*3.4. Radiation-Induced Activation of the PI3K/AKT Pathway, but Not the MAPK Pathway Modulated SOX2-Dependent Induction of Colorectal CSCs*

Next, the potential molecular mechanism involved in the SOX2-dependent induction of colorectal CSCs following irradiation was elucidated. The phenomenon of epithelialmesenchymal transition (EMT) has emerged as a feature of CSCs in recent times [6,23]. In addition, expression levels of the master regulator of EMT such as Snail and Zeb1/2 were modulated by SOX2 protein level [24,25]. Therefore, we investigated whether EMT is associated with SOX2-dependent induction of colorectal CSCs. Immunoblotting analysis showed that among EMT-associated proteins, Snail expression was decreased in SOX2 siRNA-transfected HCT116 and DLD1 cells (Figure 5A). In addition, we found that knockdown of Snail dramatically suppressed the ability of migration and invasion, a hallmark of

EMT (Figure 5B). However, Snail did not affect the induction of properties of colorectal CSCs including CD44+ population growth, resistance to radiation, and ability of tumoursphere formation (Figure 5C,D), suggesting that the Snail-mediated EMT process might not be involved in SOX2-dependent induction of colorectal CSCs upon irradiation. tochemistry further supported the AKT-dependent expression of both CD44 and SOX2 in irradiated HCT116 cells with or without AKT siRNA (Figure 6G). Collectively, these results suggested that radiation enhanced PI3K/AKT/SOX2 axis promoted the induction of colorectal CSCs in radioresistant colorectal cancer cells.

CSCs including CD44+ population growth, resistance to radiation, and ability of tumoursphere formation (Figure 5C,D), suggesting that the Snail-mediated EMT process might not be involved in SOX2-dependent induction of colorectal CSCs upon irradiation.

Since SOX2 expression is important for inducing the characteristics of CSCs as observed in our study and from previous reports [14,15], we attempted to identify the upstream regulator of SOX2. To do so, we investigated the activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways, known to regulate SOX2 under other conditions [26–29]. With western blotting, we found that radiation activated both MAPK and PI3K/AKT pathways except ERK activation (Figure 6A). Treatment with the pharmacological inhibitor of PI3K/AKT pathway (LY294002), but not the inhibitors of ERK (PD98059), p38 (SB203580), and SAPK/JNK (SP600125) pathways, dramatically suppressed radiation induced CD44 expression, which is a marker of colorectal CSCs and radioresistance in HCT116 and DLD1 cells (Figure 6B). The concentrations of the inhibitors used were referenced to previous studies, including our report [30]. Based on AKT silencing, we further confirmed the function of PI3K/AKT as an upstream regulator of SOX2-dependent induction of colorectal CSCs by observation of the reduced expression of SOX2 and CD44, as well as CSC properties, such as radioresistance, in vitro metastatic potential, and tumoursphere formation (Figure 6C–E). Moreover, immunocy-

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**Figure 3. K**nockdown of SOX2 in radioresistant colorectal cancer cells attenuated the induction of colorectal CSCs after irradiation. (**A**) Immunoblotting for the expression of CSC-related proteins on day 2 after radiation (10 Gy) in colorectal cancer cells as indicated. (**B**) siRNA-mediated SOX2 knockdown in cells was identified by western blotting (left) and CD44+ cell population (middle), or apoptotic cells were analyzed by flow cytometry (right). All experiments were performed with the SOX2 siRNA-transfected HCT116 and DLD1 cells on day 2 after radiation (10 Gy). Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 compared si-Cont + IR to si-SOX2 + IR. (**C**) The images of migration and invasion on day 2 after radiation (10 Gy) of the SOX2 siRNA-transfected HCT116 and DLD1 cells were quantified. Bars indicate measurements of migration and invasion. \* *p* < 0.05 compared si-Cont + IR to si-SOX2 + IR. (**D**) Tumoursphere-formation assay **Figure 3.** Knockdown of SOX2 in radioresistant colorectal cancer cells attenuated the induction of colorectal CSCs after irradiation. (**A**) Immunoblotting for the expression of CSC-related proteins on day 2 after radiation (10 Gy) in colorectal cancer cells as indicated. (**B**) siRNA-mediated SOX2 knockdown in cells was identified by western blotting (left) and CD44+ cell population (middle), or apoptotic cells were analyzed by flow cytometry (right). All experiments were performed with the SOX2 siRNA-transfected HCT116 and DLD1 cells on day 2 after radiation (10 Gy). Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 compared si-Cont + IR to si-SOX2 + IR. (**C**) The images of migration and invasion on day 2 after radiation (10 Gy) of the SOX2 siRNA-transfected HCT116 and DLD1 cells were quantified. Bars indicate measurements of migration and invasion. \* *p* < 0.05 compared si-Cont + IR to si-SOX2 + IR. (**D**) Tumoursphere-formation assay was performed to evaluate self-renewal ability of CSCs in SOX2 siRNA-transfected cells. Indicated cells were seeded in a non-adherent culture condition. After culturing for 7 days, the number of tumoursphere cells (>100 µm diameter) was quantified. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 versus si-Cont. IR: irradiation, Si-Cont: control siRNA, si-SOX2: SOX2 siRNA, CSCs: cancer stem-like cells.

Since SOX2 expression is important for inducing the characteristics of CSCs as observed in our study and from previous reports [14,15], we attempted to identify the upstream regulator of SOX2. To do so, we investigated the activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways, known to regulate SOX2 under other conditions [26–29]. With western blotting, we found that radiation activated both MAPK and PI3K/AKT pathways except ERK activation (Figure 6A). Treatment with the pharmacological inhibitor of PI3K/AKT pathway (LY294002), but not the inhibitors of ERK (PD98059), p38 (SB203580), and SAPK/JNK (SP600125) pathways, dramatically suppressed radiation induced CD44 expression, which is a marker of colorectal CSCs and radioresistance in HCT116 and DLD1 cells (Figure 6B). The concentrations of the inhibitors used were referenced to previous studies, including our report [30]. Based on AKT silencing, we further confirmed the function of PI3K/AKT as an upstream regulator of SOX2-dependent induction of colorectal CSCs by observation of the reduced expression

of SOX2 and CD44, as well as CSC properties, such as radioresistance, in vitro metastatic potential, and tumoursphere formation (Figure 6C–E). Moreover, immunocytochemistry further supported the AKT-dependent expression of both CD44 and SOX2 in irradiated HCT116 cells with or without AKT siRNA (Figure 6G). Collectively, these results suggested that radiation enhanced PI3K/AKT/SOX2 axis promoted the induction of colorectal CSCs in radioresistant colorectal cancer cells. was performed to evaluate self-renewal ability of CSCs in SOX2 siRNA-transfected cells. Indicated cells were seeded in a non-adherent culture condition. After culturing for 7 days, the number of tumoursphere cells (>100 μm diameter) was quantified. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 versus si-Cont. IR: irradiation, Si-Cont: control siRNA, si-SOX2: SOX2 siRNA, CSCs: cancer stem-like cells.

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**Figure 4.** SOX2 overexpression in radiosensitive colorectal cancer cells facilitated the induction of colorectal CSCs following irradiation. (**A**) Immunoblotting of SOX2 and CD44 in SOX2-overexpressing radiosensitive colorectal cancer cells (SW480 and LoVo) on day 2 after irradiation with 10 Gy. (**B**) Analysis of CD44+ cell population (left panel) and apoptotic cells (right panel) by flow cytometry in SOX2-overexpressing SW480 and LoVo cells. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 compared pcDNA + IR to WT-SOX2 + IR. (**C**) Colony formation assay was performed with SOX2-overexpressing SW480 and LoVo cells, and graph showing the quantification of relative colony numbers at different doses of radiation. Data are shown as mean ± SD (*n* = 3). (**D**) The images of migration and invasion on day 2 after radiation (10 Gy) of SOX2 overexpressing SW480 and LoVo cells were quantified. Bars indicate the measurements of migration and invasion. \* *p* < 0.05 compared pcDNA + IR to WT-SOX2 + IR. (**E**) Tumoursphere-formation assay was performed to evaluate self-renewal of CSCs in SOX2-overexpressing SW480 and LoVo cells. Indicated cells were seeded in a non-adherent culture condition. After culturing for 7 days, the images and size of tumoursphere cells were analyzed. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 versus Cont. **Figure 4.** SOX2 overexpression in radiosensitive colorectal cancer cells facilitated the induction of colorectal CSCs following irradiation. (**A**) Immunoblotting of SOX2 and CD44 in SOX2-overexpressing radiosensitive colorectal cancer cells (SW480 and LoVo) on day 2 after irradiation with 10 Gy. (**B**) Analysis of CD44+ cell population (left panel) and apoptotic cells (right panel) by flow cytometry in SOX2-overexpressing SW480 and LoVo cells. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 compared pcDNA + IR to WT-SOX2 + IR. (**C**) Colony formation assay was performed with SOX2-overexpressing SW480 and LoVo cells, and graph showing the quantification of relative colony numbers at different doses of radiation. Data are shown as mean ± SD (*n* = 3). (**D**) The images of migration and invasion on day 2 after radiation (10 Gy) of SOX2-overexpressing SW480 and LoVo cells were quantified. Bars indicate the measurements of migration and invasion. \* *p* < 0.05 compared pcDNA + IR to WT-SOX2 + IR. (**E**) Tumoursphere-formation assay was performed to evaluate self-renewal of CSCs in SOX2 overexpressing SW480 and LoVo cells. Indicated cells were seeded in a non-adherent culture condition. After culturing for 7 days, the images and size of tumoursphere cells were analyzed. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 versus Cont.

**Figure 5.** SOX2-dependent induction of colorectal CSCs was not associated with Snail-promoted ability of migration and invasion after irradiation. (**A**) Immunoblotting for SOX2 and EMT regulator (Snail, Slug, Twist, and Zeb1) in SOX2 siRNAtransfected radioresistant colorectal cancer cells (HCT116 and DLD1). (**B**) siRNA-mediated Snail knockdown cells were identified by western blotting (left) and the migration and invasion (right) on day 2 after radiation (10 Gy), of the Snail siRNA-transfected HCT116 and DLD1 cells were quantified. Bars indicate measurements of migration and invasion. \* *p* < 0.05 compared si-Cont + IR to si-Snail + IR. (**C**) Analysis of CD44+ cell population (left panel) and apoptotic cells (right panel) by flow cytometry from irradiated cells with 10 Gy on day 2. Data are shown as mean ± SD (*n* = 3). (**D**) Tumoursphere-formation assay was performed to evaluate self-renewal of CSCs in Snail siRNA-transfected cells. Indicated cells were seeded in a non-adherent culture condition. After culturing for 7 days, the size of tumoursphere cells was measured. Data are shown as mean ± SD (*n* = 3). EMT: epithelial-mesenchymal transition, si-Cont: control siRNA, si-Snail: Snail siRNA, CSCs: cancer stem-like cells. **Figure 5.** SOX2-dependent induction of colorectal CSCs was not associated with Snail-promoted ability of migration and invasion after irradiation. (**A**) Immunoblotting for SOX2 and EMT regulator (Snail, Slug, Twist, and Zeb1) in SOX2 siRNA-transfected radioresistant colorectal cancer cells (HCT116 and DLD1). (**B**) siRNA-mediated Snail knockdowncells were identified by western blotting (left) and the migration and invasion (right) on day 2 after radiation (10 Gy), of the Snail siRNA-transfected HCT116 and DLD1 cells were quantified. Bars indicate measurements of migration and invasion. \* *p* < 0.05 compared si-Cont + IR to si-Snail + IR. (**C**) Analysis of CD44+ cell population (left panel) and apoptotic cells (right panel) by flow cytometry from irradiated cells with 10 Gy on day 2. Data are shown as mean ± SD (*n* = 3). (**D**) Tumoursphere-formation assay was performed to evaluate self-renewal of CSCs in Snail siRNA-transfected cells. Indicated cells were seeded in a non-adherent culture condition. After culturing for 7 days, the size of tumoursphere cells was measured. Data are shown as mean ± SD (*n* = 3). EMT: epithelial-mesenchymal transition, si-Cont: control siRNA, si-Snail: Snail siRNA, CSCs: cancer stem-like cells.

**Figure 6.** SOX2-dependent induction of colorectal CSCs was modulated by radiation-activated PI3K/AKT pathway, but not MAPK pathway. (**A**) Immunoblotting for mitogen-activated protein kinase (MAPK) pathway activation (p-ERK, ERK1/2, p-p38, p38, p-SAPK/JNK, JNK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation (p-AKT, AKT) on day 2 after radiation (10 Gy) in radioresistant colorectal cancer cells (HCT116 and DLD1). (**B**) Analysis of CD44+ cell population by flow cytometry in HCT116 (left panel) and DLD1 (right panel) cells. The cells were exposed to radiation in the absence or presence of a pharmacological inhibitor of ERK pathway (PD98059, 10 μm), p38 pathway (SB203580, 10 μm), SAPK/JNK pathway (SP600125, 10 μm) and PI3K/AKT pathway (LY294002, 10 μm) for 2 days. Data are shown as mean ± SD (*n* = 3) with \* *p* < 0.05 for the pairwise comparisons of CD44+ cell population between irradiated cells with inhibitors and the reference group (i.e., 10 Gy). (**C**) Immunoblotting for the expression of CSC-related proteins (SOX2, Notch2, OCT3/4, and Nanog) on day 2 after radiation (10 Gy) in AKT siRNA-transfected HCT116 cells. (**D**) Analysis of CD44+ cell population (left panel) and apoptotic cells (right panel) by flow cytometry in AKT siRNA-transfected HCT116 cells. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 compared si-Cont + IR to si-AKT + IR. (**E**) The migration and invasion on day 2 after irradiation (10 Gy) of the AKT siRNA-transfected HCT 116 cells were quantified. Bars indicate the measurements of migration and invasion. \* *p* < 0.05 compared si-Cont + IR to si-AKT + IR. (**F**) Tumoursphere-formation assay was performed to evaluate self-renewal ability of CSCs in AKT siRNA-transfected cells. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 versus si-Cont. (**G**) Cells were stained with an anti-CD44 (green) and anti-SOX2 (red) antibody. Nuclei were counterstained with DAPI (blue). Si-Cont: control siRNA, si-AKT: AKT siRNA, CSCs: cancer stem-like cells. **Figure 6.** SOX2-dependent induction of colorectal CSCs was modulated by radiation-activated PI3K/AKT pathway, but not MAPK pathway. (**A**) Immunoblotting for mitogen-activated protein kinase (MAPK) pathway activation (p-ERK, ERK1/2, p-p38, p38, p-SAPK/JNK, JNK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation (p-AKT, AKT) on day 2 after radiation (10 Gy) in radioresistant colorectal cancer cells (HCT116 and DLD1). (**B**) Analysis of CD44+ cell population by flow cytometry in HCT116 (left panel) and DLD1 (right panel) cells. The cells were exposed to radiation in the absence or presence of a pharmacological inhibitor of ERK pathway (PD98059, 10 µm), p38 pathway (SB203580, 10 µm), SAPK/JNK pathway (SP600125, 10 µm) and PI3K/AKT pathway (LY294002, 10 µm) for 2 days. Data are shown as mean ± SD (*n* = 3) with \* *p* < 0.05 for the pairwise comparisons of CD44+ cell population between irradiated cells with inhibitors and the reference group (i.e., 10 Gy). (**C**) Immunoblotting for the expression of CSC-related proteins (SOX2, Notch2, OCT3/4, and Nanog) on day 2 after radiation (10 Gy) in AKT siRNA-transfected HCT116 cells. (**D**) Analysis of CD44+ cell population (left panel) and apoptotic cells (right panel) by flow cytometry in AKT siRNA-transfected HCT116 cells. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 compared si-Cont + IR to si-AKT + IR. (**E**) The migration and invasion on day 2 after irradiation (10 Gy) of the AKT siRNA-transfected HCT 116 cells were quantified. Bars indicate the measurements of migration and invasion. \* *p* < 0.05 compared si-Cont + IR to si-AKT + IR. (**F**) Tumoursphere-formation assay was performed to evaluate self-renewal ability of CSCs in AKT siRNA-transfected cells. Data are shown as mean ± SD (*n* = 3). \* *p* < 0.05 versus si-Cont. (**G**) Cells were stained with an anti-CD44 (green) and anti-SOX2 (red) antibody. Nuclei were counterstained with DAPI (blue). Si-Cont: control siRNA, si-AKT: AKT siRNA, CSCs: cancer stem-like cells.
