CREB3L2-Pparg Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

Research Article

CREB3L2-PPARg Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

Weng-Onn Lui,3 Lingchun Zeng,1 Victoria Rehrmann,1 Seema Deshpande,5 Maria Tretiakova,1 Edwin L. Kaplan,2 Ingo Leibiger,3 Barbara Leibiger,3 Ulla Enberg,3 Anders Ho¨o¨g,4 Catharina Larsson,3 and Todd G. Kroll1

Departments of 1Pathology and 2Surgery, University of Chicago Medical Center, Chicago, Illinois; Departments of 3Molecular Medicine and Surgery and 4Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden; and 5Department of Pathology, Emory University School of Medicine, Atlanta, Georgia

Abstract cancer in patients; and (c) the implementation of effective molecular-targeted chemotherapies that have relatively few side The discovery of gene fusion mutations, particularly in effects. The discovery of fusion mutations is therefore important, leukemia, has consistently identified new cancer pathways particularly in carcinoma, the most common cancer group in and led to molecular diagnostic assays and molecular-targeted which few gene fusions have been identified (1). The recent chemotherapies for cancer patients. Here, we report our discoveries of ERG (2) and ALK (3) gene fusions in prostate and discovery of a novel CREB3L2-PPARg fusion mutation in lung carcinoma, respectively, increase the prospect that new thyroid carcinoma with t(3;7)(p25;q34), showing that a family diagnostic and therapeutic strategies based on gene fusions will of somatic PPARg fusion mutations exist in thyroid cancer. The be applicable to common epithelial cancers. CREB3L2-PPARg fusion encodes a CREB3L2-PPAR; fusion Families of gene fusions tend to characterize specific cancer protein that is composed of the transactivation domain of types. For example, the RUNX1-ETO and CBFb-SMMHC gene CREB3L2and all functional domains of PPAR ;1. CREB3L2- fusions underlie acute myeloid leukemia and deregulate the RUNX/ PPARg was detected in <3% of thyroid follicular carcinomas. CBFh transcription factor complex by distinct molecular mecha- Engineered overexpression of CREB3L2-PPAR; induced pro- nisms (4). In a similar fashion, the PML-RARa, PLZF-RARa, NPM- liferation by 40% to 45% in primary human thyroid cells, RARa, NuMA-RARa, and Stat5b-RARa gene fusions underlie acute consistent with a dominant oncogenic mechanism. Wild-type promyelocytic leukemia. The PML-RARa gene fusion is present in CREB3L2was expressed in the thyroid as a bZIP transcription >95% of acute promyelocytic leukemia patients, whereas the other factor with a transmembrane domain that has flanking S1P RARa fusions are observed at low incidence. Even so, the low and S2P proteolytic cleavage sites. Native CREB3L2 was cleaved incidence gene fusions have revealed molecular mechanisms that to nuclear CREB3L2by regulated intramembrane proteolysis were unapparent from investigation of PML-RARa alone (5, 6) and in normal thyroid cells that expressed the S1P and S2P have identified biological pathways that are important in both proteases. Nuclear CREB3L2stimulated transcription 8-fold acute promyelocytic leukemia and other cancer types. from the EVX1 cyclic AMP (cAMP) response element in the PPARg is a lipid-binding nuclear receptor that has been absence of cAMP, whereas CREB3L2-PPAR; inhibited tran- implicated in physiologic processes, including adipogenesis and scription 6-fold from EVX1 in the same experiments. CREB3L2- obesity (7, 8), insulin sensitivity and diabetes (9, 10), and inflam- PPAR; also inhibited 4-fold the expression of thyroglobulin, a mation and atherosclerosis (11, 12). The activities of PPARg have native cAMP-responsive gene, in primary thyroid cells treated been studied most thoroughly in fat, the development of which with thyroid-stimulating hormone. Our findings identify a requires PPARg to induce differentiation and regulate the novel CREB3L2-PPARg gene fusion mutation in thyroid expression of fat-specific genes (7, 8, 13). PPARc has also been carcinoma and reveal a thyroid signaling pathway that is implicated in cancer, in part by the discovery of PPARc mutations regulated by intramembrane proteolysis and disrupted in in colon (14) and thyroid (15) carcinoma tissues. Such mutations cancer. [Cancer Res 2008;68(17):7156–64] seem to impair ligand-mediated transcription by PPARg (14, 15), which induces differentiation and inhibits proliferation in many, Introduction but not all, cancer cell lines (16–20). Even so, the interpretation of Gene fusion mutations underlie a significant subset of human these experiments is not straightforward because data are cancers. Scientific investigations of gene fusions, particularly in conflicting as to whether PPARg suppresses or promotes leukemia, have led to (a) the identification of biological pathways tumorigenesis in mouse tumor models (21–24). Thus, fundamental that are deregulated in many cancer types; (b) the development of mechanisms of PPARg in cancer remain to be elucidated. specific molecular diagnostic assays that identify and monitor Regulated intramembrane proteolysis is a physiologic process that cleaves transcription factors from cell membranes to activate gene expression (25). For example, SREBP1 and SREBP2 (26), Notch, and activating transcription factor-6 undergo intramem- Note: Supplementary data for this article are available at Cancer Research Online brane proteolysis in cholesterol, fatty acid, protein folding, and (http://cancerres.aacrjournals.org/). W-O. Lui and L. Zeng contributed equally to this work. intracellular signaling pathways. Cyclic AMP (cAMP) response Requests for reprints: Todd G. Kroll, Department of Pathology, University of element binding protein 3 (CREB3) and CREB4are bZIP Chicago Medical Center, 5841 South Maryland Avenue, AMB P323 (MC1089), Chicago, transcription factors with transmembrane domains that are IL 60637. Phone: 773-702-3017; Fax: 773-834-5251; E-mail: [email protected]. I2008 American Association for Cancer Research. thought to be cleaved by intramembrane proteolysis in response doi:10.1158/0008-5472.CAN-08-1085 to endoplasmic reticulum stress (27, 28). CREB3-like 2 (CREB3L2) is

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. CREB3L2-PPARg in Thyroid Cancer a CREB3-related protein that has domains fused to FUS in human (35). Primers for the amplification of S1P were 5¶-TACCACAACCTCCGC- fibromyxoid sarcoma with t(7;16) (29, 30). An oncogenic role for TATCC-3¶ (1,882–1,902, D42053) and 5¶-CCCATGTTCCACACAGACAG-3¶ the FUS-CREB3L2 fusion protein is predicted but not yet (2,303–2,283, D42053). Primers for the amplification of S2P were 5- ¶ ¶ determined. TGATGGCTGACTCTCCCTCT-3 (413–433, AF019612) and 5 -AGTGATGAG- CACCCCAACTC-3¶ (876–856, AF019612). Automated nucleotide sequencing Here, we report our discovery of a novel CREB3L2-PPARc gene was done using Big Dye Terminators (Perkin-Elmer). mRNA insitu fusion mutation in thyroid follicular carcinoma. Our experiments hybridization was done as described (36) on tumor and normal thyroid show that the encoded CREB3L2-PPARg fusion protein stimulates tissues. In brief, 5-Am sections were cut from frozen thyroid tissues, proliferation and inhibits cAMP-responsive transcription in normal hybridized with 35S end-labeled synthetic oligonucleotides, and washed thyroid cells treated with thyroid-stimulating hormone (TSH). Our under stringent conditions. Expression was determined by autoradiography discovery of CREB3L2-PPARg identifies a thyroid signaling after exposure in photographic emulsion. The antisense probe for CREB3L2- pathway that is regulated by intramembrane proteolysis and PPARg (5¶-CTGTGTCAACCATGGTCATTTCGTCATTGAAGCTGTCACTGGTG- disrupted in thyroid cancer. 3¶; 297–340, AY222643) was designed to span the CREB3L2-PPARg fusion breakpoint. An 18s rRNA antisense probe (5¶-CGCCTGCTGCCTTCCTTGG- ATGTGGTAGCCGTTTCTCAGG-3¶; 441–480, X03205) was used as a positive Materials and Methods control and a 17a-hydroxylase sense probe (5¶-CCCTGGAAGGCATCCCC- Tissues and cells. Thyroid tumors were diagnosed according to WHO AAGGTGGTCTTTCTGATCGACTC-3¶; 8,290–8,329, M63871) was used as a criteria (31), with the exception that Hurthle cell tumors were diagnosed negative control. separately from, and not as a variant of, thyroid follicular tumors. Some Cell growth assays and electroporation. CREB3L2-PPARg, PAX8- thyroid tumors have been described in previous reports (15, 32). Banked PPARg, and wild-type PPARg1 were expressed from the mammalian tissues were obtained from thyroidectomy specimens and frozen immedi- expression vectors pcDNA3.1 or pcDNA3.2 (Invitrogen). Electroporation was ately at À80jC or in liquid nitrogen. Tissue fragments were examined by done in primary thyroid cells using the Amaxa system (Amaxa). Growth frozen and/or permanent sections to confirm the proportion of tumor cells. experiments were repeated in three to six separate experiments with Informed consent was obtained from all patients and the study was different isolates of primary thyroid cells. Cell numbers were determined in approved by the Institutional Review Boards of the University of Chicago, duplicate or triplicate using a Z2 particle/cell counter (Beckman-Coulter). Emory University, and the Ethics Committee of the Karolinska Hospital. Troglitazone and rosiglitizone PPARg agonists were obtained from Cayman 293T human embryonic kidney cells and HepG2 hepatocellular carcinoma or Biomol. Tunicamycin and thapsigargin were purchased from Sigma- cells were obtained from American Type Culture Collection. Aldridge. Primary human thyroid cells. Cultures of primary human thyroid cells Transfection and trancription assays. Transcription assays were done were established from normal thyroid tissues that were removed by surgery with the EVX1 CRE reporter (37) in the dual luciferase system (Promega). for thyroid tumors (33). The thyroid tissues were dissected by an experienced Transfections were done for 24h with FuGene as described by the thyroid pathologist (T.G.K.), disaggregated, and digested with collagenase manufacturer (Roche). In some experiments, troglitazone or rosiglitazone and dispase (Roche) for up to 3 h. Intact thyroid follicles were maintained for was added at 24to 36 h in medium with charcoal-stripped FCS (Hyclone). 3 to 5 d in serum-free RPMI medium (Invitrogen-Life Technologies, Inc.) Luciferase activities were determined in duplicate or triplicate as the containing glutamine, penicillin-streptomycin, and primocin (Sigma). mean F SE. Thyroid cell monolayers of 85% to 90% purity grew from the follicles after Immunoblotting and immunoreagents. Total protein was extracted the addition of medium with 10% FCS. Primary thyroid cultures from >40 from tissues or cells with lysis buffers containing nonionic (NP40 or Triton different patients have been investigated. X-100) and/or ionic (SDS and sodium deoxycholate) detergents (U.S. Fluorescence in situ hybridization. Fluorescence insitu hybridization Biochemical or Sigma) and complete protease inhibitors (Roche). Total (FISH) was done with yeast artificial chromosomes (YAC) 753f7 and 932f3 cellular protein (20–50 Ag) was mixed with SDS sample buffer containing that flank the PPARc gene, a BAC 377b19 that contains the CREB3L2 gene, 50 mmol/L DTT or 2% to 2.5% 2-mercaptoethanol, heated to 95jC for 5 min, and BACs 22G20, 351B12, 29B3, 83I11, and 691P5 that flank the CREB3L2 and fractionated by SDS-PAGE. Proteins were blotted onto nitrocellulose gene, as described previously (15, 34). Briefly, touch preparations from (Protran) or polyvinylidene difluoride membranes. Membranes were frozen thyroid carcinoma tissues were prepared on glass slides, fixed in incubated sequentially with 5% to 10% nonfat dry milk, primary antibody either 70% ethanol or methanol/acetic acid (3:1), and air-dried. Slides were or antiserum, and secondary rabbit anti-mouse or donkey anti-rabbit treated with pepsin (50 Ag/mL in 0.01 N HCl) and postfixed in 10% horseradish peroxidase-conjugates (Santa Cruz or Amersham). Detection phosphate buffered formalin (Sigma). FISH probes were labeled with was carried out with enhanced chemiluminescence (Amersham). Kaleido- digoxigenin- or biotin-conjugated nucleotides by random priming (Invi- scope (Bio-Rad) and Magicmark (Invitrogen) markers were used to deter- trogen), mixed with human Cot-1 DNA (Invitrogen), denatured, and mine relative molecular weights. Protein levels were quantified on X-ray hybridized for 16 to 48 h at 37jC. Probes were visualized with fluorescent films from immunoblots using ImageJ software.6 Values were normalized to avidin (Vector) or anti-digoxigenin (Roche) using a Zeiss Axioplan2ie levels of control h-actin in the same lanes. Immunoreagents included the microscope fitted with Zeiss Neofluar and ApoFluar objectives, a megapixel E8 PPARg monoclonal antibody (Santa Cruz), the H74PPAR y antiserum charge-coupled device camera, and integrated Metasystems software (Santa Cruz), a h-actin antibody (Santa Cruz), a calreticulin antibody (Metasystems GmbH). (Abcam), and a thyroglobulin antiserum (M0781; DAKO). The CREB3L2 Rapid amplification of cDNA ends, reverse transcription-PCR, and antiserum was created by New England Peptide, Inc., against a synthetic mRNA in situ hybridization. cDNA was transcribed from total RNA using peptide, Ac-LQWDRKLSELSEPGDC-amide, from the NH2 terminus of wild- oligo dT primers and SMART rapid amplification of cDNA ends (RACE) type CREB3L2 (residues 12–27, NP_919047). The specificity of the CREB3L2 oligonucleotides, as described (SMART RACE kit, Clontech). CREB3L2- antiserum was verified by ELISA (New England Peptide), immunoblotting, PPARc was identified originally by RACE using the PPARc reverse primer and peptide blocking experiments (Supplementary Fig. S1). (5¶-GCAGGCTCCACTTTGATTGC-3¶; 319–341, NM_138712) and universal Thyroid tissue microarrays. FISH was done as described above on forward primers. Amplifications were done with Advantage II (Clontech) or thyroid tissue microarrays that were constructed from formalin-fixed, PFX (Invitrogen) Taq polymerase. Primers for cloning of the full-length paraffin-embedded human thyroid tissues selected from 103 patients by CREB3L2-PPARc cDNA were CREB3L2 forward (5¶-ATGGAGGTGCTGGA- two experienced endocrine pathologists (T.G.K. and M.T.). The tissue GAGCGG-3¶, 353–373, AJ549092, AJ549387) and PPARc reverse (5¶- GAGAGTCCTGAGCCACTGCC-3¶, 1,684–1,704, NM_138712) using Advan- tage II or HotStar Taq DNA polymerase (Qiagen). Quantitative reverse transcription-PCR (RT-PCR) for CREB3L2 was done as previously described 6 http://rsb.info.nih.gov/ij/ www.aacrjournals.org 7157 Cancer Res 2008;68: (17). September 1, 2008

Figure 1. Discovery of the CREB3L2-PPARc fusion mutation. A, dual-color FISH showed that YAC probes that flank the PPARc gene were split (white arrowheads) in interphase nuclei of thyroid carcinoma with t(3;7), showingrearrangement of PPARc. One carcinoma cell nucleus is shown. B, single-color FISH showed that a BAC that contains the CREB3L2 gene was also split (white arrowheads) in interphase nuclei of thyroid carcinoma with t(3;7), showingsimultaneous rearrangement of CREB3L2. Two carcinoma nuclei are shown. C, dual-color FISH with BAC probes that flank the CREB3L2 gene confirmed rearrangement of CREB3L2 (white arrowheads) in interphase nuclei of paraffin-embedded thyroid carcinoma with t(3;7). D, RACE and RT-PCR generated a CREB3L2-PPARc cDNA that consisted of 5¶ untranslated and codingsequences of wild-type CREB3L2 (exons 1 and 2) fused in-frame to codingand 3 ¶ untranslated sequences of PPARc (exons 1–6). The full-length CREB3L2-PPARc cDNA predicts a CREB3L2-PPARg fusion protein that consists of amino acids 1 to 106 of wild-type CREB3L2, a new glutamic acid (E) at position 107, and all 477 amino acids of wild-type PPARg1. Blue, CREB3L2 sequences; yellow, PPARc sequences; red, PAX8 sequences; gray, noncodingexons.

microarrays contained 127 benign thyroid tissues, including 18 follicular signals that are indicative of PPARc rearrangements (15, 34) in adenomas, 10 Hurthle cell adenomas, 7 multinodular goiters, 3 cases of interphase nuclei of thyroid carcinoma with t(3;7) (Fig. 1A). Single- Graves’ disease, 6 cases of Hashimoto’s thyroiditis, and 83 normal thyroid color FISH with a bacterial artificial chromosome (BAC) probe that sections. A minimum of two tissue cylinders with a diameter of 1 mm were contains the CREB3L2 gene at 7q34(377b19) showed one intact arrayed using an automated tissue microarrayer (ATA-27, Beecher Instru- B A and two split red signals in the thyroid carcinoma cells (Fig. 1 ), ments). The recipient blocks were cut into 4- m-thick sections on Surgipath CREB3L2 silane-coated positive-charged slides. consistent with simultaneous rearrangement of . Dual- CREB3L2 Statistical analyses. Data from cell growth, proliferation, and transcrip- color FISH with BAC probes that flank (22G20, 351B12, tion assays were calculated as the mean of duplicate or triplicate 29B3, 83I11, and 691P5) confirmed CREB3L2 rearrangement in measurements F SD. P values were determined using the Student t test paraffin-embedded thyroid carcinomas with t(3;7) (Fig. 1C). These for continuous variables. P < 0.05 was considered to be statistically experiments showed that both CREB3L2 and PPARc are rearranged significant. in thyroid carcinoma with t(3;7). We used rapid amplification of cDNA ends (RACE) to identify 5¶ coding sequences of PPARc fused in-frame to 5¶ untranslated and Results coding sequences of CREB3L2. RACE and RT-PCR generated a full- Discovery of the CREB3L2-PPAR; fusion mutation. We phy- length (1,832 bp) CREB3L2-PPARc cDNA (AY222643) that consisted sically mapped the breakpoints of the chromosomal rearrangement of wild-type exons 1 and 2 of CREB3L2 fused in-frame to wild-type t(3;7)(p25;q34) that we identified previously in a thyroid follicular exons 1 to 6 of PPARc (Fig. 1D). The CREB3L2-PPARc sequence is carcinoma (32). Dual-color FISH with YAC probes that flank the predicted to encode a CREB3L2-PPARg fusion protein (67 kDa) PPARc gene at 3p25 (932f3 and 753f7) showed split red and green composed of wild-type amino acids 1 to 106 of CREB3L2, a new

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Figure 3. CREB3L2-PPARg stimulates growth and induces proliferation in primary thyroid cells. A, the CREB3L2-PPARg and PAX8-PPARg fusion proteins stimulated the growth of primary human thyroid epithelial cells by 25% to 30% compared with wild-type PPARg or vector controls over 5 d. Similar results were obtained with several isolates of primary cells. Columns, mean from duplicate or triplicate culture dishes; bars, SD. *, P < 0.007. B, the CREB3L2-PPARg and PAX8-PPARg fusion proteins also induced thyroid cell proliferation by 42% and 58%, respectively, over vector controls based on the incorporation of BrdUrd duringa 5-h incubation period. The incorporation of BrdUrd was determined in duplicate culture dishes on day 3 after electroporation. Columns, mean; bars, SD. *, P < 0.0003.

glutamic acid (E) at position 107, and all 477 wild-type amino acids of wild-type PPARg1(Fig.1D). The extra glutamic acid and all wild- type PPARg1 amino acids are also present in the PAX8-PPARg fusion protein (Fig. 1D) that we previously discovered (15). FISH showed the CREB3L2-PPARc gene fusion in 1 of 42 (2.4%) additional paraffin-embedded thyroid follicular carcinomas that we have reported in a previous series (34), whereas no CREB3L2 rearrangements were detected in 127 benign thyroid tissues that included 18 follicular adenomas, 10 Hurthle cell adenomas, 7 multinodular goiters, 3 cases of Graves’ disease, 6 cases of Hashimoto’s thyroiditis, and 83 normal thyroid tissue sections. Thus, CREB3L2- PPARc is a low-incidence fusion mutation that is present in <3% of thyroid follicular carcinomas. Expression of CREB3L2-PPAR;. RT-PCR showed CREB3L2- PPARg fusion mRNA in thyroid follicular carcinoma with t(3;7) but not in normal thyroid tissues (Fig. 2A). Expression of CREB3L2- PPARg mRNA was also observed by insitu hybridization (data not shown). Immunoblots with the E8 PPARg antibody, which reacts with both PPARg and PPARy,7 showed the CREB3L2-PPARg fusion protein in thyroid carcinoma with t(3;7) (Fig. 2B, left) but not in Figure 2. Expression of CREB3L2-PPARg in thyroid carcinoma tissue. normal thyroid tissues (Fig. 2B). The CREB3L2-PPARg fusion A, RT-PCR with primers that flank the CREB3L2-PPARc breakpoint showed expression of CREB3L2-PPARc mRNA in thyroid carcinoma with t(3;7) (FC) but protein was expressed higher than native PPARg1 in thyroid not in normal thyroid tissue. mw, molecular weight markers. B, immunoblots carcinoma with t(3;7) (Fig. 2B, left) and migrated similarly to with the E8 PPARg antibody, which immunoreacts with both PPARg and PPARy, showed overexpression of native CREB3L2-PPARg compared with wild-type transfected CREB3L2-PPARg (67 kDa) and between transfected PPARg1 in thyroid carcinoma with t(3;7) (FC; left). The CREB3L2-PPARg fusion PAX8-PPARg (98 kDa) and transfected PPARg1 (54kDa) on protein was not expressed in normal thyroid tissue and comigrated with transfected immunoblots using lysates of 293T kidney cells (Fig. 2B). CREB3L2-PPARg (67 kDa) and between transfected PPARg1 (54 kDa) and transfected PAX8-PPARg (98 kDa) that were expressed in 293T epithelial cells. Nonspecific, nonimmunospecific band. C, immunohistochemistry with the E8 PPARg antibody showed overexpression of the CREB3L2-PPARg fusion protein in nuclei of thyroid carcinoma with t(3;7). Immunoreactivity is brown and the nuclear counterstain (Gil’s hematoxylin) is blue. 7 Unpublished data. www.aacrjournals.org 7159 Cancer Res 2008;68: (17). September 1, 2008

Immunohistochemistry with the E8 PPARg antibody showed TSH.7 Primary thyroid cells were transduced at 50% to 70% increased expression of CREB3L2-PPARg in the nuclei of thyroid efficiency by electroporation based on a green fluorescent protein carcinoma with t(3;7) relative to normal thyroid tissue on the same construct that was followed by fluorescence microscopy and flow slide (Fig. 2C). These experiments show that CREB3L2-PPARg cytometry.7 The CREB3L2-PPARg and PAX8-PPARg fusion proteins fusion mRNA and protein are expressed highly in thyroid increased the number of primary thyroid cells by 25% to 30% carcinoma with t(3;7). (P < 0.007) compared with vector controls over 5 d (Fig. 3A). In The CREB3L2-PPAR; fusion protein induces proliferation in contrast, wild-type PPARg, which contains the same PPARg amino primary thyroid cells. To begin to determine the oncogenic acids as these fusion proteins, had no stimulatory activity (Fig. 3A). mechanism of CREB3L2-PPARc, we measured the effects of the The addition of PPARg agonists troglitazone (1–10 Amol/L) or CREB3L2-PPARg fusion protein on the growth of primary human rosiglitazone (10–1,000 nmol/L; ref. 10) also had little effect in the thyroid cells. Cultures of primary thyroid cells were 85% to 90% presence or absence of the fusion proteins.7 Thus, the CREB3L2- pure (33) and retained differentiated functions such as the PPARg fusion protein is a potent inducer of thyroid cell growth and production of thyroperoxidase and thyroglobulin in response to seems to be unresponsive to synthetic PPARg ligands.

Figure 4. Wild-type CREB3L2 is expressed in thyroid tissue and regulated by intramembrane proteolysis. A, Northern blots showed expression of three wild-type CREB3L2 mRNA transcripts, f7.5, 2.3, and 1.1 kb, in thyroid and other human tissues. B, the 7.5- and 2.3-kb CREB3L2 mRNAs encode a CREB3L2 protein of 520 amino acids that has an NH2-terminal transactivation domain, a bZIP DNA bindingdomain, and a carboxyl transmembrane ( TM) domain (residues 379–395; light red) that is flanked by S2P (LCXn P; residues 385–402; light blue) and S1P (RXXL, residues 427–430; light green) proteolytic cleavage sites. S2P and S1P sites are often cleaved by intramembrane proteolysis to release transcription factor domains to the nucleus to activate gene expression. The transactivation domain of CREB3L2 is fused to PPARg in the CREB3L2-PPARg fusion protein. C, full-length CREB3L2-V5 was expressed in 293T epithelial cells that were treated with brefeldin A, an inducer of intramembrane proteolysis. Brefeldin A produced rapid conversion of full-length CREB3L2-V5 to cleaved CREB3L2-V5 beginning at 0.5 h. The expression of control h-actin was constant under these same conditions. Immunoblots with CREB3L2 antiserum and h-actin antibody are shown. D, cleaved CREB3L2-V5 localized to nuclear and total, but not cytoplasmic, fractions of 293T cells that had been treated with brefeldin A. In contrast, full-length CREB3L2-V5 localized to cytoplasmic, nuclear, and total fractions of untreated 293T cells. We attribute CREB3L2-V5 in the nuclear fractions to result from incomplete separation of nuclear and endoplasmic reticulum/membrane proteins, which is dependent on protein solubility. In support of this possibility, calreticulin, a protein marker of the endoplasmic reticulum, was also present in all nuclear fractions. The control transcription factor PPARy was enriched highly in nuclear fractions. Immunoblots with CREB3L2 antiserum, PPARy antiserum, and calreticulin antibody are shown.

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of CREB3L2-PPARg in thyroid cells. Both the 7.5- and 2.3-kb transcripts encode a wild-type CREB3L2 protein of 520 amino acids (AJ549387, AJ549092) that contains an NH2-terminal transactivation domain, a middle bZIP DNA binding domain, and a carboxyl transmembrane domain (amino acids 379–395) that is flanked by S1P (RXXL, 427–430) and S2P (LCXnP, 385–402) proteolytic cleavage sites (Fig. 4B). The S1P serine protease and S2P zinc metalloprotease cleave membrane-bound transcription factors in a process termed regulated intramembrane proteolysis that depends on metabolic, endoplasmic reticulum, and/or protein folding signals (26). The membrane-bound transcription factors are thereby released to the nucleus to activate gene expression. The CREB3L2-PPARg fusion protein contains only the transactivation domain of CREB3L2 (Fig. 4B). We next sought direct experimental evidence that CREB3L2 is regulated by intramembrane proteolysis in epithelial cells. CREB3L2 was tagged with V5 and expressed by transfection in 293T kidney cells that were treated with brefeldin A. Brefeldin A has been shown to induce regulated intramembrane proteolysis at S1P and S2P sites (38) by altering the endoplasmic reticulum-Golgi, endosome-lysosome, and protein secretory pathways (39, 40). Transfected 293T cells expressed full-length CREB3L2-V5 protein (78 kDa) that was lost after 3 hours of treatment with brefeldin A (Fig. 4C). The transfected 293T cells produced a smaller, cleaved CREB3L2-V5 protein (54kDa) beginning at 0.5 hour of brefeldin A treatment (Fig. 4C). Expression of control h-actin was constant in the same experiment (Fig. 4C). This precursor-product relationship between full-length and cleaved CREB3L2-V5, the rapid kinetics of CREB3L2-V5 cleavage, and the relative molecular weights of full- length and cleaved CREB3L2-V5 are all consistent with the regulated intramembrane proteolysis of CREB3L2 at its S1P and Figure 5. Native CREB3L2 is cleaved by intramembrane proteolysis in normal thyroid cells that express S1P and S2P proteases. A, treatment of primary S2P sites. human thyroid cells with brefeldin A for 3.5 h led to cleavage of native full-length We localized full-length and cleaved CREB3L2 proteins by cell CREB3L2 to cleaved CREB3L2, showingintramembrane proteolysis of CREB3L2 in normal thyroid cells. An immunoblot with CREB3L2 antiserum is fractionation. Cleaved CREB3L2-V5 was present in the nuclear and shown. B, RT-PCR showed expression of the S1P and S2P proteases in primary total, but not cytoplasmic, fractions of 293T cells that were treated human thyroid cells and control HepG2 liver carcinoma cells, consistent with with brefeldin A for 3.5 hours (Fig. 4D). In contrast, full-length the involvement of S1P and S2P in intramembrane proteolysis of CREB3L2. CREB3L2-V5 was absent from 293T cells that were treated with brefeldin A but was present in cytoplasmic, nuclear, and total fractions of untreated 293T cells (Fig. 4D). We believe that full- We also measured the effect of CREB3L2-PPARg on the length CREB3L2-V5 in the nuclear fraction of untreated 293T cells incorporation of bromodeoxyuridine (BrdUrd) into nuclear DNA resulted from incomplete separation of endoplasmic reticulum and as a measure of thyroid cell proliferation. CREB3L2-PPARg and nuclear proteins in this fractionation procedure, which is known to PAX8-PPARg increased the incorporation of BrdUrd by 42% and be influenced by protein solubility. In support of this possibility, 58% (P < 0.0003), respectively, compared with vector controls on calreticulin, a protein marker of the endoplasmic reticulum (41), day 3 after electroporation (Fig. 3B). In contrast, the expression of was also apparent in all nuclear fractions (Fig. 4D). On the other wild-type PPARg had little effect on the incorporation of BrdUrd hand, the nuclear receptor PPARy that served as a control (Fig. 3B). Thus, CREB3L2-PPARg and PAX8-PPARg each induced transcription factor was enriched highly in the nuclear fractions marked proliferation in normal thyroid cells, consistent with a (Fig. 4D). These experiments show that wild-type CREB3L2 is dominant transforming mechanism. cleaved by intramembrane proteolysis and transported to the CREB3L2is a transcription factor regulated by intramem- nucleus. brane proteolysis. To further understand the mechanism of We investigated whether native CREB3L2 was processed by CREB3L2-PPARg, we determined the expression, post-translational regulated intramembrane proteolysis in normal thyroid cells. Full- processing, and cellular localization of wild-type CREB3L2. length native CREB3L2 was synthesized in cultures of primary Nucleotide probes for wild-type CREB3L2 hybridized on Northern human thyroid cells, whereas only native cleaved CREB3L2 was blots to three main mRNA transcripts, Approximately 7.5, 2.3, and produced after treatment of thyroid cells with brefeldin A 1.1 kb (Fig. 4A). These transcripts corresponded in size to three (Fig. 5A). In addition, RT-PCR showed mRNA transcripts encoding CREB3L2 sequences in GenBank (NM_194071, AJ549387, the S1P and S2P proteases in primary thyroid cells and control BC063666). The 7.5-kb transcript was expressed at highest levels HepG2 cells (Fig. 5B), supporting the possibility that S1P and S2P in the thyroid and at lower levels in stomach, adrenal gland, lymph cleave CREB3L2 in the thyroid gland. Taken together, these node, and tracheal (salivary gland) tissues (Fig. 4A), showing that experiments indicate that wild-type full-length CREB3L2 is the native CREB3L2 promoter is sufficient to drive high expression cleaved by intramembrane proteolysis to nuclear CREB3L2 as www.aacrjournals.org 7161 Cancer Res 2008;68: (17). September 1, 2008

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Cancer Research part of a thyroid signaling pathway that is involved in growth responsive thyroid gene, in primary thyroid cells that were treated regulation and cancer. with TSH (Fig. 6C). These experiments show that the CREB3L2- CREB3L2-PPAR; inhibits cAMP-responsive transcription PPARg fusion protein inhibits transcription from an established activities. To further determine the mechanism of CREB3L2- CRE and inhibits expression of a native cAMP-responsive gene in PPARg, we measured the abilities of nuclear CREB3L2 and normal thyroid cells. CREB3L2-PPARg to activate transcription from the EVX1 cAMP response element (CRE). Nuclear CREB3L2 or CREB3L2-PPARg were transfected with the EVX1 CRE reporter into 293T epithelial Discussion cells that are responsive to cAMP (37). Nuclear CREB3L2 Our discovery of the CREB3L2-PPARc fusion mutation shows stimulated transcription from the EVX1 CRE 8-fold over vector for the first time that a family of somatic PPARc fusion mutations controls (P = 0.002) in the absence of cAMP, whereas CREB3L2- exist in thyroid cancer. An important aspect of fusion mutations PPARg inhibited transcription from the EVXI CRE 6-fold (P = 0.01) is that they identify molecular mechanisms that underlie cancer in the same experiments (Fig. 6A). Transcription from the EVX1 pathogenesis. In fact, our discovery of CREB3L2-PPARc shows that CRE was shown to be dose dependent under these conditions CREB3L2 is the target of at least two fusion mechanisms in (Fig. 6B). Furthermore, overexpression of CREB3L2-PPARg cancer. In thyroid follicular carcinoma, the NH2-terminal trans- inhibited 4-fold the expression of native thyroglobulin, a cAMP activation domain of CREB3L2 is fused to PPARg. In fibromyxoid sarcoma, the bZIP and carboxyl domains of CREB3L2 are fused to FUS (29). Thus, the identification of the CREB3L2-PPARc fusion underscores the importance of CREB3L2 deregulation in diverse cancer types and adds to a growing list of CREB-related alterations that have recently been identified in human cancer tissues (42, 43). Our experiments showed that CREB3L2-PPARc is a low-incidence PPARc gene fusion that is present in <3% of thyroid follicular carcinomas. The PAX8-PPARc gene fusion has been reported in 25% to 45% of thyroid follicular carcinomas in pathologically well- defined series that separate follicular from Hurthle cell carcinomas (34, 44, 45). Little is understood about the function of CREB3L2 in cancer, and our experiments provide new insights into CREB3L2 mechanisms by showing that (a) the CREB3L2-PPARg fusion protein stimulates proliferation in normal human thyroid cells; (b) wild-type CREB3L2 is cleaved by intramembrane proteolysis in a thyroid signaling pathway; (c) nuclear CREB3L2, but not CREB3L2- PPARg, stimulates transcription from the EVX1 CRE; and (d) the CREB3L2-PPARg fusion protein inhibits transcription from the EVX1 CRE and also inhibits expression of thyroglobulin in normal thyroid cells that have been treated with TSH, the major thyroid regulator that acts through cAMP. The CREB3L2-PPARg and PAX8-PPARg fusion proteins each induced the growth of normal human thyroid cells by stimulating cell proliferation. The 42% to 58% induction of proliferation by these PPARg fusion proteins is remarkable because other potent thyroid oncogenes such as mutant RAS or B-RAF often elicit growth arrest and/or apoptosis in primary cells (46, 47), presumably because they activate mechanisms that protect against cell transformation. In fact, expression of the B-RAF or ELE1-RET (RET-PTC3) mutants, which activate constitutive mitogen-activated protein kinase signaling in thyroid papillary carcinomas, induced cell death or little proliferation, respectively, in normal thyroid cells in our experiments.7 Thus, CREB3L2-PPARg and PAX8-PPARg induce proliferation by mechanisms that seem to abrogate or bypass B-RAF Figure 6. The CREB3L2-PPARg fusion protein inhibits cAMP-responsive antitumor pathways in a manner different from mutant (48) transcription. A, wild-type nuclear CREB3L2 activated transcription from the or ELE1-RET (49). Such molecular differences may determine, at EVX1 CRE 8-fold higher than a vector control in the absence of cAMP least in part, the morphologic and clinical differences that are (*, P = 0.002). In contrast, CREB3L2-PPARg inhibited 6-fold transcription from the EVX1 CRE (*, P = 0.01). Columns, mean from duplicate culture observed in patients with follicular versus papillary thyroid dishes; bars, SD. RLU, relative light units; lucF, firefly luciferase; lucR, carcinoma. renilla luciferase. B, transcription from the EVX1 CRE was activated in a dose-dependent manner under these conditions. Columns, mean from duplicate CREB3L2 was cleaved by regulated intramembrane proteolysis culture dishes; bars, SD. C, engineered overexpression of CREB3L2-PPARg in an unappreciated thyroid signaling pathway. CREB3L2 contains inhibited 4-fold the expression of thyroglobulin (Tg), a TSH/cAMP-responsive a transactivation domain, a bZIP DNA binding domain, and a native thyroid gene, in primary thyroid cells that were treated with TSH for 24 h. Thyroglobulin levels on immunoblots were quantified on X-ray films using transmembrane domain that has flanking S1P and S2P protease ImageJ software and normalized to levels of control h-actin in the same lanes. cleavage sites. Intramembrane proteolysis by S1P and S2P controls

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. CREB3L2-PPARg in Thyroid Cancer the SREBPs, which are key transcriptional regulators of cholesterol suggest that CRE-containing genes in thyroid carcinoma with metabolism (26), and CREB3, CREB4and CREB-H, which are CREB3L2-PPARg may be down-regulated by (a) disruption of one thought to be involved in protein folding and stress responses in CREB3L2 allele during CREB3L2-PPARg formation and (b) the endoplasmic reticulum (28, 50, 51). In our study, full-length functional inhibition of transcription at cAMP responsive genes CREB3L2 exhibited a precursor-product relationship with and by the CREB3L2-PPARg fusion protein. In fact, we have observed by rapid conversion to cleaved CREB3L2 after treatment of thyroid quantitative RT-PCR that expression of cAMP-responsive thyroid cells with brefeldin A, a known inducer of intramembrane genes such as thyroglobulin, thyroperoxidase, and the sodium- proteolysis (38). In addition, full-length CREB3L2 localized iodide transporter is lower in thyroid follicular carcinoma with predominantly to cytoplasmic fractions, whereas cleaved CREB3L2 CREB3L2-PPARg than in 95% of other thyroid follicular tumors localized to nuclear fractions, as expected if CREB3L2 is processed (n = 76), including five follicular carcinomas with the PAX8-PPARg by intramembrane proteolysis. We also showed that the S1P and fusion protein.7 These data support a model in which CREB3L2- S2P proteases are expressed in human thyroid cells. Interestingly, PPARg acts by interfering with CRE-related transcription that is treatment of thyroid cells with either thapsigargin or tunicamycin, central to the regulation of differentiation and proliferation in the two inducers of endoplasmic reticulum stress, did not generate thyroid gland (54). Interestingly, wild-type CREB3L2 mRNA is cleavage of CREB3L2 in our experiments,7 suggesting that physio- induced at least 2-fold in normal human thyroid cells by treatment logic stimuli other than endoplasmic reticulum stress may control with TSH (55), further connecting the CREB3L2 and TSH pathways. CREB3L2 proteolysis in the thyroid gland. Furthermore, antisera Thus, it will be important to determine the exact molecular that we have raised against distinct domains of CREB3L2 have interactions between the CREB3L2 and TSH pathways in future localized CREB3L2 to the plasma membrane, in addition to the studies, although it is likely that CREB3L2-PPARg has other nucleus and cytoplasm, of thyroid and other epithelial cells.7 oncogenic activities as well. In fact, fusion proteins exhibit potent These findings suggest that CREB3L2 is part of an unappreciated growth activities because they tend to target multiple cell pathways thyroid signaling pathway that is regulated by intramembrane in cancer. Systematic determination of CREB3L2-PPARg versus proteolysis and important in cancer. The data also suggest that PAX8-PPARg activities should help clarify mechanisms of PPAR- CREB3L2 may function in cancer through mechanisms that are related functions in cancer as well. different than senescence (52) or apoptosis (53) that have been connected recently to intramembrane proteolysis by S1P and S2P during endoplasmic reticulum stress. The CREB3L2-PPARc gene Disclosure of Potential Conflicts of Interest fusion provides a natural mutation model with which to determine No potential conflicts of interest were disclosed. the contribution of CREB3L2 and regulated intramembrane proteolysis in carcinoma, the most common and clinically significant cancer type. The physiologic signals that control Acknowledgments intramembrane proteolysis of CREB3L2 in the thyroid gland Received 4/3/2008; revised 6/9/2008; accepted 7/3/2008. remain to be elucidated. Grant support: NIH/National Cancer Institute grant CA75425 and the University The high expression of CREB3L2-PPARg in nuclei of thyroid of Chicago Louis Block Fund (T.G. Kroll). Additional support was provided by the Swedish Cancer Foundation (C. Larsson), the Go¨ran Gustafsson Foundation for carcinoma cells with t(3;7) suggests a transforming mechanism Research in Natural Sciences and Medicine (C. Larsson), the Cancer Society in that involves altered transcription. In fact, our experiments showed Stockholm (A. Ho¨o¨g), the Stockholm County Council, and the Swedish Research that the CREB3L2-PPARg fusion protein has little ability to Council (I. Leibiger). W-O. Lui was a recipient of a fellowship from the Swedish Society for Medical Research. stimulate transcription from the EVX1 CRE compared with wild- The costs of publication of this article were defrayed in part by the payment of page type nuclear CREB3L2. This finding is consistent with the fact that charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact. the CREB3L2 bZIP domain, which facilitates dimerization and DNA We thank Drs. Sam Refetoff and Barbara Kee for scientific discussions, Drs. Mark binding to CREs, is not retained in the CREB3L2-PPARg fusion Montminy and James Fagin for reagents, Drs. Donald Steiner and Graehm Bell for protein. Furthermore, CREB3L2-PPARg inhibited transcription sharing laboratory equipment, and Pablo Michalewicz for technical assistance. GenBank numbers: CREB3L2-PPAR;, AY222643; CREB3L2, NM_194071, from the EVX1 CRE and also inhibited expression of thyroglobulin, NP_919047, BC063666, AJ549387, AJ549092; S1P, D42053; S2P, AF019612; 18s rRNA, a cAMP-responsive gene, in normal thyroid cells. These data X03205; 17A-hydroxylase, M63871.

References the distinct nature of the leukemogenic process induced 10. Lehmann JM, Moore LB, Smith-Oliver TA, et al. An by the PML-RARa and PLZF-RARa oncoproteins. Proc antidiabetic thiazolidinedione is a high affinity ligand 1. Mitelman F, Johansson B, Mertens F. Fusion genes and Natl Acad Sci U S A 2000;97:10173–8. for peroxisome proliferator-activated receptor g rearranged genes as a linear function of chromosome 6. Licht JD. Reconstructing a disease: what essential (PPARg). J Biol Chem 1995;270:12953–6. aberrations in cancer. Nat Genet 2004;36:331–4. features of the retinoic acid receptor fusion oncopro- 11. Ricote M, Li AC, Willson TM, et al. The peroxisome 2. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent teins generate acute promyelocytic leukemia? Cancer proliferator-activated receptor-g is a negative regulator fusion of TMPRSS2 and ETS transcription factor genes Cell 2006;9:73–4. of macrophage activation. Nature 1998;391:79–82. in prostate cancer. Science 2005;310:644–8. 7. Tontonoz P, Hu E, Spiegelman BM. Stimulation of 12. Nagy L, Tontonoz P, Alvarez JG, et al. Oxidized LDL 3. Soda M, Choi YL, Enomoto M, et al. Identification of adipogenesis in fibroblasts by PPARg2, a lipid-activated regulates macrophage gene expression through ligand the transforming EML4-ALK fusion gene in non-small- transcription factor. Cell 1994;79:1147–56. activation of PPARg. Cell 1998;93:229–40. cell lung cancer. Nature 2007;448:561–6. 8. Rosen ED, Sarraf P, Troy AE, et al. PPARg is required 13. Zhang J, Fu M, Cui T, et al. Selective disruption of 4. Heilman SA, Kuo YH, Goudswaard CS, et al. Cbfh for the differentiation of adipose tissue invivo and PPARg 2 impairs the development of adipose tissue and reduces Cbfh-SMMHC-associated acute myeloid leuke- invitro . Mol Cell 1999;4:611–7. insulin sensitivity. Proc Natl Acad Sci U S A 2004;101: mia in mice. Cancer Res 2006;66:11214–8. 9. Barroso I, Gurnell M, Crowley VE, et al. Dominant 10703–8. 5. Rego EM, He LZ, Warrell RP, Jr., Wang ZG, Pandolfi PP. negative mutations in human PPARg associated with 14. Sarraf P, Mueller E, Smith WM, et al. Loss-of-function Retinoic acid (RA) and As2O3 treatment in transgenic severe insulin resistance, diabetes mellitus and hyper- mutations in PPARg associated with human colon models of acute promyelocytic leukemia (APL) unravel tension. Nature 1999;402:880–3. cancer. Mol Cell 1999;3:799–804. www.aacrjournals.org 7163 Cancer Res 2008;68: (17). September 1, 2008

15. Kroll TG, Sarraf P, Pecciarini L, et al. PAX8–1 fusion of the FUS and BBF2H7 genes in low grade fibromyxoid 43. Coxon A, Rozenblum E, Park YS, et al. Mect1-2 fusion oncogene in human thyroid carcinoma [corrected]. sarcoma. Hum Mol Genet 2003;12:2349–58. oncogene linked to the aberrant activation of cyclic Science 2000;289:1357–60. 30. Mertens F, Fletcher CD, Antonescu CR, et al. AMP/CREB regulated genes. Cancer Res 2005;65: 16. Elstner E, Muller C, Koshizuka K, et al. Ligands for Clinicopathologic and molecular genetic characteriza- 7137–44. peroxisome proliferator-activated receptorg and reti- tion of low-grade fibromyxoid sarcoma, and cloning of a 44. Dwight T, Thoppe SR, Foukakis T, et al. Involvement noic acid receptor inhibit growth and induce apoptosis novel FUS/CREB3L1 fusion gene. Lab Invest 2005;85: of the PAX8/peroxisome proliferator-activated receptor of human breast cancer cells invitro and in BNX mice. 408–15. g rearrangement in follicular thyroid tumors. J Clin Proc Natl Acad Sci U S A 1998;95:8806–11. 31. DeLellis R, Lloyd R, Heitz P, et al., editors. Pathology Endocrinol Metab 2003;88:4440–5. 17. Mueller E, Sarraf P, Tontonoz P, et al. Terminal and genetics of tumours of endocrine organs. Lyon: 45. Nikiforova MN, Biddinger PW, Caudill CM, et al. differentiation of human breast cancer through PPARg. IARC Press; 2004. PAX8-PPARg rearrangement in thyroid tumors: RT-PCR Mol Cell 1998;1:465–70. 32. LuiWO,KytolaS,AnfalkL,etal.Balanced and immunohistochemical analyses. Am J Surg Pathol 18. Sarraf P, Mueller E, Jones D, et al. Differentiation and translocation t(3;7)(p25;q34): another mechanism of 2002;26:1016–23. reversal of malignant changes in colon cancer through tumorigenesis in follicular thyroid carcinoma? Cancer 46. Shirokawa JM, Elisei R, Knauf JA, et al. Conditional PPARg. Nat Med 1998;4:1046–52. Genet Cytogenet 2000;119:109–12. apoptosis induced by oncogenic ras in thyroid cells. Mol 19. Martelli ML, Iuliano R, Le Pera I, et al. Inhibitory 33. Williams DW, Wynford-Thomas D. Human thyroid Endocrinol 2000;14:1725–38. effects of peroxisome proliferator-activated receptor g epithelial cells. Methods Mol Biol 1997;75:163–72. 47. Serrano M, Lin AW, McCurrach ME, et al. Oncogenic on thyroid carcinoma cell growth. J Clin Endocrinol 34. French CA, Alexander EK, Cibas ES, et al. Genetic ras provokes premature cell senescence associated with Metab 2002;87:4728–35. and biological subgroups of low-stage follicular thyroid accumulation of p53 and p16INK4a. Cell 1997;88: 20. Palakurthi SS, Aktas H, Grubissich LM, et al. cancer. Am J Pathol 2003;162:1053–60. 593–602. Anticancer effects of thiazolidinediones are indepen- 35. Lui WO, Foukakis T, Liden J, et al. Expression 48. Knauf JA, Ma X, Smith EP, et al. Targeted expression dent of peroxisome proliferator-activated receptor g profiling reveals a distinct transcription signature in of BRAFV600E in thyroid cells of transgenic mice results and mediated by inhibition of translation initiation. follicular thyroid carcinomas with a PAX8-PPAR(g) in papillary thyroid cancers that undergo dedifferenti- Cancer Res 2001;61:6213–8. fusion oncogene. Oncogene 2005;24:1467–76. ation. Cancer Res 2005;65:4238–45. 21. Saez E, Rosenfeld J, Livolsi A, et al. PPARg signaling 36. Farnebo F, Enberg U, Grimelius L, et al. Tumor- 49. Basolo F, Giannini R, Monaco C, et al. Potent exacerbates mammary gland tumor development. Genes specific decreased expression of calcium sensing mitogenicity of the RET/PTC3 oncogene correlates with Dev 2004;18:528–40. receptor messenger ribonucleic acid in sporadic prima- its prevalence in tall-cell variant of papillary thyroid 22. Saez E, Tontonoz P, Nelson MC, et al. Activators of ry hyperparathyroidism. J Clin Endocrinol Metab 1997; carcinoma. Am J Pathol 2002;160:247–54. the nuclear receptor PPARg enhance colon polyp 82:3481–6. 50. Zhang K, Shen X, Wu J, et al. Endoplasmic reticulum formation. Nat Med 1998;4:1058–61. 37. Conkright MD, Guzman E, Flechner L, et al. Genome- stress activates cleavage of CREBH to induce a systemic 23. Girnun GD, Smith WM, Drori S, et al. APC-dependent wide analysis of CREB target genes reveals a core inflammatory response. Cell 2006;124:587–99. suppression of colon carcinogenesis by PPARg. Proc promoter requirement for cAMP responsiveness. Mol 51. DenBoer LM, Hardy-Smith PW, Hogan MR, et al. Natl Acad Sci U S A 2002;99:13771–6. Cell 2003;11:1101–8. Luman is capable of binding and activating transcrip- 24. Lefebvre AM, Chen I, Desreumaux P, et al. Activation 38. DeBose-Boyd RA, Brown MS, Li WP, et al. Transport- tion from the unfolded protein response element. of the peroxisome proliferator-activated receptor g dependent proteolysis of SREBP: relocation of site-1 Biochem Biophys Res Commun 2005;331:113–9. promotes the development of colon tumors in C57BL/ protease from Golgi to ER obviates the need for SREBP 52. Denoyelle C, Abou-Rjaily G, Bezrookove V, et al. Anti- 6J-APCMin/+ mice. Nat Med 1998;4:1053–7. transport to Golgi. Cell 1999;99:703–12. oncogenic role of the endoplasmic reticulum differen- 25. Wolfe MS, Kopan R. Intramembrane proteolysis: 39. Lippincott-Schwartz J, Yuan L, Tipper C, et al. Brefeldin tially activated by mutations in the MAPK pathway. Nat theme and variations. Science 2004;305:1119–23. A’s effects on endosomes, lysosomes, and the TGN Cell Biol 2006;8:1053–63. 26. Goldstein JL, DeBose-Boyd RA, Brown MS. Protein suggest a general mechanism for regulating organelle 53. Moenner M, Pluquet O, Bouchecareilh M, et al. sensors for membrane sterols. Cell 2006;124:35–46. structure and membrane traffic. Cell 1991;67:601–16. Integrated endoplasmic reticulum stress responses in 27. Kondo S, Saito A, Hino S, et al. BBF2H7, a novel 40. Lippincott-Schwartz J, Yuan LC, Bonifacino JS, et al. cancer. Cancer Res 2007;67:10631–4. transmembrane bZIP transcription factor, is a new type Rapid redistribution of Golgi proteins into the ER in 54. Kimura T, Van Keymeulen A, Golstein J, et al. of endoplasmic reticulum stress transducer. Mol Cell cells treated with brefeldin A: evidence for membrane Regulation of thyroid cell proliferation by TSH and Biol 2007;27:1716–29. cycling from Golgi to ER. Cell 1989;56:801–13. other factors: a critical evaluation of invitro models. 28. Stirling J, O’Hare P. CREB4, a transmembrane bZip 41. Krause KH, Michalak M. Calreticulin. Cell 1997;88: Endocr Rev 2001;22:631–56. transcription factor and potential new substrate for 439–43. 55. van Staveren WC, Solis DW, Delys L, et al. Gene regulation and cleavage by S1P. Mol Biol Cell 2006;17: 42. Shankar DB, Cheng JC, Kinjo K, et al. The role of expression in human thyrocytes and autonomous 413–26. CREB as a proto-oncogene in hematopoiesis and in adenomas reveals suppression of negative feedbacks in 29. Storlazzi CT, Mertens F, Nascimento A, et al. Fusion acute myeloid leukemia. Cancer Cell 2005;7:351–62. tumorigenesis. Proc Natl Acad Sci U S A 2006;103:413–8.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. CREB3L2-PPARγ Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

Weng-Onn Lui, Lingchun Zeng, Victoria Rehrmann, et al.

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