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1.  Systematic Screen Identifies miRNAs that Target RAD51 and RAD51D to Enhance Chemosensitivity 
Molecular cancer research : MCR  2013;11(12):10.1158/1541-7786.MCR-13-0292.
Homologous recombination (HR) mediates error-free repair of DNA double-strand breaks (DSBs). RAD51 is an essential protein for catalyzing HR and its recruitment to DSBs is mediated by many factors including RAD51, its paralogs, and breast/ovarian cancer susceptibility gene products BRCA1/2. Deregulation of these factors leads to impaired DNA repair, genomic instability, and cellular sensitivity to chemotherapeutics like cisplatin and poly (ADP-ribose) polymerase (PARP) inhibitors. microRNAs (miRs) are short, non-coding RNAs that post-transcriptionally regulate gene expression; however, the contribution of miRs in the regulation of HR is not well understood. To address this, a library of human miR mimics was systematically screened to pinpoint several miRs that significantly reduce RAD51 foci formation in response to ionizing radiation (IR) in human osteosarcoma cells. Subsequent study focused on two of the strongest candidates, miR-103 and miR-107, as they are frequently deregulated in cancer. Consistent with the inhibition of RAD51 foci formation, miR-103 and miR-107 reduced homology-directed repair and sensitized cells to various DNA damaging agents, including cisplatin and a PARP inhibitor. Mechanistic analyses revealed that both miR-103 and miR-107 directly target and regulate RAD51 and RAD51D, which is critical for miR-103/107-mediated chemosensitization. Furthermore, endogenous regulation of RAD51D by miR-103/107 was observed in several tumor subtypes. Combined, these data show that miR-103 and miR-107 overexpression promotes genomic instability and may be used therapeutically to chemosensitize tumors.
Implications
These findings demonstrate a role for miR-103 and -107 in regulating DNA damage repair, thereby identifying new players in the progression of cancer and response to chemotherapy.
doi:10.1158/1541-7786.MCR-13-0292
PMCID: PMC3869885  PMID: 24088786
microRNA; cisplatin; PARP inhibitor; RAD51; RAD51D; homologous recombination; Fanconi anemia
2.  Tumors induce coordinate growth of artery, vein, and lymphatic vessel triads 
BMC Cancer  2014;14:354.
Background
Tumors drive blood vessel growth to obtain oxygen and nutrients to support tumor expansion, and they also can induce lymphatic vessel growth to facilitate fluid drainage and metastasis. These processes have generally been studied separately, so that it is not known how peritumoral blood and lymphatic vessels grow relative to each other.
Methods
The murine B16-F10 melanoma and chemically-induced squamous cell carcinoma models were employed to analyze large red-colored vessels growing between flank tumors and draining lymph nodes. Immunostaining and microscopy in combination with dye injection studies were used to characterize these vessels.
Results
Each peritumoral red-colored vessel was found to consist of a triad of collecting lymphatic vessel, vein, and artery, that were all enlarged. Peritumoral veins and arteries were both functional, as detected by intravenous dye injection. The enlarged lymphatic vessels were functional in most mice by subcutaneous dye injection assay, however tumor growth sometimes blocked lymph drainage to regional lymph nodes. Large red-colored vessels also grew between benign papillomas or invasive squamous cell carcinomas and regional lymph nodes in chemical carcinogen-treated mice. Immunostaining of the red-colored vessels again identified the clustered growth of enlarged collecting lymphatics, veins, and arteries in the vicinity of these spontaneously arising tumors.
Conclusions
Implanted and spontaneously arising tumors induce coordinate growth of blood and lymphatic vessel triads. Many of these vessel triads are enlarged over several cm distance between the tumor and regional lymph nodes. Lymphatic drainage was sometimes blocked in mice before lymph node metastasis was detected, suggesting that an unknown mechanism alters lymph drainage patterns before tumors reach draining lymph nodes.
doi:10.1186/1471-2407-14-354
PMCID: PMC4045915  PMID: 24886322
Melanoma; Carcinoma; Lymphangiogenesis; Lymphatic vessel; Angiogenesis; Blood vessel
3.  Arf Suppresses Hepatic Vascular Neoplasia in a Carcinogen-Exposed Murine Model 
The Journal of pathology  2012;227(3):10.1002/path.4024.
Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16INK4a tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane (i.p. 1 mg/g). Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients.
doi:10.1002/path.4024
PMCID: PMC3871210  PMID: 22430984
p19Arf; p14ARF; ethyl carbamate; liver; endothelium; haemorrhage; tumour progression
4.  Doxorubicin enhances nucleosome turnover around promoters 
Current biology : CB  2013;23(9):782-787.
Summary
Doxorubicin is an anthracycline DNA intercalator that is among the most commonly used anti-cancer drugs [1]. Doxorubicin causes DNA double-strand breaks in rapidly dividing cells, although whether it also affects general chromatin properties is unknown. Here, we use a metabolic labeling strategy to directly measure nucleosome turnover [2] to examine the effect of doxorubicin on chromatin dynamics in squamous cell carcinoma cell lines derived from genetically defined mice. We find that doxorubicin enhances nucleosome turnover around gene promoters, and turnover correlates with gene expression level. Consistent with a direct action of doxorubicin, enhancement of nucleosome turnover around promoters gradually increases with time of exposure to the drug. Interestingly, enhancement occurs both in wild-type cells and in cells lacking either the p53 tumor suppressor gene or the master regulator of the DNA damage response, Atm, suggesting that doxorubicin action on nucleosome dynamics is independent of the DNA damage checkpoint. In addition, another anthracycline drug, aclarubicin, shows similar effects on enhancing nucleosome turnover around promoters. Our results suggest that anthracycline intercalation promotes nucleosome turnover around promoters by its effect on DNA topology, with possible implications for mechanisms of cell killing during cancer chemotherapy.
doi:10.1016/j.cub.2013.03.043
PMCID: PMC3678987  PMID: 23602475
CATCH-IT; Squamous cell carcinoma; p53; Atm
5.  A Mouse Model Repository for Cancer Biomarker Discovery 
Journal of proteome research  2008;7(8):3613-3618.
Early detection of cancer using biomarkers obtained from blood or other easily accessible tissues would have a significant impact on reducing cancer mortality. However, identifying new blood-based biomarkers has been hindered by the dynamic complexity of the human plasma proteome, confounded by genetic and environmental variability, and the scarcity of high quality controlled samples. In this report we discuss a new paradigm for biomarker discovery through the use of mouse models. Inbred mouse models of cancer recapitulate many critical features of human cancer, while eliminating sources of environmental and genetic variability. The ability to collect samples from highly matched cases and controls under identical conditions further reduces variability which is critical for successful biomarker discovery. We describe the establishment of a repository containing tumor, plasma, urine, and other tissues from ten different mouse models of human cancer, including two breast, two lung, two prostate, two gastro-intestinal, one ovarian, and one skin tumor model. We present the overall design of this resource and its potential use by the research community for biomarker discovery.
doi:10.1021/pr800210b
PMCID: PMC3727967  PMID: 18624399
6.  Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System 
PLoS ONE  2013;8(11):e81426.
The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 1013) or varying titers (2.2 x 1012, 1.0 x 1013, 5.9 x 1013, or 1.0 x 1014) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 1014 titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 1013 titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 1013 and 1.0 x 1014 rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 1013 rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.
doi:10.1371/journal.pone.0081426
PMCID: PMC3842242  PMID: 24312298
7.  Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer 
Epigenetics  2013;8(12):1254-1260.
Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.
doi:10.4161/epi.26486
PMCID: PMC3933486  PMID: 24107773
cancer; DNA methylation; epigenomics; genetically engineered mouse models; medulloblastoma
8.  MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency 
Molecular cancer research : MCR  2012;10(4):535-545.
MYC-induced DNA damage is exacerbated in WRN deficient cells, leading to replication stress and accelerated cellular senescence. To determine if WRN deficiency impairs MYC driven tumor development, we utilized both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non-small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knock-down was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (WrnΔhel/Δhel) resulted in a significant delay in emergence of lethal lymphomas, extending tumor free survival by >30%. Analysis of pre-neoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc WrnΔhel/Δhel lymphomas demonstrated a marked increase in expression of the CDK inhibitor, p16Ink4a, as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies demonstrate that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in pre-neoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers.
doi:10.1158/1541-7786.MCR-11-0508
PMCID: PMC3707802  PMID: 22301954
Werner helicase; therapeutic target; Myc-driven cancer; senescence; tumor suppressors
9.  MiR-96 downregulates REV1 and RAD51 to promote cellular sensitivity to cisplatin and PARP inhibition 
Cancer research  2012;72(16):4037-4046.
Cell survival after DNA damage relies on DNA repair, the abrogation of which causes genomic instability. The DNA repair protein RAD51 and the trans-lesion synthesis DNA polymerase REV1 are required for resistance to DNA interstrand crosslinking agents such as cisplatin. In this study, we show that overexpression of miR-96 in human cancer cells reduces the levels of RAD51 and REV1 and impacts the cellular response to agents that cause DNA damage. miR-96 directly targeted the coding region of RAD51 and the 3′-untranslated region of REV1. Overexpression of miR-96 decreased the efficiency of homologous recombination and enhanced sensitivity to the poly(ADP-ribose) polymerase (PARP) inhibitor AZD2281 in vitro and to cisplatin both in vitro and in vivo. Taken together, our findings indicate that miR-96 regulates DNA repair and chemosensitivity by repressing RAD51 and REV1. As a candidate therapeutic, miR-96 may improve chemotherapeutic efficacy by increasing the sensitivity of cancer cells to DNA damage.
doi:10.1158/0008-5472.CAN-12-0103
PMCID: PMC3421071  PMID: 22761336
miR-96; DNA repair; drug resistance; cisplatin; PARP inhibitor
10.  VOLUNTARY CONSUMPTION OF ETHYL OLEATE REDUCES FOOD INTAKE AND BODY WEIGHT IN RATS 
Physiology & behavior  2007;93(4-5):912-918.
Previous studies have shown that administration of the fatty acids, linoleic and oleic acid, either by intragastric or intraintestinal infusion, suppresses food intake and body weight in rats. While still not fully understood, gut-mediated satiety mechanisms likely are potential effectors of this robust response to gastrointestinal fatty acid infusions. The objective of this study was to assess the effects of voluntary access to an oleic acid derivative, ethyl oleate (EO), on subsequent food intake and body weight in rats. Animals were randomized either to a 12.5% EO diet or a soybean oil diet as a “breakfast,” followed either by two one-hour or one five-hour access periods to standard rodent diet, and food intake and body weights were collected. Across 14 days access, rats consuming EO on both feeding schedules gained less weight and consumed less total kilocalories than rats consuming the SO diet. Further, plasma levels of glucose and insulin were comparable in both EO and SO diet groups. In summary, EO was found to increase weight loss in rats maintained on a 75% food-restriction regimen, and attenuate weight-gain upon resumption of an ad-libitum feeding regimen. These data indicate that voluntary access to EO promoted short-term satiety, compared to SO diet, and that these effects contributed to a important and novel attenuated weight gain in EO-fed animals.
doi:10.1016/j.physbeh.2007.12.008
PMCID: PMC2372161  PMID: 18234242
Linoleic Acid; Obesity; Oleic Acid; Ethyl Oleate; Satiety; Triglyceride
11.  Tumor Microenvironment-Derived Proteins Dominate the Plasma Proteome Response During Breast Cancer Induction and Progression 
Cancer research  2011;71(15):5090-5100.
Summary
Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression and regression. Mass spectrometry data derived from ∼ 1.6 million spectra identified protein networks involved in wound healing, microenvironment and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth, and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the non-cancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a non-specific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiological processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development with relevance to plasma-based strategies to detect and diagnose cancer.
doi:10.1158/0008-5472.CAN-11-0568
PMCID: PMC3148311  PMID: 21653680
12.  Subthalamic Nucleus Stimulation Increases Brain Derived Neurotrophic Factor in the Nigrostriatal System and Primary Motor Cortex 
Journal of Parkinson's disease  2011;1(1):123-136.
The mechanisms underlying the effects of long-term deep brain stimulation of the subthalamic nucleus (STN DBS) as a therapy for Parkinson’s disease (PD) remain poorly understood. The present study examined whether functionally effective, long-term STN DBS modulates glial cell line-derived neurotrophic factor (GDNF) and/or brain-derived neurotrophic factor (BDNF) in both unlesioned and unilateral 6-hydroxydopamine lesioned rats. Lesioned rats that received two weeks of continuous unilateral STN DBS exhibited significant improvements in parkinsonian motor behaviors in tests of forelimb akinesia and rearing activity. Unilateral STN DBS did not increase GDNF in the nigrostriatal system, primary motor cortex (M1), or hippocampus of unlesioned rats. In contrast, unilateral STN DBS increased BDNF protein 2–3 fold bilaterally in the nigrostriatal system with the location (substantia nigra vs. striatum) dependent upon lesion status. Further, BDNF protein was bilaterally increased in M1 cortex by as much as 2 fold regardless of lesion status. STN DBS did not impact cortical regions that receive less input from the STN. STN DBS also was associated with bilateral increases in BDNF mRNA in the substantia nigra (SN) and internal globus pallidus (GPi). The increase observed in GPi was completely blocked by pretreatment with 5-Methyl-10,11-dihydro-5 H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), suggesting that the activation of N-methyl-D-aspartate (NMDA) receptors was involved in this phenomenon. The upregulation of BDNF associated with long term STN DBS suggest that this therapy may exert pronounced and underappreciated effects on plasticity in the basal ganglia circuitry that may play a role in the symptomatic effects of this therapy as well as support the neuroprotective effect of stimulation documented in this rat model.
PMCID: PMC3275429  PMID: 22328911
Deep brain stimulation; Parkinson’s disease; basal ganglia; trophic factors
13.  Mapping tissue-specific expression of extracellular proteins using systematic glycoproteomic analysis of different mouse tissues 
Journal of proteome research  2010;9(11):5837-5847.
Due to their easy accessibility, proteins outside of the plasma membrane represent an ideal but untapped resource for potential drug targets or disease biomarkers. They constitute the major biochemical class of current therapeutic targets and clinical biomarkers. Recent advances in proteomic technologies have fueled interest in analysis of extracellular proteins such as membrane proteins, cell surface proteins, and secreted proteins. However, unlike the gene expression analyses from a variety of tissues and cells using genomic technologies, quantitative proteomic analysis of proteins from various biological sources is challenging due to the high complexity of different proteomes, and the lack of robust and consistent methods for analyses of different tissue sources, especially for specific enrichment of extracellular proteins. Since most extracellular proteins are modified by oligosaccharides, the population of glycoproteins therefore represents the majority of extracellular proteomes. Here, we quantitatively analyzed glycoproteins and determined the expression patterns of extracellular proteins from 12 mouse tissues using solid-phase extraction of N-linked glycopeptides and liquid chromatography tandem mass spectrometry. We identified peptides enclosing 1231 possible N-linked glycosites from 826 unique proteins. We further determined the expression pattern of formerly N-linked glycopeptides and identified extracellular glycoproteins specifically expressed in each tissue. Furthermore, the tissue specificities of the overexpressed glycoproteins in a mouse skin tumor model were determined by comparing to the quantitative protein expression from the different tissues. These skin tumor-specific extracellular proteins might serve as potential candidates for cell surface drug targets or disease-specific protein markers.
doi:10.1021/pr1006075
PMCID: PMC2988866  PMID: 20828161
Extracellular proteins; glycosylation; solid-phase extraction of glycopeptides; tissue specificity; different tissues; skin tumor; proteomics; and mass spectrometry
15.  Identification of glycoproteins from mouse skin tumors and plasma 
Clinical proteomics  2008;4(3-4):117-136.
Plasma has been the focus of testing different proteomic technologies for the identification of biomarkers due to its ready accessibility. However, it is not clear if direct proteomic analysis of plasma can be used to discover new marker proteins from tumor that are associated with tumor progression. Here, we reported that such proteins can be detected in plasma in a chemical induced skin cancer mouse model. We analyzed glycoproteins from both benign papillomas and malignant carcinomas from mice using our recently developed platform, solid-phase extraction of glycopeptides (SPEG) and mass spectrometry, and identified 463 unique N-linked glycosites from 318 unique glycoproteins. These include most known extracellular proteins that have been reported to play roles in skin cancer development such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, TGFβ receptor, etc. We further investigated whether these tumor proteins could be detected in plasma from tumor bearing mice using isotope labeling and 2D-LC-MALDI-MS/MS. Two tumor glycoproteins, Tenascin-C and Arylsulfatase B, were identified and quantified successfully in plasma from tumor bearing mice. This result indicates that analysis of tumor associated proteins in tumors and plasma by method using glycopeptide capture, isotopic labeling, and mass spectrometry can be used as a discovery tool to identify candidate tumor proteins that may be detected in plasma.
doi:10.1007/s12014-008-9014-z
PMCID: PMC2976030  PMID: 21072318
16.  N-linked glycoproteomic analysis of formalin-fixed and paraffin-embedded tissues 
Journal of proteome research  2009;8(4):1657-1662.
Formalin-fixed paraffin-embedded (FFPE) tissues have been used to discover disease-associated protein changes using mass spectrometry. Protein post-translational modifications such as glycosylation are known to associate with disease development. In this study, we investigated whether FFPE tissues preserve such modifications and therefore can be used as specimen of choice to identify the disease-associated modifications. We isolated the glycopeptides from the tryptic digest of frozen and FFPE lung tissues using solid-phase extraction of glycopeptides and analyzed them using mass spectrometry. The glycopeptides identified from FFPE lung tissue were compared to the ones from frozen lung tissue regarding their relative abundance, unique glycosylation sites, and subcellular locations. The results from our study confirmed that glycosylation in FFPE tissues are preserved and FFPE tissues can be used for discovery of new disease associated changes in protein modifications. Furthermore, we demonstrated the feasibility of applying the strategy of glycopeptide isolation from tryptic peptides of FFPE tissue to other tissues such as liver and heart.
PMCID: PMC2975740  PMID: 19714870
N-glycosylation; FFPE tissue; protein post-translational modifications; quantitative analysis
17.  Endocrine dysfunction in p27Kip1 deficient mice and susceptibility to Wnt-1 driven breast cancer 
Carcinogenesis  2009;30(6):1058-1063.
The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 (p27) is a marker of prognosis in many cancers, including breast cancer. Low p27 expression correlates with poor prognosis, especially in hormone receptor positive breast tumors. This association suggests a role for p27 in hormone-dependent cancer. We used the Wnt-1 transgenic mouse model to further explore the role of p27 in hormone-driven breast cancer. We found that p27 deficiency did not alter breast cancer rate in either male or female Wnt-1 mice. However, we did find p27−/− females had reduced levels of serum progesterone (P) and increased variability in estradiol (E), which could have affected their cancer susceptibility. To equalize hormone levels, an additional cohort of Wnt-1 female mice was ovariectomized and implanted with slow release pellets of E and P. Although this treatment did not alter the breast cancer rate, it did accelerate the development of pituitary and gastric tumors in p27−/− mice. This study shows that while not a significant inhibitor of Wnt-1-driven breast cancer, p27 inhibits gastric tumors, whose latency is modulated by sex steroids.
doi:10.1093/carcin/bgp089
PMCID: PMC2691144  PMID: 19380520
18.  Differential Plasma Glycoproteome of p19ARF Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform 
Clinical proteomics  2008;4(3-4):105.
A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.
doi:10.1007/s12014-008-9018-8
PMCID: PMC2821048  PMID: 20157627
Skin cancer; LC-MS; Label-free protein quantification; Biomarker discovery; Systems biology; Targeted peptide sequencing; Glycoproteomics; Plasma
20.  Palmitic acid mediates hypothalamic insulin resistance by altering PKC-θ subcellular localization in rodents 
The Journal of Clinical Investigation  2009;119(9):2577-2589.
Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity.
doi:10.1172/JCI36714
PMCID: PMC2735917  PMID: 19726875
21.  Metastasis suppressor function of TRAIL-R in mice- implications for TRAIL-based therapy in humans? 
Cancer research  2008;68(15):6035-6037.
TRAIL is a promising candidate for cancer therapy, as it can induce apoptosis specifically in tumor cells but not in normal cells. While earlier mouse tumor studies revealed a strong tissue dependency of TRAIL and its death receptor in suppressing primary tumorigenesis or experimental metastases, we recently found that TRAIL-R inhibits lymph node metastases without affecting primary tumor formation in a mouse model of multistage skin tumorigenesis. This finding uncouples the role of TRAIL in primary tumorigenesis from metastasis formation, likely by sensitization of previously TRAIL-resistant tumor cells upon detachment, an early step required for metastasis formation. Therefore, TRAIL-R is a novel metastasis suppressor, suggesting that TRAIL-related tumor therapy might be most effective in primary tumors and early metastatic cancers, before selection for TRAIL resistance occurs.
doi:10.1158/0008-5472.CAN-08-0078
PMCID: PMC2665198  PMID: 18676822
22.  Tumor suppression by p53 in the absence of Atm 
Molecular cancer research : MCR  2008;6(7):1185-1192.
Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in suppression of Ras- driven tumors we examined susceptibility to skin carcinogenesis in DMBA/TPA treated Atm and p53 deficient mice and compared these results to previous studies on Arf deficient mice. Mice with epidermal specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared to wild type littermates. In contrast, Atm deficient mice showed no increase in papilloma number, growth, or malignant progression. γ-H2AX and p53 levels were increased in both Atm+/+ and Atm−/− papillomas, while Arf−/− papillomas showed much lower p53 expression. Thus although there is evidence of DNA damage, signaling through Arf appears to regulate p53 in these Ras-driven tumors. In spontaneous and radiation induced lymphoma models, tumor latency was accelerated in Atm−/−p53−/− compound mutant mice compared to the single mutant Atm−/− or p53−/− mice, indicating cooperation between loss of Atm and loss of p53. Although p53 mediated apoptosis was impaired in irradiated Atm−/− lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm−/− mice. In conclusion, in these models of oncogene or DNA damage induced tumors, p53 retains tumor suppressor activity in the absence of Atm.
doi:10.1158/1541-7786.MCR-07-2009
PMCID: PMC2680228  PMID: 18583527
Squamous cell carcinoma; Trp53; DNA damage; Hras; apoptosis
23.  p27kip1 Deficiency Impairs G2/M Arrest in Response to DNA Damage, Leading to an Increase in Genetic Instability▿  
Molecular and Cellular Biology  2007;28(1):258-268.
p27kip1 is a cyclin-dependent kinase inhibitor and a tumor suppressor. In some tumors, p27 suppresses tumor growth by inhibition of cell proliferation. However, this is not universally observed, implying additional mechanisms of tumor suppression by p27. p27-deficient mice are particularly susceptibility to genotoxin-induced tumors, suggesting a role for p27 in the DNA damage response. To test this hypothesis, we measured genotoxin-induced mutations and chromosome damage in p27-deficient mice. Both p27+/− and p27−/− mice displayed a higher N-ethyl-N-nitrosourea-induced mutation frequency in the colon than p27+/+ littermates. Furthermore, cells from irradiated p27-deficient mice exhibited a higher number of chromatid breaks and showed modestly increased micronucleus formation compared to cells from wild-type littermates. To determine if this mutator phenotype was related to the cell cycle-inhibitory function of p27, we measured cell cycle arrest in response to DNA damage. Both normal and tumor cells from p27-deficient mice showed impaired G2/M arrest following low doses of ionizing radiation. Thus, p27 may inhibit tumor development through two mechanisms. The first is by reducing the proliferation of cells that have already sustained an oncogenic lesion. The second is by transient inhibition of cell cycle progression following genotoxic insult, thereby minimizing chromosome damage and fixation of mutations.
doi:10.1128/MCB.01536-07
PMCID: PMC2223298  PMID: 17954563
24.  TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development 
TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R–deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R–expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.
doi:10.1172/JCI33061
PMCID: PMC2129237  PMID: 18079967
25.  p19 Arf Suppresses Growth, Progression, and Metastasis of Hras-Driven Carcinomas through p53-Dependent and -Independent Pathways 
PLoS Biology  2004;2(8):e242.
Ectopic expression of oncogenes such as Ras induces expression of p19Arf, which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19Arf, and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19Arf-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19Arf-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 Arf- and p53-deficient mice. Thus, p19Arf inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19Arf also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19 Arf-null mice, and p53 mutations were more frequently seen in wild-type than in p19 Arf-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19Arf.
A squamous cell carcinoma model shows Ras mutation not only initiates tumor development but, through Arf and p53, directly influences the subsequent evolutionary trajectory of the tumors
doi:10.1371/journal.pbio.0020242
PMCID: PMC509304  PMID: 15314658

Results 1-25 (29)