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Tumour Modifier Genes 1. STRATEGY (KISS OF DEATH) 2. SOME RESULTS
3. WHY DO HUMAN ASSOCIATION STUDIES NOT WORK (WELL)?
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Mouse Modifier Program
RADIATION CHEMICAL MUTAGEN Skin Lung Prostate Lymphoma TUMOR PROMOTER SV40T Ki-ras
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GENETIC APPROACHES TO TUMOR MODIFIERS
INTRASPECIFIC MUSCULUS X MUSCULUS CROSSES backcross intercross recombinant inbred recombinant congenic INTERSPECIFIC SPRETUS X MUSCULUS CROSSES ADVANCED MULTISTRAIN INTERCROSS 4 OUTBRED POPULATIONS
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INTERSPECIFIC SPRETUS-MUSCULUS CROSS
GENETIC DIVERGENCE extreme phenotypic differences DOMINANT RESISTANCE GENES – reduced complexity 3 HIGHLY POLYMORPHIC mapping is easy ADVANTAGES DISADVANTAGES Male F1s are sterile Can be difficult to breed
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Gene Expression in Mus spretus and Mus musculus
SpGla SPRET/Ei C57/B6 C3H NIH/Ola 16 14 12 10 Log2 Control Signal Intensity 8 6 4 2
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Dominant resistance genes in mus spretus
Mus spretus are resistant to tumor development in the SKIN LUNG COLON LIVER LYMPHOID SYSTEM
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X X INTERSPECIFIC BACKCROSS: LINKAGE ANALYSIS AND HAPLOTYPING OUTBRED
SPRETUS X B F1 HYBRID: OR X
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Mouse Tumor Susceptibility Loci
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Par4 Scc6 Sluc13 Msmr1 Sluc7 Skts9 Scc8 Rmv1,2,3 10 Rmcf Pas4 Rfv1,2 Tgct1 Lyr2 Skts5 Lts1 Ter Sluc10 Rfv3 Pas3 Rrs Sluc14 20 Gct2 Skts3 Hcf1 Scc5 Hcr2 Hcs1 Skts10 Liver Pgct1 Esl1 Hcs6 Scc2 Skts1 Rgv1 30 Lyr1 Par3 Gct3 Msmr2 Gct4 Foc1 Fv4 Pas6 Foc1 Sluc4 Ccs1 Pas7 Skts11 Ril1 Scc4 40 Pas9 (Pas5) Par2 Ots1 Rfv3 Esl1 Hcr1 Pas6 Tlag1 Pas5b Sluc1 Pctr1 Scc1 Hcs5 Skts6 Par1 50 Papg1 Hcs2 Sluc11 Hcs3 Skts7 Sluc9 Sluc12 Sluc6 Sluc3 Lyr3 60 Tlsr2 Fv2 Scc9 Ssic1 Psl1 Skts4 Skts2 Mom1 Pas1 Tli1 Liver Pctr1 Tlsm1 70 Gct1 Skts12 Sluc8 Pctm Rvil1 Pctr2 80 Hcf2 Fv1 Scc7 Hcs7 Ccs2 Pas8 90 Ril3 Skts8 Skin loci Lung loci Hcs4 Scc3 Skts13 Lung + Skin loci Human 20q13
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Microsatellite Haplotyping of outbred spretus
The size of the spretus alleles present in the F1BX mice was determined for microsatellite markers spanning the entire distal region of chr 2. Subsequently, haplotypes were constructed for association studies. A single region of 1-2 cM is shared by the haplotypes that are associated with papilloma resistance. The order of all indicated microsatellites was determined and confirmed by recombinant mice.
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mouse human
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Chromosome 20q13.2 Candidate Locus
HEFL ZNF217 BCAS1 PFD4 Unknown hypothetical protein MCR3 STK6 60S Ribosomal Protein Like PIC1L CYP24 Unknown Q9NTU7 CSTEF1 OVC10-2 Unknown 23 kDA 54.5 Mb 55.5 Mb 56.5 Mb 57.5 Mb Amplicons in human tumors
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AURORA kinase INVOLVED IN CHROMOSOME SEGREGATION AND ANEUPLOIDY:
AMPLIFIED IN HUMAN CANCERS TUMOR STRONG ALLELE WEAK ALLELE “HIGH RISK” “LOW RISK”
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Aurora 2 Ile 31 stimulates tumor growth
Days Number of cells (x104) b Rat1 STK15 Phe-31.1 Rat1 STK15 Phe-31.2 Rat1 STK15 Ile-31.1 Rat1 STK15 Ile-31.2 Phe 31 Rat1 a-Myc c Ile31 Number of cells (x104) Phe 31 Days d 1 2 3 5 rat1 STK15 Phe-31 rat1 STK15 Ile-31 Ile31 Tumor volume cm3 Phe 31 Days Fig. 4 1
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STK15 Phe-31 / hsp70 STK15 Ile-31 / Hsp70 STK15 Phe-31 / UBE2N STK15 Ile-31 / UBE2N Empty vector / UBE2N P53 / T7 Fig. 1 1
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Aurora 2 Phe-31 preferentially binds UBE2N
STK15 Phe-31 Rec UBE2N IB: a-UBE2N beads Total STK15 a IP: a-His STK15 Ile-31 Aurora 2 Phe-31 preferentially binds UBE2N 293 cells IP: a-STK15 10859A lysate 10859A 07038D Total STK15 IB: a-UBE2N Total UBE2N IP: a-STK15 10859A lysate 10859A 07038D Total STK15 IB: a-UBE2N Total UBE2N IP: a-STK15 10859A lysate 10859A 07038D Total STK15 IB: a-UBE2N Total UBE2N Hom Phe-31 Hom Ile-31 Lymphoblastoid cells
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Aurora2 – Phe 31 relocalises UBE2N to the centrosome
DAPI a-HA 293 STK15 Phe-31-HA/UBE2N-Myc a-Myc Merge 10mm DAPI a-HA 293 STK15 Ile-31-HA/UBE2N-Myc a-Myc Merge 10mm Fig. 3 1
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GENETIC CHANGES AT TUMOR MODIFIER GENES?
NORMAL CELL RESISTANCE SUSCEPTIBILITY TUMOR CELL OR DELETION AMPLIFICATION
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D1Mit102 D1Mit33 D4Mit166 D4Mit12 D4Mit160 D5Mit66 D5Mit4 D5Mit78 D5Mit81 D6Mit223 D6Mit4 D6Mit9 D6Mit14 D6Mit15 D7Mit246 D7Mit69 D7Mit12 D7Mit14 D9Mit179 D9Mit9 D9Mit76 D9Mit136 D12Mit186 D12Mit245 D12Mit68 D12Mit5 D16Mit88 D16Mit2 D16Mit102 D17Mit176 D17Mit177 D17Mit139 D17Mit7 D17Mit109 Markers % of chromosomal imbalance 100% 0% 50% 10% 20% 30% 40% 60% 70% 80% 90%
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Array CGH Normal DNA-cy5 Cot-1 Tumor DNA-cy3
Co-hybridize to arrayed clones Data analysis Cot-1 Tumor DNA-cy3
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K-ras Amplification is an Early Event in the Progression of Lung Tumors in K-rasLA2 Mice
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
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Array CGH on Different Size K-ras Lung Tumors
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
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ALLELE-SPECIFIC EXPRESSION OF MOUSE AURORA2
(NIH/Ola x SPRET/Ei) F1 cell line A5 cell line B9 cDNA cDNA cDNA DNA 24 48 DNA 24 48 DNA 24 48 * CT * * * * * NORMAL TUMOR TUMOR (with amplification)
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ALLELE-SPECIFIC AMPLIFICATION OF AURORA KINASE IN COLON CANCER
Fig 5. Colon tumor amplification of Stk6 alleles
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Why are human association studies so weak?
Genetic interactions Some genes have +ve or -ve effects TGFb Growth arrest apoptosis Invasion Metastasis Immunosuppression angiogenesis -ve ve ras Growth arrest transformation -ve +ve myc apoptosis +ve -ve
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IDENTIFICATION OF MOUSE AND HUMAN TUMOR SUSCEPTIBILITY GENES
ASSOCIATION STUDY MOUSE LINKAGE DATA SUSCEPTIBILITY GENE MOUSE :ALLELE-SPECIFIC AMPLIFICATION AND EXPRESSION IN TUMORS HUMAN: ALLELE-SPECIFIC AMPLIFICATION IN TUMORS
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Spiros Linardopoulos- London John de Koning Hiroki Nagase Joe Gray
ACKNOWLEDGEMENTS Mandy Toland Jian-Hua Mao Jin-wei Yuan Jeff Hsu Di Wu Reyno Delrosario Spiros Linardopoulos- London John de Koning Hiroki Nagase Joe Gray Graeme Hodgson June Chan Jing Ma - Harvard Tyler Jacks – MIT Ponder Lab-Cambridge NCI MOUSE MODELS OF HUMAN CANCER CONSORTIUM
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Aurora Kinase and human prostate cancer
Aurora/STK6 SNP Cases Controls OR 95%C.I. Age-adjusted OR (95%CI) TT AT ( ) AA ( ) (P=0.05)
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