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TRAF-4 Expression in Breast Carcinomas

      To the Editor-in-Chief:

      I read with interest the recent paper by Krajewska et al
      • Krajewska M
      • Krajewski S
      • Zapata JM
      • Van Arsdale T
      • Gascoyne RD
      • Berern K
      • McFadden D
      • Shabaik A
      • Hugh J
      • Reynolds C
      • Clevenger CV
      • Reed JC
      TRAF-4 expression in epithelial progenitor cells.
      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.
      • Regnier CH
      • Tomasetto C
      • Moog-Lutz C
      • Chenard MP
      • Wendling C
      • Basset P
      • Rio MC
      Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.
      To examine the TRAF-4 expression, Krajewska et al used immunohistochemistry, whereas we performed Northern blotting andin 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 situhybridization 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.
      • Regnier CH
      • Tomasetto C
      • Moog-Lutz C
      • Chenard MP
      • Wendling C
      • Basset P
      • Rio MC
      Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.
      In addition, TRAF-4 was first identified by overexpression in breast cancer metastatic axillary lymph node,
      • Tomasetto C
      • Regnier CH
      • Moog-Lutz C
      • Mattei MG
      • Chenard MP
      • Lidereau R
      • Basset P
      • Rio MC
      Identification of four novel human genes amplified and overexpressed in breast carcinoma and located to the q11–q21.3 region of chromosome 17.
      a tissue in which contamination by normal breast cells would appear extremely unlikely.
      For their second explanation, Krajewska et al referred to Tomasetto et al
      • Tomasetto C
      • Regnier CH
      • Moog-Lutz C
      • Mattei MG
      • Chenard MP
      • Lidereau R
      • Basset P
      • Rio MC
      Identification of four novel human genes amplified and overexpressed in breast carcinoma and located to the q11–q21.3 region of chromosome 17.
      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 study
      • Bieche I
      • Tomasetto C
      • Regnier CH
      • Moog C
      • Rio MC
      • Lidereau R
      Two distinct amplified regions at 17q11–q21 involved in human primary breast cancer.
      including 98 human breast carcinomas and showing that in vivooverexpression 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.

      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.
      • Krajewska M
      • Krajewski S
      • Zapata JM
      • Van Arsdale T
      • Gascoyne RD
      • Berern K
      • McFadden D
      • Shabaik A
      • Hugh J
      • Reynolds C
      • Clevenger CV
      • Reed JC
      TRAF4 expression in epithelial progenitor cells.
      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,
      • Bieche I
      • Tomasetto C
      • Regnier CH
      • Moog C
      • Rio MC
      • Lidereau R
      Two distinct amplified regions at 17q11–q21 involved in human primary breast cancer.
      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.
      • Tomasetto C
      • Regnier CH
      • Moog-Lutz C
      • Mattei MG
      • Chenard MP
      • Lidereau R
      • Basset P
      • Rio MC
      Identification of four novel human genes amplified and overexpressed in breast carcinoma and located to the q11–q21.3 region of chromosome 17.
      In contrast, we observe exclusively cytosolic immunostaining, sometimes with a vesicular/organellar pattern typical of other TRAF-4 family proteins.
      • Regnier CH
      • Tomasetto C
      • Moog-Lutz C
      • Chenard MP
      • Wendling C
      • Basset P
      • Rio MC
      Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.
      • Kaye KM
      • Devergne O
      • Harada JN
      • Izumi KM
      • Yalamanchili R
      • Kieff E
      • Mosialos G
      Tumor necrosis factor receptor associated factor 2 is a mediator of NFκB activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.
      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.

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        • Reed JC
        TRAF-4 expression in epithelial progenitor cells.
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        Identification of four novel human genes amplified and overexpressed in breast carcinoma and located to the q11–q21.3 region of chromosome 17.
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        • Wendling C
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        Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.
        J Biol Chem. 1995; 270: 25715-25722
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        • Devergne O
        • Harada JN
        • Izumi KM
        • Yalamanchili R
        • Kieff E
        • Mosialos G
        Tumor necrosis factor receptor associated factor 2 is a mediator of NFκB activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.
        Proc Natl Acad Sci. 1996; 93: 11085-11090