Expression Profiles of Multiple Independent Cadmium‐ and Arsenite‐ Transformed Human Urothelial Cells: Specific Gene Signatures in a Sea of Heterogeneity. Scott Garrett Department of Pathology School of Medicine and Health Sciences University of North Dakota Grand Forks ND Problems in Cancer Research

• Environmental causation of cancer – President’s Cancer Panel 2008‐09, Reducing Environmental Cancer Risk, “Environmentally caused cancer are ggyrossly underestimated..” • Desperate need for diagnostic and prognostic markers of cancer. – Screening for common cancers – Prognostic markers for predicting cancer that need aggressive treatments from those that don’t • Also, need to understand carcinogenesis for prevention and treatment. Bladder Cancer Has a High Environmental Etiology • Aromatic amine dyes in bladder cancer was the first environmental etiology in cancer. • Nearly 50% of BC thought to be caused by cigarette smoking • Suscept ibility due to concentration of carcinogens in urine. Arsenite (As+3) & Cadmium (Cd+2)

• Arsenite in drinking water linked to BC in South Asia • Confirmed in animal models • Arsenite, 1St in priority as a toxic substance (ATSDR). • Cadmium has also been linked to BC, albeit much weaker, but: – Cigarette smoking is a major source of cadmium Overall Experimental Approach

Long‐Term Cultured Cells Transformed Cells Metal Exposure

Tranf & assess Discover Induced or tumor behavior Transfect or Repressed Gene knockdown & assess cell behavior Gene X

Human Tissues

Assess correlation to tumor grade Mechanism of gene and clinical outcome expression change General Experimental Approach

Athymic Nude Mouse Cell Culture Models In Vivo Tumor Model

Biopsy Specimens (formalin fixed PET) Immunohistochemistry Attached Clinical Outcome Bladder Model

• UROtsa Cells – Human urothelial cell line – Details Metal Transformation of UROtsa as a modldel of Bllddadder Carcinogenesis • Culture in 1 µM Cd+2 or As+3 (several months continuous culture) • Test for anchorage independent growth

Soft Agar Assay

• IjInjec t s.c itinto athym icnude mice Global Gene Expression Analysis to Assess Tumors Array Hybridization

SHG003‐U133_Plus_2_Unfiltered.TXT Array Analysis

• Thousands of • Had on our minds • KRT1666:KRT6 (6A )

Human tumors for KRT6

Heterotransplants for KRT6

Environ Health Perspect (2008) 116:434–44 Main Question

• During transformation of cell lines by a carcinogen, how reproducible are the gene expression changes? – Important because of heterogeneity of cancer. • Implications to applications of cell culture based studies • Are there carcinogen based expression changes? – Give clues to mechanism of transformation – Source of biomarkers of carcinogens Experimental Setup Note on Metal Characteristics

These two metals are +2 • Cd very divergent and excellent to test in this – No redox reaction system. – Binds SH groups – Accumulative toxin (especially kidney) • As+3 – Arsenic considered a metalloid – Mono & Dimethyl‐species – OidiOxidize to AVAsV (bl(resemble phhhtosphate in chhit)emistry) – As+3 exists as trihydroxy neutral species at pH 7 – Relatively low in total accumulation Transformed UROtsa Isolates

As+3: Five isolates

Cd+2: Six Isolates

Arrays ‐ three control (non‐transformed) ‐ one of each FDR and the 5% Problem

• In statistical analysis, p‐value of 0.05: 5% probability of the null hypothesis being true. • But in repetitive measurements have problem • 5% of 23,000 genes in genome = 1150 false pos. • RllReally want to conttlrol for 5% fflalse discovery – Thus the false discovery rate is calculated. – Requires very small p‐values 0.001 to E‐09 As+3‐Heat Map Note on Genes versus Probes

• 133 Plus 2.0 is slightly redundant – 45,000 probes – Have about 23,000 genes • About 2 fold redundancy • Multiple probes measure different exons – Complexity of probe changes can also reflect alternative splicing. • Fold 2 Change and DEGs – DEG (Differentially Expressed Genes) is a statistical entity Alterations in Gene Expression in As+3‐ Transfdformed Isolates

Number of probes with fold 2 change or greater Principal Component Analysis

Red: Parental Controls

Blue: As+3‐Transformed As+3‐ & Cd+2‐Transformants Heat Map As+3‐Cd+2 Venn Comparison

Ave of leaving one Cd+2‐ Isolate out: 346 induced, 251 repressed

Cd+2‐Isolates have many more repressed gene overlaps than that of As+3 As+3 & Cd+2 Principal Component Analysis Global Gene Overlap

Probe Redundancy Removed p << 10E‐6 for gene overlap

Overlap by random chance estimated < 20 genes Are Gene Expression Changes Random? • Single Isolate: 3871 • Overlap of two isolates: 1624 • Three isolates: 759 • Four: 414 • All five: 221 • It will probably decrease with more isolates. • Would there be any gene overlap left over with many isolates? Or will it be zero? Mathematical Modeling Gene Overlap with Increasing Number of Isolates

4 parameter Linear Model Cadmium logistic model Induced gene overlap stabilizes at about 230 genes at higher Induced numbers of isolates.

Repressed gene overlap stabilizes Repressed at 198 The Linear Model • Considers random variation and statistics • Log(y‐t)=α + βx + ε – X= number of isolates – Y= number of genes – t= constant integer – α and β are pp,arameters, ε is the random error. • Y converges to t as number of isolates (x) increases. • Using y= 285 at x=6, t ranges (0 to 284) • R2 can be calculated for each line at every value t • t=233 yields best R2 value (0.993) • Minimum no of isolates: 14 to give CI <1 Mathematical Modeling Gene Overlap with Increasing Number of Isolates 4 parameter logistic model Arsenite

Stabilizes at 265 Induced genes

Stabilizes at Repressed 63 genes What Does This Mean?

• Thousands of gene expression changes in transformed isolates. • Small but statistically significant number of gene expression changes common to all isolates. • Hypothesis: this reflects the “driver”‐ ”””passenger” mutation concept of carcinogenesis. Gene Validation using PCR‐Array

• SA Biosciences • Custom primers targeted to exons near to array target sequence. • All assays were valida te d • Normalized to geometric mean of five reference genes (house‐keeping) Gene Validation using PCR‐Array

• SA Biosciences • Custom primers designed to nearest exons of array target sequence • 338 genes, five house keeping genes – All on one 384 tray. – One sample per tray, 36 trays in all. Validation Heat Map

24 out of 338 failed to validate (7.1 %) KEGG Pathways As+3 Induced Repressed Pathway P‐Value Pathway P‐Value Glyco lysi s / Gluconeogenesi s 0040.04 Thyroid cancer 0.025 Biosynthesis of phenylpropanoids <0.001 Biosynthesis of terpenoids and steroids <0.001 Apoptosis 0.015 Biosynthesis of alkaloids derived from shikimate pathway <0.001 Endocytosis <0.001 Biosynthesis of alkaloids derived from ornithine, lysine and Acute myeloid leukemia 0.045 nicotinic acid <0.001 Antigen processing and presentation <0.001 Biosynthesis of alkaloids derived from histidine and purine 0.015 Biosynthesis of alkaloids derived from terpenoid and Type I diabetes mellitus <0.001 polyketide <0.001 Autoimmune thyroid disease <0.001 Alanine, aspartate and glutamate metabolism <0.001 Allograft rejection <0.001 Arginine and proline metabolism 0.005 Graft-versus-host disease <0.001 Nitrogen metabolism 0.035 Primary immunodeficiency <0.001 Cysteine and methionine metabolism <0.001 Fructose and mannose metabolism 0.03 Galactose metabolism 0.015 Starch and sucrose metabolism 0.015 Streptomycin biosynthesis 0 Selenoamino acid metabolism <0.001 Glyoxylate and dicarboxylate metabolism 0.015 Tropane, piperidine and pyridine alkaloid biosynthesis 0.01 D-Glutamine and D-glutamate metabolism 0.03 Glyc ine, ser ine an d threon ine me ta bo lism 0.005 Aminophosphonate metabolism 0.025 Taurine and hypotaurine metabolism 0.02 KEGG Pathway Cd+2

Induced Repressed Pathway P‐value Pathway P‐Value Alanine, aspartate and glutamate metabolism 0.03 Jak-STAT signaling pathway <0.001 Amino sugar and nucleotide sugar metabolism 0.015 beta-Alanine metabolism 0.03 Cytokine-cytokine receptor interaction <0.001 DNA replication <0.001 Apoptosis <0.001 Fatty acid biosynthesis <0.001 Fructose and mannose metabolism 0.04 Wnt signaling pathway <0.001 Galactose metabolism 0.005 Tyrosine metabolism <0.001 Glycolysis / Gluconeogenesis 0.045 Pentose and glucuronate interconversions <0.001 Glycosphingolipid biosynthesis - lacto and neolacto series 0.015 Cytosolic DNA-sensing pathway 0.005 Hematopoietic cell lineage 0.02 Chemokine signaling pathway 0.025 Nucleotide excision repair 0.015 Pentose phosphate pathway 0.005 BllliBasal cell carcinoma 0.025 Propanoate metabolism 0.03 Primary immunodeficiency 0.025 Purine metabolism 0.04 Pyrimidine metabolism 0.03 Hedgehog signaling pathway 0.025 metabolism 0.04 Metabolism of xenobiotics byyy cytochrome P450 0.045 Starch and sucrose metabolism <0.001 RIG-I-like receptor signaling pathway 0.045 KEGG Pathways Both As & Cd

Induced RdRepressed Pathway P‐ P‐Value Pathway P‐Value P‐Value Value Cd+2 As+3 Cd+2 As+3 Alan ine, aspartate andld glutamate Apoptosis 0. 015 <0.001 metabolism <0.001 0.03 Primary immunodeficiency <0.001 0.025 Fructose and mannose metabolism 0.03 0.04

GlGalactose metablibolism 0010.015 0000.005

Glycolysis / Gluconeogenesis 0.04 0.045

Mismatch repair 0.03 0.015

Starch and sucrose metabolism 0.015 <0.001 As+3‐Induced Specific Genes

Symbol Gene Name Fold Chg FDR Validated CPA6 carboxypeptidase A6 32.14 0.0001Yes DOCK11 dedicator of cytokinesis 11 18.51 0.0137Yes C17orf96 17 open reading frame 96 13.97 0.0252Yes HRASLS HRAS‐like suppressor 13.89 0.0030Yes actin related 2/3 complex, subunit 4, ARPC4 20kDa 12.49 0.0145Yes LOC729296 similar to hCG2017976 12.13 0.0429Yes FREM2 FRAS1 related protein 2 10.17 0.0453Yes protein kinase (cAMP‐dependent, catalytic) PKIA inhibitor alpha 9.83 0.0303Yes TFPI2 tissue factor pathway inhibitor 2 8.46 0.0428Yes RS1 retinoschisin 1 8.43 0.0139No disabled homolog 2, mitogen‐responsive DAB2 phosphoprotein (Drosophila) 8.38 0.0367Yes PDE9A phosphodiesterase 9A 8.03 0.0497Yes solute carrier family 2 (facilitated glucose SLC2A13 transporter), member 13 7.62 0.0253Yes PIK3AP1 phosphoinositide‐3‐kinase adaptor protein 1 7.39 0.0379Yes SIGLEC8 sialic acid binding Ig‐like lectin 8 7.12 0.0348No LPHN1 1 6.41 0.0320Yes PGF placental growth factor 6.34 0.0444Yes LOX 5.71 0.0160Yes DCLK1 doublecortin‐like kinase 1 5.49 0.0207Yes CSPG4 chondroitin sulfate proteoglycan 4 5.12 0.0356Yes ANG angiogenin, ribonuclease, RNase A family, 5 5.06 0.0347Yes ZBTB7C zinc finger and BTB domain containing 7C 5.03 0.0489Yes DPYSL2 dihydropyrimidinase‐like 2 4.56 0.0108Yes ATP6V0E2 ATPase, H+ transporting V0 subunit e2 4.08 0.0167Yes C14orf128 chromosome 14 open reading frame 128 3.87 0.0486Yes PPIL2 peptidylprolyl (cyclophilin)‐like 2 3.77 0.0433N/A As+3 Repressed Specific Genes

Fold Symbol Gene Name Change FDR Validated MUC4 mucin 4, cell surface associated 0.0665 0.0488 Yes MAL mal, T‐cell differentiation protein 0.0942 0.0372 Yes RABGAP1L RAB GTPase activating protein 1‐like 0.2197 0.0170 Yes XRN1 5'‐3' exoribonuclease 1 0.2829 0.0253 Yes AQP3 aquaporin 3 (Gill blood group) 0.2839 0.0314 Yes HLA‐Gmajor histocompatibility complex, class I, G 0.2852 0.0245 No SCAMP4 secretory carrier membrane protein 4 0.2872 0.0389 No C5orf41 chromosome 5 open reading frame 41 0.2875 0.0427 Yes REEP3 receptor accessory protein 3 0.2966 0.0234 Yes C1R complement component 1, r subcomponent 0.3032 0.0373 Yes KHSRP KH‐type splicing regulatory protein 0.3212 0.0499 N/A EPB41L1 erythrocyte membrane protein band 4.1‐like 1 0.3268 0.0485 N/A RABGAP1L RAB GTPase activating protein 1‐like 0.3311 0.0366 N/A KIAA1618 KIAA1618 0.3431 0.0477 N/A

QKI quaking homolog, KH domain RNA binding (mouse) 0.3636 0.0398 N/A HLA‐Gmajor histocompatibility complex, class I, G 0.3646 0.0462 N/A RIF1 RAP1 interacting factor homolog (yeast) 0.3812 0.0194 N/A FAM46A family with sequence similarity 46, member A 0.3821 0.0484 N/A

LOC286434 hypothetical protein LOC286434 0.3840 0.0365 N/A C5orf41 chromosome 5 open reading frame 41 0.3858 0.0375 N/A RNF19B ring finger protitein 19B 0.3907 0.0368 N/A C6orf106 open reading frame 106 0.3926 0.0272 N/A SRGAP1 SLIT‐ROBO Rho GTPase activating protein 1 0.3933 0.0488 N/A intercellular adhesion molecule 4 (Landsteiner‐ ICAM4 Wiener blood group) 0.3938 0.0380 N/A Cd+2 Induced Specific Genes

Fold Symbol Gene Name Change FDR Validated EEF1A2 Eukaryotic translation elongation factor 1 alpha 2 62.87 0.00 Yes TLR4 Toll‐like receptor 4 54.50 0.00 Yes FN1 Fibronectin 1 42.43 0.00 Yes LIMCH1 LIM and calponin homology domains 1 41.18 0.00 Yes Chemokine (C‐X‐C motif) ligand 1 (melanoma growth CXCL1 stimulating activity, alpha) 32.64 0.01 Yes LPHN2 29.81 0.00 Yes TSPAN12 Tetraspanin 12 29.26 0.00 Yes PTN Pleiotrophin 28.90 0.00 Yes TMEM56 Transmembrane protein 56 28.79 0.00 Yes UGT8 UDP 8 28.33 0.00 Yes UGT8 UDP gllflycosyltransferase 8 27.43 0000.00 N/A SHOX2 Short stature homeobox 2 23.96 0.00 Yes EPHA4 EPH receptor A4 23.80 0.01 Yes FN1 Fibronectin 1 22.32 0.00 N/A RUNDC3B RUN domain containing 3B 22.21 0.00 Yes CNTN1 Contactin 1 9.98 0.02 Yes GPC4 Glypican 4 9.85 0.00 Yes Serpin peptidase inhibitor, clade A (alpha‐1 SERPINA1 antiproteinase, antitrypsin), member 1 9.72 0.01 Yes TLR4 Toll‐like receptor 4 9.47 0.01 N/A NTF3 Neurotrophin 3 9.42 0.00 Yes FLJ42709 Hypothetical LOC441094 9.11 0.00 Yes Transmembrane and tetratricopeptide repeat containing TMTC1 1 9.01 0.01 N/A SLITRK6 SLIT and NTRK‐like family, member 6 8.94 0.00 Yes NA NA 8.84 0.04 N/A NA NA 8.74 0.00 N/A HRASLS HRAS‐like suppressor 8.64 0.01 Yes TNC C 8.38 0.00 Yes CTHRC1 triple helix repeat containing 1 8358.35 0000.00 Yes Pleckstrin homology domain containing, family A PLEKHA5 member 5 8.22 0.02 Yes DHX34 DEAH (Asp‐Glu‐Ala‐His) box polypeptide 34 7.95 0.00 N/A EPB41L2 Erythrocyte membrane protein band 4.1‐like 2 7.90 0.00 N/A SOX18 SRY (sex determining region Y)‐box 18 7.87 0.01 Yes Cd+2 Repressed Specific Genes

Fold Symbol Gene Name Change FDR Validated ISG20 Interferon stimulated exonuclease gene 20kDa 0.0259 0.0001 N/A RSAD2 Radical S‐adenosyl methionine domain containing 2 0.0295 0.0022 Yes IL20RB ItInterl eu kin 20 receptor btbeta 0.0345 0.0000 Yes HS3ST3B1 Heparan sulfate (glucosamine) 3‐O‐sulfotransferase 3B1 0.0367 0.0030 N/A RSAD2 Radical S‐adenosyl methionine domain containing 2 0.0373 0.0019 N/A TSPYL5 TSPY‐like 5 0.0423 0.0039 Yes Solute carrier family 16, member 4 (monocarboxylic acid SLC16A4 transporter 5) 0.0463 0.0019 Yes PSG5 Pregnancy specific beta‐1‐glycoprotein 5 0.0465 0.0005 Yes FYB FYN binding protein (FYB‐120/130) 0.0529 0.0027 Yes SLC15A3 Solute carrier family 15, member 3 0.0578 0.0004 Yes NA NA 0.0580 0.0054 N/A BLMH Bleomycin 0.0666 0.0046 Yes IL20RA Interleukin 20 receptor, alpha 0.0778 0.0049 Yes C1orf161 open reading frame 161 0.0929 0.0123 Yes NA NA 0.0939 0.0007 N/A ZNF703 Zinc finger protein 703 0.1006 0.0006 N/A FABP4 Fatty acid binding protein 4, adipocyte 0.1008 0.0074 Yes RASGEF1A RasGEF domain family, member 1A 0.1013 0.0089 Yes KRT4 Keratin 4 0.1076 0.0141 Yes MAP2 Microtubule‐associated protein 2 0.1094 0.0352 Yes IFIT2 Interferon‐induced protein with tetratricopeptide repeats 2 0.1114 0.0027 Yes IFIT2 Interferon‐induced protein with tetratricopeptide repeats 2 0.1170 0.0012 N/A SERPINB2 Serpin peptidase inhibitor, clade B (ovalbumin), member 2 0.1194 0.0135 Yes FKBP5 FK506 binding protein 5 0.1228 0.0082 Yes NA NA 0.1262 0.0082 N/A ZBP1 Z‐DNA binding protein 1 0.1282 0.0127 Yes NA NA 0.1303 0.0024 N/A NAP1L2 Nucleosome assembly protein 1‐like 2 0.1308 0.0388 Yes PSG3 Pregnancy specific beta‐1‐glycoprotein 3 0.1395 0.0037 Yes IQCA1 IQ motif containing with AAA domain 1 0.1401 0.0299 Yes TSLP Thymic stromal lymphopoietin 0.1406 0.0105 Yes Common Induced (As+3 & Cd+2) Gene As Fold Cd Fold

Symbol Gene Name Change FDR Change FDR Validation CDH2 Cadherin 2, type 1, N‐cadherin (neuronal) 56.9 0.0121 34.98 0.0007 Yes UGT8 UDP glycosyltransferase 8 45.07 0.0004 28.33 1.65E‐05 Yes LIMCH1 LIM and calponin homology domains 1 23.72 0.0221 41.18 6.01E‐05 Yes LPHN2 Latrophilin 2 32.19 0.0499 29.81 0.0013 Yes TSPAN12 Tetraspanin 12 26.27 0.0312 29.26 0.0019 Yes NR2F1 Nuclear receptor subfamily 2, group F, member 1 22.64 0.0498 6.02 0.0257 Yes CNTN1 Contactin 1 7.22 0.0155 9.98 0.0174 Yes ITM2C Integral membrane protein 2C 9.37 0.0242 6.97 0.0048 Yes DSEL Dermatan sulfate epimerase‐like 6.92 0.0231 7.05 0.0004 No SLITRK6 SLIT and NTRK‐like family, member 6 4.98 0.0398 8.94 0.0005 Yes EFNB2 Ephrin‐B2 7.61 0.0308 5.49 0.0014 Yes TFDP1 Transcription factor Dp‐1 7.95 0.0402 4.12 0.021 Yes Solute carrier family 16, member 14 (monocarboxylic acid SLC16A14 transporter 14) 7.17 0.0189 4.08 0.0046 Yes CALCRL ‐like 4.33 0.0294 6.8 0.0267 Yes EGLN3 Egl nine homolog 3 (C. elegans) 7.1 0.0491 3.84 0.0173 Yes TCF4 Transcription factor 4 5285.28 0.0326 5415.41 0.0108 Yes ASS1 Argininosuccinate synthase 1 5.29 0.0348 5.06 2.53E‐05 Yes DPM3 Dolichyl‐phosphate polypeptide 3 5.21 0.0142 5.07 0.0012 Yes DDHD1 DDHD domain containing 1 5.35 0.0137 4.86 0.001 Yes FZD3 homolog 3 (Drosophila) 6.82 0.0448 3.29 0.0231 Yes ODF2 Outer dense fiber of sperm tails 2 6.72 0.0215 3.24 0.0393 No PCOLCE2 Procollagen C‐endopeptidase enhancer 2 4974.97 0.0283 4914.91 4.70E ‐05 Yes GLT8D4 Glucoside 2 4.71 0.0273 5 0.0017 Yes TEX15 Testis expressed 15 3.98 0.0124 5.71 9.95E‐05 Yes EPB41L2 Erythrocyte membrane protein band 4.1‐like 2 3.11 0.0357 5.96 0.0005 Yes ZIC2 Zic family member 2 (odd‐paired homolog, Drosophila) 5.5 0.0315 3.5 0.0004 Yes TRIB2 Tribbles homolog 2 (Drosophila) 5.52 0.0262 3.18 0.0264 Yes MYB v‐Myb myeloblastosis viral oncogene homolog (avian) 3.6 0.0398 4.9 0.001 Yes SLC39A8 Solute carrier family 39 (zinc transporter), member 8 4.52 0.0497 3.92 0.0097 Yes IL27RA Interleukin 27 receptor, alpha 5.81 0.0136 2.55 0.017 Yes TWIST1 Twist homolog 1 (Drosophila) 4.62 0.0202 3.49 0.0143 Yes PRICKLE1 Prickle homolog 1 (Drosophila) 4.62 0.0194 3.43 0.0054 Yes KIAA0114 KIAA0114 4.55 0.0448 3.26 0.0059 Yes TGFB2 Transforming growth factor, beta 2 4.9 0.0423 2.47 0.0139 Yes Common Repressed(As+3 & Cd+2) Gene Fold Fold Symbol Gene Name Change FDR Change FDR Validation SPARC Secreted protein, acidic, cysteine‐rich (osteonectin) 0.002 0.0315 0.0075 0.0028 Yes PSG5 Pregnancy specific beta‐1‐glycoprotein 5 0.0161 0.0127 0.0465 0.0005 Yes FYB FYN binding protein 0.0727 0.0376 0.0529 0.0027 Yes SERPINB2 Serpin peptidase inhibitor, clade B (ovalbumin), member 2 0.0563 0.0452 0.1194 0.0135 Yes ZBP1 Z‐DNA binding protein 1 0140.14 0.0404 0.1282 0.0127 Yes MAP2 Microtubule‐associated protein 2 0.2253 0.0445 0.1094 0.0352 Yes ZFP36L2 Zinc finger protein 36, C3H type‐like 2 0.1623 0.0282 0.2255 0.0072 Yes HERC6 Hect domain and RLD 6 0.1528 0.0365 0.2503 0.0176 Yes KIAA0892 KIAA0892 0.219 0.0484 0.2148 0.0212 Yes CA12 Carbonic anhydrase XII 0.2317 0.0233 0.2111 0.0188 Yes ISG20 Interferon stimulated exonuclease gene 20kDa 0.2422 0.0476 0.2106 0.003 Yes RBPMS RNA bin ding protein wihith muliltip le splic ing 0.200 1 0.0398 0.283 4 0000.001Yes THBD Thrombomodulin 0.2716 0.0467 0.2286 0.0062 Yes ETV7 Ets variant 7 0.1769 0.0491 0.3246 0.0079 Yes IL1RN Interleukin 1 receptor antagonist 0.2108 0.0475 0.3111 0.0128 Yes COL12A1 Collagen, type XII, alpha 1 0.3272 0.0499 0.2474 0.0409 Yes Solute carrier family 6 (neurotransmitter transporter, glycine), SLC6A9 member 9 0.2475 0.0486 0.372 0.0062 Yes C6orf145 Chromosome 6 open reading frame 145 0.2831 0.0137 0.3467 0.0035 Yes Solute carrier family 1 (glutamate/neutral amino acid SLC1A4 transporter), member 4 0.3085 0.0234 0.3609 0.0019 Yes

PPP1R12B Protein phosphatase 1, regulatory (inhibitor) subunit 12B 0.2974 0.0346 0.3853 0.0088 Yes CFLAR CASP8 and FADD‐like apoptosis regulator 0.3299 0.0284 0.3617 0.0022 Yes NFAT5 Nuclear factor of activated T‐cells 5, tonicity‐responsive 0.2167 0.0499 0.488 0.0308 No TYMP Thymidine 0.3701 0.0346 0.3501 0.0034 Yes UDP‐Gal:betaGlcNAc beta 1,4‐ , B4GALT4 polypeptide 4 0.3306 0.0426 0.4169 0.001 Yes PHACTR2 Phosphatase and actin regulator 2 0.3541 0.0448 0.3934 0.0007 Yes NR2F2 Nuclear receptor subfamily 2, group F, member 2 0.2517 0.0369 0.4985 0.0242 Yes OAF OAF homolog (Drosophila) 0.3349 0.0368 0.4154 0.0067 Yes WDR66 WD repeat domain 66 0.3067 0.0447 0.4665 0.0107 No CCND1 Cyclin D1 0.385 0.0138 0.4102 0.0094 Yes RPS27L Ribosomal protein S27‐like 0.4955 0.0316 0.3008 0.003 Yes C1orf116 Chromosome 1 open reading frame 116 0.3838 0.0284 0.4321 0.0091 Yes INO80D INO80 complex subunit D 0.4203 0.0344 0.4005 0.0007 Yes Current Markers in Pursuit

• SPARC • SLC39A8 • ENO2 • MT‐3 • KRT6 SPARC

Normal Human Bladder

Stromal Staining Only in Heterotransplants

Lack of staining in low grade tumor

Toxicol Lett (2010) 199:166‐172 Cadmium exposure represses SPARC in parent Enolase‐2

As+3‐Transformed Normal Bladder

Toxicol Lett (2012) 212:66‐74 MT‐3 Not Found on Arrays But..

MT‐3 Not Expressed in the Bladder

MTMT‐‐33 Immunostain Protein Increases with the Grade of the Tumor

Cd+2‐Transformed Sens MA et al., (2000) Environ Perspect 108:413‐8 New Approaches

• Global DNA Methylation • Global Histone Modifications • GiGenomic SiSequencing • Gene Expression Changes in Formalin‐Fixed, Paraffin embedded Tissues • Mechanistic Studies of Gene Expression Changes. – Have intermediates frozen down. Funding Sources

• NORTH DAKOTA INBRE: HEALTH AND THE ENVIRONMENT – P20GM103442

• SEERING UNDERGRADUATE INTEREST IN ENVIRONMENTAL HEALTH SCIENCES AT THE UNIVERSITY – ST5R25ES016250

• METALLOTHIONEIN ISOFORM 3 AS A URINARY MARKER FOR BLADDER CANCER – R01ES015100 Collaborators

• BioInformatics: Kurt Zhang • Cell Culture Models & KRT6: Seema Somji • Experimental Pathology: Xudong Zhou • INBRE Administrative: Don Sens • PthlPathology: Mary Ann Sens • SPARC and Confocal: Jane Dunlevy • SPARC: Jenna Larson • ENO2: Maureen Soh