GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A

Abstract

Excessive expression of subunit 1 of GIRK1 in ER⁺ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10A^GIRK1 with the control (MCF10A^eGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Figure 1

Quantification of GIRK1 mRNA and protein in the different cell lines. (A) GIRK1 mRNA levels normalized to MCF10A^WT. WT, eGFP and GIRK1 denote wildtype, eGFP (control) and GIRK1 overexpressors, respectively. The median value is represented by the black line within the box, box margins represent 75% and 25% percentiles, whiskers indicate 90% and 10% percentiles. The red line represents the mean value. Individual values are shown as grey circles. The number of individual experiments is given in parenthesis above each box. Statistically significant differences between groups are indicated above brackets (n.s.: statistically not significant). (B) WB analysis of cell lysates: upper panel: MCF10A lysates (3 µg protein per slot was applied) probed with GIRK1^CT Ab. #1: MCF10A^WT, #2: MCF10A^eGFP and #3: MCF10A^GIRK1. Lower panel: MCF7 lysates (3 µg protein per slot) probed with GIRK1^CT Ab. #1: MCF7^WT, #2: MCF7^eGFP and #3: MCF7^GIRK1. (C)WB analysis of immunoprecipitates derived using GIRK1^CT Ab on MCF7^WT cell lysates. Left: membrane probed using GIRK1^CT Ab. #a1: lysate (30 µl), #a2: IP (30 µl). Middle: #b1: IP (30 µl) membrane probed using GIRK1^NT Ab (N-terminal) Ab. Right: #c1: IP (30 µl). Membrane probed using monoclonal GIRK1 Ab.

Figure 2

Membrane resting potentials in the different MEC lines. (A) Membrane resting potentials of MCF10A cells. WT: MCF10A^WT, eGFP: MCF10A^eGFP, GIRK1: MCF10A^GIRK1 and GIRK1 + Tertiapin: MCF10A^GIRK1 treated with 200 nmole/L tertiapin-Q. (B) Membrane resting potentials of MCF7 cells. WT: MCF7^WT, eYFP: MCF7^eYFP, eGFP: MCF7A^eGFP, GIRK1^eYFP: MCF7^GIRK1/eYFP, GIRK1: MCF7^GIRK1 and GIRK1 + Tertiapin: MCF7^GIRK1 treated with 200 nmole/L tertiapin-Q. Number of experiments is given in parenthesis above each bar. *,(***): The group differs statistically significant from GIRK1 + Tertiapin at the p < 0.05 (<0.001) level. #: The group differs statistically significant from GIRK1 at the p < 0.05 level. + , (++,+++): The group differs statistically significant from GIRK1^eYFP at the p < 0.05 (<0.01, <0.001) level.

Figure 3

Effect of GIRK1 overexpression on transcriptome of MCF10A cells. Number of significantly up- or downregulated transcripts when MCF10A^eGFP are compared to MCF10A^GIRK1. Red: upregulated transcripts, green: downregulated transcripts. (A) Top nine gene ontology clusters derived by DAVID functional clustering. (B) Heat maps displaying the fold changes of expression levels of the top 50 genes of selected GO terms. IF γ: Interferon-γ response. ECM: extracellular matrix interaction. Migration: cell migration and WH: wound healing. Bottom right: color coding for the log2 fold change.

Figure 4

Cellular migration of MCF10A cells. (A) Migration of 5 selected MCF10A^GIRK1 cells over the entire observation interval. Left: flower plots showing cellular trajectories. Starting position of each individual cell was set to the same position, indicated by grey circle. Colored circle indicates the positon of a cell after 72 h. Right: squared distance as a function of time for the five cells shown to the left (circles; bars indicate standard error). Lines represent linear fits through the data. (B) Same as (A), but MCF10A^eGFP. (C) Statistical analysis of motility coefficients derived from the different experimental groups. WT: MCF10A^WT, eGFP: MCF10A^eGFP and GIRK1: MCF10A^GIRK1. The median value is represented by the black line within the box, box margins represent 75% and 25% percentiles, whiskers indicate 90% and 10% percentiles. The red line represents the mean value. Individual values are shown as dots. The number of individual cells is given in parenthesis besides each box. Statistically significant differences between groups are indicated by brackets.

Figure 5

Cellular velocities of MCF10A cells. (A) Cellular velocities for five representative cells during the entire observation interval. Left: MCF10A^GIRK1, right: MCF10A^GFP. Red x denotes times were the cell was aggregated, green asterisk indicates cell division. (B) Statistical analysis of average cellular velocities of free moving cells derived from the different experimental groups. WT: MCF10A^WT, eGFP: MCF10A^eGFP and GIRK1: MCF10A^GIRK1. The median value is represented by the black line within the box, box margins represent 75% and 25% percentiles, whiskers indicate 90% and 10% percentiles. The red line represents the mean value. Individual values are shown as dots. The number of individual cells is given in parenthesis besides each box. Statistically significant differences between groups are indicated by brackets. (C) Similar to (B) but cellular velocities of aggregated cells.

Figure 6

Cellular migration of MCF7 cells. (A) Migration of 5 selected MCF7^GIRK1 cells over the entire observation interval. Left: flower plots showing cellular trajectories. Starting position of each individual cell was set to the same position, indicated by grey circle. Colored circle indicates the positon of a cell after 72 h. Right: squared distance as a function of time for the five cells shown to the left (circles; bars indicate standard error). Lines represent linear fits through the data.(B) Same as (A), but MCF7^eGFP. (C) Statistical analysis of motility coefficients derived from the different experimental groups. Top: WT: MCF7^WT, eGFP: MCF7^eGFP and GIRK1: MCF7^GIRK1. The median value is represented by the black line within the box, box margins represent 75% and 25% percentiles, whiskers indicate 90% and 10% percentiles. The red line represents the mean value. Individual values are shown as dots. The number of individual cells is given in parenthesis besides each box. Bottom: Difference in MCs (ΔMC = MC_eGFP-MC_GIRK1) calculated for 10% percentile intervals vs. MC_eGFP. Whiskers represent 95% confidence intervals. *The average MCs of the 70%, 80% and 90% percentile differs statistically significant between MCF7^eGFP and MCF7^GIRK1.

Figure 7

Cellular velocities of MCF7 cells. (A) Cellular velocities for five representative cells during the entire observation interval. Left: MCF7^GIRK1, right: MCF7^GFP. Red x denotes times were the cell was aggregated, green asterisk indicates cell division. (B) Statistical analysis of average cellular velocities of free moving cells derived from the different experimental groups. WT: MCF7^WT, eGFP: MCF7^GFP and GIRK1: MCF7^GIRK1. The median value is represented by the black line within the box, box margins represent 75% and 25% percentiles, whiskers indicate 90% and 10% percentiles. The red line represents the mean value. Individual values are shown as dots. The number of individual cells is given in parenthesis besides each box. There was no statistically significant difference between the individual groups. (C) Similar to (B) but cellular velocities of aggregated cells.

Figure 8

Wound healing rates in the different MEC lines. (A) Representative frames recorded at different time points during the wound healing process. eGFP: MCF10A^eGFP and GIRK1: MCF10A^GIRK1. (B) Similar to (A), but MCF7. (C) Statistical analysis of wound healing rates of MCF10A cells. WT: MCF10A^WT, eGFP: MCF10A^eGFP and GIRK1: MCF10A^GIRK1. The median value is represented by the black line within the box, box margins represent 75% and 25% percentiles, whiskers indicate 90% and 10% percentiles. The red line represents the mean value. Individual values are shown as grey circles. The number of individual experiments is given in parenthesis above each box. Statistically significant differences between groups are indicated above brackets (n.s.: statistically not significant). (D) Same as (C), but MCF7 cells.

Figure 9

Interaction of MCF10A with different ECM components. Cell adhesion (OD₅₆₀ values) of MCF10A^eGFP (green) and MCF10A^GIRK1 (orange) to surface coated with different extracellular matrix molecules: Col I: human Collagen Type I, Col II: human Collagen Type II, Col IV: human Collagen Type IV, FN: human Fibronectin, LN: human Laminin, TN: human Tenascin-C and VN: human Vitronectin. Mean values ± SEM for three or four experiments for each experimental group are shown. *(**): The mean values differ statistically significant at the p < 0.05 (p < 0.001) level.

Figure 10

Proliferation of MEC lines in cell culture. (A) Representative original results from cell cycle assessment by gated cell sorting according to fluorescence intensities (x-axis: PerCP-A; y-axis: PacificBlue). Left: MCF10A^eGFP (top) and MCF10^GIRK1 (bottom). Right: MCF7^eGFP (top) and MCF7^GIRK1 (bottom). Individual cells (represented as dots), attributed to a particular phase of the cell cycle are surrounded by black line (green dots: G₀/G₁ phase; purple dots: G₂/M phase and blue dots: S phase). (B) Statistical analysis of the percentage of time spend at different stages of the cell cycle. Left: MCF10A^WT, MCF10A^eGFP and MCF10A^GIRK1. Right: MCF7^WT, MCF7^eGFP and MCF7^GIRK1. The differences between WT, eGFP and GIRK1 do not differ statistically significant for either MCF10A nor MCF7.

Figure 11

Neovascularization and growth of MCF10A lines planted on chicken embryo. (A) Representative image of onplant taken on day 4 after beginning. Left: MCF10A^eGFP cells. Right: MCF10A^GIRK1 cells. White scalebar is 2 mm. (B) Neovascularization of MCF10A lines as assessed by macroscopic vascularization score (MVS). WT: MCF10A^WT, eGFP: MCF10A^eGFP and GIRK1: MCF10A^GIRK1. Box plots represent median value, 25% and 75% percentiles, respectively. Mean values are indicated by red line. Whiskers denote 10% and 90% percentiles. Single scores outside the 10% and 90% percentiles are depicted as black circles. Number of individual onplants is given in parenthesis above each box. Differences were not significant at the statistical level. (C) Onplant area after 4 days of growth. WT: MCF10A^WT, eGFP: MCF10A^eGFP and GIRK1: MCF10A^GIRK1. Box plots represent median value, 25% and 75% percentiles, respectively. Mean values are indicated by red line. Whiskers denote 10% and 90% percentiles. Black circles denote single values outside the 10% and 90% percentiles. Number of individual onplants is given in parenthesis above each box. P-values for statistically significant differences are indicated above brackets (n.s.: statistically not significant).