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1.  Development of a Colon Cancer GEMM-Derived Orthotopic Transplant Model for Drug Discovery and Validation 
Purpose
Effective therapies for KRAS mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described GEMMs for sporadic Kras mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation.
Experimental Design
Cell lines were derived from Kras mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis.
Results
Kras mutant cell lines displayed increased proliferation, MAPK signaling, and PI3K signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biological pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras mutant CRC on oncogenic signaling and on aerobic glycolysis.
Conclusions
We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts.
doi:10.1158/1078-0432.CCR-12-2307
PMCID: PMC3951107  PMID: 23403635
Kras; MAPK; PI3K; colorectal cancer; GEMM; orthotopic model
2.  Concomitant BRAF and PI3K/mTOR Blockade is Required for Effective Treatment of BRAFV600E Colorectal Cancer 
Purpose
BRAFV600E mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Whereas selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAFV600E CRC.
Experimental Design
We examined phosphatidyl inositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling in BRAFV600E CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAFV600E CRC.
Results
Western blot revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAFV600E GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression.
Conclusions
We have established a novel GEMM to interrogate BRAFV600E CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials.
doi:10.1158/1078-0432.CCR-12-2556
PMCID: PMC3815598  PMID: 23549875
colon cancer; mouse models; targeted therapy
3.  Contrasting roles of dietary selenium and selenoproteins in chemically induced hepatocarcinogenesis 
Carcinogenesis  2013;34(5):1089-1095.
Selenium (Se) has long been known for its cancer prevention properties, but the molecular basis remains unclear. The principal questions in assessing the effect of dietary Se in cancer are whether selenoproteins, small molecule selenocompounds, or both, are involved, and under which conditions and genotypes Se may be protective. In this study, we examined diethylnitrosamine-induced hepatocarcinogenesis in mice lacking a subset of selenoproteins due to expression of a mutant selenocysteine tRNA gene (Trsp A37G mice). To uncouple the effects of selenocompounds and selenoproteins, these animals were examined at several levels of dietary Se. Our analysis revealed that tumorigenesis in Trsp A37G mice maintained on the adequate Se diet was increased. However, in the control, wild-type mice, both Se deficiency and high Se levels protected against tumorigenesis. We further found that the Se-deficient diet induced severe neurological phenotypes in TrspA37G mice. Surprisingly, a similar phenotype could be induced in these mice at high dietary Se intake. Overall, our results show a complex role of Se in chemically induced hepatocarcinogenesis, which involves interaction among selenoproteins, selenocompounds and toxins, and depends on genotype and background of the animals.
doi:10.1093/carcin/bgt011
PMCID: PMC3643414  PMID: 23389288
4.  Altered social behavior and neuronal development in mice lacking the Uba6-Use1 ubiquitin transfer system 
Molecular cell  2013;50(2):172-184.
The Uba6 (E1)-Use1 (E2) ubiquitin transfer cascade is a poorly understood alternative arm of the ubiquitin proteasome system (UPS) required for mouse embryonic development, independent of the canonical Uba1-E2-E3 pathway. Loss of neuronal Uba6 during embryonic development results in altered patterning of neurons in the hippocampus and the amygdala, decreased dendritic spine density, and numerous behavioral disorders. The levels of the E3 ubiquitin ligase Ube3a (E6-AP) and Shank3, both linked with dendritic spine function, are elevated in the amygdala of Uba6-deficient mice, while levels of the Ube3a substrate Arc are reduced. Uba6 and Use1 promote proteasomal turnover of Ube3a in mouse embryo fibroblasts (MEFs) and catalyze Ube3a ubiquitylation in vitro. These activities occur in parallel with an independent pathway involving Uba1-UbcH7, but in a spatially distinct manner in MEFs. These data reveal an unanticipated role for Uba6 in neuronal development, spine architecture, mouse behavior, and turnover of Ube3a.
doi:10.1016/j.molcel.2013.02.014
PMCID: PMC3640669  PMID: 23499007
5.  Elucidating distinct roles for NF1 in melanomagenesis 
Cancer discovery  2012;3(3):338-349.
BRAF mutations play a well-established role in melanomagenesis; however, without additional genetic alterations tumor development is restricted by oncogene-induced senescence (OIS). Here we show that mutations in the NF1 tumor suppressor gene cooperate with BRAF mutations in melanomagenesis by preventing OIS. In a genetically engineered mouse model, Nf1 mutations suppress Braf-induced senescence, promote melanocyte hyperproliferation, and enhance melanoma development. Nf1 mutations function by deregulating both PI3K and ERK pathways. As such, Nf1/Braf mutant tumors are resistant to BRAF inhibitors but are sensitive to combined MEK/mTOR inhibition. Importantly, NF1 is mutated or suppressed in human melanomas that harbor concurrent BRAF mutations, NF1 ablation decreases the sensitivity of melanoma cell lines to BRAF inhibitors, and NF1 is lost in tumors from patients following treatment with these agents. Collectively, these studies provide mechanistic insight into how NF1 cooperates with BRAF mutations in melanoma and demonstrate that NF1-inactivation may impact responses to targeted therapies.
doi:10.1158/2159-8290.CD-12-0313
PMCID: PMC3595355  PMID: 23171796
RAS; RAF; senescence; NF1; neurofibromin; melanoma; PI3K; mTOR
6.  Mutations in Hedgehog pathway genes in fetal rhabdomyomas 
The Journal of pathology  2013;231(1):10.1002/path.4229.
Ligand-independent, constitutive activation of Hedgehog signalling in mice expressing a mutant, activated SmoM2 allele results in the development of multifocal, highly differentiated tumours that express myogenic markers (including desmin, actin, MyoD and myogenin). The histopathology of these tumours, commonly classified as rhabdomyosarcomas, more closely resembles human fetal rhabdomyoma (FRM), a benign tumour that can be difficult to distinguish from highly differentiated rhabdomyosarcomas. We evaluated the spectrum of Hedgehog (HH) pathway gene mutations in a cohort of human FRM tumours by targeted Illumina sequencing and fluorescence in situ hybridization testing for PTCH1. Our studies identified functionally relevant aberrations at the PTCH1 locus in three of five FRM tumours surveyed, including a PTCH1 frameshift mutation in one tumour and homozygous deletions of PTCH1 in two tumours. These data suggest that activated Hedgehog signalling contributes to the biology of human FRM.
doi:10.1002/path.4229
PMCID: PMC3875333  PMID: 23780909
fetal rhabdomyoma; hedgehog signalling; PTCH1
7.  Brain and Testicular Tumors in Mice with Progenitor Cells Lacking BAX and BAK 
Oncogene  2012;32(35):4078-4085.
The pro-apoptotic BCL-2 family proteins BAX and BAK serve as essential gatekeepers of the intrinsic apoptotic pathway and, when activated, transform into pore forming homo-oligomers that permeabilize the mitochondrial outer membrane. Deletion of Bax and Bak causes marked resistance to death stimuli in a variety of cell types. Bax−/−Bak−/− mice are predominantly nonviable and survivors exhibit multiple developmental abnormalities characterized by cellular excess, including accumulation of neural progenitor cells in the periventricular, hippocampal, cerebellar, and olfactory bulb regions of the brain. To explore the long-term pathophysiologic consequences of BAX/BAK deficiency in a stem cell niche, we generated Bak−/− mice with conditional deletion of Bax in Nestin-positive cells. Aged NestinCreBaxfl/flBak−/− mice manifest progressive brain enlargement with a profound accumulation of NeuN- and Sox2-positive neural progenitor cells within the subventricular zone. One-third of the mice develop frank masses comprised of neural progenitors, and in 20% of these cases, more aggressive, hypercellular tumors emerged. Unexpectedly, 60% of NestinCreBaxfl/flBak−/− mice harbored high-grade tumors within the testis, a peripheral site of Nestin expression. This in vivo model of severe apoptotic blockade highlights the constitutive role of BAX/BAK in long-term regulation of Nestin-positive progenitor cell pools, with loss of function predisposing to adult-onset tumorigenesis.
doi:10.1038/onc.2012.421
PMCID: PMC3529761  PMID: 22986529
BAX; BAK; neural progenitor cell; apoptosis; tumorigenesis
8.  A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma 
Background
Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer, their production and administration is costly and labor-intensive. We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN), which is overexpressed on ovarian cancer and mesothelioma cells, to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is a potent immune activator that stimulates monocytes and DCs, enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs.
Methods
Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (i.p.) treatments with experimental or control proteins post i.p. injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses, a time-matched study was performed. Splenocytes were stimulated with peptides, and IFNγ- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs.
Results
We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells, and induced maturation of bone marrow-derived DCs. Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs.
Conclusions
This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.
doi:10.1186/1756-8722-7-15
PMCID: PMC3943805  PMID: 24565018
Mycobacterial Hsp70; Mesothelin; Single chain variable fragment; Cancer immunotherapy; Murine tumor model
9.  Role of aberrant PI3K pathway activation in gallbladder tumorigenesis 
Oncotarget  2014;5(4):894-900.
The PI3K/AKT pathway governs a plethora of cellular processes, including cell growth, proliferation, and metabolism, in response to growth factors and cytokines. By acting as a unique lipid phosphatase converting phosphatidylinositol-3,4,5,-trisphosphate (PIP3) to phosphatidylinositol-4,5,-bisphosphate (PIP2), phosphatase and tensin homolog (PTEN) acts as the major cellular suppressor of PI3K signaling and AKT activation. Recently, PI3K mutations and loss/mutation of PTEN have been characterized in human gallbladder tumors; whether aberrant PTEN/PI3K pathway plays a causal role in gallbladder carcinogenesis, however, remains unknown. Herein we show that in mice, deregulation of PI3K/AKT signaling is sufficient to transform gallbladder epithelial cells and trigger fully penetrant, highly proliferative gallbladder tumors characterized by high levels of phospho-AKT. Histopathologically, these mouse tumors faithfully resemble human adenomatous gallbladder lesions. The identification of PI3K pathway deregulation as both an early event in the neoplastic transformation of the gallbladder epithelium and a main mechanism of tumor growth in Pten heterozygous and Pten mutant mouse models provides a new framework for studying in vivo the efficacy of target therapies directed against the PI3K pathway, as advanced metastatic tumors are often addicted to “trunkular” mutations.
PMCID: PMC4011591  PMID: 24658595
PI3K; PTEN; gallbladder tumorigenesis; mouse model
10.  Dysferlin overexpression in skeletal muscle produces a progressive myopathy 
Annals of neurology  2010;67(3):384-393.
Objective
The dose-response effects of dysferlin transgenesis were analyzed to determine if the dysferlin-deficient myopathies are good candidates for gene replacement therapy.
Methods
We have generated three lines of transgenic mice, expressing low, mid and high levels of full-length human dysferlin from a muscle-specific promoter. Transgenic skeletal muscle was analyzed and scored for morphological and functional deficits.
Results
Overexpression of dysferlin in mice resulted in a striking phenotype of kyphosis, irregular gait and reduced muscle mass and strength. Moreover, protein dosage correlated with phenotype severity. In contrast to dysferlin-null skeletal muscle, no evidence of sarcolemmal impairment was revealed. Rather, increased levels of Ca2+-regulated, dysferlin-binding proteins and ER stress chaperone proteins were observed in muscle lysates from transgenic mice as compared to controls.
Interpretation
Expression levels of dysferlin are important for appropriate function without deleterious or cytotoxic effects. As a corollary, we propose that future endeavors in gene replacement for correction of dysferlinopathy should be tailored to take account of this.
doi:10.1002/ana.21926
PMCID: PMC3900233  PMID: 20373350
11.  Non-alloreactive T Cells Prevent Donor Lymphocyte Infusion-Induced Graft-vs.-Host Disease by Controlling Microbial Stimuli 
In mice, graft-versus-host reactions (GVHR), associated with powerful graft-versus-tumor effects, can be achieved without graft-versus-host disease (GVHD) by delayed administration of donor lymphocyte infusions (DLI) to established mixed chimeras (MCs). However, GVHD sometimes occurrs after DLI in established mixed chimeric patients. In contrast to mice, in which T cell recovery from the thymus occurs prior to DLI administration, human T cell reconstitution following T cell-depleted hematopoietic cell transplantation is slow, resulting in lymphopenia at the time of DLI. We demonstrate here that T cell lymphopenia is an independent risk factor for GVHD following DLI in the absence of known inflammatory stimuli. DLI-induced GVHD was prevented in lymphopenic recipients by prior administration of a small number of non-alloreactive polyclonal T cells, insufficient to prevent lymphopenia-associated expansion of subsequently administered T cells, through a Treg-independent mechanism, but not by T cells with irrelevant specificity. Moreover, administration of antibiotics reduced the severity of GVHD in lymphopenic hosts. Accumulation of DLI-derived effector T cells and host hematopoietic cell elimination were markedly diminished by Treg-depleted, non-alloreactive T cells. Finally, thymectomized mixed chimeras showed increased GVHD following delayed DLI. Collectively, our data demonstrate that in the absence of known conditioning-induced inflammatory stimuli, T cell lymphopenia is a risk factor for GVHD in MCs receiving delayed DLI and suggest that the predisposition to GVHD can at least in part be explained by the presence of occult inflammatory stimuli due to the absence of T cells to control microbial infections.
doi:10.4049/jimmunol.1200045
PMCID: PMC3524834  PMID: 23136200
12.  Near-Infrared Laser Adjuvant for Influenza Vaccine 
PLoS ONE  2013;8(12):e82899.
Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants.
doi:10.1371/journal.pone.0082899
PMCID: PMC3859633  PMID: 24349390
13.  DOK2 Inhibits EGFR-Mutated Lung Adenocarcinoma 
PLoS ONE  2013;8(11):e79526.
Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.
doi:10.1371/journal.pone.0079526
PMCID: PMC3821857  PMID: 24255704
14.  Correction: Stochastic Model of Tsc1 Lesions in Mouse Brain 
PLoS ONE  2013;8(11):10.1371/annotation/6a5b0a50-27e4-49bc-b82a-9267dd63af53.
doi:10.1371/annotation/6a5b0a50-27e4-49bc-b82a-9267dd63af53
PMCID: PMC3821742  PMID: 24244248
15.  A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway 
Cancer research  2006;66(20):10171-10178.
We report a novel mouse model for the generation of sporadic tumors and show the efficiency of this approach by surveying Hedgehog (Hh)–related tumors. Up-regulation of the Hh pathway is achieved by conditionally regulated expression of an activated allele of Smoothened (R26-SmoM2) using either sporadic leakage or global postnatal induction of a ubiquitously expressed inducible Cre transgene (CAGGS-CreER). Following postnatal tamoxifen induction, CAGGS-CreER; R26-SmoM2 mice developed tumors with short latency and high penetrance. All mice exhibited rhabdomyosarcoma and basal cell carcinoma; 40% also developed medulloblastoma. In addition, mice showed a novel pancreatic lesion resembling low-grade mucinous cystic neoplasms in humans. In contrast, widespread activation of SmoM2 in the postnatal prostate epithelium results in no detectable morphologic outcome in 12-month-old mice. Comparison of gene expression profiles among diverse tumors identified several signature genes, including components of platelet-derived growth factor and insulin-like growth factor pathways, which may provide a common mechanistic link to the Hh-related malignancies. This experimental model provides a robust tool for exploring the process of Hh-dependent tumorigenesis and the treatment of such tumors. More generally, this approach provides a genetic platform for identifying tumorigenic potential in putative oncogenes and tumor suppressors and for more effective modeling of sporadic cancers in mice.
doi:10.1158/0008-5472.CAN-06-0657
PMCID: PMC3806052  PMID: 17047082
16.  Acquired MET Expression Confers Resistance to EGFR Inhibition In a Mouse Model of Glioblastoma Multiforme 
Oncogene  2011;31(25):3039-3050.
Glioblastoma Multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type EGFR and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that a key component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.
doi:10.1038/onc.2011.474
PMCID: PMC3774279  PMID: 22020333
Glioblastoma; genetically engineered mouse model; EGFR; PTEN; c-MET
17.  Crucial Role for Early Growth Response-1 in the Transcriptional Regulation of miR-20b in Breast Cancer 
Oncotarget  2013;4(9):1373-1387.
Transcriptional regulation of miRNAs that control the pathogenesis of breast cancer remains largely unknown. Here, we showed that ionizing radiation, a known breast carcinogen, triggered the differential expression of miR-20b in mammary tissues. We identified several GC-rich consensus binding motifs for the zinc finger transcription factor early growth response-1 (EGR1) in miR-20b promoter. miR-20b was upregulated by IR and its upregulation correlated with EGR1 expression in the breast cancer cell line HCC1806. Therefore, we used HCC1806 cells as a model system to explore the role of EGR1 in miR-20b transcription. siRNA knockdown of EGR1 attenuated miR-20b expression. Luciferase assays showed that whereas EGR1 stimulated luciferase activity driven by the wild-type miR-20b promoter, this induction was abolished in the mutant miR-20 promoter construct. We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells. Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G 0/G 1 and S phase arrest. In vitro RNA-pull down assays indicated that miR-20b targets numerous tumor suppressors, including PTEN and BRCA1, which were downregulated in HCC1806. Conversely, suppression of miR-20b increased PTEN and BRCA1 levels. Moreover, immunohistochemical and FISH analyses showed that the miR-20b expression correlated significantly with EGR1 levels in breast cancer tissues. Our findings thus demonstrate for the first time that EGR1 is a key player in the transcriptional control of miR-20b, and miR-20b may in turn function as an oncogene by contributing to breast tumorigenesis via tumor suppressor targeting.
PMCID: PMC3824527  PMID: 23945289
EGR1; miR-20b; transcription; PTEN; BRCA1; breast cancer; proliferation; migration; cell cycle arrest
18.  The Oncogenic Lung Cancer Fusion Kinase CD74-ROS Activates a Novel Invasiveness Pathway Through E-Syt1 Phosphorylation 
Cancer research  2012;72(15):3764-3774.
Patients with lung cancer often present with metastatic disease and therefore have a very poor prognosis. The recent discovery of several novel ROS receptor tyrosine kinase molecular alterations in non-small-cell lung cancer (NSCLC) presents a therapeutic opportunity for the development of new targeted treatment strategies. Here, we report that the NSCLC-derived fusion CD74-ROS, which accounts for 30% of all ROS fusion kinases in NSCLC, is an active and oncogenic tyrosine kinase. We found that CD74-ROS expressing cells were highly invasive in vitro and metastatic in vivo. Pharmacological inhibition of CD74-ROS kinase activity reversed its transforming capacity by attenuating downstrream signaling networks. Using quantitative phosphoproteomics, we uncovered a mechanism by which CD74-ROS activates a novel pathway driving cell invasion. Expression of CD74-ROS resulted in the phosphorylation of the extended synaptotagmin-like protein E-Syt1. Elimination of E-Syt1 expression drastically reduced invasiveness both in vitro and in vivo without modifying the oncogenic activity of CD74-ROS. Furthermore, expression of CD74-ROS in non-invasive NSCLC cell lines readily confered invasive properties that paralleled the acquisition of E-Syt1 phosphorylation. Taken together, our findings indicate that E-Syt1 is a mediator of cancer cell invasion and molecularly define ROS fusion kinases as therapeutic targets in the treatment of NSCLC.
doi:10.1158/0008-5472.CAN-11-3990
PMCID: PMC3753671  PMID: 22659450
19.  Chronic Activation of mTOR Complex 1 is Sufficient to Cause Hepatocellular Carcinoma 
Science signaling  2012;5(217):ra24.
The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient sensitive protein kinase that is aberrantly activated in many human cancers. However, whether dysregulation of mTORC1 signaling in normal tissues contributes to cancer risk is unknown. Here, we focused on hepatocellular carcinoma because it is a cancer with clear links to environmental factors that affect mTORC1, including dietary influences. Genetic ablation of the mTORC1 inhibitory component Tsc1 results in constitutively elevated mTORC1 signaling, an effect similar to that of obesity on this pathway. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes known to accompany the primary etiologies of this cancer, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling caused unresolved endoplasmic reticulum stress and defects in autophagy, which contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we demonstrate a previously unrecognized role for mTORC1 in carcinogenesis, perhaps representing a key molecular link between cancer risk and environmental factors, such as diet.
doi:10.1126/scisignal.2002739
PMCID: PMC3743103  PMID: 22457330
20.  Telomere dysfunction induces metabolic and mitochondrial compromise 
Nature  2011;470(7334):359-365.
Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere–p53–PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.
doi:10.1038/nature09787
PMCID: PMC3741661  PMID: 21307849
21.  Genotype Directed Therapy in Murine Mismatch Repair Deficient Tumors 
PLoS ONE  2013;8(7):e68817.
The PI3K/AKT/mTOR pathway has frequently been found activated in human tumors. We show that in addition to Wnt signaling dysfunction, the PI3K/AKT/mTOR pathway is often upregulated in mouse Msh2−/− initiated intestinal tumors. NVP-BEZ235 is a dual PI3K/mTOR inhibitor toxic to many cancer cell lines and currently involved in clinical trials. We have treated two mouse models involving Msh2 that develop small intestinal and/or colonic tumors with NVP-BEZ235, and a subset of animals with NVP-BEZ235 and MEK inhibitor ADZ4266. The disease phenotype has been followed with pathology, 18F FDG PET imaging, and endoscopy. Intestinal adenocarcinomas are significantly decreased in multiplicity by both drug regimens. The majority of tumors treated with combined therapy regress significantly, while a small number of highly progressed tumors persist. We have examined PTEN, AKT, MEK 1&2, MAPK, S6K, mTOR, PDPK1, and Cyclin D1 and find variable alterations that include downregulation of PTEN, upregulation of AKT and changes in its phosphorylated forms, upregulation of pMEK 1&2, p42p44MAPK, pS6K, and Cyclin D1. Apoptosis has been found intact in some tumors and not in others. Our data indicate that NVP-BEZ235 alone and in combination with ADZ4266 are effective in treating a proportion of colorectal cancers, but that highly progressed resistant tumors grow in the presence of the drugs. Pathways upregulated in some resistant tumors also include PDPK1, suggesting that metabolic inhibitors may also be useful in treating these tumors.
doi:10.1371/journal.pone.0068817
PMCID: PMC3720855  PMID: 23935891
22.  p600 Plays Essential Roles in Fetal Development 
PLoS ONE  2013;8(6):e66269.
p600 is a multifunctional protein implicated in cytoskeletal organization, integrin-mediated survival signaling, calcium-calmodulin signaling and the N-end rule pathway of ubiquitin-proteasome-mediated proteolysis. While push, the Drosophila counterpart of p600, is dispensable for development up to adult stage, the role of p600 has not been studied during mouse development. Here we generated p600 knockout mice to investigate the in vivo functions of p600. Interestingly, we found that homozygous deletion of p600 results in lethality between embryonic days 11.5 and 13.5 with severe defects in both embryo and placenta. Since p600 is required for placental development, we performed conditional disruption of p600, which deletes selectively p600 in the embryo but not in the placenta. The conditional mutant embryos survive longer than knockout embryos but ultimately die before embryonic day 14.5. The mutant embryos display severe cardiac problems characterized by ventricular septal defects and thin ventricular walls. These anomalies are associated with reduced activation of FAK and decreased expression of MEF2, which is regulated by FAK and plays a crucial role in cardiac development. Moreover, we observed pleiotropic defects in the liver and brain. In sum, our study sheds light on the essential roles of p600 in fetal development.
doi:10.1371/journal.pone.0066269
PMCID: PMC3688873  PMID: 23824717
23.  Intestinal Tolerance Is Converted to Autoimmune Enteritis upon PD-1 Ligand Blockade1 
The B7 family member programmed death-1 ligand (PD-L1) has been shown to play an inhibitory role in the regulation of T cell responses in several organs. However, the role of PD-L1 in regulating tolerance to self-Ags of the small intestine has not been previously addressed. In this study, we investigated the role of PD-L1 in CD8+ T cell tolerance to an intestinal epithelium-specific Ag using the iFABP-tOVA transgenic mouse model, in which OVA is expressed as a self-Ag throughout the small intestine. Using adoptive transfer of naive OVA-specific CD8+ T cells, we show that loss of PD-1:PD-L1 signaling, by either Ab-mediated PD-L1 blockade or transfer of PD-1−/− T cells, leads to considerable expansion of OVA-specific CD8+ T cells and their differentiation into effector cells capable of producing proinflammatory cytokines. A fatal CD8+ T cell-mediated inflammatory response develops rapidly against the small bowel causing destruction of the epithelial barrier, severe blunting of intestinal villi, and recruitment and activation of myeloid cells. This response is highly specific because immune destruction selectively targets the small intestine but not other organs. Collectively, these results indicate that loss of the PD-1:PD-L1 inhibitory pathway breaks CD8+ T cell tolerance to intestinal self-Ag, thus leading to severe enteric autoimmunity.
doi:10.4049/jimmunol.0802769
PMCID: PMC3682217  PMID: 19201863
24.  Stochastic Model of Tsc1 Lesions in Mouse Brain 
PLoS ONE  2013;8(5):e64224.
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment.
doi:10.1371/journal.pone.0064224
PMCID: PMC3655945  PMID: 23696872
25.  Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1 
PLoS Genetics  2013;9(4):e1003413.
Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.
Author Summary
Alkylating agents are genotoxic chemicals that induce both toxic and mutagenic DNA damage through addition of an alkyl group to DNA. Alkylating agents are routinely and successfully used as chemotherapeutic therapies for cancer patients, with one major disadvantage being the significant toxicity induced in non-tumor tissues. Accordingly, identifying factors that modify susceptibility to alkylation-induced toxicity will provide valuable information in designing cancer therapeutic regimens. This study used mouse genetic experiments to investigate whether proteins important in the base excision repair pathway modulate susceptibility to alkylating agents. In addition to whole-animal toxicity at high doses, treatment of mice with alkylating agents resulted in severe damage to numerous tissues including the cerebellum, retina, bone marrow, spleen, thymus, and the pancreas. We illustrate that the DNA glycosylase Aag can actually confer, rather than prevent, alkylation sensitivity at both the whole-animal and tissue level; i.e., Aag transgenic animals are more susceptible than wild type, whereas Aag-deficient animals are less susceptible than wild type to alkylation-induced toxicity. Further genetic experiments show that the Aag-mediated alkylation sensitivity is dependent on Parp1. Given that we observe a wide range of human AAG expression among healthy individuals, this and other base excision repair proteins may be important factors modulating alkylation susceptibility.
doi:10.1371/journal.pgen.1003413
PMCID: PMC3617098  PMID: 23593019

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