Mect1-Maml2 Fusion Oncogene Linked to the Aberrant Activation of Cyclic AMP/CREB Regulated Genes

Research Article

Amy Coxon, Ester Rozenblum, Yoon-Soo Park, Nina Joshi, Junji Tsurutani, Phillip A. Dennis, Ilan R. Kirsch, and Frederic J. Kaye

Genetics Branch and Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute and the National Naval Medical Center, Bethesda, Maryland

Abstract the opportunity to enhance our understanding of growth Malignant salivary gland tumors can arise from a t(11;19) stimulatory and inhibitory cellular processes that may be translocation that fuses 42 residues from Mect1/Torc1, a cyclic important in a wide range of distinct human tumor types. An AMP (cAMP)/cAMP-responsive element binding protein example of a somatic mutation with an undefined biological (CREB)–dependent transcriptional coactivator, with 982 mechanism is the recent discovery that a t(11;19) chromosomal residues from Maml2, a NOTCH receptor coactivator. To rearrangement creates a fusion oncogene product between the determine if the Mect1-Maml2 fusion oncogene mediates Mect1/Torc1 gene on chromosome 19p and the Maml2 gene on tumorigenicity by disrupting cAMP/CREB signaling, we have chromosome 11q in human mucoepidermoid carcinoma (1, 2). generated in-frame deletions within the CREB-binding Because pathogenic markers are uncommon in sporadic epithelial domain of Mect1/Torc1 for testing transformation activity carcinomas (3, 4), the recognition that a fusion Mect1-Maml2 gene and have also developed a doxycycline-regulated Mect1- product underlies the most common type of malignant salivary Maml2 mammalian expression vector for global gene expres- gland tumor may offer a valuable model for defining general sion profiling. We observed that small deletions within the molecular events leading to glandular epithelial carcinogenesis. CREB-binding domain completely abolished transforming Mucoepidermoid cancer can arise from major glands, such as activity in RK3E epithelial cells. Further, we have shown that parotid and submandibular, or from minor mucous/serous glands the ectopic induction of Mect1-Maml2 in HeLa cells strongly that are scattered throughout the upper aerodigestive tract, activated the expression of a group of known cAMP/CREB- including the pulmonary bronchial tree (5). Although mucoepider- regulated genes. In addition, we detected candidate cAMP- moid carcinoma is a rare tumor, with an estimated incidence responsive element sites within 100 nucleotides of the of 1,000 cases per year in the United States, the incidence of salivary transcriptional start sites of other genes activated by Mect1- cancers may be increasing slowly (5, 6). In addition, these data do not take into account the possible misdiagnosis of cases of Maml2 expression. In contrast, we did not observe alterations pulmonary mucoepidermoid cancer, as well as the detection of of known Notch-regulated target genes in these expression Mect1-Maml2 expression in otherwise unrelated eccrine-like array profile experiments. We validated the results by reverse tumors (7). Aside from overexpression of the c-ERBB2gene product transcription-PCR in transfected HeLa, RK3E, and H2009 (8, 9) and alterations in H-ras and p53 gene expression (10, 11) in lung tumor cells and in mucoepidermoid cancer cells that a small subset of tumor samples, no other molecular marker has endogenously express the fusion oncopeptide. Whereas over- been associated with these cancers. Therefore, tumor-specific expression of components of the cAMP pathway has been expression of the Mect1-Maml2 fusion gene, which has now been associated with a subset of human carcinomas, these data detected in 18 of 24 (75%) primary and derived tumor specimens provide a direct genetic link between deregulation of cAMP/ (1, 2, 12), is the most common genetic event for these cancers. CREB pathways and epithelial tumorigenesis and suggest Recent clues to understand how Mect1-Maml2 expression may future therapeutic strategies for this group of salivary gland be linked to human tumorigenesis were provided by data on the tumors. (Cancer Res 2005; 65(16): 7137-44) function of the normal Mect1/Torc1 and Maml2 genes. For example, the Mect1/Torc1 gene was shown to be a coactivator of Introduction cyclic AMP (cAMP)/cAMP-responsive element binding protein A major emphasis in modern cancer research has focused on (CREB) signaling in two independent screens using large-scale understanding the biological implications of somatic mutational arrayed cDNA methodology (13, 14). In contrast, the Maml2 gene is events that occur sequentially during the development of specific related to the Drosophila gene, Mastermind, and to the mammalian malignant tumors. This approach has been successful in providing Mastermind-like gene, Maml1, and was shown to be an essential reagents for molecular diagnosis, in suggesting models for cancer coactivator for NOTCH receptor transcriptional activation and gene pathways, and in identifying candidate targets for new signaling (1, 15). To determine if ectopic expression of the Mect1- anticancer agents. The discovery and analysis of new and Maml2 product alters the pattern of transcription of downstream unexpected mechanisms for tumorigenesis have also provided target genes and to determine the effect of the fusion oncogene on specific transcripts normally regulated by either cAMP/CREB or NOTCH signal transduction pathways, we have developed a

Note: A. Coxon and E. Rozenblum contributed equally to this work. doxycycline-regulated mammalian expression vector for the Requests for reprints: Frederic J. Kaye, National Naval Medical Center, Building 8, Mect1-Maml2 transcript. Using global gene expression profiling, Room 5101, Bethesda, MD 20889. Phone: 301-435-5375; Fax: 301-402-2414; E-mail: we have shown that Mect1-Maml2 strongly activates the expression [email protected]. I2005 American Association for Cancer Research. of a set of genes that are largely regulated by cAMP/CREB signaling. doi:10.1158/0008-5472.CAN-05-1125 These data, therefore, support a model system for Mect1-Maml2 www.aacrjournals.org 7137 Cancer Res 2005; 65: (16). August 15, 2005

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research tumorigenesis that involves the aberrant activation of downstream arrays were sequentially washed with nonstringent (6Â saline-sodium cAMP/CREB signaling genes. Whereas overexpression of compo- phosphate-EDTA, 0.01% Tween 20 final concentration) and stringent buffers + nents of the cAMP pathway has been associated with a subset of (100 mmol/L MES, 0.1 mol/L Na , 0.01% Tween 20 final concentration). The human carcinomas (16), this functional analysis of the recurrent arrays were then sequentially stained with 2 mg/mL acetylated bovine serum A Â t(11;19) translocation now provides a direct genetic link between albumin (BSA) and 10 g/mL streptavidin phycoerythrin in 1 MES buffer and acetylated BSA and normal goat IgG in 1Â MES. deregulation of cAMP/CREB pathways and epithelial tumorigene- Microarray image acquisition and data analysis. The probe arrays sis, suggesting future therapeutic strategies for this group of sali- were scanned in an Affymetrix GCOS argon-ion Scanner at 488 nm. vary gland tumors. Statistical algorithms that consider both perfect and mismatch probes were used to create expression analysis files and the program normalized the data using a global scaling method based on a trimmed mean. Statistical Materials and Methods analyses were done using the BRB ArrayTools Version 3.2.0 (BRB-Array Materials. GM6001, AG 1478, and H89 were purchased from Calbiochem Tools Users Guide, version 3.2; R. Simon and A. Peng Lam, Biometric (San Diego, CA), and gefitinib was from obtained from AstraZeneca Research Branch Technical Report 7, National Cancer Institute).1 We did (Wilmington, DE). class comparison analyses to determine which genes were significantly Description of cells and plasmids. To generate the pTRE-Mect1-Maml2 different in expression values following 24, 48, and 72 hours of doxycycline plasmid (pTRE-M-M2), a 3.5 kb fragment containing the Mect11-Maml2 induction in independent HeLa cell clones that stably express the Mect1- full-length open reading frame was cloned in-frame into the SalI-NotI site of Maml2 fusion protein (expressor clones) compared with independent HeLa the pTRE-Myc plasmid (BD Biosciences-Clontech, Palo Alto, CA; ref. 17). cell clones that retained antibiotic resistance but expressed undetectable HeLa cells stably expressing a tetracycline (Tet)-on plasmid, which contains Mect1-Maml2 protein by reverse transcription PCR (RT-PCR) and protein the reverse Tet repressor fused to the carboxyl-terminal portion of the immunoblot analyses (nonexpressor clones). In addition, class comparison herpes simplex virus VP16 activation domain, were obtained from Clontech analysis was done on gene expression obtained from the individual Mect1- (Palo Alto, CA). H2009 and H360 (human lung carcinoma) and H292 and Maml2 expressor clones induced in the presence of doxycycline compared H3118 (human mucoepidermoid) tumor cell lines were propagated as with the same HeLa cell clones grown in the absence of doxycycline. For previously described (18). RK3E cells were obtained from the American class comparisons, random variance t test was used (19). Genes were Type Culture Collection (Manassas, VA) and propagated in DMEM considered statistically significant if their P value was <0.001, which supplemented with 10% serum. Transfection of RK3E cells for foci controlled for the number of false discoveries. That is, if N is the number of formation was done as previously described (1). genes that passed the filters, then the expected number of false discoveries Generation of stable transfected cell lines. HeLa Tet-on cells stably would be 0.001 N.IfM is the number of genes significant at the 0.001 level, expressing the Tet-on plasmid were grown in DMEM containing 10% then the proportion of false discoveries would be 0.001 Â N/M. We also did tetracycline-free fetal bovine serum (FBS) and 100 Ag/mL G418 to maintain a global test of whether the expression profiles differed between the classes expression of the plasmid. These cells were plated in 100 mm plates and by permuting the labels of which arrays corresponded to which classes. For transfected with 10 Ag of pTRE-M-M2 and 1 Ag of the pTK-Hyg selection each permutation, the P values were recomputed and the number of genes plasmid using LipofectAMINE reagent according to the manufacturer- significant at the 0.001 level was noted. The proportion of the permutations recommended protocol (Invitrogen, Carlsbad, CA). Cells were washed after that gave at least as many significant genes as with the actual data was the 3 hours and then 36 hours after transfection the media was supplemented significance level of the global test (20). Clustering of genes and samples with G418 and hygromycin. Media was changed every 3 days. After 3-week were done using the BRB clustering tools by centering the genes, using a incubation in 37jC/6% CO2, antibiotic-resistant, single-cell clones were one minus correlation, using an average linkage, and by selecting a subset of transferred to separate 12-well plates for clonal expansion. Stable clones genes that were significantly different in the class comparisons. were tested for RNA and protein expression of M-M2 in both the absence Cell proliferation assay. The H2009, H292, H3118, and H360 cells were and presence of 0.1 to 0.25 Ag/mL of doxycycline. plated at 1,000 cells/well in 96-well plate in RPMI 1640 supplemented with Paired oligonucleotide target sequences for reverse transcription- 10% (v/v) FBS overnight, and agents were added on the next day. The cells PCR reactions. Mect1-Maml2 forward: 5V-ATG GCG ACT TCG AAC AAT were subsequently incubated for 4 days and the cell viability was assessed CCG CGG AA-3V, reverse: 5V-CCA TTG GGT CGC TTG CTG TTG GCA GGA by WST-1 assay according to the instruction of the manufacturer (Roche G-3V; matrix metalloproteinase 10 (MMP10) forward: 5V-AGT CTG CTC TGC Applied Science, Indianapolis, IN). CTA TCC TCT GAG-3V, reverse: 5V-CTT CAT ACA GCC TGG AGA ATG TGA G-3V; NR4A2 forward: 5V-GCT GTT GGG ATG GTC AAA GAA G-3V, reverse: 5V- Results and Discussion TCG CCT GGA ACC TGG AAT AGT C-3V; GPI-anchored metastasis (C4.4A) sense: 5V-TCC CCG AAC AAG ATG AAG ACA G-3V, reverse: 5V-CAG GCA AGG Mect1/Torc1 domain is required for transformation in RK3E ACA CAG TCA CAT TAG-3V; NR4A3 forward: 5V-TTC CCC TCC AGG TTC cells. Insight into the functional consequences of the Mect1-Maml2 CAG TTA TGC-3V, reverse: 5V-TGG TGG TGG TGA TGG TGA TGG TAG-3V; fusion transcript can be inferred from recent data suggesting that amphiregulin forward: 5V-TGG TGC TGT CGC TCT TGA TAC TCG-3Vreverse: the chimeric gene product can potentially disrupt both cAMP/ 5V-TCA CTT TCC GTC TTG TTT TGG G-3V. CREB and NOTCH signal transduction pathways. For example, a Global gene expression profiling using oligonucleotide microarrays. cDNA (called Torc1) was isolated from high-throughput reporter Double-stranded cDNA and biotin-labeled cRNA were generated from 6 Agof assays designed to identify transcriptional cAMP/CREB coactiva- total RNA (Qiagen RNeasy Mini Kit) using protocols recommended by the tors and was found to be identical to the Mect1 gene on manufacturer (Affymetrix, Santa Clara, CA). The oligonucleotide microarray chromosome 19p13 (13, 14, 21). Importantly, the minimal region platform used was the GeneChip Human Genome U133A that has 18,400 transcripts and variants, including 14,500 known genes, which are required for CREB binding was localized to the amino terminal 42- represented by 22,000 oligonucleotide probe sets (Affymetrix GeneChip). amino-acid exon 1, which is the only domain of Mect1/Torc1 that is Prehybridization of the probe arrays was done for 10 minutes in hybridization included in the oncogenic fusion peptide (13, 21). In contrast, the buffer (1Â buffer: 100 mmol/L MES, 1 mol/L Na+, 20 mmol/L EDTA, 0.01% Maml2 gene, localized to chromosome 11q21, is a homologue of Tween 20 final concentration) at 65jC. The hybridizations were done at 65jC the Drosophila NOTCH coactivator, Mastermind, which has been with 60 rpm rotation for 16 hours in 1Â hybridization supplemented with shown to be required for normal NOTCH signaling in Drosophila 0.06 Ag/AL fragmented cRNA (15 Ag in total); 50 pmol/L control oligonucleotide B2; 1.5, 5, 25, and 100 pmol/L of 20Â eukaryotic hybridization controls (bioB, bioC, bioD, cre); and 0.1 mg/mL herring sperm DNA. The probe 1 http://linus.nci.nih.gov/brb.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Mect1-Maml2 Target Genes and mammalian cells (1, 15). Mect1-Maml2 was subsequently shown to suppress the basal NOTCH/Mastermind activation of a promoter containing tandem CSL/NOTCH binding sites, which suggested that the fusion protein may be exerting a dominant- negative effect (1). Surprisingly, Mect1-Maml2 also activated the Hes1 promoter, a NOTCH receptor target gene, in the absence of NOTCH ligand and NOTCH/CSL binding sites. This observation initially suggested an alternate hypothesis for Mect1-Maml2 that involved the constitutive deregulation of NOTCH signaling (1). Recent data, however, has revealed that the Hes1 promoter contains a cryptic cAMP/CREB response DNA binding element (13, 22), suggesting the hypothesis that the biological effect of Mect1-Maml2 may be the aberrant activation of cAMP signaling through activation of selected CREB target genes. To examine the importance of the amino-terminal Mect1/Torc1 region in Mect1-Maml2 tumorigenesis, we generated two different in-frame deletions within this 42-residue domain of the chimeric oncogene (Fig. 1). We had previously reported the ability of wild- type Mect1-Maml2 to efficiently generate foci formation in RK3E rat epithelial cells at 3 weeks, whereas full-length Maml2 or the Figure 2. Doxycycline (Dox)-induced expression of expressor and parental vector alone had no transforming activity (1). We observed nonexpressor cell clones. A, RT-PCR of independent Mect1-Maml2–transfected cell clones that either express (clones 1-3) or show trace or undetectable levels that both deletions (20 or 14 residue deletions) within the CREB- (clone 4) of Mect1-Maml2 after doxycycline exposure. Size markers are binding domain of Mect1/Torc1 exon 1 resulted in complete loss of shown on the left. B, protein immunoblot analysis showing efficient induction of Mect1-Maml2 protein following doxycycline exposure in the Mect1-Maml2 foci formation. These data support the hypothesis that the CREB- expressor clones. Molecular weight standards in kDa are shown on the right. binding domain within Mect1/Torc1 exon 1 plays an essential role in the ability of Mect1-Maml2 to transform epithelial cells and of ectopic Mect1-Maml2 protein. We transfected the pTRE-M-M2 rejects an alternate hypothesis that proposed that the role of the plasmid containing the full-length Mect1-Maml2 open reading t(11;19) chromosomal translocation was to simply truncate and frame and isolated five independent clones following selection with deregulate Maml2 activity under control of the Mect1/Torc1 G418 and hygromycin. Three of these clones expressed high or promoter. Whereas a small, in-frame deletion (residues 77-118) moderate levels of the Mect1-Maml2 protein following doxycycline within the Maml2 sequence had no effect on transformation ability, expression (expressor clones 1-3), whereas two other clones serving as a negative control, we also observed that a larger expressed undetectable (or extremely low) levels of Mect1-Maml2 deletion (residues 703-838) within the conserved carboxyl-terminal protein despite doxycycline exposure (nonexpressors clones 4-6; Maml2 transactivation domain showed loss of foci formation, Fig. 2). We selected these independent paired cell clones for further consistent with the model that Mect1-Maml2 functions to analysis because the expressor and nonexpressor clones could be aberrantly transactivate selected target genes. maintained continuously under identical antibiotic (G418, hygrom- Global gene expression profiling using doxycycline- ycin, and doxycycline) incubation conditions during the harvesting inducible Mect1-Maml2. To further study possible mechanisms of RNA. We generated total RNA from independent frozen stocks of underlying Mect1-Maml2 tumorigenesis, we have generated a these transfected clones after exposure to doxycycline for 24, 48, or doxycycline-inducible Mect1-Maml2 expression vector in HeLa 72 hours to use as labeled probes in oligonucleotide microarray cells to study the pattern of mRNA expression following induction analyses. We planned to first generate a set of candidate genes that were differentially regulated in doxycycline-treated Mect1-Maml2 expressor versus doxycycline-treated, nonexpressor cell clones. This set of genes could then be independently validated for differential expression in the individual Mect1-Maml2 expressor clones that were incubated in the presence versus the absence of doxycycline. Ectopic Mect1-Maml2 induces cAMP/CREB-regulated genes. A total of 164 genes were differentially expressed at P = 0.001 between Mect1-Maml2 expressor and nonexpressor HeLa clones of which we selected the first 100 genes with the lowest parametric P value (Fig. 3). Of the differentially expressed genes, 95% showed increased expression following Mect1-Maml2 induction consistent with a model for target gene activation by the chimeric oncogene. Strikingly, no gene was repressed >2-fold in this data set. Only five genes in this data set were induced z8-fold by ectopic Mect1- Figure 1. Mect1/Torc1 domain required for foci formation in RK3E cells. RK3E rat epithelial cells were transfected with Mect1-Maml2 expression plasmids Maml2 protein expression (Table 1). These included cytosolic and transformed foci were scored by crystal violet staining at 4 weeks as phosphoenolpyruvate carboxykinase (PEPCK1/PCK1), 171-fold previously described (1). Amino acid (aa) coordinates of the in-frame deletamer induction; amphiregulin (AREG), 27-fold; GPI-anchored metasta- plasmids are indicated. Foci formation (À) represents 0 foci/100 mm plates. Foci formation (+) represents a range of 13 to 29 foci/100 mm plate (for wild-type) sis-associated gene (C4.4A), 17-fold; MMP10, 14-fold; and nuclear and 17 to 30 foci/100 mm plate (for D77-118). receptor subfamily 4, group A, member 3 (NOR1, NR4A3), 8-fold. www.aacrjournals.org 7139 Cancer Res 2005; 65: (16). August 15, 2005

Figure 3. Gene-centered image of differentially expressed genes comparing Mect1-Maml2 expressor versus nonexpressor clones.

Importantly, three of these five genes, PEPCK1/PCK1, AREG, and cystathionase (CTH) and keratin 17 promoters shows the (TGACG) NR4A3/NOR1, are previously published cAMP/CREB target genes CRE sites at nucleotide position À150 from the CTH transcrip- (23, 24). In addition, inspection of the promoter sequences of the tional start site and at À50 and À220 from the keratin 17 start site. human GPI-anchored metastasis gene (C4.4A) revealed a variant These latter sites, however, were not conserved in the promoter cAMP responsive element (TGACG) at À82 relative to the region of the corresponding murine homologues and, therefore, transcriptional start site and alignment of the promoter region are of uncertain significance. Several other previously reported from the mouse C4.4A orthologue also showed the same cAMP- CREB-inducible genes were also detected at lower levels of gene responsive element (CRE) element sequence at À81 from the induction, including crystallin a-h (3.8-fold induction; ref. 14), c-fos mouse transcriptional start site. Many CREB-inducible genes are (3.0-fold; ref. 28), IL-8 (2.2 fold; ref. 14), and dual-specificity regulated through variant or half-palindromic sites (25) and a phosphatase-1 (1.8-fold induction; refs. 23, 29). We also recognize recent genome-wide analysis showed that 70% of CREB-binding that certain gene activations may be events that are indirectly loci were within 1 kb of these half-palindromic CRE sites (22), linked to CREB activation. For example, IL-6 is known to activate suggesting that the conservation of this sequence within the several genes, including MMP10/stromelysin 2 (30) and ID2 (31), murine and human GPI-anchored gene is functionally relevant. In which we detected in our data set. Whereas we limited our search addition, Mect1/Torc1 was recently noted to activate the for potential CRE sites to within 500 bp of the transcriptional start interleukin-8 (IL-8) promoter through a cryptic CRE-like variant site, another group recently published an algorithm for a genome- site at À69 relative to the IL-8 start site (14). wide analysis of candidate CRE sites that scored for positional Fourteen additional genes were induced z4-fold among this data characteristics, clustering of sites, and mammalian conservation set (Table 1). These genes include human chorionic gonadotropin (32). This analysis, which included 5,000 bp upstream of the (HCG), IL-6, nuclear receptor subfamily 4, group A, member 2 transcriptional start site and 300 bp of exon 1 sequences, identified (NR4A2/Nurr1), and c-MAF, which are also previously known several more genes from our list of Mect1-Maml2–induced CREB-regulated genes (22, 23, 26, 27). Moreover, inspection of the transcripts (Table 1). Of interest, we did not detect induction or

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Mect1-Maml2 Target Genes repression of Notch target genes, such as Hes gene family members, follows: PEPCK1/PCK1, P = 2.2 Â 10À5, 20-fold induction; AREG, nor did we detect repression of genes >2-fold in the data set list of 1 Â 10À5, 14-fold induction; MMP10,4Â 10À6, 12-fold induction; GPI- the 100 highest scoring genes. These observations, therefore, anchored associated,1Â 10À7, 8-fold induction; CTH, 2.2 Â 10À6, further support the model that the Mect1-Maml2 product targets 6-fold induction; NR4A2,1Â 10À7, 5.4-fold induction; HCG, CREB-binding promoters using its amino-terminal Mect1/Torc1 7 Â 10À4, 5-fold induction; crystallin a-b,1Â 10À4, 5-fold induction; domain to aberrantly activate certain cAMP-responsive genes NR4A3,1Â 10À3, 4-fold induction; and cytokeratin 2, 4.6 Â 10À5, during tumorigenesis. 4-fold induction. These observations confirmed the identification of We have taken two different approaches to confirm the global the same set of activated genes that were detected in the earlier gene expression data obtained from the comparison of the different comparison of Mect1-Maml2 expressor and nonexpressor clones. Mect1-Maml2–expressing versus Mect1-Maml2–nonexpressing cell To further confirm that these transcripts identified by gene array clones. First, we harvested RNA from each of the three individual profiling are bona fide Mect1-Maml2 targets, we harvested RNA Mect1-Maml2 expressor HeLa clones in the presence or absence of from expressor clones for direct confirmation by RT-PCR analysis doxycycline and pooled the gene expression pattern using the same using paired oligonucleotide primers (Fig. 4A). We tested a fresh 18,400 oligonucleotide array. Of 80 genes that were identified at the source of RNA from a separate stock of one of the Mect1-Maml2 P < 0.001, 10 genes were induced z4-fold. These genes were as expressor clones, which had been treated with doxycycline for 24, 48, or 72 hours, to determine if we could detect induction of the PCK1, AREG, GPI-anchored metastasis associated, and MMP10 Table 1. Differentially expressed transcripts in Mect1- candidate genes. RT-PCR analysis showed maximal gene induction Maml2 expressor clones compared with nonexpressor cell at 48 and 72 hours postexposure to doxycycline induction. We also clones with >3-fold activation ranked by P value observed no gene induction when the cells were incubated for the same time period in the absence of doxycycline, nor when RNA Fold difference Parametric P UG cluster Gene symbol from a nonexpressor Mect1-Maml2 clone was tested under of geometric identical conditions (Fig. 4A). To quantitate the induction over means time for seven cAMP-regulated genes, we pooled the combined 171 1.00 Â10À7 Hs.1872 PEPCK1* (23) oligonucleotide array hybridization data from all three Mect1- 26.6 1.00 Â10À7 Hs.270833 AREG* (24) Maml2 expressor clones at 24, 48, and 72 hours. We observed a 16.6 1.00 Â 10À7 Hs.377028 GPI-anch/C4.4A* consistent increase in gene induction between 48 and 72 hours for 13.6 1.00 Â 10À7 Hs.2258 MMP10* each of the induced genes: PEPCK (1.41-fold increase from 48 to 5.2 1.00 Â 10À7 Hs.371249 PTN 72 hours), MMP10 (1.50-fold increase), GPI-anchored/C4.4 (1.34- 5.0 1.00 Â10À7 Hs.512234 IL-6* (27, 48) fold increase), NR4A2 (1.08-fold increase), amphiregulin (1.44-fold À7 3.2 1.00 Â 10 Hs.110571 GADD45B* increase), NR4A3 (1.21-fold increase), and IL-6 (1.18-fold increase). À7 6.7 2.00 Â 10 Hs.2785 KRT17 These observations validate the experimental design and data Â À7 4.5 2.00 10 Hs.165258 NR4A2* (13, 22, 23) obtained from our global gene profiling and have shown the 4.6 3.00 Â 10À7 Hs.19904 CTH 7 identification of a group of cAMP/CREB-regulated genes that are 8.0 8.00 Â10À Hs.279522 NR4A3* (13, 22, 23) 3.8 1.70 Â 10À6 Hs.376206 KLF4* aberrantly activated by the oncogenic Mect1-Maml2 product. 3.5 3.00 Â 10À6 Hs.271809 GPR161 To test the effect of Mect1-Maml2 expression in a different cell 5.8 7.80 Â 10À6 Hs.159226 HAS2 lineage, we first obtained RNA from the immortalized RK3E rat 3.8 9.60 Â10À6 Hs.408767 Crstlln AB* (14) epithelial cell line or from RK3E foci that were generated and 3.2 1.34 Â 10À5 Hs.550529 CARD14 expanded following transfection with a Mect1-Maml2 plasmid as 4.0 1.45 Â10À5 Hs.517617 C-MAF* (22) previously described (1). Similar to our data with HeLa cells, we À5 3.4 1.80 Â 10 Hs.137154 PIGA* observed induction of NR4A3, GPI-anchored/C4.4, and MMP10 in À5 6.4 1.89 Â 10 Hs.784 EBI2 the transfected RK3E cells (Fig. 4B). We also transfected either Â À5 5.8 2.59 10 Hs.446683 HCGB Mect1-Maml2, full-length Maml2, Mect1/Torc1, or the control 4.2 4.40 Â 10À5 Hs.59093 CACNB2* parental constitutive cytomegalovirus promoter plasmid into the 3.0 5.42 Â 10À5 Hs.512727 FLJ23306* 3.5 6.49 Â 10À5 Hs.345139 GEM* H2009 lung adenocarcinoma cell line and harvested RNA at 5.8 7.63 Â 10À5 Hs.180919 ID2* 72 hours. We observed induction of the CREB-regulated genes 4.1 9.10 Â 10À5 Hs.75765 CXCL2* PCK1 and NR4A3 expression following transfection of Mect1- 4.1 0.0001109 Hs.164226 THBS1* (49) Maml2, but not with any of the other vectors, confirming the 3.0 0.0002566 Hs.118695 KCNG1* induction of these CREB target genes by exogenous Mect1-Maml2 3.9 0.0003176 Hs.145949 K6HF expression in a different cell type (Fig. 4C). The inability of Mect1/ 6.9 0.0004073 Hs.296942 KRTHA4 Torc1 to induce expression of these target genes in the transfected 3.1 0.0005153 Hs.516249 IL1R1 (50) H2009 cells may reflect a more potent transcriptional activation 3.3 0.00072 Hs.109514 RYR2 domain in the Mect1-Maml2 fusion, which was derived from the carboxyl-terminal region of the Maml2 product (1). We also tested NOTE: Previously reported CREB/cAMP–regulated genes were RNA from two different mucoepidermoid cancer cell lines, H292 depicted in bold (other known CREB-regulated genes, such as c-FOS and H3118, that we had previously shown to contain a t(11;19) and IL-8 genes, were also activated; see text). translocation with endogenous expression of Mect1-Maml2 tran- *Predicted CREB target genes by genome-wide analysis of CRE scripts (1). We detected expression of CREB-regulated genes by conservation, clustering, and positional location relative to the trans- RT-PCR in both tumor cell lines (Fig. 4D), suggesting that Mect- criptional start site (32). Maml2 may be aberrantly activating a set of cAMP inducible genes in vivo. www.aacrjournals.org 7141 Cancer Res 2005; 65: (16). August 15, 2005

Inhibition of signaling pathways in mucoepidermoid cancer. less responsive to EGFR or PKA inhibition than H292 cells, but The presence of CRE or variant, half-palindromic CRE sites in were still inhibited by 40% to 50% with these agents (Fig. 5A, top multiple target genes (22, 25) has suggested that CREB functions to right). Because responsiveness to low doses of gefitinib can be regulate energy homeostasis, growth factor signaling, cell survival, associated with certain acquired EGFR mutations (43, 44), we and cell-to-cell communications through the coordinated expres- documented that these cell lines showed a wild-type sequence in sion of distinct genes. The mechanisms that allow the selective exons 18 to 21 (data not shown). In contrast, we found that two activation of distinct target genes in different tissues, however, tumor cell lines that lack expression of Mect1-Maml2 were remains undefined and may involve chromatin modifications (33) resistant to EGFR inhibition; however, the H360 cells were still or the activity of accessory cofactors that promote either CRE sensitive to H89 treatment (Fig. 5A, bottom). In addition, a recent occupancy or promoter activation. Our global gene profiling data study showed that activation of MMPs contributes to activation of suggests that Mect1-Maml2 expression can potently induce a EGFR in H292 cells (45). Because we also detected induction of small group of CREB-inducible target genes that might serve as MMP10 in our gene array experiments, we tested whether the candidate effectors of tumorigenesis in cancers that have acquired proliferation of H292 or H3118 cells is dependent on MMP the t(11;19) chromosomal rearrangement. For example, NOR1/ activity. We observed that treatment of cells with 1 Amol/L NR4A3 is a known oncogene that is tightly linked with the GM6001, a pan-MMP inhibitor, resulted in growth inhibition in development of chondrosarcoma (34), whereas AREG, MMP10, GPI- both H292 and H3118 cells by 45% to 50% (data not shown). anchored metastasis associated gene, IL-6, and PCK1 are associated These data, whereas preliminary, suggest that the targeting of with cell proliferation, invasion, or metastasis (30, 35–39). We were either global cAMP/CREB signaling through PKA inhibition or interested in the CREB-inducible epidermal growth factor receptor CRE decoy strategies (16) or by the targeting of accessible, ligand, amphiregulin, (a) because we had determined that it was downstream Mect1-Maml2–inducible gene pathways may offer a induced >20-fold by Mect1-Maml2 expression, (b) because small new approach for the systemic treatment of these malignant molecule, competitive inhibitors for this pathway were available for salivary gland tumors. in vitro testing, and (c) because activation of EGFR signaling and In summary, evidence that Mect1-Maml2 is an important expression of amphiregulin had been previously reported in H292 oncogene that underlies the development of malignant salivary cells that express endogenous Mect1-Maml2 (1, 40, 41). To test the gland tumors includes the observations that (a) the t(11;19) sensitivity of the mucoepidermoid cancer cell lines H292 and rearrangement has been detected as the sole cytogenetic abnor- H3118, as well as the nonmucoepidermoid lung cancer lines H2009 mality in several primary tumor samples (12, 46, 47), (b) Mect1- and H360, we incubated the cells with a variety of different small Maml2 can efficiently transform rat epithelial cells in vitro whereas molecules that can inhibit either EGFR (amphiregulin) or PKA the reciprocal Maml2-Mect1 transcript was not present in primary (cAMP-dependent kinase) pathways (Fig. 5). We observed dose- or derived tumor samples (1), and (c) Mect1-Maml2 is expressed dependent decreases in proliferation of H292 and H3118 cells with in 75% of all mucoepidermoid cancers tested to date (1, 2, 12). The two different inhibitors of EGFR, AG1478 or gefitinib. At the highest present study shows that the CREB-binding amino-terminal domain dose tested (1 Amol/L), AG1478 inhibited proliferation by 78% in of Mect1/Torc1 is required for transforming activity. Further, we H292 cells. In the case of gefitinib, sensitivity in lung cancer cell have shown that ectopic expression of the chimeric Mect1-Maml2 lines has been defined as an IC50 <1 Amol/L (42), and we observed transcript induces the activation of genes that are either known that gefitinib inhibited proliferation of H292 cells by 69% at cAMP/CREB targets or genes that contain CRE sequences near their 0.1 Amol/L and 78% at 1 Amol/L. The PKA inhibitor, H89, inhibited transcriptional start sites and may be previously unrecognized proliferation by 78% (Fig. 5A, top left). H3118 cells, however, were CREB-regulated targets. These observations support the model

Figure 4. Expression of Mect1-Maml2 target genes in four different cell types. A, RT-PCR from RNA obtained from either transfected HeLa expressor (Exp)or nonexpressor (Non-Exp) cell clones at 24, 48, 72 hours postexposure to doxycycline as indicated. M, size markers. B, RT-PCR from RNA obtained from either parental RK3E cells (lane 1) or from RK3E foci transfected with Mect1-Maml2 (lane 2) showing induction of NR4A3, GPI-anchored/C4.4, and MMP10 genes. The GAPDH housekeeping gene is included as a loading control for RNA integrity. C, RT-PCR from RNA obtained from the H2009 lung tumor cell line that was transiently transfected with either vector alone (lane 1), Mect1/Torc1 (lane 2), Maml2 (lane 3), Mect1-Maml2 (lane 4), or the no DNA control (lane 5). D, RT-PCR from RNA obtained from the H292 and H3118 mucoepidermoid cancer lines or no DNA control (neg. controls). Paired oligonucleotide primers were tested for Mect1-Maml2, GPI-anchored (C4.4A), IL-6, amphiregulin, MMP10, NRA4A2, and keratin 17 (lanes 1-7).

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Figure 5. Proliferation assays of mucoepidermoid and nonmucoepidermoid cells. H292, H3118, H2009, and H360 cells were plated in 10% FBS-RPMI 1640 in 96-well plates at 1,000 cells/well overnight. The following day, the indicated compounds were added to each well and cell viability was assayed at 4 days of drug exposure (WST-1 assay, Roche Applied Science). Columns, mean cell viability obtained from each treatment that was composed of six different wells and normalized to the value of control. where Mect1-Maml2 binds to CREB-regulated promoters to Acknowledgments deregulate a subset of cAMP-inducible genes during tumorigenesis, Received 4/1/2005; revised 6/8/2005; accepted 6/10/2005. thus providing a direct genetic link between an oncogenic The costs of publication of this article were defrayed in part by the payment of page chromosomal translocation and cAMP/CREB activation. Ultimately, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. these data may provide clues for preclinical opportunities in We thank Richard Simon for his help with the statistical analysis of the data and treating this class of malignant salivary gland tumors. review of the manuscript.

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