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TELO2 Induced Progression of Colorectal Cancer by Binding with RICTOR Through Mtorc2

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ONCOLOGY REPORTS 45: 523-534, 2021 TELO2 induced progression of colorectal cancer by binding with RICTOR through mTORC2 ZHENG GUO1,2*, XIUFANG ZHANG3*, HUABIN ZHU3*, NANYING ZHONG3, XIAONING LUO1,2, YU ZHANG1,2, FUPING TU1,2, JINGHUA ZHONG1,2, XIANGCAI WANG1,2, JUE HE4,5 and LI HUANG1,2 1Department of Oncology, and 2Clinical Cancer Research Center of Jiangxi Province, First Affiliated Hospital of Gannan Medical University, Gannan Medical University; 3First School of Clinical Medicine, Gannan Medical University; 4Department of Pathology, First Affiliated Hospital of Gannan Medical University, Gannan Medical University; 5School of Basic Medicine, Gannan Medical University, Ganzhou, Jiangxi 341000, P.R. China Received May 6, 2020; Accepted October 21, 2020 DOI: 10.3892/or.2020.7890 Abstract. Colorectal cancer (CRC) is a common cancer world- RICTOR induced the degradation of TELO2 upon serum wide, and its treatment strategies are limited. The underlying deprivation in an mTOR‑independent manner. These findings mechanism of CRC progression remains to be determined. indicate that TELO2 promotes tumor progression via RICTOR Telomere maintenance 2 (TELO2) is a mTOR-interacting in a serum-dependent manner, which may be a potential thera- protein. Both the role and molecular mechanism of TELO2 peutic target for CRC. in cancer progression remain unknown. In this study, the gene expression database of normal and tumor tissue, in addition to Introduction western blot analysis, and immunohistochemistry (IHC) were used to determine the expression and location of TELO2 in Colorectal cancer (CRC) is one of the most common types CRC and normal tissues. Clinical features of a tissue array of cancer worldwide, especially in China within the last were collected and analyzed. WST‑1, soft agar, flow cytometry, decade (1). CRC progression, from adenoma to adenocarci- wound healing, and invasion assays were employed to verify noma, is a multistep process (2). CRC results from a series the role of TELO2 in the growth, cell cycle, migration, and of genetic and epigenetic alterations in key growth regulatory invasion of CRC cells. The correlation between TELO2 and genes, which endow the colorectal cells with proliferative, RICTOR (rapamycin-insensitive companion of mTOR) was survival, and metastatic advantages (3). The median overall analyzed by bioinformatics, IHC, and immunoprecipitation. survival (OS) of metastatic CRC (mCRC) is less than 3 years. Normal and serum-deprived cells were collected to detect Chemotherapy, radiotherapy, targeted therapy, and immuno- the protein level of TELO2 and its downstream effectors. therapy are mainly palliative treatments (4). Therefore, there The results revealed that TELO2 was significantly upregu- is a need for more efficacious strategies to prolong the OS of lated in CRC, and TELO2 inhibition significantly restrained mCRC. the growth, cell cycle, and metastasis of CRC cells. TELO2 Telomere maintenance 2 (TELO2, also known as tel2) was overexpression correlated with age, lymph node metastasis, first identified in Saccharomyces cerevisiae and was required and TNM stage of CRC patients. In addition, TELO2 was in telomere length regulation and telomere position (5). positively correlated with RICTOR in CRC and induced TELO2 binds with phosphatidylinositol 3-kinase-related tumor progression mainly via RICTOR with serum in culture. kinase (PIKK) family members, including ATM (ataxia telangiectasia mutated), ATR (ATM- and rad3-related), and mammalian target of rapamycin (mTOR) (6), and it maintains the stability of PIKKs, which leads to the translation, growth, Correspondence to: Dr Zheng Guo, Department of Oncology, First and autophagic regulation of cells (7,8). The PI3K/Akt/mTOR Affiliated Hospital of Gannan Medical University, Gannan Medical pathway is frequently dysregulated in CRC patients, and the University, Ganzhou, Jiangxi 341000, P.R. China therapeutic potential of targeting mTOR in the treatment E‑mail: [email protected] of CRC is rational and has been shown to improve disease progression (9). With regard to mTOR, TELO2 is not only *Contributed equally critical for protein stability, but it is also essential for the Abbreviations: RICTOR, rapamycin-insensitive companion of integrity of mTOR complexes (10,11). Degradation of TELO2 mTOR; TELO2, telomere maintenance 2 promotes the survival of multiple myeloma under growth factor withdrawal (12). Although there are several reports indi- Key words: TELO2, RICTOR, colorectal cancer progression, cating that TELO2 demonstrates an oncogenic profile in solid serum-dependent, degradation tumors, such as breast cancer (13) and high-grade gliomas (14), the role and molecular mechanism of TELO2 in CRC tumori- genesis have yet to be defined. A study on TELO2 in CRC 524 GUO et al: TELO2 PROGRESSED CRC BY RICTOR demonstrates that a germline mutation of TELO2 regulated by a pathologist with over 15 years of experience: Cells with the senescence pathway and is related to sessile serrated <10% staining were scored as negative staining (-, 1); cells adenomas and adenocarcinoma progression (15). Additionally, with 10-49% staining were scored as (+, 2); cells with 50-74% rapamycin-insensitive companion of mTOR (RICTOR), a staining were scored as (++, 3); and cells with 75-100% staining specific adaptor of mTORC2 (16), plays an important role in were scored as (+++, 4). The staining color was scored as cancer cell proliferation, autophagy, migration, and invasion light-yellow particle (1), brown-yellow particle (2), and brown via activation of protein kinase B (Akt) (17,18) and is positively particle (3). The final score was defined as staining number correlated with prognosis in CRC (19). Thus, we hypothesized score multiplied by staining color score (20). The scores of that TELO2 is important in the development of CRC via mTOR negative expression were between 0 and 5, and the scores that and RICTOR. In this study, we performed bioinformatics exceeded 5 were identified as positive expression. combined with in vitro experiments and analysis of clinical characteristics of CRC patients in order to characterize the RNA isolation and quantitative PCR. Cells were harvested effect of TELO2 on CRC progression. and total RNA was extracted from LoVo cells (transfected with scramble siRNA or RICTOR siRNA) using TRIzol Materials and methods reagent (no. 15596026; Gibco) as previously described (21). qPCR was performed using Power SYBR‑Green PCR Master Cell lines and tissue specimens. The anti-TELO2 anti- Mix (no. 4309155; Applied Biosystems). The qPCR conditions body (ab122722, 1:500 dilution) was purchased from were 5 min at 95˚C, followed by 50 cycles of 95˚C for 15 sec, Abcam (Cambridge). The anti-RICTOR antibody (cat. 56˚C for 30 sec, and 72˚C for 40 sec, 72˚C for 5 min was no. A300-459A-M, 1:1,000 dilution) was purchased from included for a final extension. The primer sequences used in Bethyl (Montgomery). Antibodies against mTOR (cat. RT-PCR were as follows: TELO2 forward, 5'-GTC CCT GAA no. 2972, 1:1,000 dilution), RAPTOR (cat. no. 4978, 1:1,000 GCG GTA TCT CG-3' and reverse, 5'-TGC TGG CAA GAC ATC dilution), phosphorylated-Akt (Ser473, cat. no. 4051, 1:500 TGA GG-3' (107 bp); GAPDH forward, 5'-GTC AAC GGA TTT dilution), Akt (cat. no. 9272, 1:500 dilution), S6K1pT389 (cat. GGT CGT ATT G-3' and reverse, 5'-CTC CTG GAA GAT GGT no. 9204, 1:500 dilution), S6K1 (cat. no. 9202, 1:500 dilution), GAT GGG-3' (216 bp). 4E-BP1pS65 (no. 9451, 1:500 dilution), 4E-BP1 (no. 9452, 1:500 dilution), tubulin (cat. no. 2146, 1:1,000 dilution), Transient transfection and generation of stably transfected ubiquitin (cat. no. 43124, 1:1,000 dilution), and Myc‑tag (cat. cells. Knockdown of RICTOR expression was performed by no. 2276, 1:1,000 dilution) were obtained from Cell Signaling. transfecting cells with siRNA duplexes (sc-61478; Santa Cruz The anti-β-actin antibody (cat. no. A1978, 1:2,000 dilution) Biotechnology) using Lipofectamine 3000 (Thermo Fischer was purchased from Sigma (Milwaukee). LoVo cells, a human Scientific). Scrambled RNA (scr‑siRNA) was used as a nega- colorectal cancer cell line (CBP60032, Cobioer, Shanghai), tive control. The TELO2 shRNA plasmid (sc-93308-SH) was selected from the GENT2 database, authenticated by STR was purchased from Santa Cruz Biotechnology. The analysis (September, 2016) and maintained in RPMI-1640 pLPC-Myc-TELO2 (no. 22802) plasmid was purchased from basic media containing 10% fetal calf serum (FBS, no. 10100, Addgene. The indicated plasmids were transfected into LoVo Gibco, Shanghai) at 37˚C with 5% CO2. Serum-deprived cells. Transfects were cultured in RPMI-1640 medium supple- conditions were defined as maintaining cells in RPMI‑1640 mented with puromycin (Medchemexpress LLC) to generate media with 0.02% FBS. Four pairs of CRC tissues (on the edge stable cell lines. of cancerous specimens without necrosis) and normal adjacent tissues (2 cm away from the cancer margin) were collected from Western blot analysis and co‑immunoprecipitation (Co‑IP). resected surgical specimens in our hospital from April, 2019 Whole cell lysates and cytoplasmic protein were prepared to December, 2019. There were 2 males and 2 females, with an following the instructions of a protein extraction kit from age range of 53 to 81. Tissue array slides (no. HColA180Su10) Sangon Biotech (C006255 and C510001). For western blot were purchased from Superchip (Shanghai, China). All analysis, 30 µg of whole protein lysates was used to detect the of the patients signed informed consent for the use of their indicated protein, 4-12% NuPAGE Bis-Tris gel (NP0322BOX, tissues, and the Institutional Review Committee of Gannan Invitrogen) was used for electrophoresis. STRING database Medical University (Ganzhou, Jiangxi) approved this study (https://string-db.org) was used for predicting the protein (no. 2018023). combination before experiments. For Co-IP assay, 1% Triton (strong lysis buffer which can depolymerize the mTOR Tissue array and immunohistochemistry (IHC). For IHC complex) or CHAPS (mild buffer which can maintain the experiments, tissue array slides were used with 100 CRC integrated mTOR complex) lysis buffer was used.
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  • Structure of the TELO2-TTI1-TTI2 Complex and Its Function in TOR Recruitment to the R2TP Chaperone

    Structure of the TELO2-TTI1-TTI2 Complex and Its Function in TOR Recruitment to the R2TP Chaperone

    bioRxiv preprint doi: https://doi.org/10.1101/2020.11.09.374355; this version posted November 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone † Mohinder Pal1, Hugo Muñoz-Hernandez2 , Dennis Bjorklund1, Lihong Zhou1, Gianluca Degliesposti3, J. Mark Skehel3, Emma L. Hesketh4, Rebecca F. Thompson4, Laurence H. Pearl1,5*, Oscar Llorca2*, Chrisostomos Prodromou1 1. Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK 2. Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain 3. MRC Laboratory of Molecular Biology. Francis Crick Avenue. Cambridge Biomedical Campus, Cambridge, CB2 0QH. UK 4. Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK 5. Division of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW1E 6BT, UK * Correspondence to : LHP - [email protected] (Lead Contact) or [email protected]; OL - [email protected] † Present address: Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel bioRxiv preprint doi: https://doi.org/10.1101/2020.11.09.374355; this version posted November 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.