SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer - PubMed (original) (raw)
SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer
Z Zhou et al. Oncogene. 2016.
Abstract
Hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) has been found in various types of human cancers, including head and neck cancer (HNC). Although smoking is critical in the development and progression of HNC, how tobacco components activate STAT3 is unclear. We demonstrated that exposure of HNC cell lines to a tobacco extract induced a rapid Y705 phosphorylation of STAT3 and a rapid increase in the SUMO protease SENP3 that depended on a simultaneous increase in reactive oxygen species. We identified that SUMOylation at the lysine 451 site facilitated STAT3 binding to the phosphatase TC45 through an SUMO-interacting motif of TC45. SENP3 could thus enhance STAT3 phosphorylation by de-conjugating the SUMO2/3 modification of STAT3. Knocking-down of SENP3 greatly impaired basal and induced STAT3 phosphorylation by tobacco extract or interleukin 6. A correlation between SENP3 protein levels and STAT3 Y705 phosphorylation levels in human laryngeal carcinoma specimens was found, which was more significant in the specimens derived from the smoker patients and with poor clinicopathological parameters. Our data identified SUMOylation as a previously undescribed post-translational modification of STAT3 and SENP3 as a critical positive modulator of tobacco- or cytokine-induced STAT3 activation. These findings provide novel insights into the hyperphosphorylation of STAT3 in development of HNC.
Figures
Figure 1
SENP3 protein level correlates with smoking and STAT3 activation in laryngeal carcinoma. (a) Immunohistochemistry for SENP3 was performed in sections derived from vocal polyp (_n_=12), laryngeal dysplasia (_n_=10), laryngeal carcinoma specimens and their corresponding para-carcinoma tissues (_n_=52). The representative images were shown. (b) The relative positive stain areas of SENP3 in each specimen were measured, and the average percentage of each group was displayed in bar chart. All data were shown with the means±s.d. of all specimen in different groups. *P<0.05, ***P<0.001. (c) Immunohistochemistry for SENP3 was performed in 159 laryngeal carcinoma specimens classified based on cigarette smoking (_n_=117) and non-smoking (_n_=42) patients. The representative images were shown. (d) The relative positive stain areas of SENP3 in two groups were measured and were displayed in scatterplot. *P<0.05. (e) Immunohistochemistry for SENP3 and pY705-STAT3 was performed in serial sections derived from specimens of different laryngeal cancers. Two representative specimens were shown. (f) The positive scores of protein levels of SENP3 and pY705-STAT3 based on the data in (e) were shown in two indexes: the percentages of positive stain areas of the both proteins in each specimen (left) and the average intensities of positive stain of the both proteins in the same areas within each specimen (right). _r_=the coefficients of correlation.
Figure 2
NNK induces STAT3 phosphorylation and increase of SENP3 protein levels in HNC cells in an ROS-dependent manner. (a) The levels of STAT3 phosphorylation at tyrosine 705 (in brief, pY705-STAT3) were determined by IB in Hep-2 cells exposed to 1 μ
m
NNK and 20 ng/ml IL-6 respectively for the indicated time (left), and in KB or HN30 cells exposed to 1 μ
m
NNK for indicated time (right). (b) The pY705-STAT3 levels were determined by IB in Hep-2 cells exposed to 1 μ
m
NNK in the presence or absence of 1 μg/ml neutralized antibody against IL-6 (left) and 10 μ
m
JAK inhibitor (right) for the indicated time. Anti-IL-6 antibody and JAK inhibitor were pre-administered for 1 h. (c) The pY705-STAT3 levels were determined by IB in Hep-2 cells exposed to lower doses of NNK (0.01 μ
m
) or IL-6 (2 ng/ml) alone or in combination for the indicated time. (d) ROS level was determined by DCFH-DA staining and flow-cytometric analysis in Hep2 cells exposed to 1 μ
m
NNK for the indicated time. Error bars represent±s.d. for triplicate experiments. ***P<0.001. (e) The SENP3 and pY705-STAT3 levels were determined by IB in Hep-2, KB and HN30 cells exposed to 1 μ
m
NNK in the presence or absence of 5 m
m
NAC for the indicated time.
Figure 3
SENP3 enhances the basal and NNK-or IL-6-induced STAT3 phosphorylation in HNC cells. (a–c) The pY705-STAT3 levels were determined by IB in the cells with various gene interferences and treatments. (a) Hep-2 cells stably expressing non-specific shRNA (sh-NC) or SENP3 shRNA (sh-SENP3) were exposed to 1 μ
m
NNK for the indicated time. (b) KB and HN30 cells with SENP3 knockdown (si-SENP3) for 48 h were exposed to 1 μ
m
NNK for the indicated time. (c) Hep-2 cells with either sh-SENP3 (left) or overexpressed SENP3 (right) were exposed to 20 ng/ml IL-6 for the indicated time.
Figure 4
SENP3 enhances STAT3 phosphorylation by suppressing the interaction between STAT3 and its phosphatase TC45 in the nucleus. (a) The pY705-STAT3 levels were examined in the fractions of cytoplasm and nucleus, respectively. Hep-2 cells with SENP3 overexpression or si-SENP3 for 48 h were exposed to 20 ng/ml IL-6 for 15 min or 1 μ
m
NNK for 30 min. Tubulin and PARP were taken as the internal controls for cytoplasm ‘C' and nucleus ‘N', respectively. (b) The pY705-STAT3 levels were examined in Hep-2 cells. Hep-2 cells were transfected with the gradient concentrations of Flag-TC45 for 24 h and then exposed to 1 μ
m
NNK and 20 ng/ml IL-6, respectively, for the indicated time. (c) The interaction of STAT3 with TC45 was detected by co-IP. HEK293 T (293T) cells were transfected with the gradient concentrations of GFP-SENP3 and Flag-STAT3 for 48 h. Immunoprecipitation of Flag-tagged STAT3 was performed to detect TC45 using anti-PTP antibody. (d) The interaction of endogenous TC45 with pY705-STAT3 was determined by co-IP in the lysates of Hep-2 cells with si-SENP3 for 48 h. IP was performed using anti-pY705-STAT3 antibody.
Figure 5
SENP3 de-conjugates SUMO2/3 at lysine 451 of STAT3 to halt its association with TC45. (a, b) SUMO3 conjugates of STAT3 were determined by co-IP in HEK293T cells. Cells were transfected with FLAG-STAT3 and RH-SUMO3 for 48 h. Co-IP was performed using Flag-M2 beads for immunoprecipitation and using anti-RH and anti-STAT3 antibodies for IB. (a) Cells were transfected with RH-SUMO3 in gradient concentrations (left). Cells were co-transfected with Flag-tagged WT or mutated STAT3 (K451R, K354R and K340R) and RH-SUMO3 (right). (b) Cells were co-transfected with GFP-tagged WT or mutated SENP3 (C532A) and FLAG-STAT3. (c) Endogenous SENP3/pY705-STAT3 or STAT3 interaction was determined by co-IP in Hep-2 cells. (d) Endogenous SUMO3 conjugates of STAT3 were determined by co-IP in the lysates of HEK293T-sh-SENP3 stable cells. (e) Endogenous SUMO3 conjugates of STAT3 were determined by co-IP in the lysates of Hep2-sh-SENP3 stable cells when GFP-tagged WT or mutated SENP3 (C532A) putting back and 1 μ
m
NNK administration for 30 min. Arrowheads indicated SUMO3-conjugated STAT3 in (a, b, d, e). (f) The pY705-STAT3 levels were determined by IB in the lysates of Hep2-sh-NC and Hep2-sh-SENP3 stable cells when co-transfected with GFP-tagged WT or mutated STAT3 (K451R). (g) The interaction of STAT3 with TC45 was determined by co-IP in the lysates of HEK293T cells transfected with Flag-tagged WT STAT3 or SUMOless mutant STAT3 (K451R) and STAT3-SUMO3 fusion vector. (h) The interaction of STAT3 with TC45 was determined by FRET analysis in Hep-2 cells transfected with Flag-tagged WT STAT3 or STAT3-SUMO3 fusion vector. Bar, 10 μm. Percentages of N-FRET values in 10 cells were showed. ***P<0.001. (i) The interaction of STAT3 with TC45 was determined by co-IP in the lysates of HEK293T cells transfected with Flag-tagged WT TC45 or SIM mutant TC45 (V109A/L112A). (j) A model illustrating the critical role of SUMOylation in the association of STAT3 with TC45.
Figure 5
SENP3 de-conjugates SUMO2/3 at lysine 451 of STAT3 to halt its association with TC45. (a, b) SUMO3 conjugates of STAT3 were determined by co-IP in HEK293T cells. Cells were transfected with FLAG-STAT3 and RH-SUMO3 for 48 h. Co-IP was performed using Flag-M2 beads for immunoprecipitation and using anti-RH and anti-STAT3 antibodies for IB. (a) Cells were transfected with RH-SUMO3 in gradient concentrations (left). Cells were co-transfected with Flag-tagged WT or mutated STAT3 (K451R, K354R and K340R) and RH-SUMO3 (right). (b) Cells were co-transfected with GFP-tagged WT or mutated SENP3 (C532A) and FLAG-STAT3. (c) Endogenous SENP3/pY705-STAT3 or STAT3 interaction was determined by co-IP in Hep-2 cells. (d) Endogenous SUMO3 conjugates of STAT3 were determined by co-IP in the lysates of HEK293T-sh-SENP3 stable cells. (e) Endogenous SUMO3 conjugates of STAT3 were determined by co-IP in the lysates of Hep2-sh-SENP3 stable cells when GFP-tagged WT or mutated SENP3 (C532A) putting back and 1 μ
m
NNK administration for 30 min. Arrowheads indicated SUMO3-conjugated STAT3 in (a, b, d, e). (f) The pY705-STAT3 levels were determined by IB in the lysates of Hep2-sh-NC and Hep2-sh-SENP3 stable cells when co-transfected with GFP-tagged WT or mutated STAT3 (K451R). (g) The interaction of STAT3 with TC45 was determined by co-IP in the lysates of HEK293T cells transfected with Flag-tagged WT STAT3 or SUMOless mutant STAT3 (K451R) and STAT3-SUMO3 fusion vector. (h) The interaction of STAT3 with TC45 was determined by FRET analysis in Hep-2 cells transfected with Flag-tagged WT STAT3 or STAT3-SUMO3 fusion vector. Bar, 10 μm. Percentages of N-FRET values in 10 cells were showed. ***P<0.001. (i) The interaction of STAT3 with TC45 was determined by co-IP in the lysates of HEK293T cells transfected with Flag-tagged WT TC45 or SIM mutant TC45 (V109A/L112A). (j) A model illustrating the critical role of SUMOylation in the association of STAT3 with TC45.
Figure 6
DeSUMOylation of STAT3 enhances its transcriptional activity and oncogenic function in HNC cells. (a–e) Hep2-sh-STAT3 stable cells were added back with Flag-tagged WT STAT3 or SUMOless mutant STAT3 (K451R) before undergoing various assays. (a) The efficiency of shRNA and overexpression of SENP3 was determined by IB. (b) The mRNA levels of STAT3 target genes c-Myc, Cyclin D1, Bcl-xL, Mcl-1, VEGF and Survivin were determined by qRT-PCR at 48 h post transfection. (c) The viable cell numbers were measured by CCK8 at 0, 24, 48 and 72 h post transfection. (d) The numbers of colonies in soft agar were measured. **P<0.01. (e) The cell migration ability was analyzed by Transwell migration assay. *P<0.05. (b, d, e) Error bars represent ±s.d. for triplicate experiments. (c) Error bars represent ±s.d. for three replication wells. The experiment repeated three times.
Figure 7
A schema for the roles of SUMOylation and SENP3 in the regulation of STAT3 activation under basal and stressed microenvironments that are deteriorated by cigarette components and excessive IL-6.
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