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.

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.

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.

WASF3 regulates miR-200 inactivation by ZEB1 through suppression of KISS1 leading to increased invasiveness in breast cancer cells.

The WASF3 gene promotes invasion and metastasis in breast cancer cells, which have undergone epithelial-to-mesenchyme transition (EMT). Overexpression of WASF3 in cells that do not show EMT increases their invasion potential as a result of increased ZEB1/2 levels, which specifically suppress the anti-invasion chromosome 1 miR-200a/200b/429 cluster. ZEB1/2 upregulation by WASF3 results from downregulation of KISS1, leading to the release of inhibition of nuclear factor (NF)κB by IκBα. We further show that ZEB1 expression is regulated by the NFκB transcription factor. Knockdown of WASF3 in breast cancer cells leads to reduced ZEB1 levels and increased miR-200 and E-cadherin levels, resulting in loss of invasion potential. The central regulation of this interactive pathway by WASF3 accounts for the increased invasion associated with increased WASF3 expression seen in aggressive breast cancer cells. WASF3, therefore, is a potential target to suppress invasion and metastasis in breast cancer cells.

Ponatinib suppresses the development of myeloid and lymphoid malignancies associated with FGFR1 abnormalities.

Myeloid and lymphoid malignancies associated with fibroblast growth factor receptor-1 (FGFR1) abnormalities are characterized by constitutively activated FGFR1 kinase and rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. Molecular targeted therapies have not been widely used for stem cell leukemia/lymphoma (SCLL). Ponatinib (AP24534), which potently inhibits native and mutant BCR-ABL, also targets the FGFR family. Using murine BaF3 cells, stably transformed with six different FGFR1 fusion genes, as well as human KG1 cells expressing activated chimeric FGFR1 and five newly established murine SCLL cell lines, we show that ponatinib (<50 nM) can effectively inhibit phosphoactivation of the fusion kinases and their downstream effectors, such as PLCγ, Stat5 and Src. Ponatinib also significantly extended survival of mice transplanted with different SCLL cell lines. Ponatinib administered at 30 mg/kg daily also significantly delayed, or even prevented, tumorigenesis of KG1 cells in xenotransplanted mice. Furthermore, we demonstrate that ponatinib specifically inhibits cell growth and clonogenicity of normal human CD34+ progenitor cells transformed by chimeric FGFR1 fusion kinases. Overall, our data provide convincing evidence to suggest that pharmacologic inhibition of FGFR1 fusion kinases with ponatinib is likely to be beneficial for patients with SCLL and perhaps for other human disorders associated with dysregulated FGFR1 activity.

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.

Analysis of the RB1 gene in children with retinoblastoma having residential connections to West Cumbria, England.

Six of eight cases of retinoblastoma previously identified as having a residential association with West Cumbria, England, in which the Sellafield nuclear installation is situated, were examined for the presence of a constitutional RB1 mutation. No mutations were detected, thus providing strong evidence against an environmental or occupational genotoxic effect causing germline mutations in the parents of these children.

Identification and characterisation of constitutional chromosome abnormalities using arrays of bacterial artificial chromosomes.

Constitutional chromosome deletions and duplications frequently predispose to the development of a wide variety of cancers. We have developed a microarray of 6000 bacterial artificial chromosomes for array-based comparative genomic hybridisation, which provides an average resolution of 750 kb across the human genome. Using these arrays, subtle gains and losses of chromosome regions can be detected in constitutional cells, following a single overnight hybridisation. In this report, we demonstrate the efficiency of this procedure in identifying constitutional deletions and duplications associated with predisposition to retinoblastoma, Wilms tumour and Beckwith-Wiedemann syndrome.

Rapid detection of allelic losses in brain tumours using microsatellite repeat markers and high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) is a recently introduced high-capacity automated method for detecting unknown mutations (denaturing HPLC) or for sizing DNA fragments under nondenaturing conditions. We have adapted the HPLC method for detection of loss of heterozygosity (LOH) and used glial tumours as a model to evaluate its sensitivity and specificity in comparison to conventional denaturing polyacrylamide gel electrophoresis. A total of 20 oligodendrogliomas (grades II and III), and five astrocytic tumours (grades III and IV) were analysed using 14 polymorphic microsatellite markers mapping to regions on chromosomes 1p, 19q, and 10q using both DNA-HPLC and denaturing gel electrophoresis. This study demonstrated complete concordance between both methods. However, unlike gel electrophoresis, HPLC is automated, does not require post-PCR processing, and does not require hazardous radioactive or expensive fluorescent labelling. Our data suggest that HPLC is a reliable and sensitive method for detection of allelic losses in tumour samples and it is a favourable alternative for high-sensitivity LOH detection in both research and diagnostic environments.

A novel member of the WD-repeat gene family, WDR11, maps to the 10q26 region and is disrupted by a chromosome translocation in human glioblastoma cells.

Allelic deletions of 10q25-26 and 19q13.3-13.4 are the most common genetic alterations in glial tumors. We have identified a balanced t(10;19) reciprocal translocation in the A172 glioblastoma cell line which involves both critical regions on chromosomes 10 and 19. In addition, loss of an entire copy of chromosome 10 has occurred in this cell line suggesting that the translocation event may provide a highly specific critical inactivating event in a gene responsible for tumorigenesis. Positional cloning of this translocation breakpoint resulted in the identification of a novel chromosome 10 gene, WDR11, which is a member of the WD-repeat gene family. The WDR11 gene is ubiquitously expressed, including normal brain and glial tumors. WDR11 is composed of 29 exons distributed over 58 kilobases and oriented towards the telomere. The translocation resulted in deletion of exon 5 and consequently fusion of intron 4 of WDR11 to the 3' untranslated region of a novel member, ZNF320, of the Krüppel-like zinc finger gene family. Since ZNF320 is oriented toward the centromere of chromosome 19, both genes appeared on the same derivative chromosome der(10). The chimeric transcript encodes the WDR11 polypeptide, which is truncated after the second of six WD-repeats. ZNF320 is also expressed in A172 cells, although it is not clear if the translocation affects the expression of the altered gene because of the presence of another unrearranged gene on chromosome 19. We suggest that, because of its localization in a region frequently showing LOH and the observation of inactivation of this gene in glioblastoma cells, WDR11 is a candidate gene for the frequently proposed tumor suppressor gene in 10q25-26 which is involved in tumorigenesis of glial and other tumors showing frequent alterations in the distal 10q region.

Molecular characterization of the breakpoint region associated with a constitutional t(2;15)(q34;q26) in a patient with multiple myeloma.

The molecular cloning of the translocation breakpoints from constitutional chromosome rearrangements in patients with a variety of human diseases has consistently led to the isolation of genes important in the development of the phenotype. We used fluorescence in situ hybridization (FISH) to analyze the breakpoint region of a constitutional chromosome translocation involving regions 2q34 and 15q26 observed in a patient with multiple myeloma (MM), a malignant disorder of plasma cells secreting monoclonal immunoglobulin. FISH analysis of this rearrangement showed that the chromosome 2-specific yeast artificial chromosome (YAC) 914E7 and the chromosome 15-specific YAC 757H6 span the translocation breakpoints, respectively. In order to characterize the location of the breakpoints further, somatic cell hybrids were constructed between mouse NIH3T3 cells and t(2;15)-bearing lymphoblastoid cells. Using these somatic cell hybrids, we have shown that the breakpoint on chromosome 2 lies between D2S3007 and D2S3004 and the chromosome 15 breakpoint lies between D15S107 and WI5967 (D15S836). YAC fragmentation has been used to define a 350 kb region containing the 15q26 breakpoint.

Fine mapping of the PTGFR gene to 1p31 region and mutation analysis in human breast cancer.

The 1p31 chromosomal region shows loss of heterozygosity (LOH) in up to 50% of human breast cancer, indicating the presence of a tumor suppressor gene at this location. Many efforts have been made to identify candidate genes responsible for breast cancer on the short arm of chromosome 1. It was shown that prostaglandins have been implicated in the tumorigenesis pathway, perhaps via interactions with their cell surface receptors. The prostaglandin F2 receptor gene (PTGFR) was tentatively mapped to 1p31 adjacent to the region undergoing LOH in human breast cancer. We undertook a mutation study in 34 sporadic human breast tumors using a variant of SSCP, incorporation PCR SSCP (IPS). Several nucleotide variants were detected in different tumors. Here we report the nature of these nucleotide changes and the possible involvement of the PTGFR gene in the etiology of human cancer.

Molecular alterations in the neurofibromatosis type 2 gene and its protein rarely occurring in meningothelial meningiomas.

OBJECT: The neurofibromatosis Type 2 (NF2) gene is the only tumor suppressor gene that has been clearly implicated in the development of benign meningiomas. Interestingly, previous data obtained by the authors indicate that reduced NF2 protein expression seldom occurs in meningothelial meningiomas, the most common histological type of meningioma. The goal of the current study was to explore further the hypothesis of NF2 gene-independent tumorigenesis of meningothelial meningiomas. METHODS: The authors performed a mutational analysis of all 17 exons of the NF2 gene by using single-stranded conformational polymorphism (SSCP). In addition, expression levels of the NF2 protein and mu-calpain, a protease suggested to inactivate the NF2 protein, were determined by immunoblotting analysis of 27 meningiomas (20 meningothelial and seven nonmeningothelial). Mutations of the NF2 gene were found in only one (5%) of 20 meningothelial meningiomas and three (43%) of seven nonmeningothelial tumors (Fisher's exact test, p = 0.042). The levels of NF2 protein were severely reduced in six (28.5%) of 21 meningothelial meningiomas, in contrast to six (86%) of seven nonmeningothelial meningiomas (Fisher's exact test, p = 0.023). Activation of IL-calpain did not correlate with the status of NF2 protein expression in the meningiomas analyzed, demonstrating that mu-calpain activation does not account for the loss of NF2 protein in meningiomas with apparently normal NF2 genes. CONCLUSIONS: These results clearly demonstrate that NF2 gene mutations and decreased NF2 protein expression rarely occur in meningothelial meningiomas compared with other histological types of meningiomas. The clinical behavior of meningothelial meningiomas, however, is similar to that of other benign meningiomas. It is likely, therefore, that the tumorigenesis of meningothelial meningiomas is the result of deleterious alterations of genes that have final phenotypical effects similar to inactivation of the NF2 gene.

A transcription map of the minimally deleted region from 13q14 in B-cell chronic lymphocytic leukemia as defined by large scale sequencing of the 650 kb critical region.

Extensive analysis of tumors has demonstrated homozygous and heterozygous deletions in chromosome region 13q14.3 in B-cell chronic lymphocytic leukemia (B-CLL), suggesting the site of a tumor suppressor gene. Since previous searches for this gene have not yielded any viable candidates, we now present the sequence of the BACs which span the minimally deleted approximately 650 kb region between markers D13S319 and D13S25. This sequence has allowed us to create the definitive transcription map for the region which reveals 93 ESTs and 12 Unigene clusters in this region. Using gene prediction programs, a further 19 potential genes are also identified. The genes show an asymmetrical distribution throughout the region with most of them clustering at the extreme ends. This sequencing effort provides for the definitive structure of the B-CLL deletion region and the identification of the vast majority of the potential candidate genes. Of all the genes identified, only three have homologies to known genes: two L1 repeat genes and rabbit epididymal protein 52. This 13q14.3 sequence provides the final substrate from which to characterize the B-CLL tumor suppressor gene.

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.

Identification of the promoter, genomic structure, and mouse ortholog of LGI1.

The human LGI1 gene is a leucine-rich, repeat-containing gene that was cloned from the t(10:19) breakpoint of the T98G glioblastoma cell line. The LGI1 gene maps to 10q24, a region of peak LOH in malignant gliomas, and is inactivated during the transition from low to high-grade brain tumors. Here we report detailed studies of the genomic structure of the LGI1 gene and its promoter. We have also cloned and characterized the mouse lgil gene, which is 97% homologous to the human gene at the amino acid level and 91% homologous at the nucleotide level. LGI1 contains 8 exons, where each of the four leucine-rich repeat units is contained in an individual 72-bp exon. The cysteine-rich regions flanking the LRR and the single trans-membrane domain do not occupy individual exons. Approximately 5-kb of the genomic region 5' to LGI1 was sequenced, but conventional CAAT and TATA motifs were not present within this sequence. A 597-bp fragment of this 5' sequence was cloned upstream of a promoterless luciferase gene and was shown to be sufficient to drive transcription. SSCP analysis of the coding region of LGI1 in 20 glioblastomas and five cell lines did not reveal any mutations. Because LGI1 expression is considerably downregulated in gliomas, we also investigated whether this was owing to changes in the methylation status of the promoter. Southern blot analysis and 5-azacytidine treatment did not show any appreciable difference in methylation status between normal brain and glioblastomas.