The cancer biology of whole-chromosome instability.

One form of chromosome instability (CIN), the recurrent missegregation of whole chromosomes during cell division (W-CIN), leads to aneuploidy. Although W-CIN is a hallmark of most cancers, mutations in genes involved in chromosome segregation are exceedingly rare. We discuss an oncogene-induced mitotic stress model that provides a mechanistic framework to explain this paradox. We also review the tumor-promoting and tumor-suppressing consequences of W-CIN. Importantly, we do this in the context of cancer as a complex systemic disease, rather than as a simple linearly progressing disorder that arises from a single abnormal cell population. Accordingly, we highlight the often neglected effects of W-CIN on key non-cell-autonomous entities, such as the immune system and the tumor microenvironment. Distinct tissue-specific susceptibilities to W-CIN-induced tumorigenesis and the clinical implications of W-CIN are also discussed.

Induction of complete regressions of oncogene-induced breast tumors in mice.

Over the past decade, mouse models of cancer have come to resemble human disease much more closely than simple subcutaneous or orthotopic systems. Intervention strategies that work on these new model systems are more likely to have an impact clinically. We have shown recently that antiangiogenic stress imposed by loss of Id protein in endothelial progenitor cells results in dramatic central necrosis in breast tumors initiated in mice by overexpression of the her2/neu oncogene. Tumor cells remain viable at the periphery, perhaps via the hypoxic response pathway which allows the lesions to expand. Inhibition of this pathway by the inactivation of the Hif-1alpha chaperone Hsp90 in combination with antiangiogenic stress leads to the first reported complete regression of these aggressive breast tumors.

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.

Role of Id proteins in embryonic and tumor angiogenesis.

It has been shown recently in mouse model systems that the Id proteins play a critical role in angiogenesis both during embryogenesis and tumor formation. The Id proteins are intracellular proteins that inhibit the activity of differentiation-promoting transcription factors and thereby block differentiation and promote cell cycle progression. Loss of Id function in mice leads to premature neural differentiation and withdrawal from the cell cycle in neurons and also the execution of an aberrant differentiation cascade in endothelial cells. This latter event is associated with a mid-gestational brain hemorrhage when Id dosage is sufficiently low. Partial reduction in Id levels spares the endothelium in the brain but adults born fail to support the vascularization of tumors. Through the analysis of these phenotypes and characterization of altered gene expression in the Id knockout endothelium, subdivisions in the angiogenic process are being defined which may lead to selective anti-angiogenic treatments in the management of human disease.

Molecular analyses of the mitotic checkpoint components hsMAD2, hBUB1 and hBUB3 in human cancer.

During the metaphase-anaphase transition, the spindle checkpoint prevents segregation of chromosomes if the spindle assembly is perturbed. Critical components of this checkpoint are the MAD and BUB families of proteins, which prevent the proteolysis of Pds1 and B cyclins, producing mitotic arrest. In the present study, we first intended to resolve the role of the hsMAD2 gene in human cancer by determining the potential presence of hsMAD2 mutations in 44 primary bladder tumors, 42 soft-tissue sarcomas and 10 hepatocellular carcinomas. The entire coding region of the hsMAD2 gene was analyzed using PCR-SSCP and sequencing. One of the bladder tumor samples showed a point mutation consisting of a transition, ATC-->GTC (Ile-->Val) in codon 190 of hsMAD2. However, no differences were found in the mitotic arrest between cells transfected with mutant and wild-type MAD2 cDNA. We also identified mobility shifts in hsMAD2 in both normal and tumor DNA in 3 bladder tumors, 3 soft-tissue sarcomas and 1 hepatocellular carcinoma, consistent with a polymorphism at codon 143, CCA-->CCG (Pro-->Pro). Another polymorphism was identified in a hepatocellular carcinoma case at codon 22, GAG-->GAA (Glu-->Glu). In addition, a subgroup of 67 primary tumors was analyzed by Southern blot hybridization. No deletion or visible re-arrangements were detected by comparing tumor and normal DNA band signals. Two other important components of the spindle mitotic checkpoint, hBUB1 and hBUB3, were also screened for mutations: hBUB1 in 43 bladder tumors and 9 bladder cell lines and hBUB3 only in the cell lines. Two polymorphisms were found in hBUB1 at positions 144, CAG-->CAA (Gln-->Gln) in 1 primary tumor and 1 bladder cell line, and 913 (ATC-->ATT, Ile-->Ile) in 1 primary tumor. We did not find sequence alterations in hBUB3. These results suggest that mutations of the hsMAD2, hBUB1 and hBUB3 genes are very rare in bladder tumors and that hsMAD2 alterations are also infrequent in soft-tissue sarcomas and hepatocellular carcinomas.

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.

Mitotic checkpoints: from yeast to cancer.

Separation of chromosomes during mitosis is monitored by a checkpoint that leads to cell-cycle arrest if the chromosomes are not properly attached to the mitotic spindle. Molecular mechanisms controlling this checkpoint have been identified. In addition, loss of this checkpoint has been shown to result in chromosome missegregation in higher eukaryotes and may contribute to the genomic instability observed in human cancers.

The Id proteins and angiogenesis.

Since the identification of the Id proteins over a decade ago, a great many cell cycle and cell fate decisions have been shown to be under the control of these proteins as described in other sections of this review issue. Perhaps the most unsuspected activity of this class of proteins has been their essential role in angiogenesis, both in the forebrain during development and during the growth and metastasis of tumors in adults. This section of the review issue will focus on the key observations which have led to these conclusions, speculations about potential mechanisms and the outlook for potential therapeutic interventions.

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.

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.

Two isoforms of protein disulfide isomerase alter the dimerization status of E2A proteins by a redox mechanism.

We have shown previously that E2A helix-loop-helix proteins spontaneously form an intermolecular disulfide cross-link that is required for stable homodimer binding to DNA (Benezra, R. (1994) Cell 79, 1057-1067). These homodimers are important for the development of B lymphocytes but are not present in other cell lineages. We have purified two proteins that are capable of regulating the formation of this disulfide bond and found them to be members of the protein disulfide isomerase (PDI) family. By regulating the formation of the disulfide cross-link, these proteins are capable of regulating the dimerization state of E proteins. PDI-mediated reduction appears to dissociate E protein homodimers and favors heterodimer formation with other basic helix-loop-helix proteins in both a purified protein system and in cellular extracts. These studies suggest that PDI may play an important role in the regulation of E2A transcription factor dimerization and the development of the B lymphocyte lineage.

Mad2 transiently associates with an APC/p55Cdc complex during mitosis.

Activation of the mitotic checkpoint pathway in response to mitotic spindle damage in eukaryotic cells delays the exit from mitosis in an attempt to prevent chromosome missegregation. One component of this pathway, hsMad2, has been shown in mammalian cells to physically associate with components of a ubiquitin ligase activity (termed the anaphase promoting complex or APC) when the checkpoint is activated, thereby preventing the degradation of inhibitors of the mitotic exit machinery. In the present report, we demonstrate that the inhibitory association between Mad2 and the APC component Cdc27 also takes place transiently during the early stages of a normal mitosis and is lost before mitotic exit. We also show that Mad2 associates with the APC regulatory protein p55Cdc in mammalian cells as has been reported in yeast. In contrast, however, this complex is present only in nocodazole-arrested or early mitotic cells and is associated with the APC as a Mad2/p55Cdc/Cdc27 ternary complex. Evidence for a Mad2/Cdc27 complex that forms independent of p55Cdc also is presented. These results suggest a model for the regulation of the APC by Mad2 and may explain how the spindle assembly checkpoint apparatus controls the timing of mitosis under normal growth conditions.

High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice.

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators which function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. We report here that nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. We further show that disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life.

MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity.

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.

Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis.

We have performed a detailed comparative in situ hybridization analysis to examine the patterns of expression of all the members of the Id gene family (Id1-4) during murine gastrulation and neurogenesis. During gastrulation, both Id1 and Id3 are expressed in the tissues derived from the inner cell mass from 5.5 dpc onward, whereas Id2 is expressed in tissues derived from trophoblasts. Id4 expression is absent during this period of development. Embryonic Id1 messages are detected during gastrulation on the proximal side of the embryonic ectoderm, which is the border between the embryo proper and the extraembryonic tissues, and the expression of Id3 is found throughout the entire embryo proper. This unique pattern of expression of the different members of the Id family suggests a nonredundant role for these genes in antagonizing the activity of bHLH transcription factors during very early mouse development. During neurogenesis, the expression of each member of the Id family is present in an unique pattern along the dorsal-ventral axis of the neural tube: In the early stages of spinal cord development, both Id1 and Id2 are expressed in the roof plate, whereas Id3 is expressed both in the roof and the floor plates. As development progresses, the expression of both Id1 and Id3 is detected in the dividing neuroblasts, whereas Id2 and 4 are expressed in presumptive neurons which are undergoing maturation. The expression patterns of all the members of the Id gene family persist throughout the entire CNS, both in the spinal cord and in the brain. In addition, the characteristic expression of Id2 and Id4 in more mature neurons is reiterated both in the PNS and in the neurons of some of the sensory organs. These data suggest that the expression of different subgroups of the Id gene family may have different physiological consequences and thereby contributes in unique ways to specify the differentiation state of neuronal cells during development.

Expression patterns of Id1, Id2, and Id3 are highly related but distinct from that of Id4 during mouse embryogenesis.

The murine dominant negative helix-loop-helix (dnHLH) proteins inhibit the activities of bHLH transcription factors in diverse cell lineages (Benezra et al. [1990] Cell 61:49-59; Christy et al [1991] Proc. Natl. Acad. Sci. U.S.A. 88:1815-1819; Sun et al [1991] Mol. Cell Biol. 11: 5603-5611; Riechmann et al. [1994] Nucleic Acids Res. 22:749-755). Currently, there are four members in the dnHLH family, Id1, Id2, Id3, and Id4. In this report, we have performed a detailed comparative in situ hybridization analysis to examine their expression pattern during post-gastrulational mouse development. Id1, 2, and 3 are expressed in multiple tissues, whereas Id4 expression can only be detected in neuronal tissues and in the ventral portion of the epithelium of the developing stomach. The regions where Id1-3 genes are expressed, such as gut, lung, kidney, tooth, whisker, and several glandular structures, are undergoing active morphogenetic activities. The expression patterns of Id1, 2, and 3 overlap in many organs, except in the tissue derived from primitive gut. In the latter, Id1 and Id3 signals are detected in the mesenchyme surrounding the epithelium, whereas Id2 is expressed within the epithelium. The difference in the patterns of expressions of Id2-3 and Id4 suggest that the dominant negative transcriptional activity of these two subclasses of the Id family may have different physiological consequences.

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.

Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1.

The expression of Id1, a helix-loop-helix protein which inhibits the activity of basic helix-loop-helix transcription factors, is down-regulated during cellular differentiation and cell cycle withdrawal both in tissue culture models and in mouse embryos. In order to study the mechanism of control of Idl expression, we have isolated a 210-bp enhancer element in the upstream region of the Id1 gene whose activity recapitulates Id1 expression in C2C12 muscle cells and C3H10T1/2 fibroblasts: i.e., this element is active in proliferating cells in the presence of serum and completely inactivated upon mitogen depletion, cell cycle withdrawal, and (in the case of C2C12) induced myoblast differentiation. Using linker-scanning mutations and site-directed mutagenesis in transient transfection experiments, we have identified two functional elements within the 210-bp enhancer which are required for proper serum responsiveness. One element (A) contains a consensus Egr-1 binding site and additional flanking sequences required for optimal activity, and the other element (B) fits no known consensus. Gel shift experiments demonstrate that the protein complex binding to the A site contains Egr-1 and other proteins. This complex as well as a protein complex that binds to the B site is lost within 24 h of serum depletion, correlating with the down-regulation of Id1 expression. On the basis of these findings, we propose that the regulation of the Id1 response to serum is mediated in part by the early response gene Egr-1 and as such provides a signaling link between the early-growth-response transcription factors and dominant-negative helix-loop-helix proteins.