The E3 ubiquitin ligase specificity subunit ASB2beta is a novel regulator of muscle differentiation that targets filamin B to proteasomal degradation.

Ubiquitin-mediated protein degradation is the main mechanism for controlled proteolysis, which is crucial for muscle development and maintenance. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 gene (ASB2) encodes the specificity subunit of an E3 ubiquitin ligase complex involved in differentiation of hematopoietic cells. Here, we provide the first evidence that a novel ASB2 isoform, ASB2beta, is important for muscle differentiation. ASB2beta is expressed in muscle cells during embryogenesis and in adult tissues. ASB2beta is part of an active E3 ubiquitin ligase complex and targets the actin-binding protein filamin B (FLNb) for proteasomal degradation. Thus, ASB2beta regulates FLNb functions by controlling its degradation. Knockdown of endogenous ASB2beta by shRNAs during induced differentiation of C2C12 cells delayed FLNb degradation as well as myoblast fusion and expression of muscle contractile proteins. Finally, knockdown of FLNb in ASB2beta knockdown cells restores myogenic differentiation. Altogether, our results suggest that ASB2beta is involved in muscle differentiation through the targeting of FLNb to destruction by the proteasome.

Signaling revisited in acute promyelocytic leukemia.

Although transcription factors are still the main focus to understanding leukemogenesis, recent results strongly suggest that alteration of a receptor and/or subsequent signaling plays a critical and co-operative role in the pathogenesis of acute myeloid leukemia (AML). The t(15;17) translocation, found in 95% of APL, encodes a PML-RARalpha fusion protein. A main model proposed for acute promyelocytic leukemia (APL) is that PML-RARalpha exerts its oncogenic effects by repressing retinoic acid-inducible genes critical to myeloid differentiation. Dysregulation of these genes may result in abnormal signaling, thereby freeing pre-leukemic cells from controls which normally induce the onset of differentiation. It is also likely that treatment of APL cells by retinoic acid induces de novo up-regulation of the same genes which are dominantly repressed by PML-RARalpha and whose expression is required for reactivation of the differentiation program. Identification of such genes together with the signaling pathways interrupted at the early stages of leukemia transformation and reactivated during retinoic acid-induced differentiation in APL cells will contribute to the development of new molecular targets for treatment of leukemia.

Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid.

A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mechanisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is expressed early during hematopoiesis and is a granulocyte colony-stimulating factor-responsive gene. Myeloblastin can confer factor-independent growth to hematopoietic cells, an early step in leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb binding sites, each of which are critical for constitutive expression in myeloid cells. Inhibition of myeloblastin expression in leukemia cells growth-arrested by retinoic acid is demonstrated to depend on Myb down-regulation. Myb is shown to induce myeloblastin expression and abolish its down-regulation by retinoic acid. Altogether, the data offer a clue as to how a myeloid-specific transcriptional machinery can be accessible to regulation by retinoic acid and point to myeloblastin as a novel target of Myb. This link between Myb and myeloblastin suggests a previously nonidentified Myb pathway through which growth arrest is induced by retinoic acid in myeloid leukemia cells.

PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor alpha in acute promyelocytic leukemia cells.

The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.

Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells.

Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.

Proteinase 3 mRNA expression is induced in monocytes but not in neutrophils of patients with cystic fibrosis.

Proteinase 3 (PR3), a serine proteinase which can degrade lung tissue, is present in the cystic fibrosis (CF) sputum. In the present study, PR3 protein and mRNA expression was determined in circulating neutrophils and monocytes. CF neutrophils contained similar PR3 concentrations as healthy controls and poorly expressed PR3 mRNA. In contrast, CF monocytes showed significantly higher PR3 concentrations than controls, together with an upregulation of PR3 mRNA expression especially during pulmonary exacerbation. Interestingly, antibiotic treatment fully abrogated PR3 mRNA expression and decreased PR3 protein in monocytes. Our findings highlight a potential role of monocyte-derived PR3 in CF-associated airway inflammation.

Genomic organization and chromosomal localization of mouse proteinase 3 (Myeloblastin).

Proteinase 3 (PR3), is a matrix-degrading serine proteinase expressed in different hematopoietic cell lineages. The PR3 protein appears to regulate the myeloid differentiation and was found to be the autoantigen associated with Wegener granulomatosis. We have isolated and characterized the gene for mouse PR3 (mPR3) and determined its chromosomal location. The gene has been localized to Chromosome (Chr) 10. Comparison of mouse PR3 genomic structure with that of its human counterpart indicates that: 1) the mPR3 gene spans 7 kb organized in 5 exons and 4 introns, 2) the codons of His-Asp-Ser of the catalytic site are conserved and spread out over different exons, similar to the human gene, and 3) the gene product encodes a pre-proform of the protein. Knowledge of the structure and chromosomal location of the mPR3 gene may help better the understanding of the temporal and cell-specific expression of mouse PR3.

Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions.

The Lasp-1 gene, which has been localized to the q12-q21 region of human chromosome 17, is amplified and overexpressed in human breast cancers. In addition to the previously reported LIM and SH3 domains of Lasp-1, we report here the identification of an actin-binding domain in the core of the protein. This domain is functional as we demonstrate that Lasp-1 binds actin in vivo and in vitro. In addition, confocal analysis of the Lasp-1 subcellular distribution shows that the protein is colocalized with actin at peripheral cell extensions in individual epithelial cancer cells and in transformed fibroblastic cells. Moreover, Lasp-1 is tyrosine phosphorylated in fibroblast cell lines transformed by a constitutively active form of c-Src (c-SrcY527F). Altogether, our results show that Lasp-1 defines a new type of actin-binding protein and suggest that the protein may play a role in a signaling pathway involved in the organization of the cytoskeleton.

MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis.

MLN64 is a protein that is highly expressed in certain breast carcinomas. The C terminus of MLN64 shares significant homology with the steroidogenic acute regulatory protein (StAR), which plays a key role in steroid hormone biosynthesis by enhancing the intramitochondrial translocation of cholesterol to the cholesterol side-chain cleavage enzyme. We tested the ability of MLN64 to stimulate steroidogenesis by using COS-1 cells cotransfected with plasmids expressing the human cholesterol side-chain cleavage enzyme system and wild-type and mutant MLN64 proteins. Wild-type MLN64 increased pregnenolone secretion in this system 2-fold. The steroidogenic activity of MLN64 was found to reside in the C terminus of the protein, because constructs from which the C-terminal StAR homology domain was deleted had no steroidogenic activity. In contrast, removal of N-terminal sequences increased MLN64's steroidogenesis-enhancing activity. MLN64 mRNA was found in many human tissues, including the placenta and brain, which synthesize steroid hormones but do not express StAR. Western blot analysis revealed the presence of lower molecular weight immunoreactive MLN64 species that contain the C-terminal sequences in human tissues. Homologs of both MLN64 and StAR were identified in Caenorhabditis elegans, indicating that the two proteins are ancient. Mutations that inactivate StAR were correlated with amino acid residues that are identical or similar among StAR and MLN64, indicating that conserved motifs are important for steroidogenic activity. We conclude that MLN64 stimulates steroidogenesis by virtue of its homology to StAR.

MLN64 exhibits homology with the steroidogenic acute regulatory protein (STAR) and is over-expressed in human breast carcinomas.

The MLN64 gene, which is localized in q12-q21 of the human chromosome 17, encodes a novel protein containing 2 distinct domains. At the N-terminal, MLN64 exhibits a potential trans-membrane region, while at the C-terminal, it shares homology with the F26F4.4 protein of Coenorhabditis elegans and the steroidogenic acute regulatory (StAR) protein, a mitochondrial protein which is involved in steroid-hormone synthesis. By comparing the C-terminal part of these proteins, we defined a novel protein domain, which we termed SHD for "StAR Homology Domain". Of the 93 primary invasive breast carcinomas that were examined, 14 were found to over-express MLN64. These 14 tumors also expressed high c-erbB-2 transcript levels, which were not detected in the MLN64-negative tumors. MLN64 mRNA and protein were specifically detected in malignant cells of breast carcinomas. MLN64 protein was localized within bundle-like structures distributed throughout the cell cytoplasm and condensed in a perinuclear patch, suggesting an association with a specific cell compartment. When the N-terminal part of MLN64 was deleted, MLN64 was uniformly distributed in the cell cytoplasm, indicating that N-terminal part is involved in the specific cytoplasmic localization of MLN64. The homology between the C-terminal part of MLN64 and the functional StAR domain (SHD) suggests that MLN64 and StAR, although distributed in different cellular compartments, may both play a role in steroidogenesis. In this case, the high levels of MLN64 observed in some breast carcinomas could contribute to the progression of these tumors through increased intratumoral steroidogenesis.

Two distinct amplified regions at 17q11-q21 involved in human primary breast cancer.

Chromosomal segment 17q11-q21 is a commonly amplified region in human breast carcinomas. Several lines of evidence suggest that ERBB2 is the gene responsible for the emergence of this amplicon, but four novel genes (called MLN 50, MLN 51, MLN 62, and MLN 64) in 17q11-q21 have recently been found to be amplified and overexpressed in breast cancer cell lines. We investigated 98 primary breast tumors for amplification of these five loci. Twenty-five tumors (25.5%) showed amplification of at least one of these markers, but most amplifications did not encompass all of the tested loci. The genes most frequently amplified were ERBB2 and MLN 64 (22 of 25 amplified cases). MLN 64 was always coamplified with ERBB2, and to a similar level. Amplification of these five genes always leads to overexpression of their mRNA; we observed no cases of overexpression without amplification in any of these genes. Our results suggest that: (a) an independent, amplified region defined by MLN 62 (also called CART1 or TRAF4) is located in 17q11-q12; (b) in addition to ERBB2, MLN 64 is a major target for the 17q12-q21 amplicon; and (c) these MLN genes could be of pathogenetic significance in breast cancer.

Lasp-1 (MLN 50) defines a new LIM protein subfamily characterized by the association of LIM and SH3 domains.

MLN 50 was previously identified in a cDNA library of breast cancer metastasis. In this study, we show that MLN 50, which is expressed at a basal level in normal tissues, is overexpressed in 8% of human breast carcinomas most often together with c-erbB-2. MLN 50 cDNA encodes a putative protein of 261 residues, named Lasp-1 (LIM and SH3 protein) since it contains a LIM motif and a domain of Src homology region 3 (SH3) at the amino- and the C-terminal parts of the protein, respectively. Thus, Lasp-1 defines a new LIM protein subfamily.

Comparative expression of the psoriasin (S100A7) and S100C genes in breast carcinoma and co-localization to human chromosome 1q21-q22.

Using differential screening of a breast cancer cDNA library, we isolated a cDNA encoding the psoriasin (S100A7) protein, previously identified in psoriatic epidermis. In the present study, we demonstrate that the psoriasin gene is expressed in breast cancer cell lines and in cancer cells of some breast carcinomas but not in any non-cancerous tissues examined, except skin. Another S100 gene, S100C, which we co-localized with the psoriasin gene to human chromosome 1q21-q22, was found to be expressed in most tissues and cell lines evaluated. These findings add support to the concept that the S100 genes clustered in human chromosome 1q21-q22 are individually controlled and that some of them may be involved in the regulation of cell transformation and/or differentiation.

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.

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 domain 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.

Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17.

We have performed differential screening of a human metastatic lymph lymph node cDNA library to identify genes possibly involved during breast cancer progression. We have identified four novel genes overexpressed in malignant tiddues. They were all located on the long arm of chromosome 17, in loci located between q11 and q21.3, a region known to contain the c-erbB-2 oncogene and the BRCA1 breast carcinomas, and overexpression of three of them was dependent on gene amplification in breast cancer cell lines. These findings further support the concept that human chromosome 17 specifically carries genes possibly involved in breast cancer progression.