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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

Aurora-A modulates MMP-7 and MMP-10 expression and secretion in HNC cells. The mRNA expression levels of MMP-1, −2, −3, −7, −9, −10, −12, −13 and −14 (A) and protein expression profiles of MMP-7 and −10 (B) were examined by Q-RT-PCR and Western blotting in SAS/negative control and SAS/siAurora-A transfectants. (C and D) The endogenous mRNA and protein expression levels of MMP-7, and −10 (C) were examined by Q-RT-PCR and Western blotting in FaDu/vehicle and FaDu/Aurora-A transfectants. (E) Unsynchronized Aurora-A transfectants were fixed, and immunostained with antibodies against endogenous MMP-7 and −10 or DAPI. Representative data are shown. (F) Using the Figure 4F xenograft model. The paraffin-embedded tumor tissues were subjected to immunostainings for MMP-7 and MMP-10. (G) The migration and invasion assays were performed by Transwell chambers in Aurora-A transfectants treated with GM6001 (3 μM). D: DMSO; G: GM6001. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig6: Aurora-A modulates MMP-7 and MMP-10 expression and secretion in HNC cells. The mRNA expression levels of MMP-1, −2, −3, −7, −9, −10, −12, −13 and −14 (A) and protein expression profiles of MMP-7 and −10 (B) were examined by Q-RT-PCR and Western blotting in SAS/negative control and SAS/siAurora-A transfectants. (C and D) The endogenous mRNA and protein expression levels of MMP-7, and −10 (C) were examined by Q-RT-PCR and Western blotting in FaDu/vehicle and FaDu/Aurora-A transfectants. (E) Unsynchronized Aurora-A transfectants were fixed, and immunostained with antibodies against endogenous MMP-7 and −10 or DAPI. Representative data are shown. (F) Using the Figure 4F xenograft model. The paraffin-embedded tumor tissues were subjected to immunostainings for MMP-7 and MMP-10. (G) The migration and invasion assays were performed by Transwell chambers in Aurora-A transfectants treated with GM6001 (3 μM). D: DMSO; G: GM6001. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.

Mentions: MMPs plays a crucial role in tumor invasion and metastasis for decades [30], and many MMPs contribute to HNC progression [31,32]. According to recent report, MMP-1, −2, −3, −7, −9, −10, −12, −13 and −14 are highly expressed in HNC and involved in HNC progression through invadopodia formation [30]. Here, we assessed these MMPs mRNA expression levels in Aurora-A depleted HNC cells. The results illustrated that only MMP-7 and −10 were dramatic suppressed in Aurora-A knockdown cells by Q-RT-PCR approach, compared to negative control (p < 0.05) (Figure 6A). Our Western blotting results are consistent with the observed mRNA expressions of MMP-7 and −10 in SAS cells transfected with siRNA of Aurora-A (Figure 6B). In contrast, ectopic expression of Aurora-A in FaDu cells was not only significantly enhanced the endogenous mRNA and protein expressions of MMP-7 and −10 (Figure 6C, D and E), but also increased the matricellular-MMP-7 and −10 levels in culture media by ELISA assay (Additional file 4: Figure S4). In xenograft tumor model, the MMP-7 and MMP-10 protein expressions were diminished in MLN8237 treated group compared to DMSO by immunohistochemical staining (Figure 6F). To further illustrate whether MMP-7 and −10 involves in Aurora-A-elicited cell motility, we used an MMP inhibitor, GM6001 to inhibit MMP-7 and MMP-10 expressions [33]. The data showed that the mRNA expression profiles of MMP-7 and −10 were decreased in Aurora-A transfectants under GM6001 treatment in a dose-dependent manner (Additional file 5: Figure S5). Additionally, using the same panel, Aurora-A-raised the abilities of cell migration and invasion were also dramatic suppressed (Figure 6G). Taken together, Aurora-A gives rise to the cell migration and invasion rely on both MMP-7 and −10 expressions in HNC cells.Figure 6


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)

Aurora-A modulates MMP-7 and MMP-10 expression and secretion in HNC cells. The mRNA expression levels of MMP-1, −2, −3, −7, −9, −10, −12, −13 and −14 (A) and protein expression profiles of MMP-7 and −10 (B) were examined by Q-RT-PCR and Western blotting in SAS/negative control and SAS/siAurora-A transfectants. (C and D) The endogenous mRNA and protein expression levels of MMP-7, and −10 (C) were examined by Q-RT-PCR and Western blotting in FaDu/vehicle and FaDu/Aurora-A transfectants. (E) Unsynchronized Aurora-A transfectants were fixed, and immunostained with antibodies against endogenous MMP-7 and −10 or DAPI. Representative data are shown. (F) Using the Figure 4F xenograft model. The paraffin-embedded tumor tissues were subjected to immunostainings for MMP-7 and MMP-10. (G) The migration and invasion assays were performed by Transwell chambers in Aurora-A transfectants treated with GM6001 (3 μM). D: DMSO; G: GM6001. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
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Related In: Results  -  Collection

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Fig6: Aurora-A modulates MMP-7 and MMP-10 expression and secretion in HNC cells. The mRNA expression levels of MMP-1, −2, −3, −7, −9, −10, −12, −13 and −14 (A) and protein expression profiles of MMP-7 and −10 (B) were examined by Q-RT-PCR and Western blotting in SAS/negative control and SAS/siAurora-A transfectants. (C and D) The endogenous mRNA and protein expression levels of MMP-7, and −10 (C) were examined by Q-RT-PCR and Western blotting in FaDu/vehicle and FaDu/Aurora-A transfectants. (E) Unsynchronized Aurora-A transfectants were fixed, and immunostained with antibodies against endogenous MMP-7 and −10 or DAPI. Representative data are shown. (F) Using the Figure 4F xenograft model. The paraffin-embedded tumor tissues were subjected to immunostainings for MMP-7 and MMP-10. (G) The migration and invasion assays were performed by Transwell chambers in Aurora-A transfectants treated with GM6001 (3 μM). D: DMSO; G: GM6001. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001.
Mentions: MMPs plays a crucial role in tumor invasion and metastasis for decades [30], and many MMPs contribute to HNC progression [31,32]. According to recent report, MMP-1, −2, −3, −7, −9, −10, −12, −13 and −14 are highly expressed in HNC and involved in HNC progression through invadopodia formation [30]. Here, we assessed these MMPs mRNA expression levels in Aurora-A depleted HNC cells. The results illustrated that only MMP-7 and −10 were dramatic suppressed in Aurora-A knockdown cells by Q-RT-PCR approach, compared to negative control (p < 0.05) (Figure 6A). Our Western blotting results are consistent with the observed mRNA expressions of MMP-7 and −10 in SAS cells transfected with siRNA of Aurora-A (Figure 6B). In contrast, ectopic expression of Aurora-A in FaDu cells was not only significantly enhanced the endogenous mRNA and protein expressions of MMP-7 and −10 (Figure 6C, D and E), but also increased the matricellular-MMP-7 and −10 levels in culture media by ELISA assay (Additional file 4: Figure S4). In xenograft tumor model, the MMP-7 and MMP-10 protein expressions were diminished in MLN8237 treated group compared to DMSO by immunohistochemical staining (Figure 6F). To further illustrate whether MMP-7 and −10 involves in Aurora-A-elicited cell motility, we used an MMP inhibitor, GM6001 to inhibit MMP-7 and MMP-10 expressions [33]. The data showed that the mRNA expression profiles of MMP-7 and −10 were decreased in Aurora-A transfectants under GM6001 treatment in a dose-dependent manner (Additional file 5: Figure S5). Additionally, using the same panel, Aurora-A-raised the abilities of cell migration and invasion were also dramatic suppressed (Figure 6G). Taken together, Aurora-A gives rise to the cell migration and invasion rely on both MMP-7 and −10 expressions in HNC cells.Figure 6

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