Mutant p53 upregulates alpha-1 antitrypsin expression and promotes invasion in lung cancer.

Missense mutations in the TP53 tumor-suppressor gene inactivate its antitumorigenic properties and endow the incipient cells with newly acquired oncogenic properties that drive invasion and metastasis. Although the oncogenic effect of mutant p53 transcriptome has been widely acknowledged, the global influence of mutant p53 on cancer cell proteome remains to be fully elucidated. Here, we show that mutant p53 drives the release of invasive extracellular factors (the 'secretome') that facilitates the invasion of lung cancer cell lines. Proteomic characterization of the secretome from mutant p53-inducible H1299 human non-small cell lung cancer cell line discovered that the mutant p53 drives its oncogenic pathways through modulating the gene expression of numerous targets that are subsequently secreted from the cells. Of these genes, alpha-1 antitrypsin (A1AT) was identified as a critical effector of mutant p53 that drives invasion in vitro and in vivo, together with induction of epithelial-mesenchymal transition markers expression. Mutant p53 upregulated A1AT transcriptionally through the involvement with its family member p63. Conditioned medium containing secreted A1AT enhanced cell invasion, while an A1AT-blocking antibody attenuated the mutant p53-driven migration and invasion. Importantly, high A1AT expression correlated with increased tumor stage, elevated p53 staining and shorter overall survival in lung adenocarcinoma patients. Collectively, these findings suggest that A1AT is an indispensable target of mutant p53 with prognostic and therapeutic potential in mutant p53-expressing tumors.

Mutant p53 drives invasion in breast tumors through up-regulation of miR-155.

Loss of p53 function is a critical event during tumorigenesis, with half of all cancers harboring mutations within the TP53 gene. Such events frequently result in the expression of a mutated p53 protein with gain-of-function properties that drive invasion and metastasis. Here, we show that the expression of miR-155 was up-regulated by mutant p53 to drive invasion. The miR-155 host gene was directly repressed by p63, providing the molecular basis for mutant p53 to drive miR-155 expression. Significant overlap was observed between miR-155 targets and the molecular profile of mutant p53-expressing breast tumors in vivo. A search for cancer-related target genes of miR-155 revealed ZNF652, a novel zinc-finger transcriptional repressor. ZNF652 directly repressed key drivers of invasion and metastasis, such as TGFB1, TGFB2, TGFBR2, EGFR, SMAD2 and VIM. Furthermore, silencing of ZNF652 in epithelial cancer cell lines promoted invasion into matrigel. Importantly, loss of ZNF652 expression in primary breast tumors was significantly correlated with increased local invasion and defined a population of breast cancer patients with metastatic tumors. Collectively, these findings suggest that miR-155 targeted therapies may provide an attractive approach to treat mutant p53-expressing tumors.

Mutant p53 drives multinucleation and invasion through a process that is suppressed by ANKRD11.

Mutations of p53 in cancer can result in a gain of function associated with tumour progression and metastasis. We show that inducible expression of several p53 'hotspot' mutants promote a range of centrosome abnormalities, including centrosome amplification, increased centrosome size and loss of cohesion, which lead to mitotic defects and multinucleation. These mutant p53-expressing cells also show a change in morphology and enhanced invasive capabilities. Consequently, we sought for a means to specifically target the function of mutant p53 in cancer cells. This study has identified ANKRD11 as a key regulator of the oncogenic potential of mutant p53. Loss of ANKRD11 expression with p53 mutation defines breast cancer patients with poor prognosis. ANKRD11 alleviates the mitotic defects driven by mutant p53 and suppresses mutant p53-mediated mesenchymal-like transformation and invasion. Mechanistically, we show that ANKRD11 restores a native conformation to the mutant p53 protein and causes dissociation of the mutant p53-p63 complex. This represents the first evidence of an endogenous protein with the capacity to suppress the oncogenic properties of mutant p53.

Systematic characterisation of the rat and human CYP24A1 promoter.

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) ligands VDR (vitamin D receptor) and binds to the vitamin D response element (VDRE) located within target genes to regulate their transcription. Previously we showed that 1,25D-mediated rat CYP24A1 induction via the two critical VDREs is dependent on a short stretch of nucleotides called vitamin D stimulating element (VSE), located approximately 30bp upstream of VDRE-1 in the rat CYP24A1 promoter. We have now undertaken systematic analysis of the human CYP24A1 and rat CYP24A1 promoters to determine if the VSE is present in the human promoter. Using electrophoretic mobility shift and dual-luciferase reporter assays, we show that the VSE is absent in the human CYP24A1 promoter. In addition, we show that 1,25D-mediated induction of human CYP24A1 is dependant upon a promoter region spanning nucleotides -470 to -392 of the human CYP24A1 promoter.

Expression of ZNF652, a novel zinc finger protein, in vulvar carcinomas and its relation to prognosis.

AIMS: To determine the levels of expression of ZNF652 and its relevance to prognosis in vulvar squamous cell carcinomas. METHODS: 22 cases of vulvar intraepithelial neoplasia (VIN) and tumours from 217 patients with vulvar squamous cell carcinomas were investigated for expression of ZNF652 using immunostaining methods. The effect of ZNF652 ectopic expression was determined in the vulvar carcinoma cell line SW954 by western and cell-based assays. RESULTS: High levels of ZNF652 nuclear expression were observed in 5 (100%) of VIN I, 6 (75%) of VIN II and 109 (50.2%) of the vulvar carcinomas, whereas low levels were seen in 2 (25%) VIN II, 9 (100%) of VIN III and 108 (49.8%) of the vulvar carcinomas. High levels of ZNF652 expression in the vulvar carcinomas were significantly correlated to high expression of EphA2. However, when correcting for multiple testing this correlation was lost. No association was identified between ZNF652 expression and p16, p21, p27, p53, cyclin A, D1, D3, E, EphrinA-1 and human papillomavirus. Variations in levels of ZNF652 were not related to prognosis. Low levels of ZNF652 protein were identified in the vulvar carcinoma cell line SW954. Furthermore, SW954 cells ectopically expressing ZNF652 showed reduced cell proliferation and the ability to form colonies on plastic. CONCLUSIONS: ZNF652 protein expression is reduced in 25% of VIN II, 100% of VIN III and approximately 50% of the cases of vulvar squamous cell carcinoma, and may be an early event in the pathogenesis of vulvar squamous cell carcinoma. Variations in the levels of ZNF652 were not related to patient's prognosis.

Partial androgen insensitivity syndrome and t(X;5): are there upstream regulatory elements of the androgen receptor gene?

BACKGROUND/AIMS: Two half-brothers with similar malformed genitals, who both inherited a maternally derived t(X;5)(q13;p15) translocation, have a phenotype consistent with partial androgen sensitivity syndrome. The aim was to identify the gene disrupted by the X chromosome breakpoint. METHODS: The breakpoint was localized using fluorescence in situ hybridization to metaphase spreads of the translocation. RESULTS: The breakpoint on the X chromosome of the X;5 translocation was localized to a 30-kb region. This region does not contain any identified genes or transcripts. However, the breakpoint is approximately 134 kb from the 5' end of the androgen receptor (AR) gene. CONCLUSIONS: Genetic defects of the AR gene are collectively called androgen insensitivity syndrome and include a range of phenotypes from normal males, often with associated sterility, to XY females. The phenotype seen in the males with the t(X;5) is consistent with this syndrome. The analysis of the chromosomal abnormality suggests that this translocation may remove one or more upstream regulatory elements of the AR gene that are essential for its normal expression and its role in typical external masculinization.

CARD15/NOD2 risk alleles in the development of Crohn's disease in the Australian population.

We have previously reported strong evidence for linkage between IBD1 and Crohn's disease (CD) in Australian Crohn's disease families. Three risk alleles for Crohn's disease, (Arg702Trp (C/T), Gly908Arg (G/C) and 980fs981 (-/C), were recently identified in the CARD15/NOD2 gene on chromosome 16, implicating this as the IBD1 locus. Using a novel diagnostic PCR-RFLP, we have examined the frequency of these alleles in 205 multiplex IBD families, 107 sporadic Crohn's disease cases and 409 normal individuals. We demonstrate that the three risk alleles are more frequent in Crohn's disease, than in controls, with allelic frequencies of 0.11, 0.02 and 0.07 respectively. Heterozygosity for individual variants conferred a three-fold increase in risk for Crohn's disease while substantially higher risks were associated with being homozygous or compound heterozygous. Despite a significantly lower population allele frequency for the frameshift mutation than reported by other groups, we see a similar contribution by this allele to the risk of developing Crohn's disease. While the three risk alleles influence susceptibility to Crohn's disease in Australia, we show that these alleles do not fully explain the linkage evidence and suggest that there are very likely additional IBD1 susceptibility alleles yet to be described in Australian CD at the NOD2 locus. We also show a second linkage peak in Australian CD that provides some support for a second disease susceptibility locus on chromosome 16.

A complex rearrangement involving simultaneous translocation and inversion is associated with a change in chromatin compaction.

Detailed fluorescence in situ hybridisation analysis of a previously described translocation revealed it to be a more complex rearrangement consisting of both a translocation and a paracentric inversion with an apparent coincident breakpoint at 16p13.3, t(14;16)(p32;p13.3) inv16(p13.3p12.1). This unusual three-breakpoint rearrangement was not obvious from examination of G-banding. Such rearrangements may be undiagnosed in cytogenetic studies. The presence of an interstitial deletion of 16p was unlikely as the rearranged chromosome contained probes distributed along the short arm of chromosome 16. Fluorescence in situ hybridisation studies suggested that the inverted segment was smaller in size than that on the normal chromosome. Measurements of distances between probes on metaphase chromosomes confirmed that there was differential compaction of the inverted portion on 16p. The inverted region was significantly reduced in size by 21% compared with the same region on the normal chromosome 16. The size reduction across the region was non-uniform, with one region showing a 55% increase in compaction. The change in compaction was also associated with a change in the lateral position of a probe on the chromatids. The finding that a single chromosome breakpoint can change the compaction of chromatin over an extensive region has implications for models of the structure of metaphase chromosomes. Possible explanations are either a localized severe disruption of DNA packaging over relatively short distances (hundreds of kilobases) or a more generalized change that extends over many megabases. These results raise the important possibility that chromosome breaks may result in a more global change in DNA compaction across large segments of a chromosome.

A familial cryptic subtelomeric deletion 12p with variable phenotypic effect.

A 15-year-old-boy and his mother, both carrying a cryptic deletion within 12p13.33, are described. The proband has a mild phenotype with moderate mental retardation and severe behavioural problems. The mother had some learning difficulties at school. Conventional GTL-banded high-resolution chromosome analysis showed normal karyotypes. Subsequent analysis by fluorescence in situ hybridization using a set of probes specific for the subtelomeric regions of all chromosomes, plus a series of probes at 12p13.33 extending from the 12p telomere, showed that both mother and son carry a 1.65 Mb terminal deletion in this region. There are 10 predicted genes within the deleted region. The unanticipated familial nature of the deletion emphasizes the value of family studies in all cases with subtelomeric abnormalities. It also demonstrates the difficulty in making a clinical diagnosis of individuals with this deletion. To the best of the present authors' knowledge, the proband and his mother are the first patients described with a submicroscopic deletion at 12p13.33.

A novel Q378X mutation exists in the transmembrane transporter protein ABCC6 and its pseudogene: implications for mutation analysis in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is an inherited disorder of the elastic tissue with characteristic progressive calcification of elastic fibers in skin, eye, and the cardiovascular system. Recently mutations in the ABCC6 gene, encoding a transmembrane transporter protein, were identified as cause of the disease. Surprisingly, sequence and RFLP analysis for exon 9 with primers corresponding to flanking intronic sequence in diseased and haplotype negative members from all of our families and in a control population revealed either a homozygous or heterozygous state for the Q378X (1132C-->T) nonsense mutation in all individuals. With the publication of the genomic structure of the PXE locus we had identified the starting point of a large genomic segmental duplication within the locus in the cytogenetic interval defined by the Cy19 and Cy185 somatic cell hybrid breakpoints on chromosome 16p13.1. By means of somatic cell hybrid mapping we located this starting point telomeric to exon 10 of ABCC6. The duplication, however, does not include exon 10, but exons 1-9. These findings suggest that one or several copies of an ABCC6 pseudogene (psiABCC6) lie within this large segmental duplication. At least one copy contains exons 1-9 and maps to the chromosomal interval defined by the Cy163 and Cy11 breakpoints. Either this copy and/or an additional copy of psiABCC6 within Cy19-Cy183 carries the Q378X mutation that masks the correct identification of this nonsense mutation as being causative in pseudoxanthoma elasticum. Long-range PCR of exon 9 starting from sequence outside the genomic replication circumvents interference from the psiABCC6 DNA sequences and demonstrates that the Q378X mutation in the ABCC6 gene is associated with PXE in some families. These findings lead us to propose that gene conversion mechanisms from psiABCC6 to ABCC6 play a functional role in mutations causing PXE.

Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein.

The identification of SPG7 as the gene defective in a recessive form of spastic paraplegia has drawn attention to the yeast protein family of ATP-dependent zinc metalloproteases. The protein encoded by SPG7, paraplegin, shows high homology to members of this protein family. Recently, many mammalian ATP-dependent zinc metalloproteases have been identified and considered as possible candidates for defects in other forms of hereditary spastic paraplegia and possibly other neurodegenerative disorders. So far only a partial sequence has been available for one of those genes, ATPase family gene-3, yeast-like-1 (AFG3L1). We have carried out detailed molecular analysis of this gene and identified and characterized its mouse orthologue, Afg3l1. Our data indicate that AFG3L1 is transcribed into four mRNA isoforms that are not translated in humans. Afg3l1 encodes a protein with high homology to paraplegin and the other members of the ATP-dependent zinc metalloprotease family. Like the other ATP-dependent zinc metalloproteases, Afg3l1 localizes to the mitochondria.

Analysis of lymphoedema-distichiasis families for FOXC2 mutations reveals small insertions and deletions throughout the gene.

Lymphoedema-distichiasis (LD) is a dominantly inherited form of primary lymphoedema with onset of lower limb swelling at puberty or later. There is variable penetrance of this disorder, but the most consistently inherited feature is distichiasis, viz. fine hairs arising inappropriately from the meibomian glands. We established linkage of this disorder to 16q24.3 and the gene has recently been identified as the forkhead transcription factor FOXC2. We report the mutational analysis of 14 families with LD. All but one of these pedigrees have small insertions or deletions in the gene, which seem likely to produce haploinsufficiency. The mutation sites are scattered throughout the gene. There is one family with a mis-sense mutation in the forkhead domain of the protein. This base alteration is not a common polymorphism, is co-inherited with the disease and produces a non-conservative amino acid change.

Integration of cytogenetic landmarks into the draft sequence of the human genome.

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.

Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions.

Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.

Origins of accessory small ring marker chromosomes derived from chromosome 1.

Three patients with accessory small ring chromosomes derived from chromosome 1 are presented together with additional clinical details and cytogenetic analyses of a previously reported patient. Cytogenetic analysis was undertaken by FISH using a reverse painting probe generated from one of the patients by microdissection of the r(1) chromosome and with a BAC923C6 which maps to 1p12. Results indicated that patients with r(1) chromosomes consisting of 1q12 heterochromatin and short arm pericentric euchromatin which extends to at least the BAC923C6 were associated with a normal or mild phenotype. Patients with abnormal phenotypes possessed two types of rings. One patient had evidence for contiguous pericentric short arm euchromatin which extended from the centromere to beyond the BAC923C6. Two patients showed molecular cytogenetic results which were compatible with non-contiguous chromosome 1 euchromatin. The diversity of origin of r(1)s will hamper attempts to define phenotype/genotype relationships.

C16orf5, a novel proline-rich gene at 16p13.3, is highly expressed in the brain.

A novel gene has been characterized, designated C16orf5, with an unusually high content of proline residues (40% over 104 residues) at the N-terminus of the protein. The C-terminus of the protein is also cysteine rich with 14 cysteine residues present. Analysis using Northern and dot blots showed that the highest expression of this gene is in the brain. The gene was located on chromosome 16 at band p13.3 by FISH to metaphase chromosomes. Southern blot analysis with a human-rodent somatic cell hybrid panel showed a location between the somatic hybrid breakpoints 23HA and CY196. This gene comprises at least four exons and an open reading frame of 786 bp encoding a predicted protein of 261 amino acids. Analysis of this protein using PSORTII predicted a nuclear localization.

Characterization of copine VII, a new member of the copine family, and its exclusion as a candidate in sporadic breast cancers with loss of heterozygosity at 16q24.3.

In a search for candidate tumor suppressor genes within a 650-kb common region of loss of heterozygosity (LOH) at 16q24.3 in breast cancer tissues, a 2.6-kb cDNA, named copine VII (CPNE7), was characterized. The gene is 2654 bp and codes for a 633-residue protein with high homology to the other members of the copine family, such as copine I, copine III, and N-copine. The predicted amino acid sequence contains two copies of a C2 domain in the N-terminus. Since these domains have been found in several membrane-binding proteins involved in different intracellular processes, copine VII was viewed as a potential tumor suppressor gene. Mutation analysis was carried out by single-strand conformation polymorphism analysis of 18 breast tumor tissue samples with ascertained LOH on chromosome 16q24.3. Since only two polymorphisms were identified, no evidence was found to indicate that copine VII is the tumor suppressor gene at 16q24.3 involved in breast cancer.

Genomic structure and expression analysis of the spastic paraplegia gene, SPG7.

SPG7 is a newly identified gene involved in an autosomal recessive form of hereditary spastic paraplegia (HSP), a genetically heterogeneous group of neurodegenerative disorders. This gene encodes a protein characterized as a nuclear-encoded mitochondrial metalloprotease. The present report describes the genomic structure of the SPG7 gene. It is organized into 17 exons ranging from 78 to 242 bp and spans approximately 52 kb within three overlapping cosmids. The exon/intron boundaries and all splice junctions are consistent with the published consensus sequences for donor and acceptor sites. The provided genomic structure of SPG7 should facilitate the screening for mutations in this gene in patients with HSP and other related mitochondrial disease syndromes. SPG7 has been mapped to chromosome 16q24.3, a region of frequent loss of heterozygosity (LOH) seen in sporadic breast and prostate cancer. We have performed single-strand conformation polymorphism analysis of ten exons of this gene in a number of sporadic breast cancer samples showing LOH at 16q24.3. No mutations were detected; only single nucleotide polymorphisms were observed in exon 11, intron 7, intron 10 and intron 12. An expression analysis study has revealed the differential expression of SPG7 mRNA in various tissues and at different developmental stages.