STAT3 is activated through paracrine signaling in the breast tumor microenvironment and is mediated through IL-6

1034

A growing number of human cancers have increased levels of activated Signal Transducers and Activators of Transcription (STAT) proteins. STAT3 is a major member of this family, and phosphorylated STAT3 (at tyrosine residue 705) is frequently found elevated within breast carcinomas. To date, however, it is unknown whether the tumor microenvironment plays a role in the activation of STAT3. We are studying the effects of paracrine signaling from breast cancer cells and breast cancer associated fibroblasts with increased p-STAT3 levels on their tumor microenvironment. Media was conditioned in the presence of breast cancer cells (MDA-MB-231 and MDA-MB-468) and breast cancer associated fibroblasts (BCF#4) with high levels of p-STAT3. This condition media was then used to treat breast cell lines without this increase in phosphorylation (MDA-MB-453, a breast cancer cell line, and MCF-10A, a non-cancer immortalized breast cell line). Western blot analysis demonstrated that soluble factor(s) secreted by MDA-MB-231, MDA-MB-468, or BCF#4 cell lines were sufficient to amplify p-STAT3 (Y705) levels within MDA-MB-453 and MCF-10A. This activation was observed by 30 minutes and persisted at least 24 hours after treatment. With this increase in p-STAT3, we also observed elevated levels of the anti-apoptotic protein Bcl-x_L, a downstream target of the STAT3 pathway. Treatment of MCF-10A with 10 μM JSI-124, a Jak/STAT3 inhibitor, blocked STAT3 activation after treatment with condition media. These results also prompted us to question which specific growth factors or cytokines were responsible for this boost in p-STAT3 (Y705). ELISA analysis confirmed low levels of IL-6 secretion by MCF-10A and MDA-MB-453 and high levels of IL-6 secretion by MDA-MB-231, MDA-MB-468, and BCF#4. IL-6 antibody was added to MDA-MB-231 condition media for one hour to neutralize the IL-6 in the media prior to addition to MDA-MB-453 or MCF-10A cells. Neutralization of IL-6 was sufficient to block increased levels of p-STAT3 (Y705) protein. Blocking gp130 (a major IL-6 receptor) through antibody neutralization also prevented increased STAT3 phosphorylation in MDA-MB-453 cells after MDA-MB-231 condition media treatment. These results demonstrate the importance of IL-6 as at least one essential paracrine signal for STAT3 activation within the breast tumor microenvironment. Understanding how IL-6 and other soluble factors may lead to STAT3 activation via the tumor microenvironment will provide new therapeutic targets for breast carcinomas and other cancers with elevated p-STAT3 levels.