FGFR/RACK1 interacts with MDM2, promotes P53 degradation, and inhibits cell senescence in lung squamous cell carcinoma

Objective: FGFR is considered an important driver gene of lung squamous cell carcinoma (LSCC). Thus, identification of the biological events downstream of FGFR is important for the treatment of this malignancy. Our previous study has shown that the FGFR/RACK1 complex interacts with PKM2 and consequently promotes glycolysis in LSCC cells. However, the biological functions of the FGFR/RACK1 complex remain poorly understood. Methods: Anchorage-independent assays and in vivo tumorigenesis assays were performed to evaluate cancer cell malignancy. Distant seeding assays were performed to evaluate cancer cell metastasis. β-gal staining was used to examine cell senescence, and immunoprecipitation assays were performed to examine the interactions among FGFR, RACK1, and MDM2. Results: FGFR/RACK1 was found to regulate the senescence of LSCC cells. Treatment with PD166866, an inhibitor of FGFR, or knockdown of RACK1 induced senescence in LSCC cells (P < 0.01). A molecular mechanistic study showed that FGFR/RACK1/MDM2 form a complex that promotes the degradation of p53 and thus inhibits cell senescence. PD166866 and RG7112, an MDM2/p53 inhibitor, cooperatively inhibited the colony formation and distal seeding of LSCC cells (P < 0.01), and upregulated the expression of p53 and p21. Conclusions: Together, our findings revealed the regulatory roles and mechanisms of FGFR/RACK1 in cell senescence. This understanding should be important in the treatment of LSCC.


Introduction
Lung cancer is one of the most malignant tumors wordwide 1,2 and is the malignancy with the highest morbidity and mortality in China 3 . The FGF/FGFR tyrosine kinase signaling pathway, which regulates various biological events, including embryonic development and tissue repair 4 , plays an important role during tumorigenesis and can lead to chemotherapeutic resistance if it is overactivated 5 .
The FGFR family includes 4 members: FGFR1, FGFR2, FGFR 3, and FGFR4 6 . Each receptor contains an extracellular domain, a transmembrane domain, and an intracellular domain. The FGF ligands comprise 23 members, whose binding to FGFR mediates downstream signal transduction events 7 . FGFR abnormalities are very common in squamous nonsmall cell lung cancer [8][9][10] . Amplification of the FGFR gene can be detected in approximately 20% of lung squamous cell carcinomas 11 . Therefore, elucidation of the biological functions of FGFR and the downstream biological events is urgently needed for the treatment of lung squamous cell carcinomas. Several inhibitors targeting FGFR1 (such as PD166866) have entered clinical trials 12 . RACK1 (the receptor for activated PKC kinase) is a 7 WD40repeat-containing adaptor protein that acts as an integrated hub for different signaling pathways in functions including cell growth, migration, and tumorigenesis 13 . Recent studies have reported that RACK1 has very important roles in in lung cancer [14][15][16] , through promoting the cell growth and migration of lung cancer cells by activating Hedgehog signaling, although the exact mechanism requires further exploration 15 .
Extensive evidence indicates that senescence is a mechanism that suppresses tumourigenesis 17 . Senescence is activated by stress 18 . Cell cycle arrest may occur in the division and proliferation of cells through senescence, and the cellular morphology also changes significantly. Senescent cells are positive for β-galactosidase staining, and cell cycle arrest-related signaling pathways, such as P53-P21 and P16-Rb, become activated 19 . Chemotherapy, radiotherapy, and hypoxia are inducers of cellular senescence 20 .
We previously found that the adaptor protein RACK1 interacts with FGFR and consequently promotes the phosphorylation of PKM2 by FGFR 16 , thereby increasing tumor cell glycolysis. However, whether RACK1 mediates other downstream biological functions of FGFR remains unclear. In this article, we investigated the effect of the FGFR/RACK1 complex on cellular senescence in lung squamous cell carcinomas and determined the underlying mechanism.

Cell culture
The lung cancer cell lines NCI-H226 and H1703 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were cultured with DMEM containing 10% serum and penicillin-streptomycin solution. Cells were placed in an incubator at 37° and 5% CO 2 . Cell transfection was mainly performed with Lipofectamine 2000 and was conducted according to the manufacturer's instructions. Fortyeight hours after cell transfection, 1 μg/mL of puromycin was used for screening. Seven days later, the surviving cells were mixed, and gene expression was detected with Western blot analyses.

Western blot analysis
After the cells were washed twice with PBS, they were lysed on ice for 15 min with RIPA buffer (1% NP-40, 0.1% SDS, 0.5% deoxycholate, 50 mM Tris (pH 7.4), and protease inhibitor cocktail) and collected with a scraper. After centrifugation at 12,000 rpm at 4 °C for 15 min, the protein concentration was determined with Bradford reagent (Sigma). The protein samples were mixed with 6× loading buffer and boiled for 5 min, and electrophoresis was then performed. The proteins were subsequently transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated with TBST containing 5% skimmed milk (25 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody. After the samples were washed 3 times with TBST, horseradish peroxidase-conjugated secondary antibody was added. The cells were then incubated for 1 h at room temperature, washed 3 times with TBST, and then developed with an enhanced chemiluminescence system (Pierce).

Soft agar assays
Soft agar assays were performed in 12-well plates. The soft agar contained an upper and lower layer. The lower layer of agar, which was used to embed the 12-well plate, had an agar concentration of 0.5% and a serum concentration of 10%. The upper layer had an agar concentration of 0.35% and a serum concentration of 10% for cell resuspension. An inhibitor (PD166866 or RG7112) was mixed in the upper layer. After the agar was heated to 37 °C, the agar in the lower layer was poured. A total of 2,000 cells were added to the upper layer and seeded on the lower layer after being mixed well. The cells were cultured at 37 °C for 14 days and then photographed and counted.

Tumorigenesis
Four-week-old male nude mice were divided into 2 groups with 4 mice per group. One group was injected with cancer cells with the shRNA control (1 × 10 6 cells/point), and the other group was injected with cancer cells with shRACK1 (1 × 10 6 cells/point). The mice were euthanized 5 weeks later, the tumor tissues were removed and weighed, and RACK1 expression was detected through immunohistochemistry. The experiments and the related analysis were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (approval No. 2019-099). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Immunohistochemistry
Paraffin sections were dewaxed and hydrated with xylene and gradient ethanol, after which antigen repair was performed with sodium citrate solution. The endogenous peroxidase activity was activated with 0.3% hydrogen peroxide, and the sections were blocked with 5% BSA. The sections were then incubated with primary antibody overnight at 4 °C with the ZSGB-BIO immunohistochemistry kits. On the following day, the sections was washed 3 times with PBST, incubated with secondary antibody for 2 h at room temperature and then developed for 2 min with DAB. After development, staining was performed with hematoxylin to visualize the nuclei. The sections were treated with 1% hydrochloric acid/ethanol solution for 6 s and washed.

GST pull-down
Cells were collected, lysed, and centrifuged at 4 °C and 12,000 rpm for 20 min to obtain the supernatant, which was then incubated with 10 μg of GST and GST-RACK1 fusion protein at 4 °C overnight. Sepharose 4B GST gel beads were then added and incubated for an additional 4 h. The gel beads were washed 3 times with PBST buffer for 5 min each time; 30 μL of loading buffer was then added and boiled for 5 min at 100 °C. The supernatant was used for Western blot analysis.

Co-immunoprecipitation
NCI-H226 and H1703 cells were collected, lysed, and centrifuged at 4 °C and 12,000 rpm for 20 min to obtain the supernatant, which was then mixed with primary antibody and incubated overnight at 4 °C. Protein A beads were then added and incubated for an additional 4 h. The beads were washed 3 times with PBST buffer for 5 min each time; 30 μL of loading buffer was then added and boiled for 5 min at 100 °C. The supernatant was used for Western blot analysis.

Senescence analysis
Cultured cells were plated in 6-well dishes 1 day before staining. Cells were fixed and stained according to the manufacturer's protocol (Senescence β-Galactosidase Staining Kit, Cell Signaling Technology #9860). Briefly, cells were fixed in a 2% formaldehyde and 0.2% glutaraldehyde solution for 15 min, washed in PBS, and stained overnight at 37 °C in an X-gal staining solution. Stained cells were imaged with inverted microscopy. Ten random images were collected, and cells were counted.

Ubiquitination assays
The lysate was first prepared by suspension of cells in IP lysis buffer for 30 min at 4 °C. Immunoprecipitation was performed with 1 mg of protein and 1 μg of primary antibody in 500 μL of IP lysis buffer at 4 °C overnight. The reaction mixtures were incubated with Protein A and Protein G Magnetic Beads (50 μL) at 4 °C for 1 h on a rotator. The immunoprecipitated complexes were washed 3 times with IP wash buffer. The washed beads were incubated with 5× reducing loading buffer and boiled at 100 °C for 5 min. The proteins released from components of the complexes were examined by SDS-polyacrylamide gel electrophoresis and Western blot with anti-ubiquitin antibodies (Cell Signaling Technology, #3933).

Distal seeding assays
Six-week-old male nude mice were injected with 1 × 10 6 cells/ animal. Two days later, the intervention was performed with inhibitors. Eight weeks later, the mice were sacrificed, lung tissue samples were collected, and the number of lung metastases was detected through HE staining. All animal experiments were approved by the GMU Biomedical Ethics Committee.

The FGFR inhibitor PD166866 induces senescence in lung squamous cell carcinoma cells
To reveal the function of FGFR in lung squamous cell carcinomas, we first treated the lung squamous cell carcinoma cell lines NCI-H226 and H1703 with the FGFR inhibitor PD166866 and then detected the effect of PD166866 treatment on the phenotypes of the NCI-H226 and H1703 cells. The PD166866 treatment significantly inhibited the FGFR tyrosine phosphorylation level (an important marker of FGFR activation) (Figure 1A). In addition, PD166866 treatment inhibited the colony formation of the NCI-H226 and H1703 cells on soft agar ( Figure 1B). Further studies revealed that PD166866 treatment promoted the senescence of the NCI-H226 and H1703 cells (Figure 1C-1D). Consistently, knockdown of FGFR1 induced cell senescence in NCI-H226 cells (Figure 1E). These observations indicated that the inhibition of FGFR activity caused the senescence of lung squamous cell carcinoma.

RACK1 knockdown induces senescence in lung squamous cell carcinoma cells
In our previous studies, we have found that FGFR interacts with RACK1 16 . Therefore, we studied the functions of RACK1 expression in FGFR-mediated cellular senescence. First, we knocked down the expression of RACK1 in NCI-H226 and H1703 cells (Figure 2A), and detected RACK1 function with soft agar assays ( Figure 2B). RACK1 knockdown inhibited the formation of colonies of NCI-H226 and H1703 cells in soft agar. In agreement with the results of PD166866 treatment, RACK1 knockdown promoted the senescence of NCI-H226 and H1703 cells (Figure 2C). In the in vivo experiments, RACK1 knockdown inhibited the in vivo tumorigenesis of NCI-H226 and H1703 cells, as shown by the tumor size and tumor weight (Figure 2D). Moreover, lower intensity Ki67 staining was observed in the tumors derived from cells with knockdown of RACK1 ( Figure 2E).

FGFR forms a complex with RACK1 and MDM2 in lung squamous cell carcinoma
The MDM2-P53 signaling pathway has an important role in cellular senescence. We detected the interaction between FGFR/ were treated with PD166866, and anchorage-independent growth was detected with soft agar colony assays. (C, D) NCI-H226 and H1703 cells were treated with PD166866, and β-gal staining was used to detect cell senescence. Statistical analyses were performed. (E) FGFR 1 was knocked down in NCI-H226 cells, and β-gal staining was used to detect cell senescence. Statistical analyses were performed. **P < 0.01.
RACK1 and important components of the MDM2-P53 signaling pathway. In GST pull-down experiments, the fusion proteins GST-RACK1 and MDM2 formed a complex (Figure 3A).

Inhibition of P53 protein levels by the FGFR/ RACK1 complex
We investigated the effect of the FGFR/RACK1 complex on P53 protein levels. In NCI-H226 cells, the upregulation of RACK1 expression promoted the degradation of P53 through the ubiquitination pathway ( Figure 4A). In NCI-H226 and H1703 cells, the protein levels of P53 and P21 were downregulated by overexpression of RACK1 (Figure 4B). NCI-H226 and H1703 cells treated with PD166866 showed upregulated P53 and P21 protein levels ( Figure 4C). In addition, PD166866 treatment inhibited the interaction between RACK1 and MDM2 ( Figure 4D). These results indicated that the FGFR/RACK1/ MDM2 complex inhibits P53 protein levels by promoting P53 ubiquitination.

Treatment with FGFR and P53-MDM2 inhibitors suppresses lung cancer progression
We investigated the therapeutic effects of combining FGFR and P53-MDM2 inhibitors in lung cancer treatment. The FGFR inhibitor PD166866 and the P53-MDM2 inhibitor RG7112 cooperatively inhibited the colony formation of NCI-H226 and H1703 cells ( Figure 5A). Consistently, PD166866 and RG7112 jointly induced the expression of P53 and P21 proteins ( Figure 5B). In model mice, PD166866 and RG7112 together inhibited the distal implantation ability of NCI-H226 cells ( Figure 5C).

Discussion
FGFR amplification is very common in lung squamous cell carcinoma. FGFR is considered an important driver gene in lung squamous cell carcinoma 8,9,21 . Moreover, FGFR amplification is also found in cholangiocarcinoma 22 . Currently, the treatment strategies for lung squamous cell carcinoma and are limited. Therefore, elucidation of the biological events mediated by the FGFR pathway is important. In this study, we found that FGFR inhibitors induced cell senescence by increasing P53 protein levels, thereby inhibiting the formation of lung carcinoma cell colonies in soft agar and their distal implantation in mice. These findings expand understanding of the downstream biological functions of FGFR.
Notably, we found that the RACK1/MDM2 complex regulates the stability of the P53 protein. In previous studies, we have found that FGFR and RACK1 forms a complex that promotes the phosphorylation of PKM2 16 . In this study, the FGFR/RACK1 complex was found to interact with MDM2, thereby enhancing the interaction between MDM2 and P53, and inhibiting the P53 protein levels. These observations suggested that the complex of FGFR/RACK1 exerts multiple functions through interacting with different effectors. The FGFR inhibitor PD166866 inhibited the phosphorylation of FGFR1, thus decreasing the interaction between RACK1 and MDM2 and weakening the interactions among RACK1, MDM2 and P53. The accumulation of P53 protein activated P21 expression and induced cellular senescence (Figure 6). These experimental results indicated that the FGFR tyrosine kinase functions as a regulator of senescence.
Multiple signaling pathways are mediated by FGFR 23 . Another interesting result in this study was the therapeutic   effect resulting from combining an FGFR inhibitor with an MDM2-P53 inhibitor. Together, these inhibitors synergistically induced cellular senescence and inhibited the distal seeding of lung squamous cell carcinoma cells. Our results suggest that this combination of inhibitors may be an effective method for treating lung cancer with FGFR amplification in clinical trials.

Conclusions
In this study, we uncovered the effects and related mechanisms underlying the induction of senescence in lung squamous cell carcinoma by FGFR inhibitors, thus potentially providing a new strategy for the treatment of lung squamous cell carcinoma.