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The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy - PubMed

  • ️Wed Jan 01 2014

The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy

Yan Wang et al. Mol Cancer. 2014.

Abstract

It is well known that the activation of Aurora A/B (Aur A/B) or inactivation of BRCA1/2 induces tumor formation. Others and we have reported that the mutual suppression between Aur A/B and BRCA1/2 may manipulate cancer cell growth and tumorigenesis, however, the interactive regulation and mechanism between these molecules are still elusive. In this study, by consecutive silencing of Aur A/B or/and BRCA1/2 with specific shRNAs, we showed that, in BRCA2-deficient pancreatic cancer cell line Capan-1 and in ovarian cancer cell line OVCA433, Aur A/B and BRCA1/2 inversely regulated the expression of each other likely through proteasome-mediated proteolysis but not through gene transcription. Aur A/B and BRCA1/2 conversely regulated cell cycle progression mainly through control of p53 and cyclin A. Moreover, the disruption of Aur A/B blocked abnormal cytokinesis and decreased cell multinuclearity and chromosome tetraploidy, whereas the deprivation of BRCA1/2 promoted the abnormal cytokinesis and enhanced the cell multinuclearity and tetraploidy. Furthermore, we showed by animal assays that the depletion of Aur A/B inhibited tumor growth of both cell lines, while the knockdown of BRCA1/2 promoted the tumor growth. However, the concurrent silencing of Aur A/B and BRCA1/2 diminished the effects of these molecules on the regulation of cell cycle, cytokinesis, and tetraploidy, leading to the burdened tumor sizes similar to those induced by scrambled shRNA-treated control cells. In summary, our study revealed that the negative interplay between Aur A/B and BRCA1/2 inversely controls the cell proliferation, cell cycle progression, cell multinuclearity, and tetraploidization to modulate tumorigenesis.

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Figures

Figure 1
Figure 1

Expression levels of Aur A/B and BRCA1/2. A-B, Protein expressions detected by Western Blotting showing the interactive regulation of Aur A/B and BRCA1/2 before and after treatment with various shRNAs in BRCA2 deficient pancreatic cancer cells Capan-1 (A) and in ovarian cancer cells OVCA433 (B). β-actin is used as a loading control. C-D, Relative mRNA levels of Aur A/B and BRCA1/2 in Capan-1 cells (C) and OVCA433 cells (D) detected by qRT-PCR compared with corresponding control cells (Scr cells). E-F, The protein levels of Aur A/B and BRCA1/2 in Capan-1 cells (E) and OVCA433 cells (F) treated with 20 μM MG132 for 3 h. β-actin is used as a loading control.

Figure 2
Figure 2

Effects of Aur A/B and BRCA1/2 on cell proliferation and colony formation. A-B, Alteration of cell growth curves before and after silencing of Aur A/B or/and BRCA1/2 in Capan-1 cells (A) and OVCA433 cells (B). C-D, Colony formation rates of cells before and after disruption of Aur A/B or/and BRCA1/2. Data were collected from three independent experiments, and analyzed for statistic significance. Error bars = 95% CIs. E-F, Representative images of plate colony formation.

Figure 3
Figure 3

Morphological changes of OVCA433 cells. Selected images showing phenotypic changes of OVCA433 cells before or after the interruption of Aur A/B or/and BRCA1/2. Compared with OVCA433-Scr cells, the silencing of BRCA1 or/and BRCA2 induced cells scattered, whereas the interruption of Aur A and/or Aur B stimulated cells thinner and more dispersed compared with control cells. The disruption of Aur A and/or Aur B following double knockdown of BRCA1 and BRCA2 stimulated cells growing thinner and flatter than cells with intact Aur A and/or Aur B expressions. However, cells with the concomitant silencing of four proteins turned oblate. The images were taken at 200 × magnification by an Olympus microscope.

Figure 4
Figure 4

Regulation of cell cycle progression and cell cycle-associated proteins by Aur A/B and BRCA1/2. A-B, Cell cycle distributions detected by flow cytometry before or after silencing of Aur A/B and/or BRCA1/2. C-D, Alteration of cell cycle regulatory proteins tested by immunoblotting. Inverse regulation of p53 and cyclin A by Aur A/B and BRCA1/2 in both Capan-1 and OVCA433 cells is conceived. β-actin is used as a loading control.

Figure 5
Figure 5

Regulation of polyploidy and cytokinesis by Aur A/B and BRCA1/2. A-B, Quantification of cells with multiple nuclei before or after the depletion of Aur A/B and BRCA1/2. Error bars = 95% CIs, indicating data were collected from three independently repeated experiments. C-D, Representative images showing the localization and accumulation of Aur A/B and BRCA1/2 at the midbody during cytokinesis (×1000). Blue dye DAPI indicates nucleus. Scale bars, 5 μm for the merged images and 1 μm for other images. E-F, Representative images of karyotypes before and after the silencing of Aur A/B or/and BRCA1/2 in Capan-1 cells (E) and OVCA433 cells (F).

Figure 6
Figure 6

Xenograft tumor growth in animals. A-B, The mean volume of tumors burdened in mice receiving Capan-1 or OVCA433 cells expressing scrambled shRNA or different shRNAs against Aur A/B or BRCA1/2. Error bars = 95% CIs. *P < 0.05. C-D, The average weight of each tumor formed in different groups of mice. Error bars = 95% CIs. *P < 0.05, **P < 0.01.

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