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MPT0B098, a Microtubule Inhibitor, Suppresses JAK2/STAT3 Signaling Pathway through Modulation of SOCS3 Stability in Oral Squamous Cell Carcinoma.

Peng HY, Cheng YC, Hsu YM, Wu GH, Kuo CC, Liou JP, Chang JY, Jin SL, Shiah SG - PLoS ONE (2016)

Bottom Line: In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC).The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity.Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan.

ABSTRACT
Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC.

No MeSH data available.


Related in: MedlinePlus

MPT0B098 modulates JAK2/STAT3 pathway via SOCS3 accumulation.(A) OEC-M1 and HSC-3 cells were treated with 0.25 μM of MPT0B098 for indicated times. The protein level of PIAS1, PIAS3, SOCS1, SOCS2, and SOCS3 were determined by Western blotting. GAPDH was used as loading control. (B) OEC-M1 and HSC-3 cells were tranfected with control vector (VC) and construct containing the SOCS3-coding region (SOCS3 cDNA #1, #2) for 48 hrs. The phosphorylated proteins (p-JAK2, p-TYK2 and p-STAT3) and total level of proteins (SOCS3, JAK2, TYK2 and STAT3) were determined by Western blotting. GAPDH was used as loading control. (C) Western blot analysis of SOCS3 levels after 48 hrs transfection of OEC-M1 and HSC-3 cells with control vector (VC) or two SOCS3 constructs (SOCS3 #1, #2). GAPDH was used as loading control. These SOCS3 overexpression transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; **, p<0.01 versus vehicle control. (D) Western blot analysis of SOCS3 levels after 48 hrs transfection of YD-15 and DOK cells with control shRNA (NS) or two shSOCS3 constructs (shSOCS3 #1, #2). GAPDH was used as loading control. These shRNA transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; ***, p<0.001 versus vehicle control.
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pone.0158440.g004: MPT0B098 modulates JAK2/STAT3 pathway via SOCS3 accumulation.(A) OEC-M1 and HSC-3 cells were treated with 0.25 μM of MPT0B098 for indicated times. The protein level of PIAS1, PIAS3, SOCS1, SOCS2, and SOCS3 were determined by Western blotting. GAPDH was used as loading control. (B) OEC-M1 and HSC-3 cells were tranfected with control vector (VC) and construct containing the SOCS3-coding region (SOCS3 cDNA #1, #2) for 48 hrs. The phosphorylated proteins (p-JAK2, p-TYK2 and p-STAT3) and total level of proteins (SOCS3, JAK2, TYK2 and STAT3) were determined by Western blotting. GAPDH was used as loading control. (C) Western blot analysis of SOCS3 levels after 48 hrs transfection of OEC-M1 and HSC-3 cells with control vector (VC) or two SOCS3 constructs (SOCS3 #1, #2). GAPDH was used as loading control. These SOCS3 overexpression transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; **, p<0.01 versus vehicle control. (D) Western blot analysis of SOCS3 levels after 48 hrs transfection of YD-15 and DOK cells with control shRNA (NS) or two shSOCS3 constructs (shSOCS3 #1, #2). GAPDH was used as loading control. These shRNA transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; ***, p<0.001 versus vehicle control.

Mentions: The STAT signaling can be inhibited by two signal pathways that involve the SOCS family and the PIAS family members. These signal molecules act as negative feedback regulators to prevent further JAK/STAT signal activation [24, 25]. Thus, it was hypothesized that these negative feedback regulators may be involved in the MPT0B098 regulation of the JAK/STAT activity. To address this, the protein levels of the SOCS and PIAS family members in MPT0B098-treated OEC-M1 and HSC-3 cells were measured. The immunoblotting results showed that only SOCS2 and SOCS3 levels were markedly elevated in these cells, whereas the levels of PIAS1, PIAS3 and SOCS1 proteins remained unchanged (Fig 4A). We further explored the effect of SOCS2 and SOCS3 on JAK/STAT signaling pathway in OSCC cells. We found that the total protein and phosphorylation levels of JAK2, TYK2 and STAT3 were significantly decreased in the OSCC cells transfected with a SOCS3 cDNA construct (Fig 4B). However, overexpression of SOCS2 did not change the total protein and phosphorylation levels of JAK2, TYK2 and STAT3 (S2 Fig). We then investigated the role of SOCS3 in MPT0B098-induced apoptosis in OSCC cells. Overexpression of SOCS3 protein in OEC-M1 and HSC-3 cells markedly increased the MPT0B098-induced apoptosis (Fig 4C).However, knockdown of SOCS3 protein in DOK and YD-15 cells significantly attenuated the MPT0B098-induced apoptosis (Fig 4D). These results suggest that the induction of cancer cell apoptosis by MPT0B098 may be mediated by a negative modulation of SOCS3 on JAK2/STAT3 signaling pathway.


MPT0B098, a Microtubule Inhibitor, Suppresses JAK2/STAT3 Signaling Pathway through Modulation of SOCS3 Stability in Oral Squamous Cell Carcinoma.

Peng HY, Cheng YC, Hsu YM, Wu GH, Kuo CC, Liou JP, Chang JY, Jin SL, Shiah SG - PLoS ONE (2016)

MPT0B098 modulates JAK2/STAT3 pathway via SOCS3 accumulation.(A) OEC-M1 and HSC-3 cells were treated with 0.25 μM of MPT0B098 for indicated times. The protein level of PIAS1, PIAS3, SOCS1, SOCS2, and SOCS3 were determined by Western blotting. GAPDH was used as loading control. (B) OEC-M1 and HSC-3 cells were tranfected with control vector (VC) and construct containing the SOCS3-coding region (SOCS3 cDNA #1, #2) for 48 hrs. The phosphorylated proteins (p-JAK2, p-TYK2 and p-STAT3) and total level of proteins (SOCS3, JAK2, TYK2 and STAT3) were determined by Western blotting. GAPDH was used as loading control. (C) Western blot analysis of SOCS3 levels after 48 hrs transfection of OEC-M1 and HSC-3 cells with control vector (VC) or two SOCS3 constructs (SOCS3 #1, #2). GAPDH was used as loading control. These SOCS3 overexpression transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; **, p<0.01 versus vehicle control. (D) Western blot analysis of SOCS3 levels after 48 hrs transfection of YD-15 and DOK cells with control shRNA (NS) or two shSOCS3 constructs (shSOCS3 #1, #2). GAPDH was used as loading control. These shRNA transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; ***, p<0.001 versus vehicle control.
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pone.0158440.g004: MPT0B098 modulates JAK2/STAT3 pathway via SOCS3 accumulation.(A) OEC-M1 and HSC-3 cells were treated with 0.25 μM of MPT0B098 for indicated times. The protein level of PIAS1, PIAS3, SOCS1, SOCS2, and SOCS3 were determined by Western blotting. GAPDH was used as loading control. (B) OEC-M1 and HSC-3 cells were tranfected with control vector (VC) and construct containing the SOCS3-coding region (SOCS3 cDNA #1, #2) for 48 hrs. The phosphorylated proteins (p-JAK2, p-TYK2 and p-STAT3) and total level of proteins (SOCS3, JAK2, TYK2 and STAT3) were determined by Western blotting. GAPDH was used as loading control. (C) Western blot analysis of SOCS3 levels after 48 hrs transfection of OEC-M1 and HSC-3 cells with control vector (VC) or two SOCS3 constructs (SOCS3 #1, #2). GAPDH was used as loading control. These SOCS3 overexpression transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; **, p<0.01 versus vehicle control. (D) Western blot analysis of SOCS3 levels after 48 hrs transfection of YD-15 and DOK cells with control shRNA (NS) or two shSOCS3 constructs (shSOCS3 #1, #2). GAPDH was used as loading control. These shRNA transfactants were treated MPT0B098 for 24 hrs and caspases-3 activity was assessed. The data are represented as mean ± SE; ***, p<0.001 versus vehicle control.
Mentions: The STAT signaling can be inhibited by two signal pathways that involve the SOCS family and the PIAS family members. These signal molecules act as negative feedback regulators to prevent further JAK/STAT signal activation [24, 25]. Thus, it was hypothesized that these negative feedback regulators may be involved in the MPT0B098 regulation of the JAK/STAT activity. To address this, the protein levels of the SOCS and PIAS family members in MPT0B098-treated OEC-M1 and HSC-3 cells were measured. The immunoblotting results showed that only SOCS2 and SOCS3 levels were markedly elevated in these cells, whereas the levels of PIAS1, PIAS3 and SOCS1 proteins remained unchanged (Fig 4A). We further explored the effect of SOCS2 and SOCS3 on JAK/STAT signaling pathway in OSCC cells. We found that the total protein and phosphorylation levels of JAK2, TYK2 and STAT3 were significantly decreased in the OSCC cells transfected with a SOCS3 cDNA construct (Fig 4B). However, overexpression of SOCS2 did not change the total protein and phosphorylation levels of JAK2, TYK2 and STAT3 (S2 Fig). We then investigated the role of SOCS3 in MPT0B098-induced apoptosis in OSCC cells. Overexpression of SOCS3 protein in OEC-M1 and HSC-3 cells markedly increased the MPT0B098-induced apoptosis (Fig 4C).However, knockdown of SOCS3 protein in DOK and YD-15 cells significantly attenuated the MPT0B098-induced apoptosis (Fig 4D). These results suggest that the induction of cancer cell apoptosis by MPT0B098 may be mediated by a negative modulation of SOCS3 on JAK2/STAT3 signaling pathway.

Bottom Line: In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC).The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity.Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan.

ABSTRACT
Microtubule inhibitors have been shown to inhibit Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signal transduction pathway in various cancer cells. However, little is known of the mechanism by which the microtubule inhibitors inhibit STAT3 activity. In the present study, we examined the effect of a novel small-molecule microtubule inhibitor, MPT0B098, on STAT3 signaling in oral squamous cell carcinoma (OSCC). Treatment of various OSCC cells with MPT0B098 induced growth inhibition, cell cycle arrest and apoptosis, as well as increased the protein level of SOCS3. The accumulation of SOCS3 protein enhanced its binding to JAK2 and TYK2 which facilitated the ubiquitination and degradation of JAK2 and TYK2, resulting in a loss of STAT3 activity. The inhibition of STAT3 activity led to sensitization of OSCC cells to MPT0B098 cytotoxicity, indicating that STAT3 is a key mediator of drug resistance in oral carcinogenesis. Moreover, the combination of MPT0B098 with the clinical drug cisplatin or 5-FU significantly augmented growth inhibition and apoptosis in OSCC cells. Taken together, our results provide a novel mechanism for the action of MPT0B098 in which the JAK2/STAT3 signaling pathway is suppressed through the modulation of SOCS3 protein level. The findings also provide a promising combinational therapy of MPT0B098 for OSCC.

No MeSH data available.


Related in: MedlinePlus