H.-J. Su Huang

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers Is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named ΔEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active ΔEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of ΔEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.

Expression and distribution of vascular endothelial growth factor protein in human brain tumors

Abstract Marked neovascularization is a hallmark of many neoplasms in the nervous system. Recent reports indicate that the endothelial mitogen vascular endothelial growth factor (VEGF) may play a critical role in the regulation of vascular endothelial proliferation in malignant gliomas. Using novel monoclonal antibodies to the VEGF polypeptide we have determined the expression and cellular distribution of VEGF protein in a representative series of 171 human central nervous system (CNS) tumors by immunohistochemistry and immunoblotting. In agreement with previous in situ hybridization data, 19 out of 20 glioblastomas (95%) showed immunoreactivity for VEGF, whereas both the percentage of immunoreactive tumors and the extent of immunoreactivity for VEGF were significantly lower in astrocytomas. Of the pilocytic astrocytomas (WHO grade I) 44% were immunoreactive for VEGF, but we observed several cases with pronounced vascular proliferates in the absence of VEGF. In ependymomas, meningiomas, hemangioblastomas, and primitive neuroectodermal tumors, there was no correlation between VEGF expression, vascular endothelial proliferation and the grade of malignancy. Oligodendrogliomas and the oligodendroglial component of mixed gliomas lacked immunoreactive VEGF, indicating that endothelial growth factors other than VEGF may regulate tumor angiogenesis in these neoplasms. Western blot analysis showed a predominant VEGF protein species of 23 kDa and confirmed the immunohistochemical data in all cases. Our findings demonstrate that VEGF is expressed in a wide spectrum of brain tumors in which it may induce neovascularization. However, other angiogenic factors also appear to contribute to the vascularization of CNS neoplasms.

SETA: A novel SH3 domain-containing adapter molecule associated with malignancy in astrocytes

Differential display polymerase chain reaction analysis was used to compare five differentiation states of the O-2A progenitor-like cell line CG4: progenitor cells and cells at 12 h or 4 days after the induction of differentiation into oligodendrocytes or astrocytes. This led to the identification of 52 sequence tags that were expressed differentially with cellular phenotype. One sequence was upregulated during differentiation of CG4 cells and represented a novel gene that we named SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes). This gene encodes an SH3 domain-containing adapter protein with sequence similarity to the CD2AP (CD2 adapter protein) and CMS (Cas ligand with multiple Src homology) genes. SETA mRNA was expressed at high levels in the developing rat brain but was barely detectable in the normal adult rat or human brain. However, SETA mRNA was found in approximately one half of the human gliomas tested, including astrocytomas grades II, III, and IV, as well as oligodendrogliomas, mixed oligoastrocytomas, and human glioma-derived cell lines. A rat glioma generated by treatment with the alkylating carcinogen ethylnitrosourea on postnatal day 1 and a derived cell line also expressed SETA mRNA. Furthermore, in an in vitro model of astrocytoma progression based on p53-/- astrocytes, expression of SETA was restricted to cells that are tumorigenic.

CD95-mediated apoptosis of human glioma cells: Modulation by epidermal growth factor receptor activity

The death ligands CD95L and Apo2L/TRAIL are promising investigational agents for the treatment of malignant glioma. EGFR is overexpressed in a significant proportion of malignant gliomas in vivo. Here, we report that CD95L‐induced cell death is enhanced by EGFR inhibition using tyrphostine AG1478 in 7 of 12 human malignant glioma cell lines. Conversely, CD95‐mediated and Apo2L‐induced cell death are both inhibited by overexpression of EGFR in LN‐229 cells. CD95L‐induced cell death augmented by AG1478 is accompanied by enhanced processing of caspase 8. LN‐229 cells overexpressing the viral caspase inhibitor, crm‐A, are not sensitized to CD95L‐induced cell death by AG1478, indicating that EGFR exerts its antiapoptotic properties through a caspase 8‐dependent pathway. These data define a modulatory effect of EGFR‐activity on death ligand‐induced apoptosis and indicate that EGFR inhibition is likely to improve the efficacy of death ligand‐based cancer therapies. Furthermore, it is tempting to speculate that EGFR amplification protects tumor cells from death ligand‐mediated host immune responses in vivo and that EGFRs effects on death receptor‐mediated apoptosis may explain the anti‐tumor effects of non‐cytotoxic, unarmed anti‐EGFR family antibodies.

Functional analysis of mild-type and malignant glioma derived CDKN2Aβ alleles: Evidence for an RB-independent growth suppressive pathway

The tumor suppressor gene CDKN2A (p16/MTS1/INK4A), which encodes the cyclin-dependent kinase inhibitor p16INK4a, is a target of 9p21 deletions during the malignant progression of human gliomas. This gene also encodes a second protein product (human p16β, murine p19ARF), which originates from an unrelated exon of CDKN2A (exon 1β) spliced onto exon 2 in an alternate reading frame. Cell cycle arrest by p16β is caused by an as yet unidentified pathway. In order to test the candidacy of p16β as a glioma suppressor, we replaced p16INK4a, p15INK4b and p16β wild-type as well as a series of seven glioma-derived p16β alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A−/RB+ (U-87MG and U-251MG) or CDKN2A+/RB− (LN-319) endogenous backgrounds and demonstrated that p16β can act as a functional glioma cell growth suppressor. Moreover, p16β, but not p16INK4a or p15INK4b inhibited the growth of RB-negative LN-319 cells, indicating that p16β likely exerts its effects through an RB-independent pathway. In vitro and in vivo assays of pRB phosphorylation were consistent with this interpretation. Since none of the glioma-derived p16β mutations inactivated their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16INK4a and p16β) likely exclusively target p16INK4a.

Point mutations can inactivate in vitro and in vivo activities of p16 INK4a /CDKN2A in human glioma

Deletions of chromosomal region 9p21 are among the most common genetic alterations observed during the clonal evolution of high grade malignant gliomas. Structural and functional evidence has suggested that homozygous deletion involving CDKN2A (the genetic locus encoding the cyclin-dependent kinase inhibitor p16INK4a) is a mechanism of inactivation of this gene and that it can be a growth suppressor in human gliomas. However, the presence of other potential suppressor genes in the 9p21 region and the relatively large sizes of the deletions has made it difficult to be certain that the CDKN2A gene is their actual target. Here, we tested this hypothesis by determining the growth suppressive effects, cell cycle inhibitions, and the activities of seven naturally occurring glioma-derived CDKN2A alleles carrying point mutations and found that two of them were functionally compromised. To resolve discrepancies among the different existing functional assays, we developed an assay for p16INK4a function that allowed us to demonstrate that the expression of wild-type CDKN2A, but not alleles with inactivating mutations, prevents pRB phosphorylation in vivo in human glioma cells. These data suggest that CDKN2A is a critical target for mutational inactivation in human malignant gliomas.

KLF6 Gene and Early Melanoma Development in a Collagen I-Rich Extracellular Environment

BACKGROUND A putative tumor suppressor gene at chromosome 10p15, which contains KLF6 and other genes, is predicted to be lost during melanoma development, and its identity is unknown. In this study, we investigated the biological roles and identity of this tumor suppressor gene. METHODS The human UACC 903 melanoma cell line containing introduced DNA fragments from the 10p15 region with (10E6/3, 10E6/11, and 10E6/18) and without (10ER4S.2/1) the tumor suppressor gene was used. Xenograft tumors were generated in a total of 40 mice with melanoma cell lines, and tumor size was measured. Cells were cultured on plastic or a gel of type I collagen. Viability, proliferation, and apoptosis were assessed. Expression of KLF6 protein was assessed by immunohistochemistry and immunoblot analysis. Expression of phosphorylated Erk1/2 and cyclin D1 was assessed by immunoblot analysis. Protein expression of KLF6 was inhibited with small interfering RNA (siRNA). KLF6 protein expression was assessed in 17 human nevi and human melanoma specimens from 29 patients. Statistical analyses were adjusted for multiple comparisons by use of Dunnett method. All statistical tests were two-sided. RESULTS Melanoma cells containing KLF6 generated smaller subcutaneous xenograft tumors with fewer proliferating cells than control cells. When grown on collagen 1, viability of cells with ectopic KLF6 expression (72%) was lower than that of control cells (100%) (group difference = -28%, 95% confidence interval = -31.3% to -25.2%, P < .001). Viability of melanoma cells with or without the KLF6 tumor suppressor gene on plastic dishes was similar. When KLF6 expression was inhibited with KLF6 siRNA, viability of cells with the tumor suppressor gene on collagen I gel increased compared with that of control cells carrying scrambled siRNA. KLF6 protein was detected in all nevi examined but not in human metastatic melanoma tissue examined. Ectopic expression of KLF6 protein in melanoma cells grown on collagen I decreased levels of phosphorylated Erk1/2 and cyclin D1 in the mitogen-activated protein kinase signaling pathway. CONCLUSIONS In melanoma cells, the tumor suppressor gene at 10p15 appears to be KLF6. Signaling from the collagen I-rich extracellular matrix appears to be involved in the tumor suppressive activity of KLF6 protein.

A novel seven transmembrane receptor induced during the early steps of astrocyte differentiation identified by differential expression.

The rat glial progenitor cell line CG‐4 can be induced to differentiate into either oligodendrocytes or type‐2 astrocytes. In order to identify genes whose expression varies coincident with such phenotypic differentiation, we employed representational difference analysis (RDA) of mRNA. Here, we report 38 cDNAs induced in type‐2 astrocytes, oligodendrocytes, or both differentiated states. Among these were known transcription factors, membrane receptors, extracellular matrix proteins, secreted signaling modulators, chromatin regulators and myelin sheath components. In addition several novel genes were identified; among these was a gene induced during the very early stages of astrocyte differentiation that we have named Ieda (induced early in differentiating astrocytes). Several Ieda transcripts were detected by RT‐PCR, and appeared to be produced by alternative splicing and promoter usage. The protein deduced from the longest Ieda mRNA exhibited sequence features characteristic of G‐protein coupled receptors, including seven putative transmembrane domains, while the shorter Ieda transcripts encoded proteins that lacked several transmembrane segments. In the adult rat, Ieda transcripts were found exclusively in brain and testis. In the developing rat brain, Ieda expression was first detected at embryonic day 16, that is two days before the first appearance of mature astrocytes. Thus, this approach has yielded a potential source of markers for differentiation states of these two cellular types as well as genes predicted to be functionally involved in the differentiation process itself.