Quest for the missing gene:

Tumor suppressor on 3p21?

Tatiana Dracheva

LPG

Introduction

• 3p21 region is often missing in Lung Cancer

• Our previous data (LOH) show a significant

correlation between the loss of the specific

part of 3p21 (D3S1581 - D3S1289) and the

early onset of cancer.

• We limited the region of loss to ~2Mb (48.5-

50.5 Mb)

• Possible telomerase suppressor gene is

located in this region.

Telomerase and Cancer

• Telomeres are the repeated sequences located on both ends of chromosomes in eukaryotes

• In normal cells telomeres are shorten with each cell division• Tumor cells show activation of telomerase, a ribonucleoprotein

enzyme that maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres.

• Tanaka et al suggested the possible telomerase supressor gene located on 3p21 (Tanaka H, Shimizu M, Horikawa I, Kugoh H, Yokota J, Barrett JC, Oshimura M. Evidence for a putative telomerase repressor gene in the 3p14.2-p21.1 region. Genes Chromosomes Cancer. 1998 )

Map of LOH on 3p

LOH at Chr. 3p markers with age of lung cancer onset (20pairs <50, 20pairs>79)

p-valAge<50 Age>79 <50 vs.>79 Position on 3p

D3S2338 36% 18% 0.38 16,824,410D3S3659 29% 22% 1 22,913,782D3S1266 41% 29% 0.71 27,932,355D3S1609 55% 38% 0.65 29,915,040D3S1277 46% 14% 0.1 34,627,486D3S3521 40% 24% 0.45 38,830,323D3S3685 36% 25% 0.7 42,429,724D3S1581 63% 19% 0.03 48,557,902D3S1289 54% 12% 0.02 54,436,829D3S1300 43% 27% 0.45 60,467,242D3S1600 50% 42% 0.72 63,277,204D3S3697 44% 29% 0.47 66,307,532D3S1566 43% 21% 0.42 70,232,419

LOH %

LOH at 3p21 with age of lung cancer onset (19pairs<50, 20pairs>79)

p-valMarker on3p21 Position Mb Age<50 Age>79 <50 vs.>70

d3s1478 46.4 53% 33% 0.236294082d3s1581 48.5 59% 20% 0.025649822d3s3615 50.5 82% 30% 0.016552781d3s3688 51.8 33% 10% 0.193471885d3s1578 53.6 50% 24% 0.114150518d3s1289 54.4 41% 20% 0.197156383d3s3719 55.4 67% 45% 0.279425968d3s1295 57.9 69% 38% 0.11558194

LOH %

Goals of the study

• Narrow down the LOH region on 3p21

• Find candidates for the tumor supressor gene on 3p21

• Find candidates for the telomerase supressor gene

TaqMan® Low Density Array - micro fluidic card

• Uses TaqMan® Gene Expression Products, pre-designed TaqMan probe and primer sets for human, mouse, and rat gene expression research using real-time PCR

• Provides convenient access to 384-assay format without liquid handling robotics

• Screens multiple samples against custom-selected gene panels

• Minimizes reagent consumption

                                                                                         

Schematic representation of the TaqMan principle

A: Primers and probe anneal to the target gene. Fluorescence emission does not occur because the probe is still intact. B: During the extension phase of the PCR reaction, the probe is cleaved by the 5'–3' exonuclease activity of the Taq polymerase, allowing fluorescence emission. FW, forward; RV, reverse; F, fluorophore; Q, quencher dye.

PCR amplification plot

Fluorescence emission is measured continuously during the PCR reaction and Rn (increase in fluorescence emission, from which the background fluorescence signal is subtracted) is plotted against cycle number. The threshold cycle (Ct) is the cycle at

which the fluorescence exceeds a chosen threshold.

Samples/Arrays

Experiment “LOH”:• 39 tumor-normal pairs used for the LOH analysis• After transcription and quality control : 17 pairs + 9tumors

+1normal +4 repeats = 48 arrays we put on the cards . Among 17 pairs: 7 pairs belong to the “young” group, 10 - to the “old”

Experiment “Telomerase repressor”:• 3 samples or RCC23 cell line: 1 parental (active telomerase), 2

with transferred normal human chromosome 3 (repressed teromerase activity)

Card layout: 68 probes from 3p21, 26 control probes from the “162 list”, 18S control, telomerase reverse transcriptase (TERT)

TaqMan® Low Density Array Format

Analysis

• Normalization: 18S Ct extraction or Average of 26 probes from “162 list” extraction.

• Paired T-test Normal vs. Tumor to confirm up- or down-regulation of 26 probes

• Cluster -type representation in order to see LOH region• T-test Tumor Old vs Young• Search for genes absent in parental RCC23 but present in

modified RCC23+3

Paired T-test Normal vs. Tumor to confirm up- or down-regulation of 26 probes

GDF10 growth differentiation factor 10 10q11.22 1 T_down T_down_AffyGDF10 growth differentiation factor 10 10q11.22 1 T_down T_down_AffyCDC2 cell division cycle 2, G1 to S and G2 to M10q21.1 1 T_up T_up_AffyPAK1 p21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast)11q13-q14 1 T_up T_up_AffyVWF von Willebrand factor 12p13.3 1 T_down T_down_AffyPTPRB protein tyrosine phosphatase, receptor type, B12q15-q21 1 T_down T_down_AffyPTPRB protein tyrosine phosphatase, receptor type, B12q15-q21 1 T_down T_down_Affy13CDNA73hypothetical protein CG003 13q12.3 0 T_down T_down_AffyRASGRF1 Ras protein-specific guanine nucleotide-releasing factor 115q24 1 T_down T_down_AffyCDH5 cadherin 5, type 2, VE-cadherin (vascular epithelium)16q22.1 1 T_down T_down_AffyRAMP2 receptor (calcitonin) activity modifying protein 217q12-q21.1 1 T_down T_down_AffyNME1 non-metastatic cells 1, protein (NM23A) expressed in17q21.3 1 T_up T_up_AffyNME1 non-metastatic cells 1, protein (NM23A) expressed in17q21.3 1 T_up T_up_AffySERPINB5serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 518q21.3 0 T_up T_up_AffyCCNE1 cyclin E1 19q12 1 T_up T_up_AffyHCK hemopoietic cell kinase 20q11-q12 0 T_down T_down_AffyCISH cytokine inducible SH2-containing protein3p21.3 ND NDTGFBR2 transforming growth factor, beta receptor II (70/80kDa)3p22 1 T_down T_down_AffyTGFBR2 transforming growth factor, beta receptor II (70/80kDa)3p22 1 T_down T_down_AffyKIAA1102 KIAA1102 protein 4p14 1 T_down T_down_AffyLDB2 LIM domain binding 2 4p16 1 T_down T_down_AffyTERT telomerase reverse transcriptase 5p15.33 0 T_up NDCCNB1 cyclin B1 5q12 1 T_up T_up_AffyTFAP2A transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)6p24 1 T_up T_up_AffyTCF21 transcription factor 21 6pter-qter 1 T_down T_down_AffyTTK TTK protein kinase 6q13-q21 1 T_up T_up_AffyDLC1 deleted in liver cancer 1 8p22 1 T_down T_down_AffyTEK TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal)9p21 1 T_down T_down_AffyTEK TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal)9p21 1 T_down T_down_AffyMAOB monoamine oxidase B Xp11.23 1 T_down T_down_AffyFIGF c-fos induced growth factor (vascular endothelial growth factor D)Xp22.31 1 T_down T_down_AffyKAL1 Kallmann syndrome 1 sequence Xp22.32 1 T_down T_down_Affy

Cluster -type representation of all samples

Cluster -type representation of Tumor-Normal (paired only)

Cluster -type representation in order to see LOH region (Tumors only)

T-test Tumor Old vs Young

NAME ID Average (Yuong-Old) Ttestsolute carrier family 26, member 6 SLC26A6 1.50 0.004cadherin, EGF LAG seven-pass G-type receptor 3 CELSR3 1.54 0.029ubiquitin specific protease 4 (proto-oncogene) USP4 0.51 0.043nicolin 1 NICN1 0.89 0.015ubiquitin-activating enzyme E1-like UBE1L 0.69 0.015macrophage stimulating 1 receptor MST1R 0.90 0.043sema domain, 3F SEMA3F 0.93 0.008hyaluronoglucosaminidase 1 HYAL1 1.85 0.012

RCC23 RCC23+3I RCC+3II Neg.Control ABI_Design_Name Location dCt Fold LOH-noLOH31.8 29.9 30.2 UndeterminedUCN2 urocortin 2 3p21.3 1.82 3.52 IN -1.4726.2 24.9 25.4 UndeterminedCOL7A1 collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive)3p21.1 1.06 2.09 -0.2624.5 24.2 24.2 UndeterminedUQCRC1 ubiquinol-cytochrome c reductase core protein I3p21.3 0.26 1.20 -0.0428.7 27.1 28.4 UndeterminedSLC26A6 solute carrier family 26, member 63p21.3 0.97 1.96 -0.0126.9 25.9 26.3 UndeterminedSLC26A6 solute carrier family 26, member 63p21.3 0.78 1.72 -0.1631.5 30.6 30.7 UndeterminedCELSR3 cadherin, EGF LAG seven-pass G-type receptor 3 (flamingo homolog, Drosophila)3p24.1-p21.2 0.86 1.81 -0.7426.7 27.2 26.6 UndeterminedNCKIPSD NCK interacting protein with SH3 domain3p21 -0.23 0.85 -0.4126.1 24.9 25.3 UndeterminedIHPK2 inositol hexaphosphate kinase 23p21.31 1.03 2.05 -0.2325.1 24.4 24.6 UndeterminedNULL inositol hexaphosphate kinase 23p21.31 0.55 1.47 -0.9826.3 25.0 25.4 UndeterminedPRKAR2A protein kinase, cAMP-dependent, regulatory, type II, alpha3p21.3-p21.2 1.11 2.16 0.1226.7 26.0 26.0 UndeterminedSLC25A20 solute carrier family 25 (carnitine/acylcarnitine translocase), member 203p21.31 0.71 1.63 0.5225.3 24.3 24.4 UndeterminedARIH2 ariadne homolog 2 (Drosophila) 3p21.2-p21.3 0.96 1.94 -0.1825.8 26.5 26.1 UndeterminedPH hypoxia-inducible factor prolyl 4-hydroxylase3p21.31 -0.47 0.72 -0.5928.5 27.7 28.5 UndeterminedWDR6 WD repeat domain 6 3p21.31 0.37 1.29 0.0024.7 24.0 23.6 UndeterminedDKFZP564J0123nuclear protein E3-3 3p21.31 0.84 1.79 -0.0124.3 22.8 23.1 UndeterminedDKFZP564J0123nuclear protein E3-3 3p21.31 1.32 2.50 -0.2027.1 28.1 28.6 UndeterminedIMPDH2 IMP (inosine monophosphate) dehydrogenase 23p21.2 -1.25 0.42 -0.0225.7 24.8 24.9 UndeterminedFLJ20259 FLJ20259 protein 3p21.31 0.93 1.91 0.1224.6 24.8 24.9 UndeterminedQARS glutaminyl-tRNA synthetase 3p21.3-p21.1 -0.28 0.82 -0.1224.9 23.6 23.4 UndeterminedLAMB2 laminin, beta 2 (laminin S) 3p21 1.47 2.76 -0.09

29.0 25.7 25.8 UndeterminedMGC35097 hypothetical protein MGC35097 3p21.31 3.24 9.46 IN 0.72Undetermined 28.5 28.5 UndeterminedLOC339834 hypothetical protein LOC3398343p21.31 >11.5 2896.31 IN 1.17

31.1 31.0 30.7 UndeterminedFLJ43654 FLJ43654 protein 3p21.31 0.20 1.15 1.0625.5 24.9 25.0 UndeterminedUSP4 ubiquitin specific protease 4 (proto-oncogene)3p21.3 0.50 1.41 0.0826.0 25.1 25.3 UndeterminedUSP4 ubiquitin specific protease 4 (proto-oncogene)3p21.3 0.81 1.76 0.2721.6 20.6 20.9 UndeterminedRHOA ras homolog gene family, member A3p21.3 0.89 1.85 -0.3925.9 25.5 25.5 UndeterminedTCTA T-cell leukemia translocation altered gene3p21 0.44 1.36 -0.8430.5 29.8 30.3 UndeterminedAMT aminomethyltransferase (glycine cleavage system protein T)3p21.2-p21.1 0.52 1.43 -0.2527.0 26.9 26.9 UndeterminedNICN1 nicolin 1 3p21.31 0.06 1.04 0.1924.1 23.4 23.1 UndeterminedDAG1 dystroglycan 1 (dystrophin-associated glycoprotein 1)3p21 0.82 1.77 0.2535.8 35.0 36.0 UndeterminedBSN bassoon (presynaptic cytomatrix protein)3p21.31 0.27 1.20 -1.8225.9 25.9 25.8 UndeterminedAPEH N-acylaminoacyl-peptide hydrolase3p21.31 -0.01 0.99 -0.0728.9 29.7 29.5 UndeterminedMST1 macrophage stimulating 1 (hepatocyte growth factor-like)3p21 -0.63 0.64 -0.5325.9 25.8 26.1 UndeterminedRNF123 ring finger protein 123 3p24.3 -0.02 0.99 -0.3528.5 27.1 27.3 UndeterminedGMPPB GDP-mannose pyrophosphorylase B3p21.31 1.27 2.42 0.2025.8 24.8 25.1 UndeterminedIHPK1 inositol hexaphosphate kinase 13p21.31 0.82 1.77 -0.0332.5 30.8 31.0 UndeterminedLOC389119 similar to RIKEN cDNA 6530418L213p21.31 1.54 2.90 0.4128.1 28.0 27.8 UndeterminedUBE1L ubiquitin-activating enzyme E1-like3p21 0.25 1.19 0.35

Candidates for the telomerase supressor

Results/Future work

• We were not able to further limit the LOH region with the Q-PCR method. ( Tumor samples contain too much normal. Small sample set.)

• We found candidates genes for the telomerase supressor on 3p21.

• We are sequencing candidate genes looking for the mutations. • Our collaborators are introducing those genes to the RCC23 in

order to see if one of the indeed supress telomerase.

Aknolegments

• Jin Jen

• Felicia Mann

• Daoud Meerzaman

• Izumi Horikawa

• Carl Barrett

• Steve Hoffmann (NIDDK)

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