ML264 inhibits osteosarcoma growth and metastasis via inhibition of JAK2/STAT3 and WNT/β‐catenin signalling pathways

Abstract Osteosarcoma, the most common bone malignancy, has a high morbidity rate and poor prognosis. Krüppel‐like factor 5 (KLF5) is a key transcriptional regulator of cellular proliferation whose overexpression is observed in osteosarcoma cell lines (U2OS, 143B, MG63 and SAOS2). ML264, a small‐molecule inhibitor of KLF5, exerts antiproliferative effects in colorectal cancer; however, its function in osteosarcoma remains unknown. Here, we explored the possible antitumour effects of ML264 on 143B and U2OS cell lines and murine tumour xenograft model. ML264 suppressed proliferation and clonogenic ability of osteosarcoma cells in a dose‐dependent manner. Moreover, ML264 induced G0/G1 cell cycle arrest, with no influence on apoptosis, and inhibited the migratory and invasive abilities of osteosarcoma cells, as demonstrated by wound‐healing and Transwell assays. Exposure to ML264 reduced the mRNA and protein levels of molecules associated with epithelial‐mesenchymal transition phenotype, including N‐cadherin, vimentin, Snail, matrix metalloproteinase (MMP) 9 and MMP13. Inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation and Wnt signalling was also observed. In the murine model of osteosarcoma, tumour growth was efficiently suppressed following a 10‐day treatment with ML264. Collectively, our findings demonstrate the potential value of ML264 as a novel anticancer drug for osteosarcoma.

of osteosarcoma has reached a bottleneck. 6,7 Patients with recurrent and metastatic osteosarcoma have particularly poor outcome, owing to inadequate response to current conventional chemotherapeutic agents. 8,9 Therefore, there is an urgent need to identify new, more effective treatment strategies for patients with this lethal disease.
Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor involved in numerous functions in eukaryotic cells, such as proliferation, migration and differentiation. 10,11 Its overexpression is observed in many human diseases, including intestinal, colon, breast, bladder and pancreatic cancer. [12][13][14] KLF5 regulates epithelial-mesenchymal transition (EMT), 15 which is characterized by loss of cell adhesion and polarity, and acquisition of a mesenchymal-like phenotype by epithelial cells. 15,16 EMT is closely related to tumour migration and invasion 17,18 ; however, the underlying molecular mechanisms are yet to be elucidated.
ML264 is a small-molecule compound that inhibits the expression of KLF5 and its transcriptional activator, early growth response gene 1 (EGR1). 19 The drug potently suppresses proliferation of colon cancer cells and growth of xenograft tumours in nude mice. 19 Moreover, a novel derivative of ML264, YD277, inhibits proliferation of triple-negative breast cancer (TNBC) cells and growth of xenografts by inducing apoptosis and G1 cell cycle arrest. 20 Although KLF5 activity is implicated in many types of cancer, 12-14 the effects of its inhibition have not been investigated in detail in osteosarcoma.
This study explored the effects of ML264 on the viability, growth, metastasis and apoptosis of osteosarcoma cells to evaluate its role in osteosarcoma therapy. The molecular mechanisms and signalling pathways mediating the antitumour effects of ML264 were also investigated. Our study demonstrates that ML264 is a promising therapeutic drug to inhibit the development of osteosarcoma.
These cells were cultured in an incubator at a constant temperature of 37°C with 5% CO2.

| CCK-8 assay
The effect of ML264 on the cell viability of 143B and U2OS cells was examined by CCK-8 assay. Firstly, 96-well plates were used for inoculation of 143B and U2OS cells at a density of 4 × 10 3 cells/well.
Then, cells were treated using different concentrations of ML264 (0-40 μmol/L) for 48 or 96 hours, whereas the negative control was treated using DMSO. At the end of culture, each well was added 10 μL of CCK-8 buffer and incubated for another 2 hours, after which the absorbance of each well at 450 nm wavelength was measured using an ELX800 absorbance microplate reader (BioTek Instruments).

| Colony-formation assay
Six-well plates were used for inoculation of 143B and U2OS cells at a density of 1000 cells/well. After adherence for 24 hours, the cells were exposed to 0.5, 1 and 2 μmol/L ML264 and the control medium for 7-14 days. When the colonies became visible, 4% paraformaldehyde was used for fixation of cells, and 0.5% crystal violet was used for staining. After washing with PBS, the number of colonies was then counted and analysed.

| Flow cytometry
Six-well plates were used for inoculation of 143B and U2OS cells at a density of 5 × 10 5 cells/well. After adherence for 24 hours, the cells were exposed to different concentrations of ML264 and the control medium for 24 hours. For cell cycle assay, the cells were digested, centrifuged and washed twice. Then, cold 75% ethanol was used for fixation of the cells for at least 2 hours. After washed twice with PBS to eliminate remanent ethanol, the cells were then incubated with RNase A (100 μg/ mL), propidium iodide (PI; 50 μg/mL) and 0.2% Triton X-100 complexes for 15 minutes in the dark. At the end, the distribution of cells in different cell cycles was examined using the Accuri C6 (BD Biosciences). As for apoptosis analysis, the adherent cells were harvested, as well as the cells in medium. After washed twice with PBS, the cells were incubated with FITC-conjugated Annexin V and PI in the dark for 15 minutes.
Then, the samples were analysed using the Accuri C6, too.

| Wound-healing assay
Six-well plates were used for inoculation of 143B and U2OS cells at a density of 1 × 10 5 cells/well. Three perpendicular wounds were scratched using a 200-μL pipette tip when the cells were grown to about 80% confluency, after which the wells were washed twice with PBS to eliminate the detached cells. Then, the cells were exposed to 0.5, 1 and 2 μmol/L ML264 and the control medium for 24 hours. An inverted microscope (Nikon) was used to photograph the wounds at 0 and 24 hours.

| Cell migration and invasion assays
Transwell cell culture chambers were used to assess cell migration and invasion ability of 143B and U2OS cells. Whereas invasion assay needed Matrigel basement membrane matrix (BD Biosciences) coated on the membranes of upper chamber at 37°C for 6 hours, migration assay did not. 143B and U2OS cells were exposed to different concentrations of

| Immunofluorescence assay
Forty-eight-well plates were used for inoculation of 143B and U2OS cells. After exposed to 2 μmol/L ML264 and DMSO for 24 hours, cells were fixed for 20 minutes and permeabilized using 0.5% Triton X-100 for another 30 minutes. Whereafter, 5% sheep serum was used for blocking before cells were incubated with primary antibodies of EMT-related proteins at 4°C overnight. Cells were washed for three times the next day, after which Alexa Fluor 488 secondary antibody was used for incubation at room temperature for 1 hour. At the end, DAPI buffer was used to stain cell nuclei, after which cells were photographed using a fluorescence microscope.

| Quantitative real-time PCR
RT-PCR was carried out to assess KLF5, EGR1, cell cycle-associated genes and EMT-associated genes expression levels in osteosarcoma cells after ML264 treatment. Six-well plates were used for inoculation of 143B and U2OS cells at a density of 5 × 10 4 cells/well. After adherence for 24 hours, the cells were exposed to different concentrations of ML264 (0.5, 1 or 2 μmol/L) for 48 hours or 2 μmol/L of ML264 for different durations (6, 12, 24 hours). According to the manufacturer's instruction, total RNA of the cultured cells was extracted using RNeasy kit (Invitrogen), which was used to synthesize complementary DNA (cDNA) with the 5 × PrimeScript RT Master Mix (Takara Bio) for mRNA analysis. An ABI Prism 7500 system (Applied Biosystems) was subsequently used for RT-PCR with SYBR Green QPCR Master Mix (Takara Bio). The transcriptional levels of target gene were calculated using the 2 -ΔΔCt method, and the concentrations of cDNA in each sample were verified by β-actin. The specific primers used in this study were listed in Table 1.

| Western blotting analysis
Six-well plates were used for inoculation of 143B and U2OS cells at a density of 5 × 10 4 cells/well. Then, cells were exposed to different treatments, after which total proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) containing a protease and a phosphatase inhibitor. Using SDS-PAGE (8%-12%), total proteins were separated and transferred to 0.45-μm PVDF membranes (Bio-Rad). Whereafter, 5% non-fat dry milk was used for blocking of the membranes at room temperature for 1 hour, after which specific primary antibodies were used for incubation overnight at 4°C. After these membranes were washed with TBST buffer for three times, corresponding secondary antibody was used for incubation at room temperature for 1 hour. And then, a Chemiluminescence Kit (Millipore) was used to detect the protein band. The protein levels were first normalized to β-actin or α-tubulin, and then normalized to experimental controls.

| Histopathology and immunohistochemistry
The viscera, such as heart, liver, spleen, lung and kidney, were gutted from three groups of mice and fixed with formalin, after which they were embedded with paraffin. Then, these cut slices were stained with haematoxylin and eosin (H&E) before they were photographed for further histological examinations. As for tumour tissue, they were embedded for immunohistochemical analysis as described previously. 22

| Statistical analysis
All quantitative data are presented as mean ± SEM. Statistical significance was analysed with the unpaired, two-tailed Student's t test or ANOVA for multiple comparisons. The value of P < .05 was considered to be statistically significant. Significance level was presented as either *P < .05, **P < .01 or ***P < .001. cells. Additionally, the colony-forming ability of the cells was weaker following treatment with ML264 ( Figure 1D,E and Figure S1a,b).

| ML264 inhibits proliferation of 143B and U2OS cells without inducing apoptosis
However, exposure to ML264 (25, 50 and 100 μmol/L) did not influence apoptosis rates in 143B or U2OS cells ( Figure 1F,G). The above results show that ML264 inhibits the growth of osteosarcoma cells in a dose-and time-dependent manner without inducing apoptosis.

| ML264 induces G0/G1 phase arrest by regulating cell cycle-related proteins
As ML264 inhibited cell proliferation without an effect on apoptosis, we investigated its ability to induce cell cycle arrest. We as-

| ML264 inhibits invasion and migration in 143B and U2OS cells
We conducted wound-healing and Transwell assays to assess the ef-  Figure 3C,D), as well as the invasion rates ( Figure 3E,F). The above results suggest that ML264 treatment is able to suppress the metastatic capacity of osteosarcoma cells.

| ML264 reverses epithelial-mesenchymal transition (EMT) phenotype in vitro
EMT is thought to be closely associated with tumour progression and me- In line with these results, the immunofluorescence assay indicated decreased MMP9 and N-cadherin expression and increased E-cadherin levels following ML264 treatment ( Figure 4C). Hence, ML264 reversed the EMT phenotype to inhibit metastatic progression in osteosarcoma cells.

| ML264 inhibits the expression of EGR1 and KLF5
ML264 was originally synthesized as an inhibitor of KLF5; thus, we examined the expression of KLF5 and its transcriptional activator, EGR1, in 143B and U2OS cells following ML264 exposure. As expected, ML264 treatment reduced the mRNA and protein levels of both EGR1 and KLF5 in a dose-and time-dependent manner ( Figure 5). Thus, ML264 may exert its effects by altering the expression of EGR1, KLF5 and their downstream proteins.

| ML264 inhibits JAK2/STAT3 and Wnt/βcatenin signalling
As KLF5 is a positive regulator of both the JAK2/STAT3 and Wnt/β-  Figure 5D). In line with these results, ML264 inhibited mRNA expression of Runx2 in a time-dependent manner ( Figure 5E). Notably, MAPK pathway-related proteins were not influenced by ML264 treatment (Figure 5F). In conclusion, ML264 inhibited the JAK2/STAT3 and Wnt/β-catenin signalling pathways via down-regulation of KLF5 and EGR1 expression, thereby inhibiting proliferation and metastasis of osteosarcoma cells ( Figure 5G).

| ML264 inhibits the growth of osteosarcoma in vivo
We  Figure 6E). In addition, immunohistochemical analyses indicated that exposure to ML264 decreased the expression levels of EGR1, KLF5, cyclin D1, Ki67, MMP9 and phospho-STAT3 ( Figure 6F). To investigate whether ML264 is cytotoxic to nontumour cells, we performed H&E staining on tissue harvested from DMSO-and ML264-treated mice. No toxic effects were found in the major organs ( Figure 6G). Taken together, these results suggest that ML264 hinders the development of osteosarcoma and has low toxicity in nude mice in vivo. KLF5 is one of the basic transcriptional factors, ubiquitously expressed in different tissues. 28 It is associated with numerous cell functions, including proliferation, differentiation, migration and EMT. 10,11,15 In our study, ML264 reduced the expression of KLF5 at both the transcriptional and protein level, which, in turn, altered the F I G U R E 2 ML264 induces G0/G1 phase arrest by regulating cell cycle-related proteins. A and B, 143B and U2OS cells were treated with 0.1% DMSO or ML264 for 24 h, and cell cycle assays were performed with flow cytometry. C, 143B and U2OS cells were treated with different doses of ML264 (10, 20 or 40 μmol/L) for 48 h, or 20 μmol/L of ML264 for different durations (24,48 or 72 h). The expressions of cell cycle-regulated proteins were then measured using Western blotting. D, The grey levels of cell cycle-regulated proteins were quantified and normalized to that of β-tubulin using ImageJ. Data are expressed as the means ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001 compared with the control group. DMSO, dimethyl sulphoxide; PI, propidium iodide F I G U R E 3 ML264 inhibits invasion and migration in 143B and U2OS cells. A, 143B and U2OS cells were scratched using 200-μL pipette tips, after which they were treated with different concentrations of ML264 for 24 h. B, The migration area was qualified. C, Cell migration assay was performed using a Transwell chambers for 24 h. D, The migration abilities of 143B and U2OS cells were quantified. E, Cell invasion assay was performed using Matrigel-coated Transwell chambers for 24 h. F, The invasion abilities of 143B and U2OS cells were quantified. Data are expressed as the means ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001 compared with the control group  In summary, the above results indicate that ML264 displays

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.