Cyclin A2, a novel regulator of EMT

Abstract

Our previous work showed that Cyclin A2 deficiency promotes cell invasion in fibroblasts. Given that the majority of cancers emerge from epithelia, we explored novel functions for Cyclin A2 by depleting it in normal mammary epithelial cells. This caused an epithelial to mesenchymal transition (EMT) associated with loss of cell-to-cell contacts, decreased E-Cadherin expression and increased invasive properties characterized by a reciprocal regulation of RhoA and RhoC activities, where RhoA-decreased activity drove cell invasiveness and E-Cadherin delocalization, and RhoC-increased activity only supported cell motility. Phenotypes induced by Cyclin A2 deficiency were exacerbated upon oncogenic activated-Ras expression, which led to an increased expression of EMT-related transcriptional factors. Moreover, Cyclin A2-depleted cells exhibited stem cell-like properties and increased invasion in an in vivo avian embryo model. Our work supports a model where Cyclin A2 downregulation facilitates cancer cell EMT and metastatic dissemination.

Fig. 1

Induction of mesenchymal traits following Cyclin A2 depletion in NMuMG cells. a Visualization of sh Luc and sh CycA2 NMuMG cells by light microscopy. Scale bar 200 μm. b Western blot analysis of E-Cadherin in Cyclin A2-depleted cells and sh Luc NMuMG cells. E-Cadherin expression was quantified by densitometry and normalized to that of GAPDH (n = 3, *P = 0.01). c Staining of F-Actin using Phalloidin-Rhodamine, β Catenin (red) or Vinculin (green), E-Cadherin (red) and p120 Catenin (green) in sh Luc and sh CycA2 NMuMG cells. Scale bar 20μm. d qPCR analysis of N-Cadherin, Fibronectin and MMP-14 mRNA levels. The values are expressed as fold change, where sh CycA2 was compared to sh Luc and TGFβ treated cells to untreated cells. Data are represented as mean ± SEM

Fig. 2

Cyclin A2 knockdown in NMuMG cells modulates 3D invasion, along with RhoA and RhoC activities. a Invasion of sh Luc and sh CycA2 NMuMG cells in a 3D collagen matrix (n = 5, ****P < 0.0001). b–d Western blotting after pull-down of the activated forms of Rac1 (b), RhoA (c) or RhoC (d) in extracts of cells depleted of Cyclin A2. Lower panels Quantification where the amount of the GTP-bound form was normalized to total Rac1, RhoA, RhoC and GAPDH. Data are represented as mean ± SEM (*P = 0.01 for RhoA, n = 3 and *P = 0.01 for RhoC, n = 3). e Co-immunoprecipitation of endogenous Cyclin A2 with RhoC. Cell lysates obtained from NMuMG cells transfected with RhoC-GFP were incubated by GFP-Trap beads. Immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting with an anti-Cyclin A2 antibody

Fig. 3

Modulation of the levels of EMT markers following Cyclin A2 depletion in NMuMG RasV12 cells. mRNA analysis of EMT markers using quantitative real time PCR (qRT-PCR) and expressed as fold change, where the level in sh CycA2 cells was normalized to sh Luc cells with or without RasV12 expression. a mRNA levels of N-Cadherin and E-Cadherin, b EMT-related transcription factors and c metalloproteases (MMP-3 and -14)

Fig. 4

Impact of RhoA and RhoC knockdown ± that of Cyclin A2 in NMuMGRasV12 cells. a Invasion in collagen matrix (3D) of NMuMGRasV12 cells knocked down for both RhoC and Cyclin A2 (left panel) or for RhoA alone (right panel) (n = 3, ****P < 0.0001 for siRhoC and n = 3, ***P < 0.0002 for siRhoA). b Western blot analysis of E-Cadherin and RhoA depletion in NMuMGRasV12 cells. c, Analysis of E-Cadherin and p120 Catenin cellular localization in NMuMG and NMuMGRasV12 cells treated with siRNA RhoA. qPCR analysis of mRNA levels of EMT markers in NMuMGRasV12 cells knocked-down for RhoA (d) or both RhoC and Cyclin A2 (e). Results are shown as fold change of siRNA of interest knockdowns over control knockdowns (NMuMGRasV12 cells were used for the siRhoA and sh CycA2NMuMUGRasV12 cells for siRhoC). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Cyclin A2 knockdown enhances tumorosphere formation in NMuMGRasV12 cells. a Visualization of tumorosphere formation by light microscopy in sh Luc and sh CycA2 NMuMGRasV12 cells. Scale bar 100 μM. b Cell numbers after dissociation of tumorospheres formed by cells expressing RasV12 with or without Cyclin A2 knockdown (n = 3, *P = 0.03). c Tumorosphere number, diameter (d) and area (e) represented as frequency distribution in NMuMGRasV12 Cyclin A2-depleted cells relative to cells expressing the sh Luc control (n = 3, ****P < 0.001). f mRNA levels of Nanog and Oct4 by qRT-PCR and expressed as fold change, where the level in sh CycA2 cells was normalized to sh Luc infected cells with or without RasV12 expression. Data are represented as mean ± SEM. ***P < 0.001

Fig. 6

Cyclin A2 knockdown modulates extravasation and migration in avian embryos. a Extravasation of sh Luc and sh CycA2 NMuMG cells with or without RasV12 expression. The data represent the percentage of cells present in the stroma versus the total cell number detected in the ROI. b Velocity of sh Luc and sh CycA2 NMuMGRasV12 cells expressed in microns traveled per hour. c Displacement of sh Luc and sh CycA2 NMuMGRasV12 cells expressed in microns traveled away from the origin. d Migration tracks (red line) of sh Luc and sh CycA2 NMuMGRasV12 cells within CAM stroma as analyzed by Volocity software. e Image set of sh Luc and sh CycA2 NMuMGRasV12 cells present in the stroma. Images were acquired every 15 min for 12–16 h. Data are represented as mean ± SEM. *P < 0.05