High Cancer-Specific Expression of Mesothelin (MSLN)

Research Article

High Cancer-Specific Expression of Mesothelin (MSLN)Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor–Dependent MCAT Motif Tomas Hucl,1 Jonathan R. Brody,1 Eike Gallmeier,1 Christine A. Iacobuzio-Donahue,1 Iain K. Farrance,2 and Scott E. Kern1

1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University; and 2Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland

Abstract tions, respectively (1–3). Identifying altered cellular pathways by Identification of genes with cancer-specific overexpression directly searching for mutations in individual genes has become offers the potential to efficiently discover cancer-specific difficult. These difficulties can potentially be overcome by a activities in an unbiased manner. We apply this paradigm to renewed attention to the pathways having activation in cancers by study mesothelin (MSLN) overexpression, a nearly ubiquitous, studying them using biochemical methods. The search can start diagnostically and therapeutically useful characteristic of with the identification of a gene having cancer-specific over- pancreatic cancer. We identified an 18-bp upstream enhancer, expression. Mesothelin (MSLN) is such a gene, whose over- termed CanScript, strongly activating transcription from an expression characterizes pancreatic cancer. otherwise weak tissue-nonspecific promoter and operating The MSLN gene, localized to 16p21, encodes a precursor protein selectively in cells having aberrantly elevated cancer-specific of 69 kDa, which is cleaved to a 40-kDa membrane-bound protein, termed MSLN, and a soluble 31-kDa protein, megakaryocyte- MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an potentiating factor. The protein is glycosylated and glycosylphos- MCAT element. Gel retardation and chromatin immunopre- phatidylinositol-anchored to the membrane. Its expression in cipitation assays showed the MCAT element to be bound by normal tissue is limited mainly to mesothelia of peritoneal, pleural, transcription enhancer factor (TEF)-1 (TEAD1) in vitro and and pericardial cavities (4). Overexpression of MSLN RNA and in vivo. The presence of TEF-1 was required for MSLN protein protein was found in several human malignancies, with the highest overexpression as determined by TEF-1 knockdown experi- expression in ovarian, pancreatic, bronchial, gastroesophageal, ments. The cancer specificity seemed to be provided by a cervical, endometrial, and biliary carcinomas (4–7), totaling up to putative limiting cofactor of TEF-1 that could be outcompeted nearly a third of human malignancies. by exogenous TEF-1 only in a MSLN-overexpressing cell line. A The biological function of MSLN is unknown; knockout mice CanScript concatemer offered enhanced activity. These results have no distinguishable phenotype (8). Cancer-specific overexpres- identify a TEF family member as a major regulator of MSLN sion and its limited expression in normal tissue make MSLN a good overexpression, a fundamental characteristic of pancreatic candidate as a diagnostic marker and target in immunotherapy (9). Various methods of detecting MSLN are already used to aid the and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence diagnosis of mesotheliomas and ovarian and pancreatic cancer represents a modular element for cancer-specific targeting, (10, 11). potentially suitable for nearly a third of human malignancies. Investigators have repeatedly confirmed MSLN overexpression to affect the vast majority of ductal adenocarcinomas of the pancreas [Cancer Res 2007;67(19):9055–65] by serial analysis of gene expression (SAGE; ref. 12), oligonucleotide array (13), in situ hybridization, reverse transcription-PCR (RT- Introduction PCR), and immunohistochemistry (14). Strong immunolabeling was Overactive cellular pathways play a crucial role in tumorigenesis, detected in virtually all ductal carcinomas, whereas the adjacent as they represent fundamental and causative features of cancers. In normal pancreas did not label (14). Interestingly, of the patients some cases, unambiguous and high levels of cancer-specific vaccinated with granulocyte macrophage colony-stimulating factor– pathway activation can be reliably detected by reporter constructs transduced autologous pancreatic cells who developed systemic and are specific for defined DNA motifs at which transcription antitumor immunity, all had a strong MSLN-specific CD8+ T-cell factors bind. Determining the components of activated pathways immune response (15). A targeted therapy using anti-MSLN using reporter constructs was essential to specifying some monoclonal antibodies has entered a clinical trial.3 mutationally targeted genes. For example, SMO/GLI and h-catenin The nearly ubiquitous overexpression of MSLN in pancreatic were so defined after biochemical definition of the pathway cancer hints that its dysregulation relates to a fundamental activated by PTC and adenomatous polyposis coli (APC) muta- biological activity of the neoplasm. To improve our understanding of this dysregulation, we identified regulatory cis-elements and trans-acting factors involved in the control of MSLN cancer- specific overexpression. We found evidence that an upstream Requests for reprints: Scott E. Kern, Department of Oncology, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-614-3314; Fax: 443- 287-4653; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0474 3 http://clinicaltrials.gov/ct/show/NCT00325494 www.aacrjournals.org 9055 Cancer Res 2007; 67: (19). October 1, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research enhancer element was responsible for the aberrant cancer-specific Electrophoretic mobility shift assay. Single-stranded high-perfor- MSLN overexpression. The activity of this enhancer was dependent mance liquid chromatography–purified oligonucleotides corresponding to ¶ ¶ on the binding of transcription enhancer factor TEF-1 to an MCAT the enhancer element (5 -GGTCTCCACCCACACATTCCTGGGGCGTG-3 ) sequence. We propose that the specificity of MSLN aberrant were purchased (Integrated DNA Technology), as were oligonucleotides carrying the same mutations as certain reporter constructs (M4 and M6). expression in cancer was provided by a cofactor of TEF that Annealed oligonucleotides were end labeled with 10 ACi/AL[a-32P]dATP binds to specific sequences immediately flanking the core MCAT using Klenow polymerase (New England Biolabs). Radiolabeled probes motif. (20,000 cpm) were used per electrophoretic mobility shift assay (EMSA) binding reaction. Nuclear extracts were prepared following standard protocols. The binding reaction consisted of 5 Ag of nuclear extracts, 2.5 Materials and Methods Ag poly(deoxyinosinic-deoxycytidylic acid) [poly(dI-dC)], radiolabeled Cell lines and cell culture. Cell lines were obtained from the American probe, and binding buffer in a total volume of 20 AL. Free probes bound Type Culture Collection except for the lymphoblastoid cell line BJAB, which by proteins were separated on a 10% polyacrylamide nondenaturating gel in was kindly provided by B. Barnes (Johns Hopkins University, Baltimore, 0.5Â Tris-borate EDTA, vacuum dried, and exposed to film at À80jC. For MD), and the mesothelioma cell line H-513, which was kindly provided by competition experiments, the protein extracts were first incubated with up R. Salgia (University of Chicago, Chicago, IL). Cells were cultured in to a 100-fold molar excess of unlabeled competing fragments for 10 min conventional medium supplemented with 10% FCS, L-glutamine, and before adding the radioactive fragment. For supershift experiments, mouse penicillin/streptomycin. anti-TEF-1, goat anti-TEF-4, or control mouse IgG (both from Santa Cruz RNA isolation and RT-PCR. RNA was isolated from cells using RNeasy Biotechnology) antibodies were used. His-tagged recombinant TEF-1 was Mini kit (Qiagen) and converted to cDNA using SuperScript II (Invitrogen) grown in Escherichia coli and purified as described previously (19). following the manufacturers’ protocols. PCR conditions and primer Gene silencing. For transient transfections, cells were transfected twice sequences are available on request. sequentially using Lipofectamine 2000 with 100 nmol/L small interfering 5¶-random amplification of cDNA ends. RNA was treated with DNase I RNA (siRNA; Ambion). The oligonucleotide sequences used in transfections (Qiagen). The first cDNA strand was synthesized using a MSLN-specific were as follows: T1a, GCCCUGUUUCUAAUUGUGG; T1b, GGUUAACA- primer, treated with RNases H and T1, purified using QIAquick PCR CUAAUCUCCUA; T1c, CGGAGUAUGCAAGGUUUGA; and T4, GGACGGCA- Purification kit (Qiagen), and tailed with 200 Amol/L dGTP using 1 ALof GAUUUGUGUAC. terminal deoxytransferase (U.S. Biochemical). The dG-tailed cDNA was The TEF-1 cDNA was released from the pXJ40-TEF-1A plasmid using amplified and subsequently nested amplified by PCR. The obtained EcoRI and BglII and subcloned into pcDNA3.1+Zeo (Invitrogen) precut with products were separated on 1% lithium borate agarose gels (Faster Better EcoRI and BamHI, resulting in an antisense orientation of the insert in Media LLC; ref. 16), purified, and sequenced at the Johns Hopkins respect to the promoter. For stable transfections, AsPc1 and HeLa cells were Sequencing Core Facility. transfected at f60% confluency in T75 culture flasks with 10 Ag of plasmid Immunoblotting. Equal amounts of proteins were separated on 10% using Lipofectamine. Cells were selected in zeocin for 2 to 3 weeks, lysed, Bis-Tris gels and transferred onto polyvinylidene difluoride membranes. and analyzed for the presence of plasmid and TEF-1 protein expression. No After blocking, the membranes were incubated with a mouse anti-MSLN viable clones with a significant reduction of TEF-1 expression were antibody MN (1 Ag/mL; kindly provided by I. Pastan, National Cancer generated, possibly due to detrimental effects of down-regulation of Institute, Bethesda, MD; ref. 17), a mouse anti-TEF-1 antibody (1:250; BD expression of the multimember family of TEF transcription factors. Biosciences), a goat anti-TEF-4 antibody (1:200; Santa Cruz Biotechnology), Chromatin immunoprecipitation. The assay was done as previously or a goat anti-actin antibody (1:200; Santa Cruz Biotechnology) for 1 h to described (20) with no antibody, a mouse monoclonal anti-TEF-1 antibody, overnight. Testing other commercially available anti-MSLN antibodies (5B2, a goat anti-TEF-4 antibody, a rabbit TEF-5 antibody (21), or a control mouse Vector Laboratories; V-16, Santa Cruz Biotechnology) proved unsatisfactory. IgG. DNA was recovered (QIAquick PCR Purification kit) and amplified by Membranes were washed and incubated with a secondary anti-mouse PCR using primers spanning and flanking (230 bp upstream and 294 bp antibody (1:20,000; GE Healthcare) or a secondary anti-goat antibody downstream) the CanScript sequence. (1:20,000; Santa Cruz Biotechnology) for 90 min. Detection was done using Immunohistochemistry and immunocytochemistry. Immunohisto- SuperSignal Pico reagents (Pierce). chemistry was done on paraffin-embedded slides as previously described In silico analysis. Sequence analysis for potential transcription binding (14) using a mouse monoclonal anti-MSLN antibody (5B2). Immunocyto- sites was done using MatInspector software (18). chemistry was done using the LSAB+System-HRP kit (Dako) after Plasmids. All plasmids for reporter assays were constructed by cloning incubation with a primary anti-MSLN antibody MORAb-009 (1:500; kindly of PCR-amplified inserts (High Fidelity Platinum Taq DNA Polymerase, provided by N. Nicolaides, Morphotek, Exton, PA) for 90 min. Invitrogen) into plasmids pGL3-Basic or pGL3-Promoter (Promega). The structures and fidelity of the resulting constructs were confirmed by Results restriction mapping and sequencing. Plasmids were purified using the MSLN Plasmid Midi kit (Qiagen). At least two independent plasmid preparations RNA and protein overexpression in pancreatic cancer of each construct were used in reporter assays. Mutated constructs were tissues and representative cell lines. We reproduced the generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene). reported differential expression of MSLN in cancer cells of tissues The Gli1 expression plasmid was a kind gift of A. Maitra (Johns Hopkins by immunohistochemistry (Fig. 1A). MSLN was localized mainly to University). cell membranes (Fig. 1B). Using RT-PCR and immunoblotting, we Transfections and reporter assays. Transient transfections were done established an appropriate comparative cell line panel in which 5 using Lipofectamine or Lipofectamine 2000 (Invitrogen). Briefly, 2.5 Â 10 to regulation of the cancer-specific expression of the MSLN gene Â 5 4 10 cells were plated in six-well plates 24 h before transfection. The next could be studied (Fig. 1C). A A day, cells were transfected using 1.5 g of reporter construct and 0.5 gof Transcriptional start site determination by 5¶-random pRSVhgal vector and incubated for 5 h and the medium was exchanged. In amplification of cDNA ends. Sequencing of products generated cotransfection experiments, corresponding amounts of empty vector were ¶ ¶ used to equalize the amounts of transfected DNA. After 24 to 48 h, cells by 5 -random amplification of cDNA ends (5 -RACE) revealed were washed twice with PBS, lysed in reporter lysis buffer (Promega), and multiple transcript ends spanning a 400-bp region (Fig. 1D). No ¶ subjected to a cycle of freeze/thaw. Luciferase activity was determined TATA box was identified. We designated the 5 -end of the longest using the Steady-Glo System (Promega). To control for transfection effi- transcript as nucleotide +1 and submitted this previously ciency, h-galactosidase activity was determined using standard protocols. unreported transcript to Genbank under number EF420155. Three

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Figure 1. MSLN RNA and protein overexpression in tissues and representative cell lines. A and B, MSLN is labeled immunochemically. A, MSLN protein overexpression characterizes most pancreatic adenocarcinomas. Arrows, a cancerous duct and a normal duct (inset) from the same patient. B.1, MSLN protein overexpression localizes to cellular membranes. The AsPc1 cell line is depicted as a representative example. B.2, the MiaPaCa2 cell line is immunocytochemically negative for MSLN. C, MSLN overexpression is restricted to certain cell lines. AsPc1 and CAPAN2 represent pancreatic, and HeLa represents nonpancreatic cancer-specific MSLN overexpression. OVCAR3 represents overexpression in a cancer arising in a tissue normally expressing MSLN (ovary). HEK293 (human embryonic kidney), BJAB (Burkitt’s lymphoma), and HG261 (human noncancerous fibroblasts) represent cancerous and noncancerous cells not expressing MSLN. The glycosylated (slower) and deglycosylated (faster) forms of MSLN protein can be distinguished. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin are loading controls. RNA, RT-PCR detection of the MSLN and GAPDH gene. Protein, immunoblots using specific antibodies against MSLN and actin. NC, no template PCR. D, identification of promoter and transcriptional start sites. Sequence of the 5¶-end of the MSLN gene, including the MSLN promoter identified here. Sequencing of 5¶-RACE products revealed 10 different possible transcriptional start sites, 3 corresponding to the consensus initiator sequence (overlying arrows), the rest to nonconsensus start sites (arrowheads). Underlined, a cryptic intron excised at cryptic splice sites that define the borders of alternate exons 1A and 1B. The translation start site lies in exon 2, not depicted. Lower case letters, start of intron 1. www.aacrjournals.org 9057 Cancer Res 2007; 67: (19). October 1, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research of the start sites matched the consensus initiator sequence, and available transcripts from public databases revealed the presence others did not and may represent partially degraded transcripts of a putative polyadenylation signal, poly A tail, and absence of (Fig. 1D). Exons 1A and 1B were formed by splicing at cryptic splice potential destabilization signals. These features suggest that the sites located at position +86 and +336. This splicing was observed MSLN transcript would be stable. in our transcripts and in previously reported cDNA sequences Transcriptional regulation of MSLN from a response (Vega transcript OTTHUMT00000155614). Sequence analysis of element specific for aberrant expression in cancer. We used

Figure 2. Transcriptional regulation of MSLN overexpression. A, reporter constructs from vectors pGL3-Basic and pGL3-Promoter, engineered regions shown. Lines, MSLN gene fragments, numbered relative to transcriptional start site (+1). SV40, SV40 minimal promoter; LUC, luciferase gene. Construct labels are given to the right of the luciferase element. Arrows, orientation of the fragments within the vector. B, reporter activity of indicated constructs in MSLN-expressing and MSLN- nonexpressing cell lines. AsPc1, CAPAN2, and HeLa cells have high reporter activity originating in the region between À135 and À49 bp, whereas this activity is absent in MiaPaCa2, RKO, and HEK293 cells. Activities relative to that of vector pGL3-Basic are presented. C, the region downstream from the transcriptional start site, including intron 1 inserted in pGL3-Promoter, does not have enhancer activity (construct P2F). D, cell lines derived from tissues having expression of MSLN in the nonneoplastic state have very little or no enhancer activity of the region À135/À49. Activities of the construct B-135 relative to B-49 are presented. Columns, average data from two to six experiments done in duplicates; bars, SEM.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Regulation of Mesothelin promoter-reporter assays to analyze 8 kb of genomic sequence surrounding nucleotide +1 (6 kb upstream and 2 kb downstream, including intron 1; Fig. 2A) in our panel of representative cell lines. In transient transfection assays, we identified a region between À135 and À49 bp that activated transcription from a minimal promoter in all tested MSLN-overexpressing cell lines AsPc1, CAPAN2, HeLa, HS766, and PL45 but did not activate the minimal promoter in MSLN-nonexpressing lines MiaPaCa2, RKO, HEK293, and BJAB (Fig. 2B; data not shown). In cell lines having cancer- specific MSLN overexpression, the region produced a 4- to 10-fold increase in reporter activity over the core promoter. In nonexpressing lines, the basal core promoter activity remained low and unchanged across the tested region. Plasmid P2F, which contained the sequences downstream from the start of transcription, had no enhancing activity (Fig. 2C). No significant increase in reporter activity in the À135/À49 region was seen in cell lines derived from tissues that are known to express MSLN even in a nonneoplastic state (ovarian carcinoma cell line OVCAR3 and mesothelioma cell lines MSTO-211H and H-513; Fig. 2D; ref. 22). Those lines are thought to exhibit a tissue- specific, but not cancer-specific, MSLN expression. Once the longest transcript extending to nucleotide +1 was determined, we generated constructs extending only from +27 (constructs B-67b and B-49b; Fig. 2A). Reporter activity of these constructs did not differ from those extending from +413 (data not shown). To rule out the possibility that the short transcripts were generated from an alternative promoter, the region immediately upstream of the most downstream transcriptional start site (nucleotide +369) was also examined in a promoterless vector (B+183 and B+92; Fig. 2A) and had no enhanced activity in all cell lines tested (data not shown). We next attempted to further narrow the response element and found all the cancer-specific activity to originate from a region defined by plasmids B-67 and B-49 (Fig. 3A and B). The region acted like an enhancer, in that it functioned in either orientation, activated transcription independently of location, and also enhanced a heterologous SV40 promoter (Fig. 3C). Figure 3. The response element localized to the region between À67 and Mutations defining the required sequence. To vigorously À49 and acted as an enhancer. A, the response element containing the transcriptional activity in MSLN-expressing cell line AsPc1 was localized to the define the required sequence, variants created by site-directed region defined by plasmids B-67 and B-49. B, RKO cells were used as a mutagenesis were tested. Transfections of mutated constructs negative control. Activities relative to pGL3-Basic are presented. C, the response revealed two independent functional sites [termed site 1 (À53 to element acted as an enhancer. It activated transcription from a heterologous À À À promoter (construct P1) and was independent of orientation (PE1F versus 46) and site 2 ( 65 to 56); Fig. 4A] of the enhancer element that PE1R, PE3F versus PE3R) and location (PE1F versus PE3F). The construct were both required for reporter activity in MSLN-overexpressing P3F, which did not contain the enhancer element, and RKO cells were used as lines AsPc1 and HeLa, whereas no significant changes of basal negative controls. reporter activity in the MSLN-nonexpressing line RKO were observed (Fig. 4B; data not shown). inserted in front of a minimal promoter (pCanScript3.luc), the Site 1, located at the 3¶-end of the enhancer element, contained a enhancing activity increased to 30-fold in AsPc1 cells (Fig. 4C). The 100% match to an MCAT element, a binding site of the TEF family CanScript sequence is partially conserved among mammals. of transcription factors (23). Transfections of mutated constructs The human sequence is identical to the chimpanzee and rhesus M6, M6b, and M6c completely abolished enhancer activity, the monkey sequences and has one mismatch with the cow sequence. resultant sequence having the basal activity of the core promoter- Three mismatches with the mouse sequence and four mismatches construct B-49 (Fig. 4B). with the rat and dog sequences affect both CanScript sites. Site 2 at the 5¶-end of the enhancer contained a putative binding Binding of TEF-1 to the MCAT motif of the MSLN enhancer site for the Sp1 and Sp1-like Kru¨ppel family of transcription factors in vitro. EMSAs using synthetic radiolabeled oligonucleotides (24). Construct M4 removed 40% of reporter activity, construct M5 representing the enhancer element identified one specific DNA- removed 65% of reporter activity, whereas construct M1, combining protein complex, complex 1 (Fig. 5A, lane 3), which (a) was only one base from the substitutions of both M4 and M5, produced outcompeted by an excess of unlabeled probe (Fig. 5A, lane 5), (b) a 75% reduction in reporter activity (Fig. 4B). did not form when a radiolabeled site 1–mutated probe was used The defined requisite sequence for cancer-specific enhancement (Fig. 5A, lane 4), and (c) was not outcompeted by an excess of of transcription was thus À62 to À45 and was termed the CanScript mutated unlabeled probes based on sites 1 and 2 (Fig. 5A; data not sequence. When three copies of the CanScript sequence were shown). Complex 1 and its pattern of responses to competition www.aacrjournals.org 9059 Cancer Res 2007; 67: (19). October 1, 2007

Figure 4. Two distinct functional sites of the enhancer element were determined by site-directed mutagenesis. A, mutated constructs were based on the wild-type B-99 construct. The location of proposed sites 1 and 2 is depicted. The bases mutated in various constructs are in lower case and underlined, with constructs labeled at the left. B, reporter assays done in cell line AsPc1 indicated the presence of two distinct sites within the enhancer that were both required for reporter activity in AsPc1. No significant effect of the mutations was observed in RKO. C, pCanScript3.luc containing three concatemerized copies of CanScript produced a 30-fold enhancement over a SV40 minimal promoter.

experiments did not differ between MSLN-overexpressing lines members of the TEF family of transcription factors in cancer cell with high reporter activity of the enhancer, AsPc1 (Fig. 5A) and lines (Fig. 6A) did not correspond to MSLN expression (Fig. 1C). HeLa (Fig. 5B, lanes 1–4), the MSLN-expressing cell line without None of the TEF members was selectively expressed only in cell enhancer activity, OVCAR3 (Fig. 5B, lanes 5–8), and the MSLN- lines having cancer-specific MSLN overexpression. nonexpressing line with no enhancer activity, RKO (Fig. 5A, lanes Three nonoverlapping siRNAs targeting specifically the TEF-1 7–10). The presence of complex 1 was restricted to cells expressing transcript caused decreases in MSLN protein expression (Fig. 6B). TEF-1 (TEAD1) irrespective of MSLN expression (Fig. 5C, lanes Selective knockdown of TEF-4 resulted in down-regulation of 1–10). No complex was present in the TEF-1–nonexpressing line TEF-4 but MSLN protein expression was not reduced (Fig. 6B). BJAB and human fibroblasts having minimal TEF-1 expression Binding of TEF-1 to MSLN enhancer in vivo. Having shown HG261 (Fig. 5C, lanes 7 and 9). The specific complex 1 was binding of TEF-1 to the MCAT element of the CanScript sequence supershifted by an anti-TEF-1 antibody (Fig. 5D.1, lanes 2–4)but in vitro, we sought to confirm this finding in living cells. Chromatin not by an anti-TEF-4 (TEAD2) or a control mouse IgG (Fig. 5D.1, immunoprecipitation (ChIP) assays showed that a DNA sequence, lanes 5–7). When a mutated probe was used, no supershift including the enhancer element (100 bp tested), was bound by TEF-1 occurred (Fig. 5D.1, lanes 8–10). Complex 1 was reduced on in vivo in AsPc1 cells but not in BJAB cells, which expressed no TEF-1 knockdown of TEF-1, whereas no reduction was observed on (Fig. 6C). Sequences surrounding the enhancer element immediately knockdown of TEF-4 or an irrelevant protein FANCD2 (Fig. 5D.2, upstream and downstream were not bound by TEF-1 (Fig. 6C). No lanes 1–3). When a recombinant TEF-1 was added to an MCAT binding to any of the DNA sequences tested was detected when wild-type but not mutated probe, a complex formed that comi- antibodies against TEF-4 or TEF-5 (TEAD3) were used, although no grated with complex 1 (native TEF-1; Fig. 5D.2, lanes 4 and 5). positive control could be established for these two antibodies in the Complex 1 was enhanced when a recombinant TEF-1 was added ChIP assay (data not shown). to cell nuclear extracts (Fig. 5D.2, lane 6). Cancer specificity of the enhancer mediated by a cofactor. It A discrete larger complex (complex 2) above complex 1 was also has been suggested that the transactivation function of TEF-1 is identified (Fig. 5A–D). Complex 2 was not fully diminished when an mediated by a highly limiting, cell-specific, titratable transcriptional excess of wild-type unlabeled competitor, site 1–mutated compet- intermediary factor (a cofactor) that is reliably detected by a itor, or site 2–mutated competitor, was used. It was, however, squelching assay (25). To test such a possibility in our system, diminished on addition of an excess of poly(dI-dC) and was exogenous TEF-1 driven by the cytomegalovirus promoter was therefore considered nonspecific. The apparent enhancement of introduced in increasing concentrations into MSLN-overexpressing complex 2 that appeared when a site 1–mutated probe was used and MSLN-nonexpressing cell lines together with either a reporter was also considered nonspecific (Fig. 5A, lane 8; data not shown). containing the CanScript sequence or heterologous SV40 promoter- When a radiolabeled oligonucleotide having a wild-type site 1 driven luciferase. In the MSLN-overexpressing cell line AsPc1, and a mutation in site 2 was used as a probe, complex 1 was CanScript activity diminished with increasing concentrations of preserved and no other specific complex could be identified (data TEF-1 expression plasmid (Fig. 6D), interpreted as squelching. In not shown). contrast, in the MSLN-nonexpressing cell line MiaPaCa2, the TEF-1 is required but not sufficient for MSLN overexpres- background reporter activity was neither induced nor reduced sion. The graded levels of RNA and protein expression of the when TEF-1 plasmid was added (Fig. 6D, left).

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Figure 5. Binding of TEF-1 to site 1 in vitro. EMSAs used nuclear extracts from various cell lines. The constituents of the reaction assayed in each lane are depicted above the gel. PWT, probe wild-type; Pm1, probe mutated in site 1; CWT, competitor wild-type; Cm1, competitor mutated in site 1. A, lane 3, a specific retarded band termed complex 1 (C1) was present when nuclear extracts of AsPc1 were incubated with an oligonucleotide corresponding to the enhancer element; lane 4, this complex did not form when site 1 was mutated. It was outcompeted by a 100-fold molar excess of unlabeled wild-type probe (lane 5) but not site 1–mutated probe (lane 6). Lanes 7 to 10, the same pattern was observed in RKO cells. B, replication in HeLa (lanes 1–4) and OVCAR3 (lanes 5–8) cells gave the same results. C, the specific complex 1 was present in various cell lines irrespective of MSLN expression and required TEF-1. MSLN-expressing cell lines HeLa, CAPAN2, and MSTO-211H (lanes 1, 2, and 4) and MSLN-nonexpressing cell lines LNCaP(prostate cancer), MiaPaCa2,HEK293, BJAB, Cal27 (oral squamous cell cancer), HG261, and MDA-MB-480 (breast cancer; lanes 3, 5, 6, 7, 8, 9, and 10) all formed complex 1. Complex 1 failed to form in the cell line having no expression of TEF-1 (BJAB) and in the noncancerous cell line HG261. D.1, complex 1 was supershifted (SS) by increasing concentrations of TEF-1 antibody (1, 2, and 5 Ag; lanes 2–4, a¯TEF-1) but was not supershifted by increasing concentration of TEF-4 antibody (1.5 Ag; lanes 5 and 6,a¯TEF-4) or control mouse IgG (2 Ag; lane 7,a¯IgG). Lanes 8 to 10, the nonspecific complex 2 (C2) was not shifted by any of the antibodies (2 Ag). D.2, lanes 1 to 3, in HeLa cells, complex 1 was reduced on siRNA knockdown of TEF-1 (T1a used) but not TEF-4 or FANCD2; lanes 4 and 5, recombinant TEF-1 protein formed a retarded band that comigrated with complex 1 using a wild-type but not a mutated MCAT probe; lane 6, native complex 1 was enhanced by recombinant TEF-1. www.aacrjournals.org 9061 Cancer Res 2007; 67: (19). October 1, 2007

No significant decrease in reporter activity was observed in the transcription factors [TEF-1 (TEAD1), TEF-3 (TEAD4), TEF-4 cell lines when the heterologous SV40 promoter-driven luciferase (TEAD2), and TEF-5 (TEAD3); ref. 23]. TEF-1 was first identified (pGL3-Promoter) was used as a reporter (Fig. 6D, middle), as a binding factor of the SV40 enhancer in HeLa cells (25). The suggesting that the squelching effect observed in AsPc1 was members of the TEF family are broadly expressed, in tissue-specific specific to the CanScript sequence. A control Gli1-expressing patterns, suggesting a unique function for each of them (39). plasmid, used as a negative control expressing an irrelevant Here, we show that the MCAT element of the MSLN enhancer is protein, led only to a slight reduction of reporter activity in the cell bound by TEF-1. Several pieces of evidence support this conclusion. lines when transfected with the MSLN reporter (Fig. 6D, right). First, the 18-bp CanScript sequence, which acted as an enhancer, We evaluated the expression pattern of genes previously suggested lost all of its activity when the MCAT site was mutated. Similarly, by others as cofactors of TEF [the vestigial-like proteins VGLL2, the EMSA-defined complex 1 did not form when mutations in the VGLL3, and VGLL4; the related WW domain-containing YAP1 MCAT site were introduced. Second, complex 1 was supershifted by (YAP65), WWTR 1 (TAZ), and PARP; and interacting transcription a TEF-1 antibody and did not form in a cell line having no factors MAX and MEF2A; refs. 19, 26–30] by analyzing the publicly expression of TEF-1. Third, selective knockdown of TEF-1 resulted available SAGE database4 as well as by RT-PCR in our panel of cell in reductions of complex 1 and of MSLN protein expression. lines. The expression pattern of any of the proposed TEF-1 cofactors Fourth, a complex formed of sequences encompassing the MSLN did not correspond to the differential expression of MSLN in enhancer and TEF-1 protein was detected in ChIP experiments. pancreatic cancer or in our cell line model (data not shown). Our studies suggested that TEF-1 but not TEF-4 or TEF-5 interacts with the MSLN enhancer. Previous in vitro studies showed Discussion that all TEF proteins had almost identical DNA-binding domains, recognizing the MCAT motif with essentially the same affinity (39). We investigated the mechanism producing cancer-specific However, disruption of TEF-1 in mice resulted in embryonic overexpression of MSLN, a property common to nearly a third of lethality due to cardiac abnormalities, suggesting that TEF human cancers and almost ubiquitous in pancreatic ductal members are not redundant in function (40). The selective role of adenocarcinoma. An 18-bp upstream enhancer, termed CanScript, individual members of the TEF family may be explained by seemed responsible for this overexpression and used transcription differences in tissue expression, target accessibility, or the specificity factors from the TEF family as a major regulator. of cofactor/TEF interactions. Recently, Mahoney et al. (19) showed We identified multiple transcriptional start sites of the MSLN differential interaction of the TEF cofactor TAZ with TEF-1 and gene spanning hundreds of base pairs. Nevertheless, the transcrip- TEF-3. Our studies did not examine the effects of all the members of tion seemed to be regulated from one weak tissue-nonspecific the TEF family, and it is possible that TEF-1 is not the only member TATA-less promoter located upstream of nucleotide +1. The low of the family transactivating through the CanScript sequence. activity of the isolated MSLN promoter may explain the absence of Our data confirmed the results of others showing that tissue- MSLN expression in most tissues. specific expression of TEFs is by itself insufficient to direct tissue- In a group of cell lines having aberrant overexpression of MSLN, specific expression of TEF-driven genes. Cofactors have been the core promoter activity was strongly enhanced by an 18-bp proposed to direct tissue specificity (39). They were first suggested sequence, CanScript, located f60 bp from the transcriptional start by Xiao et al. (25) from experiments done in the SV40 enhancer. We site. In pancreatic and cervical carcinoma cells, the enhancer did experiments analogous to Xiao and observed that adding produced a 4- to 10-fold induction of promoter activity. In contrast, exogenous TEF-1 into a cell line lacking MSLN did not result in an in cell lines originating from tissues having physiologic expression induction of reporter activity. Furthermore, in a MSLN-expressing of MSLN (mesothelium, ovarian epithelium, and cortical inclusion cell line, the overexpression of TEF-1 resulted in a decrease in cysts; ref. 22), insignificant effects were seen. This absence of reporter activity. Such behavior is presumed to be attributable to enhancement in our analysis agrees with the previously published squelching of cell-specific cofactors (25). Thus, our experiments study by Urwin and Lake (31), who analyzed transcriptional suggested the cancer specificity to be provided by a titratable regulation of MSLN in the mesothelioma cell line JU77 and did not cofactor of TEF. observe any transcriptional activity of the CanScript-containing The cofactor controlling the activity of the MSLN enhancer region. remains to be identified. The expression pattern of proteins Mutational analysis revealed the presence of two functionally previously suggested as TEF cofactors (19, 26–30) did not distinguishable putative binding sites in the enhancer. The distal correspond to MSLN overexpression in our model. The tissue site 1 perfectly matched the MCAT element. The MCAT element specificity of the enhancer might also be provided by splicing was first identified by Mar and Ordahl (32) in the cardiac troponin isoforms of TEF-1 (41). Furthermore, TEF-1 might act as a selector gene (cTNT) and has been since implicated in the regulation of protein recruiting a transcription factor responsive to specific several cardiac and skeletal muscle-specific genes (a and h myosin signaling events, selectively present only in MSLN-overexpressing heavy chains, skeletal a-actin, and h acetylcholine receptor; refs. cells and binding independently to the enhancer (42). 33–35). In addition to muscle-specific expression, MCAT-depen- The exact role of site 2 is not certain. Mutational studies dent expression was described in the SV40 enhancer (36) and suggested that it was functional yet not to the same extent as site 1. human papillomavirus (HPV) 16 (37) oncogenes in a variety of cell Furthermore, EMSA experiments failed to show a retarded complex lines and in the chorionic somatomammotropin gene in placenta corresponding to this site. Such absence may have been caused by (38). The MCAT element is bound by members of the TEF family of a discrete and short-lived protein-DNA interaction that was undetectable under our experimental conditions. It is also possible that alterations in site 2 could cause conformational changes altering the binding of TEFs to site 1. None of the mutations 4 http://www.ncbi.nlm.nih.gov/SAGE/ introduced to site 2 resulted in an increase in reporter activity in

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MSLN-nonexpressing cell lines, ruling out the possibility of a shown to direct tissue specificity in the cTNT promoter (43). The repressor mechanism selectively absent in MSLN-overexpressing site has a high similarity to the consensus binding site for cell lines. transcription factors of the Sp1 family (8 of 10 bases; ref. 24). Sp1 The binding protein of site 2 may be the presumptive TEF and Kru¨ppel-like factor proteins have been proposed to play an cofactor. Sequences immediately flanking the MCAT element were important role in the growth and metastasis of many tumor types,

Figure 6. TEF-1 was required but not sufficient for MSLN cancer-specific expression. A, the members of the TEF family of transcription factors are widely expressed in cancer cell lines, and their expression does not correspond to expression of MSLN (compare with Fig. 1D). Left, gene and type of assay. GAPDH and actin were used as loading controls. B, down-regulation of TEF-1 was achieved by three nonoverlapping siRNAs (T1a, T1b, and T1c) and resulted in reduction of MSLN protein expression in HeLa cells. Scrambled siRNA (SCR) and siRNA directed against an irrelevant gene FANCD2 (D2) were used as negative controls. Down-regulation of TEF-4 (T4a and T4b) did not result in reduction of MSLN protein expression in HeLa cells. C, ChIPwith a TEF-1 antibody (10 Ag) resulted in amplification of a sequence containing the CanScript sequence (CS) in AsPc1 cells but not in TEF-1–nonexpressing BJAB cells. Immunoprecipitations serving as negative controls were done with normal mouse IgG and without antibody (NoIg) or used sequences immediately upstream (5¶) and downstream (3¶) of the CanScript sequence. Representative results of at least two independent immunoprecipitation experiments and multiple independent PCR analyses are shown. Inp, a constant fraction of the input; NC, control lacking template DNA. D, squelching in AsPc1 and MiaPaCa2. Left, exogenous TEF-1 expression caused a decrease in activity of the MSLN enhancer-containing reporter B-99 in MSLN-expressing cell line AsPc1 but not in MSLN-nonexpressing cell line MiaPaCa2. Much smaller effects were seen when the parental reporter containing the SV40 promoter, pGL3-Promoter, was used (middle) or the irrelevant protein Gli1 was expressed (right). www.aacrjournals.org 9063 Cancer Res 2007; 67: (19). October 1, 2007

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Cancer Research including pancreatic cancer (44). Recently, a GC box bound by Sp1 is the lack of specific targeting. One way to overcome this obstacle was shown to be required for the activity of an MCAT-dependent is to use highly cancer-specific promoter elements to selectively cTNT promoter (45). Sp1 is also a common regulator of TATA-less drive gene expression. The MSLN enhancer with its high specific promoters and might function as a nonspecific transactivating activity in a variety of cancers may serve this purpose well. factor of the TATA-less MSLN promoter. The slight decrease in the Our findings expand the role of TEF family of transcription background reporter activity in a MSLN-nonexpressing cell line factors in cancer. TEF-1 was first identified in cancer cells and is RKO, observed when site 2–mutated constructs were used, might important for expression of oncogenic viruses SV40 and HPV16. support this explanation. HPV plays a causative role in cervical cancer. Interestingly, cervical It is intriguing to consider possible similarities of the cancer- cancers express MSLN and the expression seems TEF-1 dependent. specific cofactor/TEF-driven MSLN overexpression and the cancer- Our data suggest a role for nonviral TEF-driven gene expression in specific alterations of h-catenin/T-cell factor (TCF). The first cancer that may participate in pancreatic tumorigenesis. According members of the TCF family, TCF1 and LEF1, were identified for to the proposed model, TEFs, similarly to TCF, are likely to their ability to drive tissue-specific expression of CD3q and HIV represent only a downstream tool to direct transcriptional long terminal repeat genes in T lymphocytes (46, 47). Because TCF activation from a specific upstream pathway. Identifying other factors could not directly activate transcription in reporter assays, biologically relevant targets of TEF-driven expression represents the appropriate molecular context, in which the factors acted, was another way to uncover the exact role of TEFs and the processes not determined until a binding cofactor, h-catenin, was discovered. upstream of them. Cotransfection with h-catenin strongly induced transcription from In conclusion, by analyzing a large region of genomic sequences multimerized TCF-binding motifs linked to a reporter gene (48). surrounding the MSLN gene in a representative panel of cell lines, This evidence led to completion of the diagram of the gatekeeper we discovered an upstream enhancer that seemed responsible for APC-h-catenin-TCF pathway, ubiquitously activated in colon cancer-specific MSLN overexpression. The activity of the enhancer cancer by mutations in APC or h-catenin (49). It is tempting to was dependent on the binding of TEF-1 and its specificity on a yet speculate that the cofactor/TEF regulation of MSLN aberrant unknown cofactor of TEF. A modular 18-bp responsive sequence, expression may result from a genetic alteration, a highly prevalent CanScript, has enhanced activity when arranged in a tandem mutation or amplification, yet unknown in pancreatic and other repeat and represents an optimal candidate for cancer-specific cancers, producing constitutive activation of a downstream targeting, potentially suitable for a third of human malignancies. pathway. The search for the cofactor thus represents an attractive goal as the final step of this alternative approach to identify highly Acknowledgments frequent aberrant cellular activities in cancer. Received 2/5/2007; revised 7/17/2007; accepted 7/20/2007. In the past two decades, gene therapy has been frequently Grant support: National Cancer Institute grant CA62924 and the Marjorie Kovler attempted as a potentially powerful therapeutic technique in the Fund. treatment of cancer. The recent development of new technologies The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance in gene therapy offers hope for further clinical improvement (50). with 18 U.S.C. Section 1734 solely to indicate this fact. However, the weak point of virtually all available delivery strategies We thank M. Gorospe and J.M. Winter for helpful suggestions.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. High Cancer-Specific Expression of Mesothelin (MSLN) Is Attributable to an Upstream Enhancer Containing a Transcription Enhancer Factor−Dependent MCAT Motif

Tomas Hucl, Jonathan R. Brody, Eike Gallmeier, et al.

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