Transcription Factor DLX5 As a New Target for Promising Antitumor Agents.

The crystal structure of the human transcription factor DLX5 has been used for the screening of a library consisting of 10(6 )compounds by the molecular docking technique.In vitro testsof the 14 top-rated ligands showed that compound Q12 displays the best ability to inhibit the proliferation ofDlx5 positive mouse lymphoma cells, which correlates with the down-regulation ofc-mycexpression. Compound Q12 has low toxicity on normal human ovarian epithelial cells and mouse lymphoma cells with absent expression ofDlx5, and can be used for further chemical optimization and for the development of novel, highly efficient cancer treatments.

The promyelocytic leukemia zinc-finger gene, PLZF, is frequently downregulated in malignant mesothelioma cells and contributes to cell survival.

DNA copy number analysis was performed, using single-nucleotide polymorphism mapping arrays, to fine map genomic imbalances in human malignant mesothelioma (MM) cell lines derived from primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. All 22 cell lines examined showed homozygous deletions of 9p21.3, centering at the CDKN2A/ARF and CDKN2B loci. Other commonly underrepresented segments included 1p36, 1p22, 3p21-22, 4q13, 4q34, 11q23, 13q12-13, 14q32, 15q15, 18q12, and 22q12, each observed in 55-90% of cell lines. Focal deletions of 11q23 encompassed the transcriptional repressor gene promyelocytic leukemia zinc finger (PLZF), which was validated by analysis of genomic DNA using real-time polymerase chain reaction (PCR). Semi-quantitative RT-PCR and immunoblot analysis revealed that PLZF is greatly downregulated in MM cell lines compared with non-malignant mesothelial cells. Ectopic expression of PLZF in PLZF-deficient MM cells resulted in decreased cell viability, reduced colony formation, as well as increased apoptosis, the latter based on results of various cell death assays and the observation of increased cleavage of caspase 3, PARP, and Mcl-1. These data indicate that deletions of PLZF are a common occurrence in MM and that downregulation of PLZF may contribute to MM pathogenesis by promoting cell survival.

Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK.

The neurofibromatosis type 2 NF2 gene product, merlin, is a tumor suppressor frequently inactivated in malignant mesothelioma (MM). To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a nonmalignant system, we used small interfering RNA to silence Nf2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulation of FAK activity.

Neurofibromatosis 2 and malignant mesothelioma.

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene cause the inherited disorder NF2 and are also common in malignant mesothelioma, which is not a characteristic feature of NF2. The authors report an asbestos-exposed person with NF2 and malignant mesothelioma. Immunohistochemical analysis of the mesothelioma confirmed loss of expression of the NF2 protein, and comparative genomic hybridization revealed losses of chromosomes 14, 15, and 22, and gain of 7. The authors propose that a person with a constitutional mutation of an NF2 allele is more susceptible to mesothelioma.

Activation of Akt2 Inhibits anoikis and apoptosis induced by myogenic differentiation.

Akt2, a homolog of Akt1, encodes a serine/threonine protein kinase that is amplified in ovarian and pancreatic cancers. The antiapoptotic activities of the Akt1 proto-oncogene product have been well documented, but the role of Akt2 in cellular survival is poorly understood. Here, we demonstrate that Akt2 mRNA, protein and kinase activity are upregulated during serum deprivation-induced C2C12 cell myogenic differentiation, a process that is associated with the acquisition of an apoptosis-resistant phenotype. Transient transfection of plasmids encoding wild-type and constitutively-active Akt2 conferred resistance against apoptosis in differentiating C2C12 cells, while a kinase-negative Akt2 construct did not. Adenovirus-mediated transfer of the constitutively-active Akt2 cDNA also suppressed apoptosis during serum deprivation-induced myogenic differentiation and it protected cells from apoptosis induced by cell detachment. These data indicate that Akt2 functions as an anti-apoptotic gene during cellular differentiation, a property that may contribute to its oncogenicity.

Loss of heterozygosity analysis defines a 3-cM region of 15q commonly deleted in human malignant mesothelioma.

Previous comparative genomic hybridization and allelic loss analyses demonstrated frequent deletions from 15q11.1-15 in malignant mesothelioma. Recurrent losses of 15q11-22 have also been reported in several other tumor types such as breast and colorectal cancers. To more precisely map the commonly deleted region, we have performed a high density loss of heterozygosity analysis of 46 malignant mesotheliomas, using 26 polymorphic microsatellite markers spanning the entire long arm of chromosome 15. Allelic loss from 15q was observed in 22 of 46 (48%) cases. These analyses have defined a minimally deleted region of approximately 3-cM, which was confirmed to reside at 15q15 by fluorescence in situ hybridization analysis with yeast artificial chromosome probes. No tumor suppressor genes have been reported to map to this site. The minimally deleted region identified in this investigation overlaps those observed in other kinds of cancer, and is the smallest site of recurrent 15q loss identified to date in human tumors. The identification of this commonly deleted site implicates a putative tumor suppressor gene(s) at 15q15 involved in diverse forms of human neoplasia.

Genomic imbalances in human lung adenocarcinomas and squamous cell carcinomas.

Comparative genomic hybridization analysis was performed on 67 non-small-cell lung cancers (NSCLCs), including 32 squamous cell carcinomas (SCCs) and 35 adenocarcinomas (ACs), to identify differences in the patterns of genomic imbalance between these two histologic subtypes. Among the entire tumor set, the chromosome arms most often overrepresented were 1q, 3q, 5p, and 8q, each detected in 50-55% of cases. The most frequently underrepresented arms were 9q, 3p, 8p, and 17p. The number of imbalances was similar in SCCs and ACs (median number/case: 12 and 11, respectively). Moreover, many imbalances, such as gains of 1q, 5p, and 8q, occurred at a high frequency in both histologic subgroups. Several statistically significant differences, however, were found. The most prominent difference was gain of 3q24-qter, seen in 81% of SCCs compared with 31% of ACs (P < 0.0001), with amplification at 3q25-26 being detected in eight of 32 (25%) SCCs but in only two of 35 (6%) ACs. Gain of 20p13 and loss of 4q also were seen at a significantly higher rate in SCCs than in ACs, whereas overrepresentation of 6p was more common in ACs. Gains of 7q and 8q each were associated with higher-stage tumors and either positive nodal involvement or higher tumor grade. These data suggest that genes located in several chromosomal regions, particularly 3q25-26, may be associated with phenotypic properties that differentiate lung SCCs from ACs. Furthermore, certain imbalances, prominent among them gains of 7q and 8q, may be indicative of tumor aggressiveness in NSCLCs.

Genetic-susceptibility factor and malignant mesothelioma in the Cappadocian region of Turkey.

Erionite present in stones used to build the villages of Karain and Tuzköy, Turkey, mined from nearby caves, is purported to cause mesothelioma in half of the villagers. We constructed genetic epidemiology maps to test whether some villagers were genetically predisposed to mesothelioma. Analysis of a six-generation extended pedigree of 526 individuals showed that mesothelioma was genetically transmitted, probably in an autosomal dominant way. This finding should lead to preventive strategies to lower the incidence of mesothelioma in future generations, and close monitoring of high-risk individuals might allow early detection and cure.

SV40 and cell cycle perturbations in malignant mesothelioma.

Although epidemiological findings have established that exposure to asbestos fibers is the major cause of malignant mesothelioma (MM), recent studies have implicated simian virus 40 (SV40) in the etiology of some of these tumors. Cytogenetic and molecular genetic evidence suggests that multiple somatic genetic events are required for tumorigenic conversion of a mesothelial cell. As with many other types of cancer, in MM critical oncogenic events exert their action via perturbations of the cell cycle. Interactions between the retinoblastoma (Rb) family of proteins and oncoproteins encoded by SV40 lead to cell cycle alterations. Likewise, inhibition of the p53 tumor suppressor by SV40 can inactivate a crucial cell cycle checkpoint, thereby permitting cells to undergo mitosis regardless of the presence of DNA damage. Many MMs exhibit loss and/or inactivation of the tumor suppressors p16(INK4a)and p14(ARF), components of the pRb and p53 cell cycle regulatory pathways, respectively. Recent investigations have demonstrated that SV40 large T antigen, isolated from frozen biopsies of human MM specimens, binds to and inactivates various tumor suppressor gene products such as pRb and p53. In this review, we discuss how SV40-oncosuppressor interactions can lead to functional alterations of the pRb- and p53-dependent cell cycle regulatory pathways and thereby contribute to neoplastic transformation of human mesothelial cells.

AKT activation up-regulates insulin-like growth factor I receptor expression and promotes invasiveness of human pancreatic cancer cells.

Insulin-like growth factor I receptor (IGF-IR) is frequently overexpressed in several types of human malignancy and is associated with invasion and metastasis of tumor cells. Recently, IGF-IR expression was reported to be up-regulated in the human pancreatic cancer cell line PANC-1 when cells were stably transfected with active Src. The downstream targets of Src that lead to the up-regulation of IGF-IR expression were previously unknown. We demonstrate here that AKT regulates IGF-IR expression in PANC-1 and AsPC-1 cells. Cells transfected with active Src exhibited significantly more IGF-IR protein compared with vector-transfected cells. Overexpression of wild-type or constitutively active AKT (i.e., AKT1 or AKT2) also resulted in elevated IGF-IR expression. IGF-IR protein levels were higher in cells transfected with constitutively active AKT than in cells transfected with active Src. In vitro kinase assays showed that AKT kinases are activated by active Src and inhibited by dominant negative Src or the tumor suppressor PTEN. Furthermore, AKT-induced IGF-IR expression was down-regulated by dominant-negative Src or PTEN. In addition, cells transfected with activated AKT in the presence of IGF-I were shown to have enhanced invasiveness compared with control cells. These data provide evidence for a link between AKT signaling and the regulation of IGF-IR expression and demonstrate that active AKT promotes the invasiveness of pancreatic cancer cells through the up-regulation of IGF-IR expression.

Increased fatty acid synthase is a therapeutic target in mesothelioma.

Many common human cancer tissues express high levels of fatty acid synthase (FAS), the primary enzyme for the synthesis of fatty acids, and the differential expression of FAS between normal and neoplastic tissues has led to the consideration of FAS as a target for anticancer therapy. To investigate the potential of targeting FAS for the treatment of pleural mesothelioma, we first determined whether FAS is overexpressed in human mesothelioma. By immunohistochemistry, we found 22 of 30 human mesothelioma tissue samples tested to express significantly increased levels of FAS compared with normal tissues, including mesothelium. To further explore FAS as a therapeutic target in mesothelioma, we established a nude mouse xenograft model for human mesothelioma using the H-Meso cell line. The i.p. xenografts of this cell line have high levels of FAS expression and fatty acid synthesis pathway activity and grow along mesothelial surfaces in a manner similar to the growth pattern of human mesothelioma. Growth of these tumor xenografts was essentially abolished in mice treated with weekly i.p. injections of C75, a synthetic, small molecule inhibitor of FAS, at levels that resulted in no significant systemic toxicity except for reversible weight loss. These results suggest that FAS may be an effective target for pharmacological therapy in a high proportion of human mesotheliomas.

Human hepatocellular carcinoma is characterized by a highly consistent pattern of genomic imbalances, including frequent loss of 16q23.1-24.1.

Comparative genomic hybridization (CGH) analysis was used to identify chromosomal imbalances in 52 human primary hepatocellular carcinomas (HCCs). The most prominent changes were gains of part or all of chromosome arms 8q (83% of cases) and 1q (73%) and loss of 16q (63%). Other commonly overrepresented sites were 5p, 7q, and Xq. Recurrent sites of DNA sequence amplification included 8q23--24 (five cases) and 11q13--14 (four cases). Other frequently underrepresented sites were 4q, 8p, 16p, and 17p. Taken collectively, these findings and data from other CGH studies of HCCs define a subset of chromosome segments that are consistently over- or underrepresented and highlight sites of putative oncogenes and tumor suppressor genes, respectively, involved in hepatocellular oncogenesis. Loss of heterozygosity analysis with a panel of polymorphic microsatellite markers distributed along 16q defined a minimal region of chromosomal loss at 16q23.1--24.1, suggesting that this region harbors a tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many HCCs.

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways.

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.

Analysis of the structure and expression pattern of MRP7 (ABCC10), a new member of the MRP subfamily.

The MRP subfamily of ABC transporters currently consists of at least six members, several of which have been demonstrated to transport amphipathic anions and to confer in vitro resistance to chemotherapeutic agents. In searching the data bases we identified the product of a cDNA sequencing project that bears significant similarity to MRP subfamily transporters. In this report the predicted coding sequence, protein product and expression pattern of this cDNA, termed MRP7, are analyzed. The MRP7 cDNA sequence encodes a 1492 amino acid ABC transporter whose structural architecture resembles that of MRP1, MRP2, MRP3, and MRP6, in that its transmembrane helices are arranged in three membrane spanning domains. However, in contrast to the latter transporters, a conserved N-linked glycosylation site is not found at the N-terminus of MRP7. Comparisons of the MRP7 amino acid sequence indicated that while it is most closely related to other MRP subfamily members, its degree of relatedness is the lowest of any of the known MRP-related transporters. The integrity of the predicted MRP7 coding sequence was confirmed by the synthesis of an approximately 158 kDa protein in reticulocyte lysates programmed with the MRP7 cDNA. While MRP7 transcript was detected in a variety of tissues by RT/PCR, it was not readily detectable by RNA blot analysis, suggesting that it is expressed at low levels in these tissues. Fluorescence in situ hybridization indicated that MRP7 maps to chromosome 6p12-21, in proximity to several genes associated with glutathione conjugation and synthesis. On the basis of these findings and evolutionary cluster analysis, we conclude that MRP7 is a member of the MRP subfamily of amphipathic anion transporters.

Absence of post-transcriptional RNA modifications of BCL10 in human malignant mesothelioma and colorectal cancer.

The BCL10 gene, located at 1p22, has been implicated in a number of human malignancies, including malignant mesotheliomas (MMs) and colorectal carcinomas. Subsequent reports, however, have revealed an absence of BCL10 mutations in genomic DNA from such tumors. It has been proposed that some abnormalities of this gene may be found only in RNA and not in genomic DNA, suggesting that BCL10 may be mutated post-transcriptionally, rather than at the genomic level. To explore this possibility, we performed SSCP mutation analysis and direct sequencing of cDNA from 17 MM cell lines displaying LOH in 1p22, 12 MM tumor specimens, and 11 colon carcinoma cell lines. SSCP revealed several different band shifts in these samples. The nucleotide changes observed in the cDNA samples were also seen in matched genomic DNA and corresponded to known polymorphisms in the general population. Thus, we conclude the BCL10 mutations are absent at the cDNA level, and that this gene does not undergo "molecular misreading." Since BCL10 also does not possess mutations at the genomic DNA level, it can be ruled out as a gene involved in the pathogenesis of MM and colorectal cancer.

The phosphatidylinositol 3-kinase/AKT signal transduction pathway plays a critical role in the expression of p21WAF1/CIP1/SDI1 induced by cisplatin and paclitaxel.

The cyclin-dependent kinase inhibitor p21WAF1/CIP1/SD11 (p21) plays a crucial role in DNA repair, cell differentiation, and apoptosis through regulation of the cell cycle. A2780 human ovarian carcinoma cells, which are sensitive to cisplatin and paclitaxel, express wild-type p53 and exhibit a p53-mediated increase in p21 in response to the chemotherapeutic agents. Here, we demonstrate that phosphatidylinositol 3-kinase (PI3K) and its downstream targets serine/threonine kinases AKT1 and AKT2 (AKT), are required for the full induction of p21 in A2780 cells treated with cisplatin or paclitaxel. Inactivation of the PI3K/AKT signal transduction pathway either by its specific inhibitor LY294002 or by expression of dominant negative AKT inhibited p21 expression but had no inhibitory effect on the expression of the proapoptotic protein BAX by cisplatin and paclitaxel treatment. In addition, overexpression of wild-type or constitutively active AKT in A2780 cells sustained the regulation of p21 induction or increased the level of p21 expression, respectively. Experiments with additional ovarian carcinoma cell lines revealed that PI3K is involved in the expression of p21 induced by cisplatin or paclitaxel in OVCAR-10 cells, which have wild-type p53, but not in OVCAR-5 cells, which lack functional p53. These data indicate that the PI3K/AKT signal transduction pathway mediates p21 expression and suggest that this pathway contributes to cell cycle regulation promoted by p53 in response to drug-induced stress. However, inactivation of PI3K/AKT signaling did not result in significant alteration of the drug sensitivity of A2780 cells, suggesting that the cell death induced by cisplatin or paclitaxel proceeds independently of cell protective effects of PI3K and AKT.

Identification and structural analysis of human RBM8A and RBM8B: two highly conserved RNA-binding motif proteins that interact with OVCA1, a candidate tumor suppressor.

The OVCA1 gene is a candidate for the breast and ovarian tumor suppressor gene at chromosome 17p13.3. To help determine the function(s) of OVCA1, we used a yeast two-hybrid screening approach to identify OVCA1-associating proteins. One such protein, which we initially referred to as BOV-1 (binder of OVCA1-1) is 173 or 174 amino acids in length and appears to be a new member of a highly conserved RNA-binding motif (RBM) protein family that is highly conserved evolutionarily. Northern blot analysis revealed that BOV-1 is ubiquitously expressed and that three distinct messenger RNA species are expressed, 1-, 3.2-, and 5.8-kb transcripts. The 1-kb transcript is the most abundant and is expressed at high levels in the testis, heart, placenta, spleen, thymus, and lymphocytes. Using fluorescence in situ hybridization and the 5.8-kb complementary DNA probe, we determined that BOV-1 maps to both chromosome 5q13-q14 and chromosome 14q22-q23. Further sequence analysis determined that the gene coding the 1- and the 3.2-kb transcripts (HGMW-approved gene symbol RBM8A) maps to 14q22-q23, whereas a second highly related gene coding for the 5.8-kb transcript resides at chromosome 5q13-q14 (HGMW-approved gene symbol RBM8B). The predicted proteins encoded by RBM8A and RBM8B are identical except that RBM8B is 16 amino acids shorter at its N-terminus. Molecular modeling of the RNA-binding domain of RBM8A and RBM8B, based on homology to the sex-lethal protein of Drosophila, identifies conserved residues in the RBM8 protein family that are likely to contact RNA in a protein-RNA complex. The conservation of sequence and structure through such an evolutionarily divergent group of organisms suggests an important function for the RBM8 family of proteins.

Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity.

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens.