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Kucherlapati, Melanie

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Kucherlapati

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Melanie

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Kucherlapati, Melanie
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    MAPRE1 as a Plasma Biomarker for Early-Stage Colorectal Cancer and Adenomas
    (American Association for Cancer Research (AACR), 2015-11) Taguchi, Ayumu; Rho, Jung-hyun; Yan, Qingxiang; Zhang, Yuzheng; Zhao, Yang; Xu, Hanwen; Tripathi, Satyendra Chandra; Wang, Hong; Brenner, Dean E.; Kucherlapati, Melanie; Kucherlapati, Raju; Boutin, Adam T.; Wang, Y. Alan; DePinho, Ronald A.; Feng, Ziding; Lampe, Paul D.; Hanash, Samir M.
    Blood-based biomarkers for early detection of colorectal cancer (CRC) could complement current approaches to CRC screening. We previously identified the APC-binding protein MAPRE1 as a potential CRC biomarker. Here we undertook a case-control validation study to determine the performance of MAPRE1 in detecting early CRC and colon adenoma and to assess the potential relevance of additional biomarker candidates. We analyzed plasma samples from 60 patients with adenomas, 30 with early CRC, 30 with advanced CRC, and 60 healthy controls. MAPRE1 and a set of 21 proteins with potential biomarker utility were assayed using high-density antibody arrays, and CEA was assayed using ELISA. The biologic significance of the candidate biomarkers was also assessed in CRC mouse models. Plasma MAPRE1 levels were significantly elevated in both patients with adenomas and patients with CRC compared with controls (P < 0.0001). MAPRE1 and CEA together yielded an area under the curve of 0.793 and a sensitivity of 0.400 at 95% specificity for differentiating early CRC from controls. Three other biomarkers (AK1, CLIC1, and SOD1) were significantly increased in both adenoma and early CRC patient plasma samples and in plasma from CRC mouse models at preclinical stages compared with controls. The combination of MAPRE1, CEA, and AK1 yielded sensitivities of 0.483 and 0.533 at 90% specificity and sensitivities of 0.350 and 0.467 at 95% specificity for differentiating adenoma and early CRC, respectively, from healthy controls. These findings suggest that MAPRE1 can contribute to the detection of early-stage CRC and adenomas together with other biomarkers.
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    Mesalazine and Thymoquinone Attenuate Intestinal Tumour Development in Msh2loxP/loxPVillin-Cre Mice
    (BMJ, 2015-12) Kortüm, Benedikt; Campregher, Christoph; Lang, Michaela; Khare, Vineeta; Pinter, Matthias; Evstatiev, Rayko; Schmid, Gerald; Mittlböck, Martina; Scharl, Theresa; Kucherlapati, Melanie; Edelmann, Winfried; Gasche, Christoph
    Objective Lynch syndrome is caused by germline mutations in DNA mismatch repair genes leading to microsatellite instability (MSI) and colorectal cancer. Mesalazine, commonly used for the treatment of UC, reduces MSI in vitro. Here, we tested natural compounds for such activity and applied mesalazine and thymoquinone in a Msh2loxP/loxP Villin-Cre mouse model for Lynch syndrome. Design Flow cytometry was used for quantitation of mutation rates at a CA13 microsatellite in human colon cancer (HCT116) cells that had been stably transfected with pIREShyg2-enhanced green fluorescent protein/CA13, a reporter for frameshift mutations. Mice were treated for 43 weeks with mesalazine, thymoquinone or control chow. Intestines were analysed for tumour incidence, tumour multiplicity and size. MSI testing was performed from microdissected normal intestinal or tumour tissue, compared with mouse tails and quantified by the number of mutations per marker (NMPM). Results Besides mesalazine, thymoquinone significantly improved replication fidelity at 1.25 and 2.5 µM in HCT116 cells. In Msh2loxP/loxP Villin-Cre mice, tumour incidence was reduced by mesalazine from 94% to 69% (p=0.04) and to 56% (p=0.003) by thymoquinone. The mean number of tumours was reduced from 3.1 to 1.4 by mesalazine (p=0.004) and to 1.1 by thymoquinone (p<0.001). Interestingly, MSI was reduced in normal intestinal tissue from 1.5 to 1.2 NMPM (p=0.006) and to 1.1 NMPM (p=0.01) by mesalazine and thymoquinone, respectively. Thymoquinone, but not mesalazine, reduced MSI in tumours. Conclusions Mesalazine and thymoquinone reduce tumour incidence and multiplicity in Msh2loxP/loxP Villin-Cre mice by reduction of MSI independent of a functional mismatch repair system. Both substances are candidate compounds for chemoprevention in Lynch syndrome mutation carriers.
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    Msh2 Acts in Medium-Spiny Striatal Neurons as an Enhancer of CAG Instability and Mutant Huntingtin Phenotypes in Huntington’s Disease Knock-In Mice
    (2012-09-07) Kovalenko, Marina; Dragileva, Ella; St. Clare, Jason; Gillis, Tammy; Guide, Jolene; New, Jaclyn; Dong, Hualing; Kucherlapati, Raju; Kucherlapati, Melanie; Ehrlich, Michelle E.; Lee, Jong-Min; Wheeler, Vanessa
    The CAG trinucleotide repeat mutation in the Huntington’s disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.
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    Aspirin and Low-Dose Nitric Oxide–Donating Aspirin Increase Life Span in a Lynch Syndrome Mouse Model
    (American Association for Cancer Research (AACR), 2011-05) Mcilhatton, Michael A.; Tyler, Jessica; Kerepesi, Laura A.; Bocker-Edmonston, Tina; Kucherlapati, Melanie; Edelmann, Winfried; Kucherlapati, Raju; Kopelovich, Levy; Fishel, Richard
    Non-steroidal anti-inflammatory drugs (NSAIDs) appear to be effective cancer chemopreventives. Previous cellular studies demonstrated that aspirin (acetylsalicylic acid: ASA) and nitric oxide-donating ASA (NO-ASA) suppressed microsatellite instability (MSI) in mismatch repair (MMR)-deficient cells linked to the common cancer predisposition syndrome hereditary non-polyposis colorectal cancer or Lynch syndrome (LS/HNPCC), at doses 300-3000-fold less than ASA. Using a mouse model that develops MMR-deficient intestinal tumors that appear pathologically identical to LS/HNPCC we show that ASA (400 mg/kg) and low dose NO-ASA (72 mg/kg) increased life span by 18–21%. We also note a trend where ASA treatment resulted in intestinal tumors with reduced high MSI (H-MSI) and increased low MSI (L-MSI) as defined by the Bethesda Criteria. Low dose NO-ASA had a minimal effect on MSI status. In contrast to previous studies, high dose NO-ASA (720/1500 mg/kg) treatments increased tumor burden, decreased life span and exacerbated MSI uniquely in the LS/HNPCC mouse model. These results suggest that MMR-deficient tissues/mice may be specifically sensitive to intrinsic pharmacokinetic features of this drug. It is likely that long-term treatment with ASA may represent a chemopreventive option for LS/HNPCC patients. Moreover, since low dose NO-ASA demonstrates equivalent life span increase at 10-fold lower doses than ASA, it may have the potential to significantly reduce the gastropathy associated with long-term ASA treatment.
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    An Msh2 Conditional Knockout Mouse for Studying Intestinal Cancer and Testing Anticancer Agents
    (Elsevier BV, 2010-03) Kucherlapati, Melanie; Lee, Kyeryoung; Nguyen, Andrew A.; Clark, Alan B.; Hou, Harry; Rosulek, Andrew; Li, Hua; Yang, Kan; Fan, Kunhua; Lipkin, Martin; Bronson, Roderick; Jelicks, Linda; Kunkel, Thomas A.; Kucherlapati, Raju; Edelmann, Winfried
    Background & Aims Mutations in the DNA mismatch repair (MMR) gene MSH2 cause Lynch Syndromes I & II, and sporadic colorectal cancers (CRCs). Msh2null mice predominantly develop lymphoma and do not accurately recapitulate the CRC phenotype. Methods We generated and examined mice with a conditional Msh2 disruption (Msh2LoxP), permitting tissue-specific gene inactivation. ECMsh2LoxP/LoxP mice carried an EIIa-Cre transgene and VCMsh2LoxP/LoxP mice carried a Villin-Cre transgene. We combined the VCMsh2LoxP allele with either Msh2Δ7null (VCMsh2LoxP/null) or Msh2G674D mutations (VCMsh2LoxP/G674D) to create allelic phase mutants. These mice were given cisplatin, or 5-fluorouracil/leucovorin and oxaliplatin (FOLFOX) and their tumors were measured by magnetic resonance imaging. Results Embryonic fibroblasts from ECMsh2LoxP/LoxP mice do not express MSH2 and are MMR-deficient. Reverse transcription, PCR, and immunohistochemistry from VCMsh2LoxP/LoxP mice demonstrated specific loss of Msh2 mRNA and protein from epithelial cells of the intestinal tract. Microsatellite instability (MSI) was observed in all VCMsh2 strains and limited to the intestinal mucosa. Resulting adenomas and adenocarcinomas had somatic Apc truncation mutations. VCMsh2LoxP/LoxP mice did not develop lymphoma. Comparison of allelic phase tumors revealed significant differences in multiplicity and size. When treated with cisplatin or FOLFOX, tumor size was reduced in VCMsh2LoxP/G674D but not VCMsh2LoxP/null tumors. The apoptotic response to FOLFOX was partially sustained in the intestinal mucosa of VCMsh2LoxP/G674D animals. Conclusion Msh2LoxP/LoxP mice in combination with appropriate Cre recombinase transgenes have excellent potential for preclinical modeling of Lynch Syndrome, MMR deficient tumors of other tissue types, and use in drug development.
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    Genotype Directed Therapy in Murine Mismatch Repair Deficient Tumors
    (Public Library of Science, 2013) Kucherlapati, Melanie; Esfahani, Shadi; Habibollahi, Peiman; Wang, Junning; Still, Eric R.; Bronson, Roderick; Mahmood, Umar; Kucherlapati, Raju
    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.
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    An EGFR Targeted PET Imaging Probe for the Detection of Colonic Adenocarcinomas in the Setting of Colitis
    (Ivyspring International Publisher, 2014) Turker, N. Selcan; Heidari, Pedram; Kucherlapati, Raju; Kucherlapati, Melanie; Mahmood, Umar
    Colorectal cancer is a serious complication associated with inflammatory bowel disease, often indistinguishable by screening with conventional FDG PET probes. We have developed an alternative EGFR-targeted PET imaging probe that may be used to overcome this difficulty, and successfully assessed its utility for neoplastic lesion detection in preclinical models. Cetuximab F(ab′)2 fragments were enzymatically generated, purified, and DOTA-conjugated. Radiolabeling was performed with 67Ga for cell based studies and 64Cu for in vivo imaging. Competitive binding studies were performed on CT26 cells to assess affinity (KD) and receptors per cell (Bmax). In vivo imaging using the EGFR targeted PET probe and 18F FDG was performed on CT26 tumor bearing mice in both control and dextran sodium sulfate (DSS) induced colitis settings. Spontaneous adenomas in genetically engineered mouse (GEM) models of colon cancer were additionally imaged. The EGFR imaging agent was generated with high purity (> 98%), with a labeling efficiency of 60 ± 5% and ≥99% radiochemical purity. The KD was 6.6 ± 0.7 nM and the Bmax for CT26 cells was 3.3 ± 0.1 × 106 receptors/cell. Target to background ratios (TBR) for CT26 tumors compared to colonic uptake demonstrated high values for both 18F-FDG (3.95 ± 0.13) and the developed 64Cu-DOTA-cetuximab-F(ab′)2 probe (4.42 ± 0.11) in control mice. The TBR for the EGFR targeted probe remained high (3.78 ± 0.06) in the setting of colitis, while for 18F FDG, this was markedly reduced (1.54 ± 0.08). Assessment of the EGFR targeted probe in the GEM models demonstrated a correlation between radiotracer uptake in spontaneous colonic lesions and the EGFR staining level ex vivo. A clinically translatable PET imaging probe was successfully developed to assess EGFR. The imaging agent can detect colonic tumors with a high TBR for detection of in situ lesions in the setting of colitis, and opens the possibility for a new approach for screening high-risk patients.
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    Mesalazine and thymoquinone attenuate intestinal tumour development in Msh2loxP/loxP Villin-Cre mice
    (BMJ Publishing Group, 2015) Kortüm, Benedikt; Campregher, Christoph; Lang, Michaela; Khare, Vineeta; Pinter, Matthias; Evstatiev, Rayko; Schmid, Gerald; Mittlböck, Martina; Scharl, Theresa; Kucherlapati, Melanie; Edelmann, Winfried; Gasche, Christoph
    Objective: Lynch syndrome is caused by germline mutations in DNA mismatch repair genes leading to microsatellite instability (MSI) and colorectal cancer. Mesalazine, commonly used for the treatment of UC, reduces MSI in vitro. Here, we tested natural compounds for such activity and applied mesalazine and thymoquinone in a Msh2loxP/loxP Villin-Cre mouse model for Lynch syndrome. Design: Flow cytometry was used for quantitation of mutation rates at a CA13 microsatellite in human colon cancer (HCT116) cells that had been stably transfected with pIREShyg2-enhanced green fluorescent protein/CA13, a reporter for frameshift mutations. Mice were treated for 43 weeks with mesalazine, thymoquinone or control chow. Intestines were analysed for tumour incidence, tumour multiplicity and size. MSI testing was performed from microdissected normal intestinal or tumour tissue, compared with mouse tails and quantified by the number of mutations per marker (NMPM). Results: Besides mesalazine, thymoquinone significantly improved replication fidelity at 1.25 and 2.5 µM in HCT116 cells. In Msh2loxP/loxP Villin-Cre mice, tumour incidence was reduced by mesalazine from 94% to 69% (p=0.04) and to 56% (p=0.003) by thymoquinone. The mean number of tumours was reduced from 3.1 to 1.4 by mesalazine (p=0.004) and to 1.1 by thymoquinone (p<0.001). Interestingly, MSI was reduced in normal intestinal tissue from 1.5 to 1.2 NMPM (p=0.006) and to 1.1 NMPM (p=0.01) by mesalazine and thymoquinone, respectively. Thymoquinone, but not mesalazine, reduced MSI in tumours. Conclusions: Mesalazine and thymoquinone reduce tumour incidence and multiplicity in Msh2loxP/loxP Villin-Cre mice by reduction of MSI independent of a functional mismatch repair system. Both substances are candidate compounds for chemoprevention in Lynch syndrome mutation carriers.
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    Adenomatous Polyposis Coli (APC) Is Required for Normal Development of Skin and Thymus
    (Public Library of Science, 2006) Kuraguchi, Mari; Wang, Xiu-Ping; Rothenberg, Rebecca; Ohene-Baah, Nana Yaw; Lund, Jennifer J; Bronson, Roderick; Kucherlapati, Melanie; Maas, Richard; Kucherlapati, Raju
    The tumor suppressor gene Apc (adenomatous polyposis coli) is a member of the Wnt signaling pathway that is involved in development and tumorigenesis. Heterozygous knockout mice for Apc have a tumor predisposition phenotype and homozygosity leads to embryonic lethality. To understand the role of Apc in development we generated a floxed allele. These mice were mated with a strain carrying Cre recombinase under the control of the human Keratin 14 (K14) promoter, which is active in basal cells of epidermis and other stratified epithelia. Mice homozygous for the floxed allele that also carry the K14-cre transgene were viable but had stunted growth and died before weaning. Histological and immunochemical examinations revealed that K14-cre–mediated Apc loss resulted in aberrant growth in many ectodermally derived squamous epithelia, including hair follicles, teeth, and oral and corneal epithelia. In addition, squamous metaplasia was observed in various epithelial-derived tissues, including the thymus. The aberrant growth of hair follicles and other appendages as well as the thymic abnormalities in K14-cre; ApcCKO/CKO mice suggest the Apc gene is crucial in embryonic cells to specify epithelial cell fates in organs that require epithelial–mesenchymal interactions for their development.