For additional information, see YOSHIMURA_MAPK8_TARGETS_UP listed in Table 1. similarities to human tumors, including head and neck, liver, thyroid, lung, and breast cancers. This may reflect functional similarities between cancer-associated fibroblasts (CAFs) and HD fibroblasts. This is consistent with the idea that the presence of HD fibroblasts may be a hallmark of a pre-cancerous phenotype. In these biological processes, GSEA predicts that several key signaling pathways may be involved, including JNK1, iNOS, Rho GTPase(s), FGF-R, EGF-R, and PDGF-R-mediated signal transduction, thereby creating a pro-inflammatory, pro-proliferative, cytokine, and chemokine-rich microenvironment. HD fibroblasts also showed significant overlap with gene profiles derived from smooth muscle cells under stress (JNK1) and activated/infected macrophages (iNOS). Thus, HD fibroblasts may behave like activated myofibroblasts and macrophages, to create and maintain a fibrotic and inflammatory microenvironment. Finally, comparisons between the HD fibroblast gene signature and breast cancer tumor stroma revealed that JNK1 stress signaling is the single most significant biological process that is shared between these 2 data sets (withP = 5.23E-05). Similarities between the HD fibroblast gene signature, wound healing, and the cancer-associated fibroblast phenotype were also noted. Thus, this unbiased informatics analysis of high breast density provides a novel framework for additional experimental exploration and new hypothesis-driven breast cancer research, with a focus on cancer prevention and personalized medicine. 0.05). These up-genes ( 1250 transcripts increased in HD fibroblasts, relative to LD fibroblasts) were then used to conduct gene set enrichment analysis (GSEA), to determine if there is significant overlap with existing gene sets Tirasemtiv (CK-2017357) deposited in the MSigDB (molecular signature database). HD fibroblast up-genes showed strong associations with gene sets related to cancer, the stress response, inflammation, stemness, and signal transduction (summarized in Table 1). Table 1. Gene set enrichment analysis (GSEA) for HD vs. LD breast fibroblasts valuevalue of 6.69E-06. HCC, CGB hepatocellular carcinoma. Open in a separate window Figure 3. HeatMaps for HD fibroblast transcripts related to breast cancer sub-types. For more details, see CHARAFE_BREAST_CANCER_LUMINAL_VS_BASAL_UP and SMID_BREAST_CANCER_NORMAL_LIKE_UP listed in Table 1. These associations have P values of 1 1.74E-05 and 2.89E-05, respectively. The stress response and inflammation HD fibroblasts showed a strong association with the activation of the “stress response” mediated by JNK1, also known as MAPK8 (= 5.20E-10). Consistent with the onset of a stress response, several gene sets associated with inflammation (dental caries) and activated macrophages, as well as iNOS (NOS2), showed striking similarities. Thus, external stimuli (from adjacent cells, such as abnormal breast epithelia or adipocytes) may induce a stress response, creating the HD fibroblast phenotype, leading to inflammation. Indeed, HD fibroblasts may behave like macrophages, driving and propagating inflammation, via the secretion of cytokines/chemokines and hydrogen peroxide, as well as via iNOS activation and NO production. This would be likely to generate an area or field of oxidative stress. HeatMaps for the HD fibroblast transcripts related to JNK1 signaling, the inflammatory response, and iNOS are shown in Figures 4, ?,5,5, and ?and66. Open in a separate window Figure 4. HeatMaps for HD fibroblast transcripts related to JNK1 (MAPK8) signaling. For more details, see YOSHIMURA_MAPK8_TARGETS_UP listed in Table 1. This association has aP P P P P This signature compares the transcriptional profiles of tumor stroma (from n = 53 patients) to normal stroma (from n = 38 patients). Genes transcripts that were consistently upregulated in tumor stroma were selected and assigned aP P This Tirasemtiv (CK-2017357) signature compares the transcriptional profiles of tumor stroma Tirasemtiv (CK-2017357) obtained from patients with tumor recurrence (n = 11) to the tumor stroma of patients without tumor recurrence (n = 42). Genes transcripts that were consistently upregulated in the tumor stroma of patients with recurrence were selected and assigned aP P P P valuevalueP P values are as shown. Augmented TGF- signaling also emerged as a common feature linking high breast density, with tumor stroma and breast cancer recurrence (Tables 2 Tirasemtiv (CK-2017357) and ?and3).3). Importantly, TGF- mediated signal transduction is known to converge on JNK1-signaling, via the Tirasemtiv (CK-2017357) activation of FAK and TAK1, driving the myofibroblast differentiation program and induction of -smooth muscle actin (-SMA) during fibrosis (see the “Discussion” section). A.