U.S. Science Wars Against Cancer

“Inbred mice are the troops which literally by the tens of thousands occupy posts on the firing line of investigation [into the] nature and cure of cancer.“

Clarence Cook Little, Founder of The Jackson Laboratory1937

The Jackson LaboratoryBar Harbor, Maine

• Non-profit mammalian genetics research

• Founded in 1929• 38 principal investigators• 1,300+ employees• Locations in Bar Harbor, Maine and

Sacramento, California• Expansion to Connecticut underway• NCI-designated Cancer Center

Bar Harbor, Maine Sacramento, California

Farmington, Connecticut

Mission

We discover the genetic basis for preventing, treating and curing human disease, and we enable research and education for the global biomedical

community.

Mission

Research Resource Education

JAX Quick Facts: Research Programs

• 38 Principal Investigator lead research groups in:

• Cancer• Neurobiology• Computational biology

and Bioinformatics• Developmental and

reproductive biology• Immunology• Complex trait genetics

ResearchResearch

JAX Quick Facts: Research

• Distribute 4,000 lines of genetically defined mice to 16,000 laboratories around the world

• Provide unique services for developing and testing in vivo mouse models to advance drug discovery and pre-clinical testing

ResourceResource

• Courses & Conferences• Precollege/College Internships• Pre- and Postdoctoral training• Graduate programs with the

University of Maine Graduate School for Biomedical Sciences (GSBS) & Tufts University Sackler School

• Visiting Investigator Program

OutreachOutreach and Education

Why Mice?

• Mice get many of the same genetic disease as humans• Mammalian physiology is highly conserved• >95% of human genes have an ortholog in mouse• Conservation of gene content and organization• Experimentally tractable• Small, “easy” to breed• Short generation time

“The mouse is thebiomedical research platform for the

21st Century.”- Harold Varmus, Director, National

Cancer Institute

genomics of normal lung development as a framework for understanding disease

processes

Carol Bult, Ph.D.Professor

Program Leader, JAX Cancer CenterSr. Advisor for Research IT

A National Cancer Institute Designated Cancer Center

development and disease

• is there a gene expression signature that is common to lung development and lung cancer?

• are developmental gene expression signatures tissue specific?

Rudolf Virchow, 1859

old question; new approaches

genomes

genomics

informatics

• Embryonic (E9 – E12)– Primitive lung buds emerge from

ventral gut epithelium• Pseudoglandular (E12-E15)

– Stereo-specific branching of the lung bronchi. Differentiation of epithelial cells to form prealveolate saccules

• Canalicular (E15-E17)– Formation of terminal sacs and

vasculature • Saccular (Terminal Sac) (E17 – P0)

– Expansion in the numbers of terminal sacs and capillaries. Differentiation of Type I and II alveolar cells

• Alveolar (P1-P30)– terminal sacs develop into mature

alveolar ducts and alveoli

lung development in mouse

http://www.cincinnatichildrens.org/research/div/pulmonary-biology/faculty-research/whitsett-lab/projects.htm

http://www.aups.org.au/Proceedings/36/9-13/

lung development stages are similar in mouse and human

Developmental Stage

Timeline for mouse

Timeline for human

approach:compare global gene expression during of normal development (mouse) to global gene expression of

tumors (human)

transcriptional profiling of lung development

E11.5 E13.5 E14.5 E16.5 P5

Images from Malpel S, Development (2000) 127:3057-67

E = embryonicP = postnatal

Extract total mRNA from the target cell/tissue- presence of mRNA means a gene is “active”

Modify the mRNA with a tag

Hybridize modified mRNAs to the probes on the gene chip- successful hybridization generates a fluorescent signal

Scan for fluorescence

Quantitate expression

1. Bright spots indicates positive hybridization events (i.e. active genes)

2. The analysis software “knows” the address of every gene and generates a report of gene activity (expression)

time course analysis

Which genes have sustained transcriptional activity changes over time?

Expectations:• genes/pathways for cell division/cell cycle

should go down over time

• genes/pathways for differentiation and lung specific pathways go up over time

http://www.cs.cmu.edu/~jernst/stem/

349312 196 141 431

320 139

Number of genes that match the expression profile

28,000+ genes on chip~20,000 genes don’t change~5,000 genes assigned to profiles

1110005A23Rik, 1700009P03Rik, 1700020C11Rik, 1810058I14Rik, 2210018M11Rik, 2610301G19Rik, 2810407C02Rik, 4931406I20Rik, 4932432K03Rik, 5730467H21Rik, 5830411E10Rik, 6330581L23Rik, 9030612M13Rik, AI848100, Abca3, Abcc4, Abcd1, Acad10, Acads, Acsbg1, Acsl5, Adam12, Adamts20, Adamts5, Adamts9, Adcy3, Akap2, Alas1, Aldh1a1, Aldh1l1, Aldoc, Alg14, Alg6, Amph, Aox3, Aplp2, Appbp2, Aqp5, Arf2, Arf4, Arhgap6, Art3, Atf6, Atm, Atp1b1, Atp6v0b, Atp6v1e1, Atp7a, Atp8a1, Atp8b2, B230118G17Rik, BC016495, Bbs4, Bcat1, Bcl2l2, Bclaf1, Bid, Bpgm, Bphl, *Braf, Brunol4, Btbd4, Bzw1, C1qtnf3, C730048C13Rik, Cacna1d, Cadps2, Calm2, Camk2d, Camkk2, Cart1, Casp7, Cav1, Ccnb1, Ccni, Cd36, Cdc26, Cdca5, Cdkn1b, Cdkn1c, Cdkn3, Cdx2, Cebpg, Ches1, Cited1, Clca1, Clta, Clu, Cmpk, Cnot6, Cntn4, Col18a1, Col3a1, Col4a1, Col4a6, Col9a1, Cox6b2, Cpm, Cpne5, Crbn, Crls1, Cse1l, *Ctnnb1, Ctps2, Ctse, Cul3, Cyp11a1, D11Ertd333e, D1Ertd161e, D230025D16Rik, D830007F02Rik, Daam2, Dab1, Dach1, Dapk2, Dcamkl1, Dhfr, Dhrs8, Dnajc15, Dtymk, Dusp4, Dyrk1a, E2f7, Eda2r, Ednra, Ell2, Elmo3, Enah, Enpep, Enpp2, Epb4.1, Eps8, Esm1, Etv5, Eya1, Fabp3, Fabp5, Fank1, Fath, Fblim1, Fbxl20, Fbxl3, Fbxw7, Fem1c, Fgfr2, Fhit, Fhl2, Fkbp6, Folr1, Foxp1, Frk, Fusip1, Fxyd6, Fzd9, Gas7, Gata2, Gdpd2, Gja1, Gpc3, Gpx3, Gstk1, Gstp1, H2-Aa, H3f3a, Hdac9, Hel308, Hesx1, Heyl, Hhip, Hif3a, Hipk2, Hist1h2bc, Hnrpf, Hook1, Hoxd8, Hsd17b12, Hsp90b1, Hspa1b, Htra3, Ifitm3, Ifnar2, Igf1, Igfbp2, Igfbp3, Igfbp7, Ing3, Ipo7, Itga4, Itgb1, Itpr2, Jarid1d, Kcnab1, Kcnb1, Kcnip1, Kcnip4, Kcnj16, Kcns2, Kdr, Keap1, Kif2a, Klf6, Klf7, Klk1, Krt2-7, Krt2-8, Lama5, Lass6, Lcn2, Lgals7, Lgtn, Lhx1, Lhx9, Lmo4, Lrrc16, Lrrk1, Lsp1, Lss, Ltf, Madd, Mafa, Man1a2, Mapk1, Mapre1, Masp1, Mef2c, Mlph, Mmp19, Mod1, Morf4l1, Morf4l2, Mrpl18, Mrpl44, Mt1, Mt2, Mtdh, Mterf, Mthfd1, Mtm1, Mtr, Mtx2, Myef2, Myl1, Mylc2b, Mylk, Myo1b, Myo5b, Narg1, Nedd9, Neo1, Nfe2l2, Npc1, Npepl1, Npr2, Nr2f2, Nrg1, Nusap1, Ogt, Otx2, Pak1, Pak3, Papss2, Pard6b, Parp1, Pbx3, Pcbd1, Pcmtd1, Pcsk5, Pctk1, Pctk3, Pdcd6ip, Pdia3, Pfdn4, Pftk1, Phb2, Phca, Phf8, Phka1, Pitx2, Pja1, Pja2, Pnck, Pomgnt1, Porcn, Ppargc1a, Ppfibp1, Ppih, Ppp1r16b, Prc1, Prcp, Prkag2, Prkar2b, Prkcd, Psmb3, *Psrc1, Ptch1, Pten, Ptgds, Ptk2b, Ptp4a1, Ptp4a2, Ptp4a3, Ptpn13, Ptx3, Qscn6, Rab2b, Rab31, Rab3a, Rab3b, Rad51l3, Rec8L1, Ren2, Rims4, Rkhd3, Rnf11, Rnf20, Robo2, Rpl39, Rps6ka3, Runx1, Runx2, Rxrb, Ryr2, S100a6, S100a9, Sat1, Scd1, Scmh1, Scn3a, Scn7a, Scn8a, Scrn1, Sdk2, Sec24a, Sec61a2, Sema3a, Sept11, Serpina3g, Sesn3, Sf4, Sfrs1, Sgk3, Shb, Sin3b, Slc11a2, Slc16a10, Slc16a7, Slc18a2, Slc25a5, Slc26a1, Slc2a13, Slc38a5, Slc39a10, Slc41a2, Slc6a14, Slc6a15, Slc6a6, Slc7a4, Slc9a2, Smc2l1, Smg5, Snapap, Sncaip, Snrk, Soat1, Sorl1, Sox10, Sox11, Sox9, Spp1, Srp54, St3gal5, Star, Strbp, Stxbp1, Sulf1, Suv420h1, Sv2b, Sycp3, Syn2, Sypl, Tacc1, Tcea3, Tcf12, Tdgf1, Tesc, Tfrc, Tgfa, Tgfb2, Thap7, Timp1, Tinagl, Tm9sf3, Tmed7, Tmed9, Tmem23, Tmpo, Tmprss13, Tradd, Tram2, Trappc5, Trim23, Trim66, *Tsc1, Tspyl2, Txndc10, Txndc2, Tyro3, Uchl5, Uhrf2, Usp12, Usp7, Utp15, Uty, Vcpip1, Vim, Vldlr, *Yes1, Ywhaz, Zdhhc2, Zfhx1b, Zfp148, Zfp192, Zfp275, Zfp277, Zfp28, Zfp30, Zfp36, Zfp583, Zfp62, Zfp68

http://www.informatics.jax.org

finding common biological themes in lists of genes

analyze biological systems and processes, not individual genes.

biological systems represented by genes whose expression levels….

Cell AdhesionSystem DevelopmentAnatomical System DevelopmentVasculature DevelopmentBlood Vessel DevelopmentAngiogenesis

increase during murine lung development:

“Late expression group”

Cell CycleCell DivisionDNA RepairMitosisRegulation of transcription

decrease during murine lung development:

“Early expression group”

gene expression in human lung tumors

Which genes are differentially expressed in tumors compared to normal lung tissue?

Expectations:• genes/pathways for cell division/cell cycle

should go up over time

• genes/pathways for differentiation and lung specific pathways go down over time

transcriptional profiling in human lung tumors compared to normal human lung

Adenocarcinoma Normal

Dehan and Kaminski, 2004. (GSE1987)NCBI’s GEO http://www.ncbi.nlm.nih.gov/geo/

• Microarray Analysis of Variance (MAANOVA)**

differential gene expression: tumor vs normal

** http://www.jax.org/staff/churchill/labsite/software/anova/index.html

A vs N (q<0.05)

1110005A23Rik, 1700009P03Rik, 1700020C11Rik, 1810058I14Rik, 2210018M11Rik, 2610301G19Rik, 2810407C02Rik, 4931406I20Rik, 4932432K03Rik, 5730467H21Rik, 5830411E10Rik, 6330581L23Rik, 9030612M13Rik, AI848100, Abca3, Abcc4, Abcd1, Acad10, Acads, Acsbg1, Acsl5, Adam12, Adamts20, Adamts5, Adamts9, Adcy3, Akap2, Alas1, Aldh1a1, Aldh1l1, Aldoc, Alg14, Alg6, Amph, Aox3, Aplp2, Appbp2, Aqp5, Arf2, Arf4, Arhgap6, Art3, Atf6, Atm, Atp1b1, Atp6v0b, Atp6v1e1, Atp7a, Atp8a1, Atp8b2, B230118G17Rik, BC016495, Bbs4, Bcat1, Bcl2l2, Bclaf1, Bid, Bpgm, Bphl, *Braf, Brunol4, Btbd4, Bzw1, C1qtnf3, C730048C13Rik, Cacna1d, Cadps2, Calm2, Camk2d, Camkk2, Cart1, Casp7, Cav1, Ccnb1, Ccni, Cd36, Cdc26, Cdca5, Cdkn1b, Cdkn1c, Cdkn3, Cdx2, Cebpg, Ches1, Cited1, Clca1, Clta, Clu, Cmpk, Cnot6, Cntn4, Col18a1, Col3a1, Col4a1, Col4a6, Col9a1, Cox6b2, Cpm, Cpne5, Crbn, Crls1, Cse1l, *Ctnnb1, Ctps2, Ctse, Cul3, Cyp11a1, D11Ertd333e, D1Ertd161e, D230025D16Rik, D830007F02Rik, Daam2, Dab1, Dach1, Dapk2, Dcamkl1, Dhfr, Dhrs8, Dnajc15, Dtymk, Dusp4, Dyrk1a, E2f7, Eda2r, Ednra, Ell2, Elmo3, Enah, Enpep, Enpp2, Epb4.1, Eps8, Esm1, Etv5, Eya1, Fabp3, Fabp5, Fank1, Fath, Fblim1, Fbxl20, Fbxl3, Fbxw7, Fem1c, Fgfr2, Fhit, Fhl2, Fkbp6, Folr1, Foxp1, Frk, Fusip1, Fxyd6, Fzd9, Gas7, Gata2, Gdpd2, Gja1, Gpc3, Gpx3, Gstk1, Gstp1, H2-Aa, H3f3a, Hdac9, Hel308, Hesx1, Heyl, Hhip, Hif3a, Hipk2, Hist1h2bc, Hnrpf, Hook1, Hoxd8, Hsd17b12, Hsp90b1, Hspa1b, Htra3, Ifitm3, Ifnar2, Igf1, Igfbp2, Igfbp3, Igfbp7, Ing3, Ipo7, Itga4, Itgb1, Itpr2, Jarid1d, Kcnab1, Kcnb1, Kcnip1, Kcnip4, Kcnj16, Kcns2, Kdr, Keap1, Kif2a, Klf6, Klf7, Klk1, Krt2-7, Krt2-8, Lama5, Lass6, Lcn2, Lgals7, Lgtn, Lhx1, Lhx9, Lmo4, Lrrc16, Lrrk1, Lsp1, Lss, Ltf, Madd, Mafa, Man1a2, Mapk1, Mapre1, Masp1, Mef2c, Mlph, Mmp19, Mod1, Morf4l1, Morf4l2, Mrpl18, Mrpl44, Mt1, Mt2, Mtdh, Mterf, Mthfd1, Mtm1, Mtr, Mtx2, Myef2, Myl1, Mylc2b, Mylk, Myo1b, Myo5b, Narg1, Nedd9, Neo1, Nfe2l2, Npc1, Npepl1, Npr2, Nr2f2, Nrg1, Nusap1, Ogt, Otx2, Pak1, Pak3, Papss2, Pard6b, Parp1, Pbx3, Pcbd1, Pcmtd1, Pcsk5, Pctk1, Pctk3, Pdcd6ip, Pdia3, Pfdn4, Pftk1, Phb2, Phca, Phf8, Phka1, Pitx2, Pja1, Pja2, Pnck, Pomgnt1, Porcn, Ppargc1a, Ppfibp1, Ppih, Ppp1r16b, Prc1, Prcp, Prkag2, Prkar2b, Prkcd, Psmb3, *Psrc1, Ptch1, Pten, Ptgds, Ptk2b, Ptp4a1, Ptp4a2, Ptp4a3, Ptpn13, Ptx3, Qscn6, Rab2b, Rab31, Rab3a, Rab3b, Rad51l3, Rec8L1, Ren2, Rims4, Rkhd3, Rnf11, Rnf20, Robo2, Rpl39, Rps6ka3, Runx1, Runx2, Rxrb, Ryr2, S100a6, S100a9, Sat1, Scd1, Scmh1, Scn3a, Scn7a, Scn8a, Scrn1, Sdk2, Sec24a, Sec61a2, Sema3a, Sept11, Serpina3g, Sesn3, Sf4, Sfrs1, Sgk3, Shb, Sin3b, Slc11a2, Slc16a10, Slc16a7, Slc18a2, Slc25a5, Slc26a1, Slc2a13, Slc38a5, Slc39a10, Slc41a2, Slc6a14, Slc6a15, Slc6a6, Slc7a4, Slc9a2, Smc2l1, Smg5, Snapap, Sncaip, Snrk, Soat1, Sorl1, Sox10, Sox11, Sox9, Spp1, Srp54, St3gal5, Star, Strbp, Stxbp1, Sulf1, Suv420h1, Sv2b, Sycp3, Syn2, Sypl, Tacc1, Tcea3, Tcf12, Tdgf1, Tesc, Tfrc, Tgfa, Tgfb2, Thap7, Timp1, Tinagl, Tm9sf3, Tmed7, Tmed9, Tmem23, Tmpo, Tmprss13, Tradd, Tram2, Trappc5, Trim23, Trim66, *Tsc1, Tspyl2, Txndc10, Txndc2, Tyro3, Uchl5, Uhrf2, Usp12, Usp7, Utp15, Uty, Vcpip1, Vim, Vldlr, *Yes1, Ywhaz, Zdhhc2, Zfhx1b, Zfp148, Zfp192, Zfp275, Zfp277, Zfp28, Zfp30, Zfp36, Zfp583, Zfp62, Zfp68

Cell CycleCell DivisionMitosis

Cell AdhesionSystem DevelopmentAnatomical System DevelopmentVasculature DevelopmentBlood Vessel DevelopmentAngiogenesis

human lung tumor(adenocarcinoma)

similar biological systems;

normal mouse lung development

“Late expression group”

Cell CycleCell DivisionDNA RepairMitosisRegulation of transcription

Cell AdhesionSystem DevelopmentAnatomical System DevelopmentVasculature DevelopmentBlood Vessel DevelopmentAngiogenesis

“Early expression group”

opposite gene expression trends

• For genes regulated in lung tumors, when are the orthologous mouse genes expressed during development?

• For genes down regulated in lung tumors, when are the orthologous mouse genes expressed during development?

integrating development and cancer genomics

Divide up the developmental timeline into segments, bin the ~5,000 or so genes that have expression levels related to development.

Determine the frequency of genes in each bin where the human ortholog is overexpressed in tumors.

Determine the frequency of genes in each bin where the human ortholog is underexpressed in tumors.

Developmental Timeline (DT)Early Late

Freq

uenc

yFr

eque

ncy

Freq

uenc

y

No association

Strong association

Ambiguous association

quantifying developmental gene expression signatures

development and disease

• is there a gene expression signature that is common to lung development and lung cancer?

• are developmental gene expression signatures tissue specific?

developmental gene expression signatures for cancer are not tissue specific

not all cancers exhibit an early developmental gene expression signature

10 developmental time series32 disease data sets (mostly cancer)

Naxerova et al. 2008. Genome Biology. 9:R108

novel classification of tumors?

• group 1 – early development gene signatures– 46% of data sets tested fall into this group– lung cancer, Wilm’s tumor

• group 2 – ambiguous developmental signature– early developmental signature plus other

transcriptional programs– observed in CNS tumors, breast cancer

• group 3 – no early development signature– genes cluster on the late end of the DT– ovarian cancer, prostate cancer

• group 1 and 2 tumors (early developmental signatures)– cell cycle, RNA splicing, DNA repair– cellular location largely in nucleus

• group 3 tumors (late development signatures)– immune response, cell adhesion, – cellular location largely in extracellular matrix and cell

membrane

functional differences in expression-based classifications

summary• genomics of normal lung development is a

useful framework for identifying genes and networks that are dysregulated in lung cancer

• developmental gene signatures do not appear to be tissue specific

• not all cancers exhibit the same developmental gene signature

• the laboratory mouse allow us to investigate early genetic events in normal development and cancer that would not otherwise be possible

ongoing studies

• developmental gene signatures in other lung tumor types

• developmental signatures in other lung diseases– Interstitial lung disease/ Pulmonary fibrosis

possible clinical relevance

• correlate gene expression patterns of lung development genes with patient outcomes– Collaboration between JAX and Cancer Care of

Maine just funded by Maine Cancer Foundation to develop new models of lung cancer

• different transcriptional profiles may reflect a variety of strategies used by tumor cells for survival– New therapeutic targets?– Biomarkers?

acknowledgements

Julie WellsCecily SwinburneJill ReclaConnie BirkenmeierKyle BeaucheminAnne Peaston

Rita ThibodeauMarge StrobelRandy Babiuk

JAX Scientific Services

Isaac KohaneKamila NaxerovaAlvin Kho

Simon Kasif

Maine Cancer FoundationTJL Cancer Center Pilot Project FundsNational Cancer Institute