NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis

Abstract

Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

Comprehensive analysis of INTS family related to expression, prognosis, diagnosis and immune features in hepatocellular carcinoma

“…The expression levels of INTS members vary across cancers. Most INTS members were upregulated in tumors, such as INTS5 in osteosarcoma [ 23 ], INTS7 in LUAD [ 24 ], INT11 in esophageal adenocarcinoma [ 25 ], INTS14 in prostate cancer [ 26 ]. Besides, INTS8 has been reported to be upregulated in various human cancers, including gastric cancer (GC), tongue squamous cell carcinoma, and renal papillary cell carcinoma [ [27] , [28] , [29] ], while INTS13 shows high expression in rectum adenocarcinoma, lung cancer small cells, and cholangiocarcinoma [ 30 ].…”