Abstract
Background
CSNK1D is a member of the CK1 family and is characterized by high conservation and widespread expression. It plays a regulatory role in the maintenance of numerous cellular processes and is expressed at low levels in normal tissues but is overexpressed in several types of cancer. All tumor tissues were pathologically diagnosed as lung adenocarcinoma.
Methods
Immunohistochemistry (IHC) was used to retrospectively assess CSNK1D expression levels in 90 lung adenocarcinoma surgical tumor tissue microarrays (TMAs). All tumor tissues were pathologically diagnosed as lung adenocarcinoma. An enzyme-linked immunosorbent assay (ELISA) was employed to detect CSNK1D expression in serum samples from 22 lung adenocarcinoma patients. All tumor tissues were pathologically diagnosed as lung adenocarcinoma
Results
CSNK1D was expressed in both the cytoplasm and nucleus, with higher levels in tumor tissues than in adjacent nontumor tissues. The results of survival analysis revealed that high CSNK1D expression was significantly associated with a favorable prognosis. The results of multivariate analysis, adjusted for clinicopathological features, indicated that high CSNK1D expression was an independent predictor of cancer-specific survival.
Keywords: CSNK1D, Prognostic value, LUAD, IHC, ELISA
Lung cancer is the leading cause of cancer-related death worldwide and the most prevalent malignancy in China in terms of incidence and mortality, with an increasing trend annually. The increasing incidence and mortality rates are primarily due to the asymptomatic nature of lung cancer at its early stages, often leading to a late-stage diagnosis when surgical or radical radiotherapy is no longer feasible, resulting in a poor prognosis [1]. Traditional treatments for advanced lung cancer include surgical resection, chemotherapy, and/or radiotherapy. However, some advanced or metastatic tumors are resistant to conventional chemotherapy and radiotherapy, leading to extremely high mortality rates.
CSNK1D is a gene encoding a protein belonging to the CK1 family, also known as CK1δ [2]. It plays crucial roles in the maintenance of multiple biological processes, including cell cycle regulation, transcriptional regulation, apoptosis, cell polarity, and cell motility [3–6]. Furthermore, CSNK1D is closely associated with the development of various human diseases, such as cancer and neurodegenerative disorders [7, 8].
Researchers have made significant discoveries regarding the role of CSNK1D in cancer. Studies have indicated that CSNK1D is essential for tumor cell proliferation, metastasis, angiogenesis, and drug resistance [9, 10]. Aberrant expression and altered functions of CSNK1D are closely linked to the onset and progression of multiple cancers, including melanoma, breast cancer, glioblastoma, and colorectal cancer [11, 12]. Additionally, studies suggest that CSNK1D plays a critical role in shaping the effectiveness of immunotherapy and may serve as a promising therapeutic target.
CSNK1D promotes cancer development and progression through various mechanisms. For instance, it regulates multiple signaling pathways, such as the Wnt/β-catenin, PI3K/AKT/mTOR, and Hippo pathways, stimulating tumor cell proliferation and metastasis [6, 13]. Moreover, CSNK1D modulates tumor cell metabolism, including glycolipid metabolism, and affects tumor cell growth and survival [14, 15]. Thus, CSNK1D is a potentially valuable prognostic marker and therapeutic target in cancer.
Our study aimed to comprehensively investigate CSNK1D variants in cancer using The Cancer Genome Atlas (TCGA) and other publicly accessible datasets. We explored the expression of CSNK1D mutants in lung adenocarcinoma and their potential correlation with clinical outcomes. In summary, our research provides novel insights into the significance of CSNK1D in cancer and highlights its potential as a target for therapeutic intervention.
Methods
Patients and tissue samples
In this study, a human lung adenocarcinoma tissue microarray (HLugA180Su11, Clinical trial number: SHYJS-CP-2206001) purchased from Shanghai Outdo Biotech Co., Ltd., was used. The array contained 90 lung adenocarcinoma surgical tumor tissue samples and corresponding adjacent nontumor tissue samples. The clinical data and clinicopathological characteristics of 90 patients were retrospectively collected, with a median follow-up time of 51.92 months. Detailed clinical information on the patients is provided in Table 1.
Table 1.
Baseline clinical characteristics of patients in the TMA
| Clinical information | ||
|---|---|---|
| Example | 90 cases | |
| Age (median) | 59.68 | |
| Sex (male/female) | 48(1.1429)/42 | |
| Location of the tumor | ||
| lymphatic metastasis | 33(36.7%) | |
| T phase c (ACJJ 8th edition) | ||
| TNM by stages | ||
| T1 | 51(56.6%) | |
| T2 | 29(32.2%) | |
| T3 | 7(7.7%) | |
| T4 | 3(3.3%) |
Immunohistochemistry
CSNK1D was detected using a specific rabbit monoclonal antibody (Affiniti cat. #: DF4774).
The TMA sections were deparaffinized and subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) by heating in a microwave for 10 min. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 15 min. After the sections were blocked with goat serum, 20 µg/ml primary antibody was applied and incubated with the sections overnight at 4 °C. An incubation with the secondary antibody was performed at room temperature for 1 h, followed by DAB chromogen development. The sections were counterstained with hematoxylin. The primary antibody was omitted as a negative control. TMA sections with distinct tumor and adjacent nontumor tissues served as positive and negative controls for CSNK1D staining. Two independent researchers scored the CSNK1D staining intensity in tumor cells as absent, low, moderate, or high. Cytoplasmic and nuclear expression was analyzed separately. (We confirm that all methods were carried out in accordance with relevant guidelines and regulations.)
Statistical analysis
The distribution of CSNK1D expression in relation to clinical indicators was analyzed on the basis of counts (percentages) for categorical variables and the chi-square test, while continuous variables are reported as medians (lower quartiles, upper quartiles) and were analyzed with the Mann‒Whitney U test (P < 0.05 was considered to indicate statistical significance).
Standardization protocol for experimental data:
Staining intensity scoring: 0 points (negative), 1 point (1+), 2 points (2+), 3 points (3+).
Staining positivity rate score: Original percentage (0-100).
Total score: The total score is calculated as the product of the “staining intensity score” and the “staining positivity rate score”.
Survival analysis grouping: CSNK1D cytoplasmic total score ≤ 130 is classified as low expression group, while total score > 130 is classified as high expression group; CSNK1D nuclear total score ≤ 127.5 is classified as low expression group, while total score > 127.5 is classified as high expression group. The surv_cutpoint function in the R package survminer was used to select the score value most closely related to prognosis as the cutoff value.
Univariate and multivariate survival analyses of CSNK1D expression and clinical indicators were performed. Variables with P < 0.05 in the univariate Cox analysis were included in the multivariate Cox regression analysis (P < 0.05 was considered to indicate statistical significance).
Survival curves were created using the Kaplan‒Meier method and log-rank test method. Cancer-specific survival was calculated from the surgery date to the event date (cancer-related death), with patients censored at the last follow-up if no events occurred. The log-rank test was used to evaluate differences in survival curves between groups.
Results
CSNK1D expression in lung adenocarcinoma and adjacent nontumor tissues
By evaluating CSNK1D expression in different lung adenocarcinoma tissues, we observed higher expression in tumor tissues, which is consistent with data from TCGA database and published reports on CSNK1D expression in cancers such as breast, liver, and prostate cancer. Cytoplasmic and nuclear expression was observed in tumor cells, which were scored as absent, low, moderate, or strong.
Differences in CSNK1D expression between tumor and adjacent nontumor tissues are detailed in Table 2 and Fig. 1.
Table 2.
Differential expression of CSNK1D in tumor and adjacent tissues
| Cytoplasm | Adjacent tissue | Tumor tissue | P value |
|---|---|---|---|
| 49 ± 42 | 137 ± 62 | < 0.001 | |
| Nucleus | 5 ± 8 | 126 ± 63 | < 0.001 |
*Statistically significant (P < 0.05)
Fig. 1.
CSNK1D expression in lung adenocarcinoma and adjacent tissues (*** indicates a statistically significant difference)
Differences in CSNK1D expression in the cytoplasm and nucleus
Immunohistochemistry (IHC) revealed differential CSNK1D expression in the cytoplasm and nucleus of tumor and adjacent nontumor tissues, which correlated with cancer-specific survival (P < 0.05).
The correlations between cytoplasmic CSNK1D expression and clinical data are provided in Table 3.
Table 3.
Correlations between CSNK1D (C) expression and clinicopathological characteristics
| Variables | CSNK1D (C) expression | χ2/U | p value | ||
|---|---|---|---|---|---|
| Low | High | ||||
| Age (years) | 62 (57, 67) | 59 (50, 67) | 770 | 0.141 | |
| Size (cm) | 3.0 (2.5, 4.0) | 3.0 (2.5, 4.5) | 926 | 0.872 | |
| Sex | 2.848 | 0.091 | |||
| Male | 15 (42.9) | 33 (61.1) | |||
| Female | 20 (57.1) | 21 (38.9) | |||
| Grade | 6.216 | 0.013 | |||
| I/II | 30 (85.7) | 33 (61.1) | |||
| III | 5 (14.3) | 21 (38.9) | |||
| Site | 0.305 | 0.581 | |||
| Left | 13 (37.1) | 17 (31.5) | |||
| Right | 22 (62.9) | 37 (68.5) | |||
| T stage | 0.002 | 0.963 | |||
| T1 + T2 | 31 (88.6) | 48 (88.9) | |||
| T3 + T4 | 4 (11.4) | 5 (11.1) | |||
| N stage | 0.529 | 0.467 | |||
| N0 | 20 (57.1) | 35 (64.8) | |||
| N1 + N2+N3 | 15 (42.9) | 19 (35.2) | |||
| TNM stage | 0.202 | 0.653 | |||
| Ι/II | 28 (80.0) | 41 (75.9) | |||
| III | 7 (20.0) | 13 (24.1) | |||
The correlations between nuclear CSNK1D expression and clinical data are provided in Table 4.
Table 4.
Correlations between CSNK1D (N) expression and clinicopathological characteristics
| Variables | CSNK1D (N) expression | χ2/U | p value | ||
|---|---|---|---|---|---|
| Low | High | ||||
| Age (years) | 62 (56, 68) | 59 (50, 66) | 753 | 0.057 | |
| Size (cm) | 3.0 (2.5, 4.3) | 3.0 (2.5, 4.0) | 940 | 0.712 | |
| Sex | 1.763 | 0.184 | |||
| Male | 19 (46.3) | 29 (60.4) | |||
| Female | 22 (53.7) | 19 (39.6) | |||
| Grade | 0.209 | 0.648 | |||
| I/II | 30 (73.2) | 33 (68.8) | |||
| III | 11 (26.8) | 15 (31.3) | |||
| Site | 2.954 | 0.086 | |||
| Left | 10 (24.4) | 20 (41.7) | |||
| Right | 31 (75.6) | 28 (58.3) | |||
| T stage | 0.880 | 0.348 | |||
| T1 + T2 | 35 (85.4) | 44 (91.7) | |||
| T3 + T4 | 6 (14.6) | 4 (8.3) | |||
| N stage | 2.133 | 0.144 | |||
| N0 | 22 (53.7) | 33 (68.8) | |||
| N1 + N2+N3 | 19 (46.3) | 15 (31.3) | |||
| TNM stage | 2.016 | 0.156 | |||
| Ι/II | 29 (70.7) | 40 (83.3) | |||
| III | 12 (29.3) | 8 (16.7) | |||
CSNK1D expression and postoperative survival
High cytoplasmic CSNK1D expression was associated with prolonged cancer-specific survival.
The results of univariate and multivariate analyses of the survival of patients stratified by cytoplasmic CSNK1D expression are listed in Table 5.
Table 5.
Analysis of the association of cytoplasmic CSNK1D expression with survival
| Variables | Univariate analysis | Multivariate analysis | ||||
|---|---|---|---|---|---|---|
| HR | 95% CI | p value | HR | 95% CI | p value | |
| CSNK1D (C, high/low) | 0.583 | 0.348–0.977 | 0.041 | 0.537 | 0.31–0.93 | 0.027 |
| Sex (male/female) | 1.130 | 0.674–1.893 | 0.644 | |||
| Age (continuous) | 1.028 | 0.996–1.061 | 0.082 | |||
| Grade (III/I + II) | 1.684 | 0.970–2.922 | 0.064 | |||
| Site (right/left) | 1.283 | 0.728–2.259 | 0.389 | |||
| Size (continuous) | 1.170 | 1.077–1.271 | < 0.001 | 1.05 | 0.917–1.203 | 0.478 |
| T stage (T3 + T4/T1 + T2) | 3.473 | 1.666–7.238 | 0.001 | 1.505 | 0.461–4.911 | 0.498 |
| N stage (N1 + N2+N3/N0) | 1.558 | 0.971–2.645 | 0.101 | |||
| TNM stage (III + IV/I + II) | 2.482 | 1.382–4.460 | 0.002 | 2.079 | 0.951–4.549 | 0.067 |
The survival curves for patients with high and low cytoplasmic CSNK1D expression are shown in Fig. 2.
Fig. 2.
Survival curves for patients with high and low cytoplasmic CSNK1D expression (P = 0.036, statistically significant)
The results of univariate and multivariate analyses of the association of nuclear CSNK1D expression with survival are provided in Table 6 and Fig. 3.
Table 6.
Analysis of the association of nuclear CSNK1D expression with survival
| Variables | Univariate analysis | Multivariate analysis | ||||
|---|---|---|---|---|---|---|
| HR | 95% CI | p value | HR | 95% CI | p value | |
| CSNK1D (N, high/low) | 0.525 | 0.311–0.884 | 0.015 | 0.557 | 0.328–0.946 | 0.03 |
| Sex (male/female) | 1.130 | 0.674–1.893 | 0.644 | |||
| Age (continuous) | 1.028 | 0.996–1.061 | 0.082 | |||
| Grade (III/I + II) | 1.684 | 0.970–2.922 | 0.064 | |||
| Site (right/left) | 1.283 | 0.728–2.259 | 0.389 | |||
| Size (continuous) | 1.170 | 1.077–1.271 | < 0.001 | 1.072 | 0.941–1.221 | 0.293 |
| T stage (T3 + T4/T1 + T2) | 3.473 | 1.666–7.238 | 0.001 | 1.461 | 0.443–4.817 | 0.534 |
| N stage (N1 + N2+N3/N0) | 1.558 | 0.971–2.645 | 0.101 | |||
| TNM stage (III + IV/I + II) | 2.482 | 1.382–4.460 | 0.002 | 1.695 | 0.802–3.581 | 0.167 |
Fig. 3.
Survival curves for patients with high and low nuclear CSNK1D expression (P = 0.013, statistically significant)
Patients with high CSNK1D expression had better survival outcomes, whereas those with low or no expression experienced shorter survival (as shown in the figures). The univariate analysis revealed that cytoplasmic CSNK1D expression, the tumor size, tumor stage (T3 + T4/T1 + T2), and TNM stage (III + IV/I + II) were significantly associated with cancer-specific survival (Table 5).
The univariate analysis results indicated that nuclear CSNK1D expression, the tumor size, tumor stage (T3 + T4/T1 + T2), and TNM stage (III + IV/I + II) were significantly correlated with cancer-specific survival (Table 6). The multivariate analysis revealed that CSNK1D expression was an independent predictor of cancer-specific survival (Table 6). These findings indicate that CSNK1D expression in tumor cells is an independent predictor of survival after surgery in a combined cohort of lung adenocarcinoma patients, irrespective of their clinical or pathological features. Notably, after we adjusted for clinicopathological factors, the tumor location was not an independent predictor of cancer-specific survival.
ELISA results
Serum samples from 22 lung adenocarcinoma patients were analyzed for CSNK1D expression(Clinical trial number: PJKT2023-104), The results demonstrated statistically significant preoperative overexpression of CSNK1D in lung cancer patients compared to postoperative levels (One-way ANOVA, P < 0.05).
A standard curve for the enzyme-linked immunosorbent assay (ELISA) was created, as detailed in Table 7, and the curve is shown in Fig. 4.
Table 7.
Standard curve data
| OD450 | 0.105 | 0.2839 | 0.4784 | 0.8432 | 2.3628 |
| CSNK1D concentration (ng/ml) | 0 | 0.0375 | 0.075 | 0.15 | 0.6 |
Fig. 4.
Standard curve for CSNK1D levels generated using standard samples (y = 0.2694x − 0.047, R² = 0.9938)
The results obtained for more than 22 patient samples are provided in Table 8; Fig. 5.
Table 8.
CSNK1D concentrations in the serum of lung adenocarcinoma patients
| Sample number | Before surgery CSNK1D(ng/mL) |
SD | After surgery CSNK1D(ng/mL) |
SD |
|---|---|---|---|---|
| Sample 1 | 20.871 | 0.559 | 19.869 | 0.123 |
| Sample 2 | 35.562 | 0.054 | 16.321 | 0.454 |
| Sample 3 | 20.065 | 0.690 | 19.060 | 1.104 |
| Sample 4 | 16.578 | 0.401 | 15.004 | 0.245 |
| Sample 5 | 19.680 | 0.447 | 17.384 | 0.749 |
| Sample 6 | 25.363 | 1.231 | 21.229 | 0.324 |
| Sample 7 | 20.274 | 0.177 | 19.156 | 0.341 |
| Sample 8 | 19.599 | 0.422 | 16.719 | 0.218 |
| Sample 9 | 11.884 | 0.164 | 10.852 | 0.640 |
| Sample 10 | 31.342 | 1.798 | 25.996 | 0.095 |
| Sample 11 | 18.896 | 0.518 | 16.401 | 0.313 |
| Sample 12 | 20.360 | 1.253 | 13.203 | 1.975 |
| Sample 13 | 17.314 | 0.405 | 16.854 | 0.490 |
| Sample 14 | 20.507 | 0.698 | 16.960 | 0.150 |
| Sample 15 | 19.580 | 0.368 | 17.393 | 1.962 |
| Sample 16 | 14.368 | 0.088 | 13.238 | 0.345 |
| Sample 17 | 17.162 | 0.218 | 15.303 | 1.076 |
| Sample 18 | 19.021 | 0.014 | 17.445 | 0.106 |
| Sample 19 | 15.674 | 0.320 | 15.206 | 0.504 |
| Sample 20 | 16.266 | 0.286 | 15.287 | 0.591 |
| Sample 21 | 19.416 | 0.109 | 17.701 | 1.662 |
| Sample 22 | 16.462 | 0.078 | 15.447 | 2.207 |
Fig. 5.
CSNK1D
Relationship between CSNK1D expression and immunotherapy
Among the 22 patients with high CSNK1D expression, 12 had high PD-L1 expression levels. In clinical practice, patients with PD-L1 expression ≥ 1% are eligible for immunotherapy. The PD-L1 expression status of these 12 patients is detailed in Table 9. All 12 patients had stage II or higher cancer and high CSNK1D expression. Due to incomplete follow-up data, the impact on survival could not be assessed. However, existing data have shown that 8 patients with high CSNK1D expression also exhibit high PD-L1 expression, making them eligible for immunotherapy, which may have clinical implications.
Table 9.
PD-L1 expression in patients with high CSNK1D expression
| Sample number | PD-L1 (%) | Tumor staging | CSNK1D (ng/ml) |
|---|---|---|---|
| Sample 2 | 60 | III | 35.562 |
| Sample 4 | < 1 | III | 16.578 |
| Sample 5 | < 1 | IV | 19.680 |
| Sample 6 | 10 | III | 25.363 |
| Sample 9 | 10 | IV | 11.884 |
| Sampe 12 | 80 | III | 20.360 |
| Sample 13 | < 1 | IV | 17.314 |
| Sample 16 | 70 | IV | 14.368 |
| Sample 17 | 80 | III | 17.162 |
| Sample 19 | 3 | III | 15.674 |
| Sample 20 | < 1 | II | 16.266 |
| Sample 21 | 80 | III | 19.416 |
Conclusions
By analyzing the impact of circadian clock factors on the occurrence and progression of lung adenocarcinoma using databases such as TCGA, it was found that CSNK1D plays a significant role in the development of lung adenocarcinoma. This study detected the expression of CSNK1D in lung adenocarcinoma tissues and peripheral blood, revealing high expression in lung adenocarcinoma patients. In subsequent research, CSNK1D may serve as a detection target for lung adenocarcinoma progression or a monitoring point for immunotherapy.
Discussion
CSNK1D was proposed as a protein molecule involved in regulating circadian rhythms, with circadian rhythm proteins closely related to key regulatory factors of the cell cycle, oxidative stress, and carcinogenesis. To control the associated circadian rhythms, regulatory components are subject to post-translational modifications such as reversible phosphorylation. In general, CK1 isoforms δ and ε can phosphorylate and regulate clock proteins BMAL1 and CRY, and can also modulate the expression of period length-regulating factor prohibitin 2 (PHB2) [16, 17]. CSNK1D is considered an important regulator of circadian rhythms, but participation of other CK1 isoforms has also been identified. CSNK1D and CK1ε can influence the length of the circadian rhythm period by regulating the stability and subcellular localization of PER [18]. In circadian rhythms, protein phosphatase 5 (PP5) is a regulator that enhances CK1ε activity through dephosphorylation, thereby increasing CK1ε-mediated phosphorylation and subsequent PER degradation. Recently, CSNK1D (but not CK1ε) has been shown to be crucial for the circadian rhythm mechanism in zebrafish [19].
As a member of the CK1 family, CSNK1D participates in the cellular growth process and plays a key regulatory role in multiple signaling pathways. It first participates in mitosis as part of the cytoskeleton during the cell cycle process, related to spindle formation, and directly regulates MT (dynein-dependent transport along microtubules) through phosphorylation of α-, β-, and γ-tubulin, thereby exerting stress-induced functions in the spindle and centrosome [3, 20]. The localization of CSNK1D in the centrosome plays a crucial role in ciliogenesis: according to studies knocking down CSNK1D kinase activity (using CK1δ-specific inhibitors or specific siRNA), CSNK1D has been shown to mediate primary ciliogenesis through interaction with the AKAP450 protein, maintenance of Golgi organization, and directed protein transport [4].
Members of the CK1 family are core components of several cellular functions related to DNA modification and DNA damage. In the context of DNA damage-related signal transduction, p53 is activated, initiating pathways that ensure centrosome integrity and genomic stability. This signaling network primarily involves the synergistic action of CK1 and p53. CK1α, CSNK1D, and CK1ε can phosphorylate N-terminal target sites of p53 (Ser-4, Ser-6, Ser-9, Ser-18, Ser-20) [21, 22]. By phosphorylating p53 (primarily Ser-15 and Thr-18), CK1δ and ε reduce p53’s binding affinity to cellular counterparts, leading to release of MDM2 and activation of p53 [23, 24].
CK1α, CSNK1D, and CK1ε are components of Fas-mediated apoptosis and induce the activation of caspase 8 [9]. The Ser-64 and Ser-66 amino acids of Bid are considered major targets for CK1-mediated phosphorylation, while Thr-58 is a target for CK2. Only unphosphorylated Bid can be proteolyzed by caspase 8 and participate in cytochrome c-mediated apoptosis [9]. Therefore, inhibition of CK1 and CK2 can accelerate Fas-induced apoptosis by blocking inhibitory phosphorylation of Bid. Conversely, overexpression of CK1ε and CK2 leads to a reduction in the number of apoptotic cells due to increased Bid phosphorylation, which blocks its caspase 8-mediated process. Thus, phosphorylation of Bid by CSNK1D, CK1ε, and CK2 inhibits Fas-mediated apoptosis [9].
In the canonical Wnt/β-catenin signaling pathway, all CK1 family members are involved. CSNK1D has been shown to phosphorylate components of the Wnt pathway including dishevelled 1 (Dvl1), axin, APC, and β-catenin [25–28]. Therefore, by phosphorylating Dvl1, CSNK1D has also been observed to play an important role in neurite outgrowth and differentiation of dopaminergic neurons. In addition, CSNK1D/ CK1ε phosphorylates Dvl2 [29, 30], it plays an important role in the maintenance of intestinal stem cells (ISCs) [31]. In fact, the synergistic action of CSNK1D and CK1ε leads to the elimination of ISCs. Additionally, CSNK1D appears to have an important role in the regulation of planar cell polarity (PCP), which depends on the phosphorylation of the PCP protein Vangl2 by the atypical Wnt signaling pathway mediated by CSNK1D/ CK1ε and Wnt-5a [32]. CSNK1D can stabilize the conformation of lipoprotein receptor-related protein 6 (LRP6) after β-catenin phosphorylation as well as the complex of Axin and β-catenin, thereby preventing β-catenin phosphorylation and ubiquitination [33, 34]. Furthermore, the activity and stability of CSNK1D/ CK1ε are affected by the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate), and TPA promotes the binding of β-catenin to TCF4E in a CSNK1D/ CK1ε-dependent manner, leading to the activation of Wnt target genes [35]. The regulation of β-catenin Ser-45 phosphorylation by CSNK1D has a negative impact on the Wnt pathway, thereby initiating β-catenin-mediated GSK-3β phosphorylation and subsequent degradation [25]. Recent studies have shown that CSNK1D increases Smo activity by regulating Smo (Ser-683) phosphorylation through PKC [36]. Under Hh stimulation, CSNK1D phosphorylates the full-length Cubitus interruptus-positive transcription factor (CiA), protecting it from proteasomal degradation.CSNK1D regulates the Hh pathway by promoting SMO phosphorylation [36]. Mutations or dysregulation of the Hh pathway are associated with tumorigenesis and cancer development.
In this study, we primarily used IHC to assess CSNK1D expression in tumor and adjacent nontumor tissues from lung adenocarcinoma patients. An independent analysis of tumor and adjacent tissues revealed higher CSNK1D expression in tumors. Our results are consistent with those of previous reports showing widespread CSNK1D expression in cancers such as breast, liver, bladder, and colorectal cancer.
In this study, we analyzed 90 lung adenocarcinoma specimens (with matched adjacent nontumor tissues) from Shanghai Outdo Biotech Co., Ltd., and 30 serum samples from lung adenocarcinoma patients in the clinical biobank of Guangdong Medical University Affiliated Hospital. Compared with adjacent nontumor tissues, all tested tumor tissues exhibited higher CSNK1D expression levels, and high CSNK1D expression was detected in the serum of lung adenocarcinoma patients. The consistency between the results from tissue and blood samples supports our findings.
Data from TCGA and other databases further corroborate our results, showing upregulated CSNK1D expression in lung adenocarcinoma and other cancer types.
Our study provides the prognostic significance of CSNK1D overexpression in lung adenocarcinoma. The survival analysis indicated that high CSNK1D expression was significantly correlated with better survival rates.
The CSNK1D mRNA is overexpressed in various cancers, including bladder, brain, breast, colorectal, kidney, lung, melanoma, ovarian, pancreatic, and prostate cancers, and hematologic malignancies [37–40]. Conversely, some studies report low CSNK1D expression in tumors [41]. Reduced CSNK1D expression may induce cell cycle arrest and apoptosis in various cancer cell lines.
Studies on the correlation between CSNK1D expression and overall survival in cancer patients have shown that high CSNK1D expression is associated with shorter survival of patients with glioblastoma, lung, and colorectal cancer [42]. In contrast, high CSNK1D expression is linked to longer survival in patients with breast cancer, chronic lymphocytic leukemia, and astrocytoma [41].
CSNK1D is overexpressed in diverse cell lines and tissues, including those of the brain, prostate, colon, and ovarian cancers and in hematologic/lymphoid systems. High CSNK1D levels are also detected in pancreatic cancer. Aberrantly altered CSNK1D expression is observed in cervical squamous cell carcinoma, where increased expression correlates with deep stromal invasion.
Moreover, low CSNK1D expression is associated with prolonged survival of patients with colorectal cancer. CSNK1D is highly expressed in triple-negative breast cancer (TNBC), particularly in BRCA1-mutated cases. CSNK1D is abnormally expressed in several cancer types, with ~ 30% of TNBC showing high CSNK1D levels.
CSNK1D overexpression is not universally predictive of favorable survival of patients with various cancer types. In patients with liver cancer, for example, high CSNK1D expression predicts shorter survival. Similarly, in patients with TNBC, CSNK1D expression is linked to hormone resistance and an increased recurrence risk. Compared with nonmetastatic tumors, primary tumor cells and those invading lymph nodes highly express CSNK1D.
Although our research data indicate that CSNK1D may be a key prognostic factor for lung adenocarcinoma, it may also serve as a monitoring point for immunotherapy in lung adenocarcinoma. The small sampling area and potential false positives from using tissue microarrays may affect the results. Additionally, our samples were limited to patients with resectable tumors and preoperative serum samples, which restricts generalizability. Future studies should expand tissue sampling and include postoperative blood samples to increase the robustness of the data.
This study is the first to demonstrate the prognostic impact of CSNK1D in lung adenocarcinoma and highlights its potential as a therapeutic target in immunotherapy.
Acknowledgements
We thank the Biotissue Repository, Affiliated Hospital of Guangdong Medical University, for providing the samples.
Author contributions
Wenmei Su and Wei Zhang were responsible for the research design of the main paper. Chunyuan Chen, Shuhong Liu , and Qu Wang were responsible for the collection of relevant data.Biyun Lin , Zhu Liang , and Yinghuan Xiong were responsible for the statistical analysis of relevant data. Chunyuan Chen and Biyun Lin were responsible for the preparation of the manuscript. All authors participated in the review of the manuscript.
Funding
This project was supported by the Zhanjiang Competitive Science and Technology Project (2021A05076); Clinical Research Program, Affiliated Hospital of Guangdong Medical University (LCYJ2018B001); Clinical Research Program, Affiliated Hospital of Guangdong Medical University (LCYJ2021A004); Cohort Study Program of the Affiliated Hospital of Guangdong Medical University (LCYJ2022DL003); General Project of Guangdong Provincial Bureau of Traditional Chinese Medicine (2022107); and Wu Jieping Medical Foundation (320.6750.2024-13-75).
Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information.
Declarations
Ethics approval
This retrospective study was conducted with all protocols approved by the ethics committees of Shanghai Xinchao Biotechnology Co., Ltd. (Ethics Code: SHYJS-CP-2206001) and Guangdong Medical University Affiliated Hospital (Ethics Code: KT2023-104-01). The research adhered to the principles of the Helsinki Declaration.
Informed consent
was obtained from all subjects or, if subjects are under 18, from a parent and/or legal guardian.(This part of the content has been uploaded in the Supplementary material).
Consent to participate
In the event that the patient is unable to sign, the signature of a legal representative is necessary. The relationship between the patient and the legal representative shall be elucidated. If the patient is unable to read or write, the signature of a disinterested witness is required. This part of the content has been uploaded in the Supplementary material.
Consent for publication
The Author confirms: (1) that the work described has not been published before; (2) that it is not under consideration for publication elsewhere; (3) that its publication has been approved by all co-author; (4) that its publication has been approved (tacitly or explicitly) by there sponsible authorities at the institution where the work is carried out. The Author agrees to publication in the Journal Discover Oncology. The copyright to the English-language article is transferred to Discover Oncology effective if and when the article is accepted for publication. The author warrants that his/her contribution is original and that he/she has full powerto make this grant. The author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors.The copyright transfer covers the exclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, micro form, electronic form (offline, online) or any other reproductions of similar nature.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Chunyuan Chen, Zhu Liang, and Biyun Lin have contributed equally to this work.
Contributor Information
Yinghuan Xiong, Email: xyh821215@163.com.
Wei Zhang, Email: wzhang@must.edu.mo.
Wenmei Su, Email: suwenmei123@hotmail.com.
References
- 1.Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74:12–49. [DOI] [PubMed] [Google Scholar]
- 2.Graves PR, Haas DW, Hagedorn CH, DePaoli-Roach AA, Roach PJ. Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. J Biol Chem. 1993;268:6394–401. [PubMed] [Google Scholar]
- 3.Behrend L, Stöter M, Kurth M, Rutter G, Heukeshoven J, Deppert W, Knippschild U. Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus. Eur J Cell Biol. 2000;79:240–51. [DOI] [PubMed] [Google Scholar]
- 4.Greer YE, Westlake CJ, Gao B, Bharti K, Shiba Y, Xavier CP, Pazour GJ, Yang Y, Rubin JS. Casein kinase 1δ functions at the centrosome and Golgi to promote ciliogenesis. Mol Biol Cell. 2014;25:1629–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Lin YC, Chen MC, Hsieh TH, Liou JP, Chen CH. CK1δ as a potential therapeutic target to treat bladder cancer. Aging. 2020;12:5764–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Zhu M, Zhang J, Bian S, Zhang X, Shen Y, Ni Z, Xu S, Cheng C, Zheng W. Circadian gene CSNK1D promoted the progression of hepatocellular carcinoma by activating Wnt/β-catenin pathway via stabilizing Dishevelled Segment Polarity Protein 3. Biol Proced Online. 2022;24:21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Schittek B, Sinnberg T. Biological functions of casein kinase 1 isoforms and putative roles in tumorigenesis. Mol Cancer. 2014;13:231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Schwab C, DeMaggio AJ, Ghoshal N, Binder LI, Kuret J, McGeer PL. Casein kinase 1 delta is associated with pathological accumulation of tau in several neurodegenerative diseases. Neurobiol Aging. 2000;21:503–10. [DOI] [PubMed] [Google Scholar]
- 9.Izeradjene K, Douglas L, Delaney AB, Houghton JA. Casein kinase I attenuates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by regulating the recruitment of fas-associated death domain and procaspase-8 to the death-inducing signaling complex. Cancer Res. 2004;64:8036–44. [DOI] [PubMed] [Google Scholar]
- 10.Knippschild U, Gocht A, Wolff S, Huber N, Löhler J, Stöter M. The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal. 2005;17:675–89. [DOI] [PubMed] [Google Scholar]
- 11.Rosenberg LH, Lafitte M, Quereda V, Grant W, Chen W, Bibian M, Noguchi Y, Fallahi M, Yang C, Chang JC, Roush WR, Cleveland JL, Duckett DR. Therapeutic targeting of casein kinase 1δ in breast cancer. Sci Transl Med. 2015;7:318ra202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Thorne CA, Hanson AJ, Schneider J, Tahinci E, Orton D, Cselenyi CS, Jernigan KK, Meyers KC, Hang BI, Waterson AG, Kim K, Melancon B, Ghidu VP, Sulikowski GA, LaFleur B, Salic A, Lee LA, Miller DM, Lee E. Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α. Nat Chem Biol. 2010;6:829–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Duan S, Skaar JR, Kuchay S, Toschi A, Kanarek N, Ben-Neriah Y, Pagano M. mTOR generates an auto-amplification loop by triggering the βTrCP- and CK1α-dependent degradation of DEPTOR. Mol Cell. 2011;44:317–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Cunningham PS, Ahern SA, Smith LC, da Silva Santos CS, Wager TT, Bechtold DA. Targeting of the circadian clock via CK1δ/ε to improve glucose homeostasis in obesity. Sci Rep. 2016;6:29983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Xu P, Fischer-Posovszky P, Bischof J, Radermacher P, Wabitsch M, Henne-Bruns D, Wolf AM, Hillenbrand A, Knippschild U. Gene expression levels of Casein kinase 1 (CK1) isoforms are correlated to adiponectin levels in adipose tissue of morbid obese patients and site-specific phosphorylation mediated by CK1 influences multimerization of adiponectin. Mol Cell Endocrinol. 2015;406:87–101. [DOI] [PubMed] [Google Scholar]
- 16.Aryal RP, Kwak PB, Tamayo AG, Gebert M, Chiu PL, Walz T, Weitz CJ. Macromolecular Assemblies Mammalian Circadian Clock Mol Cell. 2017;67:770–e782776. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Lee C, Etchegaray JP, Cagampang FR, Loudon AS, Reppert SM. Posttranslational mechanisms regulate the mammalian circadian clock. Cell. 2001;107:855–67. [DOI] [PubMed] [Google Scholar]
- 18.Fustin JM, Kojima R, Itoh K, Chang HY, Ye S, Zhuang B, Oji A, Gibo S, Narasimamurthy R, Virshup D, Kurosawa G, Doi M, Manabe I, Ishihama Y, Ikawa M, Okamura H. Two Ck1δ transcripts regulated by m6A methylation code for two antagonistic kinases in the control of the circadian clock. Proc Natl Acad Sci U S A. 2018;115:5980–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Albornoz A, Yáñez JM, Foerster C, Aguirre C, Pereiro L, Burzio V, Moraga M, Reyes AE, Antonelli M. The CK1 gene family: expression patterning in zebrafish development. Biol Res. 2007;40:251–66. [DOI] [PubMed] [Google Scholar]
- 20.Stöter M, Bamberger AM, Aslan B, Kurth M, Speidel D, Löning T, Frank HG, Kaufmann P, Löhler J, Henne-Bruns D, Deppert W, Knippschild U. Inhibition of casein kinase I delta alters mitotic spindle formation and induces apoptosis in trophoblast cells. Oncogene. 2005;24:7964–75. [DOI] [PubMed] [Google Scholar]
- 21.MacLaine NJ, Oster B, Bundgaard B, Fraser JA, Buckner C, Lazo PA, Meek DW, Höllsberg P, Hupp TR. A central role for CK1 in catalyzing phosphorylation of the p53 transactivation domain at serine 20 after HHV-6B viral infection. J Biol Chem. 2008;283:28563–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Varelas X, Miller BW, Sopko R, Song S, Gregorieff A, Fellouse FA, Sakuma R, Pawson T, Hunziker W, McNeill H, Wrana JL, Attisano L. The Hippo pathway regulates Wnt/beta-catenin signaling. Dev Cell. 2010;18:579–91. [DOI] [PubMed] [Google Scholar]
- 23.Alsheich-Bartok O, Haupt S, Alkalay-Snir I, Saito S, Appella E, Haupt Y. PML enhances the regulation of p53 by CK1 in response to DNA damage. Oncogene. 2008;27:3653–61. [DOI] [PubMed] [Google Scholar]
- 24.Dumaz N, Milne DM, Meek DW. Protein kinase CK1 is a p53-threonine 18 kinase which requires prior phosphorylation of serine 15. FEBS Lett. 1999;463:312–6. [DOI] [PubMed] [Google Scholar]
- 25.Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, Alkalay I. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev. 2002;16:1066–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Gao ZH, Seeling JM, Hill V, Yochum A, Virshup DM. Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci U S A. 2002;99:1182–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ha NC, Tonozuka T, Stamos JL, Choi HJ, Weis WI. Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation. Mol Cell. 2004;15:511–21. [DOI] [PubMed] [Google Scholar]
- 28.Xing Y, Clements WK, Kimelman D, Xu W. Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex. Genes Dev. 2003;17:2753–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Martel-Pelletier J, Faure MP, McCollum R, Mineau F, Cloutier JM, Pelletier JP. Plasmin, plasminogen activators and inhibitor in human osteoarthritic cartilage. J Rheumatol. 1991;18:1863–71. [PubMed] [Google Scholar]
- 30.Greer YE, Rubin JS. Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth. J Cell Biol. 2011;192:993–1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Morgenstern Y, Das Adhikari U, Ayyash M, Elyada E, Tóth B, Moor A, Itzkovitz S. Ben-Neriah, Casein kinase 1-epsilon or 1-delta required for Wnt-mediated intestinal stem cell maintenance. Embo j. 2017;36:3046–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Yang W, Garrett L, Feng D, Elliott G, Liu X, Wang N, Wong YM, Choi NT, Yang Y, Gao B. Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development. Cell Res. 2017;27:1466–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Zeng X, Tamai K, Doble B, Li S, Huang H, Habas R, Okamura H, Woodgett J, He X. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature. 2005;438:873–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Wu G, Huang H, Garcia Abreu J, He X. Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6. PLoS ONE. 2009;4:e4926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Su Z, Song J, Wang Z, Zhou L, Xia Y, Yu S, Sun Q, Liu SS, Zhao L, Li S, Wei L, Carson DA, Lu D. Tumor promoter TPA activates Wnt/β-catenin signaling in a casein kinase 1-dependent manner. Proc Natl Acad Sci U S A. 2018;115:E7522–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Jiang K, Liu Y, Fan J, Epperly G, Gao T, Jiang J, Jia J. Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila. Proc Natl Acad Sci U S A. 2014;111:E4842–4850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Bar I, Merhi A, Larbanoix L, Constant M, Haussy S, Laurent S, Canon JL, Delrée P. Silencing of casein kinase 1 delta reduces migration and metastasis of triple negative breast cancer cells. Oncotarget. 2018;9:30821–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Lopez-Guerra JL, Verdugo-Sivianes EM, Otero-Albiol D, Vieites B, Ortiz-Gordillo MJ, De León JM, Praena-Fernandez JM, Marin JJ, Carnero A. High casein kinase 1 epsilon levels are correlated with better prognosis in subsets of patients with breast cancer. Oncotarget. 2015;6:30343–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Lin SH, Lin YM, Yeh CM, Chen CJ, Chen MW, Hung HF, Yeh KT, Yang SF. Casein kinase 1 epsilon expression predicts poorer prognosis in low T-stage oral cancer patients. Int J Mol Sci. 2014;15:2876–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Lin SH, Yeh CM, Hsieh MJ, Lin YM, Chen MW, Chen CJ, Lin CY, Hung HF, Yeh KT, Yang SF. Low cytoplasmic casein kinase 1 epsilon expression predicts poor prognosis in patients with hepatocellular carcinoma. Tumour Biol. 2016;37:3997–4005. [DOI] [PubMed] [Google Scholar]
- 41.Foldynová-Trantírková S, Sekyrová P, Tmejová K, Brumovská E, Bernatík O, Blankenfeldt W, Krejcí P, Kozubík A, Dolezal T, Trantírek L, Bryja V. Breast cancer-specific mutations in CK1epsilon inhibit Wnt/beta-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration. Breast Cancer Res. 2010;12:R30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Richter J, Rudeck S, Kretz AL, Kramer K, Just S, Henne-Bruns D, Hillenbrand A, Leithäuser F, Lemke J, Knippschild U. Decreased CK1δ expression predicts prolonged survival in colorectal cancer patients. Tumour Biol. 2016;37:8731–9. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data supporting the findings of this study are available within the paper and its Supplementary Information.