Oncogenic activity of Tiam1 and Rac1 in NIH3T3 cells.

We have recently identified the invasion-inducing Tiam1 gene by proviral insertional mutagenesis. The Tiam1 protein shares a Dbl homology (DH) domain with an increasing number of oncoproteins, some of which have been shown to function as GDP dissociation stimulators (GDS) for small GTPases of the Rho family. In vitro and in vivo analyses indicate that Tiam1 activates the Rho like GTPase Rac1. Here we have analysed the consequences of overexpression of several mutant Tiam1 proteins in NIH3T3 fibroblasts. Similar to other proteins containing a DH domain, N-terminal truncation of the Tiam1 protein activates its oncogenic potential, establishing Tiam1 as a proto-oncogene. In addition, we show the sequences N-terminal of the catalytic DH domain are required for morphological transformation accompanied by the formation of membrane ruffling, but not for the induction of an oncogenic phenotype. Overexpression of constitutively active Rac1 (V12Rac1) in NIH3T3 cells produces a similar oncogenic phenotype, suggesting that the oncogenic effects of Tiam1 are a consequence of Rac activation.

A role for Rac in Tiam1-induced membrane ruffling and invasion.

Rho-like GTPases have been implicated in the regulation of the actin cytoskeleton which controls the morphology, adhesion and motility of cells. Like Ras proteins, they become activated when bound GDP is exchanged for GTP, a process catalysed by GDP-dissociation stimulator (GDS) proteins. Several GDS proteins specific for Rho-like GTPases have been identified. Most of these contain a conserved catalytic domain, the DBL-homology (DH) domain, and activate Cdc42 or Rho but not Rac. We have isolated the invasion-inducing Tiam1 gene, which also encodes a protein with a DH domain. Here we show that Tiam1 is a GDS protein for Rho-like GTPases in vitro. In fibroblasts, Tiam1 induces a similar phenotype as constitutively activated (V12)Rac1, including membrane ruffling, and this is inhibited by dominant negative (N17)Rac1. Moreover, T-lymphoma cells expressing V12Rac1 become invasive, indicating that the Tiam1-Rac signalling pathway could be operating in the invasion and metastasis of tumour cells.

Sequence of the human invasion-inducing TIAM1 gene, its conservation in evolution and its expression in tumor cell lines of different tissue origin.

By means of proviral tagging in combination with in vitro selection for invasive T-lymphoma variants, we have previously identified the murine invasion- and metastasis-inducing Tiam1 gene. Tiam1 encodes a novel protein which shares a Dbl-homology (DH) domain with GDP dissociation stimulator-(GDS) proteins that activate Rho-like GTPases. We have cloned the human TIAM1 coding sequence and studied its evolutionary conservation and expression pattern. TIAM1 is highly conserved among vertebrates. The close similarity between human TIAM1 and the mouse homologue is indicated by 88% and 95% identity of nucleotides and predicted sequences, respectively. The murine gene is highly expressed in brain and testis and at low or moderate levels in almost all other normal tissues. Interestingly, Tiam1 transcripts were found in virtually all analysed tumor cell lines of human and rodent origin including B- and T-lymphomas, neuroblastomas, melanomas and carcinomas. The evolutionary conservation as well as the broad expression pattern of Tiam1 in most normal tissues, suggests a general function in cellular signaling processes presumably by activation of a Rho-like GTPase that regulates the cytoskeletal organization.

The invasion-inducing TIAM1 gene maps to human chromosome band 21q22 and mouse chromosome 16.

The murine invasion-inducing Tiam1 gene maps to the distal end of chromosome 16, 3.8 cM centromeric of the Ets2 gene. TIAM1, the human homolog of Tiam1, maps to the syntenic region (q22) on human chromosome 21. The gene order in 21q22 is cen-TIAM1-AML1-ERG-ETS2-tel.

Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins.

Using proviral tagging in combination with in vitro selection for invasiveness, we have identified a gene, designated Tiam-1, that affects invasion. In the selected invasive T lymphoma variants, proviral insertions were found within coding exons of the Tiam-1 gene, resulting in both truncated 5'-end and 3'-end transcripts that give rise to N- and C-terminal Tiam-1 protein fragments. In one invasive variant, amplification of the Tiam-1 locus was observed with concomitant increase in the amount of normal Tiam-1 protein. Cell clones that were invasive in vitro produced experimental metastases in nude mice, and transfection of truncated Tiam-1 cDNAs into noninvasive cells made these cells invasive. The predicted Tiam-1 protein harbors a Dbl- and Pleckstrin-homologous domain, which it shares with GDP-GTP exchangers for Rho-like proteins that have been implicated in cytoskeletal organization.

Sublocalization of an invasion-inducing locus and other genes on human chromosome 7.

By somatic cell fusion studies between noninvasive mouse T-lymphoma cells and invasive human activated normal T-cells we have previously shown that the genetic information responsible for the induction of invasive and metastatic potential in interspecies T-cell hybrids is located on human chromosome 7. Apparently, genes derived from normal activated T-cells are dominantly expressed in the hybrids and control the invasive and, as a consequence, metastatic potential of these T-lymphoma cells. To sublocalize the invasion-inducing locus on chromosome 7 we have generated hybrids that harbor only specific regions of human chromosome 7 with or without a small fragment of human chromosome 21. Analysis of these hybrids revealed that the invasion-inducing locus maps to 7p12----cen. The human DNA complement of the hybrids was confirmed by Southern blot analysis using a large panel of chromosome 7-specific DNA probes. Several of these genes could be further sublocalized. These included: ARAF2 to 7p12----cen, D7S21 to 7pter----p12, ACTB to 7p15----p12, EGFR to 7p12, MDH2 to 7cen----q22, and PDGFA to 7pter----p15.

Allele-specific detection of K-ras oncogene expression in human non-small-cell lung carcinomas.

Point mutations in codon 12 of the K-ras oncogene are frequent in human lung adenocarcinomas. To study the expression of the K-ras gene in these tumors we have developed a mRNA detection technique based on the polymerase chain reaction (PCR). By this technique, K-ras expression can be detected semi-quantitatively in samples of less than 100 ng total RNA. Hybridization of the amplified cDNA sequences with mutation-specific oligonucleotides allows separate quantification of the expression of normal and point-mutated alleles in a single sample. RNA samples from 24 human non-small-cell lung carcinomas (NSCLC), from 2 lung metastases of colonic adenocarcinomas, from 3 human lung adenocarcinoma cell lines, and from normal lung tissue were analyzed. In most tumors, expression of K-ras was detected at levels equal to or several times higher than those found in normal lung tissue. A lung metastasis from a colon adenocarcinoma, known to contain an amplified K-ras gene, highly over-expressed the K-ras gene. In those tumors in which the K-ras oncogene was activated by a point mutation, both alleles of the gene were expressed. Our results show that a high over-expression of K-ras is a rare event in human lung carcinomas, but that a certain degree of over-expression of the mutated allele can be demonstrated in tumors with an activated K-ras gene. With the technique we describe here, adequate estimation of the expression of specific genes in minimal amounts of tumor cells becomes possible.

Induction of invasive and metastatic potential in mouse T-lymphoma cells (BW5147) by treatment with 5-azacytidine.

Non-invasive, non-metastatic mouse BW5147 T-lymphoma cells were treated with non-mutagenic concentrations of the hypomethylating agent 5-azacytidine (5-aza-C). Subsequently, invasive variants were selected on monolayers of rat embryo fibroblasts. The estimated frequency of induction of invasive variants was smaller than 1 in 10(6) cells. We obtained several independent clones that were stable in the expression of the invasive phenotype. In contrast to the parental cell line, the highly invasive clones produced widespread metastases upon tail vein injection in all the syngeneic AKR mice tested, whereas clones with an intermediate level of invasiveness formed metastases only in part of the mice tested. DNA analysis using the methylation-sensitive and insensitive restriction enzymes, Hpa-II and Msp-I, respectively, showed that the DNA of the invasive variants remained hypomethylated, up to 6 months after 5-aza-C treatment. 5-aza-C is thus able to induce invasive and metastatic potential in the BW5147 T-lymphoma cells, similar to the activated human c-Ha-ras oncogene or human chromosome 7, as studied previously. The acquisition of invasive and metastatic potential is presumably caused by DNA hypomethylation and thus activation of one or more silent invasion controlling genes.

Genetic basis of T-lymphoma invasion.

We have discussed the use of BW5147 T-lymphoma cells as a model system to explore the genetic basis of invasion and metastasis. The degree of invasiveness of these cells in vitro is highly correlated with experimental metastasis formation in vivo upon tail vein injection in syngeneic AKR mice. A powerful in vitro selection system has been developed which allows to select rare invasive cell variants obtained by various experimental manipulations. We found that introduction of the human c-Ha-ras oncogene, the presence of human chromosome 7 from normal activated T-cells, DNA hypomethylation induced by 5-azacytidine treatment, and possibly also retrovirus insertional mutagenesis can convert noninvasive BW5147 T-lymphoma cells into invasive and metastatic cells. Several experimental approaches are discussed to identify the gene(s) involved.

Genetic analysis of invasion and metastasis.

Metastasis formation is a multistep process that probably requires a complex interplay of a large and heterogeneous group of genes, including genes involved in cellular resistance to immunorejection and genes controlling the invasive potential of cells. Transfection experiments have shown that oncogenes of the ras gene family as well as oncogenes of the kinase group are able to induce invasive and metastatic properties in non-transformed cells as well as in tumorigenic, but non-metastatic, cells. However, these findings are not in agreement with observations on spontaneous human tumours in which no correlation was found between activation or increased expression of ras genes and the invasive and metastatic properties of these cells. Further studies have indicated that in particular cell types nuclear oncogenes like N-myc and adenovirus derived E1A may influence metastasis formation by trans-regulation of other genes, including class I genes of the major histocompatibility complex and genes coding for proteolytic enzymes. The many efforts to identify additional invasion and metastasis associated genes by transfection of high molecular weight metastatic tumour DNA met with little success. Somatic cell fusion studies, however, indicate that such genes exist and that one or more of them are located on human chromosome 7.