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Contribution of TIP30 to chemoresistance in laryngeal carcinoma.

Zhu M, Yin F, Yang L, Chen S, Chen R, Zhou X, Jing W, Fan X, Jia R, Wang H, Zheng H, Zhao J, Guo Y - Cell Death Dis (2014)

Bottom Line: We showed that TIP30 expression decreased significantly in drug-selected cells (DSCs) of laryngeal carcinoma.Moreover, decreased TIP30 expression contributed to chemoresistance, self-renewal and proliferation of LSCC cells via nuclearlisation of β-catenin, a cell-cell adhesion and stem cell renewal regulator.Consistently, Kaplan-Meier and Cox proportional hazards regression modelling analyses showed that decreased TIP30 expression independently predicted poor survival in patients with LSCC.

View Article: PubMed Central - PubMed

Affiliation: 1] International Joint Cancer Research Institute, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China [2] Changhai Hospital, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China.

ABSTRACT
Laryngeal squamous cell carcinoma (LSCC) is one of the most common carcinomas of the head and neck. Despite advances in diagnosis and treatment, the survival of patients with LSCC has not improved in the past two decades. TIP30, a newly identified tumour suppressor, appears to be involved in multiple processes during tumour development. Here, we investigated the involvement of TIP30 in chemoresistance of LSCC in vitro and in vivo. We showed that TIP30 expression decreased significantly in drug-selected cells (DSCs) of laryngeal carcinoma. Suppressing TIP30 enhanced resistance capability to multiple chemotherapy drugs, cell proliferation and self-renewal in Hep2 cells. Additionally, decreased self-renewal capacity and chemotherapeutic resistance were observed in DSCs overexpressing TIP30. Furthermore, TIP30 negatively regulated tumourigenesis and chemoresistance in LSCC cells subcutaneously transplanted into nude mice. Moreover, decreased TIP30 expression contributed to chemoresistance, self-renewal and proliferation of LSCC cells via nuclearlisation of β-catenin, a cell-cell adhesion and stem cell renewal regulator. Consistently, Kaplan-Meier and Cox proportional hazards regression modelling analyses showed that decreased TIP30 expression independently predicted poor survival in patients with LSCC. Taken together, our results reveal that TIP30 has a crucial role in chemoresistance of LSCC through the AKT/glycogen synthase kinase-3β/β-catenin signalling pathway and may be a promising candidate for improving LSCC chemotherapy.

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TIP30 increases gradually in DSCs by adherent growth and is negatively involved in the regulation of self-renewal and chemotherapeutic resistance in laryngeal carcinoma cells. (a) Western blot was performed to assess TIP30 protein levels in Hep2 cells and DSCs at different time points under adherent conditions. (b) Hep2 cells were infected with short shTip30, and DSCs were infected with LVTip30 for 5 days. Comparison of self-renewal marker (Bmi1, Oct4 and Nanog) mRNA levels among Hep2-shTip30, DSCs-LVTip30 and control cells (n=3). (c) Mammospheres that formed during four in vitro serial passages were quantified in the indicated cells. Data are reported as the number of mammospheres formed/1000 seeded cells±S.E. Bars denote the S.E. (n=5). (d) ABC transporter mRNA levels (ABCG2, ABCB1 and ABCC1) were detected in the indicated cells (n=3). (e) Left: time–response survival curves of described cells treated with 30 μM cisplatin; right: dose–response survival rate curves in the cells described in (b) were generated after exposure to various cisplatin concentrations for 72 h. Data are presented as means±S.D. from three independent experiments. (f) Flow-assisted cell sorting analysis was performed to determine CD133 expression at different time points in DSCs-shTip30 and control cells in adherent culture. Corresponding TIP30 mRNA levels were also detected. Data are presented as means±S.D. from three independent experiments. (g) Cytokeratin 19 immunofluorescence analysis in freshly isolated DSCs versus Hep2 cells (left) and in DSC-shTip30 versus DSC-shNon under differentiating conditions (right). DAPI was used to stain nuclei. Scale bar, 50 μm. *P<0.05 and **P<0.01
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fig2: TIP30 increases gradually in DSCs by adherent growth and is negatively involved in the regulation of self-renewal and chemotherapeutic resistance in laryngeal carcinoma cells. (a) Western blot was performed to assess TIP30 protein levels in Hep2 cells and DSCs at different time points under adherent conditions. (b) Hep2 cells were infected with short shTip30, and DSCs were infected with LVTip30 for 5 days. Comparison of self-renewal marker (Bmi1, Oct4 and Nanog) mRNA levels among Hep2-shTip30, DSCs-LVTip30 and control cells (n=3). (c) Mammospheres that formed during four in vitro serial passages were quantified in the indicated cells. Data are reported as the number of mammospheres formed/1000 seeded cells±S.E. Bars denote the S.E. (n=5). (d) ABC transporter mRNA levels (ABCG2, ABCB1 and ABCC1) were detected in the indicated cells (n=3). (e) Left: time–response survival curves of described cells treated with 30 μM cisplatin; right: dose–response survival rate curves in the cells described in (b) were generated after exposure to various cisplatin concentrations for 72 h. Data are presented as means±S.D. from three independent experiments. (f) Flow-assisted cell sorting analysis was performed to determine CD133 expression at different time points in DSCs-shTip30 and control cells in adherent culture. Corresponding TIP30 mRNA levels were also detected. Data are presented as means±S.D. from three independent experiments. (g) Cytokeratin 19 immunofluorescence analysis in freshly isolated DSCs versus Hep2 cells (left) and in DSC-shTip30 versus DSC-shNon under differentiating conditions (right). DAPI was used to stain nuclei. Scale bar, 50 μm. *P<0.05 and **P<0.01

Mentions: TIP30 expression increased gradually after DSCs were cultured under differentiating conditions, indicating that reduced TIP30 expression may be important for DSCs to maintain an undifferentiated status (Figure 2a). To test whether TIP30 expression is important for self-renewal potential in DSCs, the Tip30 gene was either introduced into DSCs or silenced by RNA interference in Hep2 cells. Self-renewal-associated transcription factors such as Bim1, Oct4 and Nanog increased significantly when TIP30 was silenced in Hep2 cells and decreased when TIP30 was introduced into DSCs (Figure 2b and Supplementary Figure S3A). The sphere formation assay revealed that silencing TIP30 led to enhanced sphere formation in Hep2 cells, whereas introducing TIP30 inhibited sphere formation in DSCs (Figure 2c and Supplementary Figure S3B). Cell proliferation was significantly enhanced in TIP30-silenced Hep2 cells and diminished in TIP30-overexpressed DSCs (Supplementary Figure S4). Furthermore, G0/G1-phase cell cycle arrest was observed in DSC lentivirus (LV) Tip30. Conversely, the proliferation index (percentage of cells in the S and G2 phases) was confirmed to increase significantly after TIP30 silencing in Hep2 cells (Supplementary Figure S5).


Contribution of TIP30 to chemoresistance in laryngeal carcinoma.

Zhu M, Yin F, Yang L, Chen S, Chen R, Zhou X, Jing W, Fan X, Jia R, Wang H, Zheng H, Zhao J, Guo Y - Cell Death Dis (2014)

TIP30 increases gradually in DSCs by adherent growth and is negatively involved in the regulation of self-renewal and chemotherapeutic resistance in laryngeal carcinoma cells. (a) Western blot was performed to assess TIP30 protein levels in Hep2 cells and DSCs at different time points under adherent conditions. (b) Hep2 cells were infected with short shTip30, and DSCs were infected with LVTip30 for 5 days. Comparison of self-renewal marker (Bmi1, Oct4 and Nanog) mRNA levels among Hep2-shTip30, DSCs-LVTip30 and control cells (n=3). (c) Mammospheres that formed during four in vitro serial passages were quantified in the indicated cells. Data are reported as the number of mammospheres formed/1000 seeded cells±S.E. Bars denote the S.E. (n=5). (d) ABC transporter mRNA levels (ABCG2, ABCB1 and ABCC1) were detected in the indicated cells (n=3). (e) Left: time–response survival curves of described cells treated with 30 μM cisplatin; right: dose–response survival rate curves in the cells described in (b) were generated after exposure to various cisplatin concentrations for 72 h. Data are presented as means±S.D. from three independent experiments. (f) Flow-assisted cell sorting analysis was performed to determine CD133 expression at different time points in DSCs-shTip30 and control cells in adherent culture. Corresponding TIP30 mRNA levels were also detected. Data are presented as means±S.D. from three independent experiments. (g) Cytokeratin 19 immunofluorescence analysis in freshly isolated DSCs versus Hep2 cells (left) and in DSC-shTip30 versus DSC-shNon under differentiating conditions (right). DAPI was used to stain nuclei. Scale bar, 50 μm. *P<0.05 and **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: TIP30 increases gradually in DSCs by adherent growth and is negatively involved in the regulation of self-renewal and chemotherapeutic resistance in laryngeal carcinoma cells. (a) Western blot was performed to assess TIP30 protein levels in Hep2 cells and DSCs at different time points under adherent conditions. (b) Hep2 cells were infected with short shTip30, and DSCs were infected with LVTip30 for 5 days. Comparison of self-renewal marker (Bmi1, Oct4 and Nanog) mRNA levels among Hep2-shTip30, DSCs-LVTip30 and control cells (n=3). (c) Mammospheres that formed during four in vitro serial passages were quantified in the indicated cells. Data are reported as the number of mammospheres formed/1000 seeded cells±S.E. Bars denote the S.E. (n=5). (d) ABC transporter mRNA levels (ABCG2, ABCB1 and ABCC1) were detected in the indicated cells (n=3). (e) Left: time–response survival curves of described cells treated with 30 μM cisplatin; right: dose–response survival rate curves in the cells described in (b) were generated after exposure to various cisplatin concentrations for 72 h. Data are presented as means±S.D. from three independent experiments. (f) Flow-assisted cell sorting analysis was performed to determine CD133 expression at different time points in DSCs-shTip30 and control cells in adherent culture. Corresponding TIP30 mRNA levels were also detected. Data are presented as means±S.D. from three independent experiments. (g) Cytokeratin 19 immunofluorescence analysis in freshly isolated DSCs versus Hep2 cells (left) and in DSC-shTip30 versus DSC-shNon under differentiating conditions (right). DAPI was used to stain nuclei. Scale bar, 50 μm. *P<0.05 and **P<0.01
Mentions: TIP30 expression increased gradually after DSCs were cultured under differentiating conditions, indicating that reduced TIP30 expression may be important for DSCs to maintain an undifferentiated status (Figure 2a). To test whether TIP30 expression is important for self-renewal potential in DSCs, the Tip30 gene was either introduced into DSCs or silenced by RNA interference in Hep2 cells. Self-renewal-associated transcription factors such as Bim1, Oct4 and Nanog increased significantly when TIP30 was silenced in Hep2 cells and decreased when TIP30 was introduced into DSCs (Figure 2b and Supplementary Figure S3A). The sphere formation assay revealed that silencing TIP30 led to enhanced sphere formation in Hep2 cells, whereas introducing TIP30 inhibited sphere formation in DSCs (Figure 2c and Supplementary Figure S3B). Cell proliferation was significantly enhanced in TIP30-silenced Hep2 cells and diminished in TIP30-overexpressed DSCs (Supplementary Figure S4). Furthermore, G0/G1-phase cell cycle arrest was observed in DSC lentivirus (LV) Tip30. Conversely, the proliferation index (percentage of cells in the S and G2 phases) was confirmed to increase significantly after TIP30 silencing in Hep2 cells (Supplementary Figure S5).

Bottom Line: We showed that TIP30 expression decreased significantly in drug-selected cells (DSCs) of laryngeal carcinoma.Moreover, decreased TIP30 expression contributed to chemoresistance, self-renewal and proliferation of LSCC cells via nuclearlisation of β-catenin, a cell-cell adhesion and stem cell renewal regulator.Consistently, Kaplan-Meier and Cox proportional hazards regression modelling analyses showed that decreased TIP30 expression independently predicted poor survival in patients with LSCC.

View Article: PubMed Central - PubMed

Affiliation: 1] International Joint Cancer Research Institute, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China [2] Changhai Hospital, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China.

ABSTRACT
Laryngeal squamous cell carcinoma (LSCC) is one of the most common carcinomas of the head and neck. Despite advances in diagnosis and treatment, the survival of patients with LSCC has not improved in the past two decades. TIP30, a newly identified tumour suppressor, appears to be involved in multiple processes during tumour development. Here, we investigated the involvement of TIP30 in chemoresistance of LSCC in vitro and in vivo. We showed that TIP30 expression decreased significantly in drug-selected cells (DSCs) of laryngeal carcinoma. Suppressing TIP30 enhanced resistance capability to multiple chemotherapy drugs, cell proliferation and self-renewal in Hep2 cells. Additionally, decreased self-renewal capacity and chemotherapeutic resistance were observed in DSCs overexpressing TIP30. Furthermore, TIP30 negatively regulated tumourigenesis and chemoresistance in LSCC cells subcutaneously transplanted into nude mice. Moreover, decreased TIP30 expression contributed to chemoresistance, self-renewal and proliferation of LSCC cells via nuclearlisation of β-catenin, a cell-cell adhesion and stem cell renewal regulator. Consistently, Kaplan-Meier and Cox proportional hazards regression modelling analyses showed that decreased TIP30 expression independently predicted poor survival in patients with LSCC. Taken together, our results reveal that TIP30 has a crucial role in chemoresistance of LSCC through the AKT/glycogen synthase kinase-3β/β-catenin signalling pathway and may be a promising candidate for improving LSCC chemotherapy.

Show MeSH
Related in: MedlinePlus