Tumorigenicity of MCF-7 human breast cancer cells lacking the p38α mitogen-activated protein kinase

Abstract

We have generated cell lines with significantly reduced expression of the p38 mitogen-activated protein kinase (p38 MAPK), Min-p38 MAPK cells, and used these cells to investigate p38 MAPK's role in tumorigenesis of breast cancer cells. MCF-7 cells were stably transfected with a plasmid producing small interfering RNA that inhibited the expression of p38 MAPK. Control cells were stably transfected with the same plasmid producing non-interfering RNA. The reduction in the p38 MAPK activity caused a significant increase in the expressions of estrogen receptor-α (ERα) and the progesterone receptor, but eliminated the expression of ERβ. Min-p38 MAPK cells showed an enhanced overall growth response to 17β-estradiol (E₂), whereas GH plus epidermal growth factor were largely ineffective growth stimulators in these cells compared to controls. Although the long-term net growth rate of the Min-p38 MAPK cells was increased in response to E₂, their proliferation rate was lower compared to controls in short-term cultures. However, the Min-p38 MAPK cells did show a significant decreased rate of apoptosis after E₂ treatment and a reduction in the basal phosphorylation of p53 tumor suppressor protein compared to controls. When the Min-p38 MAPK cells were xenografted into E₂-treated athymic nude mice, their tumorigenicity was enhanced compared to control cells. Increased tumorigenicity of Min-p38 MAPK cells was caused mainly by a decrease in the apoptosis rate indicating that the lack of the p38 MAPK caused an imbalance to increase the ERα:ERβ ratio and a reduction in the activity of the p53 tumor suppressor protein.

Figure 1

Western blot analysis showing expression of the p38 mitogen-activated protein kinase (p38 MAPK) and β-actin (internal control) in cloned MCF-7 cells stably transfected with small interfering RNA (siRNA) to the p38 MAPK (Min-p38 MAPK1,2), and control clones (Control1,2) transfected with inert small RNA of the same size as the p38 MAPK-siRNA. Cells were plated onto 60 mm culture dishes in DME/F12 medium containing 10% FBS and cultured for 24-h, followed by serum starvation for additional 24-h, when cells were harvested, lysed, total protein extracted and measured using BCA assay. Samples were electrophoresed using 60 µg total protein per lane and western blotted (A). The protein levels of p38 MAPK were quantified using densitometric analysis and the results are expressed as p38 MAPK / β-actin ratio. Each bar represents the mean ± SEM of 4 replicates for both groups. Asterisk indicate significant difference between controls and Min-p38 MAPK1,2 cells, P<0.05 (B).

Figure 2

The effects of 100 nM 17β-estradiol (E2), and a combination of 500 ng/ml human growth hormone (GH) and 10 ng/ml epidermal growth factor (EGF) on growth of MCF-7 cells stably transfected with siRNA to the p-38 MAPK (Min-p38 MAPK1, 2) and controls with intact expression of p38 MAPK. The cells were plated onto 24-well plates in DME/F12 medium containing 10% FBS and incubated for 24-h, followed by 24-h serum starvation and then treated for 6 days. Medium was replenished every other day. At the end of the culture period, total DNA was measured to assess overall cell growth. Each bar represents mean ± SEM for four replicate wells. * Significantly different, p <0.05.

Figure 3

Proliferation rate of the MCF-7 cells expressing a low level of p38 mitogen-activated protein kinase (Min-p38 MAPK1, 2) and controls. The cells were plated onto 96-well plates in DME/F12 medium with 10% FBS and incubated overnight. The following day, the medium was changed to serum-free and the cells incubated for additional 24-h. The cells were then treated with 100 nM 17β-estradiol (E2) or vehicle (controls) and incubated overnight. Fresh media containing 10 µM 5-bromo-2’-deoxyuridine (BrdU) (treatment and control) were then added and the incubation continued for additional 90 min when cultures were terminated and BrdU uptake determined. Each bar represents mean ± SEM for 8 replicate wells. * Significantly different, p <0.05.

Figure 4

The rate of apoptosis was measured using an ELISA specific for a neo-epitope on cytokeratin 18 that is exposed after caspase cleavage. Min-p38 MAPK1, 2 and controls were plated onto 96-well plates in DME/F12 medium containing 10% FBS and incubated overnight. The cells were then serum-starved for additional 24-h and then treated overnight with 100 nM 17β-estradiol (E2), 60 µM Roscovitine, or vehicle (untreated). Experiments were terminated by lysing the cells with 10% NP-40, final concentration 0.5%, and the cell lysate was then diluted and assayed according to the manufacturer’s directions. Each bar represents mean ± SEM for 4–5 replicate wells. * Significantly different, p <0.05.

Figure 5

Western blot analysis showing basal expressions of the estrogen receptor α (ERα) and ERβ in cloned MCF-7 cells stably transfected with small interfering RNA (siRNA) to p38 MAPK (Min-p38 MAPK1, 2) and controls. Β-Actin was used as an internal standard. Cells were plated onto 60 mm culture dishes in DME/F12 medium containing 10% FBS and cultured for 24-h, followed by serum starvation for additional 24-h when cells were harvested, lysed, total protein extracted and measured using BCA assay followed by SDS-PAGE using 60 µg total protein per lane and western blotted (A). The results from the western blotting were quantified using densitometry and presented as means ± SEM of 6 (ERα both groups) and 4 (ERβ both groups) replicates. Asterisk indicates significant difference between controls and Min-p38 MAPK1,2 cells p<0.05 (B).

Figure 6

Western blot analysis showing expressions of the progesterone receptor A (PR-A) in cloned MCF-7 cells stably transfected with small interfering RNA (siRNA) to p38 MAPK (Min-p38 MAPK1, 2) and controls. Β-Actin was used as an internal standard. Cells were plated onto 60 mm culture dishes in DME/F12 medium containing 10% FBS and cultured for 24-h. The cells were then serum-starved overnight and then treated with 100 nM 17β-estradiol (E2) or vehicle (untreated) and incubated for additional 24-h when cells were harvested, lysed, total protein extracted and measured using BCA assay. Samples were then subjected to SDS-PAGE using 60 µg total protein per lane and western blotted (A). The results from the western blotting were quantified using densitometry and are shown as means ± SEM of 4 replicates for both groups. Asterisk indicates significant difference between controls and Min-p38 MAPK1,2 cells p<0.05 (B).

Figure 7

The basal activity (phosphorylation) of the p53 tumor suppressor protein was measured using ELISA. The Min-p38 MAPK1, 2 and controls cells were plated onto 60 mm culture plates in 10% FBS and incubated overnight, followed by 24-h serum starvation. Medium was then removed and fresh serum-free medium applied and incubation continued for additional 10 min, when the cells were washed and harvested by scraping in ice-cold PBS. After centrifugation for 5 min, the cells were lysed and the assays carried out. Total protein concentrations of the cell lysates were used for normalizing the assay results. Each bar represents the mean ± SEM of 3 replicate plates for each cell line. * Significant difference between Min-p38 MAPK1, 2 and controls, p <0.05. The results for the two Min-p38-MAPK cell lines were combined for the statistical analysis and are presented combined in the graph (A). The total protein expression levels of p53 were determined using western blot analysis and densitometric quantification. The results are expressed as p53/β-actin ratio. Each bar represents mean ± SEM of 4 replicates for both groups (B).

Figure 8

The tumorigenicity of MCF-7 cells lacking p38 MAPK was studied by subcutaneously injecting Min-p38 MAPK cells into immunodeficient mice fitted with silastic capsule containing 30 µg 17β-estradiol (E2). Each animal was injected with 1×10⁷ Min-p38 MAPK cells in BD Matrigel (150 µl) and control cells in the same medium. Each animals received Min-p38 MAPK cells in one flank and control cells with scrambled siRNA vector in the other flank, that is each animal was inoculated with both Min-p38 MAPK cells and control cells with scrambled siRNA vector. Tumor size was measured weekly for 4 weeks beginning one month after inoculation. Two months after inoculation, animals were sacrificed, tumors were harvested and weighed. Photograph showing a mouse bearing Min-p38 xenograft and control cells with scrambled siRNA vector (A). Photograph showing xenografts of Min-p38 MAPK and control cells with scrambled siRNA vector at the time of harvest (B). Bar graph showing average xenograft weight for Min-p38 MAPK and control cells with scrambled siRNA vector after 8 weeks of growth. Each bar represents the mean ± SEM of tumors from 5 animals. Asterisk indicates significant difference between control cells with scrambled siRNA vector and Min-p38 MAPK cells, p <0.05 (C). Microphotograph showing immunohistochemical detection of p38 MAPK in tumor xenografts obtained from immunodeficient mice. (1) Positive immunostaining for p38 MAPK in control tumor with scrambled siRNA vector. (2) Section from the same control tumor with scrambled siRNA vector where the primary antibody was omitted (negative control). (3) Immunostaining for p38 MAPK of Min-p38 MAPK tumor. (4) Negative control (primary antibody omitted) for Min-p38 MAPK tumor. Notice the weak immunostaining for p38 MAPK in the Min-p38 MAPK xenograft compared to the control xenograft tumors with scrambled siRNA vector.