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. 2010 Sep;9(9):1982-90.
doi: 10.1074/mcp.M110.000943. Epub 2010 May 28.

In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo

Bart Baan  1 , Evangelia Pardali, Peter ten Dijke, Hans van Dam
Affiliations

In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo

Bart Baan et al. Mol Cell Proteomics. 2010 Sep.

Abstract

Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material.

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Figures

Fig. 1.
Fig. 1.
In situ PLA detection of endogenous c-Jun-c-Fos and c-Jun-ATF2 interactions. A, Ad-HER cells were left untreated (−) or were stimulated (+) with 100 nm TPA. After 2 h, protein extracts were prepared and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Equal loading was confirmed with Ponceau S staining. B, schematic representation of in situ PLA detection of a c-Jun heterodimer using secondary proximity probes. Green and red Ys indicate the primary and oligo-conjugated secondary antibodies specific for c-Jun (red) and its dimer partner (green) depicted at the top of the figure. The long curved black line represents the RCP, including the detection sequence (yellow bars). The full circle at the end of the RCP represents the circular DNA template that resulted from proximity-dependent ligation and was amplified by rolling circle amplification. The detection probes are represented by asterisk-arrows. For a further description, see the text. C, Ad-HER cells were grown on collagen-coated microchamber slides and treated as described above. After fixation, in situ PLA for c-Jun/c-Fos and c-Jun/ATF2 dimers was performed with c-Jun-, c-Fos-, and ATF2-specific antibodies. The detected dimers (cJun/cFos PROX and cJun/ATF2 PROX) are represented by the fluorescent rolling circle products (red dots). PROX, PLA-detected proximity.
Fig. 2.
Fig. 2.
Validation of PLA detection of c-Jun/c-Fos and c-Jun/ATF2 dimers with a partner-preferring c-Jun mutant. Ad-HER cells were transfected with HA-tagged wild type c-Jun (cJunHA wt), the ATF2-preferring c-Jun mutant m1 (cJunHA m1), or the control vector (−). A histone-GFP vector was cotransfected to identify transfected cells. After 2 days, cells were treated with TPA for 2 h. Subsequently, in situ PLA for c-JunHA-c-Fos interactions (cJunHA/cFos PROX) (A) and c-JunHA-ATF2 interactions (cJunHA/ATF2 PROX) (B) was performed using antibodies against HA and c-Fos (A) or HA and ATF2 (B). Cell nuclei were stained with DAPI (blue); transfected GFP-positive cells (green) are indicated with dashed circles. C and D, in situ PLA signals (red dots) in the GFP-positive cell population (n > 75) were quantified by semiautomated image analysis with BlobFinder software. The average number of RCPs per cell is shown ±S.E. (****, p < 1 × 10−4). E, protein extracts from cells treated as described above were analyzed by SDS-PAGE and immunoblotting with c-Fos-, ATF2-, and HA-specific antibodies. Equal loading of the gel was confirmed with Ponceau S staining. PROX, PLA-detected proximity.
Fig. 3.
Fig. 3.
Reduction of endogenous c-Jun/c-Fos and c-Jun/ATF2 PLA signals by an interfering c-Jun mutant. A, schematic representation of PLA detection of c-Jun (red) interacting with one of its dimer partners (green) in the absence (−) or presence (+) of the competing c-Jun deletion mutant ΔJHA, which lacks the epitope recognized by the c-Jun antibody used in PLA. B–E, Ad-HER cells were transfected with ΔJHA or the control vector (−) and a histone-GFP vector to identify transfected cells. After 2 days, cells were treated with TPA for 2 h. Subsequently, in situ PLA for c-Jun-c-Fos (cJun/cFos PROX) (B) and c-Jun-ATF2 interactions (cJun/ATF2 PROX) (C) was performed. Cell nuclei were stained with DAPI (blue); transfected GFP-positive cells (green) are indicated with dashed circles. D and E, in situ PLA signals in the GFP-positive cell population (n > 75) were quantified by semiautomated image analysis with BlobFinder software. The average number of RCPs per cell is shown ±S.E. (****, p < 1 × 10−4; ***, p < 0.001). F, protein extracts from cells treated as described above were analyzed by SDS-PAGE and immunoblotting with c-Fos-, ATF2-, c-Jun-, and HA-specific antibodies. Equal loading of the gel was confirmed with Ponceau S staining. PROX, PLA-detected proximity.
Fig. 4.
Fig. 4.
PLA detection and validation of c-Jun/Fra1 dimers. Ad-HER cells were transfected with ΔJHA or the control vector (−) and a histone-GFP vector to identify transfected cells. After 2 days, cells were treated with TPA for 2 h when indicated. A, cell extracts were analyzed by SDS-PAGE and immunoblotting with Fra1-, c-Jun-, and HA-specific antibodies. Equal loading of the gel was confirmed with Ponceau S staining. B, in situ PLA was performed for c-Jun/Fra1 (cJun/Fra1 PROX). Cell nuclei were stained with DAPI (blue); transfected GFP-positive cells (green) are indicated with dashed circles. C, c-Jun/Fra1 PLA signals in the GFP-positive cell population (n > 100) were quantified by semiautomated image analysis with BlobFinder software. The average number of RCPs per cell is shown ±S.E. (****, p < 0.0001). PROX, PLA-detected proximity.
Fig. 5.
Fig. 5.
Enhanced levels of c-Jun/Fra1 PLA signals in mesenchymal breast cancer and melanoma cells. A, in situ PLA for c-Jun/Fra1 (cJun/Fra1 PROX) was performed for the luminal-like breast cancer cell lines MCF7, T47D, and ZR75 and the mesenchymal cell lines MDA-MB231, MDA-MB435, and MDA-MB436. Cell nuclei were stained with DAPI (blue). B–D, quantification of the c-Jun/Fra1, c-Jun/ATF2, and c-Jun/c-Fos PLA signals obtained for the cell lines shown in A by semiautomated image analysis with BlobFinder software. The average number of RCPs per cell is shown ±S.E. E, protein extracts from the cell lines described above were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Equal loading of the gel was confirmed with Ponceau S staining. PROX, PLA-detected proximity.
Fig. 6.
Fig. 6.
In situ detection of c-Jun/Fra1 complexes in MDA-MB231-derived tumor tissue. A, a hematoxylin-eosin (HE)-stained cryosection of a MDA-MB231-derived primary tumor. B–D, in situ PLA for the indicated c-Jun/AP-1 dimers in MDA-MB231-derived tumors. The tissue section was counterstained with DAPI (blue) to visualize the nuclei. PROX, PLA-detected proximity.
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