Triple-negative breast cancer (TNBC) is the most heterogeneous and aggressive breast tumor subtype defined by absence of receptor for estrogen, progesterone, or HER2. However, the biologic mechanism for TNBC phenotype is still unclear. Here, we show that expression of Calsequestrin 2 (CASQ2), a Ca2+-binding protein, correlates with increase of proliferation, migration, and invasion, suggesting that intracellular Ca2+ may contribute to tumor growth and metastatic phenotype. CASQ2 is the main Ca2+-binding protein inside the sarcoplasmic reticulum of cardiomyocytes. CASQ2 forms a complex with ryodine receptor 2 (RyR2) luminal calcium release channel in cardiac muscle. Ca2+ is a sequestor and regulator of diverse cellular processes, and specific Ca2+ channels play important roles in cell proliferation and invasiveness of cancers. To know the role of CASQ2 TNBC cells, we established CASQ2-overexpressing stable cells in Hs578T (Hs578T-CASQ2) using retrovirus. Stimulation with caffeine triggered the remarkable increase of intracellular calcium in Hs578T-CASQ2 cells; in addition, the basal level of calcium in cells had much higher amount of Hs578T-CASQ2 than Hs578T. By contrast, calcium chelator BAPTM/AM blocked CASQ2-induced calcium release. Hs578T-CASQ2 cells showed higher level of proliferation, migration and invasion rate compared to Hs578T, which indicated that overexpression of CASQ2 related with cellular functions. We also found that CASQ2 overexpression elevates extracellular signal-related kinase (ERK) expression. In epidermal growth factor (EGF)-treated cells, Hs578T-CASQ2 cells had higher phosphorylated ERK compared to Hs578T, leading to the expression of epithelial-mesenchymal transition (EMT) marker, vimentin. These results indicate that CASQ2 overexpression increases the level of calcium and induces cell proliferation, migration and invasion through ERK signaling. Our findings from this study show a possible cause of migration and invasiveness in breast cancer cells. Taken together, these findings demonstrate that CASQ2 could be a new therapeutic target for breast cancer. Citation Format: Ju Hee Kim, Bok sil Hong, Woohang Heo, Jong Min Han, Wonsik Han, Dong-Young Noh, Hyeong-Gon Moon. Calsequestrin 2 regulates proliferation, migration, and invasion in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 33.
Triple negative breast cancer (TNBC) patients often develop metastases in visceral organs including the liver but the detailed molecular mechanisms of TNBC liver metastasis is not clearly understood. In this study, we tried to dissect the process of pre-metastatic niche formation in liver by using patient-derived xenograft (PDX) models of TNBC with different metastatic propensity. RNA sequencing of TNBC PDX models that successfully metastasized to liver showed up-regulation of Cx3cr1 gene in the liver microenvironment. In syngeneic breast cancer models, the Cx3cr1 up-regulation in liver preceded the development of cancer cell metastasis and was the results of recruitment of CX3CR1-expressing macrophages. The recruitment was induced by the CX3CL1 production from the liver endothelial cells and this CX3CL1-CX3CR1 signaling in the pre-metastatic niche resulted in up-regulation of MMP9 that promoted macrophage migration and cancer cell invasion. Additionally, our data suggest that the extra-cellular vesicles derived from the breast cancer cells induced the TNF-alpha expression in liver which leads to the CX3CL1 up-regulation. Lastly, the plasma CX3CL1 levels in 155 breast cancer patients were significantly associated with development of liver metastasis. Implications: Our data provides previously unknown cascades regarding the molecular education of pre-metastatic niche in liver for TNBC.
<p>Supplementary Table 1. Information about patients derived xenograft models and derived patient. Supplementary Table 2. Ontology of differentially expressed genes in metastasis-harboring liver of PDX models. Supplementary Table 3. Differentially expressed genes in liver and lung of metastasis-harboring PDX models. Supplementary Table 4. Human ratio in lung and liver of additional PDX models identified by RNA sequencing. Supplementary Table 5. Gene ontology of co-expressed genes with Cx3cr1 in liver of PDX models identified by RNA sequencing. Supplementary Table 6. Top ranked genes having significant correlation with Cx3cr1 in liver of PDX models identified by RNA sequencing. Supplementary Table 7. Demographic and clinical characteristics of all patients. Supplementary Table 8. Information about used primer sequences for quantitative real-time PCR Supplementary Figure 1. Liver and lung H&E staining pictures of eight nonmetastatic PDX models a. Microscopic images of non-metastasis harboring PDX mouse model's liver (left) and lung (right) taken at 400x magnification. Supplementary Figure 2. Immunohistochemistry about CX3CR1 in liver and lung of breast cancer metastasis patients. Supplementary Figure 3. Hepatic metastasis after four weeks after orthotopic injection of 4T1 breast cancer cells identified by H&E staining. Supplementary Figure 4. Identification of pre-metastatic niche in liver tissues of 4T1 bearing mice a. mRNA expression of inflammation associated genes identified by real-time PCR in liver tissues of 4T1 bearing mice. Supplementary Figure 5. Identification of CX3CR1 positive cell population. Supplementary Figure 6. FACS analysis with anti-CX3CR1 of Raw264.7 macrophage cell lines. Supplementary Figure 7. Trans-well migration assay of BMDM with or without CX3CL1. Supplementary Figure 8. Mmp9 mRNA expression having significant correlation with Cx3cr1 mRNA expression in liver of PDX models. Supplementary Figure 9. MMP9 expression of macrophages with CX3CL1 treatment. Supplementary Figure 10. Signaling pathway of CX3CL1 treated macrophages. Supplementary Figure 11. Splenic injection of 4T1 cells inducing hepatic metastasis within two weeks in most cases. Supplementary Figure 12. mRNA expression about Cx3cr1 and Mmp9 in liver of vehicle or AZD8797 treated BALB/C mice identified by real-time PCR. Supplementary Figure 13. Correlation plot of Tnfa and Cx3cr1 mRNA expression in liver and lung of PDX models. Supplementary Figure 14. EVs isolation of CD63-mRuby-4T1.</p>
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