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Robert Benezra

Publications and source records attributed to Robert Benezra.

At least 19 recordsLinked to original sources
PubMed2007

Id1 restrains myeloid commitment, maintaining the self-renewal capacity of hematopoietic stem cells.

Appropriate hematopoietic stem cell (HSC) self-renewal reflects the tight regulation of cell cycle entry and lineage commitment. Here, we show that Id1, a dominant-negative regulator of E protein transcription factors, maintains HSC self-renewal by preserving the undifferentiated state. Id1-deficient HSCs show increased cell cycling, by BrdU incorporation in vivo, but fail to efficiently self-renew, leading to low steady-state HSC numbers and premature exhaustion in serial bone marrow transplant assays. The increased cycling reflects the perturbed differentiation process, because Id1 null HSCs more readily commit to myeloid differentiation, with inappropriate expression of myeloerythroid-specific genes. Thus, Id1 appears to regulate the fate of HSCs by acting as a true inhibitor of differentiation.

Animals↗
PubMed2007

Heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1) increases the incidence of tumors in mice.

Mitotic arrest-deficient protein 1 (MAD1) is a component of the mitotic spindle assembly checkpoint. We have created a knockout mouse model to examine the physiologic consequence of reduced MAD1 function. Mad1(+/-) mice were successfully generated, but repeated paired mating of Mad1(+/-) with Mad1(+/-) mice failed to produce a single Mad1(-/-) animal, suggesting that the latter genotype is embryonic lethal. In aging studies conducted for >18 months, Mad1(+/-) mice compared with control wild-type (wt) littermates showed a 2-fold higher incidence of constitutive tumors. Moreover, 42% of Mad1(+/-) (P < 0.03), but 0% of wt, mice developed neoplasia after treatment with vincristine, a microtubule depolymerization agent. Mad1(+/-) mouse embryonic fibroblasts (MEF) were found to be more prone than wt cells to become aneuploid; Mad1(+/-), but not wt, MEFs produced fibrosarcomas when explanted into nude mice. Our results indicate an essential MAD1 function in mouse development and correlate Mad1 haploinsufficiency with increased constitutive tumors.

Amino Acid Sequence↗
PubMed2006

Mad2 overexpression promotes aneuploidy and tumorigenesis in mice.

Mad2 is an essential component of the spindle checkpoint that blocks activation of Separase and dissolution of sister chromatids until microtubule attachment to kinetochores is complete. We show here that overexpression of Mad2 in transgenic mice leads to a wide variety of neoplasias, appearance of broken chromosomes, anaphase bridges, and whole-chromosome gains and losses, as well as acceleration of myc-induced lymphomagenesis. Moreover, continued overexpression of Mad2 is not required for tumor maintenance, unlike the majority of oncogenes studied to date. These results demonstrate that transient Mad2 overexpression and chromosome instability can be an important stimulus in the initiation and progression of different cancer subtypes.

Aneuploidy↗
PubMed2006

Reassessment of id1 protein expression in human mammary, prostate, and bladder cancers using a monospecific rabbit monoclonal anti-id1 antibody.

Id proteins are a class of dominant-negative antagonists of helix-loop-helix transcription factors and have been shown to control differentiation of a variety of cell types in diverse organisms. Although the importance of Id1 in tumor endothelial cells is well established, the expression and role of the Id1 protein in human cancer cells is controversial. To explore this issue, we developed and characterized a highly specific rabbit monoclonal antibody against Id1 to assess its expression in human breast, prostate, and bladder malignancies. Our results show that in usual types of human mammary carcinomas, the Id1 protein is expressed exclusively in the endothelium. Interestingly, we detected nuclear expression of the Id1 protein in the tumor cells in 10 of 45 cases of poorly differentiated and highly aggressive carcinoma with metaplastic morphology. Similarly, only 1 of 30 prostate cancer samples showed Id1-positive tumor cells, whereas in almost all, endothelial cells showed high Id1 expression. Intriguingly, whereas normal prostate glands do not show any Id1 protein expression, basal layer cells of benign prostate glands in proximity to tumors expressed high levels of the Id1 protein. In contrast to the lack of Id1 expression in the usual types of mammary and prostate cancers, the majority of transitional cell bladder tumors showed Id1 protein expression in both tumor and endothelial cells. These results suggest that further refinement of Id1 expression patterns in a variety of tumor types will be necessary to identify and study the functional roles played by Id1 in human neoplastic processes.

Animals↗
PubMed2006

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.

Angiogenesis Inhibitors↗
PubMed2006

Id4 messenger RNA and estrogen receptor expression: inverse correlation in human normal breast epithelium and carcinoma.

Id (inhibitor of DNA binding) 4 is a member of the Id family of proteins (Id1-Id4), which function as dominant-negative regulators of basic helix-loop-helix transcription factors. Id factors are involved in numerous cell processes, including cell proliferation, differentiation, and tumorigenesis. We assessed the expression of Id4 messenger RNA (mRNA) in invasive mammary carcinoma from 31 patients, as well as in 21 cases of ductal carcinoma in situ, in 9 lymph node metastases, and in the morphologically normal epithelium adjacent to the carcinoma from the same subjects. In addition, we evaluated Id4 mRNA in atypical ductal hyperplasia from 5 other women and in normal breast tissue from yet another 5 women with no history of breast malignancy or atypia. The distribution of Id4 signal was assessed in relation to that of estrogen receptor (ER) in all samples and correlated with the Her-2 status of the carcinomas. Id4 mRNA was present in the normal ER-negative mammary epithelium in all cases; in contrast, the ER-positive cells present in the normal breast were Id4 negative. Id4 mRNA was not detected in atypical ductal hyperplasia, in 22 of the 23 cases of ductal carcinoma in situ, and in 27 of the 31 invasive carcinomas (P = .0008), all of which were ER positive. Conversely, 3 of the 31 invasive carcinomas were Id4 positive and ER negative. Only 1 ER-positive invasive carcinoma showed focal reactivity for Id4. The expression of Id4 in metastatic carcinoma paralleled that of the primary tumor. No correlation was apparent between Id4 and Her-2. Our data show that Id4 is constitutively expressed in the normal human mammary epithelium but is suppressed in ER-positive breast carcinomas and preneoplastic lesions. In contrast, ER-negative carcinomas appear to be Id4 positive. These results support a possible role of Id4 as a tumor suppressor factor in the human breast and suggest that the expression of Id4 in the mammary ductal epithelium may be regulated by estrogen. Further investigations are required to define the functions of Id4 in the human normal breast and in mammary neoplasia.

Adenocarcinoma↗
PubMed2006

Embryonic stem cells prevent developmental cardiac defects in mice.

The potential therapeutic use of embryonic stem cells (ESCs) has gathered the attention of the scientific and medical communities recently. We report that in addition to their unique capacity to populate defective cardiac tissues, ESCs secrete factors that correct gene expression profiles in the defective neighboring cells. Id (inhibitor of DNA binding) gene knockout (KO) mouse embryos die at midgestation because of multiple cardiac defects, but injection of ESCs into preimplantation Id KO embryos prevents these defects and corrects gene expression profiles throughout the heart. ESCs injected into expectant mothers only partially rescue cardiac defects in the Id KO embryos. Two secreted factors are implicated in the rescue process: insulin-like growth factor I accounts for the long-range action of the ESCs, and Wnt5a, a short-range factor, corrects gene expression profiles in the Id KO hearts. Future studies are discussed.

Animals↗
PubMed2005

Oncogenes and Angiogenesis: down-regulation of thrombospondin-1 in normal fibroblasts exposed to factors from cancer cells harboring mutant ras.

The onset of angiogenesis in cancer often involves down-regulation of endogenous angiogenesis inhibitors, of which thrombospondin-1 (TSP-1) is a paradigm. As this effect is thought to occur under the influence of transforming genetic lesions (e.g., expression of the mutant ras oncogene), its nature is regarded as intrinsic to cancer cells themselves. Here, we show that ras-transformed cancer cells can also induce TSP-1 down-regulation in their adjacent nontransformed stromal fibroblasts, but not in endothelial cells, in a paracrine and distance-dependent manner. Indeed, several H-ras-expressing fibrosarcoma (528ras1, B6ras, and NIH3T3Ras) and carcinoma (DLD-1 and IEC18Ras3) cells were found to release soluble factors capable of suppressing TSP-1 protein, mRNA, and promoter activity in nontumorigenic, immortalized dermal fibroblastic cell lines in culture (e.g., in fibroblasts expressing enhanced green fluorescent protein/TSP-1 reporter). This effect was abrogated in Id1-/- fibroblasts. At least two low molecular weight (<3 kDa), heat-labile, and trypsin-resistant mediators of TSP-1 suppression were found to be released from 528ras1 cells. Their effects on normal fibroblasts were inhibited (albeit to different extents) by pertussis toxin and, in one case, by dimethylsphingosine, none of which affected TSP-1 expression by 528ras1 cells. Collectively, our study suggests that the effect of mutant ras on tumor neovascularization is not limited to changes in angiogenic properties of cancer cells themselves. Rather, mutant ras, through a different signaling mechanism, may modulate the properties of the adjacent normal stroma, thus eliciting a proangiogenic field effect.

Animals↗
PubMed2005

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.

Anaplasia↗
PubMed2004

Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells.

We report that Id knockout mouse embryos display multiple cardiac defects, but mid-gestation lethality is rescued by the injection of 15 wild-type embryonic stem (ES) cells into mutant blastocysts. Myocardial markers altered in Id mutant cells are restored to normal throughout the chimeric myocardium. Intraperitoneal injection of ES cells into female mice before conception also partially rescues the cardiac phenotype with no incorporation of ES cells. Insulin-like growth factor 1, a long-range secreted factor, in combination with WNT5a, a locally secreted factor, likely account for complete reversion of the cardiac phenotype. Thus, ES cells have the potential to reverse congenital defects through Id-dependent local and long-range effects in a mammalian embryo.

Animals↗
PubMed2004

Utilization of bone marrow-derived endothelial cell precursors in spontaneous prostate tumors varies with tumor grade.

Id1 and Id3 genes are required for vascularization, growth, and metastasis of xenograft tumors. In Id-deficient mice, tumor transplantation and proangiogenic factors fail to mobilize and recruit circulating endothelial precursor cells (CEPs) and hematopoietic cells, leading to defective tumor angiogenesis in various models. To investigate the requirement of Id genes and bone marrow incorporation in spontaneous prostate tumors, we crossbred Id mutant mice with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Id1-/- Id3+/- TRAMP mice display delayed tumor growth at 24 weeks compared with wild-type TRAMP mice. Id1 and Id3 were strongly expressed in the endothelial cells of poorly differentiated prostate adenocarcinoma but not in the vasculature of well-differentiated tumors, a finding that is corroborated in human prostate tumor samples. In Id-deficient TRAMP mice, the poorly differentiated tumors show extensive hemorrhage, whereas well-differentiated tumors exhibit none. Transplantation with Id wild-type bone marrow significantly reduced the hemorrhage in poorly differentiated prostate adenocarcinomas with bone marrow-derived endothelial cells contributing to 14% of the tumor blood vessels. However, in well-differentiated prostate adenocarcinomas, there was little evidence of bone marrow-derived endothelial cell incorporation. These differences in the expression of Id genes, the effects of Id loss, and the recruitment of bone marrow-derived endothelial precursor cells in tumor vasculature between well-differentiated and poorly differentiated prostate adenocarcinoma suggest that tumor angiogenesis varies depending on the tumor grade.

Adenocarcinoma↗
PubMed2004

MAD2 dependent mitotic checkpoint defects in tumorigenesis and tumor cell death: a double edged sword.

The failure of cell cycle regulatory checkpoints is a common event in human cancer. Defects at the G(1)-S transition have been widely characterized, but only more recently has aberrant checkpoint signaling during mitotic progression been identified as playing a role in cancer. The metaphase to anaphase transition is regulated by multiple proteins that together comprise the mitotic checkpoint. Previously it has been shown that loss of one copy of MAD2, a mitotic checkpoint gene, results in aneuploidy and tumorigenesis arising from chromosome missegregation. More recently and quite surprisingly, MAD2 has been demonstrated to be an essential gene even in tumor cells such that near complete elimination of this protein from cancer cells results in p53 independent cell death. This is the first identification of a haploinsufficient tumor suppressor gene that is also required for tumor cell survival, and suggests that targeting this checkpoint in cancer might be a viable therapeutic strategy.

Animals↗
PubMed2004

Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control.

Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers. These mutations promote tumour development by deregulating the E2F family of transcription factors leading to uncontrolled cell cycle progression. We show that the mitotic checkpoint protein Mad2 is a direct E2F target and, as a consequence, is aberrantly expressed in cells with Rb pathway defects. Concordantly, Mad2 is overexpressed in several tumour types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produces aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributes directly to these defects. These results demonstrate how chromosome instability can arise as a by-product of defects in cell cycle control that compromise the accuracy of mitosis, and suggest a new model to explain the frequent appearance of aneuploidy in human cancer.

Aneuploidy↗
PubMed2004

Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells.

MAD2 inhibits the anaphase-promoting complex when chromosomes are unattached to the mitotic spindle. It acts as a tumor suppressor gene because MAD2+/-cells enter anaphase early and display chromosome instability, leading to the formation of lung tumors in mice. Complete MAD2 inactivation has not been identified in human tumors, although partial defects are prevalent. By employing RNA interference in human somatic cells, we found that severe reduction of MAD2 protein levels results in mitotic failure and extensive cell death arising from defective spindle formation, incomplete chromosome condensation, and premature mitotic exit leading to multinucleation. Cyclin B is degraded prematurely in the MAD2 short interfering RNA-treated cells but not in MAD2+/- cells, suggesting an explanation for the spindle failure and mitotic catastrophe in the MAD2 knockdown cells. Thus, anaphase-promoting complex substrates exhibit distinct sensitivities in the presence of different MAD2 doses, which in turn determine MAD2's role as either a tumor suppressor or an essential gene.

Base Sequence↗
PubMed2004

Endostatin's endpoints-Deciphering the endostatin antiangiogenic pathway.

Up until now, the precise mechanism for endostatin's antiangiogenesis action was not known. In a recent report, have taken advantage of gene array and proteomic analysis to map the antiangiogenic pathways turned on by endostatin. This study resolves some of the controversies surrounding endostatin's biology, and provides a new direction to help dissect the molecular pathways involved in endostatin's selective tumor antiangiogenic effects.

Angiogenesis Inhibitors↗
PubMed2003

Angiogenesis impairment in Id-deficient mice cooperates with an Hsp90 inhibitor to completely suppress HER2/neu-dependent breast tumors.

Id proteins bind basic helix-loop-helix transcription factors and function as dominant negative inhibitors of gene expression. Id1 and Id3 are required for the recruitment of bone marrow-derived endothelial cell precursors and tumors transplanted into Id-deficient mice demonstrate impaired angiogenesis. Mouse mammary tumor virus-neu mice were bred with Id1-/-Id3+/- mice to ascertain the role of Id1 and Id3 in mammary tumorigenesis in a more physiologically relevant model. In mammary tumors from these mice, Id1 and Id3 expression was restricted to the vascular endothelium. Id1 and Id3 deficiency did not prevent or delay tumor formation but did alter tumor phenotype. The tumors that developed in the Id-deficient mice were larger and cystic with a viable rim of tumor cells surrounding a nonviable core of cellular debris. The Hsp90 chaperone protein is required for cellular survival under condition of environmental stress and for the stability of the neu oncogene. 17-Allylamino-17-demethoxygeldanamycin, an Hsp90 inhibitor, was used to treat these mice. Whereas 17-allylamino-17-demethoxygeldanamycin only modestly delayed the growth of established mammary tumors in WT mice for Id, tumor suppression was dramatically more effective in an Id1- or Id3-deficient background. These data suggest that tumorigenesis can occur in a background of defective angiogenesis but that tumors developing in such an environment may be especially sensitive to inhibitors of neu and stress-activated survival pathways. Thus angiogenesis inhibitors in combination with inhibitors of Hsp90 function should be evaluated for the treatment of advanced breast cancer.

Animals↗
PubMed2003

Mad2 phosphorylation regulates its association with Mad1 and the APC/C.

Improper attachment of the mitotic spindle to the kinetochores of paired sister chromatids in mitosis is monitored by a checkpoint that leads to an arrest in early metaphase. This arrest requires the inhibitory association of Mad2 with the anaphase promoting complex/cyclosome (APC/C). It is not known how the association of Mad2 with the kinetochore and the APC/C is regulated in mitosis. Here, we demonstrate that human Mad2 is modified through phosphorylation on multiple serine residues in vivo in a cell cycle dependent manner and that only unphosphorylated Mad2 interacts with Mad1 or the APC/C in vivo. A Mad2 mutant containing serine to aspartic acid mutations mimicking the C-terminal phosphorylation events fails to interact with Mad1 or the APC/C and acts as a dominant-negative antagonist of wild-type Mad2. These data suggest that the phosphorylation state of Mad2 regulates its checkpoint activity by modulating its association with Mad1 and the APC/C.

Anaphase-Promoting Complex-Cyclosome↗