We employed a genetically defined human cancer model to investigate the contributions of two genes upregulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bonerelated genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitro phenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bonerelated gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis.

Genetic studies have demonstrated that the basic helix-loop-helix protein E2A is an essential transcription factor in B lymphocyte lineage commitment and differentiation. However, the mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B cells. Gel filtration analysis of human pre-B cell nuclear extract showed that E2A coelutes with the HATs p300, CBP, and PCAF. A co-immunoprecipitation assay further demonstrated that a fraction of endogenous E2A proteins is associated with each of the three HATs. We show that these HATs acetylate E2A in vitro, enhance E2A-mediated transcription activity, and promote nuclear retention of E2A proteins. A catalytic mutation of p300 completely abrogates the ability of p300 to acetylate E2A and to promote E2A nuclear retention in 293T cells. A breeding test between E2A heterozygous mice and p300 heterozygous mice demonstrated that these two genes interact for proper B cell development. Collectively, these results suggest that E2A and HATs collaboratively regulate B cell development.The development of B lymphocytes in the bone marrow is initiated and tightly regulated by at least three transcription factors, E2A, EBF, and Pax5 (1). Mice missing any one of these transcription factors show complete block in B cell development at the pro-B cell stage (2-5). Although the expression of EBF and Pax5 are relatively restricted to the B cell lineage, E2A is found to be much broadly expressed. Both biochemical and genetic analyses have indicated that E2A is the most upstream regulator among the three transcription factors and is continuously involved in regulating the expression of B cell-specific genes through the later stages of B cell development (6, 7). It is not clear how E2A controls the broad array of tissue-specific and stage-specific gene expression during B cell development.E2A is a founding member of the basic helix-loop-helix (bHLH) 1 transcription factor family, which plays an evolutionarily conserved role in regulating the differentiation events in various tissue types including B lymphocytes in mammals (8).The E2A gene encodes two bHLH transcription factors, E12 and E47, which are generated through differential splicing to two adjacent exons that encode the bHLH domains (9). The bHLH domains are required for protein dimerization and DNA binding (10). Two transactivation domains (AD) are mapped to the amino terminus of the E2A proteins (11, 12). E2A proteins form homodimers or heterodimers through HLH interactions with other broadly expressed bHLH transcription factors such as HEB (13) or with tissue-restricted bHLH transcription factors such as MyoD (14). These bHLH protein dimers bind to DNA at the consensus sequence CANNTG, designated as the E-box. Functional E-box sites are found in the promoters and enhancers of a wide variety of tissue-specific genes including immunoglobulin genes in B cells (15). The ubiquitously exp...

In normal epithelial tissues, the multifunctional cytokine transforming growth factor-B (TGF-B) acts as a tumor suppressor through growth inhibition and induction of differentiation whereas in advanced cancers, TGF-B promotes tumor progression through induction of tumor invasion, neoangiogenesis, and immunosuppression. The molecular mechanisms through which TGF-B shifts from a tumor suppressor to a tumor enhancer are poorly understood. We now show a role for the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in repressing the protumorigenic effects of TGF-B. The TGF-B effector SMAD3 inducibly interacts with PTEN on TGF-B treatment under endogenous conditions. RNA interference (RNAi) suppression of PTEN expression enhances SMAD3 transcriptional activity and TGF-B-mediated induction of SMAD3 target genes whereas reconstitution of PTEN in a null cancer cell line represses the expression of TGF-B-regulated target genes. Targeting PTEN expression through RNAi in a PTEN wild-type cell line increases TGF-B-mediated invasion but does not affect TGF-B-mediated growth inhibition. Reconstitution of PTEN expression in a PTEN-null cell line blocks TGF-B-induced invasion but does not modulate TGF-Bmediated growth regulation. These effects are distinct from Akt and Forkhead family members that also interact with SMAD3 to regulate apoptosis or proliferation, respectively. Pharmacologic inhibitors targeting TGF-B receptors and phosphatidylinositol 3-kinase signaling downstream from PTEN cooperate to block TGF-B-mediated invasion. Thus, the loss of PTEN expression in human cancers may contribute to a role for TGF-B as a tumor enhancer with specific effects on cellular motility and invasion. (Cancer Res 2005; 65(24): 11276-81)

Monotherapies have proven largely ineffective for the treatment of glioblastomas, suggesting that increased patient benefit may be achieved by combining therapies. Two protumorigenic pathways known to be active in glioblastoma include RAS/RAF/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT/target of rapamycin (TOR). We investigated the efficacy of a combination of novel low molecular weight inhibitors LBT613 and RAD001 (everolimus), which were designed to target RAF and TOR, respectively. LBT613 decreased phosphorylation of extracellular signal-regulated kinase 1 and 2, downstream effectors of RAF, in a human glioma cell line. RAD001 resulted in decreased phosphorylation of the TOR effector S6. To determine if targeting RAF and TOR activities could result in decreased protumorigenic glioma cellular behaviors, we evaluated the abilities of LBT613 and RAD001 to affect the proliferation, migration, and invasion of human glioma cells. Treatment with either LBT613 or RAD001 alone significantly decreased the proliferation of multiple human glioma cell lines. Furthermore, LBT613 and RAD001 in combination synergized to decrease glioma cell proliferation in association with G 1 cell cycle arrest. Glioma invasion is a critical contributor to tumor malignancy. The combination of LBT613 and RAD001 inhibited the invasion of human glioma cells through Matrigel to a greater degree than treatment with either drug alone. These data suggest that the combination of LBT613 and RAD001 reduces glioma cell proliferation and invasion and support examination of the combination of RAF and TOR inhibitors for the treatment of human glioblastoma patients. [Mol Cancer Ther 2007;6(9):2449 -57]