An integrative evaluation of circadian gene TIMELESS as a pan-cancer immunological and predictive biomarker

Cancer represents one of the most substantial hazards to human existence and stands as a global public health predicament [1]. The Global Burden of Disease Study reports that in 2019, there were 10 million cancer-related deaths and 23.6 million new cancer cases worldwide [2]. Consequently, it is utmost importance to perform a comprehensive analysis of pan-cancer expression for certain selected genes and determine how their expression relates to clinical traits, survival chances, and putative molecular mechanisms. Fortunately, there now exists a basis for doing pan-cancer analysis thanks to vast-scale and multi-omics cancer data sets, such as TCGA and GEO, which provide functional genomics data sets of numerous types of tumors [3, 4].

The circadian clock protein TIMELESS was first discovered in the Drosophila melanogaster [5, 6]. Soon after, in mammals, TIMELESS also plays a variety of roles in physiological and pathological processes, including DNA replication, DNA damage response, cell cycle, and circadian rhythm [7‐9]. A universal intrinsic timekeeping system is the circadian rhythm [10]. The relationship between circadian genes—including ARNTL, CLOCK, and PER1/2/3—and the onset of cancer has been thoroughly studied over the past few decades [11‐13]. Nowadays, night shift employment has been categorized by the International Agency for Research on Cancer (IARC) Monographs Vol 124 Working Group as a potential human carcinogen (Group 2A) in June 2019 [14]. However, shift work with circadian disruption has been considered as a vital carcinogenic risk factor for various cancer [15‐18]. Similar to TIMELESS, an important component of the circadian clock, these cancers include non-small cell lung cancer [19], colorectal cancer [20], nasopharyngeal carcinoma (NPC) [21], gliomas [22], and breast cancer [23], all of which display abnormal expression of TIMELESS and exhibit increased tumor aggressiveness. Although there have been an increasing number of research linking TIMELESS to human illnesses, notably malignant tumors, there is currently no pan-cancer analysis about the role of TIMELESS in the management of cancer. Consequently, the objective of this study is to thoroughly analyze bioinformatic data to investigate the function of TIMELESS in multiple cancer types.

To examine the function and underlying mechanism in tumorigenesis, we created a pan-cancer expression study of TIMELESS using a number of open-access databases. We also compared the correlations between TIMELESS expression and clinical characteristics, survival status, genetic alteration, immune cell infiltration, microsatellite instability (MSI), Tumor mutation burden (TMB), drug sensitivity, and relevant signaling pathway of malignant tumors. In addition, using immunohistochemistry (IHC), we confirmed the expression of TIMELESS in clinical patients across several tumor types. The study of TIMELESS could help in forecasting the survival and benefits of immunotherapeutic in cancer patients, according to our data, suggesting that TIMELESS plays a critical role in the development of various human cancers.

Large-scale and multi-omics cancer data sets, such as TCGA, UCSC, and GEO, which contain functional genomics data sets of various types of tumors, have given researchers a platform for pan-cancer study. The pan-cancer analysis is essential and useful for contrasting the similarities and differences among various malignancies, which in turn serves to throw fresh light on our understanding of how cancer develops and to offer a novel understanding of tumor biomarkers. Numerous research has shed fresh light on the human pan-cancer whole-genome analysis and showed a direct connection between carcinogenesis and mutations and copy number changes, which is crucial for the detection and management of different malignancies [27‐29]. The TIMELESS gene was first discovered as a molecule in the Drosophila melanogaster circadian clock through forward genetic screening by Leslie B. Vosshall in 1994 [30]. Due to its similar sequence, mammalian TIMELESS was identified as an essential circadian clock protein in flies. As an evolutionarily conserved core circadian clock gene in primates, TIMELESS plays multifaced roles in physiological and pathological processes, including embryonic development, DNA damage response, and DNA replication. In addition to its role in circadian rhythmicity, the TIMELESS protein also plays a role in DNA replication, the DNA damage response (DDR), embryonic development, cell cycle progression, as well as the maintenance of telomere length and integrity.

The circadian clock is a molecular time-keeping system that has survived evolution and controls daily oscillations of biological processes and behaviors, including sleep and hormone secretion [13]. Circadian disturbance has a deleterious impact on physiology and poses a threat to world health, manifesting among other things in proliferative, metabolic, and immunological illnesses [31, 32]. The period (PER) gene and its protein participate in a transcription–translation negative feedback loop that causes the TIMELESS mRNA and protein to oscillate regularly with time. The products of the important clock genes CLOCK, BMAL, PER1, PER2, and PER3 interact with the TIMELESS protein as well [33]. Human TIMELESS protein interacts with the cell cycle checkpoint proteins CHK1, ATR, and the ATR-interacting protein (ATRIP), as well as the circadian proteins CRY2/1 and PER1/2/3 [8]. In addition, exposure to DNA-damaging substances including hydroxyurea (HU) and UV radiation enhanced the interaction of TIMELESS with checkpoint proteins. The G2/M and intra-S cell cycle checkpoints depend heavily on TIMELESS protein. TIMELESS protein binds to the ATRIP component on ATR, a protein kinase sensitive to DNA damage, concerning the G2/M checkpoint. Moreover, Barrio-Alonso et al. [34] found that the expression of the circadian protein TIMELESS exhibits circadian rhythmicity in the mammalian hippocampus. TIMELESS acts as a chromatin-bound protein that targets synaptic-plasticity-related genes, such as phosphodiesterase 4B (Pde4b) and promotes Pde4b transcription, and negatively regulates cAMP signaling, thereby regulating the function of the AMPA receptor GluA1 function and affecting synaptic plasticity. Consequently, TIMELESS may be crucial to the development and progression of cancer.

Our study found that TIMELESS was aberrantly expressed and related to poorer survival in various malignant tumors, such as ACC, LGG, LIHC, and SARC. Liu Sai-Nan et al. [21] discovered that TIMELESS was overexpressed and associated with poorer survival in NPC, as well as conferred resistance to cisplatin-induced apoptosis and promoted the epithelial–mesenchymal transition (EMT), which could be a valuable prognostic factor and therapeutic target in NPC. According to a study on cervical cancer, TIMELESS overexpression is associated with lymphovascular space involvement, pelvic lymph node metastases, and worse OS and DFS. This suggests that TIMELESS may be a possible prognostic biomarker for cervical cancer patients [35]. Overexpression of TIMELESS was substantially linked to a worse prognosis and a more advanced tumor stage in breast cancer [36].

Studies in genetic mice models and cell lines have outlined the interactions between the circadian clock and numerous pathways connected to oncogenes and tumor suppressors, such as c-Myc, Ras, PTEN, and p53. The circadian clock predominates metabolic signal pathways regulating—oxidation, lipogenesis, amino acid absorption, glucose consumption, and glucose utilization. Deregulation of circadian rhythms thus impacts cancer metabolism and consequent cell proliferation, opening up new therapeutic possibilities [11]. The TIMELESS mutation caused arrhythmic behavior, namely lack of ability to establish proper circadian rhythms. In this study, there are many genetic alterations of TIMELESS, including amplification, miss mutation, and deep deletion, which may bring on a worse survival prognosis.

It has been suggested that CAF have an impact on how various immune cells interact with malignancies [37, 38]. Studies have shown that circadian rhythm significantly affects immune responses [39]. Likewise, our study showed that TIMELESS was significantly associated with the CAF in various cancer, including ACC, ESCA, HNSC, LGG, MESO, UVM, STAD, and TGCT. A study performed by Xing et al. [40] showed that high expression of TIMELESS was associated with immune cells especially macrophage infiltration in ovarian cancer. In addition, TIMELESS and its constructive binding protein TIPIN (TIMELESS-interacting protein) are critical for early embryonic development. Studies showed that TIMELESS is expressed in embryonic tissues, and involved in murine lung, kidney, and urethral bud branching morphogenesis and epithelial organogenesis [41‐43]. In addition, TIMELESS is crucial for DNA replication. TIMELESS shares structural and functional similarities with a family of proteins that are crucial for DNA synthesis, S-phase-dependent checkpoint activation, and chromosomal cohesion in eukaryotes. At the level of the DNA replication fork complex, these processed are coordinated [9, 44]. Precatenation and fork rotation help unwind at hard-replicate regions, but they also fundamentally interfere with proper chromosomal duplication, and TIMELESS–TIPIN inhibits these processes [45].

According to earlier research, chemotherapy and TIMELESS are highly connected in several types of human cancer [46, 47]. TIMELESS is significantly connected with the susceptibility of numerous anti-cancer targeted treatment medications, including fludarabine, lapatinib, and 6-mercaptopurine, as this study’s findings have shown. These results suggest that in some human malignancies, TIMELESS exhibits intriguing promise as a novel therapeutic target and a predictive biomarker for anti-cancer therapy sensitivity. The probable process by which TIMELESS modulated medication sensitivity, however, is still unclear, suggesting a potential future area of research for TIMELESS. Furthermore, Shen et al. [48] found that TIMELESS was down-regulated in cellular senescence, and deletion of clock protein TIMELESS may promote the cellular senescence and exacerbate genome instability at the onset of senescence, suggesting that TIMELESS inhibitors have potential applications in cancer prevention. Moreover, there is still no relevant evidence on the relationship between TIMELESS expression and the effectiveness of immunotherapy. We, therefore, deduced that TIMELESS may take important roles in cancer immunotherapies and alter the efficacy of immunotherapeutics. Higher levels of TMB and MSI are typically regarded as significant biomarkers correlated with the high therapeutic efficacy in numerous immune checkpoint inhibitors [25, 26]. Our research provided evidence for a potential association between TIMELESS expression and MSI and TMB in several malignancies. According to our research, TMB and MSI were substantially correlated with TIMELESS expression in COAD and lung cancer, indicating that patients who express TIMELESS substantially may respond better to immune checkpoint inhibitors.

Nevertheless, this excellent study has several shortcomings. First of all, the data enrolled in our study was primarily gathered from open-access databases and clinical samples of several cancer kinds, but it has to be confirmed with further clinical patients and contrasted with corresponding normal tissues. In addition, it is yet unclear how TIMELESS affects the tumor immune microenvironment and what functions it plays in a particular form of cancer. Future research is required to confirm these findings from in vitro and in vivo trials.

Taken together, the findings of our study showed statistically significant associations between TIMELESS expression and clinical prognosis, immune cell infiltration, tumor mutational burden or microsatellite instability, and drug sensitivity across a range of tumors, which helps to explain the function of TIMELESS in tumorigenesis and immunotherapy from the viewpoint of clinical tumor samples. As a result, TIMELESS may be crucial as a new and potent immunological and prognostic biomarker across several malignancies.