CHD4 mediates proliferation and migration of non-small cell lung cancer via the RhoA/ROCK pathway by regulating PHF5A

Between January 2005 and February 2009, a total of 242 patients with histologically confirmed NSCLC were consecutively treated for NSCLC at Zhongshan Hospital of Fudan University. Specimens of both tumor and adjacent non-tumor tissue were collected at the operation. Pathologists were helping to ensure correct sampling of tissues from the tumor, adjacent non-tumor lung tissues(3–5 cm from the tumor), without adversely affecting the participant. The pathologists classified the samples as tumor and corresponding adjacent non-tumor lung tissues. The TNM status was determined according to the 8th edition staging system for NSCLC [12]. Patients with R1/R2 resection, survival < 30 days after surgery, who died due to other causes or were lost to follow-up were excluded from the study. In total, 96 patients were excluded from the analysis, including 4 patients who had previously received radiotherapy and/or chemotherapy, 51 patients with poor quality and/or quantity of tissue samples, 19 patients with incomplete clinical data, and 22 patients who died of other causes. Finally, a total of 146 patients who underwent curative surgical resection were included in this study. Of these, 73 patients were alive at the end of the follow-up, and 73 patients died from lung cancer. The clinicopathological data for each patient, including sex, age, tumor stage, nodal status, TNM stage, histological grade and overall survival, were obtained retrospectively from the clinical records and pathological reports. The pathologists who performed the immunohistochemical assessment of CHD4 were blinded to the patients’ histopathologic and follow-up data.

The survival time was defined as the duration from the date of diagnosis to the date of death or the end of the follow-up.

The following antibodies were used in this study: CHD4 (ab72418, Abcam, polyclonal, dilution: 1:1000); PHF5A (ab103075; Abcam, polyclonal, dilution: 1:1000); myosin (MY-21, M4401, Sigma, monoclonal, dilution: 1:200); phospho–myosin (sc-12,896, Santa Cruz, monoclonal, dilution:1:200); ROCK (#4035S, Cell Signaling Technology, monoclonal, dilution:1:1000); RhoA (#2117, Cell Signaling Technology, monoclonal, dilution:1:1000). E-cadherin (#3195, Cell signaling Technology, monoclonal, dilution:1:1000); ERK (#4348S,Cell Signaling Technology, monoclonal, dilution:1:1000) and p-ERK (#4370, Cell Signaling Technology, monoclonal, dilution:1:2000).

The protein concentration was measured by the Bradford assay. Cell lysates were separated by SDS-PAGE and transferred to poly vinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in TBST and incubated with specific primary antibodies. The band intensities were measured by using SuperSignal West Pico chemi-luminescent substrate (Thermo Scientific) followed by exposure to X-ray film. After that, quantification was performed using the Image J software.

Paraffin-embedded tissue blocks were sectioned (3 μm) for immunohistochemical staining. Sections were immersed in xylene, alcohol and washed with PBS for three times after each immersion. After protein denature, using microwave and non-specific biding blocking with normal goat serum for 20 min at room tempreture. The sections were then incubated with rabbit polyclonal antibody against CHD4 (ab72418, Abcam) with 1:100 dilution for experimental slides overnight at 4 °C. The secondary antibody was Bond Polymer Refine Detection (DS9800). The sections were incubated with 3,3′-Diaminobenzidine (DAB) and hematoxylin staining.

CHD4 expression was observed in the cell cytoplasm and nucleus. Staining was assessed in five high-powered fields, and three sections per specimen were assessed. The percentage of the area that was positively stained was categorized into the following four groups: < 25% of the tumor cells stained, 0; 25–50% stained, 1; 50–75% stained, 2; and > 75% stained, 3. The staining score was categorized into four groups as follows: negative, 0; weak, 1; moderate, 2; and intense, 3. The labeling score was determined by multiplying the stained area score by the intensity score, with potential scores of 0, 1, 2, 3, 4, 6 and 9. Then, the labeling score was categorized into two groups: weak/negative staining (score < 4) and strong staining (score ≥ 4) [13]. Among the three tissue sections, the highest labeling score was entered for the statistical analyses. The pathologists were blinded to the patients’ follow-up data.

The individual small interfering RNAs (siRNAs) were obtained from Shenggong, Inc., Shanghai, China. The annotations and sequences of the siRNAs were as follows (sense strands):,5′-CGGGUAUUGAAUGGUUACUTT-3′; and control siRNA, 5′-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT-3′. The siRNA transfections were performed with 100 nM siRNA duplexes using Lipofectamine RNAiMAX (Invitrogen, USA). The cells were transfected with siRNAs 24 h after plating. The samples were harvested 72 h after transfection initiation, unless stated otherwise.

Assays to measure tumor cell migration were performed in a modified Boyden chamber (Transwell, Corning Costar, MA, USA) containing a gelatin-coated polycarbonate membrane filter (8-μm pore size). Cells were seeded at a density of 40,000 cells per well, and the wells were washed with D-PBS after 24 h. The degree of tumor cell migration and was evaluated according to previous protocols [14]. Cell counting was performed following Coomassie blue staining, and the cells were subsequently visualized under a microscope (Leica, Inc., Solms, Germany).

Cells were washed with phosphate-buffered saline (PBS) and lysed with ice-cold NETN buffer containing protease inhibitor. The lysates were centrifuged to remove cell debris. The samples were incubated with antibody overnight at 4 °C and then the whole-cell extracts were precleared with pre-washed protein A/G-conjugated agarose beads for 2 h at 4 °C. Agarose beads were then washed with NETN buffer and immunoprecipitates were collected by boiling beads in 800 μ1*SDS sample buffer for 10 min. Finally, the supernatant was subjected to SDS-PAGE and subsequent western blotting analysis.

The cells were harvested and resuspended in 200 μl of propidium iodide (PI) buffer. The samples were incubated for 30 min at 37 °C before analysis. Cell cycle analysis was performed using a flow cytometer (FACSCalibur; BD Biosciences).

Real-time PCR was performed as described previously [15], with the following primer sequences: CHD4, (sense) 5′-CAAGAAGCCTAAACCCAAGAAA-3′ and (antisense) 5′-CCACATCTAAGTCATCATCCTCAC-3′; and PHF5A, (sense) 5′-GCTTGAGGAACTGACTGTGAAG-3′ and (antisense) 5′-AAACGGGAAATGCCTACAT-3′.

A total of 20 Four- to five-week-old male BALB/CA nude mice (purchased from the Shanghai Institute of Material Medicine, Chinese Academy of Science, Shanghai, China) were maintained under specific pathogen-free (SPF) conditions. CHD4-down-regulated A549 or A549-NC cells (5 × 10⁶ per mouse) were injected subcutaneously into the right lower flanks of the nude mice (n = 6 per group). Five weeks later, the mice were sacrificed by cervical dislocation and the tumors were removed and measured for analysis.

Cells were treated to create protein–DNA crosslinks, and the crosslinked sheared chromatin was used for immune-precipitation with normal IgG, or PHF5A antibodies. The immuno-precipitates were washed, eluted, and de-crosslinked, followed by quantification PCR. Total RNA isolated were used to prepare cDNA libraries that were subsequently sequenced on the Illumina HiSeq2500. Raw reads were mapped to the genome with Bowtie (version 2). Peak calling was performed by MACS. Motif analysis was performed using MEME-ChIP, and Pathway enrichment analysis were identified using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional analysis by blast2go software.

A number of clinicopathological factors were evaluated. Fisher’s exact test was used to evaluate the associations between the clinicopathological variables of the patients and the expression of CHD4. A P-value of 0.05 was considered to be significant in all analyses. The clinicopathological variables and CHD4 expression were also subjected to survival analysis using the Kaplan–Meier method, and potential heterogeneity among the studies was quantified using chi-squared test. Multivariate analysis was performed with the Cox proportional hazards regression model to examine the independent prognostic effect of CHD4 on survival by adjusting for the confounding factors. Statistical analysis of the differences between the animal or cellular groups was performed with an unpaired student’s t-test., (two-tailed; P < 0.05 was considered significant). The results are presented as mean ± s.e.m. *, P < 0.05; **, P < 0.01; ***, P < 0.001. SPSS 19.0 was used to perform all statistical analyses in this study.

To discover whether CHD4 plays an important role in NSCLC, we examined CHD4 expression in 146 paired NSCLC and adjacent non-cancerous tissues (Fig. 1a). In non-cancerous tissues, 141 of 146 (97%) samples exhibited weak or negative staining, whereas 5 of 146 (3%) samples showed moderate or strong staining. By contrast, in cancer tissues, 81 of 146 (55%) samples had weak or negative CHD4 expression, whereas 65 of 146 (45%) samples had moderate or strong CHD4 expression (Fig. 1b), demonstrating that the expression of CHD4 was higher in NSCLC tumor tissues than in adjacent non-cancerous tissues.

To further determine the clinicopathological significance of CHD4 in NSCLC, we analyzed the relation between CHD4 expression levels and clinicopathological parameters(Table 1). The results showed that the expression level of CHD4 was significantly associated with TNM stage(P = 0.001), tumor size(P = 0.002) and lymph node metastasis (P = 0.005)(Table S1). CHD4 expression levels were also significantly associated with the overall survival of NSCLC patients(P = 0.005) (Fig. 1c). The stronger the CHD4 expression, the shorter the patient survival time. In addition, the multivariate analysis revealed that CHD4 could be used as an independent factor for predicting NSCLC patient prognosis (P = 0.024)(Table 2). Taken together, these results suggested that CHD4 overexpression is a critical factor in NSCLC development and progression.

To further determine whether CHD4 represents a novel NSCLC-associated gene, we examined the roles of CHD4 in NSCLC development and progression. First, the expression levels of CHD4 in five NSCLC cell lines were determined by immunoblotting. Based on the immunoblotting results (Fig. S1), A549 and H1299 cells were selected for use in the CHD4 knockdown experiments, and successful knockdown by siRNA was confirmed by western blot analysis (Fig. 2a, Fig. S2A). This CHD4 knockdown was observed to markedly suppress the proliferation of A549 and H1299 cells (Fig. 2b). Consistently, the CHD4 knockdown was also observed to arrest the cell cycle at the G1/S phase (Fig. 2c). Using transwell assays (Fig. 2d), it was also shown that the reduced expression of CHD4 significantly inhibited cell migration. We therefore speculated that CHD4 might be a novel candidate tumor-associated gene in NSCLC.

We then stably overexpressed CHD4 in H292 and PC-9 cell lines, which were confirmed by western blot analysis (Fig. 3a, Fig. S2B). Elevation of CHD4 expression could promote the proliferation rate at 24 and 48 h (Fig. 3b). By use of transwell assays, the overexpression of CHD4 was found to significantly increase the migratory potentials of the NSCLC cells (Fig. 3c). Taken together, these data demonstrated that increased expression of CHD4 plays an important role in NSCLC progression.

We then stably suppressed CHD4 expression in the A549 cell line, and these CHD4-down-regulated A549 cells were injected subcutaneously into nude mice to investigate the effects of CHD4 on tumorigenicity. Following the in vivo analysis of tumorigenesis in nude mice, the growth properties of tumors were observed. It was shown that CHD4-down-regulated A549 cells formed much smaller (P < 0.05) tumors than A549-NC cells (Fig. 4a) (n = 6/group). Tumor volume was much decreased (P = 0.025) in the CHD4-down-regulated A549 cells (Fig. 4b), suggesting that CHD4 suppression could significantly decrease tumorigenicity in nude mice.

Given that CHD4 is overexpressed in NSCLC and functions as a novel tumor-promoting gene in NSCLC, we further sought to determine the mechanisms underlying CHD4-mediated promotion of NSCLC cell migration and proliferation. To define the cellular pathways in which CHD4 is involved, Gene Ontology (GO) functional analysis was performed. Overall, the analysis showed enrichment for cancer-dominant functions, such as DNA replication/messenger RNA (mRNA) processing, cell cycle/cell proliferation, and regulation of cytoskeleton, signaling by Rho GTPases pathway was included (Fig. S3), suggesting that these pathways may have important roles in NSCLC pathogenesis.

According to the results of the GO functional analysis, CHD4 is likely associated with the RhoA/ROCK signaling pathway. To further explore the potential interaction of CHD4 with the RhoA/ROCK signaling pathway, we used CHD4-down-regulated cells to examine the RhoA/ROCK pathway. The results revealed that when CHD4 was suppressed, the expression levels of ROCK and RhoA and the downstream factors phospho-myosin were greatly reduced, while E-cadherin expression was elevated. Interestingly, we found that CHD4 knockdown could also reduce the expression of p-ERK, rather than ERK (Fig. 5a, Fig. S4). Together, these results suggested that CHD4 may promote NSCLC cell migration and proliferation via the RhoA/ROCK signaling pathway.

We subsequently determined which factors participate in the regulation of the RhoA/ROCK pathway by CHD4. The GO functional analysis indicated that PHF5A, was likely associated with CHD4. To verify the binding of CHD4 to PHF5A, Co-IP was performed using A549 whole-cell lysates with antibodies against CHD4 and PHF5A, which identified an interaction between CHD4 and PHF5A (Fig. 5b, Fig. S5). These results suggest that PHF5A could bind to CHD4 and that it may participate in the RhoA/ROCK signaling pathway.

To identify whether PHF5A could mediate the RhoA/ROCK signaling pathway, siRNA against PHF5A was used to suppress the expression of PHF5A in A549 cells, and the expression levels of the downstream RhoA/ROCK signaling factors were determined. PHF5A down-regulation was found to reduce the expression levels of ROCK and RhoA (Fig. 5c, Fig. S6), indicating that PHF5A participates in NSCLC cell proliferation and metastasis through regulation of the RhoA/ROCK signaling pathway.