3 ERK2 BUT NOT ERK1 CONTRIBUTES TO INVASIVE CELL MIGRATION
3.2 R ESULTS
3.2.5 Knockdown of ERK2 impairs migration on cell-‐derived matrices
decided to use cell-‐derived matrix (CDM), which was previously described by Cukierman et al., and which represents a good physiological model of the ECM [38]. CDMs are generated by human telomerase-‐immortalised fibroblasts, which naturally produce extracellular matrix-‐like fibres around themselves and thereby create a relatively thick (10 µm), pliable matrix composed mainly of fibrillar collagen and fibronectin, which recapitulates key aspects of the type of matrix found in connective tissues [38, 353]. After growing these fibroblasts as a confluent monolayer for 10-‐14 days, during which ECM fibers are synthesized, the cells were removed from the surrounding ECM by treatment with a non-‐ionic detergent. Cancer cells may then be plated onto the remaining fibrillar CDM to study their migratory and morphological characteristics in this quasi-‐3D
environment. As expected, there is a striking difference in morphology and mode of migration of A2780-‐Rab25 between plastic surfaces and CDM (Figure 3-‐7). During migration on plastic and other rigid substrates many cell types migrate by forming lamellipodia and stress fibers [387, 388]. Moreover, adhesion structures on 2D surfaces, which are divided into focal and fibrillar adhesions, are rich in αvβ3 integrin, paxillin, vinculin and FAK, or α5β1 integrin and tensin, respectively [389]. In 3D, however, cells either acquire an elongated, mesenchymal-‐like morphology, which is marked by pseudopod formation at the cellular front and requires matrix remodeling, or an amoeboid, rounded shape, which is characterized by high Rho/ROCK activity and the formation of bleb-‐like protrusions [34]. In addition, cells, cultured in 3D, lose the
dorsal-‐ventral asymmetry and form 3D matrix adhesions, which are composed mainly of paxillin and α5β1 integrin [38].
We transfected cells with either NT siRNA, or single oligos targeting ERK1 or ERK2. 50,000 cells were seeded onto a 6-‐well cell culture dish coated with CDM 48 hours post
transfection and allowed to adhere to the 3D substratum for approximately four hours.
Cell migration was monitored on a time-‐lapse microscope over the course of 16 hours and images were acquired every 10 minutes. Stills of representative movies are shown in Figure 3-‐8 A. Notably, no morphological differences between NT siRNA-‐transfected, U0126-‐treated and ERK knockdown cells were observed. However, when cell movement was analysed using the ImageJ cell tracking software, we detected a significant decrease
Figure 3-7 Cell-derived matrices (CDM) represent a 3D-like environment A. Schematic diagram illustrating the protocol for generating CDM.
B. Confocal sections of CDM displaying either parallel (left) or intersecting (right) fibronectin fibres.
Fibronectin was visualised by indirect immunofluorescence using a Cy2-conjugated secondary antibody (green). Scale bar, 10 µm
C. A2780-Rab25 cells were seeded onto plastic and CDM-coated dishes. After 16 hours cells were visualised using a bright field microscope. Scale bar, 100 µm.
Confluent fibroblasts Growth
Denudation of fibroblasts
Plate A2780-Rab25s onto cell-derived matrix
A
B
C
plastic CDM
Figure 3-8 siRNA of ERK2 reduces migration of A2780-Rab25 cells on CDM
A.A2780-Rab25 cells were transfected with non-targeting siRNAs (NT), or those targeting ERK1 or ERK2 and plated onto cell-derived matrix. Images were captured every 10 minutes over a 16 hrs period using a Nikon time-lapse microscope. Still images from a representative movie are displayed. Scale bar, 100 µm.
B-C. The movement of individual cells was followed using the ImageJ cell tracking software. The overall migration velocity (B) and persistence (C) were extracted from the trackplots. Values are means ± SEM of
>75 trackplots from three independent experiments. Statistical significance of differences was determined by Mann-Whitney U test analysis.
in the migration velocity on CDM, when cells were treated with the U0126 inhibitor or ERK2 was silenced with two independent oligos (p<0.0001). In contrast, knockdown of ERK1 did not alter the migration speed when compared to control (Figure 3-‐8 B).
Moreover, we determined the persistence of cell migration and found no significant difference between NT siRNA, U0126 treatment and ERK knockdown cells (Figure 3-‐8 C).
During our cell tracking analysis, we noticed that ERK2 knockdown cells had a tendency to remain stationary for extended periods of time. Thus, the previously observed difference in the relative migration speed may be attributed to the stationary phases, which we term ‘cellular resting’ (Figure 3-‐9 B). To quantify this we defined a cell that moved less than 2 µm within 90 minutes as one that was engaged in ‘cellular resting’. ERK2 knockdown or addition of U0126 markedly increased the proportion of cells that were resting, whereas siRNA of ERK1 was ineffective in this regard (Figure 3-‐9 C). Moreover, we compared the average duration of each rest (resting time) and found no significant
difference among our various experimental conditions (Figure 3-‐9 C). Next, we determined whether silencing of ERK2 influenced cell movement during the period in which cells were not resting. To do this, we calculated frame-‐to-‐frame displacement of cells whilst they were not resting and termed the ‘momentary velocity’. We found the momentary velocity to be significantly reduced following ERK2 knockdown or addition of U0126, but it was unaffected by siRNA of ERK1 (Figure 3-‐9 B). To represent this pictorially, we generated trackplots of cells in which the migration speed is denoted by a colour code, the scale of which is indicated on the left side of the panels, and the points at which cells moved less than 2μm in 90 min (cellular resting) are indicated by white dots. These trackplots indicate that knockdown of ERK2 increases cellular resting and decreases momentary velocity whilst siRNA of ERK1 is ineffective in both these regards (Figure 3-‐9 D)
Taken together these data indicate that knockdown of ERK2 decreases cell invasiveness, and that this corresponds to a combination of reduced momentary velocity and an increased tendency of ERK2 knockdown cells to remain immobile or rest for extended periods on CDM.
Figure 3-9 Knockdown of ERK2 decreases the momentary velocity and increases cellular resting A2780-Rab25 cells were transfected with non-targeting siRNAs (NT), or those targeting ERK1 or ERK2 and plated onto cell-derived matrix. Images were captured every 10 min over a 16 hrs period. Cell movement was followed using cell-tracking software.
A. Schematic illustration on how the overall migration velocity can be affected by cellular resting and momentary velocity.
B. Momentary migration velocities were calculated for each timeframe of the time-lapse experiment giving rise to over 7,000 values for each condition. Values are represented as box and whisker plots (whiskers:
10-90 percentile) and represent three independent experiments. Statistical significance of differences was determined by Mann-Whitney U test analysis.
C. Percentage of resting cells is displayed with absolute numbers for each condition above the column. The resting time was extracted from the trackplots and represents means ± SEM of thee independent experiments.
D. Representative migration trackplots are displayed. The migration speed is denoted by a colour code, the scale of which is indicated on the left side of the panels. The points at which cells moved less than 2 μm in 90 min (cellular resting) are indicated by white dots. Scale bar 100 μm.
3.2.6 ERK2 promotes invasion in the breast cancer cell line MDA-MB-231