Abstract
The circadian gene Timeless (TIM) provides a molecular bridge between circadian and cell cycle/DNA replication regulatory systems and has been recently involved in human cancer development and progression. However, its functional role in colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide, has not been fully clarified yet. Here, the analysis of two independent CRC patient cohorts (total 1159 samples) reveals that loss of TIM expression is an unfavorable prognostic factor significantly correlated with advanced tumor stage, metastatic spreading, and microsatellite stability status. Genome-wide expression profiling, in vitro and in vivo experiments, revealed that TIM knockdown induces the activation of the epithelial-to-mesenchymal transition (EMT) program. Accordingly, the analysis of a large set of human samples showed that TIM expression inversely correlated with a previously established gene signature of canonical EMT markers (EMT score), and its ectopic silencing promotes migration, invasion, and acquisition of stem-like phenotype in CRC cells. Mechanistically, we found that loss of TIM expression unleashes ZEB1 expression that in turn drives the EMT program and enhances the aggressive behavior of CRC cells. Besides, the deranged TIM-ZEB1 axis sets off the accumulation of DNA damage and delays DNA damage recovery. Furthermore, we show that the aggressive and genetically unstable ‘CMS4 colorectal cancer molecular subtype’ is characterized by a lower expression of TIM and that patients with the combination of low-TIM/high-ZEB1 expression have a poorer outcome. In conclusion, our results as a whole suggest the engagement of an unedited TIM-ZEB1 axis in key pathological processes driving malignant phenotype acquisition in colorectal carcinogenesis. Thus, TIM-ZEB1 expression profiling could provide a robust prognostic biomarker in CRC patients, supporting targeted therapeutic strategies with better treatment selection and patients’ outcomes.
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Data availability
All datasets on which the conclusions of the paper rely are available to readers. The raw data for the Gene expression data generated in this study can be accessed through the NCBI Gene Expression Omnibus (GEO accession number GSE169576).
References
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J Clin. 2021; https://doi.org/10.3322/caac.21660.
Shibue T, Weinberg RA. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol. 2017;14:611–29.
Mazzoccoli G, Laukkanen MO, Vinciguerra M, Colangelo T, Colantuoni V. A Timeless link between circadian patterns and disease. Trends Mol Med. 2016;22:68–81.
Baretić D, Jenkyn-Bedford M, Aria V, Cannone G, Skehel M, Yeeles JTP. Cryo-EM structure of the fork protection complex bound to CMG at a replication fork. Mol Cell. 2020;78:926–940.e13.
Leman AR, Noguchi C, Lee CY, Noguchi E. Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion. J Cell Sci. 2010;123:660–70.
Kemp MG, Akan Z, Yilmaz S, Grillo M, Smith-Roe SL, Kang T-H, et al. Tipin-replication protein A interaction mediates Chk1 phosphorylation by ATR in response to genotoxic stress. J Biol Chem. 2010;285:16562–71.
Unsal-Kaçmaz K, Mullen TE, Kaufmann WK, Sancar A. Coupling of human circadian and cell cycles by the Timeless protein. Mol Cell Biol. 2005;25:3109–16.
Bianco JN, Bergoglio V, Lin Y-L, Pillaire M-J, Schmitz A-L, Gilhodes J, et al. Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner. Nat Commun. 2019;10:910.
Young LM, Marzio A, Perez-Duran P, Reid DA, Meredith DN, Roberti D, et al. TIMELESS forms a complex with PARP1 distinct from its complex with TIPIN and plays a role in the DNA damage response. Cell Rep. 2015;13:451–9.
Xie S, Mortusewicz O, Ma HT, Herr P, Poon RYC, Poon RRY, et al. Timeless Interacts with PARP-1 to promote homologous recombination repair. Mol Cell. 2015;60:163–76.
Chi L, Zou Y, Qin L, Ma W, Hao Y, Tang Y, et al. TIMELESS contributes to the progression of breast cancer through activation of MYC. Breast Cancer Res. 2017;19:53.
Zhang W, He W, Shi Y, Zhao J, Liu S, Zhang F, et al. Aberrant TIMELESS expression is associated with poor clinical survival and lymph node metastasis in early-stage cervical carcinoma. Int J Oncol. 2017;50:173–84.
Yoshida K, Sato M, Hase T, Elshazley M, Yamashita R, Usami N, et al. TIMELESS is overexpressed in lung cancer and its expression correlates with poor patient survival. Cancer Sci. 2013;104:171–7.
Liu S-L, Lin H-X, Lin C-Y, Sun X-Q, Ye L-P, Qiu F, et al. TIMELESS confers cisplatin resistance in nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway and promoting the epithelial mesenchymal transition. Cancer Lett. 2017;402:117–30.
Neilsen BK, Frodyma DE, McCall JL, Fisher KW, Lewis RE. ERK-mediated TIMELESS expression suppresses G2/M arrest in colon cancer cells. PLoS ONE. 2019;14:e0209224.
Elgohary N, Pellegrino R, Neuman O, Elzawahry HM, Saber MM, Zeneldin AA, et al. Protumorigenic role of Timeless in hepatocellular carcinoma. Int J Oncol. 2015;46:597–606.
Li B, Mu L, Li Y, Xia K, Yang Y, Aman S, et al. TIMELESS inhibits breast cancer cell invasion and metastasis by down-regulating the expression of MMP9. Cancer Cell Int. 2021;21:38.
Uhlen M, Zhang C, Lee S, Sjöstedt E, Fagerberg L, Bidkhori G, et al. A pathology atlas of the human cancer transcriptome. Science. 2017;357:eaan2507.
Rageul J, Park JJ, Zeng PP, Lee E-A, Yang J, Hwang S, et al. SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks. Nat Commun. 2020;11:5495.
Ghandi M, Huang FW, Jané-Valbuena J, Kryukov GV, Lo CC, McDonald ER, et al. Next-generation characterization of the cancer cell line encyclopedia. Nature. 2019;569:503–8.
Dey P, Li J, Zhang J, Chaurasiya S, Strom A, Wang H, et al. Oncogenic KRAS-driven metabolic reprogramming in pancreatic cancer cells utilizes cytokines from the tumor microenvironment. Cancer Discov. 2020;10:608–25.
Zhang Y, Weinberg RA. Epithelial-to-mesenchymal transition in cancer: complexity and opportunities. Front Med. 2018;12:361–73.
Tan TZ, Miow QH, Miki Y, Noda T, Mori S, Huang RY, et al. Epithelial‐mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients. EMBO Mol Med. 2014;6:1279–93.
Dontu G. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;17:1253–70.
Puisieux A, Brabletz T, Caramel J. Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol. 2014;16:488–94.
Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019;20:69–84.
Title AC, Hong S-J, Pires ND, Hasenöhrl L, Godbersen S, Stokar-Regenscheit N, et al. Genetic dissection of the miR-200–Zeb1 axis reveals its importance in tumor differentiation and invasion. Nat Commun. 2018;9:4671.
Wilson MM, Weinberg RA, Lees JA, Guen VJ. Emerging mechanisms by which EMT programs control stemness. Trends Cancer. 2020;6:775–80.
Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10:593–601.
Brabletz S, Bajdak K, Meidhof S, Burk U, Niedermann G, Firat E, et al. The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells: ZEB1 activates Notch signalling. EMBO J. 2011;30:770–82.
Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol. 2009;11:1487–95.
Zhang P, Wei Y, Wang L, Debeb BG, Yuan Y, Zhang J, et al. ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1. Nat Cell Biol. 2014;16:864–75.
Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell. 2003;3:421–9.
Panier S, Boulton SJ. Double-strand break repair: 53BP1 comes into focus. Nat Rev Mol Cell Biol. 2014;15:7–18.
di Masi A, Cilli D, Berardinelli F, Talarico A, Pallavicini I, Pennisi R, et al. PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL. Cell Death Dis. 2016;7:e2308.
Pennisi R, Antoccia A, Leone S, Ascenzi P, di Masi A. Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks. FEBS J. 2017;284:2378–95.
Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker A, Soneson C, et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015;21:1350–6.
Alonso MH, Aussó S, Lopez-Doriga A, Cordero D, Guinó E, Solé X, et al. Comprehensive analysis of copy number aberrations in microsatellite stable colon cancer in view of stromal component. Br J Cancer. 2017;117:421–31.
Cheung WKC, Nguyen DX. Lineage factors and differentiation states in lung cancer progression. Oncogene. 2015;34:5771–80.
Ferone G, Song J-Y, Sutherland KD, Bhaskaran R, Monkhorst K, Lambooij J-P, et al. SOX2 is the determining oncogenic switch in promoting lung squamous cell carcinoma from different cells of origin. Cancer Cell. 2016;30:519–32.
Yatabe Y, Mitsudomi T, Takahashi T. TTF-1 expression in pulmonary adenocarcinomas. Am J Surg Pathol. 2002;26:767–73.
Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature. 2014;511:543–50.
Cancer Genome Atlas Research N. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489:519–25.
Macheret M, Halazonetis TD. DNA replication stress as a hallmark of cancer. Annu Rev Pathol Mech Dis. 2015;10:425–48.
Baillie KE, Stirling PC. Beyond kinases: targeting replication stress proteins in cancer therapy. Trends Cancer 2020; S2405803320302843.
Liu H, Zhang H, Wu X, Ma D, Wu J, Wang L, et al. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. Nature. 2018;563:131–6.
Brabletz S, Brabletz T. The ZEB/miR‐200 feedback loop—a motor of cellular plasticity in development and cancer? EMBO Rep. 2010;11:670–7.
Cao M, Wang Y, Xiao Y, Zheng D, Zhi C, Xia X, et al. Activation of the clock gene TIMELESS by H3k27 acetylation promotes colorectal cancer tumorigenesis by binding to Myosin-9. J Exp Clin Cancer Res. 2021;40:162.
Li J, Lu Y, Akbani R, Ju Z, Roebuck PL, Liu W, et al. TCPA: a resource for cancer functional proteomics data. Nat Methods. 2013;10:1046–7.
Mendez G, Cilli D, Berardinelli F, Viganotti M, Ascenzi P, Tanzarella C, et al. Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation. IUBMB Life. 2012;64:853–61.
Hu Y, Smyth GK. ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009;347:70–78.
ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium. Pan-cancer analysis of whole genomes. Nature. 2020;578:82–93.
Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483:603–7.
Roumeliotis TI, Williams SP, Gonçalves E, Alsinet C, Del Castillo Velasco-Herrera M, Aben N, et al. Genomic determinants of protein abundance variation in colorectal cancer cells. Cell Rep. 2017;20:2201–14.
Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The molecular signatures database hallmark gene set collection. Cell Syst. 2015;1:417–25.
Acknowledgements
We would like to thank Cristiana Tiberio for technical support and Chiara Di Giorgio for English editing and paper proofreading. The Grant of Excellence Departments, MIUR (ARTICOLO 1, COMMI 314–337 LEGGE 232/2016) to the Department of Science, University Roma TRE is also gratefully acknowledged.
Funding
This study was supported by Associazione Italiana Ricerca sul Cancro [IG-22827 to F.B.], the Italian Ministry of Health [GR-2016-02363975 and CLEARLY to FB; GR-2019-12370460 to TC; Ricerca Corrente 2021 to FB and GM]. This study was supported in part by Ateneo Roma Tre and Fondo di Finanziamento per le Attività base di Ricerca (FFABR) to AdM. TC was a recipient of a fellowship from Associazione Italiana Ricerca sul Cancro (#19548) and of a fellowship from Fondazione Umberto Veronesi. RC was a recipient of a fellowship from Fondazione Umberto Veronesi and of a fellowship from Fondazione Pezcoller. The study funders had no role in the design of the study, the collection, analysis, and interpretation of the data, the writing of the paper, and the decision to submit the paper for publication.
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FB, TC, and GM conceived and designed the study; JA, GB, TC, RC, AC, NF, FM, AdM, TN, OP, and LS performed the experiments and acquired data; FB, TC, and ED performed the statistical and bioinformatics analyses; FB, TC, and GM wrote the paper; AdM, FB, TC, VC, ED, GM, and LS reviewed the scientific literature and critically revised the paper; all authors approved the final version of the paper and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved.
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Colangelo, T., Carbone, A., Mazzarelli, F. et al. Loss of circadian gene Timeless induces EMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism. Cell Death Differ 29, 1552–1568 (2022). https://doi.org/10.1038/s41418-022-00935-y
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DOI: https://doi.org/10.1038/s41418-022-00935-y
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