Importance of OPG, RANKL and TRAIL in breast tumor growth.

MARTINEZ LM. ; LABOVSKY V,; FERNANDEZ VALLONE VB.; OTAEGUI J; CHASSEING NA.

Orlando

Conferencia; Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy, Special Conference.; 2011

AACR

Accumulating evidence suggests that bone marrow (BM) mesenchymal stem cells (MSC) can be recruited into the tumor stroma, where they play an important role in the tumor progression. Our recent studies have shown that the MSC with high capacity cloning and plasticity migrate to breast tumor at the initial stages of the disease. Thus, within the tumor stroma, they are able to release growth factors and chemokines but they also turn into tumor-associated fibroblasts, differentiate an epithelial phenotype and stimulate the epithelial-mesenchymal transition. These properties contribute to tumor growth, invasion and metastasis. Recently, it has been known the importance of OPG-RANKL-TRAIL in the regulation of survival and migration of breast cancer cells (BCC). So, this study was performed to evaluated to production of OPG, RANKL, TRAIL as well as, RANK and TRAIL-Receptors (R) 1-4 in 2 human BCC lines: MCF-7 and MDA-MB-231. Simultaneously, we measured the OPG, RANKL and TRAIL levels in the conditioned mediums (CM) of colony forming unit fibroblast (CFU-F) and MSC cultures from BM of healthy volunteers (HV). At the end, we evaluated the effect of these CM over the proliferation and apoptosis of both BCC lines. Methodology: soluble factors were studied by ELISA in the CM of CFU-F (day 14) and MSC (3rd passage, 240 cells/cm2) cultures from BM of 10 HV, as well as, in the CM of BCC of both lines arrested and stimulated (10% FBS) during 48hs. The expression of them and their R in BCC were analyzed by immunofluorescence and immunocytochemistry assay. Proliferation by MTS assay: after arrest, BCC were incubated for 48hs with 100% or 50% of CM (CFU-F and MSC cultures) with or without 1.25% FBS. Apoptosis by flow cytometry with Anexin-V and Tunel: after the arrest, BCC were incubated for 24hs with 50% of these CM in TRAIL presence or not. Results: RANKL and TRAIL were bellow 31.25pg/ml in the BM-CM. However, in previous studies we have found that 100% of HV-MSC expressed membrane RANKL. The OPG values (X +SE, pg/ml) were: CFU-F(14day)=1,422+/-264 and MSC=1,269+/-161. In addition, 100% of BCC of both lines expressed OPG, cytoplasmic RANKL, membrane RANKL, TRAIL, RANK and TRAIL-R1 and R4, 30-50% TRAIL-R2 and 40-55% TRAIL-R3. Significant difference in the OPG values was found between both BCC lines ( X+SE, pg/ml/106cells): MCF-7=2,782+/-343 and MDA-MB-231=1,081+/-204. The RANKL and TRAIL values were bellow 31.25pg/ml in the BCC-CM. No proliferative effect was observed by any of the treatments over any of the BCC lines. We did observe that, when MDA-MB-231 cells were treated with CM of CFU-F (day 14) in the TRAIL presence, the % of cells in apoptosis was significantly lower compared with the control value (early and late apoptosis evaluated by flow cytometry: p=0.0015 and p=0.0339, respectively, and total apoptosis by Tunel: p=0.0270). In contrast, MSC-CM had a less anti-apoptotic effect over these BCC. MCF-7 cells did not respond to TRAIL effect. There was no association between the expression decoy receptors of TRAIL (TRAIL-R3-4) and TRAIL resistance in MCF-7 cells. Conclusion: the highest OPG levels released in BCC could be associated with hormonal receptors. Moreover, for our data of BM-CM, we think that the MSC could release OPG when migrate into breast tumor. This OPG not only is a pro-angiogenic factor, but also, it is a soluble TRAIL receptor. So, it may favor the tumor growth. Another interesting event is the apparently association between RANK present in BCC and the membrane RANKL in MSC of the tumor microenvironment, interaction that promotes the proliferation and migration of tumor cells, especially to bone.