[CANCER RESEARCH 62, 43– 47, January 1, 2002] Advances in Brief Psoriasin Expression in Mammary Epithelial Cells in Vitro and in Vivo 1 Charlotta Enerba ¨ck, 2 Dale A. Porter, Pankaj Seth, Dennis Sgroi, Justine Gaudet, Stanislawa Weremowicz, Cynthia C. Morton, Stuart Schnitt, Robert L. Pitts, Jason Stampl, Kerry Barnhart, and Kornelia Polyak 3 Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [C. E., D. A. P., P. S., K. P.]; Department of Pathology, Massachusetts General Hospital, Charlestown, Massachusetts 02129 [D. S., J. G.]; Departments of Pathology [S. W., C. C. M.] and Obstetrics [C. C. M.], Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Boston, Massachusetts 02115; Department of Pathology, Beth-Israel Deaconess Medical Center, Boston, Massachusetts 02115 [S. S.]; Harvard Medical School, Boston, Massachusetts 02115 [C. E., D. A. P., P. S., D. S., S. W., C. C. M., S. S., K. P.]; and Imgenex Corporation, San Diego, California 92121 [R. L. P., J. S., K. B.] Abstract We determined, by serial analysis of gene expression (SAGE) analysis of normal and DCIS (ductal carcinoma in situ) mammary epithelial cells, that psoriasin and several other genes implicated in psoriasis are aber- rantly expressed in high-grade, comedo DCIS. Real-time PCR, mRNA in situ hybridization, and immunohistochemical analysis of breast carcino- mas confirmed that psoriasin is frequently overexpressed in estrogen receptor-negative tumors. To gain insight into regulatory pathways that control psoriasin expression, we developed polyclonal and monoclonal antibodies and investigated mechanisms that may account for elevated levels of psoriasin in DCIS. Here, we report that loss of attachment to extracellular matrix, growth factor deprivation, and confluent conditions dramatically up-regulate psoriasin expression in MCF10A mammary ep- ithelial cells. All of these conditions are characteristic of high-grade DCIS and psoriatic skin lesions; therefore, the same mechanisms may be re- sponsible for increased expression of psoriasin in vitro and in vivo. Introduction Psoriasin was originally identified as a protein, the expression of which is increased in psoriatic keratinocytes (1). Subsequently, pso- riasin was also found to be up-regulated in abnormally differentiating primary keratinocytes, in squamous carcinoma of the bladder, and in a subset of in situ and invasive breast carcinomas (2–5). Psoriasin is a member of the S100 family of calcium-binding proteins (S100A7); it has been shown to bind calcium, and its basal expression is influ- enced by extracellular calcium levels (6, 7). S100 proteins have been implicated in diverse cellular processes including cell proliferation, apoptosis, differentiation, invasion, and metastasis (7). Among others, the expression of S100A2 and S100A6 are significantly down- and up-regulated, respectively, in breast tumors compared with corre- sponding normal epithelium, whereas the expression of S100A4 cor- relates with tumor progression and acquisition of metastatic pheno- type (7). The partially secreted nature and restricted expression pattern of psoriasin to abnormal proliferative lesions of squamous epithelia makes it a candidate diagnostic marker (5, 8). Moreover, the high expression of psoriasin in these cell types suggests that it may play a role in the regulation of cell growth, survival, or differentiation. During recent SAGE 4 analysis of the gene expression profiles of normal and DCIS mammary epithelial cells, we identified HID-5/ psoriasin as one of the most abundant transcripts in high-grade DCIS (9). To begin to elucidate the function of psoriasin as it relates to the initiation and progression of breast carcinomas, we developed poly- clonal and monoclonal antibodies and examined its expression in vivo and in vitro. Materials and Methods Cell Lines and Culture Conditions. MDA-MB468 and MCF10A cell lines were obtained from American Type Culture Collection and were main- tained in 10% fetal bovine serum in McCoy’s medium (Life Technologies, Inc.) and in 5% horse serum in DMEM/F12 medium (Life Technologies, Inc.) supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, and 500 ng/ml hydrocortisone, respectively. To determine the effect of serum deprivation on HID-5/psoriasin expression in subconfluent or confluent cultures, MCF10A cells were switched to 0.2% serum containing DMEM/F12 medium and incubated for the indicated time (see Fig. 2B). The effect of confluency was analyzed by maintaining MCF10A cells in confluent conditions for the indicated time (see Fig. 2B) with frequent (every other day) medium changes. For suspension cultures, MCF10A cells were trypsinized, resuspended in fresh medium (1.75  10 5 cells/ml medium), plated into poly-2-hydroxy-ethylmethacrylate (Aldrich)-coated (1 mg/cm 2 in 100% etha- nol) Petri dishes, and incubated for the indicated time (see Fig. 2C). Generation of Polyclonal and Monoclonal Anti-HID-5/Psoriasin Anti- bodies. Rabbit polyclonal anti-HID-5/psoriasin antibody was generated against a synthetic peptide corresponding to amino acids 83–100 of the human protein (TDYHKQSHGAAPCSGGSQ). For the generation of mouse mono- clonal antibodies, a PCR-generated BamHI-HindIII cDNA fragment of full- length human HID-5/psoriasin was subcloned into BamHI-HindIII sites of pQE-30 (Qiagen), yielding a construct that encodes HID-5/psoriasin with an NH 2 -terminal hexahistidine sequence. The protein was expressed in M15[pREP4] bacteria, purified to homogeneity using denaturing urea buffer and NiNTA beads (Qiagen). Bound protein was eluted in 50 mM Tris (pH 7.5), 500 mM imidazole, 100 mM EDTA, 1 M NaCl, 10% glycerol, and 1 mM DTT and used for hyperimmunizing Balb/c mice in collaboration with Imgenex (anti-HID-5/psoriasin monoclonal antibodies are commercially available from Imgenex). Western Blot Analysis, Immunohistochemistry, and Tissue Microar- rays. Western blot analysis of cell lysates and immunohistochemistry were performed using anti-CD45 panleukocyte (Dako), anti-estrogen receptor , anti-erbB2, and anti-HID-5/psoriasin (clone 1068-1) antibodies as described (10, 11). Tissue microarrays were purchased from Imgenex or were generated as described (12). FISH, Real-Time PCR, Northern Blots, and mRNA in Situ Hybridiza- tion. FISH analysis of metaphase chromosome preparations from peripheral blood lymphocytes obtained from normal human males was performed ac- cording to the method described previously (13). Interphase nuclei from disaggregated, formalin-fixed, paraffin-embedded tumor tissue were prepared Received 9/20/01; accepted 11/15/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Contract S98-146A from the National Cancer Institute- Cancer Genome Anatomy Project and grants from the National Cancer Institute Special- ized Program of Research Excellence in Breast Cancer, the American Society of Clinical Oncology, and by the Dana-Farber Cancer Institute (to K. P.), as well as by fellowships from the University of Go ¨tborg, Svenska Psoriasisfo ¨rbundet, Pharmacia & Upjohn, and Go ¨teborgs La ¨karesa ¨llskap (to C. E.). 2 Present address: Department of Clinical Genetics, Sahlgrenska University Hospital SU/O, S-416 85, Goteborg, Sweden. 3 To whom requests for reprints should be addressed, at Department of Adult Oncol- ogy, Dana-Farber Cancer Institute, D740C, 44 Binney Street, Boston, MA 02115. E-mail: Kornelia_Polyak@dfci.harvard.edu. 4 The abbreviations used are: SAGE, serial analysis of gene expression; DCIS, ductal carcinoma(s) in situ; HID-5, high in DCIS-5; FISH, fluorescence in situ hybridization; ER, estrogen receptor; PR, progesterone receptor. 43 Research. on December 10, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from