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. 2015;14(8):1139-47.
doi: 10.1080/15384101.2015.1007771.

Loss of GM130 in breast cancer cells and its effects on cell migration, invasion and polarity

Francesco Baschieri  1 , Edith Uetz-von Allmen, Daniel F Legler, Hesso Farhan
Affiliations

Loss of GM130 in breast cancer cells and its effects on cell migration, invasion and polarity

Francesco Baschieri et al. Cell Cycle. 2015.

Abstract

Spatially distinct pools of the small GTPase Cdc42 were observed, but the major focus of research so far has been to investigate its signaling at the plasma membrane. We recently showed that the Golgi pool of Cdc42 is relevant for cell polarity and that it is regulated by GM130, a Golgi matrix protein. Loss of GM130 abrogated cell polarity and consistent with the notion that polarity is frequently impaired in cancer, we found that GM130 is downregulated in colorectal cancer. Whether the loss of GM130 solely affects polarity, or whether it affects other processes relevant for tumorigenesis remains unclear. In a panel of breast cancer cells lines, we investigated the consequences of GM130 depletion on traits of relevance for tumor progression, such as survival, proliferation, adhesion, migration and invasion. We show that cellular assays that depend on polarity, such as chemotaxis and wound scratch assays, are only of limited use to investigate the role of polarity modulators in cancer. Depletion of GM130 increases cellular velocity and increases the invasiveness of breast cancer cells, therefore supporting the view that alterations of polarity contribute to tumor progression.

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Figures

Figure 1
Figure 1
(A) Schematic representing the mechanism of action of GM130: GM130 binds to RasGRF and blocks its function. Once Cdc42 is activated, it accumulates on membranes and the Golgi sends Cdc42 in a polarized fashion to the Leading Edge of the migrating cell, thereby conferring persistence to the migration. GM130 will therefore contribute to maintain the balance between Cdc42 and Ras signaling. When GM130 is lost, the cell cannot migrate persistently and there is an imbalance between Cdc42 and Ras signaling. (B) Box Plots of two studies comparing the mRNA levels of GM130 in normal tissues and in breast cancer tissues (obtained from Oncomine).
Figure 2
Figure 2
(A) Histogram representing the quantification of GM130 levels compared to actin, obtained from 3 independent western blots. Results are showed as averages ± SE. Below, a representative western blot. (B) mRNA levels of GM130 normalized to GAPDH mRNA. Results are shown as 1/(CtGM130-CtGAPDH). Bar graphs are averages of 3 independent experiments ± SE. GAPDH was amplified at cycle 15.8687 ± 0.3265, confirming that it can be considered an housekeeping gene also when comparing different cell lines. (C) The Golgi Compactness Index (GCI) was calculated as described in the materials and methods. More than 20 cells per experiment in three independent experiment were scored for each cell line. Results are shown as averages ± SE. (D) GCI was plotted on the x axis, the average of the protein levels of GM130 was plotted on the y axis. The linear correlation between these two parameters was assessed for all data points (black), only for luminal cell lines (blue) or only for basal cell lines (red).
Figure 3
Figure 3
Cells were plated on coverslips and processed for immunofluorescence staining using antibodies against GM130 (green) and Giantin (red) to visualize the Golgi, and DAPI to stain the nuclei. Scale bars, 10 μm. (A) Representative images of Golgi in luminal cell lines. (B) Representative images of Golgi in basal cell lines.
Figure 4
Figure 4
(See previous page). (A) The indicated cell lines were stably transduced with either a control plasmid (pLVTHM) or a plasmid encoding a shRNA against GM130 (GM130 shRNA). The cells were lysed and processed for western blot with antibodies against GM130 and actin to verify the efficiency of the shRNA against GM130. (B) The capacity of the indicated cells to adhere to a soft substrate (Collagen type IV) were assessed as described in the materials and methods. Results are shown as averages of three independent experiments ± SE. No significant difference was observed using the Student T test. (C) Cells were plated on coverslips, grown to subconfluency and processed for immunostaining against the proliferative marker Ki67 and DAPI. The percentage of cells positive for Ki67 was calculated counting at least 300 cells per experiment in three independent experiments. Results are shown as averages ± SE. No significant difference was observed using the student T test. (D) 200.000 cells were plated on 6-well plates. The next day, cells were either left untreated or treated with the indicated concentration of doxorubicin overnight. MDA-MB231 were also left overnight in the absence of FCS, in addition to the addition of doxorubicin. Cells were then processed for Annexin-V-APC as described in materials and methods. The mean fluorescence of the Annexin-V staining was measured in at least three independent experiments for every condition. Results are shown as averages ± SE. Asterisks indicate statistically significant differences calculated with ANOVA using the Newman-Keuls correction for multiple comparisons (*P < 0.05; **P < 0.01, ***P < 0.001).
Figure 5
Figure 5
(See previous page). (A) Wound healing assays were performed as described in materials and methods. The area migrated by the cells was measured. Results are expressed as percentage of migration compared to control conditions. Results are shown as averages of at least three independent experiments ± SE. Asterisks indicate statistically significant differences calculated using Student T test (*P < 0.05; **P < 0.01). (B) Cells were plated on top of Geltrex covered membranes with 8 μm pores and let invade in the presence of a chemotactic gradient (FCS) for 24 h. The number of cells which invaded through Geltrex was then counted. Results are shown as averages of at least three independent experiments ± SE. Asterisks indicate statistically significant differences calculated using Student T test (*P < 0.05). Below the bar-graph, schematic of the experimental settings. (C) Cells were plated on glass bottom slides and imaged overnight. Velocity and persistence were calculated as described in the materials and methods. Results are shown as averages of three independent experiments ± SE. Asterisks indicate statistically significant differences calculated using Student T test (*P < 0.05). (D) Cells were plated on top of Geltrex covered membranes with 8 μm pores and let invade in the absence of a chemotactic gradient for 24 h. The number of cells which invaded through Geltrex was then counted. Results are shown as averages of at least three independent experiments ± SE. Asterisks indicate statistically significant differences calculated using Student T test (*P < 0.05). Below the bar-graph, schematic of the experimental settings.
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