Francesco Acquati

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the Down critical region

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so‐called ‘Down critical region’ in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described β‐secretase enzyme (BACE‐1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

Characterization of Multiple Enhancer Regions Upstream of the Apolipoprotein(a) Gene

Plasma concentrations of the atherogenic lipoprotein(a) (Lp(a)) are predominantly determined by inherited sequences within or closely linked to the apolipoprotein(a) gene locus. Much of the interindividual variability in Lp(a) levels is likely to originate at the level of apo(a) gene transcription. However, the liver-specific apo(a) basal promoter is extremely weak and does not exhibit common functional variations that affect plasma Lp(a) concentrations. In a search for additional apo(a) gene control elements, we have identified two fragments with enhancer activity within the 40-kilobase pair apo(a)-plasminogen intergenic region that coincide with DNase I-hypersensitive sites (DHII and DHIII) observed in liver chromatin of mice expressing a human apo(a) transgene. Neither enhancer exhibits tissue specificity. DHIII activity was mapped to a 600-base pair fragment containing nine DNase I-protected elements (footprints) that stimulates luciferase expression from the apo(a) promoter 10–15-fold in HepG2 cells. Binding of the ubiquitous transcription factor Sp1 plays a major role in the function of this enhancer, but no single site was indispensable for activity. DHIII comprises part of the regulatory region of an inactive long interspersed nucleotide element 1 retrotransposon, raising the possibility that retrotransposon insertion can influence the regulation of adjacent genes. DHII enhancer activity was localized to a 180-base pair fragment that stimulates transcription from the apo(a) promoter 4–8-fold in HepG2 cells. Mutations within an Sp1 site or either of two elements composed of direct repeats of the nuclear hormone receptor half-site AGGTCA in this sequence completely abolished enhancer function. Both nuclear hormone receptor elements were shown to bind peroxisome proliferator-activated receptors and other members of the nuclear receptor family, suggesting that this enhancer may mediate drug and hormone responsiveness.

Transcriptional deregulation and a missense mutation define ANKRD1 as a candidate gene for total anomalous pulmonary venous return.

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect in which the pulmonary veins fail to enter the left atrium and drain instead into the right atrium or one of its venous tributaries. Although a genetic basis for TAPVR has long been recognized, no single gene involved in the pathogenesis of this disease has been identified to date. We previously reported a TAPVR patient bearing a de novo 10;21 balanced translocation. In this work, we cloned both translocation breakpoints from this patient and mapped the ANKRD1 gene, encoding a cardiac transcriptional regulator, 130 kb proximally to the breakpoint on chromosome 10. In situ hybridization analysis performed on murine embryos showed ANKRD1 expression in the developing pulmonary veins, suggesting a possible role for this gene in TAPVR pathogenesis. Moreover, ANKRD1 expression levels were found to be highly increased in lymphoblastoid cell lines derived from both the translocation‐bearing proband and a second independent sporadic TAPVR patient, suggesting that disruption of the normal ANKRD1 expression pattern is associated with TAPVR. Finally, a nonconservative missense mutation in the ANKRD1 gene was found in a third sporadic TAPVR patient. In vitro calpain‐mediated degradation assays, coupled to reporter gene analysis in transfected HeLa cells, strongly suggested that this mutation enhances both the stability of the ANKRD1/CARP protein and its transcriptional repression activity upon the cardiac‐specific atrial natriuretic factor (ANF) promoter. Taken together, these results define ANKRD1 as a possible candidate gene for TAPVR pathogenesis. Hum Mutat 29(4), 468–474, 2008.

Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.

Physical map of the D6S149-D6S193 region on chromosome 6Q27 and its involement in benign surface epithelial ovarian tumours

A detailed long range restriction map of the region defined by markers D6S149 and D6S193 on chromosome 6q27 has been constructed. This was achieved by YAC cloning and contig assembling of the same region. Seven YAC clones were found to span the almost 1000 Kb region flanked by the two markers which on the genetic map resulted to be 1.9 cM apart. With some of the characterized YAC clones we undertook a molecular cytogenetic analysis of 20 benign ovarian tumors. The rationale for this was the recent mapping to a region of chromosome 6q27, flanked by markers D6281 and D6S133, of a locus for the SV40-mediated immortalization of human cells (SEN6 gene). Noteworthy we found that the the D6S149-D6S193 region (comprised in the larger D6S281-D6S133 physical interval) was altered in all samples analysed adding support to the occurrence of a immortalization step in this type of tumors.

RNASET2 as a tumor antagonizing gene in a melanoma cancer model.

The RNASET2 gene, mapped in 6q27, was previously found to exert control of tumorigenesis in an ovarian cancer system. We present here results indicating a similar control in a melanoma cancer model. Thus, this gene is most likely involved in a common general pathway of tumorigenesis. Moreover, its antitumorigenic activity is manifested in vivo but not in vitro, suggesting that this gene belongs to the growing category of tumor antagonizing/malignancy suppressor genes. A possible role of RNASET2 in the activation of a senescence program, whose responsible locus was mapped in the same chromosomal 6q27 region, seems to be inconsistent with our data.

Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease.

Genetic and cytogenetic observations among different types of ovarian tumors are compatible with a progression model underlying ovarian tumorigenesis

In this report we present the characterization of ovarian neoplasms including benign tumors, borderline tumors, and invasive carcinomas in order to assess whether a sharing of cytogenetic abnormalities is present in all three types of tumors. A cohort of 114 newly diagnosed and untreated ovarian epithelial tumors were analyzed by cytogenetic and molecular cytogenetic approaches with probes specific for chromosome 6. Three groups of chromosome abnormalities were identified: the first group included abnormalities common to all tumor classes (losses of chromosomes 6, 8, 10, 11, 15, 16, 17, 18, 19, 20, 21, 22, and X; gains of chromosomes 1, 3, 5, and 12; 6q24 approximately qter deletions); the second group presented specific abnormalities present in malignant but not in benign tumors (losses of chromosomes 2, 7, 13, and 14; gains of chromosome 4 and chromosome markers); and the last group included abnormalities unique to invasive carcinomas (loss of chromosome 4; gains of chromosomes 2, 7, 8, 9, 10, 16, 17, 18, 19, 20, and 21; 6q16 approximately q24 deletions; rearrangements of 3p, 3q, 13q, and 21q regions). The presence of shared chromosomal alterations in all three types of ovarian neoplasms investigated in this report seems therefore to suggest a progression model for these types of tumors.

Intracellular trafficking of RNASET2, a novel component of P-bodies.

The ribonucleases (RNases) constitute a heterogeneous group of enzymes, which exert diverse and specific biological functions. Several RNases have been shown to control gene expression and cell differentiation. RNASET2, a novel member of the Rh/T2/S family of RNases, exerts micro‐environmental control of malignancy in different experimental models with a general onco‐suppressor activity, since it prevents cancer proliferation. Indeed, RNASET2 was found to be downregulated at the transcript level in several primary ovarian tumours or cell lines and in melanoma cell lines. Although recent works shed light on the biological role of RNASET2 in delaying tumour growth, its trafficking within the cell is still poorly understood. RNASET2 seems to play diverse biological roles including turnover of tRNA in yeast as well as rRNA degradation in zebrafish.