High AUF1 level in stromal fibroblasts promotes carcinogenesis and chemoresistance and predicts unfavorable prognosis among locally advanced breast cancer patients.

BACKGROUND: Locally advanced breast cancer (LABC), the most aggressive form of the disease, is a serious threat for women's health worldwide. The AU-rich RNA-binding factor 1 (AUF1) promotes the formation of chemo-resistant breast cancer stem cells. Thereby, we investigated the power of AUF1 expression, in both cancer cells and their stromal fibroblasts, as predictive biomarker for LABC patients' clinical outcome following neoadjuvant treatment. METHODS: We have used immunohistochemistry to assess the level of AUF1 on formalin-fixed paraffin-embedded tissues. Immunoblotting was utilized to show the effect of AUF1 ectopic expression in breast stromal fibroblasts on the expression of various genes both in vitro and in orthotopic tumor xenografts. Cytotoxicity was evaluated using the WST1 assay, while a label-free real-time setting using the xCELLigence RTCA technology was utilized to assess the proliferative, migratory and invasive abilities of cells. RESULTS: We have shown that high AUF1 immunostaining (≥ 10%) in both cancer cells and their adjacent cancer-associated fibroblasts (CAFs) was significantly associated with higher tumor grade. Kaplan-Meier univariate analysis revealed a strong correlation between high AUF1 level in CAFs and poor patient's survival. This correlation was highly significant in patients with triple negative breast cancer, who showed poor disease-free survival (DFS) and overall survival (OS). High expression of AUF1 in CAFs was also associated with poor OS of ER+/Her2- patients. Similarly, AUF1-positive malignant cells tended to be associated with shorter DFS and OS of ER+/Her2+ patients. Interestingly, neoadjuvant therapy downregulated AUF1 to a level lower than 10% in malignant cells in a significant number of patients, which improved both DFS and OS. In addition, ectopic expression of AUF1 in breast fibroblasts activated these cells and enhanced their capacity to promote, in an IL-6-dependent manner, the epithelial-to-mesenchymal transition and stemness processes. Furthermore, these AUF1-expressing cells enhanced the chemoresistance of breast cancer cells and their growth in orthotopic tumor xenografts. CONCLUSIONS: The present findings show that the CAF-activating factor AUF1 has prognostic/predictive value for breast cancer patients and could represent a great therapeutic target in order to improve the precision of cancer treatment.

High DNMT1 Expression in Stromal Fibroblasts Promotes Angiogenesis and Unfavorable Outcome in Locally Advanced Breast Cancer Patients.

Background: Active breast cancer-associated fibroblasts (CAFs) play a leading role in breast carcinogenesis through promoting angiogenesis and resistance to therapy. Consequently, these active stromal cells have significant influence on patient outcome. Therefore, we explored here the role of the DNA methyltransferase 1 (DNMT1) protein in CAF-dependent promotion of angiogenesis as well as the prognostic power of DNMT1 level in both cancer cells and their adjacent CAFs in locally advanced breast cancer patients. Methods: We applied immunohistochemistry to evaluate the level of DNMT1 in breast cancer tissues and their adjacent normal counterparts. Quantitative RT-PCR and immunoblotting were performed to investigate the role of DNMT1 in regulating the expression of pro-angiogenic genes in active CAFs and also their response to the DNMT1 inhibitors decitabine (DAC) as well as eugenol. Results: We have shown that DNMT1 controls the pro-angiogenic potential of CAFs both in vitro and in vivo through positive regulation of the expression/secretion of 2 important pro-angiogenic factors VEGF-A and IL-8 as well as their upstream effectors mTOR and HIF-1α. To confirm this, we have shown that these DNMT1-related pro-angiogenic effects were suppressed by 2 DNMT1 inhibitors decitabine and eugenol. Interestingly, in a cohort of 100 tumors from locally advanced breast cancer patients (LABC), we have shown that high expression of DNMT1 in tumor cells and their adjacent stromal fibroblasts is correlated with poor survival of these patients. Conclusion: DNMT1 upregulation in breast stromal fibroblasts promotes angiogenesis via IL-8/VEGF-A upregulation, and correlates well with poor survival of LABC patients.

Clinical and functional significance of tumor/stromal ATR expression in breast cancer patients.

BACKGROUND: Most breast cancer-associated fibroblasts (CAFs) are active and important cancer-promoting cells, with significant impact on patient prognosis. Therefore, we investigated here the role of the protein kinase ATR in breast stromal fibroblasts in the prognosis of locally advanced breast cancer patients. METHODS: We have used immunohistochemistry to assess the level of ATR in breast cancer tissues and their adjacent normal tissues. Immunoblotting as well as quantitative RT-PCR were utilized to show the role of breast cancer cells and IL-6 as well as AUF-1 in downregulating ATR in breast stromal fibroblasts. Engineered human breast tissue model was also used to show that ATR-deficient breast stromal fibroblasts enhance the growth of breast cancer cells. RESULTS: We have shown that the protein kinase ATR is downregulated in cancer cells and their neighboring CAFs in breast cancer tissues as compared to their respective adjacent normal tissues. The implication of cancer cells in ATR knockdown in CAFs has been proven in vitro by showing that breast cancer cells downregulate ATR in breast fibroblasts in an IL-6/STAT3-dependent manner and via AUF-1. In another cohort of 103 tumors from locally advanced breast cancer patients, we have shown that absence or reduced ATR expression in tumoral cells and their adjacent stromal fibroblasts is correlated with poor overall survival as well as disease-free survival. Furthermore, ATR expression in CAFs was inversely correlated with tumor recurrence and progression. CONCLUSION: ATR downregulation in breast CAFs is frequent, procarcinogenic, and correlated with poor patient survival.