Effect of SARAF-based peptides on the store-operated calcium entry and cell invasion in triple-negative breast cancer model

Abstract

Breast cancer is the most common cause of death among women. While there have been significant improvements in diagnosis and treatment, the spread of cancer to other areas of the body remains the primary reason for mortality. The triple-negative subtype of breast cancer is particularly aggressive and does not respond well to conventional therapies. Recent studies have shown that the influx of calcium through the Store-Operated Calcium Entry (SOCE) process plays a critical role in regulating cellular processes involved in the spread of breast cancer. This has made SOCE an interesting target for new therapies. The SOCE-Associated Regulatory Factor (SARAF) has been identified as the primary regulator of the calcium-selective channel Orai, which is activated by STIM proteins. SARAF participates in the activation of the Orai1-STIM1 channel and subsequently contributes to its inhibition and potentiating Ca2+-dependent inactivation mechanisms. Therefore, the interaction between SARAF and STIM1 is the primary molecular inhibitory checkpoint for SOCE regulation. However, the TNBC subtype exhibits a decrease in SARAF expression, and the C-terminal region of SARAF is sufficient to reduce the SOCE response. Based on this, we hypothesize that SARAF-based peptides can inactivate STIM1, decreasing the SOCE response and the migration and invasion of triple-negative breast cancer cells. This proposal aims to generate SARAF-based peptides that modulate the SOCE response, cell migration, and invasion in in vitro and in vivo models of triple-negative breast cancer. This proposal comprises three specific aims: Aim 1. Determine the functional effects of the C-terminal SARAF-based peptides as modulators of the SOCE response. To determine the functional effect of the C-terminal region of SARAF on the SOCE response, recordings of calcium dynamics were performed. Moreover, STIM1 clustering recording and BiFC complementation assays were performed, evidencing the effect of the C-SARAF fragments on the formation of the STIM1 cluster and STIM1-Orai1 complex respectively. In addition, the effect on the NFAT translocation from the cytoplasm to the nucleus is one of the main downstream targets activated by calcium signaling associated with the SOCE response. Taken together, the data obtained suggest that the C-SARAF and C-SARAFK326R fragments reduce the SOCE response, affecting the formation of the STIM1-Orai1 complex. Aim 2. Determine the effect of the C-terminal SARAF-based peptides during cell migration and invasion in an in vitro model of triple-negative breast cancer. Migration and invasion assays were performed to evaluate the effect of these peptides on cellular processes involved in metastasis. In addition, focal adhesion dynamics were performed. The results obtained advise that the C-SARAF and C-SARAFK326R fragments reduce cell migration and invasion affecting the dynamics of actin cytoskeleton rearrangement and assembly/disassembly of focal adhesions. Aim 3. Determine the effect of the C-terminal SARAF-based peptides in the murine in vivo model of triple-negative breast cancer. In vivo murine models of tumor growth and metastasis of TNBC were performed. The histological analyses suggest that the overexpression of the C-SARAF and C-SARAFK326R fragments reduces the invasion of tumor cells. Moreover, increases tumor lymphocytic infiltration, which constitutes a biomarker of good prognosis in the clinical context of TNBC. Therefore, this study will contribute to the understanding of novel molecular mechanisms involved in Orai1-STIM1 activity. Moreover, the data acquired in this project could be directly applied to the design of new complementary drugs for cancer treatments.