CKS1B promotes cell proliferation and invasion by activating STAT3/PD‐L1 and phosphorylation of Akt signaling in papillary thyroid carcinoma

Abstract Objective To investigate role of GKS1B and its relationship between STAT3/PD‐L1 and p‐Akt in papillary thyroid carcinoma (PTC). Methods Expression of GKS1B and PD‐L1 was determined in PTC cell lines. GKS1B was overexpressed or knocked down by transfection with overexpression plasmids or si‐CKS1B. STAT3 inhibitor WP1066 was used to suppress STAT3, and PD‐L1 inhibitor Pembrolizumab was used to block PD‐L1. Cell viability and invasion were evaluated by MTT and transwell assay, respectively. The expression of STAT3, p‐STAT3, Akt, and p‐Akt was measured using Western blotting. Results Both protein levels and mRNA levels of CKS1B and PD‐L1 were remarkably up‐regulated in PTC cell lines. Knockdown of CKS1B significantly inhibited cell viability and invasion of PTC cells and suppressed STAT3/PD‐L1 signaling and Akt phosphorylation, while overexpression of CKS1B led to opposite results. Inhibition of STAT3 or PD‐L1 reversed the effects of overexpressed CKS1B on PTC cells. Conclusion The overexpression of CSK1B could promote cell viability and invasion of PTC cells through activation of STAT3/PD‐L1 signaling and Akt phosphorylation.

CKS1B could regulate STAT3 and further influence cancer development of lung cancer and myeloma. 12,13 Besides, other studies showed STAT3 is an upstream protein of programmed death ligand 1 (PD-L1), also known as CD274. 12,14 And in a recent research, Lubin et al demonstrated PD-L1 was elevated in PTC and was related to patients' prognosis. 15 However, up to now, no study focused on the relationship between CSK1B and STAT3/PD-L1 signaling in PTC.
In the present study, we demonstrated that the overexpression of CSK1B could promote cell viability and invasion of PTC cells through activation of STAT3/PD-L1 signaling and Akt phosphorylation. This study might provide deeper understanding of CSK1B/ STAT3/PD-L1 axis in PTC development.

| MTT assay
MTT assay was used for evaluation of cell viability. Briefly, cells (3 × 10 4 ) seeded in 96-well plates were cultured for 48 hours under 37°C and 5% CO 2 . After addition of 10 μl MTT solution (5 mg/mL), cells were cultured for another 4 hours. Then, 150 μL DMSO was added after removing MTT, and the value of optical density (OD) was evaluated 490 nm.

| Transwell assay
Transwell assay was used for determination of cell invasion. For invasion, 1 × 10 5 cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). Cells were then cultured for 24 hours in serum-free media. Then, cells were stained with 0.1% crystal violet. The invaded or migrated cells were counted and photographed using light microscope (Zeiss, Germany).
The invaded or migrated cell rates were calculated as the invaded or migrated cell number in the tested group/blank control group.

| Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The expression levels of CKS1B and PD-L1 were evaluated using qRT-PCR. Total RNA was extracted using the Trizol reagent (Tiangen

| Western blotting
The protein levels of CKS1B, STAT3, p-STAT3, PD-L1, Akt, and p-Akt were determined using Western blotting. The extracted proteins were loaded on 10% SDS-PAGE, transferred to PVDF membranes, F I G U R E 1 CKS1B and PD-L1 were highly expressed in PTC cell lines. A, mRNA expression of CKS1B and PD-L1 in PTC cell lines was determined by RT-qPCR. B, Protein levels of CKS1B and PD-L1 in PTC cell lines were determined by Western blotting. *P < .05, ***P < .001 and blocked using 5% non-fat milk. Samples were then incubated with the following specific primary antibodies (Abcam) at 4°C overnight. After being incubated with corresponding secondary antibody at 37˚C for 45 min, protein bands were analyzed with the Pierce ECL Western Blotting Substrate (Pierce, Shanghai, China). GAPDH was served as an internal control.

| Statistical analysis
The measurement data were expressed by mean ± SD. Comparisons were made using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. It was considered to be statistically significant when P-value was less than 0.05. All calculations were made using SPSS 22.0.

| Knockdown of CKS1B inhibited cell viability and invasion of PTC cells
To further investigate role of CKS1B in PTC development, CKS1B was overexpressed in BCPAP cells by transfection with OE-CKS1B plasmids and was knocked down in IHH-4 cells by transfection with si-CKS1B. After successful transfection (Figure 2A), MTT assay showed cell viability was dramatically suppressed by transfection with si-CKS1B and was remarkably enhanced by transfection with OE-CKS1B ( Figure 2B). Meanwhile, knockdown of CKS1B by si-CKS1B significantly reduced the invaded and migrated cell ratios than the NC cells, and overexpression of CKS1B by transfecting OE-CKS1B led to opposite results ( Figure 2C). All these results indicated that CKS1B promoted cell proliferation and invasion of PTC cells and inhibition of CKS1B suppressed these effects.

| Inhibition of STAT3 or PD-L1 reversed the effects of overexpressed CKS1B on PTC cells
At last, we inhibited STAT3 signaling by treatment of WP1066 and inhibited PD-L1 by treatment of Pembrolizumab. As shown in Figure 4A-B, when CKS1B was overexpressed, cell viability was significantly promoted, as well as cell invasion of BCPAP cells.
However, either the inhibition of STAT3 or PD-L1 remarkably

| D ISCUSS I ON
Although PTC is a relative less malignant tumor compared with other thyroid carcinoma such as medullary thyroid carcinoma and anaplastic thyroid carcinoma, the prognosis for recurrence patients and patients with long distant metastasis is still poor. 16,17 CKS1B is reported as a cancer promoter; however, its role in PTC is rarely noticed. In this research, we demonstrated for the first time that CKS1B was elevated in PTC cell lines and could promote PTC cell proliferation and invasion through regulation of STAT3/PD-L1 signaling and Akt phosphorylation.
Role of CKS1B in cancer development has been reported in several studies. It was reported that down-regulation of miR-1258 promoted carcinogenesis and progression of hepatocellular carcinoma by activation of CKS1B. 18 Besides, overexpression of CKS1B contributed to poor prognosis of nasopharyngeal carcinoma. 7 It was also found that CKS1B could induce chemoresistance in lung cancer which could be reversed by 3-O-(Z)-coumaroyloleanolic acid. 19 In the present research, we also found that CKS1B was up-regulated in PTC cells, which was consistent with the above previous researches.
The regulation effects of CKS1B on STAT3 signaling were also reported in many researches. Generally, CKS1B/STAT3 axis accounts for cancer development. In an early study, Zhan et al showed CKS1B contributed to myeloma cell viability by activation of JAK/STAT3 signaling. 20 CKS1B could also activate STAT3 signaling in multiple myeloma by promoting the degradation of p21. 21 Another study showed elevated CKS1B promoted myeloma cell drug resistance by activation of STAT3 signaling. 13 In the present study, we observed that CKS1B could also positively regulate STAT3/PD-L1 signaling in PTC cells, and the inhibition of CKS1B could suppress cancer development of PTC by regulating STAT3/PD-L1 signaling.
PD-L1 has been noticed in PTC development in recent years. It was found that PD-L1 was up-regulated in PTC patients and patients with higher PTC level had higher risk for recurrence. 22 Another study also showed GKS1B/STAT3 axis could enhance the expression of PD-L1 and further promote cancer development of lung cancer cells. 12 Besides, the positive interaction between PD-L1 and Akt has also been reported in several cancers such as lung cancer 23 and lymphoma. 24 In another research, Abdelhamed et al found Akt/STAT3 signaling could regulate PD-L1 expression in non-small cell lung cancer. 25 In our research, we also found that activation of STAT3 by CSK1B could both facilitate the expression of PD-L1 and the phosphorylation of Akt.
The present study also has some limitations. First, how CSK1B interacts with STAT3 and PD-L1 is still unclear, and the interaction between STAT3 and PD-L1 also needs to confirm. Secondly, whether other signaling pathways are involved in this process is also unknown. Thirdly, the role of Akt in GKS1B/STAT3/PD-L1 axis is not clearly defined. All these need more studies to illuminate.
In conclusion, we investigated the role of CSK1B and its relationship between STAT3/PD-L1 signaling and Akt phosphorylation in PTC. We found the inhibition of CSK1B could suppress cell viability and invasion of PTC cells through inhibition of STAT3/PD-L1 signaling and Akt phosphorylation. This study might provide more insights into CSK1B/STAT3/PD-L1 in PTC development.