- Medical Oncology
- Cancer > Hematology
Co-lead, Cancer Biology Program, Stanford Cancer Center (2012 - Present)
Honors & Awards
Consulting Editor, JCI (2012)
American Heart Association Innovative Science Award, AHA (2012)
Research Chair, NIH Intestinal Stem Cell Consortium, NIH (2009)
Transformative R01 Award, NIH (2009)
Member, American Society for Clinical Investigation, American Society for Clinical Investigation (2007)
Samantha Janower Research Chair, Brain Tumor Society (2005)
Merck Faculty Development Award, Merck (2003)
Kimmel Foundation Scholar in Translational Science, Kimmel Foundation (2002)
Burroughs Wellcome Foundation New Investigator in Pharmacological Sciences, Burroughs Wellcome Foundation (2001)
HHMI Physician-Scientist Fellowship, HHMI (1998)
Summa cum laude, Harvard College (1987)
Fellowship:Brigham and Women's Hospital Harvard Medical School (2000) MA
Residency:Brigham and Women's Hospital Harvard Medical School (1997) MA
Medical Education:Stanford University School of Medicine (1994) CA
A.B., Harvard College, Biochemical Sciences (1987)
M.D./Ph.D., Stanford University, Cancer Biology (1994)
Internship/ Residency, Brigham and Women's Hospital, Internal Medicine (1997)
Fellowship, Dana-Farber/Partners, Adult Oncology (2000)
Current Research and Scholarly Interests
We are interested in determining functions of novel molecules regulating angiogenesis including receptors such as GPCRs, microRNAs and secreted molecules. We found that GPR124 is essential for developmental brain angiogenesis (Kuhnert et al, Science 2010) and are exploring this receptor's function in adult pathophysiology. We have several active projects in stroke and blood-brain barrier (BBB) biology. We are also exploring the functions of the endothelial miRNA miR-126 in adults using conditional ko mice (Kuhnert et al, Development 2008). We have extensive experience using adenoviral expression of soluble receptor ectodomains to inhibit diverse angiogenic pathways including VEGF and PDGFRb. Loss-of-function phenotypes would simulate the effects of pharmacologic inhibition of novel targets for anti-angiogenic therapy of cancer and ocular disorders.
We have successfully established primary organoid cultures of diverse tissues including intestine (Ootani et al, Nat Med 2009). These comprise an robust in vitro system for the functional validation of putative oncogenic loci which are identified in whole-genome cancer surveys. We collaborate extensively with systems biologists to interrogate large-scale cancer genomics datasets.
Endothelial cell regulation of physiology,
How do endothelial cells regulate physiology of their host organs? The liver hepatocyte appears particularly responsive to its host endothelial cells. We are investigating effects of VEGF inhibition on hepatocyte functions in terms of Epo synthesis, erythropoiesis (Tam et al, Nat Med 2006) and metabolic pathways. We have found that FDA-approved VEGF inhibitors such as aflibercept improve glucose tolerance and can treat animal models of diabetes, by stimulating cross-talk between the hypoxia and insulin signaling pathways, with sensitization of hepatic insulin signaling (Wei et al., Nature Med 2013). We are also correlating these changes with anti-tumor response and survival in cancer patients receiving VEGF inhibitors, as potential surrogate biomarkers of efficacy.
Intestinal stem/progenitor biology.
The complete regeneration of the epithelial lining of the intestine every 5-7 days renders the intestine a model system for studying stem cell behaviors. We are investigating the regulation of the intestinal stem cell (ISC) compartment by extracellular signals such as Wnts, using adenoviral and conditional knockout approaches, and have found that Bmi1+ ISC are strongly injury-inducible versus the homeostatic function of Lgr5+ ISC (c.f. Yan et al, PNAS 2012, Barry et al, Nature 2013). Further, we have derived robust organoid methods for prolonged culture of and ex vivo expansion of primary intestinal tissue, with preservation of ISCs and recapitulation of the Wnt- and Notch-dependent ISC niche, even allowing peristalsis (Ootani et al, Nat Med 2009).
Independent Studies (15)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr, Sum)
- Out-of-Department Directed Reading
BIO 198X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Win, Spr, Sum)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Interfollicular Epidermal Stem Cells Self-Renew via Autocrine Wnt Signaling
2013; 342 (6163): 1226-1230
The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.
View details for DOI 10.1126/science.1239730
View details for Web of Science ID 000327857900046
View details for PubMedID 24311688
Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes
2013; 19 (10): 1325-?
Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain-containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes. Although Hif regulates many metabolic pathways, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.
View details for DOI 10.1038/nm.3294
View details for Web of Science ID 000325531700033
View details for PubMedID 24037093
A liver Hif-2 alpha-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition
2013; 19 (10): 1331-?
Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.
View details for DOI 10.1038/nm.3295
View details for Web of Science ID 000325531700034
View details for PubMedID 24037094
Restriction of intestinal stem cell expansion and the regenerative response by YAP
2013; 493 (7430): 106-?
A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3? complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.
View details for DOI 10.1038/nature11693
View details for Web of Science ID 000312933800040
View details for PubMedID 23178811
beta-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis
2012; 151 (7): 1457-1473
Wnt/?-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ?-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ?-catenin in transformation, we classified ?-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ?-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ?-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ?-catenin-dependent cancers in both cell lines and animal models. These observations define a ?-catenin-YAP1-TBX5 complex essential to the transformation and survival of ?-catenin-driven cancers.
View details for DOI 10.1016/j.cell.2012.11.026
View details for Web of Science ID 000312890300012
View details for PubMedID 23245941
The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (2): 466-471
The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.
View details for DOI 10.1073/pnas.1118857109
View details for Web of Science ID 000298950200030
View details for PubMedID 22190486
Essential Regulation of CNS Angiogenesis by the Orphan G Protein-Coupled Receptor GPR124
2010; 330 (6006): 985-989
The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.
View details for DOI 10.1126/science.1196554
View details for Web of Science ID 000284118000049
View details for PubMedID 21071672
Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.
2009; 15 (6): 701-706
The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.
View details for DOI 10.1038/nm.1951
View details for PubMedID 19398967
A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2013; 305 (8): G542-G551
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
View details for DOI 10.1152/ajpgi.00481.2012
View details for Web of Science ID 000325809200002
View details for PubMedID 23928185
The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO
2012; 149 (1): 63-74
Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.
View details for DOI 10.1016/j.cell.2012.01.051
View details for Web of Science ID 000302235400010
View details for PubMedID 22464323
- PDGF-B exploits stromal EPO NATURE MEDICINE 2012; 18 (1): 22-24
Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling
2012; 18 (1): 111-119
Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes--differentiated epithelial cells crucial for filtration--are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt-β-catenin pathways in podocyte proliferation and disease.
View details for DOI 10.1038/nm.2550
View details for Web of Science ID 000299018600036
Endochondral ossification is required for haematopoietic stem-cell niche formation
2009; 457 (7228): 490-U9
Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.
View details for DOI 10.1038/nature07547
View details for Web of Science ID 000262519200049
View details for PubMedID 19078959
Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126
2008; 135 (24): 3989-3993
Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7(Delta) and miR-126(Delta) alleles revealed that Egfl7(Delta/Delta) mice were phenotypically normal, whereas miR-126(Delta/Delta) mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85beta subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.
View details for DOI 10.1242/dev.029736
View details for Web of Science ID 000261151000002
View details for PubMedID 18987025
Soluble receptor-mediated selective inhibition of VEGFR and PDGFR beta signaling during physiologic and tumor angiogenesis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (29): 10185-10190
The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.
View details for DOI 10.1073/pnas.0803194105
View details for Web of Science ID 000257913200061
View details for PubMedID 18632559
Recombinant adenovirus as a methodology for exploration of physiologic functions of growth factor pathways
JOURNAL OF MOLECULAR MEDICINE-JMM
2008; 86 (2): 161-169
The use of recombinant adenoviruses (Ad) to express secreted antagonists of growth factors represents a powerful strategy for studying physiologic functions of growth factor pathways in experimental animals. Indeed, a single adenoviral injection can produce characteristic high-level and persistent plasma expression of soluble receptor ectodomains or secreted protein antagonists, allowing highly stringent conditional inactivation of target pathways in vivo. In this review, we describe our experience using recombinant Ad to inactivate growth factor pathways in vivo and discuss their advantages and limitations. Using our studies on vascular endothelial growth factor and Wnt systems as examples, we further describe how recombinant Ad can unveil previously unknown physiological roles of signaling pathways. Finally, we discuss the potential physiological and therapeutic relevance of our findings.
View details for DOI 10.1007/s00109-007-0261-7
View details for Web of Science ID 000252799300004
View details for PubMedID 17891365
Augmented Wnt signaling in a mammalian model of accelerated aging
2007; 317 (5839): 803-806
The contribution of stem and progenitor cell dysfunction and depletion in normal aging remains incompletely understood. We explored this concept in the Klotho mouse model of accelerated aging. Analysis of various tissues and organs from young Klotho mice revealed a decrease in stem cell number and an increase in progenitor cell senescence. Because klotho is a secreted protein, we postulated that klotho might interact with other soluble mediators of stem cells. We found that klotho bound to various Wnt family members. In a cell culture model, the Wnt-klotho interaction resulted in the suppression of Wnt biological activity. Tissues and organs from klotho-deficient animals showed evidence of increased Wnt signaling, and ectopic expression of klotho antagonized the activity of endogenous and exogenous Wnt. Both in vitro and in vivo, continuous Wnt exposure triggered accelerated cellular senescence. Thus, klotho appears to be a secreted Wnt antagonist and Wnt proteins have an unexpected role in mammalian aging.
View details for DOI 10.1126/science.1143578
View details for Web of Science ID 000248624500040
View details for PubMedID 17690294
Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis
2007; 317 (5839): 807-810
The regenerative potential of skeletal muscle declines with age, and this impairment is associated with an increase in tissue fibrosis. We show that muscle stem cells (satellite cells) from aged mice tend to convert from a myogenic to a fibrogenic lineage as they begin to proliferate and that this conversion is mediated by factors in the systemic environment of the old animals. We also show that this lineage conversion is associated with an activation of the canonical Wnt signaling pathway in aged myogenic progenitors and can be suppressed by Wnt inhibitors. Furthermore, components of serum from aged mice that bind to the Frizzled family of proteins, which are Wnt receptors, may account for the elevated Wnt signaling in aged cells. These results indicate that the Wnt signaling pathway may play a critical role in tissue-specific stem cell aging and an increase in tissue fibrosis with age.
View details for DOI 10.1126/science.1144090
View details for Web of Science ID 000248624500041
View details for PubMedID 17690295
VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis
2006; 12 (7): 793-800
Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.
View details for DOI 10.1038/nm1428
View details for Web of Science ID 000238862800066
View details for PubMedID 16799557
Apc tumor suppressor gene is the "zonation-keeper" of mouse liver
2006; 10 (6): 759-770
The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.
View details for DOI 10.1016/j.devcel.2006.03.015
View details for Web of Science ID 000238244700010
View details for PubMedID 16740478
Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (1): 266-271
Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.
View details for Web of Science ID 000187937200050
View details for PubMedID 14695885
Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (8): 4605-4610
Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in approximately 80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.
View details for Web of Science ID 000168059700062
View details for PubMedID 11274374
RAPAMYCIN SELECTIVELY INHIBITS INTERLEUKIN-2 ACTIVATION OF P70 S6 KINASE
1992; 358 (6381): 70-73
The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase. In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression. Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases. Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM). The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11). The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.
View details for Web of Science ID A1992JB34100056
View details for PubMedID 1614535
A TRANSCRIPTIONAL HIERARCHY INVOLVED IN MAMMALIAN CELL-TYPE SPECIFICATION
1992; 355 (6359): 457-461
Although transcriptional hierarchies have been extensively studied in invertebrates, their involvement in mammalian cell-type specification is poorly understood. Here we report a hepatocyte transcriptional cascade suggested by the expression patterns of hepatic transcription factors in dedifferentiated hepatomas and hepatocyte: fibroblast hybrids in which the liver phenotype was extinguished. These results indicated that the homeoprotein hepatocyte nuclear factor-1 alpha (HNF-1 alpha), and HNF-4, a member of the steroid hormone receptor superfamily, were regulated coordinately or in a hierarchy by a higher-order locus, independently of other hepatic transactivators. HNF-4 was implicated as an essential positive regulator of HNF-1 alpha, as deletion of an HNF-4 binding site in the HNF-1 alpha promoter abolished promoter activity, and HNF-4 potently transactivated the HNF-1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF-4 complementary DNA rescued expression of endogenous HNF-1 alpha messenger RNA and DNA-binding activity. Our studies therefore define an HNF-4----HNF-1 alpha (4----1 alpha) transcriptional hierarchy operative in differentiated hepatocytes but selectively inhibited by an extinguishing locus and somatic mutations which antagonize the liver phenotype.
View details for Web of Science ID A1992HB53000073
View details for PubMedID 1734282