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Cathepsin H Regulated by the Thyroid Hormone Receptors Associate with Tumor Invasion in Human Hepatoma Cells

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Cathepsin H Regulated by the Thyroid Hormone Receptors Associate with Tumor Invasion in Human Hepatoma Cells Oncogene (2011) 30, 2057–2069 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells S-M Wu1, Y-H Huang2, C-T Yeh3, M-M Tsai1,4, C-H Liao1, W-L Cheng1, W-J Chen5 and K-H Lin1 1Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan, Taiwan; 2Medical Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 3Liver Research Unit, Chang-Gung Medical Center, Taipei, Taiwan; 4Department of Nursing, Chang-Gung Institute of Technology, Taoyuan, Taiwan and 5First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan, Taiwan 0 Thyroid hormone, 3, 3 , 5-triiodo-L-thyronine (T3), Introduction mediates cell growth, development and differentiation by 0 binding to its nuclear receptors (TRs). The role of TRs in Thyroid hormone (3, 3 , 5-triiodo-L-thyronine; T3)isa cancer is still undefined. Notably, hyperthyroxinemia has potent mediator of many physiological processes in- been reported to influence the rate of colon cancer in an cluding embryonic development, cell differentiation, experimental model of carcinogenesis in rats. Previous metabolism and the regulation of cell proliferation microarray analysis revealed that cathepsin H (CTSH) is (Huang et al., 2008). Two TR genes, THRA and THRB, upregulated by T3 in HepG2-TR cells. We verified that have been identified and mapped to human chromo- mRNA and protein expression of CTSH are induced by somes 17 and 3, respectively (Lazar, 1993; Yen, 2001). T3 in HepG2-TR cells and in thyroidectomized rats The liver is a typical target organ for T3. Equal amounts following administration of T3. The possible thyroid of TRa1 and TRb1 proteins are expressed in human hormone-responsive elements of the CTSH promoter hepatocytes (Chamba et al., 1996). However, the localized to the nucleotides –2038 to –1966 and –1565 mechanisms involved in the regulation of liver-specific to –1501 regions. An in vitro functional assay showed that genes by TRa1 and TRb1 have not been elucidated. CTSH can increase metastasis. J7 cells overexpressing Evidence that TRs might be involved in carcinogen- CTSH were inoculated into severe combined immune- esis came from studies on v-erbA protein (Thormeyer deficient mice and these J7-CTSH mice displayed a and Baniahmad, 1999), thus, v-erbA antagonizes TR greater metastatic potential than did J7-control mice. The function via competition for response elements or clinicopathologic significance of CTSH expression in coactivators and ligand-dependent transcriptional acti- hepatocellular carcinoma (HCC) was also investigated. vation (Yen et al., 1994). That TRs can have an The CTSH overexpressing in HCC was associated with important role in tumor transformation was further the presence of microvascular invasion (P ¼ 0.037). The verified by aberrant expression and mutations of TRs in microvascular invasion characteristic is closely related to various human cancers including pituitary tumors our in vitro characterization of CTSH function. Our (Ando et al., 2001), hepatocellular carcinomas (HCCs) results show that T3-mediated upregulation of CTSH led (Lin et al., 2001), thyroid cancers (Puzianowska- to matrix metallopeptidase or extracellular signal-regu- Kuznicka et al., 2002) and renal clear cell carcinomas lated kinase activation and increased cell migration. This (Rosen and Privalsky, 2009). Transgenic mice (TRbPV/PV), study demonstrated that CTSH overexpression in a subset harboring a PV mutation originally identified in a hepatoma may be TR dependent and suggests that this patient with thyroid hormone resistance, spontaneously overexpression has an important role in hepatoma developed thyroid cancer (Suzuki et al., 2002). This progression. study showed that TRb could act as a tumor suppressor. Oncogene (2011) 30, 2057–2069; doi:10.1038/onc.2010.585; Recently, Zhu et al. (2010) reported that mice devoid of published online 10 January 2011 functional TRs (TRa1À/ÀTRb1À/À) spontaneously devel- Keywords: cysteine proteases; receptors; migration; op follicular thyroid cancer and metastases in the lung. hepatocellular carcinoma TRs could function as tumor suppressors in such mice. Besides their suppressor role, TRs enhanced tumor progression in other studies. Thyroid hormones stimu- late the proliferation of several cancer cell types including those from the pituitary (Barrera-Hernandez Correspondence: Dr K-H Lin, Department of Biochemistry, School of et al., 1999), gliomas (Davis et al., 2006), breast cancers Medicine, Chang-Gung University, 259 Wen-Hwa 1st Road, Taoyuan (Hall et al., 2008) and prostate cancers (Tsui et al., 333, Taiwan. 2008). Thyroid hormone and the TRa1 receptor control E-mail: [email protected] Received 14 June 2010; revised 23 November 2010; accepted 26 epithelial cell proliferation and the expression of November 2010; published online 10 January 2011 components of the Wnt pathway (Plateroti et al., 2006). Thyroid hormone receptor regulates cathepsin H S-M Wu et al 2058 Various proteases are implicated in cancer progres- et al., 2008a), SPON2 (Liao et al., 2010), MATA1A (Wu sion by degrading the basement membrane and extra- et al., 2010), SULT2A1 (Huang et al., 2006) and cellular matrix in a cascade-like manner (Mohamed and AKR1B1 (Liao et al., 2009). Supplementary Table S1 Sloane, 2006; Wilson and Singh, 2008). Cysteine lists several cancer-related genes, including those encod- cathepsins are a family of lysosomal proteases that are ing cathepsin proteases positively regulated by T3. often upregulated in various human cancers, and these We first used oligonucleotide microarrays to identify proteases have been implicated in distinct tumorigenic the genes induced by T3 in HepG2-TRa cells. Upregula- processes, such as angiogenesis, proliferation, apoptosis tion of cathepsin cysteine proteases was identified and and invasion (Berdowska, 2004; Joyce and Hanahan, the result was verified by quantitative reverse transcrip- 2004; Gocheva and Joyce, 2007). Cysteine cathepsin tion–PCR. Several human cysteine cathepsins (CTSH, proteases have recently emerged as enzymes that are CTSB, CTSS and CTSL) were induced by 3.2–8.6 times active in cancer development, and cysteine cathepsin after the incubation of HepG2-TRa1#1 cells with 10 nM inhibitors have been proposed as anticancer agents T3 for 72 h (Supplementary Figure S1). However, the (Palermo and Joyce, 2008). There is increasing evidence mRNA levels of cysteine proteases, such as CTSV, were that the expression of cathepsin H (CTSH) is increased not induced by T3. in disease states including breast carcinoma (Gabrijelcic Northern blot analysis was used to assess further the et al., 1992), melanoma (Kos et al., 1997), glioma response of CTSH mRNA expression to the exogenous (Sivaparvathi et al., 1996), and colorectal and prostate addition of T3. A 1.47-kb CTSH transcript was carcinoma (del Re et al., 2000). identified in HepG2-TRa1#1 cells (Figure 1a). The Although several individual target genes have been CTSH mRNA levels were induced by 3–4 times after studied in the liver, a large-scale profile of the target incubation of HepG2-TRa1#1 cells with 10 nM T3 for genes regulated by thyroid hormone has not been 48 h. A similar induction was observed in HepG2- undertaken. CTSH gene expression was induced by T3 TRa1#2 and -TRb1 cells. As a control, HepG2 cells in HepG2-TRa cells identified from oligonucleotide were transfected with the empty vector, yielding a cell microarrays. CTSH and other proteases may be line expressing the Neo protein (HepG2-Neo cells). involved in the destruction of extracellular matrix components, leading to cancer proliferation, migration and metastasis (Tsushima et al., 1991). However, T3 upregulates expression of CTSH protein in HepG2-TR elucidation of the underlying mechanisms by which T3 cells regulated requires further study. Previous reports have The effect of TRs on the degree of CTSH protein shown that TRs are potent inducer of tumorigenesis expression in HepG2 isogenic cell lines was studied (Plateroti et al., 2006; Chen et al., 2008a; Kress et al., (Figure 1b). CTSH is synthesized as an inactive 2009). In this study, we report that the T3/TR controls proenzyme (41 kDa, upper arrow), which is then the transcription of the CTSH gene and that T3- processed proteolytically to an active single chain, mediated cell migration appears to involve CTSH. mature form (28 kDa, lower arrow) within the endo- somes/lysosomes (Turk et al., 2001). The incubation results indicated that T3 increased the amount of 28- kDa CTSH protein in the HepG2-TRa1#1, -TRa1#2 Results and -TRb1 cells. The CTSH protein levels increased after incubation of HepG2-TRa1#1, -TRa1#2 and -TRb1 Cathepsin mRNA is regulated by T3 in the HepG2-TR cell line cells with 1 or 10 nM T3 for 24–72 h. These results show We used isogenic HepG2 cell lines that consistently that the effect of T3 on the CTSH protein level in TRa1- express wild-type TRa1 (HepG2-TRa1#1, #2) and overexpressing cells is time and dose dependent. TRb1 (HepG2-TRb1). As a control, HepG2 cells were Immunoblot analysis showed that incubation of control transfected with the empty vector, yielding a cell line HepG2-Neo cells, which did not express detectable expressing the Neo protein (HepG2-Neo cells). In the endogenous TR proteins, in 10 nM T3 for 24–72 h did not four HepG2 cell lines investigated in this study, HepG2- increase the CTSH protein level significantly. However, TRa1#1, -TRa1#2, -TRb1 and -Neo, overexpression of the CTSH proenzyme was affected marginally by T3. TR protein was approximately 10-, 3.3- and 2.5-fold To analyze whether cathepsin is secreted into the that of the HepG2-Neo control cell line (data not culture medium, the HepG2-TR stable cell line was shown). Previously, complementary DNA-microarrays incubated in serum-free medium, and the medium was were performed to identify the target genes regulated collected at 24 and 48 h. Analysis using anti-CTSH by T3 in a hepatoma cell line-overexpressing TRa1 antibodies revealed a major extracellular product with (HepG2-TRa1) (Shih et al., 2004).
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