PIG3 promotes NSCLC cell mitotic progression and is associated with poor prognosis of NSCLC patients

Primary cancer tissue specimens obtained from 201 NSCLC patients were provided by the Nanjing Medical University Affiliated Suzhou Hospital (Suzhou, China). None of the patients underwent chemo- or radiotherapy prior to surgical resection. Clinicopathologic parameters and OS data were collected. Of all patients included in the study, 120 were male and 81 female. The average age of all patients was 59.7 years (range from 22 to 83 years) at the time of operation. Mean and median follow-up times after surgery were 47.3 and 38.0 months, respectively. The 5-year survival rate was 30.3%. Tumor tissue was routinely fixed in 10% phosphate-buffered formaldehyde and embedded in paraffin for immunohistochemical evaluation. This study was approved by the Ethics Committee of Nanjing Medical University Affiliated Suzhou Hospital. All patients signed informed consent.

PIG3 localization was evaluated by immunohistochemistry (IHC) as described previously [19]. The PIG3 polyclonal antibody used was from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used at a 100-fold dilution.

PIG3 expression levels were scored blindly by two examiners who were unaware of the clinical characteristics. The staining area was scored as 0, 1, 2, 3 and 4 when 0–10, 11–25, 26–50, 51–75, and > 75% cells were stained positive, respectively. The staining intensity was scored as follows: 0, no staining; 1, pale yellow staining; 2, buffy staining; 3, intense brown staining. All scores were multiplied synchronically to calculate a subjective score for each section [20]. PIG3 expression levels were defined by a final score: low expression level (score ≤ 6) and high expression level (score > 6).

RPMI-1640 medium supplemented with 10% fetal bovine serum was used to maintain NSCLC A549, H460 and H1299 cells. Cells were cultured in a humidified atmosphere at 37 °C and 5% CO₂.

PIG3-siRNA or non-targeting negative control (NC) siRNA were designed and synthesized by GenePharm (Shanghai, China). The sequences are listed in Table 1. The PIG3 constructs were generated by cloning PCR-amplified full-length human PIG3 cDNA into a pCMV-TAG-2B vector as described by Li B et al. [14]. Cells were seeded in 3.5 cm culture dishes when in logarithmic growth phase and were transiently transfected with 20 μM of PIG3-siRNA or NC siRNA or 5 μg PIG3 constructs or empty vector using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Following incubation at 37 °C for 48 h, cells were collected and lysed to verify the expression of PIG3 by Western blot analysis.

Protein extraction and Western blot were performed as previously described [19]. The following primary antibodies were used: PIG3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), PARP-1 (Cell Signaling Technology, Beverly, MA, USA) and GAPDH (Epitomics, Burlingame, CA, USA). All primary antibodies were used at a dilution of 1000-fold.

Control and PIG3 knock down cells were cultured in a 96-well plate at a density of 1 × 10⁴ cells per well. Docetaxel (Taxotere; Sanofi-Aventis, Paris, France) was supplemented for increasing times (1, 2, 3, 4 and 5 days) or at indicated concentrations (2.5, 5, 10 and 20 μg/ml) for 48 h. After incubation with docetaxel, a volume of 10 μl Cell Counting Kit-8 solution (CCK8; Dojindo Laboratories, Japan) was added to each well and incubated for 2 h. The absorbance of each well was measured at 450 nm.

PIG3 knock down and control cells were plated in 6 cm culture dishes, washed twice with PBS and fixed in 70% ethanol, diluted with PBS, for 24 h. Prior to staining, the cells were washed twice with PBS and permeabilized in 50 μl of 0.5% Triton X-100/PBS for 15 min. Cells were incubated with an anti-phosphorylated H3 (pSer10) antibody (1:100) (Cell Signaling Technology, Beverly, MA, USA) in PBS with 0.5% Triton X-100 at room temperature for 2 h and washed twice with PBS. Next, cells were incubated with an Alexa-488 conjugated anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. Cells were washed twice with PBS, treated with 1 μg/ml RNase A, stained with 25 μg/ml Propidium Iodide (PI) and analyzed by flow cytometry.

A549 cells and H460 cells were transfected with PIG3 or NC siRNAs. Cells were plated and cultured on poly-d-lysine-coated cover slides 48 h after transfection. Cells were washed twice in PBS and fixed in 4% paraformaldehyde/PBS at room temperature for 30 min. Next, the cells were permeabilized with 0.5% Triton X-100/PBS at room temperature for 15 min. After permeabilization, cells were blocked with 1% bovine serum albumin/PBS at room temperature for 30 min. Immunostaining was performed by incubating with anti-α-tubulin antibody, γ-tubulin (Sigma, St Louis, MO, USA) and phosphorylated H3 (pSer10) (Cell Signaling Technology, Beverly, MA, USA) antibodies (1:1000) for 4 h at room temperature. After incubating with the primary antibodies, cells were washed three times with PBS. Cells were then incubated with Alexa-488 conjugated anti-rabbit and Alexa-568 conjugated anti-mouse secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. For visualization of DNA, 4, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) was added to the mounting medium. Images were obtained using a LSM 510 laser-scanning confocal microscope (Zeiss, Germany).

Microtubule regrowth assays were performed as previously described [21]. PIG3 depletion and control A549 cells were cultured on cover slides coated as before and incubated with ice-cold medium supplemented with 1 μg/ml nocodazole (Sigma, St Louis, MO, USA) for 1 h. Fresh medium without nocodazole was added after washing with PBS. Cells were fixed in 4% paraformaldehyde/PBS and subjected to immunostaining as described above.

For the detection of apoptosis, the FITC Annexin V Apoptosis Detection Kit (BD, Pharmingen, San Diego, CA, USA) was used according to the manufacturer’s protocol. PIG3 silenced or control cells were treated with or without docetaxel as described above and harvested. The cells were washed thrice with PBS and resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/ml. The cell suspension (100 μl) was transferred to a new tube and 5 μl of FITC-conjugated Annexin V was added. Cells were incubated for 15 min at room temperature. After addition of 400 μl of 1× binding buffer, cells were analyzed by flow cytometry.

To detect senescent cells, the senescence-associated β-galactosidase (SA-β-Gal) assay was performed. PIG3 silenced or control cells were cultured in 6-well plates at a density of 20,000 cells/well and treated with or without docetaxel at indicated concentrations and times. Cells were fixed and stained following the Senescence β-gal Staining Kit manufacturer’s protocol (Cell Signaling Technology, Beverley, MA, USA). Cells were incubated for 16 h at 37 °C in a dry incubator without CO₂ after which blue stained cells were detected under a bright field microscope (Leica Corporation, Germany).

To investigate the potential role of PIG3 in the progression of NSCLC, we performed PIG3 immunohistochemistry (IHC) in NSCLC tissue obtained from 201 patients (Fig. 1a). The clinicopathologic characteristics of all patients are listed in Table 2. Our results showed that an upregulated PIG3 expression level significantly correlated with tumor size (P = 0.0003), differentiation degree (P = 0.004), pathological stage (P = 0.004) and distant metastasis (P = 0.001). In addition, no association could be observed between PIG3 expression and age, gender or relapse.

To evaluate the relationship between PIG3 expression and patient prognosis, Kaplan-Meier survival analysis for OS and DFS were performed (Fig. 1b, c). Our results indicated that patients with a higher PIG3 expression (high PIG3) demonstrate a significantly shorter OS (P = 0.008) and DFS (P = 0.026) compared to patients with low PIG3 expression (low PIG3). Moreover, multivariate Cox regression analysis showed high PIG3 to be an independent prognostic marker for low survival that was associated with a relative risk of 1.742 (Table 3; 95% CI 1.023–2.976; P = 0.041). Together, these findings suggested that PIG3 may have an oncogenic role in the progression of lung cancer.

To determine the role of PIG3 on the progression of NSCLC, two different siRNA constructs that target PIG3 and a NC siRNA were transfected into A549 NSCLC cells. Western blot analysis verified that both siRNAs significantly suppress endogenous PIG3 protein expression in A549 cells (Fig. 2a). We used the siRNA with the highest efficacy to transfect H460 cells and found that PIG3 expression was significantly silenced in H460 cells (Additional file 1: Figure S1a). Compared with corresponding NC groups, depletion of PIG3 significantly reduced the proliferation rates of A549 and H460 cells (Fig. 2b and Additional file 1: Figure S1b). The PIG3 is one of p53 target genes. Consistent with this, PIG3 expression is suppressed in H1299 cells which have the homozygous partial deletion of the TP53 gene. We observed that overexpression of PIG3 dramatically promotes growth speed of H1299 cells (Additional file 2: Figure S2a and b). Interestingly, we found an increase in the generation of giant cells in PIG3 deficient NSCLC cells. After staining with an anti-α-tubulin antibody and DAPI to visualize DNA, numerous giant cells with bi- or multi-nuclei were observed in PIG3 deficient-A549 cells (Fig. 2c, d). Bi- and multi-nucleated cells may arise from cytokinesis failure, which is commonly induced by abnormality of chromosomal segregation [22, 23]. Consistent with this observation, we identified a significant increase of chromosomes lagging during anaphase in PIG3 deficient NSCLC cells as compared to NC cells (Fig. 2e, f). In a recent study, it was shown that failure in cell cleavage induces cellular senescence [24]. Here, we found that giant cells induced by PIG3 deficiency exhibited activated SA-β-galactosidase (Fig. 5e, f), which is indicative of a senescent phenotype.

The observation of increased abnormality of chromosome segregation in PIG3 deficient cells strongly indicated that PIG3 may play an important role in the regulation of spindle organization. During the transition from prometaphase to metaphase, kinetochores are held by microtubules that are released from the opposite sites of centrosomes and allow chromosomes to align along the metaphase plate of the spindle apparatus. To define whether or not PIG3 plays a role in mitotic spindle organization, NSCLC cells were stained with antibodies directed against α-tubulin and a mitosis marker, phosphorylated histone 3, at the Ser10 site. We found that, in both A549 and H460 cells, PIG3 depletion resulted in a noticeable increase of chromosome misalignment (Fig. 3a, b and Additional file 1: Figure S1c, d). Consistent with this observation, we found an increase in mitotic index in NSCLC cells lacking PIG3 as compared to the control cells (Fig. 3c, d). Thus, aberrant spindle assembly and mitotic progression may potentially lead to mitotic arrest.

Abnormal microtubule dynamics regulation has been reported to be correlated with aberrant mitotic organization [25]. To identify whether or not PIG3 is involved in microtubule organization, PIG3 silenced and control A549 cells, PIG3 overxpressed and empty vector control H1299 cells were incubated with ice-cold medium including high levels of nocodazole to depolymerize microtubules. Microtubules started to regrow after removal of the nocodazole-containing medium. Microtubule nucleation speed was analyzed by the diameter of the microtubule asters that were grown from centrosomes (Fig. 4a, c). We observed that, in A549 cells, loss of PIG3 significantly inhibited microtubule nucleation (Fig. 4b), whereas increased PIG3 in H1299 cells promotes microtubule regrowth rate (Fig. 4d). Consistent with its role in microtubule dynamic regulation, we determined that PIG3 co-localized with tubulin and accumulated at the spindle apparatus during mitosis (Fig. 4e). PIG3 localizes at both nucleus and cytoplasm in interphase cells and can be recruited at DNA damage sites when cells were exposed to γ ray (Additional file 3: Figure S3), which is consistent with the previous report [13]. In conclusion, our data revealed for the first time a novel role of PIG3 in microtubule regulation.

Our data showed that PIG3-depleted cells exhibit aberrant mitosis, which may be associated with dysregulation of the dynamics of microtubules, similar to that of drug-treated microtubule cells [26]. Next, we investigated whether silencing of PIG3 expression may modulate the sensitivity of NSCLC cells to docetaxel, a well-established anti-mitotic chemotherapeutic drug that is used to treat advanced NSCLC [18]. The proliferation of PIG3 knock down and control NSCLC cells was accessed by CCK8 assay at 48 h after docetaxel treatment (2.5, 5, 10 and 20 μg/ml). Our data showed that PIG3-depleted cells were more sensitive to docetaxel treatment compared to NC cells, and PIG3-overexpression increased NSCLCs resistance to docetaxel (Fig. 5a, Additional file 2: Figure S2c and Additional file 4: Figure S4). Docetaxel-induced apoptosis was determined by flow cytometry. As shown in Fig. 5b, treatment with 5 μg/ml docetaxel dramatically induces apoptosis in PIG3-silenced but not in control A549 cells. Docetaxel-induced apoptosis was verified by cleaved PARP-1 immunoblot analysis. We found that docetaxel treatment caused severe PARP-1 cleavage and apoptosis in PIG3-depleted cells but not in control cells (Fig. 5c, d). In addition, PIG3 knock down and control A549 cells were subjected to SA-β-gal staining. Our results indicated that following docetaxel treatment PIG3 depletion significantly enhanced senescence (Fig. 5e, f), whereas PIG3 overexpression prevents docetaxel induced senescence in H1299 cells (Additional file 2: Figure S2d).