Overexpression of Aiolos promotes epithelial-mesenchymal transition and cancer stem cell-like properties in lung cancer cells

Aiolos/Ikaros family zinc finger 3 (IKZF3), a member of the Ikaros family of lymphocyte maturation-driving transcription factors, is highly expressed in hematopoietic malignancies. However, its role in epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties in lung cancer remains unknown. Human lung cancer cell lines H1299 with overexpressing Aiolos (H1299-Aiolos) and A549 with overexpressing Aiolos (A549-Aiolos) were generated by stable transfection. Cell migration and invasion assays were done to demonstrate their invasion and migration ability. Sphere formation assay was used to determine their tumor-initiating capability. Aiolos overexpression induced EMT and increased migration/invasiveness in H1299 and A549 cells. Aiolos overexpression also increased metastatic ability in vivo. Aiolos overexpression upregulated the expression of Twist and matrix metalloproteinase 16 (MMP16). By using knockdown of Twist or an inhibitor of phosphatidylinositol (PI) 3-kinase, EMT, migration/invasiveness ability, and MMP16 expression were reversed in H1299-Aiolos and A549-Aiolos cells. Overexpression of Aiolos upregulated the CSC-like properties in lung cancer cells, and were also reversed by an inhibitor of PI 3-kinase. For lung cancer cells, Aiolos overexpression promotes EMT and CSC-like properties through upregulating the PI 3-kinase/Akt pathway. The information is helpful for developing therapeutic strategies targeting Aiolos expression for lung cancer treatment.

www.nature.com/scientificreports www.nature.com/scientificreports/ preserved cell-cell adhesion and polarity. However, H1299-Aiolos and A549-Aiolos cells showed high percentage of spindle cell-like appearance and more dispersed (data not shown). Then Boyden chamber migration assay was performed to demonstrate whether migration increases in H1299-Aiolos and A549-Aiolos cells. The results revealed that the migration increased in H1299-Aiolos (Fig. 1C,D) and A549-Aiolos cells (Fig. 1E). Matrigel invasion assay was also performed to demonstrate whether invasiveness increases in H1299-Aiolos and A549-Aiolos cells. The results demonstrated that the invasiveness increased in H1299-Aiolos (Fig. 1D) and A549-Aiolos cells (Fig. 1E). We further performed tail vein metastasis assay to demonstrate whether Aiolos overexpression increased metastasis in vivo. The mice being injected with H1299-Aiolos cells had significantly more pulmonary nodules than did those with H1299-Mock cells sixteen weeks after injection ( Fig. 2A,B). H&E stain has been done to confirm the histology of the pulmonary metastatic nodules (Fig. 2C). All the above results showed that Aiolos overexpression induces EMT and increases metastatic ability in lung cancer cells.
Aiolos overexpression upregulates Twist/MMP16 expression. The protein expression of EMT markers, including Twist, Snail, and Slug, were examined in H1299-Aiolos vs. H1299-Mock cells and in A549-Aiolos vs. A549-Mock cells using Western blot analysis. Twist protein expression was upregulated by Aiolos overexpression (Fig. 3A). Twist mRNA expression was also upregulated by Aiolos overexpression (Supplementary Figs 2D and 3D). We have performed microarray of H1299-Mock and H1299-Aiolos cells (data not shown). MMP16 was among the top uregulated genes in H1299-Aiolos as compared with H1299-Mock cells. We then confirmed the expression of MMP16 in H1299-Aiolos and A549-Aiolos cells by Western blot analysis. The expression of MMP16 was upregulated in H1299-Aiolos and A549-Aiolos cells (Fig. 3A). All these results showed that Aiolos overexpression upregulates the Twist/MMP16 expression, and leads to induction of EMT in lung cancer cells.
We further examined whether the PI 3-kinase-specific inhibitor, LY294002, blocked the upregulation of Twist/ MMP16 in H1299-Aiolos and A549-Aiolos cells. The results demonstrated that inhibition of PI 3-kinase lead to decreased expression of phosphorylated-Akt (Ser473) and Twist, reversion of EMT markers, and decreased expression of MMP16 in H1299-Aiolos cells (Fig. 4A). Inhibition of PI 3-kinase reversed migration/invasion www.nature.com/scientificreports www.nature.com/scientificreports/ ability increased by Aiolos overexpression in H1299-Aiolos cells (Fig. 4B). In A549-Aiolos cells, inhibition of PI 3-kinase also lead to repression of phosphorylated-Akt (Ser473) and Twist expression, reversion of EMT markers, and decreased expression of MMP16 (Fig. 4C). Inhibition of PI 3-kinase also reversed migration/invasion ability increased by Aiolos overexpression in A549-Aiolos cells (Fig. 4D). All the above results demonstrated that the EMT induced by Aiolos overexpression was regulated through the PI 3-kinase/Akt/Twist axis in lung cancer cells.
To demonstrate the association between Aiolos and Twist expression in human lung adenocarcinoma, immunohistochemical analyses of Aiolos and Twist expression were performed in 93 lung adenocarcinoma samples. Representative immunohistochemical staining of Aiolos and Twist is shown in Supplementary Fig. 4. Expression of Aiolos was shown in 47 (50.5%) of the 93 lung tumor samples. Aiolos expression was significantly associated with Twist expression (P = 0.005) ( Supplementary Fig. 4).

overexpression of Aiolos upregulates the CsC-like properties in lung cancer cells.
We further investigated the impact of Aiolos on tumor-initiating capability of lung cancer cells. First, we performed sphere formation assay to evaluate the tumor-initiating capability of the cells when Aiolos was overexpressed. In H1299-Aiolos cells, there was a significant increase in formation of spheroids as compared with H1299-Mock cells, which indicates that the cells were capable of initiating tumors when Aiolos was overexpressed (Fig. 5A). There was also a significant increase in formation of spheroids in A549-Aiolos cells as compared with A549-Mock cells (Fig. 5A). Next, we focused to determine the expression of lung cancer CSC surface markers CD44 and CD133 in these lung cancer cells. Both of CD44 and CD133 have been reported to be enriched in lung cancer CSCs. Although we have performed microarray of H1299-Mock and H1299-Aiolos cells, CD44 and CD133 were not available in the microarray data. Therefore, the expression levels of CD44 and CD133 were analyzed by flow cytometry and qRT-PCR. Flow cytometric analysis revealed that ectopic Aiolos expression in H1299 cells increased the CD44 + and CD133 + populations (Fig. 5B). Fractions of CD44 + /CD133 + cells also increased when Aiolos was overexpressed. Ectopic Aiolos expression in A549 cells also increased the CD44 + and CD133 + www.nature.com/scientificreports www.nature.com/scientificreports/ populations (Fig. 5B). qRT-PCR also revealed that the expression levels of CSC surface markers (CD44 and CD133) were significantly increased in H1299-Aiolos cells compared with H1299-Mock cells and in A549-Aiolos cells compared with A549-Mock cells (Fig. 5C). We further investigated the effects of Aiolos overexpression on in vitro resistance to irradiation. Clonogenic cell survival assay revealed that the resistance to irradiation was significantly increased when Aiolos was overexpressed in H1299-Aiolos (Fig. 5D) and A549-Aiolos cells (Fig. 5E). We further examined the effect of Aiolos on anchorage-independent proliferation. Aiolos significantly increased anchorage-independent growth in soft agar ( Supplementary Fig. 5). Li et al. 31 have shown that Aiolos expression negatively correlated with p66 Shc in human lung cancers. Our results also showed that expression of Aiolos in A549 cells repressed p66 Shc expression ( Supplementary Fig. 5). Similar results were also demonstrated in H1299-Aiolos cells (data not shown).
Finally, we demonstrated the effects of Aiolos overexpression on the levels of CSC transcription factors, including Oct4, Nanog and Sox2. Western blot analysis showed that Nanog, Oct4, and Sox2 proteins were upregulated in H1299-Aiolos and A549-Aiolos cells (Fig. 6A). Nanog, Oct4, and Sox2 mRNA expressions were also upregulated in A549-Aiolos (Supplementary Fig. 6) and in H1299-Aiolos cells (data not shown). We further examined whether the PI 3-kinase-specific inhibitor, LY294002, repressed the upregulation of Oct4, Nanog, and Sox2 in H1299-Aiolos and A549-Aiolos cells. The results demonstrated that inhibition of PI 3-kinase lead to decreased expression of Oct4, Nanog, and Sox2 proteins in H1299-Aiolos (Fig. 6B) and A549-Aiolos cells (Fig. 6C). Inhibition of PI 3-kinase also reversed the increased number of sphere formation induced by Aiolos overexpression in H1299-Aiolos (Fig. 6D) and A549-Aiolos cells (Fig. 6E). All the above results indicated that Aiolos overexpression promotes the ability of lung cancer cells to develop CSC-like properties, and is regulated through the PI 3-kinase/Akt pathway.

Discussion
This study showed that overexpression of Aiolos induces EMT, increases migration and invasiveness ability in lung cancer cells through up-regulation of the PI 3-kinase/Akt/Twist axis. Aiolos overexpression also up-regulates CSC-like properties through up-regulating the PI 3-kinase/Akt pathway. www.nature.com/scientificreports www.nature.com/scientificreports/ The impact of Aiolos overexpression in hematopoietic malignancies has been reported in the literature [25][26][27][28][29] . In chronic lymphocytic leukemia, Aiolos overexpression has been reported to promotes cell survival by regulation of Bcl2 family proteins [25][26][27] . Aiolos regulates the survival of multiple myeloma cells by promoting the binding of Blimp-1 to target genes and thereby enhances Blimp-1-dependent transcriptional repression 29 . IKZF1 expression was significantly associated with advanced stage and distant metastasis in ovarian cancer patients 30 . Aiolos overexpression has also been reported to be a poor prognostic factor in patients with NSCLC 31 . Since EMT has been shown to be associated with tumor recurrence, metastasis and poor prognosis in different types of human cancers 10-15 , we focused to demonstrate the regulating mechanisms that Aiolos overexpression promotes EMT and CSC-like properties in the current study.
The relationship between Ikaros family members and EMT has not been well demonstrated in the literature. He et al. 30 have shown that overexpression of IKZF1 significantly upregulated Slug expression and led to increase of migration and invasion in ovarian cancer cells. Li et al. 31 have demonstrated that Aiolos downregulated expression of a number of integrin and tight junction genes and disrupted cell-cell and cell-matrix interactions. In our study, we showed that Aiolos overexpression promoted EMT and metastasis through control of Twist and MMP16. The EMT phenotype could be reversed and the migration/invasiveness ability could be repressed by inhibition of the PI 3-kinase/Akt/Twist axis. Our study was the first in the literature to demonstrate the role of Aiolos in EMT and its regulating mechanisms through control of the PI 3-kinase/Akt/Twist pathway.
The relationship between Aiolos expression and CSC-like properties remains unknown. Li et al. 31 have shown that Aiolos promotes anchorage independence by silencing SHC1 gene in lung cancer cells. In our study, we showed that Aiolos overexpression increased tumor-initiating capability of lung cancer cells. Ectopic Aiolos expression significantly increased CSC surface markers (CD44 and CD133) in lung cancer cells. Furthermore, the resistance to irradiation was significantly increased when Aiolos was overexpressed. Ectopic Aiolos expression also increased Nanog, Oct4, and Sox2 proteins expression. We further showed that the increased CSC-like properties by overexpression of Aiolos were reversed by inhibition of the PI 3-kinase/Akt pathway. Our results www.nature.com/scientificreports www.nature.com/scientificreports/ demonstrated that increased Aiolos expression promotes the ability of lung cancer cells to develop CSC-like properties through regulation of PI 3-kinase/Akt pathway.
Some limitations of this study should be mentioned. Since Aiolos expression in lung cancer cell lines in our lab was relatively low, we performed overexpression experiments instead of knockdown experiments. Further knockdown or knockout of Aiolos expression in cell lines with Aiolos expression or determine the correlation of EMT characteristics in tumors with Aiolos expression in human lung cancer specimens will be helpful to further determine the regulating mechanisms. Furthermore, subcutaneous inoculation of different numbers of lung cancer cells mixed with matrigel into BALB/c nude mice will help to further confirm the effect of Aiolos on lung cancer cell stemness. In conclusion, we have shown that Aiolos overexpression promotes transformation activity and promotes EMT through control of PI 3-kinase/Akt/Twist axis in lung cancer. Aiolos overexpression also promotes CSC-like properties through control of PI 3-kinase/Akt pathway. The information is helpful for developing therapeutic strategies targeting Aiolos expression for lung cancer treatment.  www.nature.com/scientificreports www.nature.com/scientificreports/ Vector control cell lines (H1299-Mock and A549-Mock) were generated by transfecting pcDNA3.1(+) into H1299 and A549 cells. The plasmid pSUPER-Twisti was established by inserting the oligonucleotide of 5′-GATCCCCAGGGCAAGCGCGGCAAGAATTCAAGAGATTCTTGCCGCGCTTGCCCTTTTTTA-3′ into the pSUPER plasmid. By inserting the oligonucleotide of 5′-GATCCCCGTGTCTGTAGGAGTCATCCTT CAAGAGAGGATGACTCCTACAGACACTTTTTA-3′ into the pSUPER plasmid, the plasmid pSUPER-scramble was established. The H1299-Aiolos-Twisti cell lines were established by transfection of the pSUPER-Twisti plasmid into H1299-Aiolos cells, and were selected under puromycin (4 ug/mL). By transfection of the pSUPER-Twisti plasmid into A549-Aiolos cells and being selected under puromycin (4 ug/mL), the A549-Aiolos-Twisti cell lines were also established. The H1299-Aiolos-scramble cell lines were established by transfection of the pSUPER-scramble plasmid into H1299-Aiolos cells. By transfection of the pSUPER-scramble plasmid into A549-Aiolos cells, the A549-Aiolos-scramble cell lines were also established.
www.nature.com/scientificreports www.nature.com/scientificreports/ Detection Kit (Ventana Medical System, Inc. Tucson), according to the manufacturer's instruction. The immunoreactivity of Aiolos and Twist was graded from 0 to 2+ (0, no staining; 1+ , weak staining; 2+ , strong staining) according to nuclear expression and only 2+ was considered as a Aiolos or Twist expression immunohistochemistry result.
sphere formation assay. Cell suspensions were plated on ultra-low adherent 6 well plates (Corning, Manassas, VA, USA) at 3 × 10 3 cells per well in 3 mL medium (DMEM supplemented with 5 mM HEPES, 0.1% sodium bicarbonate, and 0.4% BSA). After 14 days, the spheres were counted under a light microscope at high magnification. The assays were independently repeated at least three times.
Radiation treatment and clonogenic cell survival assay. Cells were trypsinized and plated on dishes 16 h before irradiation. The Caesium radiation was delivered by a Model 143-68 137Cs irradiator (JL Shepherd and Associates, San Fernando, CA, USA) at a dose rate of 4 Gy min −1 . Colonies were stained with crystal violet and counted 14 days after irradiation. A colony was defined as having >50 cells. The surviving fraction was calculated by dividing the number of colonies formed by the number of cells plated, multiplied by plating efficiency. soft agar clonogenicity assay. Anchorage-independent growth of H1299-Mock, H1299-Aiolos, A549-Mock, and A549-Aiolos cells was examined by survival of colonies on soft agar as described previously 33 . Cell numbers of 750 were used. The dishes were incubated for 14 days and colonies were counted. statistical analysis. The independent Student's t-test was used for comparison of the continuous variables between two groups, and the χ 2 test was applied for comparison of dichotomous variables. Statistical significance was defined as P < 0.05.

Data Availability
All materials and data in this manuscript are available to Editorial Board Members and referees.