Osteoprotegerin (OPG) mediates the anti-carcinogenic effects of normal breast fibroblasts and targets cancer stem cells through inhibition of the ß-catenin pathway.

Normal breast fibroblasts (NBFs) support and maintain the architecture of the organ, and can also suppress tumorigenesis. However, the mechanisms involved are not fully understood. We have shown here that NBFs suppress breast carcinogenesis through secretion of osteoprotegerin (OPG), a soluble decoy receptor for the Receptor Activator of NF-κB ligand (RANKL). Indeed, NBFs and human recombinant OPG (rOPG), suppressed breast cancer cells proliferation and motility through inhibition of the epithelial-to-mesenchymal transition (EMT) process both in vitro and in vivo. Additionally, rOPG inhibited the IL-6/STAT3 and NF-κB pathways as well as the OPG gene, which turned out to be STAT3-regulated. This was confirmed using denosumab, a RANKL-targeted antibody, which also inhibited NF-κB, down-regulated OPG and repressed EMT in breast cancer cells grown in 2D and 3D. Importantly, both rOPG and denosumab targeted cancer stem cells (CSCs). This was mediated through inhibition of the CSC-related pathway ß-catenin. Moreover, rOPG reduced tumor growth and inhibited breast CSC biomarkers in orthotopic humanized breast tumors. Therefore, normal mammary fibroblasts can suppress carcinogenesis through OPG, which constitutes great potential as preventive and/or therapeutic molecule for breast carcinomas.

Let-7b inhibits cancer-promoting effects of breast cancer-associated fibroblasts through IL-8 repression.

Cancer-associated fibroblasts (CAFs) are major players in the development and spread of breast carcinomas through non-cell-autonomous signaling. These paracrine effects are under the control of several genes and microRNAs. We present here clear evidence that let-7b, a tumor suppressor microRNA, plays key roles in the persistent activation of breast stromal fibroblasts and their functional interplay with cancer cells. We have first shown that let-7b is down-regulated in CAFs as compared to their corresponding normal adjacent fibroblasts, and transient specific let-7b inhibition permanently activated breast fibroblasts through induction of the IL-6-related positive feedback loop. More importantly, let-7b-deficient cells promoted the epithelial-to-mesenchymal transition process in breast cancer cells in an IL-8-dependent manner, and also enhanced orthotopic tumor growth in vivo. On the other hand, overexpression of let-7b by mimic permanently suppressed breast myofibroblasts through blocking the positive feedback loop, which inhibited their paracrine pro-carcinogenic effects. Furthermore, we have shown that let-7b negatively controls IL-8, which showed higher expression in the majority of CAF cells as compared to their adjacent normal counterparts, indicating that IL-8 plays a major role in the carcinoma/stroma cross-talk. These findings support targeting active stromal fibroblasts through restoration of let-7b/IL-8 expression as a therapeutic option for breast carcinomas.

The inflammatory/cancer-related IL-6/STAT3/NF-κB positive feedback loop includes AUF1 and maintains the active state of breast myofibroblasts.

The IL-6/STAT3/NF-κB positive feedback loop links inflammation to cancer and maintains cells at a transformed state. Similarly, cancer-associated myofibroblats remains active even in absence of cancer cells. However, the molecular basis of this sustained active state remains elusive. We have shown here that breast cancer cells and IL-6 persistently activate breast stromal fibroblasts through the stimulation of the positive IL-6/STAT3/NF-κB feedback loop. Transient neutralization of IL-6 in culture inhibited this signaling circuit and reverted myofibrobalsts to a normalized state, suggesting the implication of the IL-6 autocrine feedback loop as well. Importantly, the IL-6/STAT3/NF-κB pro-inflammatory circuit was also active in cancer-associated fibroblasts isolated from breast cancer patients. Transient inhibition of STAT3 by specific siRNA in active fibroblasts persistently reduced the level of the RNA binding protein AUF1, blocked the loop and normalized these cells. Moreover, we present clear evidence that AUF1 is also part of this positive feedback loop. Interestingly, treatment of breast myofibroblasts with caffeine, which has been previously shown to persistently inhibit active breast stromal fibroblasts, blocked the positive feedback loop through potent and sustained inhibition of STAT3, AKT, lin28B and AUF1. These results indicate that the IL-6/STAT3/NF-κB positive feedback loop includes AUF1 and is responsible for the sustained active status of cancer-associated fibroblasts. We have also shown that normalizing myofibroblasts, which could be of great therapeutic value, is possible through the inhibition of this procarcinogenic circuit.

The cytokine IL-6 reactivates breast stromal fibroblasts through transcription factor STAT3-dependent up-regulation of the RNA-binding protein AUF1.

The development and spread of mammary carcinomas require synergetic interplay between tumor cells and their microenvironment through paracrine secretions, which are still not well defined. We have shown here that interleukin-6 (IL-6), either recombinant or secreted from highly invasive breast cancer cells, down-regulates the tumor suppressor proteins p16(INK4A), p21(WAF1), and p53 and activates breast stromal fibroblasts in a paracrine manner. The formation of myofibroblasts requires p16(INK4A) down-regulation and the activation of the JAK2/STAT3 pathway. Indeed, the transcription factor STAT3 positively controls the expression of the three major myofibroblast markers, SDF-1, α-smooth muscle actin (α-SMA), and TGF-ß1, and mediates IL-6-related down-regulation of p16(INK4A), p21(WAF1), and p53 as well as the activation of stromal fibroblasts. Importantly, these effects were mediated through STAT3-dependent up-regulation of the mRNA-binding protein AUF1, whose promoter contains three canonical STAT3 binding sites. AUF1 binds the SDF-1, α-SMA, TGF-ß1, and IL-6 mRNAs and reduces their turnover. Consequently, specific AUF1 down-regulation inhibits IL-6-dependent activation of breast stromal fibroblasts, whereas AUF1 ectopic expression of p37(AUF1) activated these cells and enhanced their paracrine induction of epithelial-to-mesenchymal transition in breast cancer cells, which shows a non-cell-autonomous oncogenic function of AUF1. Together, these results demonstrate a major role of IL-6 in activating breast stromal fibroblasts through STAT3-dependent AUF1 induction.

Curcumin triggers p16-dependent senescence in active breast cancer-associated fibroblasts and suppresses their paracrine procarcinogenic effects.

Activated cancer-associated fibroblasts (CAFs) or myofibroblasts not only facilitate tumor growth and spread but also affect tumor response to therapeutic agents. Therefore, it became clear that efficient therapeutic regimens should also take into account the presence of these supportive cells and inhibit their paracrine effects. To this end, we tested the effect of low concentrations of curcumin, a pharmacologically safe natural product, on patient-derived primary breast CAF cells. We have shown that curcumin treatment upregulates p16(INK4A) and other tumor suppressor proteins while inactivates the JAK2/STAT3 pathway. This reduced the level of alpha-smooth muscle actin (α-SMA) and the migration/invasion abilities of these cells. Furthermore, curcumin suppressed the expression/secretion of stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), MMP-9, and transforming growth factor-ß, which impeded their paracrine procarcinogenic potential. Intriguingly, these effects were sustained even after curcumin withdrawal and cell splitting. Therefore, using different markers of senescence [senescence-associated ß-galactosidase (SA-ß-gal) activity, Ki-67 and Lamin B1 levels, and bromodeoxyuridine incorporation], we have shown that curcumin markedly suppresses Lamin B1 and triggers DNA damage-independent senescence in proliferating but not quiescent breast stromal fibroblasts. Importantly, this curcumin-related senescence was p16(INK4A)-dependent and occurred with no associated inflammatory secretory phenotype. These results indicate the possible inactivation of cancer-associated myofibroblasts and present the first indication that curcumin can trigger DNA damage-independent and safe senescence in stromal fibroblasts.

p16INK4A represses breast stromal fibroblasts migration/invasion and their VEGF-A-dependent promotion of angiogenesis through Akt inhibition.

Stromal fibroblasts, the most abundant and probably the most active cellular component of breast cancer-associated stroma, become active and promote angiogenesis through paracrine effects. However, it still unclear how these processes are regulated. Here, we have shown that down-regulation of the tumor suppressor p16(INK4A) protein enhances the migration/invasion abilities of breast stromal fibroblasts, which form dendritic network of extensions into matrigel. Furthermore, we present clear evidence that p16(INK4A) represses the expression/secretion of the proangiogenesis protein vascular endothelial growth factor A (VEGF-A). Consequently, p16(INK4A)-deficient breast stromal fibroblasts and mouse embryonic fibroblasts enhanced endothelial cell differentiation into capillary-like structures in a paracrine manner. This effect was suppressed by adding bevacizumab, a specific VEGF-A inhibitor. Additionally, p16(INK4A)-defective mouse embryonic fibroblasts enhanced angiogenesis in breast cancer xenografts in mice. Furthermore, we have shown that p16(INK4A) suppresses the Akt/mammalian target of rapamycin (mTOR) signaling pathway and its downstream effector hypoxia-inducible factor 1-alpha (HIF-1α), which transactivates VEGF-A. Consequently, Akt inactivation suppressed both the p16(INK4A)-dependent autocrine effect on fibroblast migration/invasion and the paracrine effect on angiogenesis, showing the important role of this protein kinase in mediating the various effects related to p16(INK4A) deficiency. These results indicate that p16(INK4A) is an efficient inhibitor of the migration/invasion abilities of breast stromal fibroblasts and also their paracrine proangiogenic effects, through inhibition of Akt. Therefore, pharmacologic restoration of p16(INK4A) level in stromal fibroblasts may be exploited as therapeutic strategy to help eradicate tumor cells and/or prevent their recurrence, through suppressing cell non-autonomous procarcinogenic mediators.