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. 2019 May 15;202(10):3076-3086.
doi: 10.4049/jimmunol.1801152. Epub 2019 Apr 1.

Progesterone Receptor Attenuates STAT1-Mediated IFN Signaling in Breast Cancer

Merit L Goodman  1   2   3 , Gloria M Trinca  1   2   3 , Katherine R Walter  1   2   3 , Evangelia K Papachristou  4 , Clive S D'Santos  4 , Tianbao Li  5 , Qi Liu  5 , Zhao Lai  5   6 , Prabhakar Chalise  7 , Rashna Madan  8 , Fang Fan  8 , Mary A Markiewicz  9 , Victor X Jin  5 , Jason S Carroll  4 , Christy R Hagan  10   2   3
Affiliations

Progesterone Receptor Attenuates STAT1-Mediated IFN Signaling in Breast Cancer

Merit L Goodman et al. J Immunol. .

Abstract

Why some tumors remain indolent and others progress to clinical relevance remains a major unanswered question in cancer biology. IFN signaling in nascent tumors, mediated by STAT1, is a critical step through which the surveilling immune system can recognize and destroy developing tumors. In this study, we have identified an interaction between the progesterone receptor (PR) and STAT1 in breast cancer cells. This interaction inhibited efficient IFN-induced STAT1 phosphorylation, as we observed a decrease in phospho-STAT1 in response to IFN treatment in PR-positive breast cancer cell lines. This phenotype was further potentiated in the presence of PR ligand. In human breast cancer samples, PR-positive tumors exhibited lower levels of phospho-STAT1 as compared with their PR-negative counterparts, indicating that this phenotype translates to human tumors. Breast cancer cells lacking PR exhibited higher levels of IFN-stimulated gene (ISG) RNA, the transcriptional end point of IFN activation, indicating that unliganded PR alone could decrease transcription of ISGs. Moreover, the absence of PR led to increased recruitment of STAT1, STAT2, and IRF9 (key transcription factors necessary for ISG transcription) to ISG promoters. These data indicate that PR, both in the presence and absence of ligand, attenuates IFN-induced STAT1 signaling, culminating in significantly abrogated activation of genes transcribed in response to IFNs. PR-positive tumors may use downregulation of STAT1-mediated IFN signaling to escape immune surveillance, leading to the development of clinically relevant tumors. Selective immune evasion of PR-positive tumors may be one explanation as to why over 65% of breast cancers are PR positive at the time of diagnosis.

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Figures

Figure 1.
Figure 1.. PR interacts with STAT1.
A. STRING network of the top 30 PR interactors identified in RIME. The size of the node increases proportionally to the number of identified peptides and thick edges denote high confidence STRING interactions (0.7-0.99). B. Sequence coverage of PR and STAT1 in both replicate RIME experiments. Green highlights high confidence peptides at FDR<1%. PR and STAT1 have been identified by 27 and 19 unique peptides, respectively. C. Left: PR was immunoprecipitated from starved T47D-YB cell lysates (+/− R5020 60min) and the resulting associated protein complexes were analyzed by Western blotting. Bottom panels represent total input cell lysates. Species-specific (rabbit or mouse) IgG was used as a control for the IP. Right: STAT1 was immunoprecipitated from T47D-co cell lysates (+/− R5020) and the resulting associated protein complexes were analyzed by Western blotting. Bottom panels represent total input cell lysates. PR-B (upper) and PR-A (lower) isoforms are both recognized by the PR antibody. Species-specific IgG was used as a control for the IP; the band visible in the IgG only lanes represents non-specific binding between PR/STAT1 and the IgG antibody. These experiments were performed in triplicate, and a representative experiment is shown here.
Figure 2.
Figure 2.. PR attenuates interferon-induced STAT1 phosphorylation.
A. T47D cells that are PR-negative (T47D-Y) or PR-positive (T47D-co) were treated with interferon-alpha (IFNα) for 0-2hrs. Isolated protein lysates were analyzed by Western blotting. Densitometry of the ratio of p-STAT1/total STAT1, as determined using ImageJ analysis, is shown to the right of the immunoblot. B. T47D-co cells were starved for 18hrs in serum-free media, followed by treatment with interferon IFNα and R5020 or vehicle (EtOH) for 0-2hrs. Isolated protein lysates were analyzed by Western blotting. Densitometry of the ratio of p-STAT1/total STAT1, as determined using ImageJ analysis, is shown to the right of the immunoblot. C. T47D-co cells were treated as in B. Whole cell lysates were subjected to nuclear/cytoplasmic fractionation, and resulting subcellular lysates were analyzed by Western blotting. B-tubulin (cytoplasmic) and topoisomerase II (topo II; nuclear) are shown as fractionation markers. These experiments were performed in triplicate, and a representative experiment is shown here.
Figure 3.
Figure 3.. Regulation of p-STAT1/STAT1 is disrupted in PR-positive tumors.
A. Tissue microarray analysis was performed using immunohistochemical staining with phospho-STAT1 (p-STAT1), STAT1 and PR antibodies. Boxplots and Stripcharts show the distribution of p-STAT1 (left) and STAT1 (right) staining intensities in PR-negative and PR-positive breast cancer samples. B. Select PR-negative (left) and PR-positive (right) breast cancer (BrCa) cases stained for PR and p-STAT1 are shown here at 20x magnification. Negative control isotype-only control staining is shown. C. Scatter plot of pSTAT1 and STAT1 with different colors for PR-positive (red) and PR-negative (blue) with trend lines for each. The Spearman’s correlation coefficient and p-values for each group are shown on top. Only tumors with greater than zero total STAT1 staining were included in the analysis.
Figure 4.
Figure 4.. TYK2 phosphorylation is attenuated by activated PR.
A. T47D-co cells were starved for 18hrs in serum-free media, followed by treatment with interferon-alpha (IFNα) and R5020 or vehicle (EtOH) for 0-30min. Isolated protein lysates were immunoprecipitated with the total TYK2 antibody, and the resulting protein complex was blotted with phospho-TYK2 antibody. Species-specific IgG was used as a control for the IP. Densitometry of the ratio of p-TYK2/total TYK2, as determined using ImageJ analysis, is shown to the right of the immunoblot. B. TYK2 was immunoprecipitated from T47D-co cell lysates (+/− R5020; 60min) and the resulting associated protein complexes were alanyzed by Western blotting. Bottom panels represent total input cell lysates. PR-B (upper) and PR-A (lower) isoforms are both recognized by the PR antibody. Species-specific IgG was used as a control for the IP. These experiments were performed in triplicate, and a representative experiment is shown here.
Figure 5.
Figure 5.. PR activation disrupts the ISGF3 complex.
STAT2 and IRF9 (or a species-specific IgG control) was immunoprecipitated from interferon-alpha (IFNα)-treated (2 hrs) starved T47D-co cell lysates (+/− R5020 60min) and the resulting associated protein complexes were analyzed by Western blotting. This experiment was performed in triplicate, and a representative experiment is shown here.
Figure 6.
Figure 6.. PR decreases interferon-induced gene expression.
A. RNA-seq was performed on RNA isolated from interferon-treated (20IU/ml IFNα for 18hrs) T47D PR-null or PR-positive cell lines. GSEA analysis was performed using the c2 MSigDB collection comparing RNA-seq gene expression datasets obtained from interferon-treated PR-null and PR-positive cells. Shown here are the top most significantly-enriched gene sets (FDR < 0.05); select enrichment examples are shown in (B). C. Top 20 ranking genes as identified using Leading Edge (LE) analysis on 29 gene sets referred to in (A). D. T47D PR-null or PR-positive cell lines were treated as in (A). Isolated RNA was analyzed for multiple ISGs using qPCR. Gene values were normalized to an internal control (β-actin). Error bars represent standard deviation between biological triplicates. Asterisks represent statistical significance between groups; p < 0.01, as determined using an unpaired Student’s t-test. E. Cells were treated as in (D), and isolated protein was analyzed via Western blotting with respective antibodies. The experiments presented in (D) and (E) were performed in triplicate, and a representative experiment is shown here.
Figure 7.
Figure 7.. PR decreases ISGF3 recruitment to ISG promoters.
T47D-co NS and PR shRNA cells were serum-starved for 18hr, and then treated with 1000IU/ml IFNα (or vehicle) for 4 hrs. Fixed lysates were subjected to ChIP with antibodies against STAT1, STAT2, IRF9 or a species-specific IgG (control; not shown), and qPCR was performed on the isolated DNA using primers designed to amplify select ISG promoters. A percentage of ChIP’d DNA over input DNA is shown. All ChIP experiments were performed in triplicate; a representative experiment is shown here. Fold-recruitment in IFNα–treated conditions, as compared to vehicle treatment, is displayed above each bar. Error bars represent standard deviation of technical replicates.
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References

    1. Cavallo F, De Giovanni C, Nanni P, Forni G, and Lollini PL. 2011. 2011: the immune hallmarks of cancer. Cancer Immunol Immunother 60: 319–326. - PMC - PubMed
    1. Schreiber RD, Old LJ, and Smyth MJ. 2011. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 331: 1565–1570. - PubMed
    1. Matzinger P 1994. Tolerance, danger, and the extended family. Annu Rev Immunol 12: 991–1045. - PubMed
    1. Cheon H, Borden EC, and Stark GR. 2014. Interferons and their stimulated genes in the tumor microenvironment. Semin Oncol 41: 156–173. - PMC - PubMed
    1. Ivashkiv LB, and Donlin LT. 2014. Regulation of type I interferon responses. Nat Rev Immunol 14: 36–49. - PMC - PubMed

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