Inactivation of the Dlc1 gene cooperates with downregulation of p15INK4b and p16Ink4a, leading to neoplastic transformation and poor prognosis in human cancer.

The tumor suppressor gene deleted in liver cancer-1 (DLC1), which encodes a protein with strong RhoGAP (GTPase activating protein) activity and weak Cdc42GAP activity, is inactivated in various human malignancies. Following Dlc1 inactivation, mouse embryo fibroblasts (MEF) with a conditional Dlc1 knockout allele reproducibly underwent neoplastic transformation. In addition to inactivation of Dlc1 and increased activity of Rho and Cdc42, transformation depended on the subsequent decreased expression of the Cdk4/6 inhibitors p15(Ink4b) and p16(Ink4a) together with increased expression and activation of Cdk4/6. The level of expression of these cell-cycle regulatory genes was relevant to human tumors with low DLC1 expression. Analysis of publicly available annotated datasets of lung and colon cancer with gene expression microarray profiles indicated that, in pairwise comparisons, low DLC1 expression occurred frequently together (P < 0.01) with downregulation of p15(Ink4b) or p16(Ink4a) or upregulation of CDK4 or CDK6. In addition, an unfavorable prognosis (P < 0.05) was associated with low DLC1 and low p15(Ink4b) in lung cancer and colon cancer, low DLC1 and low p16(Ink4a) in lung cancer, low DLC1 and high CDK4 in lung cancer, and low DLC1 and high CDK6 in colon cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression.

Functional interaction of tumor suppressor DLC1 and caveolin-1 in cancer cells.

Deleted in liver cancer 1 (DLC1), a tumor suppressor gene frequently inactivated in non-small cell lung cancer (NSCLC) and other malignancies, encodes a multidomain protein with a RhoGTPase-activating (RhoGAP) domain and a StAR-related lipid transfer (START) domain. However, no interacting macromolecule has been mapped to the DLC1 START domain. Caveolin-1 (CAV-1) functions as a tumor suppressor in most contexts and forms a complex with DLC1. Here, we have mapped the region of DLC1 required for interaction with CAV-1 to the DLC1 START domain. Mutation of the DLC1 START domain disrupted the interaction and colocalization with CAV-1. Moreover, DLC1 with a START domain mutation failed to suppress neoplastic growth, although it negatively regulated active Rho. CAV-1 and DLC1 expression levels were correlated in two public datasets of NSCLC lines and in two independent publicly available mRNA expression datasets of NSCLC tumors. Clinically, low DLC1 expression predicted a poor clinical outcome in patients with lung cancer. Together, our findings indicate that complex formation between the DLC1 START domain and CAV-1 contributes to DLC1 tumor suppression via a RhoGAP-independent mechanism, and suggest that DLC1 inactivation probably contributes to cancer progression.

Full activity of the deleted in liver cancer 1 (DLC1) tumor suppressor depends on an LD-like motif that binds talin and focal adhesion kinase (FAK).

The deleted in liver cancer 1 (DLC1) tumor suppressor gene, which is frequently inactivated in cancer, encodes a Rho-GAP (GTPase activating protein) focal adhesion protein whose negative regulation of Rho-GTPases is necessary but not sufficient for its full tumor suppressor activity. Here, we report that DLC1 forms a complex with two prooncogenic focal adhesion proteins, talin and the focal adhesion kinase (FAK). We identified an 8-aa sequence (residues 469LDDILYHV476) in DLC1 and designated it an LD-like motif, because it shares homology with the LD motifs of paxillin. This motif was necessary for DLC1 binding to talin and FAK, because a DLC1 mutant, from which six of the residues have been deleted, and another mutant carrying amino acid substitutions in three of the residues are deficient for binding both proteins and localization of DLC1 to focal adhesions. FAK binding was independent of talin and vice versa. In bioassays, both DLC1 mutants were less active than wild-type (WT) DLC1, although the ability of the mutants to negatively regulate overall Rho-GTP was not impaired. We conclude that the LD-like motif, which binds talin and FAK, is required for the full tumor suppressor activity of DLC1 and contributes to the association of DLC1 with focal adhesions.

The Tensin-3 protein, including its SH2 domain, is phosphorylated by Src and contributes to tumorigenesis and metastasis.

In cell lines from advanced lung cancer, breast cancer, and melanoma, endogenous tensin-3 contributes to cell migration, anchorage-independent growth, and tumorigenesis. Although SH2 domains have not been reported previously to be phosphorylated, the tensin-3 SH2 domain is a physiologic substrate for Src. Tyrosines in the SH2 domain contribute to the biological activity of tensin-3, and phosphorylation of these tyrosines can regulate ligand binding. In a mouse breast cancer model, tensin-3 tyrosines are phosphorylated in a Src-associated manner in primary tumors, and experimental metastases induced by tumor-derived cell lines depend on endogenous tensin-3. Thus, tensin-3 is implicated as an oncoprotein regulated by Src and possessing an SH2 domain with a previously undescribed mechanism for the regulation of ligand binding.

Mndal, a new interferon-inducible family member, is highly polymorphic, suppresses cell growth, and may modify plasmacytoma susceptibility.

The human HIN-200 gene cluster and its mouse counterpart, the interferon inducible-200 (Ifi200) family, both on Chr 1, are associated with several diseases, including solid tumors and lupus. Our study was initiated to identify the modifier gene(s) encoded by the Pctm locus, in which mouse B-cell plasmacytomas induced by pristane are associated with heterozygosity of Chr 1 genes near the Ifi200 cluster. A screen for differentially expressed genes in granulomatous tissues induced by pristane in resistant and susceptible strains identified a new Ifi200 member whose expression was 1000-fold higher in the strain carrying the resistant allele of Pctm and was the most highly expressed Ifi200 gene. The gene, designated Mndal (for MNDA-like, myeloid nuclear differentiation antigen-like), was absent in the susceptible genome, as were genomic sequences upstream of Ifi203, the gene adjacent to Mndal. Ectopic expression of MNDAL suppressed cell growth, which, together with the disease susceptibility of heterozygotes at the Pctm locus, suggests that Mndal, perhaps with Ifi203, acts as a tumor suppressor and display(s) haploinsufficiency. Mndal is highly polymorphic among inbred mouse strains, because it is absent in 10 of 24 strains. This polymorphism may have implications for other disease modifiers mapping to the same region.

ASAP3 is a focal adhesion-associated Arf GAP that functions in cell migration and invasion.

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.

Oncogenic inhibition by a deleted in liver cancer gene requires cooperation between tensin binding and Rho-specific GTPase-activating protein activities.

The three deleted in liver cancer genes (DLC1-3) encode Rho-GTPase-activating proteins (RhoGAPs) whose expression is frequently down-regulated or silenced in a variety of human malignancies. The RhoGAP activity is required for full DLC-dependent tumor suppressor activity. Here we report that DLC1 and DLC3 bind to human tensin1 and its chicken homolog. The binding has been mapped to the tensin Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains at the C terminus of tensin proteins. Distinct DLC1 sequences are required for SH2 and PTB binding. DCL binding to both domains is constitutive under basal conditions. The SH2 binding depends on a tyrosine in DCL1 (Y442) but is phosphotyrosine-independent, a highly unusual feature for SH2 binding. DLC1 competed with the binding of other proteins to the tensin C terminus, including beta 3-integrin binding to the PTB domain. Point mutation of a critical tyrosine residue (Y442F) in DLC1 rendered the protein deficient for binding the tensin SH2 domain and binding full-length tensin. The Y442F protein was diffusely cytoplasmic, in contrast to the localization of wild-type DLC1 to focal adhesions, but it retained the ability to reduce the intracellular levels of Rho-GTP. The Y442F mutant displayed markedly reduced biological activity, as did a mutant that was RhoGAP-deficient. The results suggest that DLC1 is a multifunctional protein whose biological activity depends on cooperation between its tensin binding and RhoGAP activities, although neither activity depends on the other.

Specific regulation of the adaptor protein complex AP-3 by the Arf GAP AGAP1.

Arf1 regulates membrane trafficking at several membrane sites by interacting with at least seven different vesicle coat proteins. Here, we test the hypothesis that Arf1-dependent coats are independently regulated by specific interaction with Arf GAPs. We find that the Arf GAP AGAP1 directly associates with and colocalizes with AP-3, a coat protein complex involved in trafficking in the endosomal-lysosomal system. Binding is mediated by the PH domain of AGAP1 and the delta and sigma3 subunits of AP-3. Overexpression of AGAP1 changes the cellular distribution of AP-3, and reduced expression of AGAP1 renders AP-3 resistant to brefeldin A. AGAP1 overexpression does not affect the distribution of other coat proteins, and AP-3 distribution is not affected by overexpression of other Arf GAPs. Cells overexpressing AGAP1 also exhibit increased LAMP1 trafficking via the plasma membrane. Taken together, these results support the hypothesis that AGAP1 directly and specifically regulates AP-3-dependent trafficking.

Regulation of cell morphology and adhesion by the tuberous sclerosis complex (TSC1/2) gene products in human kidney epithelial cells through increased E-cadherin/beta-catenin activity.

We investigated the effects of overexpression of the tuberous sclerosis-1 and -2 (TSC1/2) gene products (hamartin and tuberin, respectively) in the human kidney epithelial cell line 293 with an inducible expression system. As we had observed previously in fibroblasts, 293 cells overexpressing hamartin and/or tuberin grew more slowly in vitro. However, here we also observed that the 293 overexpressing cells underwent a dramatic morphological change in which groups of cells formed compact clusters. The overexpressing cells also displayed decreased dissociation and increased reaggregation in vitro. These changes were found to be associated with an increased level of E-cadherin, which is known to regulate cell-cell interactions in epithelial cells, and of its binding partner beta-catenin. Consistent with the role of E-cadherin in these effects, we found that the observed changes in 293 cell morphology, dissociation, and adhesion were calcium-dependent, and were reproduced by overexpression of E-cadherin. In contrast, overexpression of TSC1 in rat embryo fibroblasts, which lack E-cadherin, failed to elicit the same changes as in 293 cells. We conclude that the hamartin/tuberin complex exerted a direct effect on the morphology and adhesive properties of 293 cells through regulation of the level and/or activity of cellular E-cadherin/beta-catenin.

Epidermal growth factor receptor signaling pathways are associated with tumorigenesis in the Nf1:p53 mouse tumor model.

The human disease neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene, and is characterized by the formation of benign and malignant tumors of the peripheral nervous system. We have shown previously that aberrant expression of the epidermal growth factor receptor (EGFR) is a common feature of human NF1-related tumor development in humans and in NF1 animal models. One recent approach taken to investigate the changes associated with NF1 tumor formation is the development of the Nf1:p53 mouse tumor model. Here, we examined a series of tumor cell lines derived from Nf1:p53 mice for their expression of EGFR family members. Immunoblotting analyses revealed that 23 of the 24 cell lines examined express the EGFR, and 24 of 24 express the related tyrosine kinase erbB2, whereas erbB3 was detected in only 6 of 24. All of the cell lines expressing EGFR responded to epidermal growth factor (EGF) by activation of the downstream signaling pathways, mitogen-activated protein (MAP)/extracellular signal-regulated kinase kinase/MAP kinase, and phosphatidylinositol 3'-kinase (PI3k)/AKT. Growth of the cell lines was greatly stimulated by EGF in vitro and could be blocked by an antagonist of the EGFR. In addition, inhibition of the PI3k pathway potently inhibited the EGF-dependent growth of these cell lines, whereas inhibition of the MAP/extracellular signal-regulated kinase kinase/MAP kinase pathway had more limited effects. We conclude that EGFR expression is a common feature of the Nf1:p53 tumor cell lines and that inhibition of this molecule or its downstream target PI3k, may be useful in the treatment of NF1-related malignancies.