Inflammatory regulatory network mediated by the joint action of NF-kB, STAT3, and AP-1 factors is involved in many human cancers Zhe Jia,b,1,2, Lizhi Hea,1, Aviv Regevb,c,d,e, and Kevin Struhla,3 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bBroad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142; cHoward Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 20140; dDavid H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 20140; and eDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 20140 Contributed by Kevin Struhl, February 25, 2019 (sent for review December 12, 2018; reviewed by Myles Brown and Michael R. Green) Using an inducible, inflammatory model of breast cellular trans- STAT3 (6, 11). By integrating motif analysis of DNase hypersensitive formation, we describe the transcriptional regulatory network regions with transcriptional profiling, we found that >40 transcription mediated by STAT3, NF-κB, and AP-1 factors on a genomic scale. factors are important for transformation and identified putative These proinflammatory regulators form transcriptional complexes target sites directly bound by these factors (12). that directly regulate the expression of hundreds of genes in on- A few transcriptional regulatory circuits involved in this cogenic pathways via a positive feedback loop. This transcriptional transformation model have been identified, and these are important feedback loop and associated network functions to various ex- in some other cancer cell types and human cancers (6, 11, 13, 14). tents in many types of cancer cells and patient tumors, and it is During transformation, STAT3 acts through preexisting nucleosome- the basis for a cancer inflammation index that defines cancer types depleted regions bound by FOS, and expression of several AP-1 factors is altered in a STAT3-dependent manner (15). However, by functional criteria. We identify a network of noninflammatory κ genes whose expression is well correlated with the cancer inflam- the connection between STAT3, NF- B, and AP-1 factors as well matory index. Conversely, the cancer inflammation index is nega- as the underlying transcriptional regulatory circuits has not been described on the whole-genome level. CELL BIOLOGY tively correlated with the expression of genes involved in DNA Here, we define the transcriptional network mediated by the metabolism, and transformation is associated with genome insta- combined action of NF-κB, STAT3, and AP-1 factors (JUN, JUNB, bility. We identify drugs whose efficacy in cell lines is correlated and FOS) on a genomic scale in this breast transformation model. In with the cancer inflammation index, suggesting the possibility of contrast to previous studies (12, 15), this network is defined by genes using this index for personalized cancer therapy. Inflammatory that are induced during transformation by the binding of NF-κb, tumors are preferentially associated with infiltrating immune cells STAT3, and AP-1 factors to common target sites either through their that might be recruited to the site of the tumor via inflammatory molecules produced by the cancer cells. Significance epignetic switch | inflammation | cancer | gene regulatory network In a breast cellular transformation model, we describe the gene regulatory network that is mediated by joint and direct action nflammation is a hallmark of cancer and plays important reg- of three inflammatory transcription factors on hundreds of Iulatory roles during cell transformation, invasion, metastasis, – target genes in various oncogenic pathways. This inflammatory and treatment resistance (1 3). An inflammatory reaction during feedback loop and associated network functions in many types tumor development occurs in nearly all solid malignancies (4). of cancer cells and patient tumors. It forms the basis for a Preexisting inflammation promotes subsequent cancer development, “ ” ∼ cancer inflammation index that defines cancer types by func- accounting for 15 20% of cancer deaths (3). Inflammation also tional criteria, and that may be useful for choosing drugs for per- plays critical roles in immunosuppression (2). For example, STAT3 sonalized cancer treatment. Inflammatory tumor cells, via secreted promotes the expression of PD-L1 and PD-L2 in cancer cells, inflammatory molecules, might help recruit immune cells to the suppressing immune cell activity (5). site of the tumor. Thus, detailed analysis of an artificial trans- Tumor-associated inflammation results from both an intrinsic formation model uncovers the molecular basis of an inflam- pathway, in which mutations in cancer cells activate inflamma- matory network relevant for many forms of human cancer. tory gene expression, and an extrinsic pathway, in which cytokines andchemokinessecretedbytumor-associated immune cells create Author contributions: Z.J. and K.S. designed research; Z.J. and L.H. performed research; inflammatory microenvironments (3). Oncogenes can trigger a gene Z.J. and L.H. contributed new reagents/analytic tools; Z.J., L.H., A.R., and K.S. analyzed expression cascade, resulting in activation or overexpression of data; and Z.J., A.R., and K.S. wrote the paper. proinflammatory transcription factors such as NF-κB, STAT3, and Reviewers: M.B., Dana–Farber Cancer Institute; and M.R.G., University of Massachusetts AP-1, with the resulting production of cytokines and chemokines (1, Medical School. 2, 6). For example, NF-κB signaling pathway is activated upon The authors declare no conflict of interest. activation of oncogenes RAS and MYC, through activation of IL- Published under the PNAS license. β – 1 (6 8), and the proto-oncoprotein Src kinase can directly phos- Data deposition: The data reported in this paper have been deposited in the Gene Ex- phorylate and activate STAT3 (9). pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. In previous work, we have described an inducible model of GSE115597, GSE115598, and GSE115599). cellular transformation in which transient activation of v-Src onco- 1Z.J. and L.H. contributed equally to this work. protein converts a nontransformed breast epithelial cell line into a 2Present addresses: Department of Pharmacology, Feinberg School of Medicine, Northwestern stably transformed state within 24 h (6, 10). The stably trans- University, Chicago, IL 60611; and Department of Biomedical Engineering, McCormick School formed cells form foci and colonies in soft agar, show increased of Engineering, Northwestern University, Evanston, IL 60628. motility and invasion, form mammospheres, and confer tumor 3To whom correspondence should be addressed. Email: [email protected]. formation in mouse xenografts (6, 10). This epigenetic switch This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. between stable nontransformed and transformed states is mediated 1073/pnas.1821068116/-/DCSupplemental. by an inflammatory positive feedback loop involving NF-κBand Published online March 25, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1821068116 PNAS | May 7, 2019 | vol. 116 | no. 19 | 9453–9462 Downloaded by guest on September 28, 2021 cognate motifs or via protein–protein interactions. Based on this replicates (SI Appendix, Fig. S2B). Based on this, we grouped the common NF-κb, STAT3, and AP-1 network, we develop a cancer AP-1 factors together in most subsequent analyses. Also as inflammation index to define cancer types, both in cell lines and expected, STAT3 binding sites strongly overlap with FOS binding in patients, by functional criteria. As this inflammation index is based sites (15) and with the other AP-1 factors (89% of STAT3 binding on the common regulatory network,itisdistinctfrom,andmore sites) (SI Appendix,Fig.S3A). In addition, 89% of NF-κBbind- specific than, indices based simply on gene expression profiles that ing sites overlap with AP-1 factors (SI Appendix,Fig.S3A). Thirty- arise from multiple regulatory inputs. In addition, we identify many eight percent of STAT3, NF-κB, and AP-1 sites are located in the noninflammatory genes whose expression is positively or negatively same cis-regulatory regions (CRRs; SI Appendix,Fig.S3A), which is − correlated with the cancer inflammation index, leading to the ob- far more frequent than expected by chance (P < 10 200; χ2 test). De- servation that inflammation is linked functionally to other aspects of spite the strong colocalization, STAT3, NF-κb, and AP-1 factors cancer as well as genomic instability. Lastly, we show that inflam- can independently bind to specific sites (SI Appendix, Fig. S3A), matory tumor samples preferentially contain contaminating immune and the pairwise correlation values are lower than the correla- and stromal cells from the tumor microenvironment, consistent tion values between biological replicates (SI Appendix, Fig. S3B). with the idea that immune cells might be recruited to the site of Additional results suggest that STAT3, NF-κB, and AP-1 the tumor via inflammatory molecules produced by the cancer cells. factors cobind to target sites as a multiprotein complex. Most impor- tantly, the median distance of peak summits for all pairwise com- Results binations of AP-1 factors, STAT3, and NF-κB ranges between Colocalization of STAT3, NF-κB, and AP-1 Factors on Target Sites in Vivo. 15 and 30 bp, and these values are comparable to those obtained During transformation,