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Correspondence |
IGBMC-INSERM, Strasbourg, France
To the Editor-in-Chief:
I read with interest the recent paper by Krajewska et al1 concerning the pattern of TRAF-4 expression, notably in breast carcinomas. This constitutes the second study in this field, the first one having been performed in our laboratory.2 To examine the TRAF-4 expression, Krajewska et al used immunohistochemistry, whereas we performed Northern blotting and in situ hybridization for TRAF-4 mRNA. They conclude that "breast cancers down-regulate expression of TRAF-4 as part of the malignant transformation processes." In contrast, we found that TRAF-4 mRNA can be overexpressed in some breast carcinomas. In order to account for this discrepancy, Krajewska et al proposed two explanations.
The first is that "contaminating nonmalignant TRAF-4 expressing cells may have contributed to the presence of TRAF-4 mRNA in (our) breast cancer specimens." To our knowledge, in situ hybridization permits, as efficiently as immunohistochemistry, discernment between normal and cancerous cells. In breast carcinomas, cells that are overexpressing TRAF-4 mRNA are malignant cells, as also shown by Regnier et al.2 In addition, TRAF-4 was first identified by overexpression in breast cancer metastatic axillary lymph node,3 a tissue in which contamination by normal breast cells would appear extremely unlikely.
For their second explanation, Krajewska et al referred to Tomasetto et al3 to argue that we failed to observe TRAF-4 gene amplification in tumors. This is misleading because the paper reports TRAF-4 gene amplification in breast cancer cell lines. However, it seems the authors are not aware of our study4 including 98 human breast carcinomas and showing that in vivo overexpression of TRAF-4 mRNA is always associated with TRAF-4 gene amplification.
We have no doubt that the TRAF-4 gene is amplified and overexpressed in some breast carcinomas, at least at the transcriptional level. Why this overexpression was not observed by Krajewska et al using immunohistochemistry remains unexplained.
References
The Burnham Institute La Jolla, California
Authors' Reply:
We thank Drs. Tomasetto, Regnier, and Rio for their thoughtful comments about our paper on TRAF-4 expression in normal tissues and cancers.1 Some of the points they have raised appeared in the draft Discussion section of our paper but were removed during revision in an effort to shorten it.
The principal issue is the discrepancy between levels of TRAF-4 mRNA and protein in breast cancers. Though the Rio group has found abnormally high levels of TRAF-4 mRNA in about one-quarter of adenocarcinomas of the breast,2 we failed to find evidence of TRAF-4 protein up-regulation by immunostaining in our analysis of 84 cases of primary breast cancer.
Several potential explanations can be imagined for the apparent discrepancy between the Rio group's TRAF-4 mRNA data and our TRAF-4 immunostaining results.
First, because both groups have not analyzed the same breast cancer, we do not know whether differences in patient sample selection may explain the differences in our findings. Dr. Rio and colleagues have verified amplification of the chromosomal locus where the TRAF-4 gene resides in their specimens by Southern blotting. We, in contrast, have no information about the status of the 17q11–21 region in the tumors analyzed for our paper.
Second, it is possible that translation of the TRAF-4 mRNA is highly regulated or that the stability (half-life) of the TRAF-4 protein is a point of regulation, resulting in TRAF-4 mRNA production without accumulation of TRAF-4 protein. Certainly, many samples exist in which the presence of mRNA does not guarantee the presence of the corresponding protein.
Third, as we mentioned in our paper, our anti-TRAF-4 antiserum was generated using a synthetic peptide corresponding to first 18 amino acids of the predicted TRAF-4 protein. Therefore, we cannot exclude the possibility that other forms of TRAF-4 protein could be produced that are undetectable with our antiserum through such means as alternative mRNA splicing mechanisms or proteolytic processing. In this regard, Dr. Rio and col- leagues have reported that TRAF-4 protein is nuclear.3 In contrast, we observe exclusively cytosolic immunostaining, sometimes with a vesicular/organellar pattern typical of other TRAF-4 family proteins.4,5 Thus, although unusual for TRAF-family proteins, we cannot exclude the possibility that alternative forms of the TRAF-4 protein exist that are targeted to the nucleus.
References
B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein. Proc Natl Acad Sci 1996, 93:11085-11090
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