The formation of a stiff lump in soft tissue is one of the most common symptoms that people associate with cancer. This is a sign of abnormal tissue stiffness, which derives from changes in the extracellular matrix (ECM) driven by cancer and stromal cells. This physical property is known to be a consequence and promoter of tumor development. While it is well established that high stiffness promotes invasive behavior of cancer cells, the role of abnormal stiffness on endothelial cells (ECs) is still largely unknown. Here we use unbiased mass spectrometry (MS)-based proteomics to ascertain how increased tissue stiffness affects ECs, and the impact of these alterations in cancer. To do this we used an in vitro system of polyacrylamide gels representing ECM stiffness of normal and a tumor tissue. Using high resolution MS combined with stable isotope labelling of amino acids in cell culture (SILAC), we quantified the global proteome of ECs cultured at each ECM stiffness and unveiled that many cell adhesion proteins were upregulated at tumor stiffness, including N-Cadherin and the matricellular protein CCN1. By silencing and overexpressing CCN1 in ECs, we demonstrated that stiffness-induced CCN1 induces a β-catenin mediated increase in N-Cadherin expression. N-Cadherin is known to play a key role in trans-endothelial migration of cancer cells through the blood vessels, and we show that CCN1 induces cancer cell adhesion to ECs via N-Cadherin in vitro. This indicates that CCN1 can increase cancer cell metastasis by aiding the exit of cancer cells from the primary tumor and entry at distant sites. We confirmed this hypothesis in vivo, by using C57Bl/6 Ccn1loxP/loxP mice and syngeneic B16 melanoma model. Ccn1 was acutely knocked out in the vasculature by injecting the Tat-Cre fusion protein intravenously, and this showed that knocking out Ccn1 decreased the number of circulating tumor cells and subsequent metastases in the lungs. Thus, CCN1 loss in the vasculature can decrease cancer cell intravasation. Our work determines an unprecedented mechanism through which CCN1 in the stromal cells promotes invasion of cancer cells. This complements the findings that CCN1 can induce invasion in the cancer cells, and provides evidence that targeting CCN1 therapeutically in the clinic may decrease the spread of cancer from the perspective of the endothelial cells.
This abstract is also presented as Poster C34.
Citation Format: Steven Reid, Jens Serneels, Lisa J. Neilson, Anne-Theres Henze, Juan R. Hernández-Fernaud, Ralf H. Adams, Karen Blyth, Massimiliano Mazzone, Sara Zanivan. Increased matrix stiffness induces CCN1 mediated upregulation of N-Cadherin in endothelial cells: Implications for cancer cell metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR11.