KLF6, a candidate tumor suppressor gene mutated in prostate cancer.

Kruppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function. Here, we show that the KLF6 gene is mutated in a subset of human prostate cancer. Loss-of-heterozygosity analysis revealed that one KLF6 allele is deleted in 77% (17 of 22) of primary prostate tumors. Sequence analysis of the retained KLF6 allele revealed mutations in 71% of these tumors. Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in a p53-independent manner and significantly reduces cell proliferation, tumor-derived KLF6 mutants do not. Our data suggest that KLF6 is a tumor suppressor gene involved in human prostate cancer.

Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival.

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.

Regulation of PTEN binding to MAGI-2 by two putative phosphorylation sites at threonine 382 and 383.

We have reported previously that the PTEN COOH-terminal 33 amino acids play a role in the maintenance of PTEN protein stability (Tolkacheva and Chan, Oncogene, 19: 680-689, 2000). By site-directed mutagenesis, we identified two threonine residues within this COOH-terminal region at codon 382 and 383 that may be targets for phosphorylation events. Interestingly, PTEN mutants rendered phosphorylation-incompetent at these two sites, T382A/T383A, and were found to have drastically reduced expression in cultured cells. The enhanced degradation of PTEN was most likely mediated by the proteosome-dependent pathway, we have evidence that PTEN was polyubiquitinated. More interestingly, the non-phosphorylated forms of PTEN displayed significantly greater binding affinity than the wild-type protein to a previously identified PTEN interacting partner, MAGI-2/ARIP1. On the basis of all these data, we propose that PTEN recruitment to the cell-cell junction may be regulated through the phosphorylation of its COOH terminus.

R-Ras3, a brain-specific Ras-related protein, activates Akt and promotes cell survival in PC12 cells.

The GTP-binding protein, R-Ras3/M-Ras, is a novel member of the Ras subfamily of GTPases which shows highest sequence similarity to the TC21 gene. R-Ras3 is highly expressed in both human and mouse brain and ectopic expression of a constitutively active mutant of R-Ras3 induces cellular transformation in NIH3T3 cells. To gain further insight into the normal cellular function of R-Ras3, we examined the ability of R-Ras3 in activating several known intracellular signaling cascades. We observed that R-Ras3 is a relatively weak activator of the mitogen-activated protein kinase/extracellular-signal-regulated kinases (MAPK/ERKs) when compared to the H-Ras oncogene. On the contrary, both R-Ras3 and H-Ras activated the Jun N-terminal kinase (JNK) to a similar extent. Under similar experimental conditions, R-Ras3 significantly stimulated one of the phosphatidylinositol 3-kinase (PI3-K) downstream substrates, Akt/PKB/RAC (Akt), which has been extensively implicated in mediating cell survival signaling. The activation of Akt by R-Ras3 was most likely to be PI3-K-dependent since this biochemical event was blocked by the pharmacological inhibitors, Wortmannin and LY294002, as well as by a dominant negative mutant of PI3-K. More importantly, R-Ras3 affinity-precipitated PI3-K from cell extracts in a GTP-dependent manner, and associated lipid kinase activity was readily detectable in R-Ras3 immune complexes. The biological significance of R-Ras3 in inducing Akt kinase activity is evidenced by the ability of an activated R-Ras3 to confer cell survival in the rat pheochromocytoma cell line, PC12. As expected, this biological activity of R-Ras3 was also abrogated by the addition of LY294002. Thus, R-Ras3 represents a novel G-protein which may play a role in cell survival of neural-derived cells.

Identification and characterization of R-ras3: a novel member of the RAS gene family with a non-ubiquitous pattern of tissue distribution.

Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins.

Familial neurogenic tumor syndromes.

Cancer caused more than 0.5 million deaths in the United States in 2000. This estimate includes patients who have a genetic predisposition to neoplastic disease, including brain neoplasms. Familial tumor syndromes are important to identify clinically because family members require high degrees of monitoring and genetic counseling. Study of these individuals and families has led to the discovery of genes that are an intrinsic aspect of cell regulation and will continue to be relevant in defining mechanisms of neoplastic development in brain and other tissues.

Pten and p53 converge on c-Myc to control differentiation, self-renewal, and transformation of normal and neoplastic stem cells in glioblastoma.

Glioblastoma (GBM) is a highly lethal primary brain cancer with hallmark features of diffuse invasion, intense apoptosis resistance and florid necrosis, robust angiogenesis, and an immature profile with developmental plasticity. In the course of assessing the developmental consequences of central nervous system (CNS)-specific deletion of p53 and Pten, we observed a penetrant acute-onset malignant glioma phenotype with striking clinical, pathological, and molecular resemblance to primary GBM in humans. This primary, as opposed to secondary, GBM presentation in the mouse prompted genetic analysis of human primary GBM samples that revealed combined p53 and Pten mutations as the most common tumor suppressor defects in primary GBM. On the mechanistic level, the "multiforme" histopathological presentation and immature differentiation marker profile of the murine tumors motivated transcriptomic promoter-binding element and functional studies of neural stem cells (NSCs), which revealed that dual, but not singular, inactivation of p53 and Pten promotes cellular c-Myc activation. This increased c-Myc activity is associated not only with impaired differentiation, enhanced self-renewal capacity of NSCs, and tumor-initiating cells (TICs), but also with maintenance of TIC tumorigenic potential. Together, these murine studies have provided a highly faithful model of primary GBM, revealed a common tumor suppressor mutational pattern in human disease, and established c-Myc as a key component of p53 and Pten cooperative actions in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal, and tumorigenic potential.

Loss of heterozygosity of chromosome 10p in human gliomas.

Molecular loss of heterozygosity studies on human gliomas have shown several regions on chromosome 10 frequently deleted in higher grade tumors, suggesting that chromosome 10 may contain several tumor suppressor genes. We assessed loss of heterozygosity with microsatellite markers in 20 gliomas, consisting of various grades and containing two chromosome 10 copies. The locus that exhibited the most loss (69%) was the region bordered by D10S249 and D10S558 and inclusive of D10S594, with a linkage distance of 3 cM. This region was noted to be deleted in various grades of tumor, including low- and high-grade tumors. These results suggest that chromosome region 10p15 is involved in human gliomas of diverse grades and that this region may harbor genes important in the development of and progression to the malignant phenotype.

Loss of heterozygosity in human ovarian cancer on chromosome 19q.

Abnormalities in the function of oncogenes and tumor suppressor genes have been associated with many human malignancies. The recognition of sites of loss of heterozygosity (LOH) has led to the identification of such genes. We previously reported that abnormalities of mRNA expression of ERCC1 and ERCC2 may be characteristic of epithelial ovarian carcinoma and brain tumors. This led to an investigation of chromosome 19q13.2-q13.4 which contains these DNA repair genes. A 7-Mb region was analyzed using six microsatellite repeats. Loss of heterozygosity has been identified in 53% (8/15) of cases at marker D19S246 which lies in a 2-Mb segment between HRC and KLK1. The genetic material both centromeric and telomeric to the region of loss was conserved. This area is telomeric to three DNA repair genes where LIG1 is 1-Mb centromeric and ERCC1 and ERCC2 are 3.5- and 4.0-Mb centromeric, respectively. These findings represent the first report of a biologically significant rate of LOH on chromosome 19q13.2-q13.4 in human ovarian carcinoma.