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Macrophage activation is controlled by a balance between activating and inhibitory receptors 1-7 , which protect normal tissues from excessive damage during infection 8,9 but promote tumour growth and metastasis in cancer 7,10 . Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer., (© 2024. The Author(s).)

ID proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. ID proteins inhibit basic-HLH transcription factors often blocking differentiation and sustaining proliferation. A small-molecule, AGX51, targets ID proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impairs cell growth and viability that results from an increase in reactive oxygen species (ROS) production upon ID degradation. In mouse models, AGX51 treatment suppresses breast cancer colonization in the lung, regresses the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduces tumor burden in sporadic colorectal neoplasia. Furthermore, in cells and mice, we fail to observe acquired resistance to AGX51 likely the result of the inability to mutate the binding pocket without loss of ID function and efficient degradation of the ID proteins. Thus, AGX51 is a first-in-class compound that antagonizes ID proteins, shows strong anti-tumor effects and may be further developed for the management of multiple cancers.

UBR5 is a nuclear phosphoprotein of obscure functions. Clinical analyses reveal that UBR5 amplifications and overexpression occur in over 20% cases of human breast cancers. Breast cancer patients carrying UBR5 genetic lesions with overexpression have significantly reduced survival. Experimental work in vitro and in vivo demonstrates that UBR5, functioning as an oncoprotein, plays a profound role in breast cancer growth and metastasis. UBR5 drives tumor growth largely through paracrine interactions with the immune system, particularly through inhibiting the cytotoxic response mediated by CD8 + T lymphocytes, whereas it facilitates metastasis in a tumor cell-autonomous manner via its transcriptional control of key regulators of the epithelial-mesenchymal transition, ID1 and ID3. Furthermore, simultaneous targeting of UBR5 and PD-L1 yields strong therapeutic benefit to tumor-bearing hosts. This work significantly expands our scarce understanding of the pathophysiology and immunobiology of a fundamentally important molecule and has strong implications for the development of novel immunotherapy to treat highly aggressive breast cancers that resist conventional treatment., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

This corrects the article DOI: 10.1038/ncomms15987.

Whole chromosome gains or losses (aneuploidy) are a hallmark of ~70% of human tumors. Modeling the consequences of aneuploidy has relied on perturbing spindle assembly checkpoint (SAC) components, but interpretations of these experiments are clouded by the multiple functions of these proteins. Here, we used a Cre recombinase-mediated chromosome loss strategy to individually delete mouse chromosomes 9, 10, 12, or 14 in tetraploid immortalized murine embryonic fibroblasts. This methodology also involves the generation of a dicentric chromosome intermediate, which subsequently undergoes a series of breakage-fusion-bridge (BFB) cycles. While the aneuploid cells generally display a growth disadvantage in vitro , they grow significantly better in low adherence sphere-forming conditions and three of the four lines are transformed in vivo , forming large and invasive tumors in immunocompromised mice. The aneuploid cells display increased chromosomal instability and DNA damage, a mutator phenotype associated with tumorigenesis in vivo Thus, these studies demonstrate a causative role for whole chromosome loss and the associated BFB-mediated instability in tumorigenesis and may shed light on the early consequences of aneuploidy in mammalian cells., (© 2017 The Authors.)

The widespread application of high-throughput sequencing methods is resulting in the identification of a rapidly growing number of novel gene fusions caused by tumour-specific chromosomal rearrangements, whose oncogenic potential remains unknown. Here we describe a strategy that builds upon recent advances in genome editing and combines ex vivo and in vivo chromosomal engineering to rapidly and effectively interrogate the oncogenic potential of genomic rearrangements identified in human brain cancers. We show that one such rearrangement, an microdeletion resulting in a fusion between Brevican (BCAN) and Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1), is a potent oncogenic driver of high-grade gliomas and confers sensitivity to the experimental TRK inhibitor entrectinib. This work demonstrates that BCAN-NTRK1 is a bona fide human glioma driver and describes a general strategy to define the oncogenic potential of novel glioma-associated genomic rearrangements and to generate accurate preclinical models of this lethal human cancer.

The mitotic checkpoint ensures proper segregation of chromosomes by delaying anaphase until all kinetochores are bound to microtubules. This inhibitory signal is composed of a complex containing Mad2, which inhibits anaphase progression. The complex can be disassembled by p31 comet and TRIP13; however, TRIP13 knockdown has been shown to cause only a mild mitotic delay. Overexpression of checkpoint genes, as well as TRIP13, is correlated with chromosomal instability (CIN) in cancer, but the initial effects of Mad2 overexpression are prolonged mitosis and decreased proliferation. Here, we show that TRIP13 overexpression significantly reduced, and TRIP13 reduction significantly exacerbated, the mitotic delay associated with Mad2 overexpression, but not that induced by microtubule depolymerization. The combination of Mad2 overexpression and TRIP13 loss reduced the ability of checkpoint complexes to disassemble and significantly inhibited the proliferation of cells in culture and tumor xenografts. These results identify an unexpected dependency on TRIP13 in cells overexpressing Mad2., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance., Methods: Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM., Results: In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1., Conclusions: In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Treatment with celecoxib, a selective COX-2 inhibitor, reduces formation of premalignant adenomatous polyps in the gastrointestinal tracts of humans and mice. In addition to its chemopreventive activity, celecoxib can exhibit antimicrobial activity. Differing bacterial profiles have been found in feces from colon cancer patients compared with those of normal subjects. Moreover, preclinical studies suggest that bacteria can modulate intestinal tumorigenesis by secreting specific metabolites. In the current study, we determined whether celecoxib treatment altered the luminal microbiota and metabolome in association with reducing intestinal polyp burden in mice. Administration of celecoxib for 10 weeks markedly reduced intestinal polyp burden in APC(Min/+) mice. Treatment with celecoxib also altered select luminal bacterial populations in both APC(Min/+) and wild-type mice, including decreased Lactobacillaceae and Bifidobacteriaceae as well as increased Coriobacteriaceae Metabolomic analysis demonstrated that celecoxib caused a strong reduction in many fecal metabolites linked to carcinogenesis, including glucose, amino acids, nucleotides, and lipids. Ingenuity Pathway Analysis suggested that these changes in metabolites may contribute to reduced cell proliferation. To this end, we showed that celecoxib reduced cell proliferation in the base of normal appearing ileal and colonic crypts of APC(Min/+) mice. Consistent with this finding, lineage tracing indicated that celecoxib treatment reduced the rate at which Lgr5-positive stem cells gave rise to differentiated cell types in the crypts. Taken together, these results demonstrate that celecoxib alters the luminal microbiota and metabolome along with reducing epithelial cell proliferation in mice. We hypothesize that these actions contribute to its chemopreventive activity. Cancer Prev Res; 9(9); 721-31. ©2016 AACR., Competing Interests: Edward D. Karoly is an employee of Metabolon, Inc. The other authors disclosed no potential conflicts of interest., (©2016 American Association for Cancer Research.)

Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia., (© 2016 by The American Society of Hematology.)

We have previously shown a novel antimetastatic role for neutrophils in the premetastatic lung of mice in models of breast cancer. Here we expand on those findings in the context of human breast cancer. We assessed the cytotoxicity of neutrophils from 90 newly diagnosed breast cancer patients, 24 ductal carcinoma in situ patients, 56 metastatic breast cancer patients, and 64 women with no history of cancer. We report that neutrophils from metastatic and newly diagnosed breast cancer patients are significantly more cytotoxic than neutrophils from cancer-free individuals. We hypothesized that tumor-secreted factors 'prime' neutrophils to become cytotoxic. To identify these factors we assayed for cytokines in serum from 54 breast cancer patients and 35 cancer-free controls. Tumor necrosis factor (TNFα), MCP-1 (CCL2), and IL1RA significantly correlated with cytotoxicity and directly stimulated neutrophil cytotoxicity ex vivo . RNA-seq analyses found protein kinase C iota ( PRKCI) to be over expressed in patient neutrophils relative to neutrophils from cancer-free individuals. PRKCI has been implicated in NADPH oxidase assembly, required for neutrophil-mediated cell cytotoxicity. Treatment of human neutrophils with TNF-induced PRKCI expression and cytotoxicity in samples that had low basal levels of PRKCI expression. To date, this work is the first to demonstrate the cytotoxic role of neutrophils in the peripheral blood of a large cohort of breast cancer patients, and that select cytokines appear to mediate the stimulation of neutrophil cytotoxicity. Further functional studies are necessary to identify clinically relevant means of stimulating neutrophil cytotoxicity as an effective barrier against disease progression and metastasis., Competing Interests: The authors declare no conflict of interest.

Inhibitor of DNA binding (ID)-1 is important for angiogenesis during embryogenesis and tumor development. Whether ID1 expression in endothelial cells of the colon is required for normal response to injury is unknown. We demonstrate that Id1 is up-regulated in colonic endothelial cells in an experimental model of colitis and in the inflamed mucosa of patients with inflammatory bowel disease. Because prostaglandin E2 and tumor necrosis factor-α are also elevated in colitis, we determined whether these factors could induce ID1 transcription in cultured endothelial cells. Tumor necrosis factor-α stimulated ID1 transcription via early growth response 1 protein (Egr-1). By contrast, the induction of ID1 by prostaglandin E2 was mediated by cAMP response element-binding protein (CREB). To determine whether the increased ID1 levels in the endothelial cells of inflamed mucosa were an adaptive response that modulated the severity of tissue injury, Id1 was conditionally depleted in the endothelium of mice, which sensitized the mice to more severe chemical colitis, including more severe diarrhea, bleeding, and histological injury, and shorter colon compared with control mice. Moreover, depletion of Id1 in the vasculature was associated with increased CD31(+) aggregates and increased vascular permeability in inflamed mucosa compared with those in Id1 wild-type control mice. These results suggest that endothelial ID1 up-regulation in inflamed colonic mucosa is an adaptive response that modulates the severity of tissue injury., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Bladder cancer is one of the most common malignant genitourinary diseases worldwide. Despite advances in surgical technique, medical oncology and radiation therapy, cure of invasive tumors remains elusive for patients with late stage disease. Therefore, new therapeutic strategies are needed to improve the response rates with regard to recurrence, invasion and metastasis. Objective: Inhibitor of DNA binding (Id) proteins have been proposed as therapeutic targets due to the key regulatory role they exert in multiple steps of cancer. We aimed to explore the role of Id proteins in bladder cancer development and the pattern of expression of Id proteins in bladder carcinomas. Methods: We used a well-established chemically induced model of bladder carcinogenesis. Wild type and Id-deficient mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in the drinking water and urinary bladder lesions were analyzed histopathologically and stained for Id1. We assessed the effects of Id1 inactivation in cultured bladder cancer cells and in a model of metastatic lung colonization. We also performed Id1 staining of human urothelial carcinoma samples and matched lymph node metastases. Results: Id1 protein was overexpressed in the BBN-induced model of bladder cancer. Id1 deficiency resulted in the development of urinary bladder tumors with areas of extensive hemorrhage and decreased invasiveness when compared to wild type mice. Id1 inactivation led to decreased cell growth in vitro and lung colonization in vivo of human bladder cancer cells. Immunohistochemistry performed on human urothelial carcinoma samples showed Id1 positive staining in both primary tumors and lymph node metastases. Conclusions: In summary, our studies reveal the physiological relevance of Id1 in bladder cancer progression and suggest that targeting Id1 may be important in the development of novel therapies for the treatment of bladder cancer.

Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia., (© 2015 by The American Society of Hematology.)

Background: Malignant transformation requires the interaction of cancer cells with their microenvironment, including infiltrating leukocytes. However, somatic mutational studies have focused on alterations in cancer cells, assuming that the microenvironment is genetically normal. Because we hypothesized that this might not be a valid assumption, we performed exome sequencing and targeted sequencing to investigate for the presence of pathogenic mutations in tumor-associated leukocytes in breast cancers., Methods: We used targeted sequencing and exome sequencing to evaluate the presence of mutations in sorted tumor-infiltrating CD45-positive cells from primary untreated breast cancers. We used high-depth sequencing to determine the presence/absence of the mutations we identified in breast cancer-infiltrating leukocytes in purified tumor cells and in circulating blood cells., Results: Capture-based sequencing of 15 paired tumor-infiltrating leukocytes and matched germline DNA identified variants in known cancer genes in all 15 primary breast cancer patients in our cohort. We validated the presence of mutations identified by targeted sequencing in infiltrating leukocytes through orthogonal exome sequencing. Ten patients harbored alterations previously reported as somatically acquired variants, including in known leukemia genes ( DNTM3A , TET2 , and BCOR) . One of the mutations observed in the tumor-infiltrating leukocytes was also detected in the circulating leukocytes of the same patients at a lower allele frequency than observed in the tumor-infiltrating cells., Conclusions: Here we show that somatic mutations, including mutations in known cancer genes, are present in the leukocytes infiltrating a subset of primary breast cancers. This observation allows for the possibility that the cancer cells interact with mutant infiltrating leukocytes, which has many potential clinical implications., Competing Interests: The authors declare no conflict of interest.

Different mechanisms contribute to the development of sporadic, hereditary and colitis-associated colorectal cancer. Inhibitor of DNA binding/differentiation (Id) proteins act as dominant-negative antagonists of basic helix-loop-helix transcription factors. Id1 is a promising target for cancer therapy, but little is known about its role in the development of colon cancer. We used immunohistochemistry to demonstrate that Id1 is overexpressed in human colorectal adenomas and carcinomas, whether sporadic or syndromic. Furthermore, elevated Id1 levels were found in dysplasia and colon cancer arising in patients with inflammatory bowel disease. Because levels of PGE2 are also elevated in both colitis and colorectal neoplasia, we determined whether PGE2 could induce Id1. PGE2 via EP4 stimulated protein kinase A activity resulting in enhanced pCREB-mediated Id1 transcription in human colonocytes. To determine the role of Id1 in carcinogenesis, two mouse models were used. Consistent with the findings in humans, Id1 was overexpressed in tumors arising in both Apc(Min) (/+) mice, a model of familial adenomatous polyposis, and in experimental colitis-associated colorectal neoplasia. Id1 deficiency led to significant decrease in the number of intestinal tumors in Apc(Min) (/+) mice and prolonged survival. In contrast, Id1 deficiency did not affect the number or size of tumors in the model of colitis-associated colorectal neoplasia, likely due to exacerbation of colitis associated with Id1 loss. Collectively, these results suggest that Id1 plays a role in gastrointestinal carcinogenesis. Our findings also highlight the need for different strategies to reduce the risk of colitis-associated colorectal cancer compared with sporadic or hereditary colorectal cancer., (©2015 American Association for Cancer Research.)

LGR5 and BMI1 mark intestinal stem cells in crypt base columnar cells and +4 position cells, respectively, but characterization of functional markers in these cell populations is limited. ID1 maintains the stem cell potential of embryonic, neural, and long-term repopulating hematopoietic stem cells. Here, we show in both human and mouse intestine that ID1 is expressed in cycling columnar cells, +4 position cells, and transit-amplifying cells in the crypt. Lineage tracing revealed ID1+ cells to be self-renewing, multipotent stem/progenitor cells that are responsible for the long-term renewal of the intestinal epithelium. Single ID1+ cells can generate long-lived organoids resembling mature intestinal epithelium. Complete knockout of Id1 or selective deletion of Id1 in intestinal epithelium or in LGR5+ stem cells sensitizes mice to chemical-induced colon injury. These experiments identify ID1 as a marker for intestinal stem/progenitor cells and demonstrate a role for ID1 in maintaining the potential for repair in response to colonic injury.

Inhibitor of DNA binding (ID) proteins are transcriptional regulators that control the timing of cell fate determination and differentiation in stem and progenitor cells during normal development and adult life. ID genes are frequently deregulated in many types of human neoplasms, and they endow cancer cells with biological features that are hijacked from normal stem cells. The ability of ID proteins to function as central 'hubs' for the coordination of multiple cancer hallmarks has established these transcriptional regulators as therapeutic targets and biomarkers in specific types of human tumours.

ID genes are required for breast cancer colonization of the lungs, but the mechanism remains poorly understood. Here, we show that Id1 expression induces a stem-like phenotype in breast cancer cells while retaining epithelial properties, contrary to the notion that cancer stem-like properties are inextricably linked to the mesenchymal state. During metastatic colonization, Id1 induces a mesenchymal-to-epithelial transition (MET), specifically in cells whose mesenchymal state is dependent on the Id1 target protein Twist1, but not at the primary site, where this state is controlled by the zinc finger protein Snail1. Knockdown of Id expression in metastasizing cells prevents MET and dramatically reduces lung colonization. Furthermore, Id1 is induced by transforming growth factor (TGF)-β only in cells that have first undergone epithelial-to-mesenchymal transition (EMT), demonstrating that EMT is a prerequisite for subsequent Id1-induced MET during lung colonization. Collectively, these studies underscore the importance of Id-mediated phenotypic switching during distinct stages of breast cancer metastasis., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetic and genomic aberrations are the primary cause of cancer. Chromosome missegregation leads to aneuploidy and provides cancer cells with a mechanism to lose tumor suppressor loci and gain extra copies of oncogenes. Using cytogenetic and array-based comparative genomic hybridization data, we analyzed numerical chromosome aneuploidy in 43,205 human tumors and found that 68% of solid tumors are aneuploid. In solid tumors, almost all chromosomes are more frequently lost than gained with chromosomes 7, 12 and 20 being the only exceptions with more frequent gains. Strikingly, small chromosomes are lost more readily than large ones, but no such inverse size correlation is observed with chromosome gains. Because of increasing levels of proteotoxic stress, chromosome gains have been shown to slow cell proliferation in a manner proportional to the number of extra gene copies gained. However, we find that the extra chromosome in trisomic tumors does not preferentially have a low gene copy number, suggesting that a proteotoxicity-mediated proliferation barrier is not sustained during tumor progression. Paradoxically, despite a bias toward chromosome loss, gains of chromosomes are a poor prognostic marker in ovarian adenocarcinomas. In addition, we find that solid and non-solid cancers have markedly distinct whole-chromosome aneuploidy signatures, which may underlie their fundamentally different etiologies. Finally, preferential chromosome loss is observed in both early and late stages of astrocytoma. Our results open up new avenues of enquiry into the role and nature of whole-chromosome aneuploidy in human tumors and will redirect modeling and genetic targeting efforts in patients., (Copyright © 2012 UICC.)

High-grade gliomas (HGGs) are incurable brain tumors that are characterized by the presence of glioma-initiating cells (GICs). GICs are essential to tumor aggressiveness and retain the capacity for self-renewal and multilineage differentiation as long as they reside in the perivascular niche. ID proteins are master regulators of stemness and anchorage to the extracellular niche microenvironment, suggesting that they may play a role in maintaining GICs. Here, we modeled the probable therapeutic impact of ID inactivation in HGG by selective ablation of Id in tumor cells and after tumor initiation in a new mouse model of human mesenchymal HGG. Deletion of 3 Id genes induced rapid release of GICs from the perivascular niche, followed by tumor regression. GIC displacement was mediated by derepression of Rap1gap and subsequent inhibition of RAP1, a master regulator of cell adhesion. We identified a signature module of 5 genes in the ID pathway, including RAP1GAP, which segregated 2 subgroups of glioma patients with markedly different clinical outcomes. The model-informed survival analysis together with genetic and functional studies establish that ID activity is required for the maintenance of mesenchymal HGG and suggest that pharmacological inactivation of ID proteins could serve as a therapeutic strategy.

BRCA1 germline mutation carriers usually develop ER, PR and HER2 negative breast carcinoma. Somatic BRCA1 mutations are rare in sporadic breast cancers, but other mechanisms could impair BRCA1 functions in these tumors, particularly in triple-negative breast carcinomas (TNBCs). Id4, a helix-loop-helix DNA binding factor, blocks BRCA1 gene transcription in vitro and could downregulate BRCA1 in vivo. We compared Id4 immunoreactivity in 101 TNBCs versus 113 non-TNBCs, and correlated the results with tumor morphology and immunoreactivity for CK5/6, CK14, EGFR, and androgen receptor (AR). Id4 was present in 76 out of 101 (75 %) TNBCs: 40 (40 %) TNBCs displayed Id4 positivity in >50 % of neoplastic cells, 23 (23 %) in 5-50 %, and 13 (13 %) in <5 %. In contrast, only 6 (5 %) of 113 non-TNBCs showed focal Id4 positivity, limited to fewer than 5 % of the tumor (p < 0.0001). Id4 expression significantly associated with high histologic grade (p = 0.0002) and mitotic rate (p = 0.006). Id4 decorated all 12 TNBCs with large central acellular zone of necrosis in our series, with positive staining in 10-90 % of the cells. Id4 signal strongly correlated with cytokeratin CK14 reactivity (p < 0.0001), but not with CK5/6 and EGFR. All apocrine carcinomas in our series were positive for AR and most for EGFR, but they were negative for CK5/6, CK14, and Id4, with only two exceptions. Our results document substantial expression of Id4 in most TNBCs, which could result in functional downregulation of BRCA1 pathways in these tumors.

Stem-cell functions require activation of stem-cell-intrinsic transcriptional programs and extracellular interaction with a niche microenvironment. How the transcriptional machinery controls residency of stem cells in the niche is unknown. Here we show that Id proteins coordinate stem-cell activities with anchorage of neural stem cells (NSCs) to the niche. Conditional inactivation of three Id genes in NSCs triggered detachment of embryonic and postnatal NSCs from the ventricular and vascular niche, respectively. The interrogation of the gene modules directly targeted by Id deletion in NSCs revealed that Id proteins repress bHLH-mediated activation of Rap1GAP, thus serving to maintain the GTPase activity of RAP1, a key mediator of cell adhesion. Preventing the elevation of the Rap1GAP level countered the consequences of Id loss on NSC-niche interaction and stem-cell identity. Thus, by preserving anchorage of NSCs to the extracellular environment, Id activity synchronizes NSC functions to residency in the specialized niche.

Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26(low) fast-dividing cells, whereas PKH26(high) slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26(high)Id1(high) population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26(high)Id1(high) population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.

Understanding the mechanism by which embryonic stem (ES) cells self-renew is crucial for the realization of their therapeutic potential. Earlier, overexpression of Id proteins was shown to be sufficient to maintain mouse ES cells in a self-renewing state even in the absence of serum. Here, we use ES cells derived from Id deficient mice to investigate the requirement for Id proteins in maintaining ES cell self-renewal. We find that Id1(-/-) ES cells have a defect in self-renewal and a propensity to differentiate. We observe that chronic or acute loss of Id1 leads to a down-regulation of Nanog, a critical regulator of self-renewal. In addition, in the absence of Id1, ES cells express elevated levels of Brachyury, a marker of mesendoderm differentiation. We find that loss of both Nanog and Id1 is required for the up-regulation of Brachyury, and ectopic Nanog expression in Id1(-/-) ES cells rescues the self-renewal defect, indicating that Nanog is the major downstream target of Id1. These results identify Id1 as a critical factor in the maintenance of ES cell self-renewal and suggest a plausible mechanism for its control of lineage commitment.

Within high-grade gliomas, the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis, we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity, while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly, Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further, eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival, while knockdown of Olig2 within Id1(low) cells has a significant survival benefit, underscoring the importance of non-self-renewing lineages in disease progression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Primary tumors have been shown to prepare distal organs for later colonization of metastatic cells by stimulating organ-specific infiltration of bone marrow derived cells. Here we demonstrate that neutrophils accumulate in the lung prior to the arrival of metastatic cells in mouse models of breast cancer. Tumor-entrained neutrophils (TENs) inhibit metastatic seeding in the lungs by generating H(2)O(2) and tumor secreted CCL2 is a critical mediator of optimal antimetastatic entrainment of G-CSF-stimulated neutrophils. TENs are present in the peripheral blood of breast cancer patients prior to surgical resection but not in healthy individuals. Thus, whereas tumor-secreted factors contribute to tumor progression at the primary site, they concomitantly induce a neutrophil-mediated inhibitory process at the metastatic site., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Multiple mechanisms have been proposed to explain how Rb and p53 tumor suppressor loss lead to chromosome instability (CIN). It was recently shown that Rb pathway inhibition causes overexpression of the mitotic checkpoint gene Mad2, but whether Mad2 overexpression is required to generate CIN in this context is unknown. Here, we show that CIN in cultured cells lacking Rb family proteins requires Mad2 upregulation and that this upregulation is also necessary for CIN and tumor progression in vivo. Mad2 is also repressed by p53 and its upregulation is required for CIN in a p53 mutant tumor model. These results demonstrate that Mad2 overexpression is a critical mediator of the CIN observed upon inactivation of two major tumor suppressor pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Antiangiogenic therapies are frequently used with concomitantly administered cancer chemotherapy to improve outcomes, but the mechanism for the benefit of the combination is uncertain. We describe a mechanism by which a specific, cytotoxic antivascular agent causes vascular remodeling and improved chemotherapy results. By selectively killing tumor neovasculature using short-ranged α-particles targeted to vascular endothelial (VE)-cadherin on vascular endothelial cells (by use of 225Ac-labeled E4G10 antibody) we were able both to reduce tumor growth and to increase the efficacy of chemotherapy, an effect seen only when the chemotherapy was administered several days after the vascular targeting agent, but not if the order of administration was reversed. Immunohistochemical and immunofluorescence studies showed that the vasculature of 225Ac-E4G10-treated tumors was substantially depleted; the remaining vessels appeared more mature morphologically and displayed increased pericyte density and coverage. Tumor uptake and microdistribution studies with radioactive and fluorescent small molecule drugs showed better accumulation and more homogenous distribution of the drugs within 225Ac-E4G10-treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and improvement of the tumor vascular architecture, and also show that the resulting vascular remodeling can increase tumor delivery of small molecules, thus providing a process for the improved outcomes observed after combining antivascular therapy and chemotherapy. This study directly shows evidence for what has long been a speculated mechanism for antiangiogenic therapies. Moreover, targeting the vessel for killing provides an alternative mode of improving chemotherapy delivery and efficacy, potentially avoiding some of the drawbacks of targeting a highly redundant angiogenic pathway., (Copyright © 2010 AACR.)

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however, the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here, we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM, blood, and tumor stroma, as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally, use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors, confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs, our results establish a strategy to track and target EPCs in vivo, clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis., (©2010 AACR.)

BACKGROUND Blood vessels are formed either by sprouting of resident tissue endothelial cells (angiogenesis) or by recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs, vasculogenesis). Neovascularization has been implicated in tumor growth and inflammation, but its roles in graft-vs-host disease (GVHD) and in tumors after allogeneic BM transplantation (allo-BMT) were not known. METHODS We analyzed neovascularization, the contribution of endothelial cells and EPCs, and the ability of anti-vascular endothelial-cadherin antibody, E4G10, to inhibit neovascularization in mice with GVHD after allo-BMT using immunofluorescence microscopy and flow cytometry. We examined survival and clinical and histopathologic GVHD in mice (n = 10-25 per group) in which GVHD was treated with the E4G10 antibody using immunohistochemistry, flow cytometry, and cytokine immunoassay. We also assessed survival, the contribution of green fluorescent protein-marked EPCs to the tumor vasculature, and the ability of E4G10 to inhibit tumor growth in tumor-bearing mice (n = 20-33 per group) after allo-BMT using histopathology and bioluminescence imaging. All statistical tests were two-sided. RESULTS We found increased neovascularization mediated by vasculogenesis, as opposed to angiogenesis, in GVHD target tissues, such as liver and intestines. Administration of E4G10 inhibited neovascularization by donor BM-derived cells without affecting host vascularization, inhibited both GVHD and tumor growth, and increased survival (at 60 days post-BMT and tumor challenge with A20 lymphoma, the probability of survival was 0.29 for control antibody-treated allo-BMT recipients vs 0.7 for E4G10-treated allo-BMT recipients, 95% confidence interval = 0.180 to 0.640, P < .001). CONCLUSIONS Therapeutic targeting of neovascularization in allo-BMT recipients is a novel strategy to simultaneously ameliorate GVHD and inhibit posttransplant tumor growth, providing a new approach to improve the overall outcome of allogeneic hematopoietic stem cell transplantation.

Inhibition of an initiating oncogene often leads to extensive tumour cell death, a phenomenon known as oncogene addiction. This has led to the search for compounds that specifically target and inhibit oncogenes as anticancer agents. However, there has been no systematic exploration of whether chromosomal instability generated as a result of deregulation of the mitotic checkpoint pathway, a frequent characteristic of solid tumours, has any effect on oncogene addiction. Here we show that induction of chromosome instability by overexpression of the mitotic checkpoint gene Mad2 in mice does not affect the regression of Kras-driven lung tumours when Kras is inhibited. However, tumours that experience transient Mad2 overexpression and consequent chromosome instability recur at markedly elevated rates. The recurrent tumours are highly aneuploid and have varied activation of pro-proliferative pathways. Thus, early chromosomal instability may be responsible for tumour relapse after seemingly effective anticancer treatments.

The stepwise progression from an early dysplastic lesion to full-blown metastatic malignancy is associated with increases in genomic instability. Mitotic chromosomal instability - the inability to faithfully segregate equal chromosome complements to two daughter cells during mitosis - is a widespread phenomenon in solid tumours that is thought to serve as the fuel for tumorigenic progression. How chromosome instability (CIN) arises in tumours and what consequences it has are still, however, hotly debated issues. Here we review the recent literature with an emphasis on models that recapitulate observations from human disease.

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell-specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)beta at day 7 of differentiation increases hVPr-GFP(+) cells by tenfold. In phase 2, TGFbeta inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1(high)VEGFR2(high)VE-cadherin(+) ephrinB2(+). Using an Id1-YFP hESC reporter line, we showed that TGFbeta inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

Mechanisms by which whole chromosome instability lead to tumorigenesis have eluded the cancer research field. In this issue of Cancer Cell, Baker et al. show that CIN induced by a defective mitotic checkpoint, under certain genetic and tissue contexts, leads to accelerated loss of heterozygosity of a tumor suppressor gene.

Defining the molecular identity of stem cells may be critical for formulating a rational strategy for the therapeutic intervention of stem cell dysfunction. We find that high expression of Id1, a dominant-negative helix-loop-helix transcriptional regulator, identifies a rare population of GFAP(+) astrocytes with stem cell attributes among the subventricular astrocytes in the adult brain. The rare, long-lived, and relatively quiescent Id1(high) astrocytes with morphology characteristic of B1 type astrocytes self-renew and generate migratory neuroblasts that differentiate into olfactory bulb interneurons. Cultured Id1(high) neural stem cells can self-renew asymmetrically and generate a stem and a differentiated cell expressing progressively lower levels of Id1, revealing an Id1 gradient in unperturbed cells of subventricular neurogenic lineages. Moreover, Id genes are necessary to confer self-renewal capacity, a characteristic of stem cell identity. We suggest that high expression of a single transcriptional regulator, Id1, molecularly defines the long-sought-after B1 type adult neural stem cells.

Emerging evidence indicates that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated growth of certain tumors in mice and human. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. While the contributions of EPCs to neovessel formation in spontaneous and transplanted tumors and to the metastatic transition have been reported to be relatively low, remarkably, specific EPC ablation in vivo has resulted in severe angiogenesis inhibition and impaired primary and metastatic tumor growth. The existence of a BM reservoir of EPCs, and the selective involvement of EPCs in neovascularization, have attracted considerable interest because these cells represent novel target for therapeutic intervention. In addition, EPCs are also being used as pharmacodynamic surrogate markers for monitoring cancer progression, as well as for optimizing efficacy of anti-angiogenic therapies in the clinic. This review will focus primarily on recent advances and emerging concepts in the field of EPC biology and discuss ongoing debates involving the role of EPCs in tumor neovascularization. For detailed information on the in vitro characterization of EPCs contribution to non-tumor pathologies, the reader is directed towards several excellent reviews and publications [F. Bertolini, Y. Shaked, P. Mancuso and R.S. Kerbel, Nat. Rev., Cancer 6 (2006) 835-845. [1]] [J.M. Hill, T. Finkel and A.A. Quyyumi, Vox Sang. 87 Suppl 2 (2004) 31-37. [2]] [A.Y. Khakoo and T. Finkel, Annu. Rev. Med. 56 (2005) 79-101. [3]] [H.G. Kopp, C.A. Ramos and S. Rafii, Curr. Opin. Hematol. 13 (2006) 175-181. [4]; K.K. Hirschi, D.A. Ingram and M.C. Yoder, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1584-1595. [5]; F. Timmermans, J. Plum, M.C. Yoder, D.A. Ingram, B. Vandekerckhove and J. Case, J. Cell. Mol. Med. 13 (2009) 87-102. [6]] and reviews by Bertolini, Voest and Yoder in this issue.

Cyclooxygenase-2 (COX-2) and Id-1 are overexpressed in a variety of human malignancies. Recently, each of these genes was found to play a role in mediating breast cancer metastasis to the lungs, but their potential interdependence was not evaluated. Hence, the main objective of the current study was to determine whether COX-2-derived prostaglandin (PGE(2)) activated Id-1 transcription, leading in turn to increased invasiveness of mammary epithelial cells. In MDA-MB-231 cells, treatment with PGE(2) induced Id-1, an effect that was mimicked by an EP(4) agonist. PGE(2) via EP(4) activated the epidermal growth factor receptor (EGFR) --> ERK1/2 pathway, which led to increased expression of Egr-1. PGE(2) stimulated EGFR signaling by inducing the release of amphiregulin, an EGFR ligand. The ability of PGE(2) to activate Id-1 transcription was mediated by enhanced binding of Egr-1 to the Id-1 promoter. Silencing of COX-2 or pharmacological inhibition of COX-2 led to reduced PGE(2) production, decreased Id-1 expression, and reduced migration of cells through extracellular matrix. A similar decrease in cell migration was found when Id-1 was silenced. The interrelationship between COX-2, PGE(2), Id-1, and cell invasiveness was also compared in nontumorigenic SCp2 and tumorigenic SCg6 mammary epithelial cells. Consistent with the findings in MDA-MB-231 cells, COX-2-derived PGE(2) induced Id-1, leading in turn to increased cell invasiveness. Taken together, these results suggest that PGE(2) via EP(4) activated the EGFR --> ERK1/2 --> Egr-1 pathway, leading to increased Id-1 transcription and cell invasion. These findings provide new insights into the relationship between COX-2 and Id-1 and their potential role in metastasis.

Hec1 (Highly Expressed in Cancer 1) is one of four proteins of the outer kinetochore Ndc80 complex involved in the dynamic interface between centromeres and spindle microtubules. Its overexpression is seen in a variety of human tumors and correlates with tumor grade and prognosis. We show here that the overexpression of Hec1 in an inducible mouse model results in mitotic checkpoint hyperactivation. As previously observed with overexpression of the Mad2 gene, hyperactivation of the mitotic checkpoint leads to aneuploidy in vitro and is sufficient to generate tumors in vivo that harbor significant levels of aneuploidy. These results underscore the role of chromosomal instability as a result of mitotic checkpoint hyperactivation in the initiation of tumorigenesis.

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.

Objective: Vascular progenitors of bone marrow origin participate to neovascularization at sites of wound healing and transplantation. We hypothesized that the biological purpose of this bone marrow-derived vascular component is to contribute angiogenic and survival functions distinct from those provided by the local tissue-derived vasculature., Research Design and Methods and Results: To address this hypothesis, we investigated the functional impact of bone marrow-derived vascular cells on pancreatic islets engraftment using bone marrow-reconstituted Id1(+/-)Id3(-/-) mice, a model of bone marrow-derived vasculogenesis. We show that, in this model, bone marrow-derived vasculogenic cells primarily contribute to the formation of new blood vessels within islet transplants. In contrast, graft revascularization in a wild-type background occurs by tissue-derived blood vessels only. Using these distinct transplant models in which bone marrow-and tissue-derived vasculature are virtually mutually exclusive, we demonstrate that bone marrow-derived vasculogenic cells exhibit enhanced angiogenic functions and support prompt activation of islets survival pathways, which significantly impact on islets engraftment and function. Moreover, gene profiling of vascular and inflammatory cells of the grafts demonstrate that neovascularization by bone marrow-derived cells is accompanied by the activation of a genetic program uniquely tuned to downregulate harmful inflammatory responses and to promote tissue repair., Conclusions: These studies uncover the biological significance of bone marrow-derived vasculogenic cells in the response to injury during transplantation. Enhancing the contribution of bone marrow-derived vasculogenic cells to transplantation sites may help to overcome both limited angiogenic responses of the adult tissue-derived vasculature and untoward effects of inflammation on transplant engraftment.

Transcription factors are important targets for the treatment of a variety of malignancies but are extremely difficult to inhibit, as they are located in the cell's nucleus and act mainly by protein-DNA and protein-protein interactions. The transcriptional regulators Id1 and Id3 are attractive targets for cancer therapy as they are required for tumor invasiveness, metastasis and angiogenesis. We report here the development of an antitumor agent that downregulates Id1 effectively in tumor endothelial cells in vivo. Efficient delivery and substantial reduction of Id1 protein levels in the tumor endothelium were effected by fusing an antisense molecule to a peptide known to home specifically to tumor neovessels. In two different tumor models, systemic delivery of this drug led to enhanced hemorrhage, hypoxia and inhibition of primary tumor growth and metastasis, similar to what is observed in Id1 knockout mice. Combination with the Hsp90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin yielded virtually complete growth suppression of aggressive breast tumors.

Loss of Id1 in the bone marrow (BM) severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis.