Background
Non-small cell lung cancer (NSCLC) is one of the most common malignant tumor with high mortality rate worldwide [
1]. Despite the current therapies including surgery, radiotherapy, chemotherapy and immunotherapy has notably improved patient survival, the overall survival of NSCLC patients remains unsatisfactory [
2]. Insufficient understanding of related molecular mechanisms limits the improvement in NSCLC patient prognosis. c-MYC, a key transcription factor by binding on enhancer box sequences (E-boxes), is frequently dysregulated and acts as a tumor promoting protein in numerous cancers [
3,
4]. c-MYC is essential for normal cell proliferation to occur, and its dysregulation could trigger cell malignant transformation and finally lead to carcinogenesis [
5]. The aberrant activation of c-MYC can immortalize cells, facilitate cell cycle progression and suppress differentiation [
6]. c-MYC is observed to be frequently amplified in NSCLC, promoting various kinds of tumor malignant behaviors such as proliferation, invasion, chemotherapy resistance and immune escape [
7]. Importantly, phosphorylation of c-MYC on Ser-62 is indispensable for its malignant behaviors [
8]. This phosphorylation site exerts opposing control of c-MYC degradation through the ubiquitin–proteasome pathway. In response to a growth-stimulatory signal, transcription of the c-MYC gene is increased and newly synthesized c-MYC protein is phosphorylated on the Ser-62 residue, which results in its stabilization [
9].
Bridging integrator 1 (BIN1), also known as Myc box-dependent-interacting protein 1, was identified as a tumor suppressor interacting with MYC box 1, a highly conversed region of the c-MYC N terminus [
10]. By binding with c-MYC, BIN1 could significantly inhibit proliferation and apoptosis ability while induce apoptosis of cancer cells [
11,
12]. Our previous study has revealed that BIN1 could inhibit programmed death ligand 1 (PD-L1) mediated immune suppression by neutralizing the c-MYC induced PD-L1 upregulation [
7]. A structural analysis showed a canonical interaction between the SH3 domain and the proline-rich region of c-MYC centered on two Xxx-Pro di-peptides P59-P60 and S62-P63. While the affinity of unphosphorylated Myc-55-68 for BIN1-SH3 was significant, and the peptide phosphorylated on Ser-62 was unable to bind BIN1-SH3 even at micromole concentrations [
13]. Thus, the presence of the factors inducing phosphorylation of c-MYC on Ser-62 could deactivate the tumor suppressing effect of BIN1.
Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase that functions as tumor promoter in the development and progression of multiple cancers by regulating cell proliferation, apoptosis, DNA repair and immune escape [
14‐
16]. Various researches have revealed that overexpression of CDK5 was associated with cancer progression and worse prognosis, and inhibition of CDK5 activity was determined to suppress growth in numerous cancers [
17,
18]. It is noteworthy that CDK5 is identified to directly phosphorylate c-MYC on Ser-62 [
19]. Taken above, aberrant activation of CDK5 might be able to limit the tumor suppressing effect of BIN1 by blocking the BIN1/c-MYC interaction, consequently, overcoming the overexpression of CDK5 might further improve the prognosis of NSCLC patients.
Here, we showed that the high expression of CDK5 neutralized the tumor suppressing effect of BIN1 by phosphorylating c-MYC at Ser-62 site. In addition, knockdown of CDK5 inhibited clone formation, proliferation, invasion and migration ability of NSCLC cells by restoring BIN1/c-MYC interaction. Meanwhile, CDK5 inhibitor Dinaciclib restored the function of BIN1 to suppress cell proliferation via interrupting phosphorylation of c-MYC at Ser-62 site. Furthermore, high expression of CDK5 along with low expression of BIN1 associated with worse prognosis of NSCLC patients, indicating a novel role of CDK5 in NSCLC.
Materials and methods
Patients and specimens
The specimens of NSCLC tissues and matched para-carcinoma tissues were collected from 153 patients who underwent radical operation at the Fourth Hospital of Hebei Medical University (Shijiazhuang, China) between Jan. 2015 and Feb. 2016. The median patient age at the time of surgery was 54 years (range: 25–72 years). None of the NSCLC patients received preoperative radiotherapy, chemotherapy and immunotherapy. The clinical stage and histological tumor type were determined according to the Uion of International Cancer Control (UICC) Classification of 2017 (8th edition). Patient’s clinical information, such as gender, age, TNM stage, invasion range, lymph node metastasis, was collected and stored in a database. All participant information was updated every 3 months by telephone follow-up. Complete follow-up was updated until death or Mar. 2019. Carcinoma tissue and para-carcinoma tissue specimens were collected and treated promptly after the surgery. The data of this research was approved for use by the ethic committee of the Hebei Medical University (approval number: 2018MEC065) and all informed consents were signed by patients.
Reagents and materials
Antibodies to CDK5 (EP715), c-MYC (ab32072), phosphorylated c-MYC at Ser-62 (ab185656), GAPDH (ab9485) were purchased from Abcam. (Cambridge, MA, US). Antibodies to CDK5/p35 (PR4026C) was purchased from Thermo Fisher (Carlsbad, CA, US). The CDK5-expressing lentivirus vector pCDH-CDK5 (pCDH-CMV-MCS-EF1-copGFP) and the control vector pCDH (pCDH-CMV-MCS-EF1-puro) and CDK5-siRNA were purchased from Gene Pharma Company (Shanghai, China). GoTaq qPCR Master Mix was purchased from Promega (Madison, WI, USA). Revert Aid First Strand cDNA Synthesis Kits was purchased from MBI Fermentas (Hanover, MD, USA). CDK5 inhibitor Dinaciclib (sc-364483A) was purchased from Santa Cruz Biotechnology (Dallas, TX).
RNA extraction and quantitative reverse-transcriptase PCR
Reverse-transcriptase PCR primers for human CDK5 mRNA on the basis of the sequence in NCBI (accession number U68485). CDK5: forward: 5′-GTCCATCGACATGTGGTCAG-3′; reverse: 5′-CTGGTCATCCACATCATTGC-3′, GAPDH forward: 5′-ACCACAGTCCATGCCATCACT-3′, reverse: 5′-TCCACCACCCTGTTGCTGTA-3′. The experiments were triplicated. The comparative threshold cycle (Ct) method was used to calculate the relative expression. The amount of target relative to a calibrator is given by 2−∆∆Ct [∆Ct = Ct (target gene) − Ct (GAPDH), ∆∆Ct = ∆Ct (carcinoma specimen) − ∆Ct (matched para-carcinoma specimen)].
Western blotting
Cells were lysed in lysis buffer, and protein concentrations were measured with the BCA protein assay kit (ThermoFisher). Proteins were separated by 10% SDS-PAGE and transferred electrophoreticly onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated in PBS containing 5% bovine serum albumin for 2 h at room temperature, followed by overnight incubation at 4 °C with different dilutions of the primary antibodies, including antibodies to CDK5, c-MYC, p-c-MYC (Ser-62). The membranes were developed with the Odyssey infrared imaging system according to the manufacturer’s instructions. The levels of protein in each sample were normalized relative to those of GAPDH. Each experiment contained triplicate wells of each sample, and all experiments were repeated at least three times.
Expression plasmid construction and transient transfections
A eukaryotic expression plasmid of the human CDK5 gene was constructed using a pCDH vector (Invitrogen, China). We constructed CDK5 cDNA expression vector on the basis of the sequence in NCBI. The empty vector was used as negative control. PC9 cells were cultured in six-well plates until they reached 80–90% confluence, and then transient transfections were performed using Lipofectamine 2000 (Invitrogen, China) according to the manufacturer instructions. At 48 h after transfection, gene expression was confirmed via immunoblotting analysis and qRT-PCR.
Knockdown of CDK5 with siRNA
To further investigate the mechanisms of immunomodulation by CDK5, the CDK5 gene was silenced with siRNA. The siRNA sequences were as follows: CDK5-siRNA-1: 5′-GCGUCCAGAAUUUCAACAATT-3′; CDK5-siRNA-2: 5′-CCACUACGAGUCCCUUCAATT-3′; CDK5-siRNA-3: 5′-GCGUAGGUUUCUACGUCAATT-3′; negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′. The siRNAs were synthesized by GenePharma Company (Shanghai, China). A total of 400 pmol siRNA was transfected into 4 × 105 H460 cells using Lipofectamine RNAi MAX reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s protocol.
Co-immunoprecipitation
In order to obtain total protein, transfected cells were harvested and lysed using lysis buffer containing protease inhibitors. Co-immunoprecipitation (Co-IP) assay was performed using a Co-IP kit (Pierce, Rockford, USA) according to the manufacturer’s instructions. Briefly, 3 mg of total protein for each treatment was used in this study, pre-cleared with Sepharose resins. The supernatants were divided equally into 2 tubes and incubated into columns containing 1 µg immobilized anti-c-MYC or anti-BIN1 antibody (Abcam). The immunocomplexes were covalently associated with Sepharose resins. The resins were eluted 5 times and the co-immunoprecipitate were separated on an SDS-PAGE gel before transferring to a PVDF membrane for analysis with anti-c-MYC or anti-BIN1 antibodies.
Cell proliferation assay
The proliferation of cells was measured by cell-counting kit-8 (CCK-8) assay according to the manufacturer’s protocol. Briefly, approximately 2 × 103 cells were plated into 96-well plates. When cells adhered, 10 μl of CCK8 (Solarbio, Beijing, China) was added to each well and incubated for 2 h in a humidified incubator containing 5% CO2 at 37 °C. The absorbance of each well was detected at a wavelength of 450 nm. Proliferation rates were determined at 0, 24, 48, 72, 96 h after transfection. Experiments were performed in triplicate.
Wound-healing experiments
5 × 105 H460 and PC9 cells were seeded in 24-well plates. After scraping the cell monolayer with a sterile micropipette tip, the wells were washed with serum-free medium in triplicate. The first image of each scratch was acquired at time zero. After 24 h, each scratch was examined and captured at the same location. Then, the healed area was measured for calculating the index of cell migration.
Transwell invasion assay
Tumor cell migration assay was performed in a 24-well transwell chamber (Corning, NY, USA), which contained an 8 μm pore size polycarbonate membrane filter and was precoated with 100 μg Matrigel for invasion assay (Becton–Dickinson, Bedford, USA). Briefly, the cells were seeded in the upper chambers and incubated in 500 μl RPMI 1640 medium without FBS, while 500 μl medium with 10% FBS was placed in the lower chambers. The plates were incubated for 24 h in a 5% CO2 humidified incubator at 37 °C. Cells on the upper side of the filters were removed by cotton-tipped swabs, and the filters were washed with PBS. Then the cells on the lower side were fixed in 4% formaldehyde and stained with 1% crystal violet in PBS for 5 min at room temperature. The cells on the lower side of the filters were defined as migration cells and counted at 200× magnification in 5 random fields of each filter.
Treatment with Dinaciclib
NSCLC cell lines H460 and PC9 were grown in RPMI supplemented with 10% FBS. A stock solution of Dinaciclib (1 nM) or vehicle control-DMSO was diluted in fresh tumor medium and added to samples to achieve a final concentration of 10 μM or 20 μM. After 4 days of treatment for 24 h and then harvested for cell proliferation analysis.
In vivo tumor growth assay
BALB/c nude mice were used for tumor growth assay in vivo. Care was performed according to the National Research Council Guide for the Care and Use of Laboratory Animals, and was approved by the Institutional Animal Care and Use Committee (IACUC) of Hebei Medical University, Shijiazhuang, China. Tumor cells were harvested with trypsin solution and resuspended in PBS. Cells (1 × 106 cells/mouse) in 0.1 ml were injected subcutaneously into BALB/c nude mice. The mice were divided randomly to 2 groups: Dinaciclib treated group and control group. All mice were sacrificed by cervical dislocation on the day 32.
Immunohistochemistry assay
Immunohistochemistry (IHC) analysis was performed as we previously described [
20]. The rabbit polyclonal antibody against CDK5 was used for detection. For evaluating expression of CDK5 in NSCLC tissues, the staining was visualized and classified based on the percentage of positive cells and the intensity of staining according to the 0–4 semi-quantitative system described in our previous study [
20]. The total scores were determined by multiplying the percentage score and intensity score and graded as low for score of 0–4 and high for score of 5–12. Each section was scored independently by two pathologists and a third pathologist determined the final score if there was any inconsistency.
Immunofluorescence
PC9 and H460 cells harvested and incubated with rabbit to human BIN1, mouse to human CDK5 mAb at 4 °C overnight. The cells were then stained by FITC conjugated goat anti-mouse and PE conjugated goat anti-rabbit antibody, followed by DAPI staining of the nucleus. The fluorescence was observed and analyzed with a fluorescence microscope at high magnification (200×).
Statistical analysis
Statistical analysis was performed by using SPSS statistics software, version 25.0 (SPSS, Chicago, IL, USA). Spearman rank correlation was used to analyze the association of CDK5 and BIN1 expression in NSCLC specimens. Survival analysis was carried out using the log-rank test in association with Kaplan–Meier analysis and Cox proportional hazards model analysis. A P value < 0.05 was considered to be statistically significant, and all P values were two-tailed.
Discussion
NSCLC is the one of the most common malignant tumor with unsatisfactory prognosis [
24]. Aberrant activation of oncogenes is one of most important cancer hallmarks, which is closely related to worse prognosis [
25‐
27]. c-MYC is known to be a frequent crucial oncoprotein in NSCLC, which is associated with poor prognosis and recurrence [
28]. Therefore, interrupting of c-MYC regulatory units might be a potential alternative to indirectly inactivate c-MYC in tumors. BIN1 has been identified as a crucial tumor suppressor, which is important in numerous biological behaviors such as proliferation, apoptosis, DNA repair and immune escape [
29]. Elliott et al. demonstrated that BIN1 could reverse c-MYC-mediated malignant transformation via directly interacting the N-terminus of c-MYC [
11,
12]. However, aberrant phosphorylation of c-MYC at Ser-62 site could block the BIN1/c-MYC interaction [
13]. In addition, CDK5 overexpression can directly phosphorylate c-MYC on Ser-62 [
19]. Taken above, demonstrating whether CDK5 is involved in blocking BIN1/c-MYC interaction is beneficial for comprehending the biological function of CDK5 to establish novel therapeutic strategies in treating NSCLC.
In the present study, we evaluated the effect of CDK5 on BIN1/c-MYC interaction in NSCLC. CDK5, an aberrantly overexpressed protein in multiple cancers, could significantly promote proliferation, invasion and migration abilities of tumor cells [
14,
15]. In hepatocellular carcinoma (HCC), CDK5 overexpression could promote proliferation of HCC cells to exert an oncogenic activity [
30]. In prostate cancer, CDK5 increased cell growth ability in an androgen receptor (AR)-independent manner [
31]. As for NSCLC, Wei et al. reported that CDK5 was significantly correlated with poor prognosis [
32]. However, the concrete effects of CDK5 and related mechanisms in NSCLC still remain unclear. CDK5 phosphorylates a diverse list of substrates, implicating that it can regulate various cellular processes including the function of c-MYC, which is a crucial transcription factor in cancer [
33,
34]. Aiming at further realizing the effect of CDK5 on BIN1/c-MYC interaction, we then detected its expression in NSCLC cell lines, and found that H460, H1299, H1975, and A549 cells showed high CDK5 expression, while PC9 and H1972 cells showed low CDK5 expression. Meanwhile, PC9, H460 and H1972 cells showed high BIN1 expression while H1299, H1975 and A549 cells showed low BIN1 expression. Thus, we chose PC9 and H460 cells for further studying the function of CDK5.
Knockdown of CDK5 expression in H460 cells significantly promoted the interaction of BIN1 and c-MYC. We found that knockdown of CDK5 could inhibit cell growth, invasion, migration and EMT ability of H460 cells. Meanwhile, we transfected CDK5 into PC9 cell which highly expressed BIN1, and the results showed that the growth, invasion, migration and EMT ability could be significantly enhanced by CDK5 overexpression. Furthermore, with Co-IP experiment, we found that CDK5 was correlated with the anergy of BIN1/c-MYC interaction, suggesting that CDK5 could neutralize the tumor-suppressing effect of BIN1 via inducing phosphorylation of c-MYC at Ser-62 site.
Due to the oncogenic function of CDK5, Dinaciclib, an effective inhibitor of CDK5, exerted potent tumor-suppressing effect in relapsed multiple myeloma and hepatocellular carcinoma [
35,
36]. However, the function of Dinaciclib in NSCLC remains unclear. In present study, Dinaciclib could inhibit the growth, migration and invasion ability of NSCLC cells. We subsequently investigated the effect of Dinaciclib in vivo, and we found that mice injected with Dinaciclib-treated group developed significantly smaller tumors than mice injected with control group. These results provided some supporting evidence for its clinical implication in NSCLC.
Conclusion
Taken together, to our best knowledge, despite of the studies on the carcinogenesis role of CDK5, this is the first study to analyze its effect on BIN1/c-MYC interaction. Knockdown of CDK5 significantly inhibited the phosphorylation of c-MYC at Ser-62 site to facilitate the interaction of BIN1 and c-MYC, and consequently suppressed the invasion and migration ability of NSCLC cells. High expression of CDK5 along with low expression of BIN1 could predict poor postoperative prognosis for NSCLC patients. Taken above, CDK5 might be used as a potential biomarker in predicting prognosis of NSCLC patients, and regarded as a novel target for anti-cancer therapies.
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