Olig2 regulates p53-mediated apoptosis, migration and invasion of melanoma cells

Melanoma is a disease with a high recurrence rate and poor prognosis; therefore, the need for targeted therapeutics is steadily increasing. Oligodendrocyte transcription factor2 (Olig2) is a basic helix-loop-helix transcription factor that is expressed in the central nervous system during embryonic development. Olig2 is overexpressed in various malignant cell lines such as lung carcinoma, glioma and melanoma. Olig2 is known as a key transcription factor that promotes tumor growth in malignant glioma. However, the role of Olig2 in melanoma is not well characterized. We analyzed the role of Olig2 in apoptosis, migration, and invasion of melanoma cells. We confirmed that Olig2 was overexpressed in melanoma cells and tissues. Reduction of Olig2 increased apoptosis in melanoma cells by increasing p53 level and caspase-3/-7 enzyme activity. In addition, downregulation of Olig2 suppressed migration and invasion of melanoma cells by inhibiting EMT. Reduction of Olig2 inhibited expression of MMP-1 and the enzyme activity of MMP-2/-9 induced by TGF-β. Moreover, Olig2 was involved in the downstream stages of MEK/ERK and PI3K/AKT, which are major signaling pathways in metastatic progression of melanoma. In conclusion, this study demonstrated the crucial roles of Olig2 in apoptosis, migration, and invasion of melanoma and may help to further our understanding of the relationship between Olig2 and melanoma progression.

www.nature.com/scientificreports/ Olig2 is a basic helix-loop-helix factor that is expressed in the central nervous system during embryonic development 17,18 . Olig2 is expressed in multipotent neurospheres from the murine embryonic forebrain, where it promotes proliferation of neural progenitors 19 . Although Olig2 was thought to be expressed only in neural tissue, Ying-Wei Lin et al. found that it is expressed in various tissues including kidney, thymus, lung, brain, and skin. Additionally, they found overexpression of Olig2 in malignant cell lines from lung carcinoma, breast cancer, and melanoma 20 . However, the role of Olig2 in melanoma is not well characterized. Our data provide the first evidence supporting the role of Olig2 in invasive growth of melanoma. Here, we investigated whether Olig2 regulates melanoma progression. We show that Olig2 is highly expressed in various melanoma cell lines and contributes to invasive growth of melanoma cells.

Results
Olig2 expression is elevated in human melanoma. To investigate the clinical significance of Olig2 in melanoma tissue, we analyzed published microarray data of Olig2 from the GSE7553 data set. We found that expression of Olig2 was elevated in primary and metastatic melanoma tissues compared with normal melanocyte tissues. However, there was no significant difference in the expression level of Olig2 between primary and metastatic melanoma (Fig. 1a). We performed Olig2 IHC analysis on a melanoma tissue microarray (TMA). Olig2 staining was higher in stage 2 and 3/4 melanoma tissue compared with normal skin tissue (Fig. 1b,c). To examine the basal expression of Olig2, we performed immunoblotting in normal human epidermal melanocytes (NHEM) and four human metastatic melanoma cell lines (MNT-1, 501mel, A375, and HM3KO). Olig2 was expressed significantly higher in the four metastatic melanoma cell lines compared with NHEM. There was no significant difference in the expression of Olig2 between the four melanoma cell lines (Fig. 1d).

Reduction of Olig2 inhibits growth of melanoma cells.
We examined Olig2 localization using western blot analysis of nuclear and cytoplasmic fractions from melanoma cells. Olig2 predominantly localized in the nucleus in both A375 and 501mel melanoma cells (Fig. 2a).
To identify the roles of Olig2 in melanoma, we used a CRISPR/Cas9 system to precisely create a loss-offunction mutation in the Olig2 gene. Our results showed successful dose-dependent inhibition of CRISPR-Olig2 in A375 and 501mel melanoma cells (Fig. 2b). Next, we examined cell viability after transfection of CRISPR-Olig2 to examine the role of Olig2 in melanoma survival. Down-regulation of Olig2 decreased cell viability in A375 and 501mel cells (Fig. 2c). We further validated these results by cell counting assays. As shown in Fig. 2, knockdown of Olig2 decreased the number of melanoma cells (Fig. 2d).

Olig2 inhibition induces apoptosis of melanoma cells.
To examine the mechanism by which silencing of Olig2 suppressed melanoma cell viability, we measured cell apoptosis following Olig2 knockdown. As shown in Fig. 3a, reduction of Olig2 induced apoptosis of both A375 and 501mel melanoma cells. Next, we detected apoptosis-related factors such as p53, p21 and Bcl-2 after transfection of CRISPR-Olig2 in two melanoma cell lines. We found that reduction of Olig2 increased p53 and p21 expression, and decreased Bcl-2 expression (Fig. 3b,c). To determine whether downregulation of Olig2 activates caspases involved in apoptosis, we assessed caspase-3/-7 enzymatic activity using a fluorogenic substrate that can detect this enzymatic reaction in cells. When Olig2 level was decreased, caspase-3/-7 activity increased in melanoma cells (Fig. 3d). Subsequently, we observed cleavage of PARP, which is considered an apoptotic marker. Western blot analysis revealed that downregulation of Olig2 induced cleavage of PARP (89 KD) in A375 and 501mel cells (Fig. 3e). Taken together, these findings indicate that reduction in Olig2 induces apoptosis in melanoma cells.

Silencing of Olig2 inhibits migration and invasion of melanoma cells by regulating EMT-related factors.
Metastasis, which is the main characteristic of malignant melanoma, is a phenomenon in which cells are separated from a primary tumor and relocate 21 . To investigate whether Olig2 regulates migration of melanoma cells, we evaluated cell migration using an in vitro scratch assay. As shown in Fig. 4a,b, cells closed the wound more slowly in the CRISPR-Olig2 transfection groups than in the control groups. To further examine the metastatic role of Olig2, we examined the migration and invasion of cells by performing a transwell assay using a chamber with 8-µm pores. The results revealed that the number of cells that migrated or invaded the underside of the chamber decreased in the CRISPR-Olig2 transfection groups compared to the control groups ( Fig. 4c-e).
EMT is a biological mechanism of cancer cell recurrence and metastasis, accompanied by a loss of epithelial proteins such as E-cadherin and occludin, and an increase in mesenchymal proteins including N-cadherin, vimentin, and smooth muscle actin 22 . To further define the role of Olig2 as an EMT regulator, we examined the expression level of EMT-related factors by western blot analysis. Knockdown of Olig2 led to simultaneous downregulation of Snail (Snail1), Slug (Snail2), Smuc (Snail3) and N-cadherin, and upregulation of E-cadherin in A375 cells. In addition, these changes in protein expression were dependent on the concentration of CRISPR-Olig2 treatment (Fig. 5a). In 501mel cells, unlike A375 cells, Slug and Smuc expression did not change when Olig2 was decreased. However, Snail and N-cadherin expression was reduced and E-cadherin was increased in a concentration-dependent manner (Fig. 5b).
Olig2 regulates MMP-2/-9 activation and MMP-1 expression. Since matrix metalloproteinase-2/-9 (MMP-2/-9) are known to play an important role in melanoma metastasis, we estimated their enzymatic activity with gelatin zymography. MMP-2/-9 activity was increased following treatment with transforming growth factor-β (TGF-β). However, the levels of MMP-2/-9 were reduced in the Olig2-deficient group compared to the TGF-β treatment group in both A357 and 501mel cells (Fig. 6a,b). In addition, reduction of Olig2 inhibited mRNA levels of MMP-2 and MMP-9 (Fig. 6c). We further verified that Olig2 influences the expression of MMP- www.nature.com/scientificreports/ 1, another MMP that is important for invasion and growth of melanoma. An ELISA assay showed that expression of MMP-1 was decreased in the CRISPR-Olig2 transfection group (Fig. 6d).
Active MEK/ERK and PI3K/AKT signaling controls Olig2 protein expression. Hyperactivation of the RAS-RAF-MAPK pathway is closely associated with development of melanoma. Extracellular signal-regulated kinase (ERK) activity in cutaneous melanoma is mostly caused by mutations in BRAF, the most common genetic variation in melanoma 23 . To determine whether Olig2 is a downstream effector of mitogen-activated protein kinase kinase (MEK)/ERK signaling, we treated A375 cells with PD98059, an MEK inhibitor. PD98059 markedly suppressed activation of ERK and expression of Olig2 and Snail (Fig. 7a). In melanoma, the Phosphoinositide 3-kinase (PI3K)/AKT pathway is implicated in mutations in phosphatase and tensin homologue www.nature.com/scientificreports/ (PTEN) and NRAS, which occur most frequently after BRAF mutations. Because NRAS is located upstream of BRAF, NRAS mutations activate mitogen-activated protein kinase (MAPK) and the PI3K/AKT/ mammalian target of rapamycin (mTOR) pathway 24 . Therefore, we next identified whether Olig2 is a downstream effector of PI3K/AKT. We found that phosphorylation of AKT and expression of Olig2 and Snail decreased after treatment with the PI3K inhibitor LY294002 (Fig. 7b). Additionally, we identified the roles of Olig2 in ERK and AKT activation. However, when cells were transfected with CISRPR-Olig2, there was no change in the activity of ERK or AKT (Fig. 7c,d).
Olig2 silencing further increased the inhibition of cell viability by dabrafenib in 501mel cells. We next investigated whether combined treatment of BRAF inhibitor (dabrafenib) or MEK inhibitor (trametinib) with CRISPR-Olig2 could be effective in reducing melanoma cell viability. Dabrafenib and trametinib inhibited phosphorylation of ERK or AKT, and decreased expression of Olig2 ( Fig. 8a,b) Co-treatment of CRISPR-Olig2 and dabrafenib slightly reduced cell viability compared to the treatment of dabrafenib alone in 501mel cells. However, combined treatment with CRISPR-Olig2 and trametinib showed no significant difference from trametinib alone in 501mel cells. Also, no synergistic effect was observed in A375 cells (Fig. 8c,d).

Discussion
Malignant melanoma develops more frequently than any other tumor and can spread to most parts of the body. Therefore, prevention and treatment of melanoma are increasing in importance; accordingly, studies on the cell biology of tumor development and immune evasion of melanoma are ongoing 25 . Olig2 has been studied in certain types of neuronal and glial cells for its roles in tumorigenicity. In glioma stem cells, Olig2 promotes cell proliferation through direct inhibition of p21 WAF1/CIP1 , a tumor suppressor. Phosphorylation of the serine motif of Olig2 promotes proliferation of neural progenitor cells and induces potential astrocytoma malignancies 19,26 .
Recently, Olig2 was shown to increase in recurrent medulloblastoma after radiation therapy and in drug-resistant regions 27 . However, the role of Olig2 in melanoma is still not clearly understood.
Here, we identified that Olig2 mRNA expression was high in primary and metastatic melanoma tissues compared with normal human melanocyte tissues by microarray analysis. In addition, we verified that metastatic melanoma has higher Olig2 protein level than normal human melanocytes and normal skin ( Fig. 1a-d).
To investigate the function of Olig2 in melanoma progression, we used CRISPR/Cas9 to knockdown the Olig2 gene in A375 and 501mel metastatic melanoma cells. Previous studies found that Olig2 controls cell cycling and growth in malignant neural progenitors and plays an important role in glioma formation 19 . We found that knockdown of Olig2 suppressed survival of A375 and 501mel cells and decreased the number of cells (Fig. 2c,d). These findings suggest that Olig2 might be positively correlated with melanoma progression.
Previously, it was reported that Olig2 suppressed p53 response in neural progenitors and gliomas 28 . Consistent with this result, our study showed that knockdown of Olig2 increased expression of p53 and induced apoptosis of two melanoma cell lines (Fig. 3a,b). Suppression of Olig2 increased the protein expression of p21, confirming the possibility that Olig2 may be associated with p21 in melanoma (Fig. 3c), as in Ligon, K. L. et al.'s study that Olig2 directly inhibits p21 19 . The Bcl-2 gene family, which is normally involved in anti-apoptotic activities, can potentially cause cancer, and cancer-related proteins such as Myb and Ras induce high levels of Bcl-2 in hematopoietic cells. Therefore, Bcl-2 is considered an inhibitor of apoptotic proteins and may be an important target for cancer treatment 29 . We found that reduction of Olig2 in melanoma cells inhibited the expression of Bcl-2 (Fig. 3b). In addition, Olig2 knockdown induced activation of caspase-3/-7 (Fig. 3d) and PARP cleavage (89 KD) (Fig. 3e). Taken together, these findings suggest that Olig2 downregulation promoted a p53-mediated apoptosis pathway, suggesting that low expression of Olig2 is associated with unfavorable survival in melanoma cells. Microphthalmia-associated transcription factor (MITF) is a key transcription factor that regulates survival and invasiveness of melanoma 30 . To determine whether Olig2 regulates MITF expression, we inhibited Olig2 in two melanoma cells and then identified the protein expression of MITF by immunoblotting. However, Olig2 downregulation didn't induce significant change in expression of MITF. Thus, Olig2 is expected to regulate survival and migration independently of MITF (Fig. S1).
Along with overgrowth, the most dangerous aspect of cancer is metastasis. Melanoma penetrates deep into the skin layer and spreads to lymph nodes, blood vessels, and even distal parts of the body such as bone, brain, liver, and lung 31 . The association between Olig2 and metastasis is unknown. Therefore, we examined the wound healing assay after suppressing Olig2 expression to determine whether Olig2 is involved in melanoma migration. We confirmed that movement of cells into the wound slowed when Olig2 expression was inhibited in A375 and 501mel cells (Fig. 4a,b). Additionally, in the transwell migration and invasion assay, reduction of Olig2 inhibited Figure 2. Downregulation of Olig2 decreased viability of melanoma cells. (a) Nuclear and cytoplasmic extracts were analyzed by western blot analysis using Olig2 (AF2418) antibody. (b) Cells were transfected with negative control (Con) or CRISPR/Cas9-Olig2 plasmid (CRISPR-Olig2) (1 μg and 2 μg, respectively). Western blot analysis of Olig2 protein expression was performed with Olig2 (H-10) antibody. Original images of blots were provided in supplementary file. (c) Cells were transfected with negative control (Con) or CRISPR/Cas9-Olig2 plasmid (CRISPR-Olig2) (1 μg and 2 μg, respectively). Cell viability was determined by EZ-Cytox assay 24 h, 48 h and 72 h after transfection respectively. Results are presented as percentages of the Con and the data were analyzed using Student's unpaired t-tests (*p < 0.05; **p < 0.01; ***p < 0.001 versus Con cells, n = 3). (d) 48 and 72 h after transfection, cell number was measured by trypan blue exclusion. Results are presented as percentages of the Con and the data were assessed using Student's unpaired t-tests (*p < 0.05; **p < 0.01; ***p < 0.001 versus Con cells, n = 3). www.nature.com/scientificreports/ the migration and invasion of A375 and 501mel cells (Fig. 4c-e). These results indicate that Olig2 is required for migration and invasion in melanoma cells.
In addition to embryonic development, EMT provides a model for studies in which carcinoma is dedifferentiated and more malignant. If the EMT program is pathologically activated in a cancerous environment, it can lead to cancer metastasis 12 . As the transcription factor Snail can inhibit E-cadherin expression, it has been recognized as a key factor in EMT. E-cadherin and N-cadherin are important regulators of morphogenesis in mouse embryonic formation and are considered representative cadherins in epithelial and mesenchymal tissues 22 . We further investigated the mechanistic roles of Olig2 in melanoma invasion through the expression of EMT markers. Western blot analysis revealed that decreased Olig2 inhibited the expression of EMT-inducing markers including Snail, Slug, Smuc, and N-cadherin and conversely increased the expression of E-cadherin in A375 cells. Depending on the cell type, the regulation of Slug and Smuc by Olig2 may appear differently. In 501mel cells, only the expression of Snail, N-cadherin, and E-cadherin changed when Olig2 decreased (Fig. 5). Because melanoma cells are derived from embryonic neural crest, not epithelial cell origin, they do not undergo traditional EMT. Instead, melanoma cells appear EMT-like process by which they acquire mesenchymal or epithelial features. Our results suggest that Olig2 control EMT-like process in melanoma cells. www.nature.com/scientificreports/ MMPs are endogenous factors that regulate proteolysis and play an important role in degradation of extracellular matrix. They are normally involved in important functions such as wound healing or embryonic development; however, they are also involved in pathological processes such as cancer metastasis and tumor growth. In particular, MMP-2/-9 are key regulators of cancer invasion 32,33 . They break down type IV collagen and fibronectin, which are important constituents of the basement membrane, and contribute to angiogenesis, which provides nutrients for tumor growth 34 . Additionally, MMP-1, known as pro-collagenase-1, is overexpressed in BRAF-mutated melanoma and contributes to melanoma invasion and growth by activating the ERK pathway 35 . Blackburn et al. also found that the MMP-1/protease activated receptor-1 (PAR1) axis promotes progression of melanoma by inducing the transition from the non-invasive radial-growth-phase (RGP) phenotype to the invasive vertical growth phase (VGP) phenotype 36 . MMP-1 expression is associated with poor prognosis in colorectal cancer. Specific inhibitors of MMP-1 suppressed tumor induction in mouse model of colon cancer 37,38 . Therefore, inhibiting the expression of MMP-2, MMP-9, and MMP-1 is important to prevent tumor invasion and metastasis. In our study, Olig2 knockdown suppressed mRNA expression of MMP-2/-9 and TGF-β-induced MMP-2/-9 activity (Fig. 6a-c). Furthermore, the expression level of MMP-1 was attenuated in Olig2 knockdown of melanoma cells (Fig. 6d). These results suggest that Olig2 can regulate melanoma invasion through activation of MMPs and might be associated with TGF-β-induced EMT in melanoma cells.
BRAF and NRAS mutations are representative genetic mutations that cause skin melanoma. They primarily activate MAPK and the PI3K/AKT/mTOR signaling pathway 39,40 and cause various transcription factors such as cAMP-response element binding protein (CREB) and Brn-2 (POU3F2) to become increasingly active, resulting in excessive cell proliferation 41,42 . Therefore, the BRAF inhibitors vemurafenib and dabrafenib have been approved by the FDA for treatment of melanoma. However, the leading cause of death in patients with advanced melanoma is persistent recurrence and chemotherapy resistance. We demonstrated that Olig2 is associated with MEK/ERK and PI3K/AKT signaling pathways in melanoma cells. Treatment with PD98059 significantly suppressed expression of Olig2 and Snail in melanoma cells (Fig. 7a). Also, LY294002 decreased the levels of Olig2 and Snail (Fig. 7b). However, CRISPR-Olig2 had no effect on ERK or AKT activation (Fig. 7c,d). These results suggest that Olig2 is a downstream target of MAPK and PI3K pathway and may be involved in EMT regulation through activation of MAPK and PI3K signaling pathways. Cell viability slightly decreased from combining CRISPR-Olig2 and dabrafenib compared to dabrafenib alone in 501mel cells. However, it appears that block of Olig2 and BRAF&MEK inhibition do not show a distinct synergistic effect in reducing the viability of melanoma cells (Fig. 8c,d). This is probably the result of suppressing Olig2 expression by inhibitors treatment as in Fig. 8a,b. Further in vivo studies are necessary to confirm the roles of Olig2 in metastatic progression of melanoma.
In conclusion, this study is the first to investigate the molecular mechanism of Olig2 in melanoma cells. We revealed oncogenic roles of Olig2 in melanoma cells by inhibiting p53-mediated apoptosis and promoting migration and invasion of cells through the MAPK and PI3K pathways (Fig. 9). Our findings suggest that Olig2 may be a novel potential regulator of melanoma progression.

Materials and methods
Cell culture. A735

Ez-CyToX cell viability assay. Cell viability was examined by Ez-Cytox assay kit (Daeil Lab Service Co
Ltd, Seoul, Korea). Cells were seeded at 1 × 10 4 cells/well in a 96-well plate. Cell viability was detected at 24 h, 48 h and 72 h after transfection of CRISPR-Olig2. 10 μl of Ez-Cytox solution was added to each well and 96-well plates were incubated for an additional 1 h at 37 °C in a 5% CO 2 incubator. Cell viability was detected at 490 nm by ELISA reader (Tecan, Mannedorf, Switzerland). Relative cytotoxicity was measured by expressing the viability of the cells as a percentage.
Cell counting assay. Cells were trypsinized after 72 h of transfection of CRISPR/Cas9 plasmid. Equally mix 10 μl of trypan blue dye (Sigma Aldrich, St. Louis, MO, USA) with the cell suspension. After staining cells with trypan blue, the cell number was counted using the hemocytometer. Each measurement was repeated in triplicate.

Apoptosis assay. Cell apoptosis was analyzed 24 h after transfection using RealTime-Glo Annexin V
Apoptosis Assay (Promega Corporation). Cells were seeded in white 96-well polystyrene microplate (Corning, Inc., Corning, NY, USA). Detection reagent was treated according to the manufacturer's instructions. Annexin V-based luminescence was measured using ELISA reader (Tecan, Mannedorf, Switzerland). The fluorescence intensity of each sample is expressed as a percentage. Gelatin zymography. The gelatinolytic activities of MMP-2/-9 were assessed by gelatin zymography assay.
24 h after transfection of 2 μg of CRISPR/Cas9 plasmid, the conditioned media were collected and centrifuged for concentration using the 50 K Amicon Ultra Centrifugal Filters. Filtered media was assayed by electrophoresis on 10% zymogram gel (Novex 10% Zymogram Plus (Gelatin) Protein Gels, Thermo Fisher Scientific, MA, USA) with 1 mg/ml gelatin. The gel was run at 120 V for 1.5 h at 4 °C. Novex Tris-Glycine SDS Running Buffer (10X) was used as electrophoresis reagent. After electrophoresis, incubate the gel with renaturing buffer (Novex Zymogram Renaturing Buffer (10X), Thermo Fisher Scientific, MA, USA) under gentle agitation for 30 min. Replace with developing buffer (Novex Zymogram Developing Buffer (10X), Thermo Fisher Scientific, MA, USA) and incubate the gel at 37 °C overnight. After washing with distilled water three times, the gels were stained with SimplyBlue safe stain (Thermo Fisher Scientific, MA, USA) overnight under gentle agitation at room temperature. The gel was de-stained with solution of 10% acetic acid, 40% methanol and 50% distilled water to visualize the zymogen bands. The zymography gels were scanned and analyzed using the ImageJ software 1.  Olig2 knockdown decreased migration and invasion of melanoma cells. Wound healing assay was performed to evaluate the migration abilities of (a) A375 cells and (b) 501mel cells after silencing of Olig2 expression. The cell migration into the wound was measured at 24 h and 48 h after scratch. Representative images (magnification, × 40) of cells were taken celenas digital imaging system. Scale bar: 100 µm. The migration was determined by the rate of cells filling the scratched area (*p < 0.05; **p < 0.01; ***p < 0.001 versus Con cells, n = 3). Transwell assay was performed to evaluate the migration ability of (c) A375, and invasion ability of (d) A375 and (e) 501mel cells after silencing of Olig2 expression. The cells which migrated or invaded through the membrane were stained with DAPI for visualization of the nuclei. Representative images and quantitative analysis of migrating or invading cells (*p < 0.05; **p < 0.01 versus Con cells, n = 3).   with negative control (Con) or CRSPR/Cas9-Olig2 plasmid (CRISPR-Olig2) followed by TGF-β (10 ng/ ml) treatment. The fold-change in MMP-2 and MMP-9 activities was assessed by densitometry compared to Con group. The band intensity of MMP-2 and MMP-9 was quantified using NIH ImageJ software. (*p < 0.05; **p < 0.01versus Con group and #p < 0.05 versus TGF-β group, n = 3). (c) MMP-2 and MMP-9 mRNA were evaluated using each primer by RT-PCR. The band intensity was quantified using NIH ImageJ software and normalized relative to β-actin. Original images of gels were provided in supplementary file. (d) The concentration of MMP-1 was measured by ELISA using the supernatant of two melanoma cells. (**p < 0.01; ***p < 0.001 versus Con group, n = 3).