared to matched normal tissues and in 6 of 11 breast cancer cell lines and in HEK293 cells [5
]. In our study, we confirmed RAI3
mRNA overexpression by cDNA dot blot analysis, showing RAI3
upregulation in 60% of a large collection of 50 matched human breast carcinomas compared to the corresponding normal breast tissues. However, we did not observe similar upregulation at the protein level based on immunohistochemical analysis of a TMA. Here we analysed a large cohort of 157 breast cancer specimens and 44 normal breast tissue specimens that were not matched. The median IRSs for normal and cancerous breast tissues showed no statistically significant difference. A possible explanation for the discrepancy between the mRNA and protein results may be the presence of larger amounts of connective tissue in normal tissue samples compared with the corresponding tumour tissue samples. Thus, RNA preparations from such samples would contain an excess of mRNA originating from RAI3-negative connective tissue, resulting in lower overall signal levels compared to the tumour samples, a phenomenon that could also have affected previous RNA-based studies of breast cancer. In an immunohistochemical approach like the TMA, only normal and cancerous epithelial cells are compared directly, so the presence of connective tissue does not influence the result. Furthermore, we cannot exclude the possibility of posttranscriptional regulation of the RAI3 gene. In such a case, translation of mRNA into protein may be inhibited which would result in discordant mRNA and proteins levels [23
]. This could also explain the fact that we could not detect endogenous RAI3 in HEK 293T by western blot and immunocytochemistry analysis of untransfected cells (see Additional file 3
). Another explanation for this discrepancy may be clonal differences in the used cell lines from different laboratories. There is a tremendous number of different factors that could account for changes of the physiological status of cells and can affect the expression levels of certain proteins, e.g. age of the cells, numbers of passages, cell culture medium, supplements, etc. [24
]. In addition, it should be noted that RAI3
expression data derived from cells cultured in vitro
is liable to misinterpretation because RAI3
is induced in cancer cells by the presence of serum in the culture medium. This could introduce a bias if normal cells are cultivated under different conditions, e.g. without serum [25
]. However, such factors can be excluded from our analysis because we used tissue samples rather than cell lines.
A cDNA microarray and RT-PCR study of 20 oestrogen receptor-negative breast cancers identified RAI3
as one of the genes indicating poor patient survival (RAI3
was upregulated in the group of 10 patients who had died of breast cancer within 5 years after surgery [9
]). Genuine RAI3
upregulation has to be questioned as the authors used a very small collection of breast cancer specimens. In our study, using a TMA containing a much larger set of breast carcinomas, we found no significant correlation between RAI3
expression and overall or recurrence-free survival.
The ambiguous role of RAI3 in oncogenesis is underlined by studies that indicate RAI3
is a tumour suppressor gene in lung cancer, based on increased lung tumour prevalence in RAI3
knock-out mice [8
]. Additionally, RAI3
mRNA levels were reduced in 11 of 18 human lung cancer samples compared to adjacent normal tissue samples. In our study, we found no evidence in either the CPA or TMA experiments of RAI3
downregulation in human breast carcinoma. The RAI3 protein was distributed heterogeneously throughout the normal breast tissue and tumour samples. Therefore, whether RAI3 could be a target for future breast cancer therapy is still unclear. Further studies in larger cohorts of matched normal breast and tumour tissues may provide more detailed information about the upregulation of RAI3
in breast tumours because the induction of RAI3 was demonstrated in the CPA, which comprised matched tissue samples. Further functional studies are also required to determine the receptor's mode of action and its ligand.
In summary, the novel monoclonal anti-RAI3 antibody Mab 24 2.3 may be useful in further studies to determine the potential of RAI3 as a tumour marker. It would be particularly beneficial to study the expression of the RAI3 protein in large TMAs representing a range of different tissues to gain better insight into the role of RAI3 in human cancer and to evaluate its potential as a target for antibody-based cancer therapy.