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Identification of Salt-Inducible Kinase 3 As a Novel Tumor Antigen

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Identification of Salt-Inducible Kinase 3 As a Novel Tumor Antigen Oncogene (2011) 30, 3570–3584 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Identification of salt-inducible kinase 3 as a novel tumor antigen associated with tumorigenesis of ovarian cancer S Charoenfuprasert1,2,9, Y-Y Yang3,10, Y-C Lee3,10,11, K-C Chao4,10, P-Y Chu5, C-R Lai6, K-F Hsu7, K-C Chang8, Y-C Chen9, L-T Chen9, J-Y Chang9, S-J Leu1,2,12 and N-Y Shih9,12 1Graduate Institute of Medical Science, College of Medicine, Taipei Medical University, Taipei, Taiwan; 2Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 3School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; 4Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei, Taiwan; 5Department of Surgical Pathology, Changhua Christian Hospital, Changhua, Taiwan; 6Department of Pathology, Taipei Veterans General Hospital, Taipei, Taiwan; 7Department of Obstetrics and Gynecology, National Cheng Kung University, Tainan, Taiwan; 8Department of Pathology, National Cheng Kung University, Tainan, Taiwan and 9National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan Existence of humoral immunity has been previously novel ovarian TAA. Overexpression of SIK3 promotes demonstrated in malignant ascitic fluids. However, only G1/S cell cycle progression, bestows survival advantages a limited number of immunogenic tumor-associated to cancer cells for growth and correlates the clinicopatho- antigens (TAAs) were identified, and few of which are logical conditions of patients with ovarian cancer. associated with ovarian cancer. Here, we identified salt- Oncogene (2011) 30, 3570–3584; doi:10.1038/onc.2011.77; inducible kinase 3 (SIK3) as a TAA through screening of a published online 14 March 2011 random peptide library in the phage display system. Overexpression of SIK3 markedly promoted cell prolif- Keywords: tumor-associated antigen; ovarian cancer; eration, attenuated p21Waf/Cip1 and p27Kip expressions in CA125; malignant ascitic fluid; phage display low-grade OVCAR3 cells, and permitted the cells to grow in mice. Decrease in SIK3 expression in high-grade SK- OV3 cells consistently demonstrated its tumorigenic potency by modulating the protein levels of cell cycle Introduction regulators. When the expressions of SIK3 and CA125 were compared in cancer tissues, immunohistochemical Ovarian carcinoma represents one of the most insidious (IHC) studies indicated that cytoplasm-localized SIK3 and aggressive cancers and is also the most lethal was highly expressed in 55% of the ovarian cancer gynecological malignancy in the United States and samples. In contrast, it was rarely detected in adenomyo- Taiwan. The high mortality rate of ovarian cancer is sis, leiomyoma and normal ovary tissues, showing its a consequence of the fact that 70–75% of women with higher specificity (97%) to CA125 (65%) in ovarian this disease are diagnosed with stage III or IV disease cancer. Moreover, experiments using pharmacological (Runnebaum and Stickeler, 2001). Thus, discovery of inhibitors to block SIK3-induced p21Waf/Cip1 expression new relevant biomarkers to increase specificity or revealed that activation of c-Src and phosphoinositide-3- sensitivity for early diagnosis and prognosis of ovarian kinase were critically required for its biological activity, cancer is an important and urgent need. Besides their suggesting that they are the downstream signaling potential clinical use for diagnosis and prognosis of this mediators of SIK3. These data were further supported disease, study of their pathological functions in tumori- by IHC studies, showing coexpression of c-Src with SIK3 genesis or in tumor malignancy is not only provide in 85% of the ovarian tumor samples stained positive for molecule-basis understanding in fundamental cancer SIK3. Collectively, our findings indicate that SIK3 is a biology, but also may identify potential signaling mediators of the biomarkers in cancer cells for targeting therapy in future. Correspondence: Dr S-J Leu, Department of Microbiology and Immuno- One promising approach in this field is to study logy, School of Medicine, College of Medicine, Taipei Medical University, the immune responses to cancer and identify more Taipei 110, Taiwan. tumor-associated antigens (TAAs) for clinical applica- E-mail: [email protected] or Professor N-Y Shih, National Institute of Cancer Research, NHRI, Tainan 706, Taiwan. tions. The presence of humoral response to TAAs has E-mail: [email protected] been well demonstrated in patients with various cancers. 10These authors contributed equally to this work. The responses are frequently mounted against tumor 11Current address: The Genomic Research Center, Academia Sinica, cells expressing altered or abnormally high levels of Taipei 115, Taiwan. 12S-J Leu and N-Y Shih co-correspond to this work. normal self-antigens with inappropriate timing (Brewer Received 7 September 2010; revised 29 January 2011; accepted 9 et al., 2009), cellular compartmentalization (Chang February 2011; published online 14 March 2011 et al., 2006) or tissue expression (Old, 2008) that result SIK3 is involved in ovarian tumorigenesis S Charoenfuprasert et al 3571 in increased immunogenicity. Therefore, high levels of loss of the cell polarity is closely associated with tumor-associated autoantibodies can be also observed in development of the epithelial–mesenchymal transition of some cancer patients throughout their disease progres- epithelial-derived cancer cells during tumor metastasis sions (Gagnon et al., 2008), and some of them are (Guarino, 2007), it may represent a key mechanism associated with their disease outcomes (Reuschenbach by which LKB1 and SIK3 are related to cancers. Here, we et al., 2009). Currently, over 100 immunogenic TAAs report that SIK3 is an ovarian TAA. Increasing expres- have been reported in patients with a variety of cancers; sion of SIK3 promoted G1/S cell cycle progression via however, few of them are associated with ovarian activation of c-Src-phosphoinositide-3-kinase (PI3K) cancer, including CA125 (Reuschenbach et al., 2009; linkage to downregulate p21Waf/Cip1 and led to tumorigen- Taylor et al., 2009). esis in mice. As SIK3 is highly and preferentially expressed CA125 is the best-characterized tumor marker for in ovarian tumors but not in adenomyosis and leiomyo- advanced epithelial ovarian cancer. CA125 is a type I ma, it can be a potential diagnostic marker for ovarian transmembrane protein expressed on the cell surface cancers. (O’Brien et al., 2002). Its soluble proteolytic fragment can be released into the blood stream. Thus, serum CA125 has been studied intensively as a potential biomarker for diagnosis of epithelial ovarian cancer or Results to monitor disease recurrence after therapy (Baron et al., 2005; Rosenthal et al., 2006). However, elevated Identification of KIAA0999 gene product, SIK3, as the CA125 expressions are not only observed in >80% of target of the CA502 ascitic antibody patients with advanced epithelial ovarian cancer, but are Although tumor-associated antibodies have been found also found in various physiological or pathological in the ascitic fluids of ovarian cancer patients (Giancotti conditions (Cannistra, 2004). In this study, we used et al., 1990), only a few ovarian TAAs were identified. serological screening of a phage library with malignant In this study, the ascitic tumor cells of 12 patients were ascitic antibodies and identified salt-inducible kinase 3 first purified with Ficoll–Percoll gradient centrifugation, (SIK3) as a novel TAA for ovarian cancers. lyzed and immunoblotted with their ascitic auto- SIKs have their kinase domain sequences closely antibodies as described previously (Chang et al., 2006). homologous to AMP-activated protein kinase (AMPK) The ascitic antibodies of patient CA502 were highly (Lizcano et al., 2004; Bright et al., 2009), and may have immunoreactive to a major protein band with an a role in steroidogenesis, adipogenesis or regulation of approximate molecular mass of 150 kDa in the auto- tumor malignancy. Three isoforms in the SIK family logous tumor lysates (Supplementary Figure 1A). have been identified so far, namely, SIK1 (SNF1LK), To identify the target of the autoantibody, a 7-mer SIK2 (QIK or SNF1LK2) and SIK3 (KIAA0999 or random peptide phage library was screened as illustrated QSK) (Katoh et al., 2004; Lizcano et al., 2004). SIK1 is in Figure 1a. The library was pre-absorbed with the total an important regulator for steroidogenic gene expres- immunoglobulin G (IgG) mix of 10 healthy donors to sion, and can be activated by increased intracellular remove the phages recognized by the IgG. Subsequently, sodium leading to stimulation of Na þ /K þ -ATPase the resultant library was applied onto enzyme-linked activity and activation of calmodulin kinase (Sjostrom immunosorbent assay wells pre-coated with the et al., 2007). In addition, it is also reported to participate CA502 ascitic IgG. After four biopanning cycles, the in a negative feedback program initiated by transform- bound phages were eluted and allowed to infect their ing growth factor-b (Kowanetz et al., 2008). Loss of Escherichia coli host cells. Direct sequence analyses of SIK1 expression facilitated lung metastatic spread in 15 random clones revealed 3 residues highly conserved mice and was closely correlated with the development of in the displayed peptides,
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  • Supplementary Data
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