Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell-conditioned media (TCM) from human TNBC cells (MDA-MB-231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible spontaneous metastases in lymph nodes (LN) and lungs within 4-5 weeks after orthotropic tumor inoculation. We have discovered that the TNBC tumor cells secrete large amounts of interleukin-6 (IL-6) that “educates” lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis.
In this report, using a human antibody array, we identified factors secreted by fibroblasts and macrophages upon induction by MDA-MB-231 TCM. We ranked the expression level of each factor by real time qRT-PCR and determined that interleukin 8 (IL-8) was the top candidate. We confirmed by ELISA that IL-8 secreted from either fibroblasts or macrophages treated with MDA-MB-231 TCM was upregulated compared to treatment with serum free media (SFM). Our data showed that the proliferation of MDA-MB-231 cells co-cultured with fibroblasts or macrophages was enhanced compared to monoculture. Furthermore, MDA-MB-231 cell migration, a key step in tumor metastasis, was promoted by CM from TCM-induced fibroblasts or macrophages. Knockdown of CXCR2, IL-8 receptor, expression by CRISPR-Cas9 system reduces MB231 cell proliferation and migration compared to wild type. In a mouse xenograft tumor model, the tumor growth of MB231-CXCR2-/- cell was significantly decreased. In addition, the incidence of thoracic metastasis of MB231-CXCR2-/- tumor was reduced compared to WT.
We found that the auto- and paracrine loop of IL-8 exists between TNBC cells and stroma, which results in enhanced IL-8 secretion from the stromal components. Significantly, inhibition of the IL-8 signaling pathway by Reparixin, an inhibitor of the IL-8 receptor CXCR1/2, abrogated MDA-MB-231 tumor growth and metastasis.
These findings implicate IL-8 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk with stromal components. Further, these studies suggest that IL-8 acts as a key regulator orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional antagonism of the IL-8 signaling pathway has the potential to circumvent TNBC breast cancer growth and metastasis.
Citation Format: Kideok Jin, Niranjan B. Pandey, Aleksander S. Popel. IL-8 signaling enhances TNBC growth and metastasis via crosstalk with stromal components [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5896. doi:10.1158/1538-7445.AM2017-5896