Hedgehog is activated in LMS with increased GLI nuclear translocation
The constitutive (basal) expression levels of HH signaling components in UTSM, HuLM, and LMS cells were evaluated by qRT-PCR. Higher expressionof SMO, SUFU, and GLI1 were detected in LMS compared to UTSM (P<0.05) and HuLM. The expression of PTCH1 was down-regulated in LMS. GLI2 did not show difference among the cells, while GLI3 was up-regulated in HuLM (Figure 1A). The protein expression levels of SMO and GLI1 were highest in LMS among three detected cell lines. The protein levels of GLI2 and GLI 3 were also upregulated in HuLM. However, SUFU did not show difference among the cell lines (Figure 1B). The expression of HH ligands (IHH, DHH, and SHH) were not detected in these cells.
We further evaluated the level of GLIs nuclear translocation in UTSM, HuLM and LMS cells (Figure 1C). The expression levels of GLI1 were highest in the nucleus of the LMS cells, low in UTSM and undetected in HuLM cells. GLI2 and GLI3 were mainly expressed in the nucleus of all cell lines, with the highest expression levels in LMS. (Figure 1C). These results showed that HH pathway was activated in LMS due to higher expression of SMO and GLI1 with increased GLI nuclear translocation.
Inhibition of Hedgehog pathway using SMO and GLI inhibitors in LMS cells
SMO inhibitors (LDE225 and GDC0449) and GLI inhibitors (Gant58 and Gant61) were selected to determine their effect on LMS cells. MTT assay was performed using three-time points (24, 48 and 72 h) with varying drug concentrations. Treatment with SMO inhibitor (LDE225) for 72 hours showed a dose-dependent inhibitory effect on cell proliferation (Figure S3 A). 10 µM was used in the following experiments. LMS cells treated with GDC0449 did not show any inhibitory effect using different concentrations (Figure S3 B). GLI1 inhibitor (Gant61) showed a dose-dependent inhibitory effect on LMS after 72 hours of treatment. 30 µM was chosen for the next experiments. While Gant58 did not show an effect on proliferation, Based on MTT results, LDE225 (SMO inhibitor) and Gant61 (GLI inhibitor) were selected for the next experiments (Figure S3 C and D).
To verify the specificity of the effect of SMO (LDE225) and GLI inhibitors (Gant61) on LMS, we performed MTT assay using the same doses and duration in both UTSM and HuLM cells (Figure S4). After 72 hours of treatment, LDE225 and Gant61 showed inhibitory effects on UTSM cells proliferation. SMO inhibitor did not show a significant growth inhibition in UTSM cells (Figure S 4A). While GLI inhibitor showed a significant effect (p<0.05) in UTSM (Figure S4 B), however, the proliferation decrease was more potent in LMS cells when compared with UTSM cells in response to GLi inhibitor. SMO or GLI inhibitors did not show any inhibitory effect on HuLM cells proliferation. These results showed that the HH inhibitors exhibited a dominantly inhibitory effect on LMS. (Figure S4 C and D)
LMS cells were treated with SMO inhibitor, LDE225, for 24, 48 and 72 hours, with drug replacement every 24 hours. RNA and protein expression of HH components were evaluated for all time points. qRT-PCR showed that the expression levels of SMO, GLI1, GLI2, and GLI3 were significantly downregulated after 72 hours LDE225 treatment (p<0.05), while alteration of expression levels after 24 and 48 hours treatment was not observed (Figure 2A). The protein expression levels of these key HH members were decreased in a time-dependent manner in response to the treatment of SMO inhibitor (LDE225) (Figure 2B). For GLI inhibitor, qRT-PCR analysis demonstrated that RNA expression of SMO, GLI1, GLI2, and GLI3 was not altered after Gant61 treatment (Figure 2 C). However, protein expression of GLI1, GLI2, and GLI3 were decreased in response to Gant61 treatment (Figure 2D).
The GLIs nuclear translocation was evaluated in LMS cells treated with LDE225 or Gant61 inhibitor for 24, 48, 72 hours, respectively. The results showed that translocation of GLIs into the nucleus was markedly decreased in three-time point treatments with SMO or GLI inhibitor as compared to the untreated control (Figure 2 E and F).
To determine the effects of treatment with LDE225 or Gant61 inhibitor on phenotype of LMS, MTT assay was performed to determine the effect of the treatment on cell proliferation. Our data demonstrated that the proliferation was decreased after treatment with SMO or GLI inhibitor (Figure 2 G and H).
Cell migration is a multi-step process that plays an important role in tumorigenesis. To evaluate the effect of SMO and GLI inhibitors on the migration of LMS cells, would-healing assay was performed. The results showed that the SMO and GLI inhibitor significantly decreased the migration capacity in LMS cells (Figure 3A) (p<0.05). Apoptosis assay was performed and demonstrated that SMO and GLI inhibitors induced apoptosis in LMS cells (Figure 3B), the GLI inhibitor showed a more potent effect compared to SMO inhibitor.
To explore the possible synergistic or additive effect of combination treatment with SMO and GLI inhibitors. We evaluated their inhibitory effect on LMS cells using MTT assay. The results showed no synergism or additive effect using a combination of the treatment (Figure S5)
Treatment with SMO or GLI inhibitors individually showed an inhibitory effect on proliferation, migration, and invasion while induced apoptosis in LMS cells. The inhibition of GLI nuclear translocation was more potent using SMO inhibitor. Thus, the SMO gene was selected for knockdown. The knockdown of the SMO gene was performed using interference RNA and the HH components were evaluated in LMS cells. SMO protein expression was evaluated to verify the efficiency of the knockdown and was highly decreased after the knockdown (Figure 3C). Figure 3D shows that knockdown of SMO decreased the expression of SMO, GLI1, GLI2, and GLI3 (p<0.05).
Inhibition of DNA methyltransferase regulated HH signaling in LMS cells
To understand the molecular mechanism of activation of the HH signaling pathway in LMS in the context of epigenetic regulation, we determined the expression of DNA methyltransferases (DNMT) in LMS and UTSM cells. Our studies showed that RNA expression of DNMT1, DNMT3a, and DNMT3b was upregulated in LMS compared to UTSM cells (p<0.05) (Figure 4A). Accordingly, the protein expression of DNMT1 and DNMT3a were also increased in the LMS compared to UTSM cells (Figure 4B). Next, we determined whether inhibition of DNA methyltransferases with 5’-Aza–2’-Deoxycytidine (5’-Aza-dc) suppressed LMS phenotype. We performed a MTT assay using a different dose at three-times point (24, 48 and 72 hours). After 72 hours of treatment, 5’-Aza-dc at the concentration of 2 µM showed 50% of inhibition in proliferation (Figure S6 A). PTCH1 DNA methylation was evaluated to verify if the treatment with DNA methyltransferase inhibitor was able to reverse the methylation profile in LMS cells. The basal level of the percentage of PTCH1 DNA methylation in LMS was 2.3%. The percentage of PTCH1 DNA methylation after 72 hours of 5’-Aza-dc treatment was decreased to 1%.
RNA and protein expression levels of DNMTs were evaluated in LMS cells in response to 5’-Aza-dc treatment. The RNA expression of DNMT1, DNMT3a, and DNMT3b was decreased in 5’-Aza-dc-treated LMS cells compared to the control (Figure 4C). The protein expression of DNMT1 and DNMT3a in LMS cells was also decreased after treatment with 5’-Aza-dc (Figure 4D).
To evaluate the impact of DNA methyltransferase inhibition on the HH signaling pathway, the components of the HH signaling were evaluated in the presence or absence of 5’-Aza-dc in LMS cells. Although the RNA expression of PTCH1, SMO, SUFU, GLI2, and GLI3 was not altered after5’-Aza-dc treatment, the decreased RNA expression of GLI1 was observed in response to 5’-Aza-dc treatment (Figure 5A). WB analysis exhibited decreased expression levels of SMO and GLI1 (Figure 5B). Moreover, GLI1 and GLI2 nuclear translocation were decreased in response to 5’-Aza-dc treatment. On the other hand, 5’-Aza-dc treatment increased the nuclear translocation of GLI3 (Figure 5C).
Next, determine the effect of DNMT inhibition on proliferation, migration, and apoptosis in LMS cells. The results showed that 5’-Aza-dc decreased proliferation (Figure 5D), concomitantly with decreased expression of PCNA proliferation marker in LMS cells (Figure S 6B). Migration capacity was decreased after 5’-Aza-dc treatment (p<0.05) (Figure 5E). Moreover, 5’-Aza-dc treatment was capable of inducing apoptosis in LMS cells (Figure 5F).
Inhibition of both DNA Methylation and Hedgehog Signaling in Human LMS cell Lines
The treatment with DNA methyltransferase and HH inhibitors showed an inhibitory effect on HH signaling via decreasing GLI1 transcription and protein expression, as evidenced by decreasing proliferation, migration and invasion while inducing apoptosis. Next, we performed experiments to explore whether the DNA methyltransferase inhibitor, in combination with HH inhibitors, can exhibit an additive or synergistic effect in LMS cells.
MTT assay was performed to evaluate the combined effect of DNA methylation and HH inhibitors on LMS proliferation. The results showed that the combination treatment with DNA methylation and SMO inhibitors did not show synergism or additive effect. However, the combination of 5’-Aza-dc with GLI inhibitor showed a synergistic effect (Figure S7A). Since the treatment of 1 µM 5’-Aza-dc with 30 µM GLI inhibitor exhibited a most potently inhibitory effect on LMS proliferation, this combination treatment was used for further studies (Figure S7B).
The RNA expression of key HH members including of SMO, GLI1, GLI2, and GLI3 was measured with and without combination treatment, our data demonstrated that combination treatment decreased the RNA expression of SMO, GLI1, GLI2, and GLI3 (p<0.05) compared to the control (Figure 6A). The combination treatment also resulted in decreased protein levels of GLI1 (Figure 6B). Moreover, the combination treatment decreased GLI1 nuclear translocation in a time-dependent manner compared to the control (Figure 6C).
To explore this combination effect on LMS cells phenotype, the cells were treated with both inhibitors for 72 hours, and proliferation was evaluated every 24 hours. Figure 6D showed that combination treatment decreased the proliferation of LMS cells. In addition, the wound-healing assay demonstrated that combination treatment decreased the migration capacity in the LMS cells (p<0.05) (Figure 6E). The combination showed a more potent effect comparing to the single treatment, with decrease expression of HH signaling components, proliferation, decreasing GLI1 nuclear translocation and migration capacity in LMS.