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// Takuya Yoshimura 1 , Taiji Hamada 2 , Hiroshi Hijioka 1 , Masakazu Souda 2 , Kazuhito Hatanaka 2 , Takako Yoshioka 3 , Sohsuke Yamada 2 , Masato Tsutsui 4 , Yoshihisa Umekita 5 , Norifumi Nakamura 1 , Akihide Tanimoto 2 1 Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan 2 Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan 3 Department of Pathology, Children’s Cancer Center, National Center for Child Health and Development, Tokyo, Japan 4 Department of Pharmacology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan 5 Division of Organ Pathology, Faculty of Medicine, Tottori University, Yonago, Japan Correspondence to: Akihide Tanimoto, email: akit09@m3.kufm.kagoshima-u.ac.jp Keywords: PCP4/PEP19, breast cancer, EMT, migration, invasion Abbreviations: Purkinje cell protein 4/peptide 19, PCP4/PEP19; epithelial-mesenchymal transition, EMT Received: September 02, 2015 Accepted: January 22, 2016 Published: February 20, 2016 ABSTRACT Purkinje cell protein (PCP) 4/peptide (PEP) 19 is expressed in Purkinje cells where it has a calmodulin-binding, anti-apoptotic function. We recently demonstrated that PCP4/PEP19 is expressed and inhibit apoptosis in human breast cancer cell lines. In the present study we investigated the role of PCP4/PEP19 in cell morphology, adhesion, migration, and invasion in MCF-7 and T47D human breast cancer cell lines. Knockdown of PCP4/PEP19 reduced the formation of filopodia-like cytoplasmic structures and vinculin expression, and enhanced E-cadherin expression. Activities of migration, invasion, and cell adhesion were also decreased after the knockdown of PCP4/PEP19 in MCF-7 and T47D cells. These results suggested that PCP4/PEP19 promotes cancer cell adhesion, migration, and invasion and that PCP4/PEP19 may be a potential target for therapeutic agents in breast cancer treatment which act by inhibiting epithelial-mesenchymal transition and enhancing apoptotic cell death.

The Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is a novel breast cancer cell expressing peptide, originally found in the neural cells as an anti-apoptotic factor, could inhibit cell apoptosis and enhance cell migration and invasion in human breast cancer cell lines. The expression of PCP4/PEP19 is induced by estrogens in estrogen receptor-positive (ER+) MCF-7 cells but also highly expressed in ER- SK-BR-3 cells. In this study, we investigated the effects of PCP4/PEP19 on aromatase gene expression in MCF-7 and SK-BR-3 human breast cancer cells. In SK-BR-3 cells but not in MCF-7 cells, PCP4/PEP19 knockdown by siRNA silencing decreased the aromatase expression in gene transcriptional level. When PCP4/PEP19 was overexpressed by CMV promoter-driven PCP4/PEP19 expressing plasmid transfection, aromatase gene transcription increased in SK-BR-3 cells. This aromatase gene transcription is mainly mediated through promoter region PI.1, which is usually active in the placental tissue but not in the breast cancer tissue. These results indicate a new function of PCP4/PEP19 that would enhance aromatase gene upregulation to supply estrogens in heterogeneous cancer microenvironment.

// Taiji Hamada 1 , Masakazu Souda 1 , Takuya Yoshimura 2 , Shoko Sasaguri 1 , Kazuhito Hatanaka 1 , Takashi Tasaki 1 , Takako Yoshioka 1 , Yasuyo Ohi 3 , Sohsuke Yamada 4 , Masato Tsutsui 5 , Yoshihisa Umekita 6 and Akihide Tanimoto 1 1 Department of Molecular and Cellular Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. 2 Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. 3 Department of Pathology, Sagara Hospital, Social Medical Corporation Hakuaikai, Kagoshima, Japan. 4 Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. 5 Department of Pharmacology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan. 6 Division of Organ Pathology, Faculty of Medicine, Tottori University, Yonago, Japan. Correspondence to: Akihide Tanimoto, e-mail: akit09@m3.kufm.kagoshima-u.ac.jp Key words: PCP4/PEP19, breast cancer, apoptosis, CaMKK, Akt Received: April 23, 2014 Accepted: June 23, 2014 Published: July 08, 2014 ABSTRACT The PCP4/PEP19 is a calmodulin-binding anti-apoptotic peptide in neural cells but its potential role in human cancer has largely been unknown. We investigated the expression of PCP4/PEP19 in human breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 cells, and found that estrogen receptor (ER)-positive MCF-7 and ER-negative SK-BR-3 cells expressed PCP4/PEP19. In the MCF-7 cells, cell proliferation was estrogen-dependent, and PCP4/PEP19 expression was induced by estrogen. In both cell lines, PCP4/PEP19 knockdown induced apoptosis and slightly decreased Akt phosphorylation. Knockdown of calcium/calmodulin-dependent protein kinase kinase 1 (CaMKK1), resulting in decreased phospho-Akt Thr308 , enhanced apoptosis in SK-BR-3 but not in MCF-7 cells. CaMKK2 knockdown moderately decreased phospho-Akt Thr308 and increased apoptosis in MCF-7 cells but not in SK-BR-3 cells. These data indicated that PCP4/PEP19 regulates apoptosis but exact mechanism is still unknown. PCP4/PEP19 can therefore potentially serve as independent oncotarget for therapy of PCP4/PEP19-positive breast cancers irrespective of ER expression.