Abstract
The pathogenesis of thymic epithelial tumors (TETs) is poorly understood. Recently we reported the frequent occurrence of a missense mutation in the GTF2I gene in TETs and hypothesized that GTF2I mutation might contribute to thymic tumorigenesis. Expression of mutant TFII-I altered the transcriptome of normal thymic epithelial cells and upregulated several oncogenic genes. Gtf2i L424H knockin cells exhibited cell transformation, aneuploidy, and increase tumor growth and survival under glucose deprivation or DNA damage. Gtf2i mutation also increased the expression of several glycolytic enzymes, cyclooxygenase-2, and caused modifications of lipid metabolism. Elevated cyclooxygenase-2 expression by Gtf2i mutation was required for survival under metabolic stress and cellular transformation of thymic epithelial cells. Our findings identify GTF2I mutation as a new oncogenic driver that is responsible for transformation of thymic epithelial cells.
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Introduction
Thymic epithelial tumors (TETs) are uncommon, primary neoplasms of the anterior mediastinum derived from the thymic epithelium, with an annual incidence ranging from 1.3 to 3.2/million [1, 2]. The World Health Organization histologic classification distinguishes thymomas (A, AB, B1, B2, and B3), from thymic carcinomas (TC) [3]. However, the complex and frequently mixed histomorphology renders TETs one of the most challenging tumors to classify histologically [4]. Prognosis worsens progressively from type A to TC: 10-year survival is 90–95% in types A through B1, and 5-year survival rates for B2, B3, and TC are 75%, 70%, and 48%, respectively [5]. The etiology of TETs is obscure, aside from the controversial reports of an association with EB virus infection [6], and thymomas can be commonly associated with autoimmune diseases such as myasthenia gravis. In addition, the genetic/molecular aberrations associated with TETs are largely unknown and a lack of appropriate preclinical models hinders investigation of the cause of TETs.
Using exome and RNA sequencing, we recently reported a highly recurrent unique somatic mutation of GTF2I in the least aggressive thymomas, and predicted that GTF2I mutation is necessary for the founder tumor clone in thymus [7]. These findings were later confirmed by the The Cancer Genome Atlas (TCGA) program [8]. GTF2I encodes TFII-I, a transcription factor, and there are currently at least five known splice variants of GTF2I recognized in the human genome. We discovered that the β and δ isoforms of TFII-I are predominantly expressed in TETs; these isoforms are known as negative or positive regulators in gene transcription with distinct subcellular localization in response to mitogen signal [9]. The observed GTF2I T1211A (L424H) missense mutation at chr.7:74146970 leads to p.Leu404His and p.Leu383His substitution in the β and δ isoforms of TFII-I, respectively.
The human TFII-I protein belongs to a family of transcription factors, consisting of three related genes (GTF2I and its pseudogenes GTF2IP1, and LOC100093631) that are located in close proximity on the long arm of chromosome 7. A portion of the long arm of chromosome 7 is deleted in a haplo-insufficient manner in Williams–Beuren syndrome, a genetic disorder characterized by marked defects in neurological and cardiovascular development [10]. Originally TFII-I was identified as a novel type of transcription initiation factor that binds to multiple promoter elements including a pyrimidine-rich initiator (Inr). TFII-I is a multifunctional protein associated with the transcriptional regulation of several genes that control cell proliferation, cell cycle, angiogenesis, cellular stress response, and development, through sequence-specific DNA binding [11]. Despite the many functions of TFII-I, alterations of GTF2I in tumors have not been reported until recently.
Here, we investigated the effects of GTF2I mutation on transcriptional regulation, and several biological and metabolic processes involved in tumorigenesis. We demonstrated that the mutant TFII-I (mtTFII-I) expression in thymic epithelial cells (TECs) results in upregulation of oncogenes and cell transformation. Gtf2i mutation conferred cell transformation and survival advantage through COX-2 expression in response to metabolic stress and promoted tumor growth in vivo.
Materials and methods
Cell culture
All mouse normal TEC lines (TEC71, TEC100.4, TEC41.2, TEC301, and Z210r; a kind gift from Dr. Julien Sage, Stanford University) were maintained in RPMI-1640 (Thermo Fisher; Waltham, MA, USA), and 293T cells were grown in DMEM supplemented with 10% FBS (Thermo Fisher). NIH-3T3 cells were maintained in DMEM supplemented with 10% bovine calf serum (Thermo Fisher). All cell lines were cultivated as monolayer in each optimal medium with 2% penicillin–streptomycin (Thermo Fisher). Doxycycline Hydrochloride was purchased from Thermo Fisher and solubilized in distilled water. For metabolic studies, RPMI-1640 with no glucose, dialyzed FBS, and d-glucose from Thermo Fisher were used. Cisplatin and Doxorubicin were purchased from Selleckchem (Houston, TX, USA).
Production of lentiviral particles
293T cells were plated in 10 cm culture dishes and were transfected with 6 µg lentiviral vector, 0.6 µg lenti-Rev/-PM2/-Tat, and 1.2 µg lenti-Vsv-G at the ratio 10:1:1:1:2 using X-tremeGENE 9 (SIGMA; St. Louis, MO, USA), the next day. Growth medium was changed after 24 h and virus-containing medium was collected between 36 and 48 h after transfection and filtered through 0.45 μm low-binding PES membrane. Virus particles were stored at −80 °C until use.
Doxycycline inducible system
Mutated GTF2I [7] was amplified by PCR using the following primers: 5′-CACCATGGCCCAAGTTGCAATGTC-3′, and 5′-AAACCACGTGGGGTCTGGTTCTTG-3′. PCR fragments were clone into the pENTR/D-TOPO (Thermo Fisher), and subsequently moved into a pLIX402 lentiviral vector (Addgene; Cambridge, MA, USA) using Gateway LR Clonase II Enzyme mix (Thermo Fisher) according to the manufacturer’s instructions. The insert sequence in the plasmid was sequenced, and confirmed HA-tag expression under the control of doxycycline in 293T cells first. Mouse thymic epithelial cells (mTECs) were seeded (3–4 × 104) in six-well plates and infected with lentivirus the next day. After 48 h infection, 3–5 μg/mL puromycin was used for selection and puromycin-resistant single clones were picked by pyrex cloning cylinder (SIGMA). Five micrograms of the pLIX402 plasmids with mutated GTF2I isoforms were transfected into TEC100.4 cells using the Nucleofector Kit V (Lonza; Salisbury, MD, USA) with Nucleofector 2b device under T-30 program. After 48 h transfection, 3 μg/mL puromycin was used for selection and puromycin-resistant single clones were picked by Pyrex cloning cylinder.
Establishment of stable knockdown (KD) cells
pLKO.1 from Addgene (#8453), two COX-2 shRNA (TRCN0000067941 and TRCN0000067939), PFKFB2 shRNA (TRCN0000361450 and TRCN0000361451), PFKP (TRCN0000025962 and TRCN0000025916), and ENO2 shRNA (TRCN0000340138 and TRCN0000340139) were purchased from Sigma. Cells were infected with shRNA lentiviral particles and selected by 10 μg/ml of puromycin. Cells transfected with pLKO.1 were used as a control.
Western blot analysis and antibodies
Cells were harvested and lysed in 1 × lysis buffer [60 mM Tris (pH 7.4), 25 mM HEPES, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 5 mM Na3VO4, 50 mM NaF, two pills protease inhibitor cocktail tablets] on ice for 45 min and centrifuged at 15,000 rpm for 15 min. Cell lysates were separated by 4–20% SDS-PAGE and transferred to a PVDF membrane. Subsequently, the membrane was incubated in TBST supplemented with 5% nonfat dry milk or BSA and probed with the appropriate primary antibodies. The bound antibodies were visualized with a suitable secondary antibody conjugated with horseradish peroxidase using enhanced chemiluminescence reagent WESTSAVE up by P** with our findings, pathway analysis demonstrated lower expression of genes involved in apoptosis, cell cycle, and DNA damage response in GTF2I mutant tumors compared with GTF2I wild-type tumors [8].
In summary, here we propose that GTF2I mutation can directly contribute to tumor formation in TECs. Since we have identified the missense mutation of GTF2I, other groups confirmed this specific mutation [41, 42] including TCGA project, which suggested to be a founder mutation occurring early in tumor development [8]. Moreover, thymus specific GTF2I L424H mutation was recently identified as one of 1165 statistically significant recurrent mutations in 24,592 human tumor samples [43]. Taken together, all our observations underscore the GTF2I mutation as a specific molecular marker for TETs. The study of the precise mechanisms by which mtTFII-I regulates target gene expression and the exact functional differences between TFII-I-βmt and TFII-I-δmt will need to be addressed.
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Acknowledgements
We acknowledge the assistance provided by the Flow Cytometry and Proteomics & Metabolomics Shared Resource of Lombardi Comprehensive Cancer Center for FACS and Metabolomics. We would like to thank Dr. Uimook Choi (NIAID, NIH) and Jung-hyun Kim (NCI, NIH) for valuable discussions on CRISPR system. This work was supported by Lombardi Comprehensive Cancer Center grant (P30-CA051008 to GG).
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Conceptualization, I-KK, YW, Y-WZ and GG; methodology, I-KK, YW, and Y-WZ; validation, I-KK, GR and XZ; formal analysis, I-KK and RF; investigation and data curation, I-KK; writing—original draft, I-KK; writing—review and editing, I-KK, GR, XZ, RF, YW, MLA, Y-WZ and GG.
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Kim, IK., Rao, G., Zhao, X. et al. Mutant GTF2I induces cell transformation and metabolic alterations in thymic epithelial cells. Cell Death Differ 27, 2263–2279 (2020). https://doi.org/10.1038/s41418-020-0502-7
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DOI: https://doi.org/10.1038/s41418-020-0502-7
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