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

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1 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 DOI /s RESEARCH Open Aess PIG3 promotes NSCLC ell mitoti progression and is assoiated with poor prognosis of NSCLC patients Ming Li 1, Shanhu Li 2, Biao Liu 3, Meng-Meng Gu 1, Shitao Zou 3, Bei-Bei Xiao 1, Lan Yu 4, Wei-Qun Ding 5, Ping-Kun Zhou 6, Jundong Zhou 3* and Zeng-Fu Shang 1,4* Abstrat Bakground: Non-small ell lung aner (NSCLC) is the most ommonly diagnosed type of lung aner that is assoiated with poor prognosis. In this study we explored the potential role of p53-indued gene 3 (PIG3) in the progression of NSCLC. Methods: Immunohistohemistry was used to determine the expression levels of PIG3 in 201 NSCLC patients. We performed in vitro studies and silened endogenous PIG3 by using speifi sirnas that speifi target PIG3. Immunofluoresent staining was performed to determine the effet of PIG3 on mitoti progression in NSCLC ells. The growth rates of mirotubules were determined by mirotubule nuleation analysis. Cell proliferation and hemosensitivity were analyzed by CCK8 assays. Annexin V staining and β-galatosidase ativity analysis were used to evaluate PIG3 defiieny-related apoptosis and senesene, respetively. Results: PIG3 expression levels negatively orrelated with overall survival and disease-free survival of NSCLC patients. Knok down of PIG3 resulted in repressed proliferation of NSCLC ells and inreased aberrant mitosis, whih inluded misaligning and lagging hromosomes, and bi- or multi-nuleated giant ells. In addition, PIG3 ontributed to mitoti spindle assembly by promoting mirotubule growth. Furthermore, loss of PIG3 sensitized NSCLC ells to doetaxel by enhaning doetaxel-indued apoptosis and senesene. Conlusions: Our results indiate that PIG3 promotes NSCLC progression and therefore suggest that PIG3 may be a potential prognosti biomarker and novel therapeuti target for the treatment of NSCLC. Keywords: Non-small ell lung aner (NSCLC), p53-indued gene 3 (PIG3), Mitoti progression, Mirotubule assembly, Chemoresistane Bakground Worldwide, lung aner is the deadliest type of aner among both men and women [1, 2]. The most ommonly diagnosed type of lung aner is non-small ell lung aner (NSCLC), whih aounts for nearly 80% of lung aner-related mortalities [1]. Despite reent advanes in * Correspondene: zhoujundong330@163.om; ziyu_620@163.om Equal ontributors 3 Suzhou Caner Center Core Laboratory, Nanjing Medial University Affiliated Suzhou Hospital, Suzhou, Jiangsu , People s Republi of China 1 Shool of Radiation Mediine and Protetion, Medial College of Soohow University, Collaborative Innovation Center of Radiation Mediine of Jiangsu Higher Eduation Institutions, Suzhou, Jiangsu , People s Republi of China Full list of author information is available at the end of the artile early diagnosis [3, 4], hemotherapy, and targeted biologial moleular therapies [5, 6], the overall survival (OS) rate of patients with NSCLC is still signifiantly lower than that of many other solid tumors. Consequently, identifying novel biomarkers and eluidating the underlying mehanisms, whih promote malignant progression of lung aner, are desperately needed to improve lung aner outomes and provide personalized treatment. The p53-indued gene 3 (PIG3 or TP53I3) was initially identified through serial analysis of p53-target genes, whih may be orrelated with p53-mediated apoptosis [7]. The PIG3 gene loates at hromosome 2p23.3 and omprises 5 exons [8]. The promoter of the PIG3 gene inludes a polymorphie pentanuleotide mirosatellite The Author(s) Open Aess This artile is distributed under the terms of the Creative Commons Attribution 4.0 International Liense ( whih permits unrestrited use, distribution, and reprodution in any medium, provided you give appropriate redit to the original author(s) and the soure, provide a link to the Creative Commons liense, and indiate if hanges were made. The Creative Commons Publi Domain Dediation waiver ( applies to the data made available in this artile, unless otherwise stated.

2 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 2 of 11 sequene ((TGYCC) n, Y = C or T) that is the p53- binding is-element and mediates p53-indued transativation of PIG3 [9]. (TGYCC) 15 is the most ommon wild-type allele whih loalizes at PIG3 promoter and has been reported to be orrelated with a dereased risk of squamous ell arinoma of the head and nek (SCCHN) [10]. Given that the PIG3 protein shares high sequene identity with NADH quinine oxidoredutase 1 (NQO1), it was implied that PIG3 may ontribute to p53-indued ell apoptosis by promoting the prodution of reative oxygen speies (ROS) [7]. Consistent with this hypothesis, Porte and olleagues further investigated PIG3 3-D struture, substrate and ofator speifiity, and determined that PIG3 exhibits a NADPH-dependent redutase ativity with orthoquinones [11]. PIG3 also ats as a ROS generator through diret assoiation with and suppression of atalase in response to DNA damage [12]. The same group revealed that PIG3 is a novel regulator of DNA damage response [13]. Loss of PIG3 impairs reruitment of 53BP1, Mre11, Rad50, Nbs1 proteins to DNA break sites and attenuates DNA damage-indued phosphorylation of H2AX, Chk2 and Chk1 in response to UV treatment [13]. Our previous study found that PIG3 ould enhane DNA-PKs expression and ontribute to Chk2, Chk1 phosphorylation after γ-ray exposure [14]. Given its established involvement in p53-indued apoptosis and DNA damage response, it seems reasonable to propose that PIG3 ats as a tumor suppressor to prevent aner development and progression. In a reent study it was found that the tumor suppressor gene BRCA1 promotes transription of PIG3 by p53 and that PIG3 expression status in breast aner samples is positively orrelated with OS rate of patients [15]. Researh from other groups has demonstrated that PIG3 inhibits HIF-1α expression in renal ell arinoma in addition to several other types of aner ells in a mtor pathwaydependent manner. Defiieny of PIG3 also promotes renal aner ell migration by failitating HIF-1α-VEGF signal pathway ativation [16]. PIG3 is known to be highly expressed in papillary thyroid arinoma (PTC) tissues and plays an onogeni role by ativating the PI3K/Akt pathway [17]. Although these seemingly ontraditory reports indiated the potential importane of PIG3 in tumor progression, its role(s) in NSCLC still remains unknown and further investigation is warranted. In the urrent study, we revealed that the expression levels of PIG3 in NSCLC tissues are inversely assoiated with OS and disease-free survival (DFS) of patients. To further explore the role of PIG3 in lung aner development, we suppressed PIG3 expression in NSCLC ells and found that depletion of PIG3 leads to mitosis defets and an inrease in the generation of bi- and multinuleus whih might be due to the dysregulation of mirotubule dynami. Furthermore, we demonstrated that loss of PIG3 signifiantly inreases NSCLC ells hemosensitivity to doetaxel, one of the most ommonly used hemotherapeuti drugs against multiple aners inluding advaned NSCLC [18], via enhaning doetaxel-indued apoptosis and senesene. Methods Patients and tissue speimens Primary aner tissue speimens obtained from 201 NSCLC patients were provided by the Nanjing Medial University Affiliated Suzhou Hospital (Suzhou, China). None of the patients underwent hemo- or radiotherapy prior to surgial resetion. Cliniopathologi parameters and OS data were olleted. Of all patients inluded 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, respetively. The 5-year survival rate was 30.3%. Tumor tissue was routinely fixed in 10% phosphate-buffered formaldehyde and embedded in paraffin for immunohistohemial evaluation. This study was approved by the Ethis Committee of Nanjing Medial University Affiliated Suzhou Hospital. All patients signed informed onsent. Immunohistohemistry and immunohistohemial evaluation PIG3 loalization was evaluated by immunohistohemistry (IHC) as desribed previously [19]. The PIG3 polylonal antibody used was from Santa Cruz Biotehnology (Santa Cruz, CA, USA) and used at a 100-fold dilution. PIG3 expression levels were sored blindly by two examiners who were unaware of the linial harateristis. The staining area was sored as 0, 1, 2, 3 and 4 when 0 10, 11 25, 26 50, 51 75, and > 75% ells were stained positive, respetively. The staining intensity was sored as follows: 0, no staining; 1, pale yellow staining; 2, buffy staining; 3, intense brown staining. All sores were multiplied synhronially to alulate a subjetive sore for eah setion [20]. PIG3 expression levels were defined by a final sore: low expression level (sore 6) and high expression level (sore > 6). Cell ulture RPMI-1640 medium supplemented with 10% fetal bovine serum was used to maintain NSCLC A549, H460 and H1299 ells. Cells were ultured in a humidified atmosphere at 37 C and 5% CO 2. PIG3 small interfering RNA and onstrut and transfetion PIG3-siRNA or non-targeting negative ontrol (NC) sirna were designed and synthesized by GenePharm (Shanghai, China). The sequenes are listed in Table 1. The PIG3

3 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 3 of 11 Table 1 SiRNA sequenes for PIG3 and non-targeting negative ontrol sirna name Sequenes Sense (5-3 ) Antisense (5-3 ) sipig3 #1 AAAUGUUCAGGCUGGAGACUATT UAGUCUCCAGCCUGAACAUUUTT sipig3 #2 GGAAGUCUGAUCACCAGUUTT AACUGGUGAUCAGACUUCCTT sinc UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT NC negative ontrol onstruts were generated by loning PCR-amplified fulllength human PIG3 DNA into a pcmv-tag-2b vetor as desribed by Li B et al. [14]. Cells were seeded in 3.5 m ulture dishes when in logarithmi growth phase and were transiently transfeted with 20 μm of PIG3-siRNA or NC sirna or 5 μg PIG3 onstruts or empty vetor using Lipofetamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufaturer s instrutions. Following inubation at 37 C for 48 h, ells were olleted and lysed to verify the expression of PIG3 by Western blot analysis. Protein extration and Western blot analysis Protein extration and Western blot were performed as previously desribed [19]. The following primary antibodies were used: PIG3 (Santa Cruz Biotehnology, Santa Cruz, CA, USA), PARP-1 (Cell Signaling Tehnology, Beverly, MA, USA) and GAPDH (Epitomis, Burlingame, CA, USA). All primary antibodies were used at a dilution of 1000-fold. CCK8 ell proliferation assay Control and PIG3 knok down ells were ultured in a 96-well plate at a density of ells per well. Doetaxel (Taxotere; Sanofi-Aventis, Paris, Frane) was supplemented for inreasing times (1, 2, 3, 4 and 5 days) or at indiated onentrations (2.5, 5, 10 and 20 μg/ml) for 48 h. After inubation with doetaxel, a volume of 10 μl Cell Counting Kit-8 solution (CCK8; Dojindo Laboratories, Japan) was added to eah well and inubated for 2 h. The absorbane of eah well was measured at 450 nm. Mitoti index analysis PIG3 knok down and ontrol ells were plated in 6 m ulture dishes, washed twie with PBS and fixed in 70% ethanol, diluted with PBS, for 24 h. Prior to staining, the ells were washed twie with PBS and permeabilized in 50 μl of 0.5% Triton X-100/PBS for 15 min. Cells were inubated with an anti-phosphorylated H3 (pser10) antibody (1:100) (Cell Signaling Tehnology, Beverly, MA, USA) in PBS with 0.5% Triton X-100 at room temperature for 2 h and washed twie with PBS. Next, ells were inubated with an Alexa-488 onjugated anti-rabbit seondary antibody (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. Cells were washed twie with PBS, treated with 1 μg/ ml RNase A, stained with 25 μg/ml Propidium Iodide (PI) and analyzed by flow ytometry. Immunofluoresent staining and onfoal mirosopy A549 ells and H460 ells were transfeted with PIG3 or NC sirnas. Cells were plated and ultured on poly-dlysine-oated over slides 48 h after transfetion. Cells were washed twie in PBS and fixed in 4% paraformaldehyde/pbs at room temperature for 30 min. Next, the ells were permeabilized with 0.5% Triton X-100/PBS at room temperature for 15 min. After permeabilization, ells were bloked with 1% bovine serum albumin/pbs at room temperature for 30 min. Immunostaining was performed by inubating with anti-α-tubulin antibody, γ-tubulin (Sigma, St Louis, MO, USA) and phosphorylated H3 (pser10) (Cell Signaling Tehnology, Beverly, MA, USA) antibodies (1:1000) for 4 h at room temperature. After inubating with the primary antibodies, ells were washed three times with PBS. Cells were then inubated with Alexa-488 onjugated anti-rabbit and Alexa-568 onjugated anti-mouse seondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at 37 C. For visualization of DNA, 4, 6-diamidino-2-phenylindole (DAPI, Vetor Laboratories, Burlingame, CA, USA) was added to the mounting medium. Images were obtained using a LSM 510 laser-sanning onfoal mirosope (Zeiss, Germany). Mirotubule regrowth assay Mirotubule regrowth assays were performed as previously desribed [21]. PIG3 depletion and ontrol A549 ells were ultured on over slides oated as before and inubated with ie-old medium supplemented with 1 μg/ ml noodazole (Sigma, St Louis, MO, USA) for 1 h. Fresh medium without noodazole was added after washing with PBS. Cells were fixed in 4% paraformaldehyde/pbs and subjeted to immunostaining as desribed above. Apoptosis detetion For the detetion of apoptosis, the FITC Annexin V Apoptosis Detetion Kit (BD, Pharmingen, San Diego, CA, USA) was used aording to the manufaturer s protool. PIG3 silened or ontrol ells were treated with or without doetaxel as desribed above and harvested. The ells were washed thrie with PBS and resuspended in 1 binding buffer at a onentration of

4 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 4 of ells/ml. The ell suspension (100 μl) was transferred to a new tube and 5 μl of FITC-onjugated Annexin V was added. Cells were inubated for 15 min at room temperature. After addition of 400 μl of 1 binding buffer, ells were analyzed by flow ytometry. Senesent ells detetion To detet senesent ells, the senesene-assoiated β- galatosidase (SA-β-Gal) assay was performed. PIG3 silened or ontrol ells were ultured in 6-well plates at a density of 20,000 ells/well and treated with or without doetaxel at indiated onentrations and times. Cells were fixed and stained following the Senesene β-gal Staining Kit manufaturer s protool (Cell Signaling Tehnology, Beverley, MA, USA). Cells were inubated for 16 h at 37 C in a dry inubator without CO 2 after whih blue stained ells were deteted under a bright field mirosope (Leia Corporation, Germany). Statistial analysis Statistial analyses were onduted using SPSS 19.0 software (SPSS In., Chiago, IL, USA). For ontinuous or disrete data analysis, the hi-square test was used. The assoiation between PIG3 expression and OS or DFS rates was estimated by Kaplan-Meier survival analysis and assessed using a log-rank test. The effet of liniopathologi variables on survival was assessed with a Cox regression model. One-way ANOVA was performed for multiple omparisons. The data were presented as the mean ± standard deviation (SD) of three independent experiments. All tests were 2-sided, and differenes were onsidered signifiant when P < Results Inreased expression of PIG3 is assoiated with poor prognosis of NSCLC patients To investigate the potential role of PIG3 in the progression of NSCLC, we performed PIG3 immunohistohemistry (IHC) in NSCLC tissue obtained from 201 patients (Fig. 1a). The liniopathologi harateristis of all patients are listed in Table 2. Our results showed that an upregulated PIG3 expression level signifiantly orrelated with tumor size (P = ), differentiation degree (P = 0.004), pathologial stage (P = 0.004) and distant metastasis (P = 0.001). In addition, no assoiation ould 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, ). Our results indiated that patients with a higher PIG3 expression (high PIG3) demonstrate a signifiantly shorter OS (P = 0.008) and DFS (P =0.026) ompared to patients with low PIG3 expression (low PIG3). Moreover, multivariate Cox regression analysis showed high PIG3 to be an independent prognosti marker for low survival that was assoiated with a relative risk of (Table 3; 95% CI ; P = 0.041). Together, these findings suggested that PIG3 may have an onogeni role in the progression of lung aner. Suppression of PIG3 results in bi- and multi-nuleated ells and retarded growth of NSCLC ells To determine the role of PIG3 on the progression of NSCLC, two different sirna onstruts that target PIG3 and a NC sirna were transfeted into A549 NSCLC ells. Western blot analysis verified that both sirnas a b Fig. 1 PIG3 expression is assoiated with poor prognosis of NSCLC patients. a Immunohistohemial analysis of PIG3 in 201 tumor tissue samples (100 ): a), Negative expression; b), Low expression; ), Moderate expression; d), High expression. b Kaplan-Meier survival analysis between PIG3 expression levels and overall survival of NSCLC patients (P = 0.008). The assoiation between PIG3 expression levels and disease-free survival of NSCLC patients (P = 0.026). All groups were ranked aording to PIG3 staining intensity

5 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 5 of 11 Table 2 Correlation between PIG3 expression and liniopathologial parameters of 201 patients with non-small ell lung aner (NSCLC) Charateristis Total PIG3 protein expression P-value (n = 201) Low High Age >0.05 > Gender Male >0.05 Female Smoking history Yes <0.05 No Tumor size <3 m < m Differentiation degree Poorly <0.01 Well, moderately Pathologial stage IA <0.01 IB Distant metastasis Yes <0.01 No Relapse Yes >0.05 No signifiantly suppress endogenous PIG3 protein expression in A549 ells (Fig. 2a). We used the sirna with the highest effiay to transfet H460 ells and found that PIG3 expression was signifiantly silened in H460 ells (Additional file 1: Figure S1a). Compared with orresponding NC groups, depletion of PIG3 signifiantly redued the proliferation rates of A549 and H460 ells (Fig. 2b and Additional file 1: Figure S1b). The PIG3 is one of p53 target genes. Consistent with this, PIG3 Table 3 Multivariate analysis of aner speifi survival HR 95% CI P-value Gender Age Smoking Reurrene Metastasis PIG3 expression (high vs. low) HR hazard ratio, CI onfidene interval expression is suppressed in H1299 ells whih have the homozygous partial deletion of the TP53 gene. We observed that overexpression of PIG3 dramatially promotes growth speed of H1299 ells (Additional file 2: Figure S2a and b). Interestingly, we found an inrease in the generation of giant ells in PIG3 defiient NSCLC ells. After staining with an anti-α-tubulin antibody and DAPI to visualize DNA, numerous giant ells with bi- or multi-nulei were observed in PIG3 defiient-a549 ells (Fig. 2, d). Bi- and multi-nuleated ells may arise from ytokinesis failure, whih is ommonly indued by abnormality of hromosomal segregation [22, 23]. Consistent with this observation, we identified a signifiant inrease of hromosomes lagging during anaphase in PIG3 defiient NSCLC ells as ompared to NC ells (Fig. 2e, f). In a reent study, it was shown that failure in ell leavage indues ellular senesene [24]. Here, we found that giant ells indued by PIG3 defiieny exhibited ativated SA-β-galatosidase (Fig. 5e, f), whih is indiative of a senesent phenotype. PIG3 ontributes to mitoti spindle assembly in NSCLC ells The observation of inreased abnormality of hromosome segregation in PIG3 defiient ells strongly indiated that PIG3 may play an important role in the regulation of spindle organization. During the transition from prometaphase to metaphase, kinetohores are held by mirotubules that are released from the opposite sites of entrosomes and allow hromosomes to align along the metaphase plate of the spindle apparatus. To define whether or not PIG3 plays a role in mitoti spindle organization, NSCLC ells were stained with antibodies direted against α-tubulin and a mitosis marker, phosphorylated histone 3, at the Ser10 site. We found that, in both A549 and H460 ells, PIG3 depletion resulted in a notieable inrease of hromosome misalignment (Fig. 3a, b and Additional file 1: Figure S1, d). Consistent with this observation, we found an inrease in mitoti index in NSCLC ells laking PIG3 as ompared to the ontrol ells (Fig. 3, d). Thus, aberrant spindle assembly and mitoti progression may potentially lead to mitoti arrest. Lak of PIG3 inhibits mirotubule dynamis in NSCLC ells Abnormal mirotubule dynamis regulation has been reported to be orrelated with aberrant mitoti organization [25]. To identify whether or not PIG3 is involved in mirotubule organization, PIG3 silened and ontrol A549 ells, PIG3 overxpressed and empty vetor ontrol H1299 ells were inubated with ie-old medium inluding high levels of noodazole to depolymerize mirotubules. Mirotubules started to regrow after removal of the noodazole-ontaining medium. Mirotubule nuleation

6 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 6 of 11 a b d e f Fig. 2 Loss of PIG3 leads to inreased outome of bi-/multi-nuleated ells and aberrant hromosomes segregation in normal ultured ells. a Western blot analysis demonstrating the effiay of two different sirnas against PIG3 in A549 ells at 48 h post-transfetion. b ells were seeded in 96-well plates at day 0, and CCK8 assay was used to determine ell proliferation rates at indiated days (1, 2, 3, 4 and 5 days). Absorbane values at 450 nm were normalized by the value measured on day 1 ( ** P < 0.01). Representative images showing bi- and multinuleated ells (arrowheads). d Quantitative analysis of bi- and multi-nuleated ells from PIG3-defiient and ontrol A549 ells ( ** P < 0.01). e Representative images showing normal and aberrant anaphase ells with lagging hromosome (arrowheads). f Perentages of mitoti ells showing lagging hromosome were ounted from three independent experiments, ** P < 0.01, * P <0.05 speed was analyzed by the diameter of the mirotubule asters that were grown from entrosomes (Fig. 4a, ). We observed that, in A549 ells, loss of PIG3 signifiantly inhibited mirotubule nuleation (Fig. 4b), whereas inreased PIG3 in H1299 ells promotes mirotubule regrowth rate (Fig. 4d). Consistent with its role in mirotubule dynami regulation, we determined that PIG3 oloalized with tubulin and aumulated at the spindle apparatus during mitosis (Fig. 4e). PIG3 loalizes at both nuleus and ytoplasm in interphase ells and an be reruited at DNA damage sites when ells were exposed to γ ray (Additional file 3: Figure S3), whih is onsistent with the previous report [13]. In onlusion, our data revealed for the first time a novel role of PIG3 in mirotubule regulation. Depletion of PIG3 sensitizes NSCLC ells to doetaxel Our data showed that PIG3-depleted ells exhibit aberrant mitosis, whih may be assoiated with dysregulation of the dynamis of mirotubules, similar to that of drugtreated mirotubule ells [26]. Next, we investigated whether silening of PIG3 expression may modulate the sensitivity of NSCLC ells to doetaxel, a well-established anti-mitoti hemotherapeuti drug that is used to treat advaned NSCLC [18]. The proliferation of PIG3 knok down and ontrol NSCLC ells was aessed by CCK8

7 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 7 of 11 a b d Fig. 3 PIG3 is required for normal mitoti progression. a Exponentially growing PIG3 depleted and ontrol A549 ells were subjeted to immunofluoresent analysis using the indiated antibody. The representative images show aberrant mitoti ells with misalignment hromosomes (arrowheads). b Perentages of mitoti ells showing misaligned hromosomes were ounted from three independent experiments ( ** P < 0.01). A549 ells were transfeted with ontrol sirna or PIG3 sirnas for 48 h and stained with an anti-phosphorylated H3 antibody to determine the perentage of mitosis ell population by flow ytometry. d Quantitative analysis of mitoti index. Results were generated from three independent experiments ( ** P <0.01) assay at 48 h after doetaxel treatment (2.5, 5, 10 and 20 μg/ml). Our data showed that PIG3-depleted ells were more sensitive to doetaxel treatment ompared to NC ells, and PIG3-overexpression inreased NSCLCs resistane to doetaxel (Fig. 5a, Additional file 2: Figure S2 and Additional file 4: Figure S4). Doetaxel-indued apoptosis was determined by flow ytometry. As shown in Fig. 5b, treatment with 5 μg/ml doetaxel dramatially indues apoptosis in PIG3-silened but not in ontrol A549 ells. Doetaxel-indued apoptosis was verified by leaved PARP-1 immunoblot analysis. We found that doetaxel treatment aused severe PARP-1 leavage and apoptosis in PIG3-depleted ells but not in ontrol ells (Fig. 5, d). In addition, PIG3 knok down and ontrol A549 ells were subjeted to SA-β-gal staining. Our results indiated that following doetaxel treatment PIG3 depletion signifiantly enhaned senesene (Fig. 5e, f), whereas PIG3 overexpression prevents doetaxel indued senesene in H1299 ells (Additional file 2: Figure S2d). Disussion Due to its important role in ROS generation and p53- mediated apoptosis, PIG3 is well-known as an inhibitory fator of aner ell survival [27]. It has been reported that PIG3 promotes proliferation of PTC ells by ativating the PI3K/Akt signaling pathway [17]. Here, we examined the expression of PIG3 in 201 NSCLC samples and found that expression levels of PIG3 positively assoiated with poor prognosis of NSCLC patients (Fig. 1b, ). Consistent with these results, knoking down PIG3 signifiantly inhibited proliferation of NSCLC ells (Fig. 2b, Additional file 1: Figure S1b). Furthermore, we revealed an important role of PIG3 in spindle stability maintenane and mitoti progression regulation. It is well known that numerous mitoti regulators are overexpressed in various tumors due to the inreased proliferation of aner ells ompared to healthy ells [28 30]. Overall, our study provides evidene that PIG3 may ontribute to NSCLC development by promoting mitoti progression. We have previously reported the positive orrelation of DNA-PKs protein levels with PIG3 expression [14]. DNA- PKs is a ritial kinase that is involved in the nonhomologous end joining (NHEJ) pathway and repairs DNA double strand breaks (DSBs). We and others have identified a novel role of DNA-PKs in maintaining normal spindle formation or entrosomes stability [21, 31 33]. In addition, DNA-PKs ontributes to mitoti entry and ytokinesis progression by ativating Plk1 in a temporal and spatial fashion [34, 35]. During mitosis, the Chk2-Bra1 signaling asade is downstream of DNA-PKs and has an effet on mitoti mirotubule assembly [21]. Consistent with these important roles, inativation of DNA-PKs results in a

8 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 8 of 11 a b d e Fig. 4 PIG3 is required for mirotubule growth in NSCLC ells. a Knok down of PIG3 by sirna markedly inhibits the regrowth of mirotubules from entrosomes in A549 ells. PIG3 and ontrol sirnas were transfeted into A549 ells and 48 h post transfetion the ells were treated with hilled medium + 1 μm noodazole for an additional 1 h on the ie. Cells were fixed and stained with an anti-α-tubulin antibody. b The length of the mirotubule emanating from the entrosomes was measured (n 50, ** P <0.01). Overexpressing PIG3 signifiantly inreases mirotubules growth rates in H1299 ells. d The length of the mirotubule emanating from the entrosomes was measured (n 50, ** P <0.01). e The loalization of PIG3 in the ells deteted by immunofluoresent staining phenotype of abnormal mitoti spindle organization and mitoti atastrophe, whih is aompanied by dysregulation of Plk1 and Chk2 in mitosis [21, 34, 36]. Similar to the loss of DNA-PKs, we found that there is an inrease in misaligned and lagged hromosomes in PIG3 defiient ells ompared to normal ells, whih implies a funtional link between these two moleules (Figs. 2 and 3). In onlusion, further investigation is warranted to delineate the impat of PIG3 on the mitoti funtion of Chk2, Plk1 and whether expression of DNA-PKs will restore the defets found during mitosis that were indued by PIG3 defiieny. Appropriate mirotubule assembly is ruial for mitoti spindle organization, proper hromosome alignment and their segregation. In a reent study, Ertyh et al. demonstrated that inreased assembly rates by Aurora A-overexpressing or loss of Chk2 in olon aner ells resulted in abnormal mirotubule spindle geometry and a subsequent inrease of hromosomal instability (CIN), whih is one of the harateristis of tumor ells and a driving fore for aner development [37, 38]. In addition, CIN plays a ritial role in lung aner progression and fluoresene in situ hybridization (FISH) analysis revealed a lose assoiation between CIN in NSCLC and poor prognosis of patients [39, 40]. Consistent with these findings, our results indiate that PIG3 positively regulated mirotubule growth rate (Fig. 4). These studies support the speulation that inreased PIG3 expression promoted aggressive progression of NSCLC by aelerating mirotubule assembly and the generation of CIN ells. As desribed previously, PIG3 is homologous with NADH quinine oxidoredutase 1 (NQO1) [7]. In human ells, NQO1 has been reported to loalize to the mitoti spindle [41]. Furthermore, NQO1 has a diret physial interation with the mitoti fator Aurora A and promotes Aurora A degradation by antagonizing the protetive funtion of TPX2 [42]. Further investigation of the potential interation between PIG3 and the TPX2-Aurora A signal pathway during mitosis as well as its potential role(s) in regulating mirotubule assembly and hromosomal stability is learly warranted. Mirotubule dynamis-targeted hemotherapeuti agents suh as taxanes are widely used, alone or in ombination with other drugs, to treat various solid tumors suh as advaned NSCLC [43 45]. Unfortunately, primary

9 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 9 of 11 a b d f Fig. 5 Depletion of PIG3 sensitizes NSCLC ells to doetaxel through promoting apoptosis and senesene. a Forty-eight hrs following transfetion with PIG3 and ontrol sirnas, A549 ells were exposed to various onentrations of doetaxel. Cell proliferation was determined by CCK8 assay 48 h post treatment. The data are expressed as the mean and standard deviations from three independent experiments ( ** P < 0.01). b Forty-eight hrs following transfetion with PIG3 and ontrol sirnas, A549 ells were treated with 5 μg/ml doetaxel or DMSO for 24 h. Apoptoti ells were deteted using the Annexin V staining method. The data are expressed as mean and standard deviations from three independent experiments, ** P <0.01asomparedto ontrol sirna transfeted ells under similar treatment onditions of doetaxel. and d Forty-eight hrs following transfetion with PIG3 and ontrol sirnas, A549 ells were treated with different onentrations (0, 5 and 10 μg/ml) of doetaxel for 24 h or 5 μg/ml of doetaxel for indiated time intervals (0,6,16and24h).ApoptosiswasdeterminedbyPARP-1leavage(indiatedbyanarrow) following Western blot analysis. e SA-β-gal staining of NSCLC ells. PIG3 depleted and ontrol A549 ells were treated with 0, 5 and 10 μg/ml of doetaxel for 48 h and then stained for SA-β-gal ativity. f Quantitative analysis of senesent ells. The results were generated from three independent experiments ( * P < 0.05, ** P < 0.01) resistane to these agents is a major hallenge for the use of taxanes in linial appliations. Numerous studies have shown that targeting the mitoti fators may be a suessful strategy to overome the hemoresistane to taxanes in aner ells [30, 45]. In the present study, we found that a lak of PIG3 signifiantly sensitizes NSCLC ells to doetaxel, whih at least an partially be attributed to the inreased indution of apoptosis and senesene. This suggested that doetaxel may be onsidered an effetive targeted therapy against PIG3-low expressing lung aners. In this ontext, it is important to further examine whether low PIG3 expression is assoiated with linial benefits patients with NSCLC following doetaxel-based hemotherapy. Conlusion In summary, in our work we revealed that PIG3 expression levels positively orrelated with poor prognosis of NSCLC patients. Our study also demonstrated that PIG3 is a novel mitoti fator that regulated mirotubule dynamis and mitoti spindle assembly. In addition, loss of PIG3 indued phenotypes of mitoti atastrophe, whih exhibited misaligned and lagged hromosomes and led to multi-nuleated and senesent ells. Loss of PIG3 onferred sensitivity of NSCLC ells to doetaxel-based hemotherapy. These findings demonstrated a role for PIG3 in the progression of NSCLC, indiating that PIG3 may be a potential prognosti marker and novel therapeuti target for NSCLC.

10 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 10 of 11 Additional files Additional file 1: Figure S1. Loss of PIG3 inhibites ell proliferation and leads to inreased outome of misalignment hromosomes in H460 NSCLC ells. a Western blot analysis demonstrating the effiay of PIG3 sirna #1 in H460 ells at 48 h post-transfetion. b ells were seeded in 96-well plates at day 0, and CCK8 assay was used to determine ell proliferation rates at indiated days (1, 2, 3, 4 and5 days). Absorbane values at 450 nm were normalized by the value measured on day 1 ( * P <0.05, ** P <0.01). Exponentially growing PIG3 depleted and ontrol H460 ells were subjeted to the immunofluoresent staining using the indiated antibody. The representative images showing aberrant mitoti ells with misalignment hromosomes (arrowheads). d Perentages of mitoti ells showed misaligned hromosomes were ounted from three independent experiments. ** P < (PPT 3533 kb) Additional file 2: Figure S2. Overexpression PIG3 promotes NSCLC ells proliferation and inreases resistane of NSCLC ells to doetaxel. a Western blot analysis demonstrating the level of PIG3 in PIG3-overexpressed and ontrol H1299 ells. b ells were seeded in 96-well plates at day 0, and CCK8 assay was used to determine ell proliferation rates at indiated days (1, 2, 3, 4 and 5 days). Absorbane values at 450 nm were normalized by the value measured on day 1 ( * P <0.05, ** P < 0.01). PIG3 overexpressed and ontrol H1299 ells were exposed to various onentrations of doetaxel. Cell proliferation was determined by CCK8 assay 48 h post treatment. The data are expressed as the mean and standard deviations from three independent experiments ( ** P <0.01).d PIG3 overexpressed and ontrol H1299 ells were treated with 0, 5 and 10 μg/ml of doetaxel for 48 h and then stained for SA-β-gal ativity. Quantitative analysis of senesent ells. The results were generated from three independent experiments ( * P < 0.05). (PPT 298 kb) Additional file 3: Figure S3. The loalization of PIG3 in the ells deteted by immunofluoresent staining. A549 ells were treated or untreated with 4Gy γ ray irradiation. One hour post irradiation, ells were fixed and stained using anti-pig3, KAP-1 and phosphorylated H2AX antibody. (PPTX 1444 kb) Additional file 4: Figure S4. Depletion of PIG3 sensitized NSCLC ells to doetaxel. Forty eight hrs following transfetion with PIG3 and ontrol sirnas, H460 ells were exposed to various onentrations of doetaxel. Cell proliferation was determined by CCK8 assay 48 h post treatment. The data are expressed as the mean and standard deviations from three independent experiments ( ** P < 0.01). (PPT 475 kb) Aknowledgements This work was supported by Jiangsu Provinial Key Laboratory of Radiation Mediine and Protetion and the Priority Aademi Program Development of Jiangsu Higher Eduation Institutions (PAPD). Funding This work was supported by grants from the National Natural Siene Foundation of China ( , , and ) and China Postdotoral Siene Fund Program (2015 M580463) and Suzhou Key Medial Center (SZZX201506). Availability of data and materials Raw and proessed data are available from the orresponding author on reasonable request. Authors ontributions ZFS, JZ and PKZ oneived and designed the experiments. ML, SL and BL performed the experiments. MMG, SZ, and BBX oordinated the researh and analyzed the data. WQD and LY supported the experiments and helped to draft the manusript. ZFS and JZ performed the statistial analysis. ZFS wrote the manusript. All authors read and approved the final manusript. Competing interests The authors delare that they have no ompeting interests. Consent for publiation Not appliable. Ethis approval and onsent to partiipate Ethis approval was given by the Ethis Committee of Nanjing Medial University Affiliated Suzhou Hospital (Suzhou, China) for the use of linial materials for researh purposes. Author details 1 Shool of Radiation Mediine and Protetion, Medial College of Soohow University, Collaborative Innovation Center of Radiation Mediine of Jiangsu Higher Eduation Institutions, Suzhou, Jiangsu , People s Republi of China. 2 Laboratory of Medial Moleular Biology, Beijing Institute of Biotehnology, Beijing , People s Republi of China. 3 Suzhou Caner Center Core Laboratory, Nanjing Medial University Affiliated Suzhou Hospital, Suzhou, Jiangsu , People s Republi of China. 4 Department of Radiation Onology, Simmons Comprehensive Caner Center at UT Southwestern Medial Center, Dallas 75390, TX, USA. 5 Department of Pathology, University of Oklahoma Health Siene Center, Oklahoma City, OK 73104, USA. 6 Department of Radiation Toxiology and Onology, Beijing Institute of Radiation Mediine, Beijing , People s Republi of China. Reeived: 1 Otober 2016 Aepted: 21 February 2017 Referenes 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global aner statistis. CA Caner J Clin. 2011;61: Soerjomataram I, Lortet-Tieulent J, Parkin DM, Ferlay J, Mathers C, Forman D, et al. Global burden of aner in 2008: a systemati analysis of disabilityadjusted life-years in 12 world regions. Lanet. 2012;380: Mao L. Reent advanes in the moleular diagnosis of lung aner. Onogene. 2002;21: Seo AN, Yang JM, Kim H, Jheon S, Kim K, Lee CT, et al. Cliniopathologi and prognosti signifiane of -MYC opy number gain in lung adenoarinomas. Br J Caner. 2014;110: Rooney C, Sethi T. Advanes in moleular biology of lung disease: aiming for preision therapy in non-small ell lung aner. Chest. 2015;148: Rosell R, Karahaliou N. Lung aner in 2014: optimizing lung aner treatment approahes. Nat Rev Clin Onol. 2015;12: Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53- indued apoptosis. Nature. 1997;389: Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, et al. Diversifiation of transriptional modulation: large-sale identifiation and haraterization of putative alternative promoters of human genes. Genome Res. 2006;16: Contente A, Dittmer A, Koh MC, Roth J, Dobbelstein M. A polymorphi mirosatellite that mediates indution of PIG3 by p53. Nat Genet. 2002;30: Guan X, Liu Z, Wang L, Wang LE, Sturgis EM, Wei Q. Funtional repeats (TGYCC)n in the p53-induible gene 3 (PIG3) promoter and suseptibility to squamous ell arinoma of the head and nek. Carinogenesis. 2013;34: Porte S, Valenia E, Yakovtseva EA, Borras E, Shafqat N, Debrezeny JE, et al. Three-dimensional struture and enzymati funtion of proapoptoti human p53-induible quinone oxidoredutase PIG3. J Biol Chem. 2009;284: Kang MY, Kim HB, Piao C, Lee KH, Hyun JW, Chang IY, et al. The ritial role of atalase in prooxidant and antioxidant funtion of p53. Cell Death Differ. 2013;20: Lee JH, Kang Y, Khare V, Jin ZY, Kang MY, Yoon Y, et al. The p53-induible gene 3 (PIG3) ontributes to early ellular response to DNA damage. Onogene. 2010;29(10): Li B, Shang ZF, Yin JJ, Xu QZ, Liu XD, Wang Y, et al. PIG3 funtions in DNA damage response through regulating DNA-PKs homeostasis. Int J Biol Si. 2013;9: Zhang W, Luo J, Chen F, Yang F, Song W, Zhu A, et al. BRCA1 regulates PIG3-mediated apoptosis in a p53-dependent manner. Onotarget. 2015;6: Chen G, Xu JY, Chen J, Zhang JX, Zhou J, Liang Y, et al. Loss of PIG3 inreases HIF-1alpha level by promoting protein synthesis via mtor pathway in renal ell arinoma ells. Onotarget. 2016;7: Xu J, Cai J, Jin X, Yang J, Shen Q, Ding X, et al. PIG3 plays an onogeni role in papillary thyroid aner by ativating the PI3K/AKT/PTEN pathway. Onol Rep. 2015;34:

11 Li et al. Journal of Experimental & Clinial Caner Researh (2017) 36:39 Page 11 of Zhao J, Kim JE, Reed E, Li QQ. Moleular mehanism of antitumor ativity of taxanes in lung aner. Int J Onol. 2005;27: Li M, Ma Y, Huang P, Du A, Yang X, Zhang S, et al. Lentiviral DDX46 knokdown inhibits growth and indues apoptosis in human oloretal aner ells. Gene. 2015;560: Chen P, Zhu J, Liu DY, Li HY, Xu N, Hou M. Over-expression of survivin and VEGF in small-ell lung aner may predit the poorer prognosis. Med Onol. 2014;31: Shang Z, Yu L, Lin YF, Matsunaga S, Shen CY, Chen BP. DNA-PKs ativates the Chk2-Bra1 pathway during mitosis to ensure hromosomal stability. Onogenesis. 2014;3:e Ganem NJ, Storhova Z, Pellman D. Tetraploidy, aneuploidy and aner. Curr Opin Genet Dev. 2007;17: Mason PJ, Bessler M. Cytokinesis failure and attenuation: new findings in Fanoni anemia. J Clin Invest. 2011;121: Sharpless NE, Sherr CJ. Forging a signature of in vivo senesene. Nat Rev Caner. 2015;15: Rao CV, Yamada HY, Yao Y, Dai W. Enhaned genomi instabilities aused by deregulated mirotubule dynamis and hromosome segregation: a perspetive from geneti studies in mie. Carinogenesis. 2009;30: Yvon AMC, Wadsworth P, Jordan MA. Taxol suppresses dynamis of individual mirotubules in living human tumor ells. Mol Biol Cell. 1999;10: Kotsinas A, Aggarwal V, Tan EJ, Levy B, Gorgoulis VG. PIG3: a novel link between oxidative stress and DNA damage response in aner. Caner Lett. 2012;327: Takai N, Hamanaka R, Yoshimatsu J, Miyakawa I. Polo-like kinases (Plks) and aner. Onogene. 2005;24: Gavriilidis P, Giakoustidis A, Giakoustidis D. Aurora Kinases and Potential Medial Appliations of Aurora Kinase Inhibitors: A Review. J Clin Med Res. 2015;7: Shang ZF, Yu L, Li B, Tu WZ, Wang Y, Liu XD, et al. 4E-BP1 partiipates in maintaining spindle integrity and genomi stability via interating with PLK1. Cell Cyle. 2012;11: Shang ZF, Huang B, Xu QZ, Zhang SM, Fan R, Liu XD, et al. Inativation of DNA-dependent protein kinase leads to spindle disruption and mitoti atastrophe with attenuated hekpoint protein 2 Phosphorylation in response to DNA damage. Caner Res. 2010;70: Lee KJ, Lin YF, Chou HY, Yajima H, Fattah KR, Lee SC, et al. Involvement of DNA-dependent protein kinase in normal ell yle progression through mitosis. J Biol Chem. 2011;286: Douglas P, Ye R, Trinkle-Mulahy L, Neal JA, De Wever V, Morrie NA, et al. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNAdependent protein kinase atalyti subunit (DNA-PKs) phosphorylation in mitosis. Biosi Rep. 2014;34:e Lee KJ, Shang ZF, Lin YF, Sun J, Morotomi-Yano K, Saha D, et al. The Catalyti Subunit of DNA-Dependent Protein Kinase Coordinates with Polo- Like Kinase 1 to Failitate Mitoti Entry. Neoplasia. 2015;17: Huang B, Shang ZF, Li B, Wang Y, Liu XD, Zhang SM, et al. DNA-PKs assoiates with PLK1 and is involved in proper hromosome segregation and ytokinesis. J Cell Biohem. 2014;115: Yu L, Shang ZF, Hsu FM, Zhang Z, Tumati V, Lin YF, et al. NSCLC ells demonstrate differential mode of ell death in response to the ombined treatment of radiation and a DNA-PKs inhibitor. Onotarget. 2015;6: Ertyh N, Stolz A, Stenzinger A, Weihert W, Kaulfuss S, Burfeind P, et al. Inreased mirotubule assembly rates influene hromosomal instability in oloretal aner ells. Nat Cell Biol. 2014;16: Nowak MA, Komarova NL, Sengupta A, Jallepalli PV, Shih Ie M, Vogelstein B, et al. The role of hromosomal instability in tumor initiation. Pro Natl Aad Si U S A. 2002;99: Testa JR, Liu Z, Feder M, Bell DW, Balsara B, Cheng JQ, et al. Advanes in the analysis of hromosome alterations in human lung arinomas. Caner Genet Cytogenet. 1997;95: Nakamura H, Saji H, Idiris A, Kawasaki N, Hosaka M, Ogata A, et al. Chromosomal instability deteted by fluoresene in situ hybridization in surgial speimens of non-small ell lung aner is assoiated with poor survival. Clin Caner Res. 2003;9: Siegel D, Kepa JK, Ross D. NAD(P)H:quinone oxidoredutase 1 (NQO1) loalizes to the mitoti spindle in human ells. PLoS One. 2012;7:e Park MT, Oh ET, Song MJ, Lee H, Choi EK, Park HJ. NQO1 prevents radiationindued aneuploidy by interating with Aurora-A. Carinogenesis. 2013;34: Janne PA, Smith I, MWalter G, Mann H, Dougherty B, Walker J, et al. Impat of KRAS odon subtypes from a randomised phase II trial of selumetinib plus doetaxel in KRAS mutant advaned non-small-ell lung aner. Br J Caner. 2015;113: Ham SY, Kwon T, Bak Y, Yu JH, Hong J, Lee SK, et al. Muin 1-mediated hemo-resistane in lung aner ells. Onogenesis. 2016;5:e Brodie SA, Li G, Harvey D, Khuri FR, Vertino PM, Brandes JC. Small moleule inhibition of the CHFR-PARP1 interation as novel approah to overome intrinsi taxane resistane in aner. 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