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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

Reinforced FLJ10540 expression resists the cytotoxicity to cisplatin in depletion and inactivation cells of Aurora-A. (A) SAS/negative control or SAS/siAurora-A cells co-transfected with vehicle or FLJ10540 were incubated with increasing concentrations of cisplatin for 48 hour, and their percentage viability was measured and compared to that of untreated respective cells. (B) The cells were cultured in the presence of cisplatin (15 μM) for 48 hour, and their viability was measured. Vehicle and FLJ10540 transfectants were cultured in the presence or absence of cisplatin (15 μM) and/or MLN8237 for 48 hour (C) or 15 days (D) and total cell viability were assessed by MTT assay and soft agar. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig5: Reinforced FLJ10540 expression resists the cytotoxicity to cisplatin in depletion and inactivation cells of Aurora-A. (A) SAS/negative control or SAS/siAurora-A cells co-transfected with vehicle or FLJ10540 were incubated with increasing concentrations of cisplatin for 48 hour, and their percentage viability was measured and compared to that of untreated respective cells. (B) The cells were cultured in the presence of cisplatin (15 μM) for 48 hour, and their viability was measured. Vehicle and FLJ10540 transfectants were cultured in the presence or absence of cisplatin (15 μM) and/or MLN8237 for 48 hour (C) or 15 days (D) and total cell viability were assessed by MTT assay and soft agar. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: A recent studies have shown that Aurora-A contributes to the resistance to cisplatin in breast, pancreatic, esophageal squamous cell carcinoma, Acute myeloid leukemia, medulloblastoma, and ovarian cancer cells [7,27-29]. To gain insight the biological function of both Aurora-A and FLJ10540 on chemoresistance to cisplatin, we carried out MTT assay for assessing cell viability in siAurora-A transfected cells and siAurora-A/FLJ10540-overexpressed cells under cisplatin treatment. As shown in Figure 5A, the viability of siAurora-A cells was significantly more sensitive to cisplatin than negative control group in a concentration-dependent manner. Notably, in the presence of cisplatin, siAurora-A transfectants with FLJ10540 exhibited a lower sensitivity than siAurora-A transfectants or negative control to the cytotoxic effects of cisplatin (Figure 5A). In addition, the viability of siAurora-A cells was significantly lower than that of siAurora-A with FLJ10540-overexpressing cells after incubation with cisplatin (15 μM) for 48 hour (Figure 5B), indicating that increased FLJ10540 decreases the sensitivity of Aurora-A-depleted cells to cisplatin. Next, we used NLM8206 to investigate the effects of cisplatin in FLJ10540-overexpressing cells. As shown in Figure 5C, FLJ10540 transfectants incubated with MLN8206 (0.025nM) and cisplatin (15 μM) for 48 hour were significantly less viable than the same cells treated with cisplatin alone or MLN8206 alone. In addition, an assay of in vitro colony formation was used to confirm the result of MTT assay. As expected, FLJ10540 transfectants treated with MLN8237 and cisplatin in combination increased the colony-formation ability than that the vehicle control (Figure 5D). Together, these results demonstrate that the function of FLJ10540 modulated by Aurora-A expression or Aurora-A activity contributes to cisplatin resistance in HNC cells.Figure 5


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)

Reinforced FLJ10540 expression resists the cytotoxicity to cisplatin in depletion and inactivation cells of Aurora-A. (A) SAS/negative control or SAS/siAurora-A cells co-transfected with vehicle or FLJ10540 were incubated with increasing concentrations of cisplatin for 48 hour, and their percentage viability was measured and compared to that of untreated respective cells. (B) The cells were cultured in the presence of cisplatin (15 μM) for 48 hour, and their viability was measured. Vehicle and FLJ10540 transfectants were cultured in the presence or absence of cisplatin (15 μM) and/or MLN8237 for 48 hour (C) or 15 days (D) and total cell viability were assessed by MTT assay and soft agar. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403844&req=5

Fig5: Reinforced FLJ10540 expression resists the cytotoxicity to cisplatin in depletion and inactivation cells of Aurora-A. (A) SAS/negative control or SAS/siAurora-A cells co-transfected with vehicle or FLJ10540 were incubated with increasing concentrations of cisplatin for 48 hour, and their percentage viability was measured and compared to that of untreated respective cells. (B) The cells were cultured in the presence of cisplatin (15 μM) for 48 hour, and their viability was measured. Vehicle and FLJ10540 transfectants were cultured in the presence or absence of cisplatin (15 μM) and/or MLN8237 for 48 hour (C) or 15 days (D) and total cell viability were assessed by MTT assay and soft agar. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: A recent studies have shown that Aurora-A contributes to the resistance to cisplatin in breast, pancreatic, esophageal squamous cell carcinoma, Acute myeloid leukemia, medulloblastoma, and ovarian cancer cells [7,27-29]. To gain insight the biological function of both Aurora-A and FLJ10540 on chemoresistance to cisplatin, we carried out MTT assay for assessing cell viability in siAurora-A transfected cells and siAurora-A/FLJ10540-overexpressed cells under cisplatin treatment. As shown in Figure 5A, the viability of siAurora-A cells was significantly more sensitive to cisplatin than negative control group in a concentration-dependent manner. Notably, in the presence of cisplatin, siAurora-A transfectants with FLJ10540 exhibited a lower sensitivity than siAurora-A transfectants or negative control to the cytotoxic effects of cisplatin (Figure 5A). In addition, the viability of siAurora-A cells was significantly lower than that of siAurora-A with FLJ10540-overexpressing cells after incubation with cisplatin (15 μM) for 48 hour (Figure 5B), indicating that increased FLJ10540 decreases the sensitivity of Aurora-A-depleted cells to cisplatin. Next, we used NLM8206 to investigate the effects of cisplatin in FLJ10540-overexpressing cells. As shown in Figure 5C, FLJ10540 transfectants incubated with MLN8206 (0.025nM) and cisplatin (15 μM) for 48 hour were significantly less viable than the same cells treated with cisplatin alone or MLN8206 alone. In addition, an assay of in vitro colony formation was used to confirm the result of MTT assay. As expected, FLJ10540 transfectants treated with MLN8237 and cisplatin in combination increased the colony-formation ability than that the vehicle control (Figure 5D). Together, these results demonstrate that the function of FLJ10540 modulated by Aurora-A expression or Aurora-A activity contributes to cisplatin resistance in HNC cells.Figure 5

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