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Involvement of Elevated Expression of Multiple Cell-Cycle Regulator, DTL

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Involvement of Elevated Expression of Multiple Cell-Cycle Regulator, DTL Oncogene (2008) 27, 5672–5683 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Involvement of elevated expression of multiple cell-cycle regulator, DTL/RAMP (denticleless/RA-regulated nuclear matrix associated protein), in the growth of breast cancer cells T Ueki1,2, T Nishidate1, JH Park1, ML Lin1, A Shimo1, K Hirata2, Y Nakamura1 and T Katagiri1,3 1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan and 2Department of Surgery, Sapporo Medical University, Chuo-ku, Sapporo, Japan To investigate the detailed molecular mechanism of mam- Introduction mary carcinogenesis and discover novel therapeutic targets, we previously analysed gene expression profiles of breast Breast cancer is the most common cancer in women, cancers. We here report characterization of a significant role with estimated new cases of 1.15 million worldwide of DTL/RAMP (denticleless/RA-regulated nuclear matrix in 2002 (Parkin et al., 2005). Incidence rates of breast associated protein) in mammary carcinogenesis. Semiquanti- cancer are increasing in most countries, and the tative RT–PCR and northern blot analyses confirmed increasing rate is much higher in countries where its upregulation of DTL/RAMP in the majority of breast incidence was previously low (Parkin et al., 2005). Early cancer cases and all of breast cancer cell lines examined. detection with mammography as well as development Immunocytochemical and western blot analyses using anti- of molecular targeted drugs such as tamoxifen and DTL/RAMP polyclonal antibody revealed cell-cycle-depen- trastuzumab reduced the mortality rate and made the dent localization of endogenous DTL/RAMP protein in quality of life of the patients better (Bange et al., 2001; breast cancer cells; nuclear localization was observed in cells Navolanic and McCubrey, 2005). However, still very at interphase and the protein was concentrated at the limited treatment options are available to patients at an contractile ring in cytokinesis process. The expression level of advanced stage, particularly those with a hormone- DTL/RAMP protein became highest at G1/S phases, independent tumor. Hence, development of novel drugs whereas its phosphorylation level was enhanced during to provide better management to such patients is still mitotic phase. Treatment of breast cancer cells, T47D and eagerly expected. HBC4, with small-interfering RNAs against DTL/RAMP Gene expression profiles obtained by cDNA micro- effectively suppressed its expression and caused accumulation array analysis have been proven to provide detailed of G2/M cells, resulting in growth inhibition of cancer cells. characterization of individual cancers and such informa- We further demonstrate the in vitro phosphorylation of DTL/ tion should contribute to choose more appropriate RAMP through an interaction with the mitotic kinase, clinical strategies to individual patients through Aurora kinase-B (AURKB). Interestingly, depletion of development of novel drugs and providing the basis of AURKB expression with siRNA in breast cancer cells personalized treatment (Petricoin et al., 2002). Through reduced the phosphorylation of DTL/RAMP and decreased the genome-wide expression analysis we have isolated a the stability of DTL/RAMP protein. These findings imply number of genes that function as oncogenes in the process important roles of DTL/RAMP in growth of breast cancer of development and/or progression of breast cancers cells and suggest that DTL/RAMP might be a promising (Park et al., 2006; Lin et al., 2007; Shimo et al., 2007), moleculartargetfortreatmentofbreastcancer. synovial sarcomas (Nagayama et al., 2004, 2005) Oncogene (2008) 27, 5672–5683; doi:10.1038/onc.2008.186; and renal cell carcinomas (Togashi et al., 2005; Hirota published online 9 June 2008 et al., 2006). Such molecules are considered to be candidate targets for development of new therapeutic Keywords: cDNA microarray; molecular target; modalities. AURKB; multiple cell-cycle regulator; cytokinesis In an attempt to identify novel molecular targets for breast cancer therapy, we previously analysed the detailed gene-expression profiles of breast cancer cells, which were purified by laser microbeam microdissection, by means of cDNA microarray (Nishidate et al., 2004). In this Correspondence: Dr T Katagiri, Laboratory of Molecular Medicine, report we focused on characterization of a DTL/RAMP Human Genome Center, Institute of Medical Science, The University (denticleless/RA-regulated nuclear matrix associated of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. protein) gene that was previously isolated as one of E-mail: [email protected] downregulated genes during retinoic acid-induced differ- 3Current address: Division of Genome Medicine, Institute for Genome Research, The University of Tokushima, Tokushima, Japan entiation of human embryonal carcinoma cell line, NT2 Received 23 May 2007; revised 6 May 2008; accepted 12 May 2008; cells (Cheung et al., 2001) to elucidate its pathophysio- published online 9 June 2008 logic roles in growth of breast cancer cells. DTL/RAMP as a molecular-target for breast cancer T Ueki et al 5673 Results To examine the expression of endogenous DTL/ RAMP protein, we generated a polyclonal antibody Overexpression of DTL/RAMP in breast cancer cells against DTL/RAMP protein and performed western We validated that the elevated the DTL/RAMP blot analysis using cell lysates from eight breast cancer (denticleless/RA-regulated nuclear matrix associated cell lines, HBC4, HBC5, HCC1937, MCF-7, MDA-MB- protein) gene (GenBank accession no. NM_016448) 231, SK-BR-3, T47D and YMB-1. We confirmed a high expression in 10of 12 clinical breast cancer cases by level of DTL/RAMP protein expression in all the eight semiquantitative RT–PCR analysis (Figure 1a) as well breast cancer cell lines examined, but its expression was as cDNA microarray data (Nishidate et al., 2004). hardly detectable in human mammalian epithelial cell Subsequent northern blot analysis showed that an (HMEC) cells (Figure 2a). Additionally, we noticed two approximately 4.5-kb transcript of DTL/RAMP was bands that reacted with anti-DTL/RAMP protein when significantly elevated in all of eight breast cancer cell a longer electrophoresis was performed. Therefore, lines examined (Figure 1b), compared with normal to examine a possible modification of DTL/RAMP, breast. This transcript was most highly expressed in we treated cellular extracts from T47D cells with l- testis, and weakly expressed in bone marrow, thymus phosphatase and analysed the molecular weight of and placenta, but its expression was hardly detectable DTL/RAMP protein by western blot analysis. As the in any of the remaining normal organs we examined upper band was not observed when the cell extracts were (Figure 1c). incubated with phosphatase, we considered that the 12 breast cancer cases 16 102 247 252 302 473 478 502 552 646 769 779 normal MG DTL/RAMP FDFT1 kb HBC 4 HBC 5 HCC1937 MCF-7 MDA-MB-231 SK-BR-3 T47D YMB-1 Breast Lung Heart Liver Kidney Bone marrow 9.5 7.5 4.4 2.4 kb Small intestine P.B.L. Heart Brain Placenta Lung Liver muscle Skeletal Kidney Pancreas Spleen Thymus Prostate Testis Ovary Colon Stomach Thyroid Spinal cord node Lymph Trachea Adrenal gland Bone marrow 9.5 7.5 4.4 2.4 Figure 1 Expression pattern of DTL/RAMP in breast cancers and normal human organs. (a) Expression of DTL/RAMP in microdissected tumor cells from breast cancer tissues (16, 102, 247, 252, 302, 473, 478, 502, 552, 646, 769 and 779), compared with normal human tissues (normal, microdissected normal breast ductal cells; MG, mammary gland) by semiquantitative RT–PCR. FDFT1 served as a loading control. (b) Northern blot analysis of the DTL/RAMP in eight breast cancer cell lines (HBC4, HBC5, HCC1937, MCF-7, MDA-MB-231, SK-BR-3, T47D and YMB-1) and normal human tissues including breast, lung, heart, liver, kidney and bone marrow. (c) Northern blot analysis of the DTL/RAMP transcript in various human tissues. PBL indicates peripheral blood leukocytes. Oncogene DTL/RAMP as a molecular-target for breast cancer T Ueki et al 5674 DTL/RAMP (1-730) ∆ 1 (1-401) ∆ 2 (388-590) HBC4 HBC5 HCC1937 MCF-7 MDA-MB-231 SK-BR-3 T47D YMB-1 HMEC ∆ 3 (438-730) DTL/RAMP IB : HA ACTB lambda-phosphatase -+ ∆ lambda-phosphatase 1 -+ kD ∆ 2 p-DTL/RAMP DTL/RAMP p-DTL/RAMP ∆ 3 DTL/RAMP 75 Figure 2 Expression of DTL/RAMP protein in breast cancers. (a) Expression of endogenous DTL/RAMP protein in eight breast cancer cell lines (HBC4, HBC5, HCC1937, MCF7, MDA-MB-231, SK-BR-3, T47D and YMB-1) in comparison with human mammary epithelial cell line (HMEC) cells by western blot analysis using anti-DTL/RAMP polyclonal antibody. (b) Phosphorylation of DTL/RAMP protein in T47D cells. The cell lysates were incubated with/without treatment of l-phosphatase for 2 h at 30 1C. A phosphorylated DTL/RAMP protein is observed as a slowly migrated band as indicated by arrow (p-DTL/RAMP). (c) Determination of a phosphorylated region in the DTL/RAMP protein. The upper panel shows schematic representation of DTL/RAMP fragments (D1, 1–401; D2, 388–590and D3, 438–730amino acids) and full length of DTL/RAMP (1–730amino acids) used for l-phosphatase experiments. The six black boxes in full length of DTL/RAMP indicate WD40repeat domains. The lower panel shows l-phosphatase assay results. The C-terminal region of DTL/RAMP (438–730amino acid) was phosphorylated. The cell lysates were incubated with/without treatment of l-phosphatase as described in (b). DTL/RAMP protein was possibly phosphorylated in mammary tissues including ductal cells (no. 10441N; breast cancer cells (Figure 2b). Subsequently, to Figure 3b). Furthermore, we performed breast cancer determine the phosphorylated region of the DTL/ tissue-array analysis and verified positive staining of RAMP protein, we made three constructs, each of DTL/RAMP in 33 of 39 infiltrating ductal carcinomas, which was designed to express a part of DTL/RAMP, whereas no staining was observed in 5 normal mammary and performed l-phosphatase assay (Figure 2c, upper tissues including ductal cells (data not shown).
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