JNK1 stress signaling is hyper-activated in high breast density and the tumor stroma: connecting fibrosis, inflammation, and stemness for cancer prevention

Abstract

Mammography is an important screening modality for the early detection of DCIS and breast cancer lesions. More specifically, high mammographic density is associated with an increased risk of breast cancer. However, the biological processes underlying this phenomenon remain largely unknown. Here, we re-interrogated genome-wide transcriptional profiling data obtained from low-density (LD) mammary fibroblasts (n = 6 patients) and high-density (HD) mammary fibroblasts (n = 7 patients) derived from a series of 13 female patients. We used these raw data to generate a "breast density" gene signature consisting of>1250 transcripts that were significantly increased in HD fibroblasts, relative to LD fibroblasts. We then focused on the genes that were increased by ≥ 1.5-fold (P<0.05) and performed gene set enrichment analysis (GSEA), using the molecular signatures database (MSigDB). Our results indicate that HD fibroblasts show the upregulation and/or hyper-activation of several key cellular processes, including the stress response, inflammation, stemness, and signal transduction. The transcriptional profiles of HD fibroblasts also showed striking 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 (with P values between 5.40E-09 and 1.02E-14), and is specifically associated with tumor recurrence. These results implicate "stromal JNK1 signaling" in the pathogenesis of human breast cancers and the transition to malignancy. Augmented TGF-β signaling also emerged as a common feature linking high breast density with tumor stroma and breast cancer recurrence (P = 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.

Keywords: EGF; FGF; JNK; PDGF; SAPK; TGF-beta; breast cancer; cancer associated fibroblasts; fibrosis; gene signature; inflammation; mammographic density; mammography; microenvironment; stress signaling; tumor stroma; wound healing.

Figure 1. HeatMaps for HD fibroblast transcripts related to nasopharyngeal carcinoma. For more details, see DODD_NASOPHARYNGEAL_CARCINOMA_UP listed in Table 1. This association has a P value of 8.21E-16. This association may be due to functional similarities between cancer-associated fibroblasts (CAFs) and HD fibroblasts; CAFs are relatively abundant in most solid tumors, where the tumor stroma can represent up to or greater than 50% of the tumor mass.

Figure 2. HeatMaps for HD fibroblast transcripts related to metastatic liver cancer. For more details, see LIAO_METASTASIS listed in Table 1. This association has a P value of 6.69E-06. HCC, hepatocellular carcinoma.

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 1.74E-05 and 2.89E-05, respectively.

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 a P value of 5.20E-10.

Figure 5. HeatMaps for HD fibroblast transcripts related to the macrophage inflammatory response and infection. For more details, see ZHOU_INFLAMMATORY_RESPONSE_FIMA_UP and MCLACHLAN_DENTAL_CARIES_UP listed in Table 1. These associations have P values of 1.56E-06 and 5.16E-07, respectively.

Figure 6. HeatMaps for HD fibroblast transcripts related to iNOS. For more details, see MORF_NOS2A listed in Table 1. This association has a P value of 4.53E-05. Other HeatMaps related to FLT1, phospholipase C, and Rho GTPases are also shown.

Figure 7. HeatMaps for HD fibroblast transcripts related to TCF3 signaling. For more details, see CAGGTG_V$E12_Q6 listed in Table 1. This association has a P value of 3.18E-06.

Figure 8. HeatMaps for HD fibroblast transcripts related to FGF-R and EGF-R signaling. For more details, see the GO TERMS listed in Table S2. These associations have P values ranging from 2.22E-04 to 9.18E-06.

Figure 9. Venn diagrams for the intersection of HD fibroblast transcripts with tumor stroma from breast cancer patients. Intersection of the HD fibroblast gene signature with tumor stroma yields 471 common gene transcripts, with a P value of 6.69E-04. Intersection of the HD fibroblast gene signature with recurrence stroma yields 257 common gene transcripts, with a P value of 7.42E-05.

Figure 10. Biological processes that are shared between HD breast fibroblasts and the tumor stroma: Similarities to wound healing. These five shared biological processes include stress signaling, stemness, angiogenesis, inflammation and fibrosis. Many of these processes also commonly occur during wound healing. However, JNK1 stress kinase signaling is the single most significant signal transduction network that is activated (P = 1.02E-14; Table 2).

Figure 11. Understanding the role of JNK1 stress signaling in high mammographic density and breast cancer risk. Here, we postulate that microenviornmental stressors (ROS; hydrogen peroxide; acidic pH), TGF-β, and FGF signaling networks all converge on the JNK1 stress kinase, in mammary stromal fibroblasts. Then, hyper-activated JNK-signaling drives the onset of a myofibroblastic phenotype, characterized by chronic inflammation, stemness, and catabolic metabolism. This, in turn, leads to local fibrosis and high mammographic density. As such, HD fibroblasts with activated JNK1 signaling would generate a pro-tumorigenic environment, similar to what is currently observed in cancer-associated fibroblasts (CAFs) and the tumor stroma. HD fibroblasts would provide a "pre-fertilized" local microenvironment (the soil), for the successful engraftment and expansion of epithelial cancer cells (the seeds). Thus, "stromal" cancer prevention would involve the use of available JNK-inhibitors in new clinical trials, to reverse or prevent the HD fibroblast phenotype. The clinical response to JNK-therapy could be monitored by using FDG-PET-imaging, which allows the functional visualization of fibrotic and inflammatory areas of dense breast tissue.