癌症的發生有很明顯的地域差別, 如惡性淋巴瘤在西方比東方常見, 而鼻咽癌 肝癌等則在東方亞洲特別多發 許多病毒相關的癌症都特殊地好發於台灣及中國人,

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2 中文摘要 癌症的發生有很明顯的地域差別, 如惡性淋巴瘤在西方比東方常見, 而鼻咽癌 肝癌等則在東方亞洲特別多發 許多病毒相關的癌症都特殊地好發於台灣及中國人, 如 HBV 與肝癌,HPV( 乳頭瘤病毒 ) 與子宮頸癌, 以及 EBV 與鼻咽癌及 NK/T 細胞淋巴瘤等 其中的原因除了病毒及環境因素外, 遺傳背景及基因的改變一直被認為是重要的相關因素 近幾年來分子醫學的快速發展使我們瞭解癌症的發生 惡化和轉移的機轉, 是一連串致癌基因 (oncogene) 放大及抑癌基因 (tumor suppressor gene) 失活累積而成 尤其是抑癌基因的結構性改變及功能喪失被認為可能是癌症發生早期最重要的變化 雖然目前至少有四十種以上的抑癌基因已經被發現, 但在肝癌 鼻咽癌等實心性癌症研究上, 則因變化複雜, 目前尚未有突破性的發現 近年來的癌症研究發現體內小微星標竿在癌細胞內, 因基因缺失而有結構上的改變或產生 DNA 複製時不穩定的現象 (Microsatelliet instability; MIN) 藉著聚合脢連鎖反應放大特定的小微星標竿再利用電泳分析正常細胞及癌細胞內 DNA 片段異質性喪失 (Lossofheterozygosity; LOH) 同質性定位缺失(homozygosity mappingofdeletion, HOMOD) 或不穩定性的現象, 可以成功的找出抑癌基因, 幫助癌症診斷或早期偵測腫瘤的復發 本計劃主要是以好發於台灣及中國人中 EBV 相關之 NK/T 細胞淋巴癌為研究對象 先前的研究結果顯示此淋巴癌有高頻率的染色體 6q2125 缺失現象, 極可能存有與 NK/T 細胞淋巴癌相關的重要致癌基因 我們藉著分子遺傳的技術, 利用小微星標竿標記來進行異質性喪失分析及同質性定位缺失分析 我們已經完成 50 個檢體的分析工作, 並決定出最小重複範圍 (smallest region of overlapping, SRO) 的缺失片段約是 3.4 Mb 此外, 進一步利用 sequence tagged sites (STS) 定量 PCR 分析, 再將 SRO 縮小至 2.6 Mb 在本篇報告中, 已完成四個候選基因的功能性分析, 包括了 :Insulinlike growth factor 2 receptor (IGF2R; a tumor suppressor gene), Tcell lymphoma invasion and metastasis 2 (TIAM2), MAS1 (an oncogene) 和 an uncharacterized bone marrow protein (BM033, also known as C6orf35) 關鍵詞 :EB 病毒,NK/T 細胞淋巴癌, 分子遺傳 I

3 Abstract Nasal natural killer (NK)/T cell lymphoma is a special subtype of malignant lymphoma that is more prevalent in Asia than in America and Europe. This type of tumor had been classified as Tcell lymphoma that tends to affect nasal and paranasal regions and pursues a highly aggressive clinical course. The exact cellular origin and detailed tumorgenesis are not yet understood though a closed association with EpsteinBarr virus (EBV) has been well documented. Previous studies have identified a common region of deletion at 6q21q25 in several CD56+ putative NK lymphoma/leukemia patients thus suggesting that this chromosome aberration may be a nonrandom event. To further explore the molecular background in chromosomal region related to the carcinogenesis of NK/T cell lymphoma/leukemia, we have refined the possible TS genecontaining region to a 2.6Mb interval on chromosome 6q by loss of heterozygosity (LOH), homozygosity mapping of deletion (HOMOD), and quantitative PCR of sequence tagged sites analyses. There are eighteen known genes/unigene clusters and 25 hypothetical genes located in this region. Studies of function and sequence mutations of candidate genes including insulinlike growth factor 2 receptor (IGF2R; a tumor suppressor gene), Tcell lymphoma invasion and metastasis 2 (TIAM2), MAS1 (an oncogene) and an uncharacterized bone marrow protein (BM033, also known as C6orf35) have been completed to determine whether they are involved in the pathogenesis of nasal NK/T cell lymphoma. Keywords: EpsteinBarr virus, NK/Tcell lymphoma, LOH analysis II

4 TABLE OF CONTENTS Specific Aims. 1 Progress Summary Reference.12 Appendix. 13 III

5 Specific Aims This project is designed mainly base on our previous identification of novel chromosomal abnormalities of 6q2125 in EBVassociated nasal NK/T cell lymphoma. This chromosomal abnormality is consistently demonstrated for nasal NK/T cell lymphoma and some types of EBVassociated NK/T cell lymphomas in other regions. To further explore the molecular background in chromosomal region related to the carcinogenesis of NK/T cell lymphoma, our previous works (NSC912318B006010M51) have narrowed down the potential tumor suppressor (TS) regions to a 10 cm (approximately 10 Mb) interval on 6q25 region. The first year s study under this project enabled us to further refine the interval to a 2.6 Mb region on 6q (Sun et al, 2003). Studies of function and sequence mutations of candidate genes within this region have been completed to determine whether they are involved in the pathogenesis of nasal NK/T cell lymphoma. The overall objectives of this proposal are to identify the putative TS genes and study the mechanism related to carcinogenesis of NK/T cell lymphoma. The specific aims of this study are: 1. Establish cell line of nasal NK/T cell lymphoma and to examine the tumor clonality and tumor biology 2. Apply bioinformatics tools to identify candidate genes for functional analysis. 3. Perform sequence analysis for identifying sequence variation that alters function of the candidate gene. 4. Perform immunostaining on paraffinembedded tissue section to examine the level of target protein production. 5. Conduct functional studies to characterize the function of the candidate genes in general as well as in related to tumorigenesis of NK/T cell lymphoma. 1

6 1. Progress Summary I. Refine the potential TSG containing region To refine the smallest region of overlapping (SRO) deletion for localization of potential tumor suppressor (TS) genes, we performed lossofheterozygosity (LOH) and homozygositymappingofdeletion (HOMOD) analyses on 37 nasal and nasal type NK/T cell lymphoma cases using a panel of 25 microsatellite markers covering the 6q21q25 region. In all cases studied, LOH was detected in 8 (89%) pairedsample cases, while hemizygous deletion was detected in 3 (11%) singlesample cases. HOMOD results defined a distinct 3Mb SRO on chromosome 6q25. Combination of the LOH and Quantitative multiplex PCR analysis of 10 sequencetagged sites further refined the putative TS genecontaining region to a 2.6Mb interval between TIAM2 and SNX9. Results from the first year s study have been published in the British Journal of Hematology this year (Sun et al, 2003). Eighteen known genes/unigene clusters and 25 hypothetical genes are located within this 2.6Mb region, but none are previously identified TS genes. We started our functional analysis from three strong candidates: Insulinlike growth factor 2 receptor (IGF2R; a tumor suppressor gene), Tcell lymphoma invasion and metastasis 2 (TIAM2), and MAS1 (an oncogene) and an uncharacterized bone marrow protein (BM033, also known as C6orf35). We have accomplished the following tasks during the past few months. II. Primerextension preamplification (PEP) of nasal NK/T cell lymphoma cases DNA Since the original DNA isolated from nasal NK/T cell lymphoma cases are in very limited amount, it may not be enough for further application. To circumvent the limitation, we apply the Primerextension preamplification (PEP) method to amplification the genomic material (Zhang et al, 1992). The authors suggested that PEP can copy at least 30 times from a haploid cell like sperm or oocyte, and almost 78% of the genomic sequence would be covered. We have modified some procedures to amplify the sample from 10 ng of DNA template instead of single cell. contains many shot fragments of genomic DNA. dilution of PEP product would feasible for reaction. After multiple rounds of extension, the final product For the use of PCR, the 10~100 times III. Sequence variant screen of candidate genes Four candidate genes include IGF2R, MAS1, TIAM2 and C6orf35 were selected for further mutation screen. No mutation has been identified but sequence variants have been found in paired nasal NK/T cell lymphoma samples (Table 1). 2

7 Table 1. Sequence variants of the candidate genes identified on nasal NK/T cell lymphoma patients* Patients P4 P5 P6 P7 P9 P11 P12 Genomic Position N T N T N T N T N T N T N T IGF2R: Exon33(G4755A) G/G Exon34(G5002A) Intron34 C/T C/C Intron35 A/C A/C TIAM2: Intron5 NA G/G A/G Intron5 T/C C/C C6orf35: 5 UTR(C46G) CG CG CG CG CG CG 3 UTR(G30857A) NA GG AA NA 3 UTR(G30972A) GG AA NA 3 UTR(A31002G) AG AG AG AA NA * P: Nasal NK/T cell lymphoma patients; N: Normal sample; T: Tumor sample; : Homozygote; NA: Not available; Gray color: Genotype change in normal and tumor sample 3

8 IV. Expression profile of candidate genes As IGF2R is a wellstudied multifunctional gene, we established expression profiles for TIAM2 L TIAM2 C MAS1, TIAM2 and C6orf35 by isoformspecific PCR using commercial cdna panels from different tissues. The PCR products were electrophoresized on agarose gel (Figure 1, take TIAM2 as an example) to determine the relative expression level (Table 2). From the analysis of expression profile, we observed different expression patterns of different isoforms. study. A B TIAM2 S TIAM2 C positive TIAM2 L positive Brain Liver Kidney Figure 1. tissues. Brain Liver It suggests that the cellular functions of different isoforms are worthy of further Testis Thymus Breast Testis Thymus Breast Kidney Ovary Prostate Pancreas NK cell Ovary Prostate Pancreas NK cell Lung Colon Lung Colon Heart Placenta Muscle Heart Placenta Muscle Spleen Lympho node Tonsil Leukocyte Spleen Lympho node Tonsil Leukocyte Bone Marrow Fetal liver Bladder Brain tumor Liver tumor Kidney tumor Testis tumor Thymus tumor Breast tumor Ovary tumor Bone Marrow Fetal liver Bladder Brain tumor Liver tumor Kidney tumor Testis tumor Thymus tumor Breast tumor Ovary tumor Prostate tumor Pancreas tumor NK92 (lymphoma) Lung tumor(lx1) Lung tumor(gi117) Colon tumor(cx1) Colon tumor(gi112) 300bp 200bp Prostate tumor Pancreas tumor NK92 (lymphoma) Lung tumor(lx1) Lung tumor(gi117) Colon tumor(cx1) Colon tumor(gi112) The expressed cdna level of TIAM2 isoforms in human normal and tumor PCR analyses were carried out by using TIAM2 isoformspecific primers on cdna panels from human normal and tumor tissues. (A) The 293bp and 221bp cdna fragments were amplified from TIAM2L (30 cycles) and TIAM2C (30 cycles), respectively in 2.5% agarose gel electroporesis. (B) The 279bp cdna fragments were amplified from TIAM2S (30 cycles). 300bp 200bp 4

9 Table 2. tissue a Expression profile of three candidate genes in cdna panel from various 5

10 IV. Functional study of IGF2R gene The IGF2R functions in the intracellular trafficking of lysosomal enzymes, the activation of the potent growth inhibitor, transforming growth factor beta, and the degradation of IGF2, a mitogen often overproduced in tumors. Previous studies have provided evidence that the IGF2R gene functions as a tumor suppressor in human liver carcinogenesis (De Souza et al, 1995a; De Souza et al, 1995b). To illustrate the role of IGF2R in human NK/T cell lymphoma, we conducted the following experiments: Pattern of IGF2R gene expression in adult leukocyte: Imprinting of IGF2R gene is a polymorphic trait in the human with most studies indicating biallelic expression patterns (Kalscheuer et al, 1993; Killian et al, 2001a; Oudejans et al, 2001; Xu et al, 1993). involvement of this receptor in fetal organogenesis, cellular growth suppression and Tcell mediated programmed cell death predicts that those individuals who inherit an imprinted IGF2R gene would be predisposed to fetal overgrowth, and carcinogenesis because of gene haploinsufficiency (Killian et al, 2001b). To clarify the imprinting status of the human IGF2R gene, we examine the expression pattern of IGF2R gene in adult leukocyte from health population. We isolate the genomic DNA and total RNA from the same individual and examine the sequence of polymorphic SNP in DNA as well as cdna. Only those individuals who are heterozygote in selected csnp marker will be further genotyping with their cdna sample. Each csnp marker was tested in 150 population genomic DNA samples and we have observed 28 samples are A/G heterozygotes at rs marker, 31 samples are A/G heterozygotes at rs marker, and 9 samples are polymorphic at both markers. In cdna sequencing of those 50 informative cases, all showed two alleles as seen in their genomic DNA thus we conclude that the human IGF2R gene is bialleleic expression in adult leukocyte. The IGF2R Immunohistochemistry: The IGF2R Ab was purchased from commercial company (Santa Cruz Inc, Santa Cruz, CA, USA) and used for western blot as well as immunohistochemistry. The optimal condition for western blot analysis has been determined (data not shown). The optimal condition for immunohistochemistry on frozen tissue sections has been examined using hepatocellular carcinomas and normal liver tissues (Figure 2). The tissue sections were counterstained with hematoxylin after immunohistochemical staining. The Ab will be used to study the IGF2R protein production on paraffinembedded sections from NK/T cell lymphoma cases. 6

11 A B C D E F Figure 2. Immunohistochemical staining of M6P/IGF2R monoclonal antibody in hepatocellular carcinomas and normal liver tissues. (A) Normal tissue and (B) tumor tissue are stained by IgG as blank control. (C) M6P/IGF2R expression is positive in normal liver tissue but it is negative in hepatocellular carcinoma (D). (E) and (F) are HE staining in normal liver tissue and hepatocellular carcinoma; they show the morphology of cells. All figures are in magnification of 200x. 7

12 V. Functional study of TIAM2 gene Sequence analysis of TIAM2: Two isoforms had been identified and the cdna for the larger message encodes a deduced 1,077amino acid protein (TIAM2 L ), whereas that for the smaller transcript encodes a deduced 626amino acid protein (TIAM2 S ). isoform of TIAM2 in testis (TIAM2 C ) (Chen, et al 2004). We have identified a novel The functional domains encoded by TIAM2 were predicted by using NCBI Conserved Domain Search ( and found that TIAM2 consisted of Raflike ras binding domain (RDB), a PDZ domain, and a RhoGEF (DH) domain (Figure 3). Cellular localization of TIAM2: Examining by transient transfection of TIAM2GFP fusion protein into culture cells, the cellular localization of all TIAM2 isoforms are localized in the cytoplasm (Figure 4). Protein expression of TIAM2: Expression constructs of three TIAM2 isoforms in pcdna3.1 have been cloned, and the effects of protein overexpression are on going RBD 8 93 PDZ RhoGEF TIAM2 L RBD 8 93 PDZ RhoGEF TIAM2 C RhoGEF TIAM2 S Figure 3. Diagram of three TIAM2 isoforms. The numbers on the top of red, blue and black boxes indicate the sizes of exons. RBD, PDZ and RhoGEF domains are in yellow, light blue and purple colors. 8

13 pegfpn1 pegfpn1tiam2 L pegfpn1tiam2 C pegfpn1tiam2 S A B C D HeLa E F G H NIH3T3 Figure 4. Subcellular localization of TIAM2 isoforms expressed in HeLa and NIH3T3 cells. Cells were plated in culture dishes and transfected with expression plasmids encoding GFP alone, GFPTIAM2 L, GFPTIAM2 C, GFPTIAM2 S as indicated. Replaced transfectedmedium with fresh culture medium after 12 h, cells were examined using inverted fluorescence microscopy after 24h following by the medium change. From (A) to (D) are HeLa cells transfected with empty vector, TIAM2 L GFP, TIAM2 C GFP and TIAM2 S GFP. From (E) to (H) are NIH3T3 cells transfected with empty vector, TIAM2 L GFP, TIAM2 C GFP and TIAM2 S GFP. Results from these experiments indicated all three isoforms are expressed in cytoplasm. 9

14 VI. Functional study of C6orf35 gene Sequence analysis of C6orf35: Sequence analysis revealed the two transcripts could be encoded by alternative splicing of five exons within 32 Kb genomic region; the longer transcript contains a 1799 nucleotides ORF encoding a protein of 141 amino acid, while the shorter one contains a 944 nucleotide ORF encoding a protein of 128 amino acid (Figure 5). Cellular localization of C6orf35: Examining by transient transfection of C6orf35GFP fusion protein into culture cells, the long form is localized in the cytoplasm and short form is presented in the whole cell (Figure 6). The biological functions played by these two transcripts will be further studied. Protein expression of C6orf35: Both isoforms have been cloned into an expression vector to examine the cellular function of the candidate protein. E1 E2 ae3 ae5 E1 E2 ae4 0.5 kb Figure 5. The genomic structures of two transcripts of C6orf35. The boxes, lines and gray boxes indicate exons, introns and untranslated regions (UTR). The upper represents the long form containing exon 1 (E1), exon 2 (E2), alternative exon 3 (ae3) and alternative exon 5 (ae5). The lower represents the short form containing exon 1 (E1), exon 2 (E2) and alternative exon 4 (ae4). 10

15 pegfpn1 C6orf35L_GFP C6orf35S_GFP Figure 6. Cellular localization of C6orf35. Different constructs (from left to right: pegfpn1, C6orf35L_GFP, C6orf35S _GFP) have been transfected into HEK293 cells and examined under fluorescent microscope. The results show that C6orf35 long form is in the cytoplasm and short form in the whole cell. 11

16 Reference De Souza, A.T., Hankins, G.R., Washington, M.K., Fine, R.L., Orton, T.C. & Jirtle, R.L. (1995a) Frequent loss of heterozygosity on 6q at the mannose 6phosphate/insulinlike growth factor II receptor locus in human hepatocellular tumors. Oncogene, 10, De Souza, A.T., Hankins, G.R., Washington, M.K., Orton, T.C. & Jirtle, R.L. (1995b) M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity. Nat Genet, 11, Kalscheuer, V.M., Mariman, E.C., Schepens, M.T., Rehder, H. & Ropers, H.H. (1993) The insulinlike growth factor type2 receptor gene is imprinted in the mouse but not in humans. Nat Genet, 5, Killian, J.K., Nolan, C.M., Wylie, A.A., Li, T., Vu, T.H., Hoffman, A.R. & Jirtle, R.L. (2001a) Divergent evolution in M6P/IGF2R imprinting from the Jurassic to the Quaternary. Hum Mol Genet, 10, Killian, J.K., Oka, Y., Jang, H.S., Fu, X., Waterland, R.A., Sohda, T., Sakaguchi, S. & Jirtle, R.L. (2001b) Mannose 6phosphate/insulinlike growth factor 2 receptor (M6P/IGF2R) variants in American and Japanese populations. Hum Mutat, 18, Oudejans, C.B., Westerman, B., Wouters, D., Gooyer, S., Leegwater, P.A., van Wijk, I.J. & Sleutels, F. (2001) Allelic IGF2R repression does not correlate with expression of antisense RNA in human extraembryonic tissues. Genomics, 73, Sun, H.S., Su, I.J., Lin, Y.C., Chen, J.S. & Fang, S.Y. (2003) A 2.6 Mb interval on chromosome 6q25.2q25.3 is commonly deleted in human nasal natural killer/tcell lymphoma. Br J Haematol, 122, Xu, Y., Goodyer, C.G., Deal, C. & Polychronakos, C. (1993) Functional polymorphism in the parental imprinting of the human IGF2R gene. Biochem Biophys Res Commun, 197, Zhang, L., Cui, X., Schmitt, K., Hubert, R., Navidi, W. & Arnheim, N. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A, 89,

17 Appendix 13