Oncogene (2009) 28, 4162–4174 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Thyroid hormone receptor mutants implicated in human hepatocellular carcinoma display an altered target gene repertoire IH Chan and ML Privalsky Department of Microbiology, College of Biological Sciences, University of California at Davis, Davis, CA, USA Thyroid hormone receptors (TRs) are hormone-regulated lapping, biological roles (Lazar, 1993; Murata, 1998; transcription factors that control multiple aspects of Yen, 2001; Harvey and Williams, 2002). Both TRa1 and normal physiology and development. Mutations in TRs TRb1 bind to specific DNA sequences (response have been identified at high frequency in certain cancers, elements), and regulate the expression of adjacent target including human hepatocellular carcinomas (HCCs). The genes up or down (Katz and Koenig, 1993; Lazar, 1993, majority of HCC–TR mutants bear lesions within their 2003; Yen, 2001). On ‘positively regulated’ target genes, DNA recognition domains, and we have hypothesized that TRs recruit corepressors and repress gene transcription these lesions change the mutant receptors’ target gene in the absence of T3, but release corepressors, recruit repertoire in a way crucial to their function as oncoproteins. coactivators and activate gene transcription in the Using stable cell transformants and expression array presence of T3 (Cheng, 2000; Zhang and Lazar, 2000; analysis, we determined that mutant TRs isolated from Harvey and Williams, 2002; Tsai and Fondell, 2004). two different HCCs do, as hypothesized, display a target There is less understanding of ‘negatively regulated’ gene repertoire distinct from that of their normal TR target genes that are activated by TRs in the absence, progenitors. Only a subset of genes regulated by wild-type but repressed in the presence, of hormone; this inverted TRs was regulated by the corresponding HCC–TR T3 response presumably arises because of alterations in mutants. More surprisingly, the HCC–TR mutants also coregulator recruitment or function, perhaps reflecting gained the ability to regulate additional target genes not combinatorial interactions with other transcription recognized by the wild-type receptors, and were not simply factors also arrayed on the same promoter (Meyer restricted to repression, but could also activate a subset of et al., 1997; Tagami et al., 1997; Berghagen et al., 2002; their target genes. We conclude that the TR mutants Nygard et al., 2003; Matsushita et al., 2007). isolated from HCC have sustained multiple alterations Disruptions of TR function lead to disease (Yen and from their normal progenitors that include not only changes Cheng, 2003; Cheng, 2005). Inherited mutations in the in their transcriptional outputs, but also changes in the TRb locus result in human resistance to thyroid genes they target; both are likely to contribute to neoplasia. hormone syndrome, an endocrine disorder (Refetoff, Oncogene (2009) 28, 4162–4174; doi:10.1038/onc.2009.265; 1993; Refetoff et al., 1993; DeGroot, 1996; Kopp et al., published online 14 September 2009 1996; Yen, 2003). A virally transduced mutant form of TRa contributes to leukemogenesis by the avian Keywords: hepatocellular carcinoma; thyroid hormone erythroblastosis retrovirus (Graf and Beug, 1983; Sap receptors; gene expression; mutant; dominant negative et al., 1986; Weinberger et al., 1986; Damm et al., 1989; Privalsky, 1992). Spontaneous mutations in TRa and TRb are found at high frequency in human hepatocel- lular carcinomas (HCCs), renal clear cell carcinomas, Introduction and certain thyroid malignancies and are believed to participate in the initiation or progression of these Thyroid hormone receptors (TRs) have key roles in malignancies (Lin et al., 1996, 1999; Kamiya et al., 2002; normal physiology and development (Brent, 2000; Yen, Puzianowska-Kuznicka et al., 2002; Cheng, 2003; 2001; Harvey and Williams, 2002; Buchholz et al., 2006; Gonzalez-Sancho et al., 2003; Chan and Privalsky, Flamant et al., 2006). Two distinct genetic loci encode 2006). Virtually, all of these TR mutants are impaired in TRs, denoted a and b, each of which is alternatively the ability to exchange coactivator for corepressor in spliced to generate additional receptor diversity. TRa1 response to physiological concentrations of T3. As a and TRb1 are among the most abundant of these result, the mutant receptors retain corepressor inappro- receptor isoforms, and exert distinct, if partially over- priately and can function as dominant-negative inhibi- tors of wild-type receptor function (Lin et al., 1996, Correspondence: Professor ML Privalsky, Department of Microbio- 1997; Chan and Privalsky, 2006; Privalsky, 2008). logy, One Shields Avenues, University of California at Davis, Davis, Given this commonality, why do certain dominant- CA 95616, USA. negative TRs produce primarily endocrine disease, E-mail: [email protected] Received 13 April 2009; revised 3 July 2009; accepted 31 July 2009; whereas others are closely associated with neoplasia? published online 14 September 2009 Notably, the mutant TRs found in resistance to thyroid Altered gene recognition by TR mutants in HCC IH Chan and ML Privalsky 4163 hormone syndrome represent single mutational events, whereas the mutant TRs found in neoplasia are typically aggregates of multiple genetic lesions, often including mutations in the DNA recognition domain of these receptors (Refetoff, 1993; Lin et al., 1999; Kamiya et al., 2002; Puzianowska-Kuznicka et al., 2002; Yen and Cheng, 2003). We have proposed that single TR mutations give rise to simple dominant-negative recep- tors that produce endocrine disorders, whereas addi- tional mutations further modify the dominant-negative phenotype, at least in part by altering target gene recognition, to generate the neoplastic phenotype (Chan and Privalsky, 2006; Privalsky, 2008). To test this hypothesis, we used a microarray analysis to compare the target gene profiles of wild-type and HCC mutant TRs. We report that the HCC–TR mutants tested showed widespread alterations in their target gene repertoire compared with the corresponding wild-type controls, and we discuss how these changes may contribute to oncogenesis. Figure 1 The human hepatocellular carcinoma–thyroid hormone receptor (HCC–TR) mutants are impaired in transcriptional Results activation. (a) Schematic of wild-type and mutant TRs. The different TRs are depicted with the locations of the DNA binding The HCC–TR mutants are impaired in transcriptional (DBD), hormone-binding domain (HBD) and HCC mutations activation and act as dominant-negative inhibitors of indicated. (b) Impaired reporter activation in HCC mutant TR transformants. HepG2 cells stably integrated with TRa1-WT, wild-type TRs in reporter assays TRb1-WT, TRa1-I, TRb1-N or an empty-plasmid control (no- HepG2 are a well-characterized human HCC cell line receptor (NR)) were transiently transfected with a DR4-TK- that lacks known TR mutations and expresses modest luciferase reporter and pCH110 as an internal control. The cells levels of wild-type TRa1andTRb1 (Chamba et al., were treated 24 h later with T3 or vehicle alone, harvested 48 h after 1996; Lin et al., 1996). We generated stable HepG2 transfection, and the luciferase activity was determined relative to b-galactosidase activity (mean þ s.e.m., n ¼ 3). transformants expressing a representative HCC–TRa1 mutant (denoted TRa1-I), a representative HCC-TRb1 mutant (denoted TR-b1-N), wild-type TRa1 (TRa1- WT), wild-type TRb1 (TRb1-WT), or an empty expres- microarray analysis. The TRa1-I and TRb1-N mutants sion vector control (Figure 1a). We first examined the have been suggested to operate in cancer by constitu- overall T3 response in these cells using a luciferase tively mimicking the repressive functions mediated by reporter containing an archetypal DR4 positive- the wild-type receptors in the absence of T3 (Chan and response element (Figure 1b). The HepG2 transformants Privalsky, 2006). Therefore, we first identified the containing the empty expression plasmid showed a low cellular genes repressed by the mutant TRs relative to level T3-mediated regulation of this reporter mediated the empty-vector transformants, and compared these by the endogenous TRs present in these cells (Darby genes to those repressed by the wild-type TRs relative to et al., 1991; Theriault et al., 1992). Transformants the empty-vector transformants, all in the absence of T3 bearing the TRa1-WT constructs showed a significantly (symbolized as kTR–T3) (Figures 2a and b). Representa- enhanced T3 response (Figure 1b); a similar, although tive target genes were confirmed by reverse transcrip- more modest T3 response was also observed for TRb1- tase–PCR (RT–PCR) (Figure 2c and data not shown). WT (Figure 1b). In contrast, HepG2 transformants Notably, several genes, such as BICC1, were strongly expressing the TRa1-I or TRb1-N mutants showed repressed by all TRs tested (Figures 2b and c). However, reduced levels of reporter expression in the absence of other genes were repressed by the wild-type TR, but not T3 compared with the empty-vector controls, and a by the corresponding mutant under these conditions. severely attenuated reporter gene activation in response For example, DOCK9 was repressed by TRa1-WT but to T3. These results support previous findings that these not TRa1-I, and PROM1 was repressed by both HCC–TR mutants are impaired for T3-induced tran- TRa1-WT and TRb1-WT, but not by either mutant scriptional