Robert Benezra

The protein Id: A negative regulator of helix-loop-helix DNA binding proteins

We have isolated a cDNA clone encoding a novel helix-loop-helix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodimeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal differentiation, Id RNA levels decrease upon induction. Transfection experiments indicate that over-expression of Id inhibits the trans-activation of the muscle creatine kinase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a basic region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimeric complexes.

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/−Id3−/− host, which were associated with VEGF-receptor-1–positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.

Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts

Id proteins may control cell differentiation by interfering with DNA binding of transcription factors. Here we show that targeted disruption of the dominant negative helix–loop–helix proteins Id1 and Id3 in mice results in premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers. The Id1–Id3 double knockout mice also display vascular malformations in the forebrain and an absence of branching and sprouting of blood vessels into the neuroectoderm. As angiogenesis both in the brain and in tumours requires invasion of avascular tissue by endothelial cells, we examined the Id knockout mice for their ability to support the growth of tumour xenografts. Three different tumours failed to grow and/or metastasize in Id1+/-Id3-/- mice, and any tumour growth present showed poor vascularization and extensive necrosis. Thus, the Id genes are required to maintain the timing of neuronal differentiation in the embryo and invasiveness of the vasculature. Because the Id genes are expressed at very low levels in adults, they make attractive new targets for anti-angiogenic drug design.

MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells.

The mitotic checkpoint protein hsMad2 is required to arrest cells in mitosis when chromosomes are unattached to the mitotic spindle. The presence of a single, lagging chromosome is sufficient to activate the checkpoint, producing a delay at the metaphase–anaphase transition until the last spindle attachment is made. Complete loss of the mitotic checkpoint results in embryonic lethality owing to chromosome mis-segregation in various organisms. Whether partial loss of checkpoint control leads to more subtle rates of chromosome instability compatible with cell viability remains unknown. Here we report that deletion of one MAD2 allele results in a defective mitotic checkpoint in both human cancer cells and murine primary embryonic fibroblasts. Checkpoint-defective cells show premature sister-chromatid separation in the presence of spindle inhibitors and an elevated rate of chromosome mis-segregation events in the absence of these agents. Furthermore, Mad2+/- mice develop lung tumours at high rates after long latencies, implicating defects in the mitotic checkpoint in tumorigenesis.

Identification of a Human Mitotic Checkpoint Gene: hsMAD2

In Saccharomyces cerevisiae, MAD2 is required for mitotic arrest if the spindle assembly is perturbed. The human homolog of MAD2 was isolated and shown to be a necessary component of the mitotic checkpoint in HeLa cells by antibody electroporation experiments. Human, or Homo sapiens, MAD2 (hsMAD2) was localized at the kinetochore after chromosome condensation but was no longer observed at the kinetochore in metaphase, suggesting that MAD2 might monitor the completeness of the spindle-kinetochore attachment. Finally, T47D, a human breast tumor cell line that is sensitive to taxol and nocodazole, had reduced MAD2 expression and failed to arrest in mitosis after nocodazole treatment. Thus, defects in the mitotic checkpoint may contribute to the sensitivity of certain tumors to mitotic spindle inhibitors.

Therapy-Induced Acute Recruitment of Circulating Endothelial Progenitor Cells to Tumors

The contribution of bone marrow–derived circulating endothelial progenitor cells (CEPs) to tumor angiogenesis has been controversial, primarily because of their low numbers in blood vessels of untreated tumors. We show that treatment of tumor-bearing mice with vascular disrupting agents (VDAs) leads to an acute mobilization of CEPs, which home to the viable tumor rim that characteristically remains after such therapy. Disruption of this CEP spike by antiangiogenic drugs or by genetic manipulation resulted in marked reductions in tumor rim size and blood flow as well as enhanced VDA antitumor activity. These findings also provide a mechanistic rationale for the enhanced efficacy of VDAs when combined with antiangiogenic drugs.

Id proteins in development, cell cycle and cancer.

Id proteins are important parts of signaling pathways involved in development, cell cycle and tumorigenesis. They were first shown to act as dominant negative antagonists of the basic helix-loop-helix family of transcription factors, which positively regulate differentiation in many cell lineages. The Id proteins do this by associating with the ubiquitous E proteins and preventing them from binding DNA or other transcription factors. Id proteins also associate with Ets transcription factors and the Rb family of tumor suppressor proteins, and are downstream targets of transforming growth factor beta and bone morphogenic protein signaling. Thus, the Id proteins have become important molecules for understanding basic biological processes as well as targets for potential therapeutic intervention in human disease.

Chromosome Missegregation and Apoptosis in Mice Lacking the Mitotic Checkpoint Protein Mad2

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome-microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.

Id family of helix-loop-helix proteins in cancer

Over the past few decades, biologists have identified key molecular signatures associated with a wide range of human cancers. Recently, animal models have been particularly useful in establishing whether such signatures have functional relevance; the overexpression of pro-oncogenic or loss of anti-oncogenic factors have been evaluated for their effects on various tumour models. The aim of this review is to analyze the potential role of the inhibitor of DNA binding (Id) proteins in cancer and examine whether deregulated Id activity is tumorigenic and contributes to hallmarks of malignancy, such as loss of differentiation (anaplasia), unrestricted proliferation and neoangiogenesis.

Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents

Several hypotheses have been proposed to explain how antiangiogenic drugs enhance the treatment efficacy of cytotoxic chemotherapy, including impairing the ability of chemotherapy-responsive tumors to regrow after therapy. With respect to the latter, we show that certain chemotherapy drugs, e.g., paclitaxel, can rapidly induce proangiogenic bone marrow-derived circulating endothelial progenitor (CEP) mobilization and subsequent tumor homing, whereas others, e.g., gemcitabine, do not. Acute CEP mobilization was mediated, at least in part, by systemic induction of SDF-1alpha and could be prevented by various procedures such as treatment with anti-VEGFR2 blocking antibodies or paclitaxel treatment in CEP-deficient Id mutant mice, both of which resulted in enhanced antitumor effects mediated by paclitaxel, but not by gemcitabine.