CANCER GENOMICS & PROTEOMICS 14 : 69-74 (2017) doi:10.21873/cgp.20019

Customized Array Comparative Genomic Hybridization Analysis of 25 -encoding in Colorectal Tissues IZABELA LACZMANSKA 1, PAWEL SKIBA 1, PAWEL KARPINSKI 1, MAREK BEBENEK 2 and MARIA M. SASIADEK 1

1Genetics Department, Wroclaw Medical University, Wroclaw, Poland; 21st Department of Surgical Oncology, Lower Silesian Oncology Center, Wroclaw, Poland

Abstract. Background/Aim: Molecular mechanisms of functioning mainly as tumour suppressor genes has been alterations in tyrosine (PTPs) genes in described in many , such as colorectal, gastric, cancer have been previously described and include breast, ovarian, cervical, pancreatic, prostate, thyroid and chromosomal aberrations, , and epigenetic many others (2). Colorectal cancer (CRC) is one of the silencing. However, little is known about small intragenic common causes of cancer-related deaths and therefore is gains and losses that may lead to either changes in once of the most intensively studied types (1, 3). Thus, it expression or activity and even loss of protein has been proven that genetic pathways that are altered function. Materials and Methods: The aim of this study was during CRC development and progression involve genes to investigate 25 phosphatase genes using customized array regulated to and differentiation, and comparative genomic hybridization in 16 sporadic colorectal other processes crucial for cell homeostasis (2). Major cancer tissues. Results: The analysis revealed two unique genetic mechanisms underlying either hereditary or small alterations: of 2 kb in PTPN14 intron 1 and of 1 kb in sporadic CRC are well known, but intensive studies on PTPRJ intron 1. We also found gains and losses of whole molecular mechanisms influencing CRC development and PTPs gene sequences covered by large progression are still ongoing. CRC is characterized by aberrations. Conclusion: In our preliminary studies using chromosomal instability, loss of heterozygosity, high-resolution custom microarray we confirmed that PTPs microsatellite instability, multiple gene mutations and also are frequently subjected to whole-gene rearrangements in epigenetic alterations, making CRC an extremely colorectal cancer, and we revealed that non-polymorphic heterogeneous disease (3). intragenic changes are rare. Protein tyrosine phosphatases were also revealed as being altered in CRCs. While searching for molecular mechanisms The role of protein tyrosine phosphatases (PTPs) is widely of these alterations, chromosomal aberrations (deletions/ described in both normal and cancer tissues. PTPs play an duplications of whole regions including PTPs), gene important role in many cellular pathways, co-acting with mutations (small intragenic deletions, point mutations), and protein via phosphorylation and dephosphorylation epigenetic deregulation (promoter hypermethylation) have in activation and deactivation of a variety of . been observed (1, 2, 4-6). Therefore, their impact on cancer development and The PTP superfamily is divided into four families progression seems undeniable (1). To date the role of PTPs depending on the amino acid sequence in their catalytic domain (1). Receptor PTPs (RPTPs), non-receptor PTPs (NRPTPs) and dual specificity phosphatases (DUSPs) that can dephosphorylate not only tyrosine but also serine and This article is freely accessible online. threonine residues belong to the largest class (class I), possessing a cysteine residue in their catalytic centre. Correspondence to: Izabela Laczmanska, Genetics Department, Moreover, cycle 14 (Cdc14) (serine and Medical University, Wroclaw, Poland. Marcinkowskiego 1, 50-368 Wroclaw, Poland. Tel: +48 0717841258, Fax: +48 0717840063, e- threonine specific), MAP phosphatases (MPKs), mail: [email protected] slingshots (serine specific) as well as phosphatase and tensin homologs (PTENs) and (both D3- Key Words: aCGH, colorectal cancer, protein tyrosine phosphatases. phosphoinositide specific) are also included in this class (1).

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Cysteine-based classes include class II, constituting the Table I. Molecular characteristic of examined tissues. smallest class with low molecular weight protein tyrosine Tissue Age Gender Tumor MSI BRAF KRAS Epigenotype phosphatases (LMW PTPs), and class III, consisting of location mutation HME/LME tyrosine/threonine specific phosphatases for cell division cycle 25 () enzymes. Aspartate-based class IV with 1 57 F P MSS + - HME tyrosine and serine phosphatase activity contains Eyes absent 2 70 M D MSS - + LME homolog (Eya) enzymes (1). 3 62 F P MSS + - HME 4 69 M P MSS - - LME Array comparative genomic hybridization (aCGH) is a 5 61 M P MSS - + LME molecular cytogenetic technique that is very useful and 6 67 F P MSS - + LME widely applied in the examination of genome imbalances. 7 53 F D MSS - + LME The possibility of construction of the unique aCGH design 8 72 M P MSS - + LME makes the aCGH technique a precise tool for searching for 9 64 F D MSS - - LME 10 78 M D MSS - - LME deletions and duplications of specific genome parts such as 11 52 M D MSS - - LME chromosomal regions, particular genes or exons/introns. 12 71 M P MSI - + LME Moreover, aCGH allows for discovery of mosaic genetic 13 80 M P MSS - + LME alterations if these alterations are present in at least 20% of 14 65 F D MSS - + LME tested cells, that is crucial for cancer analysis in which copy 15 59 F P MSS - + LME 16 55 F P MSS - - LME number alternations are often present in only a fraction of examined cells (7). F, Female; M, male; P, proximal; D, distal; MSS, microsatellite stability; The aim of this study was to investigate 25 protein tyrosine MSI, microsatellite instability; HME, high methylation epigenotype; phosphatases belonging to class I. We focused on studying LME, low methylation epigenotype. receptor-like protein tyrosine phosphatases, PTEN and three DUSPs. In this group, four genes, PTPRT, PTPRZ1, PTPRQ and PTPRM , have been examined by our group recently and promoter hypermethylation along with copy number changing DNA isolation. DNA was isolated from cancer tissues using a of chromosomal regions encoding PTPRT, PTPRZ1, PTPRQ QIAamp DNA mini (Qiagen, Hilden, Germany) following the and PTPRM has been revealed (5). recommended protocol. All tissues were examined histopathologically In the present study the aCGH custom array, covering 25 as described earlier (4, 5). Concentration and purity of DNA samples were checked using a NanoDrop spectrophotometer. Integrity was genes, that were reported earlier to be additionally checked by agarose gel electrophoresis. Only non- altered in different cancers, was designed to enable for degraded samples with an A260/A280 ratio between 1.8 and 2.0 and precise analysis of exonic/intronic deletions/duplications. We an A260/A230 ratio greater than 2.0 were used for further analysis. hypothesize that besides large aberrations involving whole and chromosome arms, as shown earlier, small Sample labelling and microarray processing. Three hundred intragenic duplication/deletions including single exons or nanograms of each DNA sample was labelled with Cy-5 fluorescent introns could play a role in mechanisms of colorectal cancer dye using the CGH Labelling Kit for Oligo Arrays (Enzo Life Sciences Farmingdale, New York) according to the manufacturer’s development and progression. protocol. As a reference genotype for hybridization 300 ng of Human Reference DNA – Male or Female (Agilent Technologies, Materials and Methods Santa Clara, California) was simultaneously labelled with Cy-3 dye. Hybridization of samples and processing of microarrays were Microarray comparative genomic hybridization (aCGH) analysis performed with the standard Agilent Oligonucleotide Array-Based was performed on 16 colorectal cancer samples. All samples were CGH protocol. obtained from patients of the First Department of Surgical Oncology of the Lower Silesian Oncology Centre in Wroclaw, diagnosed with Microarray slide design. The examined group of phosphatases colon adenocarcinoma. The patients underwent surgery, and before included 25 genes: PTPRT, PTPRM, PTPRZ1 and PTPRQ (examined tissue collection no chemo- or radiotherapy was applied. All earlier) and also PTPRA, PTPRE, PTPRF, PTPRG, PTPRH, PTPRJ, subjects had a negative family history in regard to both hereditary PTPRS, DUSP1, DUSP4, DUSP26, PTEN, PTP4A3, PTPN1, cancer syndromes and accumulation of cancers in the family. The PTPN3, PTPN5, PTPN7, PTPN13, PTPN14, PTPN21, PTPN22 and group consisted of 8 females and 8 males with a mean age of PTPN23 . Two slides containing 8 custom microarrays each with 60 64.69±8.14 years (Table I). K probes for CGH analysis were obtained from Agilent Technologies All samples were molecularly characterized including analysis and designed using the Agilent SureDesign platform. The design was of: the BRAF V600E (exon 15) and K-ras (codons 12 and 13) generated with Standard Design Wizard without modification of mutations, methylator phenotype (CIMP) and microsatellite default parameters for probes selected from database. For the 25 instability (MSI) (8). The study was approved by the Wroclaw targets probe groups were generated and extended with additional Medical University Ethical Committee (Approval number 3,000 base regions flanking 3’ and 5’ end of all targets. For high 328/2009). resolution and accuracy the target-specific probes were designed in

70 Laczmanska et al : Customized aCGH Analysis of Phosphatases in CRC

Table II. Microarray design details–target phosphatases coverage in Table III. Chromosomal aberrations revealed in aCGH analysis. accordance to Genome Reference Consortium , build 37 (GRCh37/hg19, March 2009). Sample Gains on chromosomes Losses on chromosomes

Target ID Interval Target Total Median 1 2, 7, 12, 20 6q21q22.31 (chromosomal localization) coverage probes probe 2 - - spacing 3 2, 7, 8q, 20q 1q32.1q43, 17p, 18, 21 4 - - DUSP1 chr5:172,192,093-172,201,203 97.5% 37 223 5 12, 13, 20 18 DUSP26 chr8:33,445,854-33,460,624 82.0% 39 247 6 7, 8, 12, 13, 19 - DUSP4 chr8:29,187,579-29,211,267 96.3% 101 194 7 1q, 6p, 7q, 8q11.21q21.13, 8p, 17p, 18, 20p PTEN chr10:89,619,870-89,734,687 87.2% 379 223 8q23.3q24.13, 12p, 13, 16, 20q PTP4A3 chr8:142,399,093-142,445,554 94.5% 176 212 8 - - PTPN1 chr20:49,123,858-49,204,300 95.1% 282 240 9 7q, 20 8p, 18 PTPN13 chr4:87,512,468-87,739,328 84.2% 712 230 10 1p, 3, 7, 8, 12q21.31q22, - PTPN14 chr1:214,519,039-214,728,792 94.5% 731 236 12q24.31q24.33, 13, 20q PTPN21 chr14:88,929,122-89,024,123 85.1% 295 237 11 13, 20q 1q31.1q44, 8p12p23.3, PTPN22 chr1:114,353,433-114,417,381 85.0% 181 245 11q, 17p, 18q, 22 PTPN23 chr3:47,419,491-47,457,931 81.5% 111 242 12 8p23.3q21, 8q21q24.3 8q21.3q22.1 PTPN3 chr9:112,134,974-112,263,593 94.0% 459 227 13 - - PTPN5 chr11:18,746,475-18,817,268 95.3% 249 242 14 13, Xp - PTPN7 chr1:202,113,141-202,133,716 93.2% 72 226 15 8p22, 8q, 10p - PTPRA chr20:2,841,830-3,022,320 83.8% 509 255 16 7p, 11p, 13, 20q11.21q13.33 - PTPRE chr10:129,702,325-129,887,164 95.6% 679 229 PTPRF chr1:43,987,858-44,092,343 97.1% 401 232 PTPRG chr3:61,544,243-62,286,288 95.4% 2654 232 PTPRH chr19:55,689,615-55,723,874 69.7% 79 268 PTPRJ chr11:47,999,110-48,195,394 90.3% 623 245 PTPRM chr18:7,564,314-8,409,859 89.1% 2914 231 PTPRQ chr12:80,835,126-81,076,968 84.3% 799 230 16 (1/16), 19 (1/16), 20 (8/16) and 19 losses in 1 (2/16), 6 PTPRS chr19:5,170,977-5,343,814 81.0% 482 244 (1/16), 8 (4/16), 11 (1/16), 17 (3/16), 18 (5/16), 20 (1/16), 21 PTPRT chr20:40,698,392-41,821,610 91.9% 3922 231 (1/16) and 22 (1/16). Most aberrations covered either the PTPRZ1 chr7:121,510,143-121,705,090 96.3% 723 232 whole chromosome or a whole arm (54/67) (Table III).

Microarray analysis (gene level). The analysis of targeted genes encoding tyrosine phosphatases revealed gains and losses of whole gene sequences covered by large chromosome triplicate with median spacing of 233 bp. The final design of the aberrations for 22 out of 25 genes (Figure 1). No changes microarray included 17 609 target-specific probes for 25 selected were observed for phosphatases: PTPN22, DUSP1, PTPN21 . genes covering 5.2 Mbp of the genome. The targets coverage details are shown in Table II. An additional standard set of controls, Also 9 different small intragenic alterations for 8 out of normalization and backbone probes gives a whole-genome backbone 25 genes were discovered. Seven of them were classified as with approximately 1 probe per 1-1,5 Mbp. copy number variations (CNVs) according to the Database of Genomic Variants (Structural Variants, July 2015), while Data analysis. Analysis of the results was performed with one small deletion: of 2 kb in PTPN14 intron 1 and one CytoGenomics software (Agilent Technologies) with the default small gain of 1 kb in PTPRJ intron 1 were classified as CGH analysis method. The protocol calls an aberration as gain or unique (Table IV). loss for at least 3 continuous probes with mean signal (sample to reference) Log Ratio of at least 0.25 or –0.25 respectively. With this No aberration with Log Ratio greater than 1 or below -1 algorithm and microarray design the practical resolution of detected indicating DNA amplification (above 4 copies) or aberrations was approximately 700 bp for targeted genes and 3-5 homozygous deletion respectively was observed. Mb for the background (similar to karyotype resolution for whole chromosome analysis). Discussion

Results The role of receptor-type PTPs has been intensively studied in recent years because of their important role in fundamental Microarray analysis (chromosomal level). The analysis of cellular processes, crucial also for cancer development and chromosomal regions revealed 48 chromosomal gains in progression. Different alterations in PTPs genes including chromosomes: 1 (2 of 16 probes), 2 (2/16), 3 (1/16), 6 (1/16), whole-gene deletions, point-mutations, aberrant methylation 7 (7/16), 8 (9/16), 10 (1/16), 11 (1/16), 12 (6/16), 13 (7/16), pattern and changes in mRNA expression levels show that

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gene (2 kb, 9 probes). In two other tissues the PTPN14 whole gene deletion together with chromosomal region 1q31.1q44 covering this gene was observed. Also, we found a gain of this region in one sample. PTPN14 is a potential tumour suppressor gene whose function is not well characterized yet. It is a non-receptor PTP that was shown to be involved in cell adhesion, proliferation and angiogenesis regulation (11-13). Moreover, several PTPN14 mutations were found in breast and colorectal cancer (6). The function of PTPN14 still remains unknown, but it has been shown that this protein mediates beta- dephosphorylation and epithelial-mesenchymal transition and also promotes transfer of oncoprotein yes-associated protein 1 (YAP1) from the nucleus to cytoplasm and thus inhibits its transcriptional activity (14, 15). Also, the loss of PTPN14 in mammary epithelium cells led to their transformation