Women with metastatic breast cancer have a disheartening 5-year survival rate of only 28%. CREB3L1 (cAMP responsive element binding protein 3 like 1) is a transcription factor and tumor suppressor which is downregulated in ~ 30% of human breast cancers, with higher frequencies in more advanced metastatic breast tumors.

To identify new targets contributing to metastatic properties, we carried out a differential gene expression analysis between highly metastatic breast cancer cells with low CREB3L1 and the corresponding lines expressing HA-CREB3L1. This analysis was carried out across three different subtypes of breast cancer cells (T47D, HCC1954 and HCC1806; all CREB3L1-low). Key signaling pathways and cell functions most impacted by CREB3L1 expression were identified using a bioinformatics analysis. Specific genes consistently upregulated in the metastatic cells were knocked down to assess their impact on cell migration, cell invasion, growth in soft agar across in multiple breast cancer lines. The effect of knocking down the top metastatic gene identified in this study was further tested using in vivo mouse model of primary breast tumor growth in the mammary fat pad, and metastatic colonization of the lung.

Breast cancer cells with low CREB3L1 expression showed upregulation of metastasis and integrin signaling pathways and enhanced cell movement, migration, invasion functions, consistent with its known role as a tumor suppressor. CLIC3 (chloride intracellular channel 3), a protein with roles in integrin recycling, cell migration and invasion, was found to be consistently upregulated in CREB3L1-low cells and in all subtypes of breast tumors. Increased CLIC3 expression was associated with poor patient survival. Knockdown of CLIC3 in several cell lines reduced cell migration, invasion and anchorage-independent growth in soft agar, effects that could be rescued by co-transfection of an shRNA-insensitive CLIC3 plasmid. CLIC3 knockdown also decreased tumor growth and blocked metastases in a mouse xenograft model of breast cancer.

These results suggest that CLIC3 has a key role in promoting cell migration, invasion and growth in soft agar, and CLIC3 inhibitors may be a viable treatment option for breast cancer.