The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

Abstract

Activating protein-1 (AP-1) family members, especially Fra-1 and c-Jun, are highly expressed in invasive cancers and can mediate enhanced migration and proliferation. The aim of this study was to explore the significance of elevated levels of AP-1 family members under conditions that restrict growth. We observed that invasive MDA-MB-231 cells express high levels of Fra-1, c-Jun, and Jun-D during serum starvation and throughout the cell cycle compared to non-tumorigenic and non-invasive cell lines. We then analyzed Fra-1 levels in additional breast and other cancer cell lines. We found breast and lung cancer cells with higher levels of Fra-1 during serum starvation had relatively higher ability to proliferate and migrate under these conditions. Utilizing a dominant negative construct of AP-1, we demonstrated that proliferation and migration of MDA-MB-231 in the absence of serum requires AP-1 activity. Finally, we observed that MDA-MB-231 cells secrete factors(s) that induce Fra-1 expression and migration in non-tumorigenic and non-metastatic cells and that both the expression of and response to these factors require AP-1 activity. These results suggest the presence of an autocrine/paracrine loop that maintains high Fra-1 levels in aggressive cancer cells, enhancing their proliferative and metastatic ability and affecting neighbors to alter the tumor environment.

Keywords: AP-1; autocrine; cancer; paracrine; self-sufficient growth.

Figure 1. Analysis of Fos and Jun family members in non-tumorigenic and tumorigenic breast epithelial cell lines

(A) MCF10A, MDA-MB-468, and MDA-MB-231cells were subjected to serum starvation, then stimulated with serum for the times indicated in the figure (h). Cells were harvested and protein levels were detected using western blot. X = exponentially growing cells with serum. The figure is representative of more than three independent experiments. (B) Fra-1 protein levels were analyzed in a panel of TNBC cell lines. X = exponential growth with serum 0 = serum starvation for 48 hours. 8 = 8 hours of serum stimulation.

Figure 2. Analysis of Fra-1 expression in cancer cell lines of different origins

Colon, lung, prostate, and melanoma cancer cell lines were analyzed for Fra-1 protein by western blotting. X = exponential growth in complete medium. 0 = serum starvation for 48 hours. 8 = 8 hours of serum stimulation.

Figure 3. Fra-1 expression during serum starvation correlates with the ability of cells to progress through the cell cycle in the absence of serum

(A) Cells were collected either during exponential growth with serum (X), after 48 hours in 0.05% serum (SF), or following serum starvation (0.05%) plus treatment with nocodazole (250 ng/ml) for 30 hours (NCD). Cells were stained with propidium iodide and analyzed for cell cycle distribution by flow cytometry. Data shown is the average of one experiment conducted in triplicate. (B) Cells were treated as in (A) then stained with both anti-Fra-1 antibody and PI and analyzed by flow cytometry. Numbers are the mean of 2 independent experiments.

Figure 4. Breast and lung cell lines with higher Fra-1 during serum starvation exhibit higher ability to grow and migrate in the absence of serum

(A and B) Cell growth in 10% or 0.05% (SF) serum was determined using manual cell counting as described in materials and methods in breast (A) and lung (B) cancer cell lines. The relative cell number was calculated as indicated in the materials and method section. (C) Wound healing assays were performed in the presence and absence (0.05%) (SF) of serum in breast cancer cell lines. ^*= significant (p<0.05) using Student's t test, # = non-significant. All numbers are the average of 3 independent experiments.

Figure 5. Inhibition of AP-1 activity in MDA-MB-231 cells reduces Fra-1 expression and suppresses the ability of the cells to progress through the cell cycle in the absence of serum

MDA-MB-231 cells were infected with a retroviral vector encoding a doxycycline inducible Flag-AFos gene as described in material and methods. Transfected cells were divided into two groups; non-induced Dox (-) or induced Dox (+). (A) A-Fos and Fra-1 protein levels were examined by western blotting. The figure is representative of 3 independent experiments (B) Cells were treated +/- Dox then subjected to serum starvation and nocodazole treatment as described in Figure 3. The data shown is representative of three independent experiments.

Figure 6. Inhibition of AP-1 activity and Fra-1 expression reduces MDA-MB-231 cell growth and migration

(A) The effects of A-Fos expression on the ability of the MDA-MB-231 cells to grow in the presence (10%) and absence (0.05%) of serum were analyzed for several passages. Cells were plated at density of 3×10⁵ with and without Dox and/or serum. After 72 hours cells were counted and re-plated at the same density, and this process was repeated for 3 passages. (B) The effect of A-Fos on the migration of MDA-MB-231 cells in the presence and absence of serum using the wound healing assay ^*= significant (p<0.05), the supplementary image represents migration in presence of serum. (C) Cells were infected with a retroviral vector encoding a scrambled sequence (MDA231_S) or Fra-1 shRNA (MDA231_ShFra-1). Protein lysates were analyzed by western blot to detect Fra-1 expression (left). Cell proliferation was compared in the absence (72 hour SF) and presence (72 hours of serum) of serum using manual cell counting. The white bar to the left (plated cells) shows the number of cells plated in each well (3X 10⁵) (Right). For all serum starvation experiments 0.05% of serum was used. The numbers are the mean of three independent experiments.

Figure 7. AP-1 dependent soluble factors from MDA-MB-231 cells induce Fra-1 expression in MCF10A and MDA-MB-468 cells

(A) Using a transwell system MCF10A cells were cultured in the upper chamber with MDA-MB-231 cells or serum free medium in the lower chamber, then the levels of Fra-1 protein in MCF10A cells were examined. (B) Conditioned medium from serum starved MDA-MB-231 cells was added to serum starved MCF10A cells for 6 hours then Fra-1 protein levels were examined. Blots were quantified using Image J software. (C) MDA-MB-468 cells were incubated with CM or co-cultured with MDA-MB-231 cells in a transwell chamber as described in materials and methods, then Fra-1 expression was examined. (D) CM from MDA-MB-231/Flag-AFos cells incubated in the presence (Dox (+)) and absence (Dox (-)) of doxycycline was added to MCF10A cells, and Fra-1 protein levels were examined. The results shown are representative of 3 experiments. Induction with serum for 6 hours was added as a positive control (10% FBS – 6h). (E) MCF10A cells were incubated with MDA-MB-231 CM with and without PD98059 and U0126 (MEK inhibitors). The cells were harvested after 6 hours and the level of Fra-1 was detected by western blotting. For all these experiments 0% serum was used.

Figure 8. AP-1 dependent soluble factors from MDA-MB-231 cells increase migration of MCF10A and MDA-MB-468 cells in an AP-1 dependent manner

(A) MCF10A cells were cultured on an 8μm pore transwell insert with or without MDA-MB-231 cells in the lower chamber for 24 hours and migrated MCF10A cells on the lower side of the membrane were fixed and stained as described in materials and methods. (B) MCF10A cells were cultured for 12 hours in the upper chamber of transwell inserts in MDA-MB-231 CM versus serum free medium (left graph), or MCF10A cells were cultured in the upper chamber in serum free medium with the lower chamber filled with either CM or serum free medium (right graph). Cells were counted per HPF. from 3 independent experiments and a representative picture of the migrated cells is shown. (C) MDA-MB-468 cells were co-cultured with MDA-MB-231/Flag-AFos cells in a transwell system in the presence (Dox (+)) and absence (Dox (-)) of doxycycline for 24 hours. (D) A wound healing migration assay was used to measure the effect of CM on MCF10A cell migration. The graph shows percent closure after 24 hours. (E) MCF10A cells were infected with scrambled shRNA virus (vector) or two different Fra-1 shRNA viruses and Fra-1 levels were compared (Left). A wound healing assay was carried out to measure the effect of conditioned medium on MCF10A migration (Right). For all these experiments 0% serum was used.

Figure 9. A summary of the role of AP-1 in auto/paracrine loops that mediate cancer progression and the associated secreted factors [–79]

AP-1 plays a central role in auto/paracrine loops by regulating the expression of soluble extracellular factors to control functions that enhances the ability of cancer cells to grow, survive and metastasize. These soluble factors are able at the same time to re-induce the expression and/or activity of different AP-1 members through different signaling pathways to keep the loop firing.