Publications by authors named "John E Wilkinson"

42 Publications

A gene-environment-induced epigenetic program initiates tumorigenesis.

Nature 2021 02 3;590(7847):642-648. Epub 2021 Feb 3.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Tissue damage increases the risk of cancer through poorly understood mechanisms. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-03147-xDOI Listing
February 2021

Acarbose has sex-dependent and -independent effects on age-related physical function, cardiac health, and lipid biology.

JCI Insight 2020 Nov 5;5(21). Epub 2020 Nov 5.

Department of Pathology, UM Medical School, Ann Arbor, Michigan, USA.

With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat type 2 diabetes mellitus (T2DM), can extend mouse life span with greater effect in males than in females. Using a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological, and biochemical changes that occur during aging, and we determined which of the effects of age and drug were sex dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength test. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth-promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids - changes that have previously been associated with aging, cardiac dysfunction, and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging antiaging effects in multiple tissues, and it may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.137474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710286PMC
November 2020

An Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants.

Cancer Discov 2020 Nov 12;10(11):1654-1671. Epub 2020 Aug 12.

Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, New York.

is the most frequently mutated oncogene in cancer, yet there is little understanding of how specific KRAS amino acid changes affect tumor initiation, progression, or therapy response. Using high-fidelity CRISPR-based engineering, we created an allelic series of new mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRAS), pancreas (KRAS), and colon (KRAS) cancers. Induction of each allele in either the murine colon or pancreas revealed striking quantitative and qualitative differences between KRAS mutants in driving the early stages of transformation. Furthermore, using pancreatic organoid models, we show that KRAS mutants are sensitive to EGFR inhibition, whereas KRAS-mutant organoids are selectively responsive to covalent G12C inhibitors only when EGFR is suppressed. Together, these new mouse strains provide an ideal platform for investigating KRAS biology and for developing preclinical precision oncology models of KRAS-mutant pancreas, colon, and lung cancers. SIGNIFICANCE: KRAS is the most frequently mutated oncogene. Here, we describe new preclinical models that mimic tissue-selective KRAS mutations and show that each mutation has distinct cellular consequences and carries differential sensitivity to targeted therapeutic agents...
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-0442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642097PMC
November 2020

Pregnancy reprograms the epigenome of mammary epithelial cells and blocks the development of premalignant lesions.

Nat Commun 2020 05 27;11(1):2649. Epub 2020 May 27.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, 11724, USA.

Pregnancy causes a series of cellular and molecular changes in mammary epithelial cells (MECs) of female adults. In addition, pregnancy can also modify the predisposition of rodent and human MECs to initiate oncogenesis. Here, we investigate how pregnancy reprograms enhancer chromatin in the mammary epithelium of mice and influences the transcriptional output of the oncogenic transcription factor cMYC. We find that pregnancy induces an expansion of the active cis-regulatory landscape of MECs, which influences the activation of pregnancy-related programs during re-exposure to pregnancy hormones in vivo and in vitro. Using inducible cMYC overexpression, we demonstrate that post-pregnancy MECs are resistant to the downstream molecular programs induced by cMYC, a response that blunts carcinoma initiation, but does not perturb the normal pregnancy-induced epigenomic landscape. cMYC overexpression drives post-pregnancy MECs into a senescence-like state, and perturbations of this state increase malignant phenotypic changes. Taken together, our findings provide further insight into the cell-autonomous signals in post-pregnancy MECs that underpin the regulation of gene expression, cellular activation, and resistance to malignant development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16479-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253414PMC
May 2020

Somatic Tissue Engineering in Mouse Models Reveals an Actionable Role for WNT Pathway Alterations in Prostate Cancer Metastasis.

Cancer Discov 2020 Jul 6;10(7):1038-1057. Epub 2020 May 6.

Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York.

To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer. SIGNIFICANCE: Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-19-1242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334089PMC
July 2020

Heterochronic parabiosis regulates the extent of cellular senescence in multiple tissues.

Geroscience 2020 06 13;42(3):951-961. Epub 2020 Apr 13.

Institute on the Biology of Aging and Metabolism, University of Minnesota, Minneapolis, MN, 55455, USA.

An increase in the burden of senescent cells in tissues with age contributes to driving aging and the onset of age-related diseases. Genetic and pharmacologic elimination of senescent cells extends both health span and life span in mouse models. Heterochronic parabiosis in mice has been used to identify bloodborne, circulating pro- and anti-geronic factors able to drive or slow aging, respectively. However, whether factors in the circulation also regulate senescence is unknown. Here, we measured the expression of senescence and senescence-associated secretory phenotype (SASP) markers in multiple tissues from 4- to 18-month-old male mice that were either isochronically or heterochronically paired for 2 months. In heterochronic parabionts, the age-dependent increase in senescence and SASP marker expression was reduced in old mice exposed to a young environment, while senescence markers were concurrently increased in young heterochronic parabionts. These findings were supported by geropathology analysis using the Geropathology Grading Platform that showed a trend toward reduced hepatic lesions in old heterochronic parabionts. In summary, these results demonstrate that senescence is regulated in part by circulating geronic factors and suggest that one of the possible mediators of the rejuvenating effects with heterochronic parabiosis is through the reduction of the senescent cell burden.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11357-020-00185-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286998PMC
June 2020

Senescence-Induced Vascular Remodeling Creates Therapeutic Vulnerabilities in Pancreas Cancer.

Cell 2020 04 31;181(2):424-441.e21. Epub 2020 Mar 31.

Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

KRAS mutant pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic response that promotes hypovascularity, immunosuppression, and resistance to chemo- and immunotherapies. We show that a combination of MEK and CDK4/6 inhibitors that target KRAS-directed oncogenic signaling can suppress PDAC proliferation through induction of retinoblastoma (RB) protein-mediated senescence. In preclinical mouse models of PDAC, this senescence-inducing therapy produces a senescence-associated secretory phenotype (SASP) that includes pro-angiogenic factors that promote tumor vascularization, which in turn enhances drug delivery and efficacy of cytotoxic gemcitabine chemotherapy. In addition, SASP-mediated endothelial cell activation stimulates the accumulation of CD8 T cells into otherwise immunologically "cold" tumors, sensitizing tumors to PD-1 checkpoint blockade. Therefore, in PDAC models, therapy-induced senescence can establish emergent susceptibilities to otherwise ineffective chemo- and immunotherapies through SASP-dependent effects on the tumor vasculature and immune system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278897PMC
April 2020

Validation of a geropathology grading system for aging mouse studies.

Geroscience 2019 08 29;41(4):455-465. Epub 2019 Aug 29.

Department of Comparative Medicine, University of Washington, Seattle, WA, USA.

An understanding of early-onset mechanisms underlying age-related changes can be obtained by evaluating changes that precede frailty and end of life using histological characterization of age-related lesions. Histopathology-based information as a component of aging studies in mice can complement and add context to molecular, cellular, and physiologic data, but there is a lack of information regarding scoring criteria and lesion grading guidelines. This report describes the validation of a grading system, designated as the geropathology grading platform (GGP), which generated a composite lesion score (CLS) for comparison of histological lesion scores in tissues from aging mice. To assess reproducibility of the scoring system, multiple veterinary pathologists independently scored the same slides from the heart, lung, liver, and kidney from two different strains (C57BL/6 and CB6F1) of male mice at 8, 16, 24, and 32 months of age. There was moderate to high agreement between pathologists, particularly when agreement within a 1-point range was considered. CLS for all organs was significantly higher in older versus younger mice, suggesting that the GGP was reliable for detecting age-related pathology in mice. The overall results suggest that the GGP guidelines reliably distinguish between younger and older mice and may therefore be accurate in distinguishing between experimental groups of mice with more, or less, age-related pathology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11357-019-00088-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815299PMC
August 2019

The PHLPP2 phosphatase is a druggable driver of prostate cancer progression.

J Cell Biol 2019 06 15;218(6):1943-1957. Epub 2019 May 15.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Metastatic prostate cancer commonly presents with targeted, bi-allelic mutations of the and tumor suppressor genes. In contrast, however, most candidate tumor suppressors are part of large recurrent hemizygous deletions, such as the common chromosome 16q deletion, which involves the AKT-suppressing phosphatase PHLPP2. Using RapidCaP, a genetically engineered mouse model of mutant metastatic prostate cancer, we found that complete loss of paradoxically blocks prostate tumor growth and disease progression. Surprisingly, we find that Phlpp2 is essential for supporting Myc, a key driver of lethal prostate cancer. Phlpp2 dephosphorylates threonine-58 of Myc, which renders it a limiting positive regulator of Myc stability. Furthermore, we show that small-molecule inhibitors of PHLPP2 can suppress MYC and kill mutant cells. Our findings reveal that the frequent hemizygous deletions on chromosome 16q present a druggable vulnerability for targeting MYC protein through PHLPP2 phosphatase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201902048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548123PMC
June 2019

POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer.

Genes Dev 2018 07 26;32(13-14):915-928. Epub 2018 Jun 26.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells. Using chromatin- and RNA-profiling experiments, we provide evidence that POU2F3 is a master regulator of tuft cell identity in a variant form of SCLC. Moreover, we show that most SCLC tumors can be classified into one of three lineages based on the expression of POU2F3, ASCL1, or NEUROD1. Our CRISPR screens exposed other unique dependencies in POU2F3-expressing SCLC lines, including the lineage TFs SOX9 and ASCL2 and the receptor tyrosine kinase IGF1R (insulin-like growth factor 1 receptor). These data reveal POU2F3 as a cell identity determinant and a dependency in a tuft cell-like variant of SCLC, which may reflect a previously unrecognized cell of origin or a differentiation event in this disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.314815.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075037PMC
July 2018

Asparagine bioavailability governs metastasis in a model of breast cancer.

Nature 2018 02 7;554(7692):378-381. Epub 2018 Feb 7.

Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature25465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5898613PMC
February 2018

A TFIID-SAGA Perturbation that Targets MYB and Suppresses Acute Myeloid Leukemia.

Cancer Cell 2018 01;33(1):13-28.e8

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Electronic address:

Targeting of general coactivators is an emerging strategy to interfere with oncogenic transcription factors (TFs). However, coactivator perturbations often lead to pleiotropic effects by influencing numerous TFs. Here we identify TAF12, a subunit of TFIID and SAGA coactivator complexes, as a selective requirement for acute myeloid leukemia (AML) progression. We trace this dependency to a direct interaction between the TAF12/TAF4 histone-fold heterodimer and the transactivation domain of MYB, a TF with established roles in leukemogenesis. Ectopic expression of the TAF4 histone-fold fragment can efficiently squelch TAF12 in cells, suppress MYB, and regress AML in mice. Our study reveals a strategy for potent MYB inhibition in AML and highlights how an oncogenic TF can be selectively neutralized by targeting a general coactivator complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2017.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764110PMC
January 2018

Apoptosis-induced CXCL5 accelerates inflammation and growth of prostate tumor metastases in bone.

J Clin Invest 2018 01 27;128(1):248-266. Epub 2017 Nov 27.

Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.

During tumor progression, immune system phagocytes continually clear apoptotic cancer cells in a process known as efferocytosis. However, the impact of efferocytosis in metastatic tumor growth is unknown. In this study, we observed that macrophage-driven efferocytosis of prostate cancer cells in vitro induced the expression of proinflammatory cytokines such as CXCL5 by activating Stat3 and NF-κB(p65) signaling. Administration of a dimerizer ligand (AP20187) triggered apoptosis in 2 in vivo syngeneic models of bone tumor growth in which apoptosis-inducible prostate cancer cells were either coimplanted with vertebral bodies, or inoculated in the tibiae of immunocompetent mice. Induction of 2 pulses of apoptosis correlated with increased infiltration of inflammatory cells and accelerated tumor growth in the bone. Apoptosis-induced tumors displayed elevated expression of the proinflammatory cytokine CXCL5. Likewise, CXCL5-deficient mice had reduced tumor progression. Peripheral blood monocytes isolated from patients with bone metastasis of prostate cancer were more efferocytic compared with normal controls, and CXCL5 serum levels were higher in metastatic prostate cancer patients relative to patients with localized prostate cancer or controls. Altogether, these findings suggest that the myeloid phagocytic clearance of apoptotic cancer cells accelerates CXCL5-mediated inflammation and tumor growth in bone, pointing to CXCL5 as a potential target for cancer therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI92466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749545PMC
January 2018

fusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma.

Proc Natl Acad Sci U S A 2017 12 21;114(50):13076-13084. Epub 2017 Nov 21.

Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

A segmental deletion resulting in gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC. Notably, overexpression of the wild-type PRKACA was unable to fully recapitulate the oncogenic activity of , implying that FL-HCC does not simply result from enhanced PRKACA expression. Tumorigenesis was significantly enhanced by genetic activation of β-catenin, an observation supported by evidence of recurrent Wnt pathway mutations in human FL-HCC, as well as treatment with the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which causes tissue injury, inflammation, and fibrosis. Our study validates the fusion kinase as an oncogenic driver and candidate drug target for FL-HCC, and establishes a practical model for preclinical studies to identify strategies to treat this disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1716483114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740683PMC
December 2017

R-Spondin chromosome rearrangements drive Wnt-dependent tumour initiation and maintenance in the intestine.

Nat Commun 2017 07 11;8:15945. Epub 2017 Jul 11.

Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, New York 10021, USA.

Defining the genetic drivers of cancer progression is a key in understanding disease biology and developing effective targeted therapies. Chromosome rearrangements are a common feature of human malignancies, but whether they represent bona fide cancer drivers and therapeutically actionable targets, requires functional testing. Here, we describe the generation of transgenic, inducible CRISPR-based mouse systems to engineer and study recurrent colon cancer-associated EIF3E-RSPO2 and PTPRK-RSPO3 chromosome rearrangements in vivo. We show that both Rspo2 and Rspo3 fusion events are sufficient to initiate hyperplasia and tumour development in vivo, without additional cooperating genetic events. Rspo-fusion tumours are entirely Wnt-dependent, as treatment with an inhibitor of Wnt secretion, LGK974, drives rapid tumour clearance from the intestinal mucosa without effects on normal intestinal crypts. Altogether, our study provides direct evidence that endogenous Rspo2 and Rspo3 chromosome rearrangements can initiate and maintain tumour development, and indicate a viable therapeutic window for LGK974 treatment of RSPO-fusion cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms15945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508203PMC
July 2017

The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein.

J Cell Biol 2017 03 13;216(3):641-656. Epub 2017 Feb 13.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724

Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact allele.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201604025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350510PMC
March 2017

A Pipeline for Drug Target Identification and Validation.

Cold Spring Harb Symp Quant Biol 2016 5;81:257-267. Epub 2017 Jan 5.

Department of Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York 10065.

Rapid and affordable tumor profiling has led to an explosion of genomic data that is facilitating the development of new cancer therapies. The potential of therapeutic strategies aimed at inactivating the oncogenic lesions that contribute to the aberrant survival and proliferation of tumor cells has yielded remarkable success in some malignancies such as BRAF-mutant melanoma and BCR-ABL expressing chronic myeloid leukemia. However, the direct inhibition of several well-established oncoproteins in some of these cancers is not possible or produces only transient benefits. Functional genomics represents a powerful approach for the identification of vulnerabilities linked to specific genetic alterations and has provided substantial insights into cancer signaling networks. Still, as inhibition of gene function can have diverse effects on both tumor and normal tissues, information on the potency of target inhibition on tumor growth as well as the toxic side effects of target inhibition are also needed. Here, we discuss our RNA interference (RNAi) pipeline for cancer target discovery based on our optimized short-hairpin RNA (shRNA) tools for negative selection screens and inducible RNAi platform that, in combination with embryonic stem cell (ESC)-based genetically engineered mouse models (GEMMs), enable deep in vivo target validation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/sqb.2016.81.031096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469697PMC
February 2018

ATDC/TRIM29 Drives Invasive Bladder Cancer Formation through miRNA-Mediated and Epigenetic Mechanisms.

Cancer Res 2015 Dec 15;75(23):5155-66. Epub 2015 Oct 15.

Translational Oncology Program, University of Michigan Medical Center, Ann Arbor, Michigan. Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan. Department of Molecular and Integrative Physiology, University of Michigan Medical Center, Ann Arbor, Michigan.

Bladder cancer is a common and deadly malignancy but its treatment has advanced little due to poor understanding of the factors and pathways that promote disease. ATDC/TRIM29 is a highly expressed gene in several lethal tumor types, including bladder tumors, but its role as a pathogenic driver has not been established. Here we show that overexpression of ATDC in vivo is sufficient to drive both noninvasive and invasive bladder carcinoma development in transgenic mice. ATDC-driven bladder tumors were indistinguishable from human bladder cancers, which displayed similar gene expression signatures. Clinically, ATDC was highly expressed in bladder tumors in a manner associated with invasive growth behaviors. Mechanistically, ATDC exerted its oncogenic effects by suppressing miR-29 and subsequent upregulation of DNMT3A, leading to DNA methylation and silencing of the tumor suppressor PTEN. Taken together, our findings established a role for ATDC as a robust pathogenic driver of bladder cancer development, identified downstream effector pathways, and implicated ATDC as a candidate biomarker and therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-15-0603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668224PMC
December 2015

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice.

PLoS One 2015 6;10(8):e0134832. Epub 2015 Aug 6.

Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone's effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134832PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4527695PMC
May 2016

A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis.

Nature 2015 Apr 8;520(7547):358-62. Epub 2015 Apr 8.

1] Watson School of Biological Sciences, Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA [2] CRUK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robininson Way, Cambridge CB2 0RE, UK.

Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extravascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature14403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634366PMC
April 2015

Successful Porcine Renal Transplantation After 60 Minutes of Donor Warm Ischemia: Extracorporeal Perfusion and Thrombolytics.

ASAIO J 2015 Jul-Aug;61(4):474-9

From the *Department of General Surgery, Henry Ford Health Systems, Detroit, Michigan; †Department of Surgery, Extracorporeal Life Support Laboratory, University of Michigan, Ann Arbor, Michigan; ‡Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Michigan; §Michigan Critical Care Consultants, Corp. Ann Arbor, Michigan; and ¶Department of Surgery, Section of Transplantation, University of Michigan, Michigan.

Donation from uncontrolled circulatory determination of death donors (uDCD) is impractical in United States because of the time needed to organize procurement before irreversible organ damage. Salvaging organs after prolonged warm ischemic time (WIT) may address this limitation. We evaluated the combination of extracorporeal support (ECS) and thrombolytics in a porcine uDCD renal transplant model. Nonanticoagulated uDCD sustained 60 min of WIT, and two groups were studied. Rapid recovery (RR)-uDCD renal grafts procured using the standard quick topical cooling and renal flush, and ECS-assisted donation (E-uDCD), 4 hr ECS plus thrombolytics for in situ perfusion before procurement. All kidneys were flushed and cold stored, followed by transplantation into healthy nephrectomized recipients without immunosuppression. Delayed graft function (DGF) was defined as creatinine more than 5.0 mg/dl on any postoperative day. Twelve kidneys in E-uDCD and 6 in RR-uDCD group were transplanted. All 12 E-uDCD recipients had urine production and adequate function in the first 48 hr, but two grafts (16.7%) had DGF at 96 hr. All six recipients from RR-uDCD group had DGF at 48 hr and were killed. Creatinine and blood urea nitrogen (BUN) levels were significantly lower in E-uDCD compared with RR-uDCD group at 24 hr (2.9 ± 0.7 mg/dl vs. 5.2 ± 0.9 mg/dl) and 48 hr (3.2 ± 0.9 mg/dl vs. 7.2 ± 1.0 mg/dl); BUN levels at 24 and 48 hr were 28.3 ± 6.7 mg/dl vs. 39.5 ± 7.5 mg/dl and 23.9 ± 5.0 mg/dl vs. 46 ± 12.9 mg/dl, respectively. Thrombolytics plus ECS precondition organs in situ yielding functional kidneys in a porcine model of uDCD with 60 min of WIT. This procurement method addresses logistical limitations for uDCD use in the United States and could have a major impact on the organ donor pool.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MAT.0000000000000228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4486602PMC
April 2016

MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2.

Cancer Discov 2015 Jun 31;5(6):636-51. Epub 2015 Mar 31.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Unlabelled: We have recently recapitulated metastasis of human PTEN/TP53-mutant prostate cancer in the mouse using the RapidCaP system. Surprisingly, we found that this metastasis is driven by MYC, and not AKT, activation. Here, we show that cell-cell communication by IL6 drives the AKT-MYC switch through activation of the AKT-suppressing phosphatase PHLPP2, when PTEN and p53 are lost together, but not separately. IL6 then communicates a downstream program of STAT3-mediated MYC activation, which drives cell proliferation. Similarly, in tissues, peak proliferation in Pten/Trp53-mutant primary and metastatic prostate cancer does not correlate with activated AKT, but with STAT3/MYC activation instead. Mechanistically, MYC strongly activates the AKT phosphatase PHLPP2 in primary cells and prostate cancer metastasis. We show genetically that Phlpp2 is essential for dictating the proliferation of MYC-mediated AKT suppression. Collectively, our data reveal competition between two proto-oncogenes, MYC and AKT, which ensnarls the Phlpp2 gene to facilitate MYC-driven prostate cancer metastasis after loss of Pten and Trp53.

Significance: Our data identify IL6 detection as a potential causal biomarker for MYC-driven metastasis after loss of PTEN and p53. Second, our finding that MYC then must supersede AKT to drive cell proliferation points to MYC inhibition as a critical part of PI3K pathway therapy in lethal prostate cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-14-1113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456272PMC
June 2015

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis.

Genes Dev 2015 Jan;29(2):171-83

Department of Surgery, Translational Oncology Program, Department of Molecular and Integrative Physiology,

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.253591.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298136PMC
January 2015

Inducible in vivo silencing of Brd4 identifies potential toxicities of sustained BET protein inhibition.

Cell Rep 2014 Sep 18;8(6):1919-1929. Epub 2014 Sep 18.

Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA; Howard Hughes Medical Institute, New York, NY 10065, USA. Electronic address:

BET family proteins are novel therapeutic targets for cancer and inflammation and represent the first chromatin readers against which small-molecule inhibitors have been developed. First-generation BET inhibitors have shown therapeutic efficacy in preclinical models, but the consequences of sustained BET protein inhibition in normal tissues remain poorly characterized. Using an inducible and reversible transgenic RNAi mouse model, we show that strong suppression of the BET protein Brd4 in adult animals has dramatic effects in multiple tissues. Brd4-depleted mice display reversible epidermal hyperplasia, alopecia, and decreased cellular diversity and stem cell depletion in the small intestine. Furthermore, Brd4-suppressed intestines are sensitive to organ stress and show impaired regeneration following irradiation, suggesting that concurrent Brd4 suppression and certain cytotoxic therapies may induce undesirable synergistic effects. These findings provide important insight into Brd4 function in normal tissues and, importantly, predict several potential outcomes associated with potent and sustained BET protein inhibition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2014.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234106PMC
September 2014

Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma.

J Immunol 2014 Oct 15;193(8):4245-53. Epub 2014 Sep 15.

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;

The role and origin of alveolar macrophages (AMs) in asthma are incompletely defined. We sought to clarify these issues in the context of acute allergic lung inflammation using house dust mite and OVA murine models. Use of liposomal clodronate to deplete resident AMs (rAMs) resulted in increased levels of inflammatory cytokines and eosinophil numbers in lavage fluid and augmented the histopathologic evidence of lung inflammation, suggesting a suppressive role for rAMs. Lung digests of asthmatic mice revealed an increased percentage of Ly6C(high)/CD11b(pos) inflammatory monocytes. Clodronate depletion of circulating monocytes, by contrast, resulted in an attenuation of allergic inflammation. A CD45.1/CD45.2 chimera model demonstrated that recruitment at least partially contributes to the AM pool in irradiated nonasthmatic mice, but its contribution was no greater in asthma. Ki-67 staining of AMs supported a role for local proliferation, which was increased in asthma. Our data demonstrate that rAMs dampen, whereas circulating monocytes promote, early events in allergic lung inflammation. Moreover, maintenance of the AM pool in the early stages of asthmatic inflammation depends on local proliferation, but not recruitment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4185233PMC
October 2014

Acquired dependence of acute myeloid leukemia on the DEAD-box RNA helicase DDX5.

Cell Rep 2014 Jun 5;7(6):1887-99. Epub 2014 Jun 5.

Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA. Electronic address:

Acute myeloid leukemia (AML) therapy involves compounds that are cytotoxic to both normal and cancer cells, and relapsed AML is resistant to subsequent chemotherapy. Thus, agents are needed that selectively kill AML cells with minimal toxicity. Here, we report that AML is dependent on DDX5 and that inhibiting DDX5 expression slows AML cell proliferation in vitro and AML progression in vivo but is not toxic to cells from normal bone marrow. Inhibition of DDX5 expression in AML cells induces apoptosis via induction of reactive oxygen species (ROS). This apoptotic response can be blocked either by BCL2 overexpression or treatment with the ROS scavenger N-acetyl-L-cysteine. Combining DDX5 knockdown with a BCL2 family inhibitor cooperates to induce cell death in AML cells. By inhibiting DDX5 expression in vivo, we show that DDX5 is dispensable for normal hematopoiesis and tissue homeostasis. These results validate DDX5 as a potential target for blocking AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2014.05.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100070PMC
June 2014

RapidCaP, a novel GEM model for metastatic prostate cancer analysis and therapy, reveals myc as a driver of Pten-mutant metastasis.

Cancer Discov 2014 Mar 20;4(3):318-33. Epub 2014 Jan 20.

1Cold Spring Harbor Laboratory, Cancer Center, Cold Spring Harbor; 2Department of Pathology and Laboratory Medicine, New York-Presbyterian Hospital, Weill Cornell Medical College; 3Mount Sinai School of Medicine, New York, New York; 4Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts; and 5Unit for Laboratory Animal Medicine, Medical School, University of Michigan, Ann Arbor, Michigan.

Genetically engineered mouse (GEM) models are a pillar of functional cancer research. Here, we developed RapidCaP, a GEM modeling system that uses surgical injection for viral gene delivery to the prostate. We show that in Pten deficiency, loss of p53 suffices to trigger metastasis to distant sites at greater than 50% penetrance by four months, consistent with results from human prostate cancer genome analysis. Live bioluminescence tracking showed that endogenous primary and metastatic disease responds to castration before developing lethal castration resistance. To our surprise, the resulting lesions showed no activation of Akt but activation of the Myc oncogene. Using RapidCaP, we find that Myc drives local prostate metastasis and is critical for maintenance of metastasis, as shown by using the Brd4 inhibitor JQ1. Taken together, our data suggest that a "MYC-switch" away from AKT forms a critical and druggable event in PTEN-mutant prostate cancer metastasis and castration resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-13-0346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4084646PMC
March 2014

Splicing factor SRSF6 promotes hyperplasia of sensitized skin.

Nat Struct Mol Biol 2014 Feb 19;21(2):189-97. Epub 2014 Jan 19.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

Many biological processes involve gene-expression regulation by alternative splicing. Here, we identify the splicing factor SRSF6 as a regulator of wound healing and tissue homeostasis in skin. We show that SRSF6 is a proto-oncogene frequently overexpressed in human skin cancer. Overexpressing it in transgenic mice induces hyperplasia of sensitized skin and promotes aberrant alternative splicing. We identify 139 SRSF6-target genes in skin and show that this SR-rich protein binds to alternative exons in the pre-mRNA of the extracellular-matrix protein tenascin C, thus promoting the expression of isoforms characteristic of invasive and metastatic cancer independently of cell type. SRSF6 overexpression additionally results in depletion of LGR6+ stem cells and excessive keratinocyte proliferation and response to injury. Furthermore, the effects of SRSF6 in wound healing assayed in vitro depend on the tenascin-C isoforms. Thus, abnormal SR-protein expression can perturb tissue homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.2756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118672PMC
February 2014

Transcription factors OVOL1 and OVOL2 induce the mesenchymal to epithelial transition in human cancer.

PLoS One 2013 4;8(10):e76773. Epub 2013 Oct 4.

Department of Urology, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America.

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076773PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790720PMC
May 2014