Abstract
Objective: Abnormalities in the STAT3 pathway are involved in the oncogenesis of several cancers. However, the mechanism by which dysregulated STAT3 signaling inhibited the progression of human retinoblastoma (RB) has not been elucidated. To investigate the role of targeting STAT3 in RB progression, we inhibited STAT3 with Y79 cells and depleted STAT3 with a siRNA. Results: Our results demonstrate that targeting STAT3 inhibited survival and induced apoptosis of Y79 cells through downregulation of Slug and upregulation of PUMA. In addition, targeting STAT3 inhibited invasion and migration of Y79 cells through downregulation of Slug and upregulation of E-cadherin. Furthermore, Slug overexpression inhibited PUMA and E-cadherin upregulation in the Y79 cells with targeting STAT3. Targeting PUMA and E-cadherin reversed the role of targeting STAT3 in the Y79 cells. Conclusions: our findings showed that targeting STAT3 inhibited survival, invasion, and migration in Y79 cells through inactivation of Slug, resulting in the upregulation of PUMA and E-cadherin, and provide novel evidence that the STAT3/Slug pathway may be a new potential target for therapy of RB.
Keywords: Retinoblastoma, apoptosis, invasion, Stat3, Slug, PUMA, E-cadherin
Introduction
Retinoblastoma is the most common intraocular tumor in children worldwide. Although it is highly curable when detected early, the mortality rate is as high as 70% in less-developed countries [1]. This poor outcome is largely due to late detection likely related to socioeconomic disparities. Additionally, 23% of eyes requiring enucleation in the United States display high-risk histopathologic features [2]. These features include invasion of the optic nerve beyond the lamina cribrosa, which is a known metastatic risk factor [3]. Advanced stage retinoblastoma is challenging to treat because retinoblastomas can rapidly fill the eye, invade the optic nerve, and eventually spread to the central nervous system, thus becoming fatal. Understanding the molecular regulation of tumor cell invasion and apoptosis helps in identifying new therapeutic targets.
STAT3 is activated in 70% of all solid and hematological tumors, where it stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation [4]. Recently, STAT3 also was found to have an important role in maintaining cancer stem cells, both in vitro and in mouse tumor models, indicating that it is integrally involved in tumor initiation, progression, and maintenance [5]. It has found that STAT3 activation in retinoblastoma mediated various cellular events including the expression of genes related with anti-apoptotic activity and migration/invasion [6]. Although targeting STAT3 activation inhibited invasion and growth of RB cells, the underling mechanisms are still very unclear.
Slug belongs to the highly conserved Slug/Snail family of zinc-finger transcription factors found in diverse species ranging from Caenorahbditis elegans to humans [7]. Slug was found to promote cell survival, and not EMT, during kidney tubulogenesis, as shown by persistent E-cadherin expression [8]. Similarly, in hematopoietic progenitor cells, where EMT is not involved, Slug was found to promote survival by blocking apoptosis in response to DNA damage [9]. Namely, Slug knockout mice succumbed to γ-irradiation, due to apoptosis of hematopoietic progenitor cells, thus impairing regenerative potential. In this case, Slug induced apoptosis by repressing the p53 pro-apoptotic target gene, Puma [9]. Puma (BBC3), or p53-upregulated modulator of apoptosis, is a BH3-only member of the Bcl-2 family and a target of p53-mediated apoptosis. It activates an apoptotic cascade by facilitating Bax activation, causing cytochrome C release from the mitochondria, caspase-3 activation and DNA fragmentation [10].
Lo et al. has reported that EGF/EGFR signaling pathways induce cancer cell EMT via STAT3-mediated TWIST gene expression [11]; Liu et al. has reported that STAT3-knockdown inhibited CRC cell aggressiveness by regulating the expression of EMT-promoting factors (ZEB1, Snail, Slug, MMP-2 and MMP-9) [12]; Kang et al. has reported that JAK2/Stat3/Slug signaling pathway affected cell growth and apoptosis [13]. We therefore suggested tha tSTAT3 mediated mechanism underlying constitutive activation of Slug pathways in human cancers.
In this present study, we studied the roles of targeting STAT3 on invasion, apoptosis and growth in RB Y79 cells in vitro. We have found that downregulation of STAT3 could reduce cell migration, invasion and proliferation significantly in Y79 cells. In addition, downregulation of STAT3 significantly induced apoptosis in Y79 cells. These results could be attributed to the functions of STAT3 in the regulation of PUMA and E-cadherin via STAT3/Slug pathway. In conclusion, our data indicated that targeting of STAT3 is the therapeutic manipulation in the future for multimodal management of RB.
Materials and methods
Cell culture
Human retinoblastoma Y79 cells were purchased from the American Type Culture Collection. Y79 cells were grown in RPMI-1640 (Thermo Scientific, Hangzhou, China) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co., Hangzhou, China). The cell line was cultured at 37°C in a 5% CO2 atmosphere.
Gene silencing and overexpression
Small inference RNA against STAT3 (STAT3 siRNA), PUMA (PUMA siRNA), E-cadherin (E-cadherin siRNA) and scrambled siRNA were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The full-length Slug open reading frames was amplified and cloned into the pcDNA3.1 vector to generate pcDNA3.1-Slug constructs. Sequences of primers used for PCR amplification were 5’-GAGTCTGTAATAGGATTTCCC-3’, 5’-GGGAAATCCTATTACAGACTC-3’. The generated constructs of Slug were then verified by sequencing (Beijing Tianyi Huiyuan Bioscience & Technology Inc., Beijing, China). The empty pcDNA3.1 construct was used as negative control.
Y79 cells were placed in a serum-free medium into six-well plates immediately before transfection. The cells were incubated with STAT3 siRNA precomplexed with LipofectamineTM 2000 (Invitrogen, Hangzhou, China) in an Opti-MEM medium (Invitrogen, United States) at 37°C for 4 h according to the manufacturer’s protocol. Then the medium was replaced with a supplemental medium containing 10% FBS, and the cells were cultivated for 24-96 h under standard conditions.
To establish Y79 cell lines stably expressing STAT3 siRNA, we transfected STAT3 siRNA plasmids into Y79 cells for 48 hrs, and cultured cells in the presence of 1 μg/ml of puromycin. Colonies resistant to puromycin appeared within 2 weeks, after which the cells were expanded for another 3 weeks to make the original stock cells.
To determine the signaling pathways of STAT3 involved in the production of Slug, PUMA and E-cadherin, the stable STAT3 siRNA transfected Y79 cells were transfected with pCDNA3.1-Slug cDNA plasmid or PUMA siRNA or E-cadherin or scrambled siRNA or pCDNA3.1 48 h using Lipofectamine 2000 according to the manufacturer’s instructions above.
Western blot analysis
Cells were washed with cold PBS, lysed with ice-cold lysis buffer and incubated on ice for 30 min. Lysates were centrifuged, supernatants were collected, and protein concentration was determined using Bio-Rad Protein Reagents (Bio-Rad, Hercules, CA). Protein lysates (30 μg) were separated by SDS-PAGE, blotted onto membranes, and probed with the appropriate dilution of each primary antibody (STAT3, Slug, PUMA and E-cadherin). Membranes were rinsed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, rinsed again, and the bound antibodies were detected using enhanced chemiluminescence (GE Healthcare, Piscataway, NJ) following by autoradiography in a FluorChemTM 8900 (Alpha Innotech Corporation, San Leandro, CA).
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total cellular RNA was isolated from each well at different time points after siRNA/cDNA transfection using an RNeasy Mini Kit (Qiagen). Up to 4 micrograms of RNA were used to make cDNA with the SuperScript III 1st strand RT kit for PCR (Invitrogen). PCR primers were designed for STAT3 (5’-GGC CCA ATG GAA TCA GCT ACA G-3’, 5’-GAA GAA ACT GCT TGA TTC TTC G-3’), Slug (5’-CACTGTGTGGACTACCGCT-3’, 5’-TGGAGGAGGTGTCAGATGG-3’), PUMA (5’-ATG GCG GAC GAC CTC AAC-3’, 5’-AGT CCC ATG AAG AGA TTG TAC ATG AC-3’), E-cadherin (5’-GAC GCG GAC GAT GTG AAC-3’, 5’-TTG TAC GTG GTG GGA TTG AAG A-3’) and β-actin (5’-GTG GGC CGC TCT AGG CAC CA-3’, 5’-CGG TTG GCC TTA GGG TTC AGG GGG G-3’) using Primerquest software and purchased from Integrated DNA Technologies (IDT). PCR was performed by the manufacture’s instruction.
Cell viability assay
The cells were seeded onto 96-well plates at a density of approximate 2×104 cells per well and incubated at 37°C in a 5% CO2 humid incubator for 24 hours. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell viability at 24, 48, 72 hours after the cells were transfected with STAT3 siRNA. To determine the role of PUMA in STAT3 siRNA induced cell viability, Y79 cells were transfected with PUMA siRNA for 24 h, then transfected with STAT3 siRNA, cell viability was detected at 24, 48, 72 hours after the cells were transfected with STAT3 siRNA. We measured absorbance at 570 nm using a Quant Universal Microplate Spectrophotometer (BioTek, Winooski, VT).
Annexin V/PI staining assay
Annexin V/PI staining was performed using Tali Apoptosis Kit (Invitrogen, Hangzhou, China). After treatment, cells were trypsinised, centrifuged for 15 min at 1000 rpm (95 rcf) and cell pellet was dissolved in 100 µl Annexin binding buffer (5×105-5×106 cells/ml). To each 100 µl of samples, 5 µl of annexin V were added and after thorough mixing, samples were incubated at RT for 20 min. Cells were then centrifuged for 15 min at 1000 rpm (95 rcf) and supernatant was discarded. Cell pellet was resuspended in 100 µl of Annexin binding buffer and 1 µl of Propidium Iodide (PI). After incubating the samples for 5 min at RT in the dark, they were transferred in Tali cellular analysis slides. Analysis of apoptosis was performed using Tali image-based cytometer (Invitrogen). The annexin V positive/PI negative cells were recognized as early apoptotic cells by the cytometer software whereas the annexin V positive/PI positive cells were identified as late apoptotic/dead cells. Similarly, the annexin V negative/PI negative cells were identified as viable cells. The baseline apoptosis varied between 5 and 15% among the various apoptosis-related experiments performed.
Boyden chamber transwell migration and invasion assay
The Y79 cell lines, stable STAT3 siRNA transfected Y79 cell, stable STAT3 siRNA/E-cadherin siRNA transfected Y79 cell and scrambled siRNA transfected Y79 cells were used in cell migration and invasion analyses performed by Boyden chamber assays. For determining cell migration, 2.5×105 cells were seeded into each transwell chamber with filter membranes of 12 μm pore size (Millipore). For invasion, filter membranes were coated with Matrigel (BD Biosciences; diluted 1:3 in growth medium) 4 hours before seeding 5×105 cells, and fresh medium was added to the bottom chamber. After 48 hours, inserts were removed and cells which had migrated through the membrane to the lower chamber were trypsinized and counted in a Neubauer chamber (LO-Laboroptik). Each well was counted ten times. Each migration or invasion experiment was performed in duplicate. The average number of migrated or invaded cells was determined from at least three independent experiments.
Statistics
Data are presented as the mean ± SD unless otherwise indicated. Statistical significance was determined by a paired or unpaired two-tailed Student’s t test, and a P value of less than 0.05 was considered statistically significant.
Results
Silencing of STAT3 gene by siRNAs
To silence STAT3 expression, we used 3 siRNAs targeting different mRNA regions of STAT3. Silencing effects of these STAT3 siRNAs were examined in Y79 cells at a concentration of 50 nM after complexation with Lipofectamine-2000. A scrambled siRNA that does not target any gene was used as the negative control. All 3 siRNAs showed a significant silencing effect (P < 0.05) and knocked down 63.5 to 71.6% of STAT3 mRNA in comparison with scrambled siRNA (Figure 1A). Among them, STAT3 siRNA2 showed the greatest suppression of STAT3 and therefore STAT3 siRNA2 (named STAT3 siRNA) was selected for subsequent biological studies. The silencing effect of STAT3 expression at the protein level was also confirmed with western blot. As shown in Figure 1B, STAT3 siRNA2 significantly inhibited the STAT3 protein expression inY79 cells, which is consistent with the silencing effect at the mRNA level.
Figure 1.
Silencing effect of STAT3 siRNAs in Y79 cells. A. Y79 cells were transfected with 3 STAT3 siRNAs and negative control siRNA (NC) at a concentration of 50 nM. Cells were harvested 48 hours after the transfection, and the silencing effect at the STAT3, Slug, PUMA and E-cadherin mRNA level was determined using RT-PCR. B. The silencing effect of STAT3 siRNA at the protein level was determined using western blot.
We also detected the mRNA and protein expression of Slug, PUMA and E-cadherin by RT-PCR and Western blot assay. As shown in Figure 1, STAT3 siRNA1-3 significantly inhibited the Slug protein expression, and promoted PUMA and E-cadherin protein expression in Y79 cells, which is consistent with the silencing effect at the mRNA level.
Silencing of STAT3 upregulates PUMA and E-cadherin expression via Slug signals
To determine whether the knockdown of STAT3 gene affects the Slug expression in Y79 cells, the Y79 cells were stably transfected with STAT3 siRNA, followed by western blot and RT-PCR assay. As shown in Figure 2, the STAT3 and Slug expression was reduced in the Y79 cells (Figure 2A, 2B). However, PUMA and E-cadherin expression was significantly increased in the STAT3 siRNA stably transfected Y79 cells compared to the control siRNA transfected Y79 cells (Figure 2A, 2B).
Figure 2.
Silencing of STAT3 leads to reduction of Slug and increase of PUMA and E-cadherin in Y79 cells. A. The mRNA of STAT3, Slug, PUMA and E-cadherin was detected by RT-PCR assay. B. The protein of STAT3, Slug, PUMA and E-cadherin was detected by western blot assay. NC, negative control siRNA.
To determine whether PUMA and E-cadherin was STAT3/Slug dependent regulation, the STAT3 siRNA stably transfected Y79 cellswere transfected with pcDNA3.1 Slug or pcDNA3.1 for 48 hours, followed by western blot and RT-PCR assay. As shown in Figure 2A, 2B, with the restoration of Slug expression, PUMA and E-cadherin expression was significantly decreased in the STAT3 siRNA stably transfected Y79 cells compared to the control siRNA transfected Y79 cells. This result therefore suggests that STAT3 regulated Slug dependent PUMA and E-cadherin expression.
Targeting STAT3 inhibits cell proliferation and induces apoptosis of Y79 cells
To determine whether STAT3 downregulation affects Y79 cell viability in vitro, Y79 cells were transiently transfected with STAT3 siRNA for 24-96 h. Cell viability was assessed by MTT assay 4 days after transfection. As shown in Figure 3A, targeting STAT3 shows the gradually decreasing growth rate of Y79 cells with 96 h STAT3 siRNA transfection, suggesting that targeting STAT3 decreased cell proliferation rate in Y79 cells.
Figure 3.
Targeting STAT3 mediates PUMA dependent pro-apoptotic and anticancer effects. Y79 cells were transfected with STAT3 siRNA or co-transfected with PUMA siRNA or control siRNA for 24-72 h. A. Cell viability was detected by MTT assay; B. Apoptosis was analyzed by annexin V/PI staining followed by flow cytometry. The percentages of annexin-positive apoptotic cells are indicated in the two right quadrants. C. The mRNA of PUMA was detected by RT-PCR assay. D. The protein of PUMA was detected by western blot assay. *P < 0.05.
Furthermore, targeting STAT3 shows the gradually increasing apoptotic rate of Y79 cells with 96 h STAT3 siRNA transfection confirmed by FCM, suggesting that targeting STAT3 decreased cell proliferation rate by inducing apoptosis in Y79 cells (Figure 3B).
Targeting STAT3 is dependent on PUMA to induce apoptosis in Y79 cells
We then investigated the role of PUMA in targeting STAT3-induced apoptosis in Y79 cell lines. Y79 cells were transfected with PUMA siRNA for 24 h, then transfected with STAT3 siRNA for 24-72 h. As shown in Figure 3C, 3D, the PUMA expression was reduced in cells treated with PUMA siRNA in comparison with cells treated with scrambled siRNA. MTT assay confirmed that cell viability was reversed in cells treated with PUMA siRNA in comparison with cells treated with scrambled siRNA (Figure 3A). Annexin V/PI staining confirmed targeting PUMA in Y79 cells abrogated STAT3 siRNA-induced apoptosis (Figure 3B).
Targeting STAT3 decreases cell invasion and migration
To further demonstrate the effect of STAT3 knockdown, Y79 cells were subjected to invasion and migration assay. Compared with control cells, the migration and invasion abilities of Y79 cells transfected with STAT3 siRNA were dramatically decreased as revealed by Boyden chamber assays (Figure 4A, 4B). The above results demonstrate that targeting STAT3 suppresses cell migration and invasion in vitro, indicating that STAT3 may play a key role in promoting cancer metastasis of Y79 cells.
Figure 4.
Targeting STAT3 mediated E-cadherin dependent migration and invasion of Y79 cells. Y79 cells were transfected with STAT3 siRNA or co-transfected with E-cadherin siRNA or control siRNA for 48 h. The migration and invasion of Y79 cells were detected by transwell migration (A) and matrigel invasion assays (B), respectively. Migrated or invaded cells were counted in five random fields of each filter under a microscope (IX71; OLYMPUS) using a 200× magnification. The scale bar indicates 50 μm. Bars represent the average number of migrated or invaded cells. *P < 0.05 compared with respective controls. The mRNA of E-cadherin was detected by RT-PCR assay (C). The protein of E-cadherin was detected by western blot assay (D).
Targeting STAT3 is dependent on activation of E-cadherin to inhibit the migration and invasion of Y79 cells
We found that the effect of targeting STAT3 inhibited cell migration and invasion, but the mechanisms remained largely unknown. We had shown above that targeting STAT3 regulated Slug-dependent E-cadherin expression. To further investigate the molecular mechanisms of targeting STAT3 on migration and invasion in Y79 cells, we targeted the E-cadherin in the STAT3 siRNA transfected Y79 cells, and detected the expression of E-cadherin was detected by Western blotting and/or RT-PCR. The results showed that knockdown of E-cadherin inhibited STAT3 siRNA-induced E-cadherin upregulation (Figure 4C, 4D). Furthermore, the migration and invasion of STAT3 siRNA transfected Y79 cells were rescued by E-cadherin siRNA transfection compared with that of the control cells (Figure 4A, 4B). The results further indicate that targeting STAT3 inhibited the migration and invasion of Y79 cells through upregulation E-cadherin expression.
Discussion
STAT3 is activated in many solid and hematological tumors, where it stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. Hence in the current study, we analyzed the importance of STAT3 in RB cells using short interfering RNA (siRNA) mediated knockdown studies. Our results showed that silencing of STAT3, the growth rate of RB cell lines was significantly reduced and the rate of cell apoptosis was significantly increased. Furthermore, silencing of STAT3 inhibited migration and invasion of RB cells. Correspondingly, the expression levels of zinc-finger transcription factor Slug was downregulated and PUMA and E-cadherin was up-regulated after silencing of STAT3. These results indicated that STAT3 knockdown had a tumor suppressive effect, potentially by an induction of cell apoptosis and inhibition of cell invasion through inhibition of Slug signals.
STAT3 is an important signaling node that is involved in multiple pathways including inflammation, differentiation, proliferation or metastasis through the activation of target genes following the translocation of p-STAT3. Our data provide the mechanisms by which STAT3 suppresses tumor invasion in RB cell lines. In Y79 cell line, targeting STAT3 suppresses invasiveness by limiting expression of Slug. Previous study has reported that overexpression of a dominant negative STAT3 mutant in a CRC cell line was reported to cause down-regulation of E-cadherin, and targeting STAT3 cause upregulation of E-cadherin [14]. In our study, targeting STAT3 downregulated Slug and upregulated E-cadherin expression in the Y79 cells. Furthermore, overexpression of Slug suppresses E-cadherin in Y79 cells after targeting STAT3.Therefore, it is possible that targeting STAT3 inhibited invasion of Y79 cells through Slug/E-cadherin signals.
In colitis-associated cancer, STAT3 activation in IEC regulates cell survival and cell cycle progression through its transcriptional activation of downstream targets such as Bcl-XL, c-Myc, and cyclin D1 [15,16]. Induction of p53 pro-apoptotic target gene PUMA can inhibit all antiapoptotic Bcl-2 family members, and activate the intrinsic apoptotic pathway [17]. Wu et al. has reported that targeting Slug induced apoptosis by inducing PUMA [9]. In our study, the observed STAT3 depletion-mediated increase in PUMA was inversely correlated with p-STAT3 levels. Furthermore, the overexpression of Slug rescued Y79 cells from the induction of PUMA with the STAT3 knockdown. Furthermore, STAT3 knockdown sensitized Y79 cells to apoptosis, effects that were reversed by PUMA knockdown. Consistent with inhibition of Puma by Slug, STAT3 knockdown in Y79 cells caused increased Puma expression, whereas silencing Puma in STAT3-knockdown cells inhibited apoptosis. Therefore, it is possible that targeting STAT3 induced apoptosis of Y79 cells through Slug/PUMA signals.
In conclusion, we have shown that targeting STAT3 induces apoptosis and inhibits invasion through the regulation of STAT3/Slug/PUMA axis and STAT3/Slug/E-cadherin axis in Y79 cells in vitro. Therefore, intervention in STAT3/Slug signaling may have potential therapeutic value in the treatment of human RB.
Acknowledgements
This study was supported by grants from National Natural Science Foundation of China (No: 81170852).
Disclosure of conflict of interest
None.
References
- 1.Kivela T. The epidemiological challenge of the most frequent eye cancer: retinoblastoma, an issue of birth and death. Br J Ophthalmol. 2009;93:1129–1131. doi: 10.1136/bjo.2008.150292. [DOI] [PubMed] [Google Scholar]
- 2.Kaliki S, Shields CL, Rojanaporn D, Al-Dahmash S, McLaughlin JP, Shields JA. High-risk retinoblastoma based on international classification of retinoblastoma: analysis of 519 enucleated eyes. Ophthalmology. 2013;120:997–1003. doi: 10.1016/j.ophtha.2012.10.044. [DOI] [PubMed] [Google Scholar]
- 3.Shields CL, Shields JA, Baez K, Cater JR, De Potter P. Optic nerve invasion of retinoblastoma. Metastatic potential and clinical risk factors. Cancer. 1994;73:692–698. doi: 10.1002/1097-0142(19940201)73:3<692::aid-cncr2820730331>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
- 4.Lavecchia A, Di Giovanni C, Cerchia C. Novel inhibitors of signal transducer and activator of transcription 3 signaling pathway: an update on the recent patent literature. Expert Opin-Ther Pat. 2014;24:383–400. doi: 10.1517/13543776.2014.877443. [DOI] [PubMed] [Google Scholar]
- 5.Xiong A, Yang Z, Shen Y, Zhou J, Shen Q. Transcription factor STAT3 as a novel molecular target for cancer prevention. Cancers (Basel) 2014;6:926–957. doi: 10.3390/cancers6020926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Jo DH, Kim JH, Cho CS, Cho YL, Jun HO, Yu YS, Min JK, Kim JH. STAT3 inhibition suppresses proliferation of retinoblastoma through downregulation of positive feedback loop of STAT3/miR-17-92 clusters. Oncotarget. 2014;5:11513–11525. doi: 10.18632/oncotarget.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Hemavathy K, Ashraf SI, Ip YT. Snail/slug family of repressors: slowly going into the fast lane of development and cancer. Gene. 2000;257:1–12. doi: 10.1016/s0378-1119(00)00371-1. [DOI] [PubMed] [Google Scholar]
- 8.Leroy P, Mostov KE. Slug is required for cell survival during partial epithelial-mesenchymal transition of HGF-induced tubulogenesis. Mol Biol Cell. 2007;18:1943–52. doi: 10.1091/mbc.E06-09-0823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Wu WS, Heinrichs S, Xu D, Garrison SP, Zambetti GP, Adams JM, Look AT. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell. 2005;123:641–53. doi: 10.1016/j.cell.2005.09.029. [DOI] [PubMed] [Google Scholar]
- 10.Hikisz P, Kiliańska ZM. PUMA, a critical mediator of cell death--one decade on from its discovery. Cell Mol Biol Lett. 2012;17:646–69. doi: 10.2478/s11658-012-0032-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y, Abbruzzese JL, Hortobagyi GN, Hung MC. Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition incancer cells via up-regulation of TWIST gene expression. Cancer Res. 2007;67:9066–9076. doi: 10.1158/0008-5472.CAN-07-0575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Liu H, Ren G, Wang T, Chen Y, Gong C, Bai Y, Wang B, Qi H, Shen J, Zhu L, Qian C, Lai M, Shao J. Aberrantly expressed Fra-1 by IL-6/STAT3 transactivation promotes colorectal cancer aggressiveness through epithelial-mesenchymal transition. Carcinogenesis. 2015;36:459–68. doi: 10.1093/carcin/bgv017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kang FB, Wang L, Jia HC, Li D, Li HJ, Zhang YG, Sun DX. B7-H3 promotes aggression and invasion of hepatocellular carcinoma by targeting epithelial-to-mesenchymal transition via JAK2/STAT3/Slug signaling pathway. Cancer Cell Int. 2015;15:45. doi: 10.1186/s12935-015-0195-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Rivat C, De Wever O, Bruyneel E, Mareel M, Gespach C, Attoub S. Disruption of STAT3 signaling leads to tumor cell invasion through alterations of homotypic cell-cell adhesion complexes. Oncogene. 2004;23:3317–27. doi: 10.1038/sj.onc.1207437. [DOI] [PubMed] [Google Scholar]
- 15.Bollrath J, Phesse TJ, von Burstin VA, Putoczki T, Bennecke M, Bateman T, Nebelsiek T, Lundgren-May T, Canli O, Schwitalla S, Matthews V, Schmid RM, Kirchner T, Arkan MC, Ernst M, Greten FR. gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell. 2009;15:91–102. doi: 10.1016/j.ccr.2009.01.002. [DOI] [PubMed] [Google Scholar]
- 16.Grivennikov SI, Karin M. Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev. 2010;21:11–19. doi: 10.1016/j.cytogfr.2009.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Yu J, Zhang L. PUMA, a potent killer with or without p53. Oncogene. 2008;27(Suppl 1):S71–83. doi: 10.1038/onc.2009.45. [DOI] [PMC free article] [PubMed] [Google Scholar]