Hamid Band

Direct presentation of nonpeptide prenyl pyrophosphate antigens to human γδ T cells

Abstract Human Vγ2Vδ2 + T cells recognize mycobacterial non-peptide antigens, such as isopentenyl pyrophosphate, and their synthetic analogs, such as monoethyl phosphate, through a TCR-dependent process. Here, we examine the presentation of these antigens. Vγ2Vδ2 + T cells recognized secreted prenyl pyrophosphate antigens in the absence of other accessory cells but, under such conditions, required T cell-T cell contact. Recognition required neither the expression of classical MHC class I, MHC class II, or CD1a, CD1b, and CD1c molecules, nor MHC class I or class II peptide loading pathways. Fixed accessory cells also presented the prenyl pyrophosphate antigens to γδ T cells. Thus, in contrast with the presentation of conventional peptide antigens, protein antigens, and superantigens to αβ T cells, prenyl pyrophosphate antigens are presented to γδ T cells through a novel extracellular pathway that does not require antigen uptake, antigen processing, or MHC class I or class II expression. This pathway allows for the rapid recognition of bacteria by γδ T cells and suggests that γδ T cells play a role in the early response to bacterial infection.

Mutation of the c-Cbl TKB Domain Binding Site on the Met Receptor Tyrosine Kinase Converts It into a Transforming Protein

The c-Cbl protooncogene is a negative regulator for several receptor tyrosine kinases (RTKs) through its ability to promote their polyubiquitination. Hence, uncoupling c-Cbl from RTKs may lead to their deregulation. In testing this, we show that c-Cbl promotes ubiquitination of the Met RTK. This requires the c-Cbl tyrosine kinase binding (TKB) domain and a juxtamembrane tyrosine residue on Met. This tyrosine provides a direct binding site for the c-Cbl TKB domain, and is absent in the rearranged oncogenic Tpr-Met variant. A Met receptor, where the juxtamembrane tyrosine is replaced by phenylalanine, is not ubiquitinated and has transforming activity in fibroblast and epithelial cells. We propose the uncoupling of c-Cbl from RTKs as a mechanism contributing to their oncogenic activation.

The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells

The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1-mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelial-mesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility. Furthermore, upregulation of Bmi-1 led to the stabilization of Snail, a transcriptional repressor associated with EMT, via modulation of PI3K/Akt/GSK-3beta signaling. Chromatin immunoprecipitation assays revealed that Bmi-1 transcriptionally downregulated expression of the tumor suppressor PTEN in tumor cells through direct association with the PTEN locus. This in vitro analysis was consistent with the statistical inverse correlation detected between Bmi-1 and PTEN expression in a cohort of human nasopharyngeal carcinoma biopsies. Moreover, ablation of PTEN expression partially rescued the migratory/invasive phenotype of Bmi-1-silenced cells, indicating that PTEN might be a major mediator of Bmi-1-induced EMT. Our results provide functional and mechanistic links between the oncoprotein Bmi-1 and the tumor suppressor PTEN in the development and progression of cancer.

REGULATION OF CXCR4-MEDIATED CHEMOTAXIS AND CHEMOINVASION OF BREAST CANCER CELLS

The chemokine-CXCL12 and its receptor, CXCR4, have recently been shown to play an important role in regulating the directional migration of breast cancer cells to sites of metastasis. In the present study, we showed that CXCL12 enhanced the chemotaxis, chemoinvasion and adhesive properties of breast cancer cells; parameters that are critical for development of metastasis. We have also evaluated the signaling mechanisms that regulate CXCL12-induced and CXCR4-mediated breast cancer cell motility and invasion. These studies revealed that CXCL12 induces the tyrosine phosphorylation of focal adhesion kinase (FAK) at residues 397 and 577, and of RAFTK/Pyk2 at residues 402 and 579/580. The cytoskeletal proteins paxillin and Crk, as well as tyrosine phosphatase SHP2 and adaptor protein Cbl, were also phosphorylated. CXCL12 induced the activation of PI 3-kinase, and increased its association with Cbl and SHP2. PI 3-kinase, RAFTK/Pyk2 and tyrosine phosphatase inhibitors significantly blocked CXCL12-induced chemotaxis and chemoinvasion. The role of SHP2 and Cbl in CXCL12-induced chemotaxis and chemoinvasion in breast cancer cells was further defined by transiently overexpressing wild-type SHP2, wild-type Cbl, dominant-negative SHP2, Cbl mutants 70Z/3 and G306E or double transfectants of the Cbl and SHP2 constructs. We found a novel role of Cbl in CXCL12-induced chemotaxis, which may be mediated through the activation and formation of a multimeric complex comprised of Cbl, SHP2 and PI 3-kinase. We also observed the activation of matrix metalloproteinases 2 and 9 upon CXCL12 stimulation. These studies provide new information regarding signaling pathways that may regulate CXCL12-induced metastasis in breast cancer cells.

Biology of the human gamma delta T-cell receptor.

The specificity of responses to foreign antigens is a hallmark of both humoral and cellular immunity in vertebrates. Although a complex interaction of many distinct cell types and a host of biologically active soluble mediators contribute to the inflammatory response, only two cell types interact in a highly specific manner with foreign antigens to initiate the immune response. B lymphocytes and T lymphocytes accomplish this through the use of highly diverse protein complexes on their cell surfaces which act as antigen receptors. After the identification of membrane-bound immunoglobulin as the B-cell receptor for antigen, it was several decades before a specific T-cell antigen receptor, the a^ TCR, was discovered (AlHson et al. 1982, Meuer et al. 1983, Haskins et al. 1983). This heterodimer was found to be associated with a complex of invariant chains called CD3 based on co-modulation (Meuer et al. 1983), co-immunoprecipitation (Borst et al. 1982) and by chemically cross-linking the subunits on the cell surface (Brenner et al. 1985, Allison & Lanier 1985). The genes encoding the a^ TCR reveal immunoglobulin-like sequences composed of separate variable (V), diversity (D) and joining (J) gene segments that are brought together into a contiguous gene during development (Yanagi et al. 1984, Hedrick et al. 1984, Chien et al. 1984, Saito et al. 1984a, Sim et al. 1984, Hannum et al. 1984, Fabbi et al. 1984). That the a^ TCR is the receptor carrying specificity for both foreign antigen and the major histocompatibility (MHC) self-restricting element was demonstrated by conferring this dual specificity to recipient cells by transfection of the genes (Dembic et al. 1986, Saito et al. 1987).

The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system.

The Cbl family of proteins are evolutionarily conserved negative regulators of activated tyrosine kinase‐coupled receptors. Antigen receptors are prominent targets of negative regulation by the Cbl family members, Cbl and Cbl‐b, which proteins function as ubiquitin ligases. Cbl and Cbl‐b contain substrate recognition domains that interact specifically with activated protein tyrosine kinases of the Src and Syk/ZAP‐70 families. Cbl‐mediated ubiquitination of these kinases leads to their degradation, resulting in attenuation of receptor signals. Cbl may also control activation‐induced monoubiquitination of antigen receptors, thus facilitating their delivery to lysosomes for subsequent degradation. Finally, the interactions of Cbl proteins with downstream targets of tyrosine kinases, such as PI‐3‐kinase and Vav, could provide an additional mechanism to attenuate receptor signaling. By targeting multiple components of antigen receptor signaling for degradation, the Cbl protein family provides a critical mechanism to ensure an appropriate immune response. The hyperresponsiveness of Cbl−/− and Cbl‐b−/− lymphocytes and the autoimmune phenotype of Cbl‐b−/− mice lend strong support for this proposal. The ability to control early receptor signals through regulated protein degradation provides a novel paradigm of immunoregulation.

The Evolutionarily Conserved N-terminal Region of Cbl Is Sufficient to Enhance Down-regulation of the Epidermal Growth Factor Receptor*

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans andDrosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1–357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1–440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.

Induction of Cancer Cell Death by Self-assembling Nanostructures Incorporating a Cytotoxic Peptide

Nanotechnology offers novel delivery vehicles for cancer therapeutics. Potential advantages of nanoscale platforms include improved pharmacokinetics, encapsulation of cytotoxic agents, enhanced accumulation of therapeutics in the tumor microenvironment, and improved therapeutic structures and bioactivity. Here, we report the design of a novel amphiphilic molecule that self-assembles into nanostructures for intracellular delivery of cytotoxic peptides. Specifically, a cationic alpha-helical (KLAKLAK)(2) peptide that is known to induce cancer cell death by membrane disruption was integrated into a peptide amphiphile (PA) that self-assembles into bioactive, cylindrical nanofibers. PAs are composed of a hydrophobic alkyl tail, a beta-sheet forming peptide, and a bioactive peptide that is displayed on the surface of the nanofiber after self-assembly. PA nanostructures that included (KLAKLAK)(2) were readily internalized by breast cancer cells, in contrast to the (KLAKLAK)(2) peptide that on its own was not cell permeable. (KLAKLAK)(2) nanostructures, but not the peptides alone, also induced breast cancer cell death by caspase-independent and Bax/Bak-independent mechanisms associated with membrane disruption. Significantly, (KLAKLAK)(2) nanostructures induced cell death more robustly in transformed breast epithelial cells than in untransformed cells, suggesting a degree of tumor selectivity. Our results provide proof-of-principle that self-assembling PAs can be rationally designed to generate nanostructures that can efficiently deliver cytotoxic peptides to cancer cells.

The Cbl protooncoprotein: a negative regulator of immune receptor signal transduction

The Cbl protooncoprotein has recently emerged as a component of tyrosine kinase-mediated signal transduction in a variety of cell types. Here, we discuss evidence that supports a role for Cbl as a novel negative regulator of immune receptor signaling, and present models for its mode of function.

Human Papillomavirus Oncoprotein E6 Inactivates the Transcriptional Coactivator Human ADA3

ABSTRACT High-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. The HPV oncoprotein E6 is essential for oncogenic transformation. We identify here hADA3, human homologue of the yeast transcriptional coactivator yADA3, as a novel E6-interacting protein and a target of E6-induced degradation. hADA3 binds selectively to the high-risk HPV E6 proteins and only to immortalization-competent E6 mutants. hADA3 functions as a coactivator for p53-mediated transactivation by stabilizing p53 protein. Notably, three immortalizing E6 mutants that do not induce direct p53 degradation but do interact with hADA3 induced the abrogation of p53-mediated transactivation and G1 cell cycle arrest after DNA damage, comparable to wild-type E6. These findings reveal a novel strategy of HPV E6-induced loss of p53 function that is independent of direct p53 degradation. Given the likely role of the evolutionarily conserved hADA3 in multiple coactivator complexes, inactivation of its function may allow E6 to perturb numerous cellular pathways during HPV oncogenesis.