Role STIM2 in Human Breast Cancer

Background: Metastasis is a multistep process that remains a major cause of death for most oncologic patients. However, the molecular mechanisms which underlie the metastatic process are still poorly understood.

Store-Operated Calcium (Ca²⁺) Entry (SOCE), a major mechanism for Ca²⁺ permeation across membranes of cells, is essential for various cellular processes including cell migration. SOCE depends on stromal interaction molecules (STIM) and Orai channels. Mammalian cells express two STIM isoforms, STIM1 and STIM2. Ablation of STIM1 expression has been shown to reduce the cellular migration, invasion and focal adhesion turnover rate and STIM1 has been implicated in epithelial-to-mesenchymal transition of breast cancer cells. In contrast, however, much less is known about the contribution of STIM2 to cancerous processes.

Materials and methods: The role of STIM2 in metastasis was studied in high metastatic human breast cancer cell line LM24 cells. We ablated the expression of STIM2 in LM24 cells by using CRISPR-Cas9 gene editing and determined STIM2 knockout (KO) clones by using DNA sequencing and Western Blot analyses. Ca²⁺ imaging was used to determine the activity of STIM2 KO cells in SOCE. Boyden chamber-based migration and invasion assays were used for testing the mobility of KO cells. Immuno-labeling and live cell-edge dynamics were used for assessing cell-extracellular matrix (ECM) adhesions. Tumor development was examined in vivo following subcutaneous injection of wild type (WT) cells or STIM1 KO or STIM2 KO cells into flanks of 8-weeks old SCID male mice.

Results and discussion: While knockout of STIM1 abolished SOCE in LM24 cells, knockout of STIM2 only mildly attenuated SOCE. Despite the mild effect of STIM2 KO on SOCE, assays of invasion and migration demonstrated a significant reduction in motility of STIM2 KO cells compared to WT indicating an essential function of STIM2 in cell motility. Assessment of cellular adhesion showed deficiency of STIM2 KO cells compared to WT cells indicating the important contribution of STIM2 to cell adhesion. Finally, we investigated tumor formation in vivo. While WT cells formed tumors, STIM2 KO or STIM1 KO cells did not, suggesting STIM2 may constitute an important marker for tumor progression and prognosis.

Conclusion: In our study, we characterized the role of STIM2 in highly metastatic human breast cancer. We found that while STIM2 is not essential for SOCE, it plays an important role in cellular migration and invasion. STIM2 cells showed deficiency in interaction with ECM and failed to form tumors in vivo. Thus, we suggest that by regulating cellular adhesion, STIM2 contributes to tumor formation and metastatic.