Background
With advancement in HPV preventative vaccines development, HPV positive cervical cancer (HPV
+ CC), accounting for about 95% of CC, have been considered as preventable for HPV naïve young women [
1]. However, HPV is not the only high-risk factor of CC, with approximately 5.5–11% CC HPV negative accompanied by poor prognosis [
1]. Besides, HPV vaccines could not cover all HPV types, even for Gardasil-9 with expanded coverage to HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) [
2,
3], and large amounts of older women with pre-existing HPV infections remain unsuitable for such vaccines since they are not therapeutic [
4]. On the other hand, late-stage CC diagnosed as distant or metastatic have limited treatment approaches and poor 5-year OS of only 20% [
5]. Considering the rather low vaccination rates worldwide and large potential risk groups with HPV infection, gene signatures with prognosis and predictive value are in urgent need for CC patients.
Circadian clock refers to an oscillation pattern of divergent gene networks that cooperate to adapt to environmental cycles and internal timing system on a 24-h-basis. Its core regulators include an activator comprised of CLOCK and BMAL1 and a repressor comprised of Per1/2 and CRY1/2 [
6]. Emerging evidence have indicated circadian rhythm as an indispensable factor for pathophysiological processes, such as in lifespan extension through caloric restriction and tumorigenesis. Recent literatures proved that chronic jetlag and chronic circadian disruption could accelerate breast tumor growth by creating an immune-suppressive TME [
7]. Furthermore, CRY1/2-/- and Per1/2-/- knockout mice experienced enhanced hepatocarcinoma metastasis along with disrupted serum bile acids and glycogen metabolism [
8]. On the other hand, CLOCK mutation mice exhibited resistance to carcinogen-induced skin carcinoma development [
9], and knockdown BMAL1 by shRNAs also interrupted the proliferation of leukemia stem cells [
10]. Pan-cancer analysis of TCGA provided transcriptional-level evidence of the heterogeneric effects of circadian clock related gene signatures in caner hallmarks as well. PER2 was found to be highly associated with activation of PI3K/AKT oncogenic pathway and cancer metabolism, while CLOCK was quite the opposite [
11]. Although disruption of circadian rhythm has been proved to be strongly correlated with cancer hallmarks including uncontrolled proliferation, genome instability, deregulation of metabolism and immune-suppressive TME, the translational value remained obscure until recently. Jiao Wang et al. designed a time-restricted regimen, that is, giving combination therapy of metformin and trastuzumab at zeitgeber time (ZT) 6 rather than ZT18, which significantly suppressed breast tumor growth compared to trastuzumab alone in murine models [
12]. To be noticed, such regimen failed to restrain tumor growth given at any other ZTs. They suggested that the underlying mechanism was that metformin-targeted HK2 fell into a circadian oscillation regulated by PPARγ and PER1 and disruption of HK2 interfered with such circadian rhythm thus leading to reversion of trastuzumab [
12].
Recently, another circadian clock core gene, TIMLESS was found to be over-expressed in ovarian cancer (OV) and was negatively correlated with B cells and DC cells infiltration in TCGA data analysis [
13]. Knockdown of TIMELESS significantly slowed tumor growth in vivo. While disruptions in circadian clock have been described in other gynecologic neoplasms, there have not been systematic bioinformatics analysis to date on circadian machinery of cervical cancer. Considering heterogeneity effects of circadian rhythm in cancer biology, knowledge of its functioning specifically in CC might benefit a lot.
Herein, to facilitate understanding into clinical relevance of circadian clock related signature in cervical cancer and illuminate its impact upon cancer metabolism and TME, we investigated TCGA-CESC dataset from the perspective of circadian oscillation thoroughly on a multi-omics level. Through K-means method, Cox regression and lasso analysis, we constructed a prognostic risk model based upon circadian-oscillation-signature and confirmed its validity by time-dependent ROC analysis. Furthermore, we discovered an ‘immune-desert’-like TME pattern in high-risk group by ESTIMATE and CIBERSORT analysis. Afterwards, to infer differences on genomic level, we conducted investigation upon SNP, CNV and TMB as well. With TIDE score and GDSC database, we then analyzed different drug sensitivity, including to immune checkpoints blockades (ICB), in high and low-risk groups. Then in combination with clinicopathological features, we built a risk prognostic model based upon independent prognostic factors and verified its solidity in GSE44001.Finally, through FISH and IHC analysis of our own real-world patients’ clinical surgery specimens, we validated CCL20 as an independent indicator for worse stages, and overexpression of CCL20 in U14 cell lines could lead to significant more M2 macrophages, Tregs and less M1 macrophages, in accordance with CIBERSORT analysis.
Discussion
Cervical cancer is the fourth most common female cancer worldwide [
5]. Besides, overall low prophylactic vaccination rates among adolescents worldwide and preexisting infections in older women demonstrate the urgent need for developing state-of-the-art early diagnostic strategies and therapeutic regimens [
42], for example, immunotherapies. Circadian disruption has been associated with tumorigenesis through effects on tumor proliferation, DNA repair and stemness, and combination therapies targeting circadian disorders with other regimens are emerging for GC [
12], NSCLC [
43], etc. However, little is known about its effects in tumor metabolism and TME, especially in cervical cancer, as well as its prognostic and therapeutic value [
44]. In this research, we systematically investigated circadian rhythm related genes genomic and transcriptional level patterns in TCGA-CESC dataset. We successfully divided CESC patients into low-risk and high-risk groups based upon their circadian clock gene signature expression modes. We further confirmed immune and metabolism related pathways enrichment of differentially expressed genes (DEGs) between high and low-risk groups, which includes response to lipopolysaccharide, fructose and mannose metabolism, and IL-17 signaling pathway. Furthermore, we also uncovered significantly different immune infiltration patterns between them. Through lasso regression analysis of DEGs, we proposed a prognosis model composed of GJB2, CCL20 and KRT24, all with independent prognostic value, and validated its predictive value on patients’ overall survival (OS) in dataset GSE44001. Finally, we illuminated that low-risk group was more sensitive to certain chemotherapy regimens and immunotherapy, providing informational clues for application of circadian gene signatures in clinical settings.
Coincidentally, circadian oscillation plays an important role in tumor-immune interaction, including antigen presentation, immunogenicity enhancement or loss and TME biology, during which these pathways might experience upregulation or downregulation depending on tumor type as well. When it comes to melanoma, higher CTL infiltration along with higher PD-1/L1 expression was found to be correlated with higher BMAL1 during anti-PD-1 immunotherapy clinical settings [
45]. While in KIRC and breast cancer, upregulation of CLOCK, ARNTL and PER3 promotes TME inflammation via modulating macrophages and neutrophils infiltration, which leads to worse prognosis [
46,
47]. However, the specific mechanisms under this myeloid-induced inflammation have not been demonstrated yet. In our research, high-risk patients’ TME is characterized as an immune-suppressed TME with more infiltration of macrophages, neutrophils, activated mast cells and activated DCs, while TME in low-risk groups has more CD8 T cells, activated CD4 Tm and rested masted cells, indicating a myeloid-dominated signature in high-risk group with worse prognosis as well. To be noticed, reasons behind this might be attributed to enhanced expression of ICs. For example, RORγ agonists, which can activate BMAL1 transcription [
48], attenuate the expression of PD-1 receptors, and LYC-55716, a RORy inhibitor, is currently under a Phase 1 trial used in combination with pembrolizumab for NSCLC (NCT03396497). Nevertheless, direct and indirect circadian control over specific immune cells subtypes should be investigated thoroughly for their potential translational clinical applications.
Besides, PPI interaction built upon hub genes (CCL20, KRT16, GJB2) in our analysis pointed to interaction network among IL1A, CXCL1, CXCL8, S100A7 and S100A9, which are important mediators of myeloid immune cells function. The chemokine CCL20 is notably overexpressed by myeloid cells, as is its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice restores CTL activity and significantly prolongs survival [
32]. CXCL8 functions as a chemotactic factor by guiding the neutrophils to the site of infection [
37]. IL1A is produced by monocytes and macrophages as a proprotein, which is proteolytically processed and released in response to cell injury, and thus induces apoptosis. Upregulated S100A7 could promote tumor proliferation through paracrine interaction with RAGE receptors [
39]. To be noticed, through KEGG analysis we also uncovered RAGE receptor pathways enrichment in DEGs between high and low-risk groups. In addition, exocrine S100A7 could promote M2 macrophage infiltration in esophageal squamous carcinoma (ESCC) [
36,
39]. SY Lim et al. demonstrated that monocytes/macrophages in the metastatic liver microenvironment induce S100A8 and S100A9 in cancer cells, and that these proteins are essential for tumor cell migration and invasion [
36].
Another major cancer hallmark, metabolism disorder, is indispensable for cancer cells proliferation requirements under TME selection pressure as well. Constitutive activation of the PI3K/PDK1/AKT pathway and HIF1a pathway under low level of oxygen contribute to increased glycolysis in tumor [
49]. Circadian clock has been proved to be essential regulators of glycolysis and oxidative phosphorylation through AKT and HIF1a pathways either [
50]. Conversely, hypoxia and HIF1α affect circadian rhythms through regulation of the circadian clock genes CRYs, RORα, Per2, and Cry1 [
51]. We also discovered enhanced expression pattern of fructose and mannose metabolism through KEGG analysis, as well as pentose phosphate and galactose metabolism pathways through GSEA analysis in high-risk groups of cervical cancer patients, further proving the potential link of glycolysis and circadian rhythm in cancer. Besides, PPAR signaling pathway and FA metabolism were also enriched in high-risk group, indicating regulation of FA metabolism by circadian oscillation as well. Recently, T Fedchenko et al. found that PPAR-γ agonist given to mice orally induced disruption of PER1/2 and BMAL1 expression in liver through regulation of NFKB and IL-6 pathways [
52]. At the same time, PPAR signaling pathway is the center of de novo synthesis of fatty acids. Sai Ma et al. characterized a mutual activation loop between PPARγ and esophageal adenocarcinoma-specific master regulator transcription factors (MRTF) in upregulation of synthesis of phospholipids [
53]. However, whether circadian clock signature could manipulate FA metabolism through PPAR pathways in cancer still lacks research till now.
Circadian rhythm interference has been attributed to uncontrolled proliferation and dampened DNA damage response, which are further linked to TMB and MSI. Emerging evidence have proved effects of circadian signature upon TMB and MSI, such as the positive regulation of growth promoter SERPINE1 via BMAL2, which promotes MSI [
54]. Bioinformatics analysis of TCGA discovered an index of core circadian genes (PER1/2/3, CRY1/2, CLOCK and BMAL1) that is negatively related to MMR pathway [
55]. However, we did not recognize significant TMB and MSI changes between high-risk and low-risk groups based upon circadian signature classification model in TCGA-CESC. Indeed, cancer heterogeneity and tissue origin specificity could make regulation of circadian clock even more complicated, indicating necessity for thorough investigation of circadian rhythm in different cancer type separately.
CCL20 was indicated as inducer of dampened anti-tumor ability of CTL when it was secreted by macrophages. We not only uncovered connection between expression of CCL20 and worse stage in clinical settings, but also uncovered disrupted CD8
+ T cells function with exhaustion phenotype and M2 infiltration triggered by ccl20 overexpression in U14 cancer cell lines in immune-competent murine tumor model. Although Hirotaka et al. found that dietary consumption of Lactobacillus-derived exopolysaccharide induced CCR6
+ CD8
+ T cells by CCL20-secreting tumor cells [
56], Wang et al. also uncovered FOXO1 promoted the migration of M2 macrophages via CCL20 secretion in esophageal squamous cell carcinoma [
57]. Besides, resently Liu et al.revealed a specific M2-like macrophages subtype with high CCL20 expression, which is associated with worse prognosis by single-cell analysis in CESC [
58]. Besides, they proposed that CCL20
+ macrrophges also expressed high levels of CXCL8, which is in accordance with our PPI analysis in TCGA-CESC as well [
59]. All in all, the regulation of CCL20 on the tumor immune microenvironment is currently controversial, our research might have shed light upon an indispensable role for M2 macrophages as well in cervical cancer.
Despite the critical role of CCL20 in our circadian-based risk model, KRT24, an OS-favorable protective marker in CESC has also been included into our model. KRT24 has also been proved to be a potential tumor suppressor. Désirée. et al. described suppression of viability and proliferation induced by KRT24 upon human HNSCC cell lines and mouse xenograft model [
60]. However, the impact of KRT24 upon TME in CESC still lacks certain research. Based upon a significant up-regulated immune-score calculated by ssGSEA with more KRT24 expression (Fig.
11A), we proposed that in-depth profiling of impacts of KRT24 upon TME in CESC, as well as its correlation with CCL20 is in urgent need.
To our knowledge, this is the first comprehensive bioinformatics analysis of circadian rhythm signatures in cervical cancer. Through multi-omics analysis of TCGA-CESC dataset and using GSE44001 as verification, we successfully constructed a prognostic risk model based upon circadian rhythm signature and discovered three independent prognostic factors, GJB2, CCL20 and KRT24, with hints upon metabolism features and suppressive myeloid cells enriched TME as poor prognostic indicators. However, due to intrinsic nature of data-mining, the underlying mechanism of impacts of circadian clocks upon macrophages and neutrophils infiltration as well as PPAR signaling pathways and glycolysis-related pathways enrichment still need to be thoroughly verified by molecular and cell biology experiments further. Nevertheless, we believed that our research could facilitate understanding of clinical value of circadian rhythm in cervical cancer, and unleash probability of digging into the prognostic value and even targetable features of circadian clock gene sets in cancer biology.
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