Abstract 4886: Caffeine normalizes breast stromal fibroblasts and suppresses their pro-carcinogenic effects.

Abstract Breast cancer is the leading cause of morbidity and second-leading cause of death in women worldwide. Breast stromal fibroblasts, the most abundant and the most reactive cells of the tumor stroma, actively promote tumor growth, invasion, migration, angiogenesis and metastasis through elevated secretion of several factors including SDF1, MMP-2 and vascular endothelial growth factor A (VEGF-A). These secretions are under the control of several tumor suppressor genes such as p16, p53 and CAV-1, which were fond to be down-regulated in most active fibroblasts. Therefore, our major aim in this study was to study the potential use of caffeine to up-regulate these tumor suppressor genes and hence reduces the secretion of these cancer-promoting factors. We have shown that low doses of caffeine up-regulate the tumor suppressor proteins p16, p21, p53 and CAV-1 in breast stromal fibroblasts. Consequently, the expression and secretion of MMP-2 and VEGF-A was reduced following caffeine treatment. Moreover, caffeine reduced the expression of SDF-1, TGF-β and α-SMA, three important markers of active fibroblast, suggesting that caffeine has the ability of “normalizing” stromal fibroblasts. To further confirm this, we have shown that caffeine suppresses stromal fibroblast-related enhancement of invasion/migration of breast cancer cells. Importantly, this effect was mediated through caffeine-dependent inactivation of the two major invasion/migration protein kinases Akt and Erk, through up-regulating PTEN in breast stromal fibroblasts. Furthermore, caffeine suppressed the expression/secretion of VEGF-A in breast stromal fibroblasts and their paracrine enhancement of angiogenesis in vitro. This effect was mediated through inhibition of the VEGF-A activator HIF-1α. Therefore, we have clearly shown here that caffeine, a natural and pharmacologically safe product, has the potential to normalize breast stromal fibroblasts and consequently suppresses their paracrine procarcinogenic effects. Citation Format: Mysoon M. Al Ansari, Abdelilah Aboussekhra. Caffeine normalizes breast stromal fibroblasts and suppresses their pro-carcinogenic effects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4886. doi:10.1158/1538-7445.AM2013-4886</jats:p

Caffeine mediates sustained inactivation of breast cancer-associated myofibroblasts via up-regulation of tumor suppressor genes.

BACKGROUND: Active cancer-associated fibroblasts (CAFs) or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects. METHODOLOGY/PRINCIPAL FINDINGS: We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2), and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/-migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine. CONCLUSION/SIGNIFICANCE: The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts

Abstract P4-04-26: Down-regulation of p16INK4a inhibits miR-146b-5p and modulates IL-6 in breast stromal fibroblasts

Abstract Breast cancer is a major health problem that threatens millions of women’s lives each year worldwide. Cancer-associated fibroblasts (CAFs), which constitute the major component of the tumor stroma, have been reported to actively contribute to tumor cells proliferation and invasion. Recently, we have shown down-regulation of the tumor suppressor p16INK4a protein in breast cancer-associated fibroblasts. Moreover, p16INK4a deficiency led to the activation of the stromal fibroblasts, which express/secrete elevated levels of IL-6, a major player in breast carcinogenesis. We have shown here that p16INK4a negatively regulates the IL-6 expression and secretion in breast stromal fibroblasts. Furthermore, we have shown that IL-6 is playing a major role in mediating the paracrine pro-carcinogenic effect of p16-deficient fibroblasts. We have also shown that p16INK4a inhibits the IL-6 expression in a miRNA-146b-5p-dependent manner. Importantly, we present clear evidence that miR-146b-5p inhibition activates breast stromal fibroblast. Indeed, miR-146b-5p inhibition increased the migration/invasion abilities of breast stromal fibroblasts, and the paracrine effect of these cells on the migration/invasion of breast cancer cells. Furthermore, miR-146b-5p-deficient stromal fibroblasts triggered epithelial to mesenchymal transition in breast cancer cells in a paracrine manner. In addition, we have shown that miR-146b-5p is down-regulated in CAFs as compared to their adjacent counterpart fibroblasts. These results indicate that p16INK4a negatively regulates IL-6 through the activation of miR-146b-5p, which plays a major role in repressing breast stromal fibroblasts and inhibiting their pro-carcinogenic effects. This indicates that miR-146b-5p has cell-non-autonomous tumor suppressor function. Therefore, this miRNA could be of great therapeutic value. Citation Format: Mysoon M Al-Ansari, Abdelilah Aboussekhra. Down-regulation of p16INK4a inhibits miR-146b-5p and modulates IL-6 in breast stromal fibroblasts [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-26.</jats:p

Osteoprotegerin (OPG) Upregulation Activates Breast Stromal Fibroblasts and Enhances Their Pro-Carcinogenic Effects through the STAT3/IL-6 Signaling

Breast carcinomas are composed of cancer cells surrounded by various types of non-cancer cells such as fibroblasts. While active cancer-associated fibroblasts (CAFs) support tumor initiation and progression, quiescent breast stromal fibroblasts (BSFs) inhibit these effects through various cytokines such as osteoprotegerin (OPG). We showed here that OPG is upregulated in CAFs as compared to their adjacent normal tumor counterpart fibroblasts. Interestingly, breast cancer cells can upregulate OPG in BSFs in an IL-6-dependent manner through the IL-6/STAT3 pathway. When upregulated by ectopic expression, OPG activated BSFs through the NF-&kappa;B/STAT3/AUF1 signaling pathway and promoted their paracrine pro-carcinogenic effects in an IL-6-dependent manner. In addition, this increase in the OPG level enhanced the potential of BSFs to promote the growth of humanized orthotopic tumors in mice. However, specific OPG knock-down suppressed active CAFs and their paracrine pro-carcinogenic effects. Similar effects were observed when CAF cells were exposed to the pure recombinant OPG (rOPG) protein. Together, these findings show the importance of OPG in the activation of stromal fibroblasts and the possible use of rOPG or inhibitors of the endogenous protein to target CAFs as precision cancer therapeutics

Caffeine inhibits the expression/secretion of SDF1, IL-6, MMP-2 and TGF-.

<p>CAF-180 cells were either sham-treated or challenged with caffeine for 1 hr. (A) Whole cell lysates were prepared and used for immunoblotting analysis using the indicated antibodies. (B) Histograms show the expression levels of the indicated proteins. The values were determined by densitometry and normalized against GAPDH. (C) Total RNA was prepared and used to assess the level of the indicated genes by qRT-PCR. The obtained values were normalized against β-actin. (D) Secreted levels of proteins were determined in SFCM by ELISA and shown in the histograms. Error bars represent means ± S.D. *: <i>p</i><0.05.</p