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.2013 Apr 22;8(4):e61353.
doi: 10.1371/journal.pone.0061353. Print 2013.

Mutant p53 attenuates the anti-tumorigenic activity of fibroblasts-secreted interferon beta

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Mutant p53 attenuates the anti-tumorigenic activity of fibroblasts-secreted interferon beta

Shalom Madar et al. PLoS One..

Abstract

Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. An in vitro model to study the tumor-stroma encounter in lung cancer.
(A). p53-null lung carcinoma cells (H1299) were introduced with the designated mutations. p53 levels were determined by Western blot analysis (A) and by QRT-PCR (B). A fluorescent microscope image of co-cultured dsRed-labeled H1299 with GFP-labeled HK3 (C, upper panel). Representative FACS analysis depicting dsRed- and GFP-labeled sub-populations following a sorting procedure (C, middle panel). Each sub-population was then re-sorted using the same sorting gates (C, lower panel).
Figure 2
Figure 2. The interferon beta pathway is up regulated in fibroblasts after co-cultivation with mutp53-bearing carcinoma cells.
(A) Following the described sorting procedure, HK3-T samples were subjected to a microarray analysis (see materials and methods). The presented cluster was obtained by the ‘CLICK’ algorithm from the ‘Expander’ package using default homogeneity (version 5.2) . The log 2 ratios were standardized to have zero mean and unit standard deviation for each gene. (B) ‘HK3-T cluster’ which contains 414 genes was compared to a previously reported ‘IFN signature’ that was induced by co-cultures of cancer and stromal cells. (C) ‘HK3-T cluster’ was compared with the ‘Interferome database’ containing 1196 IFN targets. A Fisher's exact test was utilized to compare these overlaps with those of all other clusters. * = P<0.0001 (D) To assess the significance of the physical interaction between HK3-T and H1299175 in eliciting the IFN pathway, HK3-T were introduced to conditioned media from each cell type grown alone or from a co-culture plate. mRNA levels of two known IFN targets were measured by QRT-PCR. (E) Interferon α and β mRNA levels. (F) CAFs and H1299 were grown either alone or in a co-culture. The co-cultured cells were incubated with the designated antibodies. Shown is the mRNA level of the designated IFN targets. * P<0.05. (G) HK3-T cells were subjected to conditioned media of HK3-T or that of HK3-T cultured with H1299175. Shown is a western blot of GAPDH, STAT1, pSTAT1 and STAT2. (H) The same experimental setup was used. Shown in the upper panel are microscope images of the cells. Cells were then collected and stained with Propidium Iodide (PI) and apoptotic cells (PI positive) were detected by FACS sorting and their percentage is depicted in the lower panel.
Figure 3
Figure 3. Expression patterns of H1299 in response to co-cultivation with CAFs.
(A) Three principal expression patterns of H1299 cultivated with HK3-T. (B) A heat-map depicting genes that exhibit Pearson correlation of at least 0.9 to representative bait(s) from each pattern shown in A. Beneath is the percentage of IFN targets in each list based on the ‘Interferome database’ . (C) QRT-PCR analysis of a representative gene from each expression pattern. (D) The H1299 panel was treated with the designated IFNs for 24 h. Shown is a QRT-PCR analysis of MX1 expression.
Figure 4
Figure 4. Mutant p53 counteracts IFNβ by SOCS1-mediated attenuation of STAT1 phosphorylation.
(A) Cells were treated with IFNβ for 16 h, fixed and sorted by an “Image stream” FACS. The upper panel depicts representative images from each condition and the graph represents the mean pixel intensity of STAT1 positive cells for the entire population. (B) Same as in A, here the graph shows the similarity between p-STAT and DAPI staining, thereby quantifying both the expression and localization of pSTAT1. (C) Cells were treated with IFNβ for the designated durations, shown is a graph depicting nuclear p-STAT1. (D) The cells were also collected for RNA analysis and a QRT-PCR for SOCS1 expression was performed. (E) H1299175 cells were introduced with RNAi against LacZ as a control or against SOCS1. p = 0.002 (F) Cells were then treated with IFNβ for 24 h. Shown is a QRT-PCR analysis of MX1 expression. p<0.05. (G) Cells were seeded in trans-wells in serum-free media and treated with IFNβ for 24 h. Migrating cells were collected and counted.
Figure 5
Figure 5. IFNβ reduces mutant p53 mRNA levels by inhibiting its RNA stabiliser WIG1 (ZMAT3).
Cells were treated with IFNβ for the designated time points, fixed and sorted by an “Image stream” FACS. (A) Representative images from each condition and (B) a graph representing the mean pixel intensity of mutant p53 positive cells for the entire population. (C) Cells were treated with the designated IFNs for 24 h and mutant p53 and GAPDH levels were measured by western blot. Cells were treated with IFNβ for 24 h and mutant p53 (D) and WIG1 (E) RNA levels were determined by QRT-PCR. * P<0.05. (F) HepG2 cells were introduced with mut175 plasmid and treated with IFNβ. RNA levels were determined by QRT-PCR. * P<0.05. (G) H1299 cell harbouring a TS form of mutant p53 (Wild type form at 32°C and mutant form at 37°C) were treated with IFNβ for 30 h. p53 and GAPDH levels were measured by western blot, shown in the lower panel a is normalized quantification of the bands.
Figure 6
Figure 6. A schematic representation of the interaction between mutant p53 and the IFNβ pathway.
Blue arrows denote positive effect while red arrows denote negative effect. Upon encounter with cancer cell CAFs activate the IFNβ pathway which limits cancer cells' migration. When mutant p53 is present in the cancer cells, this pathway is moderated via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ is able to reduce mutant p53 RNA levels by attenuating the expression of mutant p53 RNA stabilizer WIG1.
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