(Received for publication, March 31, 1995; and in revised form, July 28, 1995)
From the
CART1, a novel human gene, encodes a putative protein exhibiting three main structural domains: first, a cysteine-rich domain located at the amino-terminal part of the protein, which corresponds to an unusual RING finger motif; second, an original cysteine-rich domain located at the core of the protein and constituted by three repeats of an HC3HC3 consensus motif that we designated the CART motif, and which might interact with nucleic acid; third, the carboxyl-terminal part of the CART1 protein corresponds to a TRAF domain known to be involved in protein-protein interactions. Similar association of RING, CART, and TRAF domains was observed in the human CD40-binding protein and in the mouse tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), both involved in signal transduction mediated by the TNF receptor family and in the developmentally regulated Dictyostelium discoideum DG17 protein. CART1 is specifically expressed by epithelial cells in breast carcinomas and metastases. Moreover, in these malignant cells, the CART1 protein is localized in the nucleus. Altogether, these observations indicate that CART1 may be involved in TNF-related cytokine signal transduction in breast carcinoma.
Despite earlier detection and a lower size of the primary tumors
at the time of diagnosis (Nystrom et al., 1993; Fletcher et al., 1994), breast cancer mortality remains high (Frost and
Levin, 1992). Therefore, defining the molecular mechanisms involved in
cancer formation and progression is still a major challenge in breast
cancer research (Rusciano and Burger, 1992; Hoskins and Weber, 1994).
Human CART1 cDNA corresponds to the metastatic lymph node 62-cDNA clone
recently isolated from a cDNA library of breast cancer-derived
metastatic lymph nodes. 10 recombinants were differentially
screened using two subtractive probes, respectively, representative of
malignant (metastatic lymph node) and non-malignant (fibroadenoma)
breast tissues, to identify new genes that might be specifically
involved in breast cancer (Tomasetto et al., 1995). 2% of the
clones differentially expressed contained CART1 cDNA insert. CART1 has been mapped on the q11-q12 region of the long arm of
chromosome 17 (Tomasetto et al., 1995), a locus that includes
the oncogene c-erbB2 whose overexpression is correlated with a
shorter overall and disease-free survival for breast cancer patients
(Slamon et al., 1987; Muss et al., 1994).
In the present study, we characterized the CART1 cDNA, protein, and gene organization and investigated CART1 gene expression in a panel of normal and malignant human tissues. CART1 was specifically expressed in epithelial breast cancer cells. The predicted amino acid sequence of CART1 reveals a new conserved cysteine-rich domain that we name the CART domain. Moreover, the CART1 protein showed a structural organization similar to that present in the recently identified TNF receptor-associated proteins (Rothe et al., 1994; Hu et al., 1994). Finally, in breast carcinoma, CART1 protein is localized in the nucleus of the malignant cells. Altogether, these results suggest that CART1 is implicated in signal transduction by TNF-related cytokines maybe as a latent cytoplasmic transcription factor.
The mean age of the 39 patients included in the present study was 55 years. The main characteristics of the breast carcinomas were as follows: SBR grade I (13%), grade II (38%), and grade III (49%); estradiol receptor positive (25%) and negative (75%); and lymph nodes without invasion (39%) and with invasion (61%).
The CART1 probe corresponding to the
full-length human cDNA (nucleotides 1-2004), cloned into
pBluescript II SK- vector (Stratagene) (Tomasetto et al., 1995), was P labeled using random priming
(
10
cpm/ng DNA) (Feinberg and Vogelstein, 1983).
Filters were prehybridized for 2 h at 42 °C in 50% formamide, 5
SSC, 0.1% SDS, 0.5% polyvinylpyrrolidone, 0.5% Ficoll, 50
mM sodium pyrophosphate, 1% glycine, 500 µg/ml
single-stranded DNA. Hybridization was for 18 h under stringent
conditions (50% formamide, 5
SSC, 0.1% SDS, 0.1%
polyvinylpyrrolidone, 0.1% Ficoll, 20 mM sodium pyrophosphate,
10% dextran sulfate, 100 µg/ml single-stranded DNA; 42 °C).
Filters were washed 30 min in 2
SSC, 0.1% SDS at room
temperature, followed by 30 min in 0.1
SSC, 0.1% SDS at 55
°C.
Figure 1: cDNA and deduced amino acid sequences of human CART1. Nucleotide residues are numbered in the 5` to 3` direction, and amino acids in the open reading frame are designated by the one-letter code. The underlined nucleotide sequences correspond to the Kozak and poly(A) addition signal sequences. Putative NLS sequences are shown in bold type and broken underline. The two C-rich regions are boxed, and His and Cys residues are in bold type. TRAF-C domain is shown with a gray box. Arrow heads indicate the splicing sites, and the asterisk indicates the stop codon.
Figure 5: Primary structure of the TRAF-C domain and comparison with those of CD40-bp, TRAF1, and TRAF2. Alignment and conventional symbols are as described in the legend to Fig. 2. Consensus sequence is indicated.
Figure 2: Primary structure of the CART1 C3HC3D motif and comparison with RING finger proteins from various species. These sequences are aligned to each other using the PileUp program (Feng and Doolittle, 1987). Numbers in parentheses indicate the respective position of the motif in each protein. Residues identical in all sequences are in bold type, and the conservative residues (R/K, I/V/L, Y/F, D/E, N/Q, S/T) are shown with a gray box. Gaps are used to optimize alignment.
Figure 3: Pattern of AvaII digestion of the full-length CART1 cDNA. A, positions and sequence of AvaII sites (bold type) in the full-length CART1 cDNA. Protein sequence from residue 54-60 is indicated using one-letter code. Asp is in bold type. B, ethidium bromide staining of gel electrophoresis of the CART1 AvaII digest. Molecular weight (m.w.) and CART1 fragments sizes are given on the left and right sides, respectively.
Figure 4: Primary structure of the three original HC3HC3 CART motifs present in CART1 and comparison with those of CD40-bp, TRAF2, and DG17. Alignment and conventional symbols are as described in the legend to Fig. 2.
Figure 6: Organization of the human CART1 gene and protein. Schematic representation of the CART1 gene exon/intron organization. Exons are numbered from 1 to 7. The correspondence between DNA coding sequences and protein domains is indicated. B, BamHI; ORF, open reading frame; UTR, untranslated region; aa, amino acid; bp, base pairs.
Figure 7:
Northern blot analysis of CART1 mRNA in
human breast fibroadenomas, carcinomas, and lymph node metastases. Each lane contains 10 µg of total RNA. From left to right, RNA samples from breast fibroadenomas (FA, lanes 1-6), carcinomas (BC, lanes
7-16), and metastatic lymph nodes (MLN, lanes
17 and 18) are loaded. Hybridization was carried out
using P-cDNA probe for CART1. A 2000-base-long CART1
transcript is expressed, at various levels, in some carcinomas (lanes 7, 11, and 13) and in one metastatic
sample (lane 17). The 36B4 probe (Masiakowski et al.,
1982) was used as a positive internal control. Autoradiography was for
2 days for hybridization of CART1, whereas 36B4 hybridization was
exposed for 16 h.
In situ hybridization, using an antisense CART1 RNA probe, was performed on primary breast carcinomas and axillary lymph node metastases. CART1 was expressed in the malignant epithelial cells, in in situ (Fig. 8C) and invasive (Fig. 8B) carcinomas, whereas tumoral stromal cells were negative. CART1 transcripts were homogeneously distributed among the positive areas (Fig. 8, B and C). Normal epithelial cells did not express the CART1 gene, even when located at the proximity of invasive carcinomatous areas (Fig. 8A and data not shown). A similar pattern of CART1 gene expression was observed in metastatic axillary lymph nodes from breast cancer patients with expression limited to cancer cells, whereas non-involved lymph node areas were negative (Fig. 8D and data not shown).
Figure 8:
In situ hybridization of CART1
mRNA in human breast carcinoma and axillary lymph node metastasis.
Sections of normal breast (A), in situ carcinoma (C), invasive carcinoma (B), and metastatic lymph
node (D) were hybridized with antisense S-RNA
probe specific for CART1. CART1 is strongly expressed in the tumoral
epithelial cells, whereas the stromal part of the tumor is totally
negative (B). CART1 transcripts are homogeneously distributed
throughout the positive areas (B-D). Normal ducts are
devoid of CART1 signal (A). No significant labeling above
background was found when using sense human CART1 RNA probe (data not
shown). A-D, bright field
micrographs.
Figure 9:
Immunoperoxidase staining of CART1 protein
in paraffin-embedded section of invasive breast carcinoma. Sections of
human invasive breast carcinoma were stained using hematoxylin (A) and immunostained using a rabbit polyclonal antibody
directed against a CART1-specific synthetic peptide (B); the
CART1 protein is located in the nucleus of the malignant epithelial
cells. A and B, 60
magnification.
In the present study, we have characterized the products of a novel human gene that we called the CART1 gene, since it encodes a protein containing a new conserved C-rich domain associated with RING and TRAF domains (the CART domain).
The CART1 amino-terminal part contained a C-rich domain characterized by the presence of a RING finger motif giving rise to two zinc fingers (Freemont, 1993). The RING finger protein family presently comprises more than 70 members involved in the regulation of cell proliferation and differentiation (reviewed in Freemont(1993)). Interestingly, one of the recently identified members of the family is the tumor suppressor gene BRCA1, responsible for about 50% of inherited breast cancers (Miki et al., 1994). In the CART1 RING finger, the last Cys residue is substituted by an Asp residue giving rise to a C3HC3D motif instead of the usual C3HC4 motif. Since aspartic acid has already been described as a potential zinc-coordinating residue (Vallee and Auld, 1990), we assume that the C3HC3D motif may efficiently bind metal atoms through the zinc-finger structure (Borden et al., 1995). Consistent with this hypothesis, aspartic acid has already been reported to be functional in another type of zinc-finger motif, the LIM domain (Sànchez-Garcia and Rabbits(1994) and references therein). This represents the second variant of the RING finger domain since a C2THC4 motif has already been reported in the RING finger of the p53-associated oncoprotein MDM2 (Boddy et al., 1994). The CART1 RING finger is encoded by two exons coding for the C3HC2 and Cys-Asp part of the C3HC3D motif, respectively, a genomic organization slightly different from that previously described for the consensus MEL-18 RING finger, which results from two exons encoding the C3H and C4 putative zinc finger, respectively (Asano et al., 1993).
CART1 also contained an original C-rich region, located more
centrally within the protein and composed of three repeats of an HC3HC3
motif corresponding to a novel protein signature, which we designated
the CART motif. These three repeats were encoded by distinct exons
homologous with each other, suggesting that they derived from an
ancestral exon (Dorit et al., 1990). CART motifs were only
found, in variable copy numbers, in three RING finger proteins, the
human CD40-bp (two copies), the mouse TRAF2 (two copies), and the D. discoideum DG17 protein (one copy) (Hu et al.,
1994; Rothe et al., 1994; Driscoll and Williams, 1987). The
corresponding C-rich regions of CD40-bp (Hu et al., 1994;
Cheng et al., 1995), TRAF2 (Rothe et al., 1994), and
DG17 (Driscoll and Williams, 1987) have been previously reported to be
partially arranged in a pattern resembling either the CHC3H2 ``B
box'' motif or the C2H2 Xenopus laevis transcription
factor III A motif. The CART motif, as defined in the present study,
encompasses almost the totality of the corresponding C-rich region
observed in CART1, CD40-bp, TRAF2, and DG17. The function of the CART
domain remains to be determined. Preliminary protein studies ()indicate that the correct folding of the CART motif is
depending on the presence of zinc, supporting the hypothesis that CART
corresponds to a novel zinc binding motif presumably involved in
nucleic acid binding (Schwabe and Klug, 1994; Schmiedeskamp and Klevit,
1994).
The COOH-terminal part of CART1 corresponded to a TRAF domain
previously identified in TRAF1, TRAF2, and CD40-bp. This motif is
involved in protein-protein interaction; TRAF1, TRAF2, and CD40-bp have
been reported to specifically interact with the cytoplasmic domain of
two members of the TNF receptor family, TNF-R2 and CD40 (Rothe et
al., 1994; Hu et al., 1994). The TRAF domain is composed
of two structural domains, TRAF-N, which is amino-terminally located
and corresponds to a weakly conserved helix, and TRAF-C, which is
COOH-terminally located and highly conserved (Cheng et al.,
1995). Both structural domains were encoded by the same exon of the CART1 gene. Homology was also observed with the COOH-terminal
part of the protozoan DG17 protein which, although less conserved,
could be considered as a TRAF-C domain.
Thus, CART1 shared a protein organization similar to that of the human CD40-bp, the mouse TRAF 2, and the protozoan DG17, including an amino-terminal RING finger, one to three central CART motifs, and a COOH-terminal TRAF domain (Fig. 10). These results suggest that these structurally related proteins belong to the same protein family and may exhibit analogous function. DG17 is expressed during D. discoideum aggregation, which occurs under stress conditions to permit cell survival through a differentiated multicellular organism. The precise function of DG17 remains unknown (Driscoll and Williams, 1987). However, both CD40-bp and TRAF2 have been previously shown to be involved in TNF-related cytokine signal transduction (Hu et al., 1994; Rothe et al., 1994). In contrast to growth factor receptors, cytokine receptors generally do not contain kinase activity in their cytoplasmic region, and their signal transduction mechanisms remain elusive (reviewed by Taga and Kishimoto, 1993). To date, the TNF and TNF receptor families contain 8 and 12 members, respectively. The lack of sequence homology among TNF receptor cytoplasmic domains, required for signal transduction, suggests the existence of a specific signaling pathway for each receptor (reviewed in Smith et al.(1994)). Recently, it has been proposed that signal transduction through CD40 and TNF-R2 involved the interaction of their cytoplasmic domain with two cytoplasmic proteins, CD40-bp and TRAF2, respectively (Rothe et al., 1994; Hu et al., 1994). Thus, CD40-bp and TRAF2 could be latent cytoplasmic transcription factors, which would be translocated to the nucleus under receptor activation by their respective ligands. A similar model has already been proposed for the protein family of signal transducers and activators of transcription (STAT) involved in gene activation pathways triggered by interferons (Darnell et al., 1994). This system implies a direct signal transduction pathway through STAT migration from cytoplasm to nucleus, presumably triggered by STAT phosphorylation following receptor activation (Ihle et al., 1994).
Figure 10:
Comparison of CART1, CD40-bp, TRAF2, and
DG17 protein structural organization. The size and position of RING
finger, CART motif, helix, and TRAF-C domain are represented for
each of these proteins, highlighting the similarity in the organization
of these proteins. aa, amino
acids.
This is the first report of
TNF receptor-associated protein expression in vivo in
malignant tissues. In contrast to TRAF2 and CD40-bp, which have been
shown to be ubiquitously expressed in normal tissues (Rothe et
al., 1994; Hu et al., 1994; Mosialos et al.,
1995), no CART1 expression was observed in a panel of normal
human tissues including breast, lymph node, heart, brain, skin, lung,
stomach, colon, liver, kidney, and placenta. CART1 gene
expression is restricted to some primary breast carcinomas and
metastatic axillary lymph nodes. CART1 transcripts were specifically
detected in malignant epithelial cells and homogeneously distributed
throughout the carcinomatous areas. This expression pattern suggests
that CART1 could be involved in processes leading to the formation
and/or progression of primary carcinomas and metastases. Moreover,
immunostaining analysis showed that, in malignant cells, CART1 protein
was localized in the cell nucleus. This observation is consistent with
the presence of NLS consensus sequences in CART1 and constitutes the
first evidence of a possible nuclear function for a member of the TRAF
protein family. TNF ligand family members have been shown to induce
pleiotropic biological effects, including cell differentiation,
proliferation, or death, all processes involved during carcinogenesis
and tumor progression (Smith et al.(1994) and references
therein). Very little is known concerning the involvement of
TNF-related cytokines in breast cancer. TNF-R1 and TNF-R2 have been
shown to be expressed in tumoral tissues, and a dramatic increase of
the secretion of TNF has been associated with the presence of
metastases (Pusztai et al.(1994) and references therein).
From all of these observations, we assume that CART1 may participate in TNF-related cytokine signal transduction pathway, and the nature of protein(s) and/or nucleic acid(s), which may interact with CART1, is now under characterization.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X80200[GenBank].