- Open Access
PIG3 promotes NSCLC cell mitotic progression and is associated with poor prognosis of NSCLC patients
- Ming Li†1,
- Shanhu Li†2,
- Biao Liu†3,
- Meng-Meng Gu1,
- Shitao Zou3,
- Bei-Bei Xiao1,
- Lan Yu4,
- Wei-Qun Ding5,
- Ping-Kun Zhou6,
- Jundong Zhou3Email author and
- Zeng-Fu Shang1, 4Email author
© The Author(s). 2017
- Received: 1 October 2016
- Accepted: 21 February 2017
- Published: 4 March 2017
Non-small cell lung cancer (NSCLC) is the most commonly diagnosed type of lung cancer that is associated with poor prognosis. In this study we explored the potential role of p53-induced gene 3 (PIG3) in the progression of NSCLC.
Immunohistochemistry was used to determine the expression levels of PIG3 in 201 NSCLC patients. We performed in vitro studies and silenced endogenous PIG3 by using specific siRNAs that specific target PIG3. Immunofluorescent staining was performed to determine the effect of PIG3 on mitotic progression in NSCLC cells. The growth rates of microtubules were determined by microtubule nucleation analysis. Cell proliferation and chemosensitivity were analyzed by CCK8 assays. Annexin V staining and β-galactosidase activity analysis were used to evaluate PIG3 deficiency-related apoptosis and senescence, respectively.
PIG3 expression levels negatively correlated with overall survival and disease-free survival of NSCLC patients. Knock down of PIG3 resulted in repressed proliferation of NSCLC cells and increased aberrant mitosis, which included misaligning and lagging chromosomes, and bi- or multi-nucleated giant cells. In addition, PIG3 contributed to mitotic spindle assembly by promoting microtubule growth. Furthermore, loss of PIG3 sensitized NSCLC cells to docetaxel by enhancing docetaxel-induced apoptosis and senescence.
Our results indicate that PIG3 promotes NSCLC progression and therefore suggest that PIG3 may be a potential prognostic biomarker and novel therapeutic target for the treatment of NSCLC.
- Non-small cell lung cancer (NSCLC)
- p53-induced gene 3 (PIG3)
- Mitotic progression
- Microtubule assembly
Worldwide, lung cancer is the deadliest type of cancer among both men and women [1, 2]. The most commonly diagnosed type of lung cancer is non-small cell lung cancer (NSCLC), which accounts for nearly 80% of lung cancer-related mortalities . Despite recent advances in early diagnosis [3, 4], chemotherapy, and targeted biological molecular therapies [5, 6], the overall survival (OS) rate of patients with NSCLC is still significantly lower than that of many other solid tumors. Consequently, identifying novel biomarkers and elucidating the underlying mechanisms, which promote malignant progression of lung cancer, are desperately needed to improve lung cancer outcomes and provide personalized treatment.
The p53-induced gene 3 (PIG3 or TP53I3) was initially identified through serial analysis of p53-target genes, which may be correlated with p53-mediated apoptosis . The PIG3 gene locates at chromosome 2p23.3 and comprises 5 exons . The promoter of the PIG3 gene includes a polymorphie pentanucleotide microsatellite sequence ((TGYCC)n, Y = C or T) that is the p53-binding cis-element and mediates p53-induced transactivation of PIG3 . (TGYCC)15 is the most common wild-type allele which localizes at PIG3 promoter and has been reported to be correlated with a decreased risk of squamous cell carcinoma of the head and neck (SCCHN) . Given that the PIG3 protein shares high sequence identity with NADH quinine oxidoreductase 1 (NQO1), it was implied that PIG3 may contribute to p53-induced cell apoptosis by promoting the production of reactive oxygen species (ROS) . Consistent with this hypothesis, Porte and colleagues further investigated PIG3 3-D structure, substrate and cofactor specificity, and determined that PIG3 exhibits a NADPH-dependent reductase activity with orthoquinones . PIG3 also acts as a ROS generator through direct association with and suppression of catalase in response to DNA damage . The same group revealed that PIG3 is a novel regulator of DNA damage response . Loss of PIG3 impairs recruitment of 53BP1, Mre11, Rad50, Nbs1 proteins to DNA break sites and attenuates DNA damage-induced phosphorylation of H2AX, Chk2 and Chk1 in response to UV treatment . Our previous study found that PIG3 could enhance DNA-PKcs expression and contribute to Chk2, Chk1 phosphorylation after γ-ray exposure .
Given its established involvement in p53-induced apoptosis and DNA damage response, it seems reasonable to propose that PIG3 acts as a tumor suppressor to prevent cancer development and progression. In a recent study it was found that the tumor suppressor gene BRCA1 promotes transcription of PIG3 by p53 and that PIG3 expression status in breast cancer samples is positively correlated with OS rate of patients . Research from other groups has demonstrated that PIG3 inhibits HIF-1α expression in renal cell carcinoma in addition to several other types of cancer cells in a mTOR pathway-dependent manner. Deficiency of PIG3 also promotes renal cancer cell migration by facilitating HIF-1α-VEGF signal pathway activation . PIG3 is known to be highly expressed in papillary thyroid carcinoma (PTC) tissues and plays an oncogenic role by activating the PI3K/Akt pathway . Although these seemingly contradictory reports indicated the potential importance of PIG3 in tumor progression, its role(s) in NSCLC still remains unknown and further investigation is warranted.
In the current study, we revealed that the expression levels of PIG3 in NSCLC tissues are inversely associated with OS and disease-free survival (DFS) of patients. To further explore the role of PIG3 in lung cancer development, we suppressed PIG3 expression in NSCLC cells and found that depletion of PIG3 leads to mitosis defects and an increase in the generation of bi- and multi-nucleus which might be due to the dysregulation of microtubule dynamic. Furthermore, we demonstrated that loss of PIG3 significantly increases NSCLC cells chemosensitivity to docetaxel, one of the most commonly used chemotherapeutic drugs against multiple cancers including advanced NSCLC , via enhancing docetaxel-induced apoptosis and senescence.
Patients and tissue specimens
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.
Immunohistochemistry and immunohistochemical evaluation
PIG3 localization was evaluated by immunohistochemistry (IHC) as described previously . 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 . 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 small interfering RNA and construct and transfection
SiRNA sequences for PIG3 and non-targeting negative control
Protein extraction and Western blot analysis
Protein extraction and Western blot were performed as previously described . 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.
CCK8 cell proliferation assay
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.
Mitotic index analysis
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.
Immunofluorescent staining and confocal microscopy
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 assay
Microtubule regrowth assays were performed as previously described . 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.
Senescent cells detection
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).
Statistical analyses were conducted using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). For continuous or discrete data analysis, the chi-square test was used. The association between PIG3 expression and OS or DFS rates was estimated by Kaplan-Meier survival analysis and assessed using a log-rank test. The effect of clinicopathologic variables on survival was assessed with a Cox regression model. One-way ANOVA was performed for multiple comparisons. The data were presented as the mean ± standard deviation (SD) of three independent experiments. All tests were 2-sided, and differences were considered significant when P < 0.05.
Increased expression of PIG3 is associated with poor prognosis of NSCLC patients
Correlation between PIG3 expression and clinicopathological parameters of 201 patients with non-small cell lung cancer (NSCLC)
(n = 201)
PIG3 protein expression
Multivariate analysis of cancer specific survival
PIG3 expression (high vs. low)
Suppression of PIG3 results in bi- and multi-nucleated cells and retarded growth of NSCLC cells
PIG3 contributes to mitotic spindle assembly in NSCLC cells
Lack of PIG3 inhibits microtubule dynamics in NSCLC cells
Depletion of PIG3 sensitizes NSCLC cells to docetaxel
Due to its important role in ROS generation and p53-mediated apoptosis, PIG3 is well-known as an inhibitory factor of cancer cell survival . It has been reported that PIG3 promotes proliferation of PTC cells by activating the PI3K/Akt signaling pathway . Here, we examined the expression of PIG3 in 201 NSCLC samples and found that expression levels of PIG3 positively associated with poor prognosis of NSCLC patients (Fig. 1b, c). Consistent with these results, knocking down PIG3 significantly inhibited proliferation of NSCLC cells (Fig. 2b, Additional file 1: Figure S1b). Furthermore, we revealed an important role of PIG3 in spindle stability maintenance and mitotic progression regulation. It is well known that numerous mitotic regulators are overexpressed in various tumors due to the increased proliferation of cancer cells compared to healthy cells [28–30]. Overall, our study provides evidence that PIG3 may contribute to NSCLC development by promoting mitotic progression.
We have previously reported the positive correlation of DNA-PKcs protein levels with PIG3 expression . DNA-PKcs is a critical kinase that is involved in the non-homologous end joining (NHEJ) pathway and repairs DNA double strand breaks (DSBs). We and others have identified a novel role of DNA-PKcs in maintaining normal spindle formation or centrosomes stability [21, 31–33]. In addition, DNA-PKcs contributes to mitotic entry and cytokinesis progression by activating Plk1 in a temporal and spatial fashion [34, 35]. During mitosis, the Chk2-Brca1 signaling cascade is downstream of DNA-PKcs and has an effect on mitotic microtubule assembly . Consistent with these important roles, inactivation of DNA-PKcs results in a phenotype of abnormal mitotic spindle organization and mitotic catastrophe, which is accompanied by dysregulation of Plk1 and Chk2 in mitosis [21, 34, 36]. Similar to the loss of DNA-PKcs, we found that there is an increase in misaligned and lagged chromosomes in PIG3 deficient cells compared to normal cells, which implies a functional link between these two molecules (Figs. 2 and 3). In conclusion, further investigation is warranted to delineate the impact of PIG3 on the mitotic function of Chk2, Plk1 and whether expression of DNA-PKcs will restore the defects found during mitosis that were induced by PIG3 deficiency.
Appropriate microtubule assembly is crucial for mitotic spindle organization, proper chromosome alignment and their segregation. In a recent study, Ertych et al. demonstrated that increased assembly rates by Aurora A-overexpressing or loss of Chk2 in colon cancer cells resulted in abnormal microtubule spindle geometry and a subsequent increase of chromosomal instability (CIN), which is one of the characteristics of tumor cells and a driving force for cancer development [37, 38]. In addition, CIN plays a critical role in lung cancer progression and fluorescence in situ hybridization (FISH) analysis revealed a close association between CIN in NSCLC and poor prognosis of patients [39, 40]. Consistent with these findings, our results indicate that PIG3 positively regulated microtubule growth rate (Fig. 4). These studies support the speculation that increased PIG3 expression promoted aggressive progression of NSCLC by accelerating microtubule assembly and the generation of CIN cells. As described previously, PIG3 is homologous with NADH quinine oxidoreductase 1 (NQO1) . In human cells, NQO1 has been reported to localize to the mitotic spindle . Furthermore, NQO1 has a direct physical interaction with the mitotic factor Aurora A and promotes Aurora A degradation by antagonizing the protective function of TPX2 . Further investigation of the potential interaction between PIG3 and the TPX2-Aurora A signal pathway during mitosis as well as its potential role(s) in regulating microtubule assembly and chromosomal stability is clearly warranted.
Microtubule dynamics-targeted chemotherapeutic agents such as taxanes are widely used, alone or in combination with other drugs, to treat various solid tumors such as advanced NSCLC [43–45]. Unfortunately, primary resistance to these agents is a major challenge for the use of taxanes in clinical applications. Numerous studies have shown that targeting the mitotic factors may be a successful strategy to overcome the chemoresistance to taxanes in cancer cells [30, 45]. In the present study, we found that a lack of PIG3 significantly sensitizes NSCLC cells to docetaxel, which at least can partially be attributed to the increased induction of apoptosis and senescence. This suggested that docetaxel may be considered an effective targeted therapy against PIG3-low expressing lung cancers. In this context, it is important to further examine whether low PIG3 expression is associated with clinical benefits patients with NSCLC following docetaxel-based chemotherapy.
In summary, in our work we revealed that PIG3 expression levels positively correlated with poor prognosis of NSCLC patients. Our study also demonstrated that PIG3 is a novel mitotic factor that regulated microtubule dynamics and mitotic spindle assembly. In addition, loss of PIG3 induced phenotypes of mitotic catastrophe, which exhibited misaligned and lagged chromosomes and led to multi-nucleated and senescent cells. Loss of PIG3 conferred sensitivity of NSCLC cells to docetaxel-based chemotherapy. These findings demonstrated a role for PIG3 in the progression of NSCLC, indicating that PIG3 may be a potential prognostic marker and novel therapeutic target for NSCLC.
This work was supported by Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
This work was supported by grants from the National Natural Science Foundation of China (81673091, 81472919, 81573079 and 81470138) and China Postdoctoral Science Fund Program (2015 M580463) and Suzhou Key Medical Center (SZZX201506).
Availability of data and materials
Raw and processed data are available from the corresponding author on reasonable request.
ZFS, JZ and PKZ conceived and designed the experiments. ML, SL and BL performed the experiments. MMG, SZ, and BBX coordinated the research and analyzed the data. WQD and LY supported the experiments and helped to draft the manuscript. ZFS and JZ performed the statistical analysis. ZFS wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Ethics approval was given by the Ethics Committee of Nanjing Medical University Affiliated Suzhou Hospital (Suzhou, China) for the use of clinical materials for research purposes.
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