Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases.

BACKGROUND: The WASF3 protein is involved in cell movement and invasion, and to investigate its role in prostate cancer progression we studied the phenotypic effects of knockdown in primary tumors and cell lines. METHODS: ShRNA was used to knockdown WASF3 function in prostate cell lines. Cell motility (scratch wound assay), anchorage independent growth and in vivo tumorigenicity and metastasis were then compared between knockdown and wild-type cells. RESULTS: Increased levels of expression were seen in high-grade human prostate cancer and in the PC3 and DU145 cell lines. Inactivation of WASF3 using shRNAs reduced cell motility and invasion in these cells and reduced anchorage independent growth in vitro. The loss of motility was accompanied by an associated increase in stress fiber formation and focal adhesions. When injected subcutaneously into severe combined immunodeficiency (SCID) mice, tumor formation was significantly reduced for PC3 and DU145 cells with WASF3 knockdown and in vivo metastasis assays using tail vain injection showed a significant reduction for PC3 and DU145 cells. The loss of the invasion phenotype was accompanied by down-regulation of matrix metalloproteinase 9. CONCLUSIONS: Overall, these observations demonstrate a critical role for WASF3 in the progression of prostate cancer and identify a potential target to control tumorigenicity and metastasis.

Distinct molecular signatures in pediatric infratentorial glioblastomas defined by aCGH.

Glioblastomas (GBM) are rare in children, but reportedly have more varied outcome which suggests differences in tumor etiology compared to typical GBM of adults. To investigate this we performed high resolution array comparative genomic hybridization (aCGH) analysis on three pediatric infratentorial GBM, ages 3.5, 7 and 14 years. Two of these tumors occurred in the brainstem and one in the spinal cord. While histologically typical, one brainstem tumor showed mainly pleomorphic astrocytic cells, whereas the other brainstem and spinal tumors showed a GFAP positive small cell component. Whole chromosomal gains (#1 and #2) and loss (#20) were seen only in the pleomorphic brainstem GBM, which also showed a high level of segmental genomic copy number changes. Segmental loss involving chromosome 8 was seen in all three tumors (Chr8;133039446-136869494, Chr8;pter-3581577, and Chr8;pter-30480019 respectively), whereas loss involving chromosome 16 was seen in only 2 cases with small cell components (Chr16;31827239-qter and Chr16;pter-29754532). Segmental gain of chromosome 7 was shared only between the 2 brainstem cases (Chr7;17187166-qter and Chr7;69824947-qter). Chromosome 17 showed segmental gain of 17q in the backdrop of loss of 17p only in case 1. Segmental gain of chromosome 1q was seen only in case 2. The spinal GBM showed a relatively stable karyotype with a unique loss of Chr19;32848902-qter. None of the frequent losses, gains and amplifications known to occur in adult GBM were identified, suggesting that pediatric infratentorial glioblastomas show a molecular karyotype that was more characteristic of pediatric embryonal tumors than adult GBM.

The EVI5 TBC domain provides the GTPase-activating protein motif for RAB11.

The human EVI5 gene was originally isolated through its involvement with a constitutional chromosome translocation in a patient with stage 4S neuroblastoma. Recently, it has been shown that EVI5 is a centrosomal protein in interphase cells, which relocalizes to the midbody during late phases of mitosis. Disruption of its function leads to incomplete cell division and the formation of multinucleate cells. The EVI5 protein contains a TBC (TRE2/BUB/CDC16 homology) motif located in the N-terminal region. Proteins containing a TBC domain have been shown in some cases to act as GTPase-activating proteins (GAPs) and function through the interaction with Rab-like small G proteins. Despite the identification of over 50 TBC-containing proteins, and over 70 Rab-like proteins, only three combinations have been shown to have Rab/GAP activity to date. In this study, using linear ion trap mass spectroscopy, we have demonstrated that EVI5 exists in a protein complex with Rab11. Further, using a specific Rab-binding assay, we have shown that EVI5 preferentially interacts with the guanosine triphosphate-bound form of Rab11, and in a GAP activity assay, we have confirmed that EVI5 functions as a GAP for the Rab11 GTPase.

Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells.

Inhibition of the nonsense-mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene identification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations. Genes containing frameshift mutations in colon cancer cell line have been identified using a modified version of GINI. To increase the efficiency of identifying mutant genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhibiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analysed colon cancer cell lines showing microsatellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC31L1, NCOR1, BAT3, PHF14, ZNF294, C19ORF5 genes as well as genes coding for proteins with yet unknown functions.

Consistent chromosome abnormalities associated with mouse bladder epithelial cell lines transformed in vitro.

Nine epithelial tumor cell lines that were derived from inbred C57BL/lcrf-at adult male mouse bladder and were transformed in vitro were cytogenetically analyzed. Most cell lines were near-diploid and showed only minor karyotypic deviations from normal. Tetraploid cell lines showed duplication of chromosome markers, which suggested that they arose originally as near-diploid cell lines with minor karyotypic changes. Chromosome abnormalities were consistent among different cell lines and involved chromosomes #6, #3, and #15. The Y-chromosome was frequently missing. Identical chromosome abnormalities were present in cell lines that arose in control, dimethyl sulfoxide-treated, or 7, 12-dimethylbenz[a]anthracene-treated cultures. The most frequent abnormality was was the presence of three copies (trisomy) of chromosome #6. Few structural chromosome abnormalities were detected, although a marker chromosome derived from chromosome #3 was present in several cell lines. Two cell lines had excesses of chromosome #15. These results suggest that excesses of individual chromosomes or parts of chromosomes, particularly chromosome #6, may be important in the expression of the malignant phenotype in mouse bladder epithelium.

Changes in DNA content during in vitro transformation of mouse salivary gland epithelium.

DNA content was measured in single cells in situ during the in vitro development of neoplastic transformation in epithelial cells from mouse salivary glands. The earliest change in DNA content was found in stage III of the transformation process (a preneoplastic stage). In foci of these cells, the percentage of tetraploid (4C) cells was 30--60% compared with the starting tissue and early primary cultures, which were diploid. The stage III foci eventually gave rise to subtetraploid tumorigenic cell lines. The studies suggested that during in vitro development of neoplastic transformation in salivary gland epithelium, tetraploids were generated that then underwent a period of chromosome instability and loss. Evidence was also presented that a similar mechanism may be operating in transformation of salivary gland epithelium in vivo. A possible mechanism for transformation on the basis of chromosome imbalance was discussed.

Chromosome changes associated with the progression of cell lines from preneoplastic to tumorigenic phenotype during transformation of mouse salivary gland epithelium in vitro.

Two mouse salivary gland epithelial cell lines, CSG 211 and CSG 205/2B1, isolated during carcinogen-induced neoplastic transformation in vitro, were analyzed cytogenetically before and after they acquired the ability to produce carcinomas in syngeneic animals. With the use of Giemsa banding techniques, chromosome changes were identified that were associated with the transition from a preneoplastic to a fully transformed (tumorigenic) phenotype during serial passage in vitro. Results were compared with those from a third cell line of similar origin, CSG 225, which was tumorigenic at the earliest passage tested. These cell lines were found to be subtetraploid, which confirms previous data, and the tumorigenic lines showed consistent losses of copies of chromosomes 1, 4, 7, 9, and 14. Compared with their preneoplastic counterparts, the loss of no single chromosome seems to be sufficient to generate the tumorigenic phenotype, but the loss of a combination of some or all of these chromosomes appears to be important in the phenotypic transition. In CSG 211 the loss of chromosome 7 is probably more important in this respect than loss of the other chromosomes listed. The karyotype of this cell line undergoes major structural rearrangement, which suggests that loss of specific regions of chromosomes 1 and 9 is also important.

Presence of two cytogenetic forms of amplified DNA: evidence for a role in tumor growth in an intraspecific mouse hybrid cell line.

An intraspecific mouse hybrid epithelial cell line, F5/B, is described in which the homogeneously staining region (HSR)-containing marker chromosome from one parent is absent in about half of the cells. It is replaced in these cells by double minutes (DM), an alternative form of amplified DNA, which is liable to loss because of its instability at mitosis. DM probably arise from the breakdown of the HSR during clonal growth of F5/B. Subclones were derived possessing one or another cytogenetic feature, and their cloning efficiency in vitro and tumorigenicity in syngeneic animals were compared. There were no differences in in vitro tumorigenicity, but in vivo DM-containing subclones were significantly less tumorigenic than HSR-containing subclones or the F5/B parent hybrid. In tumors that developed after long latent periods, cells had increased numbers of DM compared with the inoculated population, demonstrating a selective advantage in vivo for cells with a high DM content. These results indicate a role for the amplified DNA in tumor growth.

Chromosome abnormalities associated with salivary gland epithelial cell lines transformed in vitro and in vivo with evidence of a role for genetic imbalance in transformation.

Chromosomal abnormalities associated with five in vitro-transformed male mouse salivary gland epithelial cell lines were compared with those in three cell lines derived from in vivo-induced tumors. All cell lines were hypotetraploid. Structural chromosome abnormalities were found in all cell lines, but no consistent aberration was detected. Nevertheless, losses of chromosomes 1, 4, 7, 9, and 14 were observed in all of the in vitro-transformed cell lines. With the possible exception of chromosome 1, the same chromosome losses were noted in the in vivo-transformed cell lines. In addition, a consistent feature of both in vitro- and in vivo-transformed cell lines was the presence of double minute chromosomes and homogeneously staining regions. Where both of these chromosome types were present in the same cell line, they were mutually exclusive. The Y chromosome was absent in nearly all of the cell lines. These findings are consistent with the view that, in salivary gland epithelium, the malignant phenotype may result from a genetic imbalance caused by specific chromosome losses from tetraploid cells.

Transformation of human CD34+ hematopoietic progenitor cells with DEK-NUP214 induces AML in an immunocompromised mouse model.

Acute myeloid leukemia (AML) is a heterogeneous disease comprising a large number of subtypes defined by specific chromosome abnormalities. One such subtype carries the t(6;9)(p22;q34) chromosome rearrangement, which leads to expression of the DEK-NUP214 chimeric gene, and has a particularly poor outcome. To provide a better understanding of the molecular etiology of these relatively rare individual AML variants, it is necessary to generate in vivo models, which can also serve as a means to evaluate targeted therapies based on their specific genetic abnormalities. Here, we describe the development of a human cell AML, generated in CD34+ human hematopoietic progenitor cells xenografted into immunocompromised mice that express human myeloid cell growth factors. Within 6 months, these mice develop a human cell AML with phenotypic characteristics of the primary t(6;9) disease and a CD45+CD13+CD34+CD38+ immunophenotype. Gene expression studies show that members of the HOX family of genes (HOXA9, 10, B3, B4 and PBX3) are highly upregulated in the AML from this mouse model as well as from primary human t(6;9) AML. Gene expression analysis also identified several other significantly disregulated pathways involving KRAS, BRCA1 and ALK, for example. This is the first report of a humanized model of the DEK-NUP214 disease and provides a means to study the development and treatment of this particular subtype of AML.

WASF3 provides the conduit to facilitate invasion and metastasis in breast cancer cells through HER2/HER3 signaling.

The WASF3 gene is overexpressed in high-grade breast cancer and promotes invasion and metastasis, but does not affect proliferation. The HER2/ERBB2/NEU gene is also frequently overexpressed in breast cancer, and has been shown to promote invasion and metastasis in these tumors. Here, we show that WASF3 is present in the HER2 immunocomplex and suppression of WASF3 function leads to suppression of invasion even in the presence of HER2 expression. Overexpression of both HER2 and WASF3 in non-metastatic MCF7 breast cancer cells promotes invasion and metastasis more significantly than either gene alone. HER2 forms homodimers as well as heterodimers with other HER family members and we now show that the ability of WASF3 to promote invasion is highly dependent on the HER2/HER3 heterodimer. The engagement of WASF3 with the HER2/HER3 complex facilitates its phospho-activation and transcriptional upregulation, which is facilitated by HER2/HER3 activation of JAK/STAT signaling. In breast cancer cells overexpressing HER2, therefore, WASF3 is specifically required to facilitate the invasion/metastasis response. Targeting WASF3, therefore, could be a potential therapeutic approach to suppress metastasis of HER2-overexpressing breast tumors.

Mitochondrial ATAD3A combines with GRP78 to regulate the WASF3 metastasis-promoting protein.

AAA domain containing 3A (ATAD3A) is an integral mitochondrial membrane protein with unknown function, although we now show that high-level expression is associated with poor survival in breast cancer patients. Using a mass spectrometry approach we have demonstrated that ATAD3A interacts with the WASF3 metastasis-promoting protein. Knockdown of ATAD3A leads to decreased WASF3 protein levels in breast and colon cancer cells. Silencing ATAD3A also results in loss of both cell anchorage-independent growth and invasion and suppression of tumor growth and metastasis in vivo using immuno-compromised mice. HSP70 is responsible for stabilizing WASF3 in the cytoplasm, but inactivation of HSP70 does not lead to the loss of WASF3 stability at the mitochondrial membrane, where presumably it is protected through its interaction with ATAD3A. In response to endoplasmic reticulum (ER) stress, increases in the GRP78 protein level leads to increased WASF3 protein levels. We also show that ATAD3A was present in a WASF3-GRP78 complex, and suppression of GRP78 led to destabilization of WASF3 at the mitochondrial membrane, which was ATAD3A dependent. Furthermore, ATAD3A-mediated suppression of CDH1/E-cadherin occurs through its regulation of GRP78-mediated WASF3 stability. Proteolysis experiments using isolated mitochondria demonstrates the presence of the N-terminal end of WASF3 within the mitochondria, which is the interaction site with the N-terminal end of ATAD3A. It appears, therefore, that stabilization of WASF3 function occurs through its interaction with ATAD3A and GRP78, which may provide a bridge between the ER and mitochondria, allowing communication between the two organelles. These findings also suggest that pharmacologic inhibition of ATAD3A could be an effective therapeutic strategy to treat human cancer.

Loss of ATF3 promotes hormone-induced prostate carcinogenesis and the emergence of CK5(+)CK8(+) epithelial cells.

Steroid sex hormones can induce prostate carcinogenesis, and are thought to contribute to the development of prostate cancer during aging. However, the mechanism for hormone-induced prostate carcinogenesis remains elusive. Here, we report that activating transcription factor 3 (ATF3)-a broad stress sensor-suppressed hormone-induced prostate carcinogenesis in mice. Although implantation of testosterone and estradiol (T+E2) pellets for 2 months in wild-type mice rarely induced prostatic intraepithelial neoplasia (PIN) in dorsal prostates (one out of eight mice), the loss of ATF3 led to the appearance of not only PIN but also invasive lesions in almost all examined animals. The enhanced carcinogenic effects of hormones on ATF3-deficient prostates did not appear to be caused by a change in estrogen signaling, but were more likely a consequence of elevated androgen signaling that stimulated differentiation of prostatic basal cells into transformation-preferable luminal cells. Indeed, we found that hormone-induced lesions in ATF3-knockout mice often contained cells with both basal and luminal characteristics, such as p63(+) cells (a basal-cell marker) showing luminal-like morphology, or cells double-stained with basal (CK5(+)) and luminal (CK8(+)) markers. Consistent with these findings, low ATF3 expression was found to be a poor prognostic marker for prostate cancer in a cohort of 245 patients. Our results thus support that ATF3 is a tumor suppressor in prostate cancer.

Predisposition to retinoblastoma due to a translocation within the 4.7R locus.

A candidate for the retinoblastoma (Rb) predisposition gene, known as 4.7R, has recently been cloned. Authentication of this gene depends on abnormalities detected in either the genomic sequence or transcription products (or both) in Rb tumours and cell lines. We report a constitutional chromosome translocation, with a breakpoint lying within the putative retinoblastoma gene, from a patient with retinoblastoma; there is an associated small intragenic deletion. This structural rearrangement, which confers susceptibility to tumour formation, provides strong evidence that the 4.7R cDNA represents the Rb gene.

Cloning of the human Gfi-1 gene and its mapping to chromosome region 1p22.

Recently the rat and mouse Growth Factor Independence (Gfi-1) genes have been cloned (Gilks et al., 1993; Zoring et al; 1996). This gene allows cells in culture to overcome the depletion of growth factors in the culture medium and maintain their proliferative potential. As part of a cloning strategy to isolated genes from human chromosome 1p22 which are associated with a constitutional chromosome translocation from a patient with stage 4S neuroblastoma, we have identified the human homologue of the Gfi gene and defined a 50 Kb map position within a well characterised YAC contig from the region. The full length cDNA sequence is 81% homologous with the rodent counterparts and, at the protein level, is even more highly conserved.

Independent constitutional germline mutations occurring in the RB1 gene in cousins with bilateral retinoblastoma.

The inheritance of a genetic susceptibility to the development of retinoblastoma generally follows an autosomal mode of inheritance with high penetrance. Rare families, however, show evidence of incomplete penetrance where individuals can transmit the mutant gene without being affected themselves. In these families formal proof of this dogma requires the identification of the predisposing mutation. In this study we have identified the mutations in cousins with bilateral (hereditary) disease. Using SSCP and DNA sequencing, different constitutional mutations were detected in the affected cousins in this pedigree. One cousin carries a C-->T mutation in exon 8 generating a stop codon directly which was also present in his affected mother whereas the other cousin carries an 8 base pair deletion in exon 20. Neither half of the family carried the same mutation as the other. The mother of the patient with the 8 bp deletion carried neither of the mutations. Thus, we have demonstrated that the retinoblastomas in this family have developed as a result of independent, sporadic genetic events which occurred coincidentally in the same extended family rather than being due to a common mutation which manifests as incompletely penetrant. These observations have important implications for genetic counselling in this type of family.

A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype.

We have used single strand conformation polymorphism analysis to study the 27 exons of the RB1 gene in individuals from a family showing 'mild' expression of the retinoblastoma phenotype. In this family affected individuals developed unilateral tumors and, as a result of linkage analysis, unaffected mutation carriers were also identified within the pedigree. A single band shift using SSCP was identified in exon 21 which resulted in a missense mutation converting a cys-->arg at nucleotide position 28 in the exon. The mutation destroyed an NdeI restriction enzyme site. Analysis of all family members demonstrated that the missense mutation co-segregated with patients with tumors or who, as a result of linkage analysis had been predicted to carry the predisposing mutation. These observations point to another region of the RB1 gene where mutations only modify the function of the gene and raise important questions for genetic counseling in families with these distinctive phenotypes.

A novel mutation in the promotor region in a family with a mild form of retinoblastoma indicates the location of a new regulatory domain for the RB1 gene.

We describe a family segregating the retinoblastoma phenotype where the affected individuals have only unifocal tumours and where linkage analysis has identified unaffected mutant gene carriers. DNA from members of this 'low penetrance' pedigree was subjected to an exon-by-exon SSCP analysis of the RB1 gene. No mutations were found in the 27 exons of the coding region but an SSCP band shift was seen for PCR products covering the RB1 promoter region. Sequencing identified a G-->C change within a GGGCGG motif which is the core of the recognition sequence of the SP1 transcription factor. Electromobility shift assays demonstrated that SP1 does not bind to oligomers from this region of the RB1 promoter but bandshifts were seen for an, as yet, unidentified protein(s) which was not seen using an oligomer containing the G-->C mutation. Thus, identification of a naturally occurring mutation in a family with only 'mild' phenotypes has identified another regulatory sequence in the RB1 promoter which binds an endogenous cellular protein(s). Identification of this protein should allow a better understanding of the control of expression of the RB1 gene.

Isolation of chromosome-specific DNA sequences from an Alu polymerase chain reaction library to define the breakpoint in a patient with a constitutional translocation t(1;13) (q22;q12) and ganglioneuroblastoma.

We describe the cytogenetic and molecular characterization of a t(1;13)(q22;q12) constitutional rearrangement occurring in a patient with a relatively benign form of neuroblastoma, called ganglioneuroblastoma. Somatic cell hybrids were generated between mouse 3T3 cells and a lymphoblastoid cell line from this patient, D.G. One isolated subclone, DGF27C11, contained the derivative chromosome, 1pter-q22::13q12-qter, but no other material from either chromosome 1 or 13. Using available DNA probes the 13 breakpoint was assigned proximal to all reported markers. In order to generate flanking markers to define this translocation further, an Alu polymerase chain reaction library was constructed from a somatic cell hybrid containing only the proximal, 13pter-13q14, region of chromosome 13. Seven unique sequences have been isolated from the library, three of which lie below and four of which lie above the 13q12 breakpoint. More precise mapping of the distal markers was achieved using a panel of somatic cell hybrids with overlapping deletions of chromosome 13. The paucity of probes in the 1q22 region has made a precise assignment of this breakpoint difficult, however it has been shown to lie distal to c-SKI and proximal to APOA2. This refined characterization of the breakpoint is a prerequisite for its cloning, which may yield genes important in the pathogenesis of ganglioneuroblastoma.

TTC4, a novel candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the skin.

A novel candidate tumor suppressor gene, TTC4, on chromosome 1p31 has been described recently. Since aberrations in this region have been detected in malignant melanoma, we investigated DNA of paraffin-embedded sections from 16 typical naevi, 19 atypical naevi, 32 primary melanomas (15 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases and DNA from four melanoma cell lines by PCR and direct sequencing analysis for mutations in all exons of TTC4. Tumors comprised a wide range of thickness (Breslow index) and Clark levels. No mutations could be detected in typical or atypical naevi, but we found seven different point mutations in the tumor samples, six of them causing an amino acid change. Ten melanoma samples belonging to nine patients showed one or more of these mutations. In detail, in six of 25 metastases, in two of 17 nodular melanomas and in two of 15 superficial spreading melanomas point mutations could be detected. In two cell lines, a loss of a whole exon could be demonstrated and in one cell line we found a point mutation. In addition, three polymorphisms were found. Our findings indicate that TTC4 may participate in the pathogenesis of malignant melanomas of the skin.