Decreased differentiation capacity and altered expression of extracellular matrix components in irradiation-mediated senescent human breast adipose-derived stem cells.

Radiotherapy is widely used for the treatment of breast cancer. However, we have shown that ionizing radiation can provoke premature senescence in breast stromal cells. In particular, breast stromal fibroblasts can become senescent after irradiation both in vitro and in vivo and they express an inflammatory phenotype and an altered profile of extracellular matrix components, thus facilitating tumor progression. Adipose-derived stem cells (ASCs) represent another major component of the breast tissue stroma. They are multipotent cells and due to their ability to differentiate in multiple cell lineages they play an important role in tissue maintenance and repair in normal and pathologic conditions. Here, we investigated the characteristics of human breast ASCs that became senescent prematurely after their exposure to ionizing radiation. We found decreased expression levels of the specific mesenchymal cell surface markers CD105, CD73, CD44, and CD90. In parallel, we demonstrated a significantly reduced expression of transcription factors regulating osteogenic (i.e., RUNX2), adipogenic (i.e., PPARγ), and chondrogenic (i.e., SOX9) differentiation; this was followed by an analogous reduction in their differentiation capacity. Furthermore, they overexpress inflammatory markers, that is, IL-6, IL-8, and ICAM-1, and a catabolic phenotype, marked by the reduction of collagen type I and the increase of MMP-1 and MMP-13 expression. Finally, we detected changes in proteoglycan expression, for example, the upregulation of syndecan 1 and syndecan 4 and the downregulation of decorin. Notably, all these alterations, when observed in the breast stroma, represent poor prognostic factors for tumor development. In conclusion, we showed that ionizing radiation-mediated prematurely senescent human breast ASCs have a decreased differentiation potential and express specific changes adding to the formation of a permissive environment for tumor growth.

Previous chronic exogenous glucocorticoid administration in vivo does not affect functional characteristics and cellular lifespan of human skin fibroblasts in vitro.

Excess of glucocorticoids (GCs) has been reported to lead to skin atrophy and impaired wound healing. The present study investigates whether human skin fibroblasts suffer permanent damages due to a long-term exposure to GC excess. Fibroblasts obtained from patients being under GC treatment for periods over one year were cultured under standard conditions in vitro, and studied regarding pivotal parameters involved in skin homeostasis and aging, i.e. collagen production, cell proliferation, and cellular replicative lifespan. No statistical differences were observed regarding these functions compared to those of normal human skin fibroblasts. Furthermore, no differences between normal and patient-derived cells were observed regarding their sensitivity to a supra-physiological cortisol concentration. In conclusion, the prolonged exposure of human skin fibroblasts in vivo to high concentrations of exogenously-administered GC does not lead to persistent adverse effects on their physiology.

Down-Regulation of the Proteoglycan Decorin Fills in the Tumor-Promoting Phenotype of Ionizing Radiation-Induced Senescent Human Breast Stromal Fibroblasts.

Down-regulation of the small leucine-rich proteoglycan decorin in the stroma is considered a poor prognostic factor for breast cancer progression. Ionizing radiation, an established treatment for breast cancer, provokes the premature senescence of the adjacent to the tumor stromal fibroblasts. Here, we showed that senescent human breast stromal fibroblasts are characterized by the down-regulation of decorin at the mRNA and protein level, as well as by its decreased deposition in the pericellular extracellular matrix in vitro. Senescence-associated decorin down-regulation is a long-lasting process rather than an immediate response to γ-irradiation. Growth factors were demonstrated to participate in an autocrine manner in decorin down-regulation, with bFGF and VEGF being the critical mediators of the phenomenon. Autophagy inhibition by chloroquine reduced decorin mRNA levels, while autophagy activation using the mTOR inhibitor rapamycin enhanced decorin transcription. Interestingly, the secretome from a series of both untreated and irradiated human breast cancer cell lines with different molecular profiles inhibited decorin expression in young and senescent stromal fibroblasts, which was annulled by SU5402, a bFGF and VEGF inhibitor. The novel phenotypic trait of senescent human breast stromal fibroblasts revealed here is added to their already described cancer-promoting role via the formation of a tumor-permissive environment.

Prior chronic in vivo glucocorticoid excess leads to an anabolic phenotype and an extension of cellular life span of skin fibroblasts in vitro.

Intense stress can be detrimental for tissue homeostasis and accelerates aging. On the other hand, repeated mild stresses can have beneficial and even life-prolonging effects. Hypersecretion of glucocorticoids (GCs) represents the major hormonal response to stress. However, besides its life-sustaining role, GC excess can promote a "catabolic" phenotype. Accordingly, we have studied the effect of long-lasting exposure to high GC levels in vivo on several parameters of tissue homeostasis, as well as cellular senescence, in cells removed from the high-GC milieu in vivo and then cultured in vitro. To this end, we have used human skin fibroblasts from (a) Cushing's syndrome patients that are characterized by chronic endogenous GC excess and (b) patients treated with exogenous GC administration. Interestingly, when Cushing's syndrome fibroblasts were cultured in vitro under standard conditions they express an "anabolic" phenotype, i.e., they restore their ability for collagen synthesis, secrete reduced levels of metalloproteases, and have an increased proliferative capacity and contractility. Furthermore, these cells exhibit a significant extension of their proliferative life span, while they respond better to exogenous stress by producing significantly higher levels of heat-shock protein-70 (HSP70). In addition, preliminary results with fibroblasts from patients subjected to chronic exogenous GC administration indicate that they express a similar behavior in vitro, at least with regard to the restoration of collagen expression. These data suggest that prior exposure to elevated GC concentrations is not associated with persisting adverse effects on fibroblasts and may also have a beneficial outcome in some aspects of cell physiology, including longevity in vitro.