Functional Studies on Primary Tubular Epithelial Cells Indicate a Tumor Suppressor Role of SETD2 in Clear Cell Renal Cell Carcinoma.
SET domain-containing 2 (SETD2) is responsible for the trimethylation of histone H3 lysine36 (H3K36me3) and is one of the genes most frequently mutated in clear cell renal cell carcinoma (ccRCC). It is located at 3p21, one copy of which is lost in the majority of ccRCC tumors, suggesting that SETD2 might function as a tumor suppressor gene. However, the manner in which loss of SETD2 contributes to ccRCC development has not been studied in renal primary tubular epithelial cells (PTECs). Therefore, we studied the consequences of SETD2 knockdown through lentiviral shRNA in human PTECs. Consistent with its known function, SETD2 knockdown (SETD-KD) led to loss of H3K36me3 in PTECs. In contrast to SETD2 wild-type PTECs, which have a limited proliferation capacity; the SETD2-KD PTECs continued to proliferate. The expression profiles of SETD2-KD PTECs showed a large overlap with the expression profile of early-passage, proliferating PTECs, whereas nonproliferating PTECs showed a significantly different expression profile. Gene set enrichment analysis revealed a significant enrichment of E2F targets in SETD2-KD and proliferating PTECs as compared with nonproliferating PTECs and in proliferating PTEC compared with SETD2-KD. The SETD2-KD PTECs maintained low expression of CDKN2A and high expression of E2F1, whereas their levels changed with continuing passages in untreated PTECs. In contrast to the nonproliferating PTECs, SETD2-KD PTECs showed no β-galactosidase staining, confirming the protection against senescence. Our results indicate that SETD2 inactivation enables PTECs to bypass the senescence barrier, facilitating a malignant transformation toward ccRCC.
Department of Genetics, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: j.li@umcg.nl.
Jun
J
Li
Department of Pathology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands; Department of Medical Biology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: j.l.kluiver@umcg.nl.
Joost
J
Kluiver
Department of Genetics, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: j.osinga@umcg.nl.
Jan
J
Osinga
Department of Genetics, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: h.westers@umcg.nl.
Helga
H
Westers
Department of Nephrology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: maaikevanwerkhoven@gmail.com.
Maaike B
MB
van Werkhoven
Department of Nephrology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: m.seelen@umcg.nl.
Marc A
MA
Seelen
Department of Genetics, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: r.h.sijmons@umcg.nl.
Rolf H
RH
Sijmons
Department of Pathology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands; Department of Medical Biology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: a.van.den.berg01@umcg.nl.
Anke
A
van den Berg
Department of Genetics, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands. Electronic address: k.kok@umcg.nl.
Klaas
K
Kok
D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000473:pathology*; D002460:Cell Line; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D016922:Cellular Senescence / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18 / Q000096:biosynthesis*; D050687:E2F1 Transcription Factor / Q000096:biosynthesis*; D004847:Epithelial Cells / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D057809:HEK293 Cells; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics / Q000473:pathology*; D007684:Kidney Tubules / Q000166:cytology; D008745:Methylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D006657:Histones; D034741:RNA, Small Interfering; D011495:Histone-Lysine N-Methyltransferase; C521636:SETD2 protein, human; D001616:beta-Galactosidase