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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 APCMin/+ mice. Treatment with celecoxib also altered select luminal bacterial populations in both APCMin/+ 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 APCMin/+ 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.

Pathway alterations associated with celecoxib-induced metabolite changes in WT mice.

Supplementary Figure S1. Administration of celecoxib alters the luminal metabolome of WT mice. Feces were collected at baseline (0 weeks) then again at 5 and 10 weeks after initiating celecoxib treatment.

Data are rank transformed and displayed as color intensity with low levels indicated by green color and high levels indicated by red color.

Supplementary Figure Legends.

Supplementary Figure 2 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Table 1 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Figure 4 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

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. Cancer Res; 70(18); 7273–82. ©2010 AACR.

Supplementary Figure 9 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Figure 7 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Figure 1 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Methods, Figures 1-3 from Selective Killing of Tumor Neovasculature Paradoxically Improves Chemotherapy Delivery to Tumors

Supplementary Figure 6 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Figure 8 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

Supplementary Figure 5 from Using the Transcription Factor Inhibitor of DNA Binding 1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth

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.

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.

SummaryId proteins are helix-loop-helix (HLH) transcriptional regulators frequently overexpressed in cancer. Id proteins inhibit basic HLH transcription factors through protein-protein interactions, often inhibiting differentiation and sustaining proliferation. We recently identified a small-molecule, AGX51, which targets Id proteins for degradation and impairs ocular neovascularization in mouse models. Here we show that AGX51 treatment of cancer cell lines impaired cell growth and viability that results from a dramatic increase in ROS production upon Id degradation. In mouse models, AGX51 treatment suppressed breast cancer colonization in the lung, regressed the growth of paclitaxel-resistant breast tumors when combined with paclitaxel and reduced tumor burden in a model of sporadic colorectal neoplasia. Furthermore, in cells and mice, we failed to observe acquired resistance to AGX51 likely the result of the immutability of the binding pocket 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.

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.

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.

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.

Background: In the last few decades, theoretical models of cancer growth and progression have long focused on the aberrations of cancer cells alone, such as the abnormal mitotic and invasive characteristics of cancer cells. More recent research across multiple solid tumors suggests a critical interplay between solid tumors and immune regulating cells. Mounting evidence suggests that the immune system can tip the scales of cancer progression, eliciting either an anti-tumor or pro-tumor immune response depending upon varying stimulating and inhibitory factors. Here, we are the first to demonstrate novel mutations including leukemogenic mutations among tumor infiltrating lymphocytes in breast cancer patients. Methods: We obtained 17 primary breast cancer samples from patients who presented for either a lumpectomy or mastectomy as part of an IRB approved biospecimen protocol. Of the 17 patient samples, 13 had triple negative breast cancer, 2 had ER+, HER2+ disease, and 2 had ER+, HER2- disease. In the 17 samples, we used fluorescent activated cell sorting to separate CD45-positive hematopoietic cells from CD45-negative epithelial cells. We then performed exome sequencing of tumor-infiltrating hematopoietic cells to investigate for the presence of pathogenic mutations in tumor-associated leukocytes. In this first step, we identified candidate mutations in known cancer genes, including BCOR, NOTCH2, TET2, NF1, EZH2, and JAK1. As a validation step, we then performed capture-based sequencing of tumor-infiltrating leukocytes in 20 breast cancer samples matched to each patient’s germline DNA sample (buccal swab). In 10 of the 20 patients, we identified and validated somatic mutations. Of note, 6 of these patients harbored mutations known to be associated with leukemia, including DNTM3A, TET2, and BCOR. Most of these mutations were present in at least 5-20% of reads. This suggests that these mutations were present in enriched subclones and were not rare alleles occurring in a minority of hematopoietic stem cells. Lastly, we performed 454 deep sequencing analysis of microdissected tumor DNA samples and confirmed the absence of these mutations in breast cancer cells. Conclusion: Our data demonstrate somatic mutations in tumor infiltrating leukocytes in breast tumors which were not identified in matched germline or tumor DNA samples. Notably, some of these mutations have been implicated in the pathogenesis of lymphoid and myeloid malignancies. This observation suggests a unique relationship between cancer cells and mutant infiltrating leukocytes. We are now investigating the functional interaction between cancer cells and hematopoietic cells. Our findings reframe our understanding of carcinogenesis and offer novel opportunities for cancer detection and treatment. Citation Format: Elizabeth A Comen, Maria Kleppe, Hannah Wen, Britta Weigelt, Lennart Bastian, Brian Blum, Franck T Rapaport, Matt Keller, Nicolas Socci, Agnes Viale, Daoqi You, Robert Benezra, Edi Brogi, Jorge Reis-Filho, Michael Berger, Ross Levine, Larry Norton. Somatic leukemogenic mutations associated with infiltrating white blood cells in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD1-4.

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 ApcMin/+ 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 ApcMin/+ 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. Cancer Prev Res; 8(4); 303–11. ©2015 AACR.

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 use of mouse models has been an invaluable resource in cancer research but their generation is lengthy and costly. Here the authors describe an approach to generate engineered mouse models carrying specific gene fusions and, as a proof of principle, show that Bcan-Ntrk1 fusion leads to glioblastomas.

Summary 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., Highlights • ID1 is expressed in mouse and human intestinal and colonic stem and progenitor cells • ID1+ cells are long-lived and multipotent • Deletion of Id1 in stem and progenitor cells sensitizes mice to colon injury, In this article, Benezra, Dannenberg, and colleagues show that the dominant-negative transcription factor ID1 marks the stem and progenitor cells of the gut and is required in the colon for an optimal response to injury in a mouse model of colitis.

Inhibitor of DNA binding (ID) proteins are transcriptional regulators that control the timing of cell fate determination and differentiation in stem and progenitor cells. The ability of ID proteins to function as central 'hubs' for the coordination of multiple cancer hallmarks is establishing them as therapeutic targets and biomarkers in specific types of human tumours. 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.

SUMMARY 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., Graphical Abstract, In Brief Wojnarowicz et al., describe the identification, by an in silico screen, and characterization of a small molecule, AGX51, that targets Id proteins. AGX51 treatment of cells lead to Id protein degradation, cell cycle arrest, and reduced cell viability. AGX51 inhibited pathologic ocular neovascularization in mouse models, phenocopying genetic Id loss.

Low-grade gliomas account for ~30% of primary central nervous system tumors arising in children. Although LGGs grow slowly, inoperable tumors can lead to morbidity and premature death. The most common oncogenic event observed in nearly 70% of cases of Pilocytic astrocytomas (PA) and diffuse leptomeningeal glioneuronal tumors (DLGT)—is a recurrent tandem duplication on chromosome 7. This complex rearrangement results in the fusion of BRAF C-Terminus comprising of the kinase domain with KIAA1549 N-Terminus, an uncharacterized gene. Apart from creating this fusion, the entire region between KIAA1549 and BRAF comprising of 17 protein coding genes is duplicated. Currently, there are no accurate preclinical models for KIAA1549-BRAF duplication which has hindered targeted therapy for this class of pediatric brain tumors. Although cDNA overexpression of KIAA1549-BRAF has been shown to activate the MAPK pathway, in vivo models overexpressing the fusion protein lack the physiological features of either PA or DLGT. Our hypothesis in this project is that generating the entire tandem duplication event in addition to the Kiaa1549-Braf fusion is necessary for modeling the patho-physiological characteristics of the pediatric brain tumors. We have employed CRISPR based somatic genome editing to generate the tandem duplication of Kiaa1549-Braf in p53-/- adult neural stem cells ex-vivo, which results in brain tumors predominantly growing in the subarachnoid/leptomeningeal space and displays histopathological features of human DLGT. Further, we have shown that the Kiaa1549-Braf driven tumor lines can be inhibited by next generation Braf dimer inhibitors. In summary, we have developed a new preclinical model for Kiaa1549-Braf driven brain cancer and demonstrated efficacy against a new preclinical Braf inhibitor.

SummaryID 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.

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.

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 APCMin/+ mice. Treatment with celecoxib also altered select luminal bacterial populations in both APCMin/+ 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 APCMin/+ 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.

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

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 PKH26low fast-dividing cells, whereas PKH26high 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 (PKH26highId1high population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26highId1high 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.

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. Cancer Res; 70(22); 9277–86. ©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. Cancer Res; 70(18); 7273–82. ©2010 AACR.