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. 2020 Mar 9;52(5):563-573.e3.
doi: 10.1016/j.devcel.2020.01.004. Epub 2020 Feb 6.

Oncogenic Signaling Alters Cell Shape and Mechanics to Facilitate Cell Division Under Confinement

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Free PMC article

Oncogenic Signaling Alters Cell Shape and Mechanics to Facilitate Cell Division Under Confinement

Helen K Matthews et al. Dev Cell. .
Free PMC article

Abstract

To divide in a tissue, both normal and cancer cells become spherical and mechanically stiffen as they enter mitosis. We investigated the effect of oncogene activation on this process in normal epithelial cells. We found that short-term induction of oncogenic RasV12 activates downstream mitogen-activated protein kinase (MEK-ERK) signaling to alter cell mechanics and enhance mitotic rounding, so that RasV12-expressing cells are softer in interphase but stiffen more upon entry into mitosis. These RasV12-dependent changes allow cells to round up and divide faithfully when confined underneath a stiff hydrogel, conditions in which normal cells and cells with reduced levels of Ras-ERK signaling suffer multiple spindle assembly and chromosome segregation errors. Thus, by promoting cell rounding and stiffening in mitosis, oncogenic RasV12 enables cells to proliferate under conditions of mechanical confinement like those experienced by cells in crowded tumors.

Keywords: ERK; MAPK signaling; MEK; Ras; actin; cancer; cell confinement; cell mechanics; mitosis; mitotic rounding.

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

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Figure 1
Figure 1
Ras/MEK/ERK Signaling Controls Cell Shape in Mitosis (A) Western blot showing levels of ERK1/2 phosphorylation and total ERK1/2, Ras, and E-cadherin expression in MCF10A, MCF10A ER-hRasV12, and MCF10A+hRasv12 cells. MCF10A and ER-hRasV12 cells were treated with ethanol (−) or 4-OH-tamoxifen (+) for 7 h before lysis. The ratio of pERK/tERK signal was quantified for 3 independent blots and means and standard deviation, normalized to the ethanol (−) condition, are shown. Position of molecular weight markers (kDa) is indicated on right-hand side. (B) Representative phase contrast images of the different cell types in metaphase. Metaphase is taken as 5 min before anaphase elongation or furrowing is first observed. Scale bars represent 10 μm. (C) Scatter plots of cell length (Feret diameter) and aspect ratio for the different cell types in interphase (black, taken as 15 min before nuclear envelope breakdown) and metaphase (blue, 5 min before anaphase). Cells were imaged every 5 min for 15 h using phase contrast microscopy, and the shapes were recorded for every cell division. For ER-hRasV12 cells, cells were analyzed 5–15 h post-ethanol or 4-OH-tamoxifen addition. n = 30 cells per condition. (D) Western blot showing levels of phospho-ERK1/2, total ERK1/2, phospho-p90RSK, total RSK1/2/3, phospho-Akt, and total Akt for MCF10A ER-hRasV12 cells, treated for 7 h with ethanol (−) or 4-OH-tamoxifen (+) plus DMSO or the following inhibitors: 2 μM PD 184352, 10 μM selumetinib (MEK inhibitors – MEKi), 10 μM GDC-0994 (ERK inhibitor – ERKi), and 2 μM ZSTK474 (PI3K inhibitor – PI3Ki). The position of the molecular weight markers (kDa) is indicated on the right-hand side. (E) Boxplot showing metaphase length for ER-hRasV12 cells following 5–15 h ethanol or 4-OH-tamoxifen treatment alongside addition of DMSO or the following small molecule inhibitors: 2 μM PD 184352, 10 μM selumetinib (MEK inhibitors), 10 μM GDC-0994 (ERKi), or 2 μM ZSTK474 (PI3Ki). p Values calculated using Mann-Whitney ∗∗∗p < 0.001. (F–H) Plot of the metaphase length of individual ER-hRasV12 cells dividing against time after ethanol (F) or 4-OH-tamoxifen (G) addition and following addition of 10 μM selumetinib (H).
Figure 2
Figure 2
Ras/MEK/ERK Signaling Alters Contractility in Early Mitosis (A) Representative montage time-lapse images of MCF10A ER-hRasV12 cells labeled with LifeAct-GFP following an 8 h treatment with ethanol, 4-OH-tamoxifen, or 4-OH-tamoxifen + 2 μM PD 184352 (MEKi). Time in minutes is aligned so that t = 0 is the frame after NEB (blue line). Scale bars represent 10 μm. (B) Quantification of cell area for 8 cells from conditions in (A) entering mitosis, aligned so that t = 0 is NEB (blue lines). Measurements were taken from time-lapse microscopy of cells 5–15 h after treatment. (C) Boxplot showing rate of area decrease during mitotic rounding for MCF10A ER-hRasV12 cells following 8 h treatment with ethanol or 4-OH-tamoxifen and DMSO or 2 μM PD 184352 as well as control, unsynchronized MCF10A, and MCF10A+hRasv12 cells. p Values calculated using Mann-Whitney ∗∗∗p < 0.001. (D and E) Boxplots showing metaphase cell length for ER-hRasV12 cells following 5–15 h ethanol or 4-OH-tamoxifen treatment and treated with DMSO, 25 μM Y-27632, or 2 μM GSK 269962 (ROCK inhibitors) (D) or scrambled or Ect2 siRNA (E). Ect2 knockdown was verified by analyzing the cytokinesis failure rate (shown in blue). p Values calculated using Mann-Whitney ∗∗∗p < 0.001.
Figure 3
Figure 3
Ras Activation Alters Cell Mechanical Properties (A) Schematic comparing two different techniques used to measure cell mechanical properties: atomic force microscopy (AFM) and real-time deformability cytometry (RT-DC). (B) Boxplot showing the apparent elastic modulus for rounded control MCF10A cells and MCF10A+hRasv12 in interphase and mitosis (arrested in pro-metaphase by S-trityl-L-cysteine (STLC) treatment) as determined by AFM. The number of curves analyzed are indicated in brackets. Data shown is from one representative experiment (n = 2). (C) Bar chart shows apparent elastic modulus (mean + SEM) calculated using RT-DC for interphase and mitotic cells from 10 independent experiments (n > 15,000 cells). Because of the large sample size, a linear mixed model was used to calculate the statistical significance of the results. ∗∗∗p < 0.001.
Figure 4
Figure 4
Ras Activation Promotes Mitotic Rounding under Confinement (A) Schematic of cells dividing in confinement under compliant (~5 kPa) and stiff (~ 30 kPa) polyacrylamide gels. (B) Plot of maximum metaphase length versus height for unconfined MCF10A cells and cells under soft (~5 kPa) and stiff (~ 30 kPa) gels. Measurements were taken from confocal time-lapse images as shown in (C). Metaphase was taken as the frame before anaphase. (C) XY and XZ confocal slices of metaphase MCF10A+hRasv12 cells, MCF10A cells, and MCF10A cells treated for 5–10 h with 10 μM selumetinib. Cells were pre-treated with 100 nM SiR-DNA and SiR-actin to label DNA and actin. Scale bars represent 20 μm. (D) Boxplot showing metaphase cell length for MCF10A+hRasv12, control MCF10A, and MCF10A + 2 μM PD 184352 without confinement and under soft and stiff gels. Data come from time-lapse movies for 15 h after confinement onset. The MEKi was added immediately before confinement and mitoses were analyzed 5–15 h post addition and confinement. p Values calculated using Mann-Whitney ∗∗∗p < 0.001. (E) Graphs showing cell area for control MCF10A cells and MCF10A+hRasv12 entering mitosis under soft and stiff gels, aligned so that t = 0 is NEB.
Figure 5
Figure 5
Ras Activation Reduces Mitotic Defects in Confinement (A) Boxplot showing the time taken for cells to progress through mitosis (from NEB to anaphase) for MCF10A+hRasv12, control MCF10A, and MCF10A + 2 μM PD 184352 without confinement and under soft and stiff gels. Data come from time-lapse movies for 15 h after confinement onset. MEKi was added immediately before confinement and mitoses were analyzed 5–15 h post-addition/confinement. p Values calculated using Mann-Whitney ∗∗∗p < 0.001. (B) Time-lapse montage showing an example of tri-polar mitosis and graph showing the percentage of cells (mean ± SD) that undergo tri-polar mitosis underneath a stiff gel. n = 3 independent experiments. (C) Time-lapse montage showing an example of extreme blebbing and graph showing the percentage (mean ± SD) of cells that undergo extreme blebbing underneath a stiff gel. n = 3 independent experiments. Extreme blebbing was defined as any mitotic cells having a bleb equal to or longer than cell diameter. Note that no tri-polar mitosis or extreme blebbing was observed in any condition under soft gels or without gels. Times are in minutes and scale bars represent 20 μm. (D and E) Boxplot of metaphase length (D) and mitotic duration (time from NEB to anaphase) (E) for control MCF10A and MCF10A+hRasv12 cells under soft and stiff gels, treated with DMSO or 25 μM Y-27632 (ROCK inhibitor – ROCKi). n for (E) is the same as indicated in the figure in (D).

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