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Suppression of Aurora-A-FLJ10540 signaling axis prohibits the malignant state of head and neck cancer.

Chen CH, Chang AY, Li SH, Tsai HT, Shiu LY, Su LJ, Wang WL, Chiu TJ, Luo SD, Huang TL, Chien CY - Mol. Cancer (2015)

Bottom Line: In addition, the FLJ10540-PI3K complex was destroyed by inhibition the Aurora-A kinase activity.Forced overexpression of FLJ10540 in Aurora-A-depleted or in MLN8237-treated HNC cells attenuated the effect on cytotoxicity to cisplatin.Finally, immunohistochemical analysis of human HNC specimens showed a significant positively correlation among Aurora-A, FLJ10540, MMP-7 and MMP-10 expressions.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. chench7@gmail.com.

ABSTRACT

Background: Head and neck cancer (HNC) is a highly invasive cancer. Aurora-A has been reported for a number of malignancies. However, the identity of downstream effectors responsible for its aggressive phenotype in HNC remains underinvestigated.

Methods: The mRNA and protein expression levels of Aurora-A and FLJ10540 were assessed in HNC specimens and cell lines using RT-qPCR, western blot, Oncomine, and microarray database analysis. The downstream molecular mechanisms of Aurora-A were confirmed by RT-qPCR, western blot, luciferase reporter, confocal microscopy analyses, immunoprecipitation, colony formation, cell viability, and xenograft model. Cellular functions in response to Aurora-A-modulated downstream targets such as FLJ10540 and MMPs were examined in vitro and in vivo, including cell growth, motility and chemosensitivity. Aurora-A/FLJ10540/MMPs expression was determined in cancer and adjacent normal tissues from HNC patients by immunohistochemistry approach.

Results: In the current study, Aurora-A exhibited similar gene expression profiles with FLJ10540 by using accessibly public microarray and Oncomine database analysis, raising the possibility that these molecules might coordinately participate in cancer progression and metastasis of HNC. These two molecules connection were also examined in cell lines and tissues of HNC. Aurora-A overexpression could not only bind to the promoter of FLJ10540 to induce FLJ10540 expression, but also increase both mRNA and protein levels of MMP-7 and MMP-10 in HNC cells. Conversely, depletion of Aurora-A expression by using siRNA or Aurora-A kinase inhibitor, MLN8237, suppressed FLJ10540, MMP-7 and MMP-10 mRNA and protein expressions in vitro and in vivo. In addition, the FLJ10540-PI3K complex was destroyed by inhibition the Aurora-A kinase activity. Forced overexpression of FLJ10540 in Aurora-A-depleted or in MLN8237-treated HNC cells attenuated the effect on cytotoxicity to cisplatin. Elevated Aurora-A expression in HNC cells led to the characteristics of more aggressive malignancy, including enhanced chemoresistance and increased the abilities of proliferation, migration and invasion, which was required for FLJ10540/MMP-7 or FLJ10540/MMP-10 expressions. Finally, immunohistochemical analysis of human HNC specimens showed a significant positively correlation among Aurora-A, FLJ10540, MMP-7 and MMP-10 expressions.

Conclusion: Together, our findings define a novel mechanism by which Aurora-A promotes cell malignancy, with potential implications for understanding the clinical action of Aurora-A.

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Related in: MedlinePlus

FLJ10540 expression and PI3K-FLJ10540 complex are restrained upon MLN8237 stimulation in HNC cells. (A and B) The mRNA and protein expression levels of FLJ10540 were examined by Q-RT-PCR and Western blotting in SAS cells in MLN8237 dose-dependent manner. The results were normalized against the expression level of GAPDH mRNA in each MLN8237-treated cell. Using the same panel, the total proteins were probed with antibodies against phosphorylated-Aurora-A, Aurora-A, FLJ10540 and β-actin. β-actin was used as a control. Data are representative of three independent experiments done in triplicate. (C) Luciferase assays were done to detect promoter activity of FLJ10540 in transfected FaDu cells in the presence or absence of MLN8237. (D) Unsynchronized HNC cells were fixed, and immunostained with antibodies against endogenous FLJ10540 or DAPI. Representative data are shown. (E) Co-immunoprecipitation of FLJ10540 and PI3K. Cells treated with or without MLN8237 and antibody of p110-α used for immunoprecipitation and representative western blot detection of p85-α and FLJ10540 were shown. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig3: FLJ10540 expression and PI3K-FLJ10540 complex are restrained upon MLN8237 stimulation in HNC cells. (A and B) The mRNA and protein expression levels of FLJ10540 were examined by Q-RT-PCR and Western blotting in SAS cells in MLN8237 dose-dependent manner. The results were normalized against the expression level of GAPDH mRNA in each MLN8237-treated cell. Using the same panel, the total proteins were probed with antibodies against phosphorylated-Aurora-A, Aurora-A, FLJ10540 and β-actin. β-actin was used as a control. Data are representative of three independent experiments done in triplicate. (C) Luciferase assays were done to detect promoter activity of FLJ10540 in transfected FaDu cells in the presence or absence of MLN8237. (D) Unsynchronized HNC cells were fixed, and immunostained with antibodies against endogenous FLJ10540 or DAPI. Representative data are shown. (E) Co-immunoprecipitation of FLJ10540 and PI3K. Cells treated with or without MLN8237 and antibody of p110-α used for immunoprecipitation and representative western blot detection of p85-α and FLJ10540 were shown. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: MLN8237, an ATP-competitive and reversible inhibitor has been shown to inhibit Aurora-A activity in advanced malignancies [7,26]. To further confirm the positive correlation between Aurora-A activity and FLJ10540, we examined FLJ10540 expression by Q-RT-PCR and Western blotting under the condition of Aurora-A inhibition by MLN8237 treatment in SAS cell lines. As shown in Figure 3A and B, MLN8237 significantly reduced FLJ10540 mRNA and protein expressions in a concentration-dependent manner. To determine whether Aurora-A activity induction of FLJ10540 is regulated at the level of transcription, FLJ10540 reporter-based plasmids were transfected into FaDu cells under treatment of MLN8237 in a dose-dependent manner. As expected, the luciferase activity of FLJ10540 was suppressed upon MLN8237 treatment in a dose-dependent manner (Figure 3C). Next, we further investigated the protein expression of FLJ10540 in unsynchronized SAS cells by indirect immunofluorescence. It was indicated that indeed, endogenous FLJ10540 protein expression was decreased in HNC cells treated with MLN8237 (Figure 3D). Our previous data demonstrated that FLJ10540 may act as a scaffold protein to stabilize the PI3K complex in human cancer cells [16,18,24]. According to data described above, we inspired that FLJ10540 associated with PI3K is required Aurora-A activity. The data illustrated that the associated strength of PI3K-FLJ10540 complex was depended on Aurora-A activity (Figure 3E). These data suggest that the Aurora-A activity is critical for FLJ10540 expression and PI3K-FLJ10540 interaction in HNC cells.Figure 3


Suppression of Aurora-A-FLJ10540 signaling axis prohibits the malignant state of head and neck cancer.

Chen CH, Chang AY, Li SH, Tsai HT, Shiu LY, Su LJ, Wang WL, Chiu TJ, Luo SD, Huang TL, Chien CY - Mol. Cancer (2015)

FLJ10540 expression and PI3K-FLJ10540 complex are restrained upon MLN8237 stimulation in HNC cells. (A and B) The mRNA and protein expression levels of FLJ10540 were examined by Q-RT-PCR and Western blotting in SAS cells in MLN8237 dose-dependent manner. The results were normalized against the expression level of GAPDH mRNA in each MLN8237-treated cell. Using the same panel, the total proteins were probed with antibodies against phosphorylated-Aurora-A, Aurora-A, FLJ10540 and β-actin. β-actin was used as a control. Data are representative of three independent experiments done in triplicate. (C) Luciferase assays were done to detect promoter activity of FLJ10540 in transfected FaDu cells in the presence or absence of MLN8237. (D) Unsynchronized HNC cells were fixed, and immunostained with antibodies against endogenous FLJ10540 or DAPI. Representative data are shown. (E) Co-immunoprecipitation of FLJ10540 and PI3K. Cells treated with or without MLN8237 and antibody of p110-α used for immunoprecipitation and representative western blot detection of p85-α and FLJ10540 were shown. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
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Related In: Results  -  Collection

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Fig3: FLJ10540 expression and PI3K-FLJ10540 complex are restrained upon MLN8237 stimulation in HNC cells. (A and B) The mRNA and protein expression levels of FLJ10540 were examined by Q-RT-PCR and Western blotting in SAS cells in MLN8237 dose-dependent manner. The results were normalized against the expression level of GAPDH mRNA in each MLN8237-treated cell. Using the same panel, the total proteins were probed with antibodies against phosphorylated-Aurora-A, Aurora-A, FLJ10540 and β-actin. β-actin was used as a control. Data are representative of three independent experiments done in triplicate. (C) Luciferase assays were done to detect promoter activity of FLJ10540 in transfected FaDu cells in the presence or absence of MLN8237. (D) Unsynchronized HNC cells were fixed, and immunostained with antibodies against endogenous FLJ10540 or DAPI. Representative data are shown. (E) Co-immunoprecipitation of FLJ10540 and PI3K. Cells treated with or without MLN8237 and antibody of p110-α used for immunoprecipitation and representative western blot detection of p85-α and FLJ10540 were shown. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: MLN8237, an ATP-competitive and reversible inhibitor has been shown to inhibit Aurora-A activity in advanced malignancies [7,26]. To further confirm the positive correlation between Aurora-A activity and FLJ10540, we examined FLJ10540 expression by Q-RT-PCR and Western blotting under the condition of Aurora-A inhibition by MLN8237 treatment in SAS cell lines. As shown in Figure 3A and B, MLN8237 significantly reduced FLJ10540 mRNA and protein expressions in a concentration-dependent manner. To determine whether Aurora-A activity induction of FLJ10540 is regulated at the level of transcription, FLJ10540 reporter-based plasmids were transfected into FaDu cells under treatment of MLN8237 in a dose-dependent manner. As expected, the luciferase activity of FLJ10540 was suppressed upon MLN8237 treatment in a dose-dependent manner (Figure 3C). Next, we further investigated the protein expression of FLJ10540 in unsynchronized SAS cells by indirect immunofluorescence. It was indicated that indeed, endogenous FLJ10540 protein expression was decreased in HNC cells treated with MLN8237 (Figure 3D). Our previous data demonstrated that FLJ10540 may act as a scaffold protein to stabilize the PI3K complex in human cancer cells [16,18,24]. According to data described above, we inspired that FLJ10540 associated with PI3K is required Aurora-A activity. The data illustrated that the associated strength of PI3K-FLJ10540 complex was depended on Aurora-A activity (Figure 3E). These data suggest that the Aurora-A activity is critical for FLJ10540 expression and PI3K-FLJ10540 interaction in HNC cells.Figure 3

Bottom Line: In addition, the FLJ10540-PI3K complex was destroyed by inhibition the Aurora-A kinase activity.Forced overexpression of FLJ10540 in Aurora-A-depleted or in MLN8237-treated HNC cells attenuated the effect on cytotoxicity to cisplatin.Finally, immunohistochemical analysis of human HNC specimens showed a significant positively correlation among Aurora-A, FLJ10540, MMP-7 and MMP-10 expressions.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. chench7@gmail.com.

ABSTRACT

Background: Head and neck cancer (HNC) is a highly invasive cancer. Aurora-A has been reported for a number of malignancies. However, the identity of downstream effectors responsible for its aggressive phenotype in HNC remains underinvestigated.

Methods: The mRNA and protein expression levels of Aurora-A and FLJ10540 were assessed in HNC specimens and cell lines using RT-qPCR, western blot, Oncomine, and microarray database analysis. The downstream molecular mechanisms of Aurora-A were confirmed by RT-qPCR, western blot, luciferase reporter, confocal microscopy analyses, immunoprecipitation, colony formation, cell viability, and xenograft model. Cellular functions in response to Aurora-A-modulated downstream targets such as FLJ10540 and MMPs were examined in vitro and in vivo, including cell growth, motility and chemosensitivity. Aurora-A/FLJ10540/MMPs expression was determined in cancer and adjacent normal tissues from HNC patients by immunohistochemistry approach.

Results: In the current study, Aurora-A exhibited similar gene expression profiles with FLJ10540 by using accessibly public microarray and Oncomine database analysis, raising the possibility that these molecules might coordinately participate in cancer progression and metastasis of HNC. These two molecules connection were also examined in cell lines and tissues of HNC. Aurora-A overexpression could not only bind to the promoter of FLJ10540 to induce FLJ10540 expression, but also increase both mRNA and protein levels of MMP-7 and MMP-10 in HNC cells. Conversely, depletion of Aurora-A expression by using siRNA or Aurora-A kinase inhibitor, MLN8237, suppressed FLJ10540, MMP-7 and MMP-10 mRNA and protein expressions in vitro and in vivo. In addition, the FLJ10540-PI3K complex was destroyed by inhibition the Aurora-A kinase activity. Forced overexpression of FLJ10540 in Aurora-A-depleted or in MLN8237-treated HNC cells attenuated the effect on cytotoxicity to cisplatin. Elevated Aurora-A expression in HNC cells led to the characteristics of more aggressive malignancy, including enhanced chemoresistance and increased the abilities of proliferation, migration and invasion, which was required for FLJ10540/MMP-7 or FLJ10540/MMP-10 expressions. Finally, immunohistochemical analysis of human HNC specimens showed a significant positively correlation among Aurora-A, FLJ10540, MMP-7 and MMP-10 expressions.

Conclusion: Together, our findings define a novel mechanism by which Aurora-A promotes cell malignancy, with potential implications for understanding the clinical action of Aurora-A.

Show MeSH
Related in: MedlinePlus