Selective inhibition of NF-jB suppresses bone invasion by oral squamous cell carcinoma in vivo Hiroyuki Furuta 1,2 , Kenji Osawa 1 , Masashi Shin 3 , Ayataka Ishikawa 4 , Kou Matsuo 4 , Masud Khan 5,6 , Kazuhiro Aoki 5 , Keiichi Ohya 5 , Masato Okamoto 7 , Kazuhiro Tominaga 8,9 , Tetsu Takahashi 10 , Osamu Nakanishi 2 and Eijiro Jimi 1,9 1 Division of Molecular Signaling and Biochemistry, Department of Bioscience, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Fukuoka, Japan 2 Division of Dental Anesthesiology, Department of Control of Physical Functions, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Fukuoka, Japan 3 Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka-shi, Saitama, Japan 4 Division of Oral Pathology, Department of Bioscience, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Fukuoka, Japan 5 Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan 6 Global Center of Excellence (G-COE) Program, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo Japan 7 Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo Japan 8 Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Fukuoka, Japan 9 Center for Oral Biological Research, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Fukuoka, Japan 10 Division of Oral and Maxillofacial Reconstructive Surgery, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kokurakita-ku, Kitakyushu, Fukuoka, Japan Nuclear factor-jB (NF-jB) is constitutively activated in many cancers, including oral squamous cell carcinoma (OSCC), and is involved in the invasive characteristics of OSCC, such as growth, antiapoptotic activity and protease production. However, the cellular mechanism underlying NF-jB’s promotion of bone invasion by OSCC is unclear. Therefore, we investigated the role of NF-jB in bone invasion by OSCC in vivo. Immunohistochemical staining of OSCC invading bone in 10 patients indicated that the expression and nuclear translocation of p65, a main subunit of NF-jB, was increased in OSCC compared with normal squamous epithelial cells. An active form of p65 phosphorylated at serine 536 was present mainly in the nucleus in not only differentiated tumor cells but also tumor-associated stromal cells and bone-resorbing osteoclasts. We next injected mouse OSCC SCCVII cells into the masseter region of C 3 H/HeN mice. Mice were treated for 3 weeks with a selective NF-jB inhibitor, NBD peptide, which disrupts the association of NF-jB essential modulator (NEMO) with IjB kinases. NBD peptide treatment inhibited TNFa-induced and constitutive NF-jB activation in SCCVII cells in vitro and in vivo, respectively. Treatment with NBD peptide decreased zygoma and mandible destruction by SCCVII cells, reduced number of osteoclasts by inhibiting RANKL expression in osteoblastic cells and SCCVII cells, increased apoptosis and suppressed the proliferation of SCCVII cells. Taken together, our data clearly indicate that inhibition of NF-jB is useful for inhibiting bone invasion by OSCC. Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity, head and neck. 1–3 Despite advances in our understanding of the molecular mechanisms of tumor development, prevention and treatment, the long- term survival for patients with OSCC, a cancer that, worldwide, accounts for more than 500,000 cases each year, 1 remains low. The poor prognosis for OSCC reflects a limited understanding of the mechanisms of local and regional invasion and metasta- sis present in a significant portion of patients, together with an unsatisfactory responsiveness to conventional systemic therapy in recurrent and advanced disease. 2,3 The ability of OSCC to invade the maxilla or mandibular bone is a critical factor, which, because it leads to metastasis, affects the prognosis of patients. 4 Although controversial, bone destruction that occurs with OSCC invasion is thought to be mediated by osteoclasts rather than by the carcinoma itself. 4 Recent studies have established that bone resorption by osteoclasts is an important step in the process of bone invasion and metastasis in several types of malignancy, 5 indi- cating that a full understanding of the regulation of osteoclas- togenesis by OSCC cells is necessary to prevent bone inva- sion by OSCC cells. Several in vitro and animal experiments using human OSCC cells have shown that tumor cells Key words: NF-jB, NBD peptide, bone invasion, oral squamous cell carcinoma Additional Supporting Information may be found in the online version of this article. Conflicts of interest: The authors declare no conflict of interest. Grant sponsor: Kyushu Dental College Internal Grants; Grant sponsor: The Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant numbers: 70549261 and 23592707 DOI: 10.1002/ijc.27435 History: Received 19 Sep 2011; Accepted 29 Dec 2011; Online 19 Jan 2012 Correspondence to: Eijiro Jimi, Division of Molecular Signaling and Biochemistry, Department of Biosciences, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, Japan, Tel.: þ[81-93-285-3047], Fax: þ[81-93-582-6000], E-mail: ejimi@kyu-dent.ac.jp Cancer Cell Biology Int. J. Cancer: 131, E625–E635 (2012) V C 2012 UICC International Journal of Cancer IJC