High cancer-specific expression of mesothelin (MSLN) is attributable to an upstream enhancer containing a transcription enhancer factor dependent MCAT motif.
Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies.
Hucl
Tomas
T
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD 21231, USA.
Brody
Jonathan R
JR
Gallmeier
Eike
E
Iacobuzio-Donahue
Christine A
CA
Farrance
Iain K
IK
Kern
Scott E
SE
D001483:Base Sequence; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D058851:GPI-Linked Proteins; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D000090204:Mesothelin; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D058851:GPI-Linked Proteins; C000715527:MSLN protein, human; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D012334:RNA, Neoplasm; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors; D000090204:Mesothelin