1. TGF and ERK1/2 pathways regulate homotypic breast cancer cell aggregation in 3D cultures
Angela Dittmer, Dario Schunke, Christoph Thomssen, Jürgen Dittmer
Klinik für Gynäkologie, Universität Halle, Halle (Saale), Germany
INTRODUCTION
In 3D MDA-MB-231 cell aggregates, almost all cells phosphorylate ERK1/2 and do not undergo apoptosis
siPTHrP, siKISS-1 and siITGA6 decrease average colony size of MDA-MB-231 cells in 3D by half
Cells in 3-dimensional (3D) cultures more likely resemble cells in tissues. Cells in 3D are different from cells in 2D in many aspects:
N=4-7
single experiment
morphology
gene expression
adhesion properties
resistence to drugs
a-P-ERK1/2
3D cultures of MCF-7 (epithelial cell-like) and MDA-MB-231 (mesenchymal cell-like) were analyzed
We analyzed expression of selected genes in 3D-MDA-MB-231 vs. 2D-MDA-MB-231 cells:
Expression of PTHrP (parathyroid hormone-related protein) and Kiss1 is increased in 3D
RESULTS
TGFb neutralizes the effects of siPTHrP and siKiss1 on expression and colony size
In 3D, MCF-7 cells form spheroids (mammospheres)
RNA
p < 0.02
RNA expression (3D/2D)
p <
p = 0.099
p = 0.065
p < 0.02
p < 0.01
p < 0.02
which form acinus-like structure after 5-7 days
N =
TbRI inhibitor Ly blocks TGFb effect on gene expression and 3D cell aggregation
To study the importance of PTHrP and KISS-1 for MDA-MB-231 cells in 3D, we used RNA interference:
PTHrP- and KISS-1-specific siRNA efficiently downregulate the levels of their target-RNA also in 3D
The inner cells of MCF-7 spheroids undergo apoptosis
Concomitantly, ERK1/2 phosphorylation is reduced
Ly364947
a-PARP1, 25kD
An antibody against the PARP1-25kD fragment was used to detect apoptosis
RNA
siRNA
RNA
day 0
N =3
N =3
day 3
RNA
ERK phosphorylation is decreased by siPTHrP and siKiss1 and increased by TGFb
day 7
RNA
ERK1/2 is only phosphorylated in the outer cell ring of the MCF-7 spheroid
a-P-ERK1/2
a-RhoGDIa
siPTHrP, siKISS-1 and siITGA6 inhibit cell aggregation
Direct inhibition of ERK1/2 phosphorylation by
U0126 blocks cell aggregation and induces apoptosis
Mesenchymal MDA-MB-231 breast cancer cells form asymmetrical aggregrates in 3D culture that only rarely show loss of cells in the inner core
Measurement of MDA-MB-231 colony size in 3D
CONCLUSIONS
PTHrP
TGFb
KISS-1
ERK1/2 regulates homotypic aggregation and survival of MDA-MB-231 cells in 3D
ITGA6
P-ERK1/2
survival
adhesive strength of cell-cell interaction
3 days
1 month
Zeiss AxioCam MRc 5 camera, AxioVision R 4.5 software
Dittmer et al. Cancer Lettt 260 (2008) 56-61
migration