LETTERS The ubiquitin ligase CHIP acts as an upstream regulator of oncogenic pathways Masashi Kajiro 1,6 , Ryuichi Hirota 1,6 , Yuka Nakajima 1 , Kaori Kawanowa 2 , Kae So-ma 1 , Ichiaki Ito 1 , Yuri Yamaguchi 3 , Sho-hei Ohie 1 , Yasuhito Kobayashi 2 , Yuko Seino 3 , Miwako Kawano 1 , Yoh-ichi Kawabe 1 , Hiroyuki Takei 2 , Shin-ichi Hayashi 4 , Masafumi Kurosumi 2 , Akiko Murayama 1,5 , Keiji Kimura 1 and Junn Yanagisawa 1,5,7 CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins 1–12 . The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs13–19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3. We examined the mRNA and protein levels of CHIP (carboxyl termi- nus of Hsc70-interacting protein) in human breast cancer tissues, as it controls protein levels of several oncogenic proteins 9–12 , including oes- trogen receptor α (ERα). Samples of breast cancer tumours and normal tissue were obtained from 27 patients with breast cancer (Supplementary Information, Table S1), and the expression levels of CHIP mRNA were examined. In cancerous tissues from patients clinically determined to be stage I (Fig. 1a), or from node-negative patients (Fig. 1b), we found that the level of CHIP mRNA expression was comparable to that observed in the corresponding normal breast tissue (P = 0.83, Fig. 1a and P = 0.16, Fig. 1b). Tumours from stage II patients (Fig. 1a) or those from node-positive (N1a) patients (Fig. 1b) showed decreased levels of CHIP mRNA, compared with the non-cancerous tissues (P < 0.001, Fig. 1a, b). These results demon- strate a significant negative correlation between CHIP mRNA levels and tumour malignancy in human breast cancer tissues. We also compared the correlation between CHIP mRNA levels and tumour grade using the Oncomine database, which provides publicly available cancer gene expression datasets. Nine of 21 datasets that contain gene chip profiles classified by breast tumour grade, showed an inverse correlation between CHIP mRNA expression and tumour grade, consistent with our results. Two datasets characterized by large population sizes showed a significant inverse correlation between CHIP expression levels and tumour grade (Desmedt_Breast and vantVeer_Breast, P = 0.00035 and P = 0.00093, respectively). Immunohistochemical staining indicates that expression levels of CHIP proteins also decreased in malignant tumours (Fig. 1c). Thus, these results demonstrate that CHIP protein and mRNA levels were reduced in the invasive tumour region. As CHIP is a ubiquitin ligase for ERα, we expected the inverse correlation between CHIP mRNA and ERα protein levels. We did not observe a significant correlation between CHIP expression and ERα positivity (P = 0.18; Fig. 1d). Moreover, none of the 32 datasets from the Oncomine database (each dataset contains gene chip profiles classified by ERα positivity) demonstrated an inverse correlation between CHIP expression levels and ERα positivity. We then examined whether tumour progression was enhanced when the expression level of CHIP was downregulated. Protein levels of CHIP were much higher in MCF-7 cells, a non-aggressive cell line derived from human breast cancer cells, than in MDA-MB-231 cells, a highly aggressive cell line (Fig. 2a). We generated three MCF-7 clones in which endogenous CHIP expression was knocked down by a short hairpin RNA (shRNA; Fig. 2b). To investigate the effects of CHIP knockdown in vivo, ten nude 1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan. 2 Department of Pathology, Saitama Cancer Center, 818 Komuro, Ina-machi, Kitaadachi- gun, Saitama 362-0806, Japan. 3 Research Institute for Clinical Oncology, Saitama Cancer Center, 818 Komuro, Ina-machi, Kitaadachi- gun, Saitama 362-0806, Japan. 4 Department of Medical Technology, Course of Health Sciences, School of Medicine, Tohoku University, 2-1 Seiryou-machi, Aoba-ku, Sendai, 980-8575, Japan. 5 Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan. 6 These authors contributed equally to this work. 7 Correspondence should be addressed to J.Y. (e-mail: e-mail: junny@agbi.tsukuba.ac.jp) Received 26 August 2008; accepted 25 November 2008; published online 8 February 2009; DOI: 10.1038/ncb1839 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2009 Macmillan Publishers Limited. All rights reserved.