SETD2 Haploinsufficiency for Microtubule Methylation Is an Early Driver of Genomic Instability in Renal Cell Carcinoma
Total Page：16
File Type：pdf, Size：1020Kb
Published OnlineFirst May 3, 2018; DOI: 10.1158/0008-5472.CAN-17-3460 Cancer Genome and Epigenome Research SETD2 Haploinsufficiency for Microtubule Methylation Is an Early Driver of Genomic Instability in Renal Cell Carcinoma Yun-Chen Chiang1, In-Young Park2, Esteban A. Terzo3, Durga Nand Tripathi2, Frank M. Mason3, Catherine C. Fahey1, Menuka Karki2,4, Charles B. Shuster4, Bo-Hwa Sohn2, Pratim Chowdhury2, Reid T. Powell5, Ryoma Ohi6, Yihsuan S. Tsai1, Aguirre A. de Cubas3, Abid Khan1,7, Ian J. Davis1, Brian D. Strahl1,7, Joel S. Parker1, Ruhee Dere2, Cheryl L. Walker2, and W. Kimryn Rathmell3 Abstract Loss of the short arm of chromosome 3 (3p) occurs early in human kidney cells, rescue with a pathogenic SETD2 mutant >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiqui- deficient for microtubule (aTubK40me3), but not histone tous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor (H3K36me3) methylation, replicated this phenotype. Genomic suppressors as early drivers of tumorigenesis. We previously instability (micronuclei) was also a hallmark of patient-derived reported methyltransferase SETD2, which trimethylates H3 his- cells from ccRCC. These data show that the SETD2 tumor sup- tones on lysine 36 (H3K36me3) and is located in the 3p deletion, pressor displays a haploinsufficiency phenotype disproportion- to also trimethylate microtubules on lysine 40 (aTubK40me3) ately impacting microtubule methylation and serves as an early during mitosis, with aTubK40me3 required for genomic sta- driver of genomic instability. bility. We now show that monoallelic, Setd2-deficient cells retain- Significance: Loss of a single allele of a chromatin modifier ing H3K36me3, but not aTubK40me3, exhibit a dramatic plays a role in promoting oncogenesis, underscoring the grow- increase in mitotic defects and micronuclei count, with increased ing relevance of tumor suppressor haploinsufficiency in tumor- viability compared with biallelic loss. In SETD2-inactivated igenesis. Cancer Res; 78(12); 3135–46. Ó2018 AACR. Introduction vided by a large deletion of chromosome 3p (2). Recent large- scale multiplatform sequencing techniques identified that, in Many cancer types are characterized by large-scale, character- addition to VHL, other tumor suppressor genes encoding epige- istic alterations of chromosomal copy number. Clear cell renal cell netic regulators such as Polybromo 1 (PBRM1/BAF180), BAP1, and carcinoma (ccRCC) is characterized by frequent inactivating Set domain containing 2 (SETD2) are implicated in ccRCC mutations of the Von Hippel–Lindau (VHL) tumor suppressor tumorigenesis (3–5). Importantly, all of these commonly mutat- gene (1), with accompanying loss of heterozygosity (LOH) pro- ed genes reside in the same portion of affected chromosome 3p. Loss of 3p results in LOH for all four of these genes, which individually acquire "second-hit" mutations and loss of function 1Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel of the remaining allele in subsets of ccRCC. Of these, SETD2 is of 2 Hill, North Carolina. Center for Precision Environmental Health, Department of particular interest, as this tumor suppressor has been increasingly Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas. identified as a common mutation across cancer types, including 3Vanderbilt-Ingram Cancer Center, Division of Hematology and Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, lung (6), bladder (7), glioma (8), and leukemia (9). Tennessee. 4Department of Biology, New Mexico State University, Las Cruces, SETD2 encodes a methyltransferase known to be the sole New Mexico. 5Texas A&M Health Sciences Center, Institute of Biosciences and enzyme responsible for the trimethylation of lysine 36 on Technology, Houston, Texas. 6Department of Cell and Molecular Biology, histone H3 (H3K36me3; refs. 7, 8, 10–12). Biallelic deficiency 7 Vanderbilt University, Nashville, Tennessee. Department of Biochemistry and of SETD2 via deletions and inactivating mutations occur in up Biophysics, University of North Carolina, Chapel Hill, North Carolina. to 20% of primary human RCC tumors and it is associated with Note: Supplementary data for this article are available at Cancer Research more advanced disease and the metastatic phenotype, typically Online (http://cancerres.aacrjournals.org/). lethal within 1–5 years (13). Biallelic loss of SETD2 has been Y.C. Chiang, I.Y. Park, and E.A. Terzo are co-first authors and contributed shown to result in loss of H3K36me3 in ccRCC-derived cells equally to this article. and tumors (9, 14, 15). Examination of H3K36me3 status in Corresponding Author: W. Kimryn Rathmell, Vanderbilt-Ingram Cancer Center, ccRCC cells of metastatic tumor specimens suggest that SETD2 2220 Pierce Avenue, Preston Research Building, 798C, Nashville, TN 37232. mutations may occur in over 50% of metastatic lesions (16). Phone: 615-343-0250; Fax: 615-343-0250; E-mail: Furthermore, a study of ccRCC intratumoral heterogeneity [email protected] identified distinct SETD2 mutations across subsections of an doi: 10.1158/0008-5472.CAN-17-3460 individual tumor, suggesting a selection bias for SETD2 muta- Ó2018 American Association for Cancer Research. tion in the course of ccRCC development (7). www.aacrjournals.org 3135 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 3, 2018; DOI: 10.1158/0008-5472.CAN-17-3460 Chiang et al. SETD2 is a multidomain-containing protein with distinct ing a previously unrecognized effect of SETD2 dosage. Comparing functions for each domain. The methyltransferase activity is different SETD2 mutations, we also found that the SRI domain mediated by a centrally located SET domain. Mutations in this mutation, which rescues H3K36me3, but not aTubK40me3, fails domain are common in ccRCC (10, 14), suggesting loss of to rescue micronuclei formation and mitotic defects in SETD2- catalytic activity is a critical event in tumor development. We deleted cells compared with WT SETD2. Importantly, micronuclei previously characterized a pathogenic SET domain mutation were also a common feature of 3p-deleted ccRCC cell lines and found in ccRCC, an arginine-to-cysteine mutation at residue were seen in a significant fraction of tumor cells from all patients 1625 of SETD2 (R1625C; ref. 15), which abolishes methylation with ccRCC. These data indicate that SETD2 is haploinsufficient activity. At its C-terminus, SETD2 also contains the Set2–Rpb1 for aTubK40 trimethylating activity, with loss of microtubule interaction (SRI) domain (17). This domain mediates the methylation driving early genomic instability in tumors with 3p interaction between SETD2 and the phosphorylated C-terminal loss, preceding the loss of histone H3K36 trimethylation, and domain of RNA polymerase II (RNAPII). We also identified a contributing to the progression of ccRCC. Moreover, our results recurrent mutation in the SRI domain, an arginine-to-histidine shed light on a new mechanism by which monoallelic inacti- mutation at residue 2510 (R2510H; ref. 15). This mutation vation of SETD2 or 3p loss can drive tumorigenesis in other preserves the H3 trimethylation catalytic activity of SETD2, cancers, and points to microtubule methylation as a key tumor suggesting SETD2 may have other key functions in addition suppressive activity of SETD2. to its well-characterized role as a histone methyltransferase. We recently discovered that SETD2 also functions as a micro- tubule methyltransferase, in addition to the well-characterized Materials and Methods role of SETD2 in histone methylation (18). SETD2 trimethy- Cell cultures lates a-tubulin on lysine 40 (aTubK40me3) of microtubules Human renal cell carcinoma ACHN, UMRC2, Caki-1, and A498 and loss of this marker results in genomic instability. SETD2- cells were acquired from the ATCC. RCC4 cells were generously null cells exhibit mitotic spindle and cytokinesis defects, poly- provided by Dr. William Kim (University of North Carolina, ploidy, and formation of micronuclei.Importantly,pathogenic Chapel Hill, NC). The SV-40–transformed human renal tubule SETD2 mutationsintheSETdomainaswellastheSRIdomain epithelial cell line (HKC) was obtained from Dr. Lorraine were unable to methylate microtubules, and caused an increase Racusen, (Johns Hopkins Hospital, Baltimore, MD). All cells in chromosome bridges and lagging chromosomes relative to undergo annual short tandem repeat (STR) confirmation. Human wild-type SETD2, indicating that in addition to the catalytic cell lines were cultured in DMEM (Gibco/Life Technologies) domain, a functional SRI domain was also required for supplemented with 10% FBS (Gemini Bio-Products), nonessen- aTubK40me3 (18). tial amino acids, L-glutamine, penicillin, and streptomycin. For w/f f/f These mitotic alterations caused by loss of aTubK40me3 Setd2 and Setd2 MEFs, cells were maintained in phenol can lead to chromosomal abnormalities and genomic instabil- red–free media (DMEM, high glucose, HEPES, no phenol red) ity, hallmarks of tumorigenesis, and are thought to be an supplemented with sodium pyruvate and GlutaMAX (Thermo important source of genetic diversity and development of cell Fisher Scientific), to prevent any spontaneous activation of clones during tumor progression (19). In the case of the type of the Cre recombinase by estrogenic compounds found in phenol defects observed with SETD2 mutants deficient in microtubule red–containing media. All cultures