Journal of Cancer 2010, 1 38 Journal of Cancer 2010; 1:38-50 © Ivyspring International Publisher. All rights reserved Research Paper Undetectable and Decreased Expression of KIAA1949 (Phostensin) Encoded on Chromosome 6p21.33 in Human Breast Cancers Revealed by Transcrip- tome Analysis Yan A. Su1 , Jun Yang1, Lian Tao1, and Hein Nguyen1, and Ping He2 1. GenProMarkers Inc., Rockville, Maryland 20850, USA; 2. Division of Hematology, Center for Biological Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA Corresponding author: Yan A. Su, MD, PhD, GenProMarkers Inc., 9700 Great Seneca Highway, Suite 182, Rockville, Maryland 20850. Phone: (301) 326-6523; FAX: (240) 453-6208; Email:[email protected] Published: 2010.06.21 Abstract Cytogenetic aberration and loss of heterozygosity (LOH) are documented on chromosome 6 in many cancers and the introduction of a neo-tagged chromosome 6 into breast cancer cell lines mediates suppression of tumorigenicity. In this study, we described the identification of KIAA1949 (phostensin) as a putative tumor suppressor gene. Our microarray analysis screened 25,985 cDNAs between a tumorigenic and metastatic breast cancer cell line MDA-MB-231 and the chromosome 6-mediated suppressed, non-tumorigenic and non-metastatic derivative cell line MDA/H6, resulting in the identification of 651 differentially expressed genes. Using customized microarrays containing these 651 cDNAs and 117 con- trols, we identified 200 frequently dysregulated genes in 10 breast cancer cell lines and 5 tumor tissues using MDA/H6 as reference. Our bioinformatics analysis revealed that chro- mosome 6 encodes 25 of these 200 genes, with 4 downregulation and 21 upergulation. Northern analysis validated microarray results and was used to detect the size and number of RNA species of 2 downregulated (KIAA1949, PTK7) and 2 upregulated (SFRS3, HMGN3) genes in the cell lines. Particularly, the KIAA1949 gene at 6p21.33, a band region with the frequent cytogenetic aberration and LOH encodes 4 poly(A)-RNA species (4.6-, 4-, 3- and 1.5-kb) in multiple normal and breast cancer samples. Microarray analysis revealed KIAA1949 downregulation in 86.7% (n=15) of breast cancer cell lines and tumor tissues. Northern analysis demonstrated undetectable and decreased expression of KIAA1949 in 100% (n=10) of breast cancer cell lines. Taken together, these results strongly suggest KIAA1949 is a novel candidate breast cancer suppressor gene. Key words: Breast cancer, MDA-MB-231, Microarray, Tumor suppressor gene, Phostensin, KIAA1949 Introduction Cytogenetic and loss of heterozygocity (LOH) primary breast tumor tissue (n=72) were reported on studies document frequent genetic alterations on the short arm of chromosome 6 (6p) using 13 micro- chromosome 6 in many types of human cancers in- satellite markers at 6p21 (1). On the long arm of cluding breast cancer and malignant melanoma. In chromosome 6 (6q), five distinct regions of allelic im- human breast cancer, 18-57% deletion frequencies in balance or LOH were observed in breast cancer tumor http://www.jcancer.org Journal of Cancer 2010, 1 39 tissues (2-5) and cell lines (6;7). Functionally, the mi- (phostensin) using both genome-wide microarrays of crocell-mediated introduction of a neo-tagged chro- 25,985 cDNA probes and customized microarrays of mosome 6 resulted in the suppression of transformed, 651 genes and 117 controls. The genome-wide micro- tumorigenic, and/or metastatic characteristics of arrays were used to screen differentially expressed breast cancer cell lines MCF7(8), MDA-MB-231 (8), genes between the tumorigenic and metastatic breast and CAL51 (9), and of the malignant melanoma cell cancer cell line MDA-MB-231 and the chromosome line UACC903 (10). The structural and functional 6-mediated suppressed non-tumorigenic and evidence strongly suggest the presence of chromo- non-metastatic derivative cell line MDA/H6. The some 6-encoded tumor suppressor genes that have yet customized cDNA microarray contained double sets to be identified. of 651 genes and 117 controls per glass slide and was Our study of malignant melanoma using human used to analyze expression profiles in 10 breast cancer chromosome 6-mediated suppressed melanoma cell cell lines and 5 primary breast tumor specimens with line UACC903(+6) (10) led to the identification of MDA/H6 as a reference. Microarray analysis identi- chromosome 6-encoded connexin 43 (Cx43, or gap fied 200 frequently dysregulated genes in these breast junction protein, alpha 1, 43kDa [GJA1], 6q21-q23.2) cancer samples. Out of the 25 genes that are known to as a melanoma suppressor gene (11), the transfection be encoded by chromosome 6, four were downregu- and overexpression of Cx43 cDNA in breast cancer lated but 21 were upergulated. Northern analysis va- cell line MDA-MB-231 by other investigators sup- lidated the microarray results. The KIAA1949 gene is pressed the xenograft tumor growth in athymic nude known at 6p21.33, a band region with the frequent mice (12). Cx43 may be only one of several candidate cytogenetic aberration and LOH. Our Northern anal- breast cancer suppressor genes encoded by chromo- ysis on KIAA1949 detected four poly(A)-RNA bands some 6. Others include pleiomorphic adenoma (4.6-, 4-, 3- and 1.5-kb) in multiple normal and breast gene-like 1 (PLAGL1 or Zac1/Lot1, 6q24-q25) (13), cancer samples. Microarray analysis revealed the Parkinson disease (autosomal recessive, juvenile) 2 downregulation of KIAA1949 in 86.7% (n=15) of (PARK2, 6q25.2-q27) (14), and SAM and SH3 domain breast cancer cell lines and tumor tissues. Northern containing 1 (SASH1, 6q24.3) (15). Because human analysis demonstrated 100% (n=10) downregulation breast cancers are extremely heterogeneous in bio- in the cell lines. Taken together, these results strongly logical behavior and clinical outcomes, the pheno- suggest that KIAA1949 is a novel candidate breast typic complexity may be due to the broad differences cancer suppressor gene. in gene expression that are determined by genetic and epigenetic alterations accumulated during the cancer development and by the gene-environment interac- Materials and Methods tions. In addition, none of above candidate tumor Cell lines, tumor tissues, and cell culture con- suppressor genes can fully explain chromosome ditions: Table 1 list 11 breast cancer cell lines and 5 6-mediated suppression of tumorigenicity in breast tumor tissues used in this study. Ten breast cancer cell cancer cell lines MCF7 (8), MDA-MB-231 (8) and lines were purchased from American Type Culture CAL51 (9). It is conceivable that direct comparison of Collection (www.atcc.org). The MDA/H6 cell line (8) gene expression profile between a breast cancer cell was generously provided by Dr. Negrini. Cells were line and its chromosome 6-mediated suppressed de- cultured in DMEM medium with L-glutamine sup- rivative cell line may lead to the identification of a plemented with 10% fetal bovine serum. G418 (800 putative breast cancer suppressor gene. μg/ml) was added to MDA/H6 cell culture to select The DNA microarray technology (16) has facili- for the neo-tagged chromosome 6. 100 U penicillin G tated the identification of expression profiles and as- sodium and 100 µg streptomycin sulfate were added sociation of abnormal patterns to hereditary breast into each milliliter of all cell culture media as antibio- cancer (17), sporadic breast cancer (18;19), and clinical tics. Breast cancer tissue samples with pathological outcomes (20;21). Combined with PCR subtraction, reports were purchased from the Lombardi Cancer DNA microarrays were also used for rapid isolation Center Histopathology/Tissue Core, Georgetown of differentially expressed genes in breast cancers University Medical Center. The patients’ identifica- (22;23). Using cDNA microarrays, we had successfully tions of all tissue samples had been removed by the identified the chromosome 6-encoded tumor sup- core facility personnel before the PI received the pressor gene Cx43 (11). In this study, we describe the samples. All media, serum, and antibiotics were ob- identification of another chromosome 6-encoded tained from GIBCO BRL (Gaithersburg, MD). novel candidate tumor suppressor gene KIAA1949 http://www.jcancer.org Journal of Cancer 2010, 1 40 Table 1. Human Breast Cancer Cell Lines and Tumor Tissues Name Symbol Original Clinical Diagnosis Source MDA-MB-231 MB231 Adenocarcinoma ATCC* MDA/H6 MDA/H6 Non-tumorigenic Dr. Negrini (8) MDA-MB-134 MB134 Carcinoma ATCC MDA-MB-157 MB157 Carcinoma ATCC MDA-MB-436 MB436 Adenocarcinoma ATCC MDA-MB-453 MB453 Carcinoma ATCC MDA-MB-468 MB468 Adenocarcinoma ATCC BT-20 BT20 Adenocarcinoma ATCC BT-474 BT474 Ductal carcinoma ATCC BT-549 BT549 Ductal carcinoma ATCC ZR75-1 ZR751 Ductal carcinoma ATCC Breast cancer tissue 1 BCTis-1 Poorly differentiated invasive ductal carcinoma LCC* Breast cancer tissue 2 BCTis-2 Poorly differentiated infiltrating ductal carcinoma LCC Breast cancer tissue 3 BCTis-3 Poorly differentiated infiltrating carcinoma LCC Breast cancer tissue 4 BCTis-4 Poorly differentiated infiltrating ductal carcinoma LCC Breast cancer tissue 5 BCTis-5 Poorly differentiated infiltrating ductal carcinoma LCC * ATCC: American Type Culture Collection; LCC: Lombardi Cancer Center Histology Facility. Tumorigenicity test. The test was performed by Genetics) with 5-µl Poly(dA) (POLYA.GF, Research the standard methods in the IACUC approved animal Genetics) and 5-µl Cot-1 DNA (15279-011, Life Tech- protocol and core facility. Briefly, 0.1 ml of 5 x 107 cells nologies) at 42°C for 2 to 4 hours. per ml were inoculated in subcutaneous fat pad at one The first strand cDNA was labeled as follows. site of flank of female athymic nude mice at age of 4 Total RNA (0.8μg) was suspended to 8-μl Rnase-free weeks. Three mice per cell line were tested. The mice water. Two μl of 1μg/μl 10-20 mer of Oligo-(dT) were subject to euthanasia when a tumor reaches 20 (POLYT.GF, Research Genetics) was added to the mm in diameters.