Unusual roles of caspase-8 in triple-negative breast cancer cell line MDA-MB-231

Abstract

Abstract. Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non-apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1-to S-phase transition and increasing p21, p27 and hypo-phosphorylated/ active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB-231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB-231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target.

Parthenolide prevents resistance of MDA-MB231 cells to doxorubicin and mitoxantrone: the role of Nrf2

“…Then, cells were transfected with 50 nM siNrf2 in the presence of 5 μ l Lipofectamine 2000 (Invitrogen Life Technologies, Monza, Italy) in a final volume of 1 ml serum-free medium. 67 The reaction was stopped after 5 h replacing the culture medium with fresh DMEM + 10% FBS. After 24 h from transfection, silenced cells were employed for the experiments.…”

Loss of MCL1 function sensitizes the MDA‐MB‐231 breast cancer cells to rh‐TRAIL by increasing DR4 levels

“…Cell lysates and protein samples were prepared as previously reported (De Blasio et al, 2016). Briefly, proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad) for detection with primary antibodies against MCL1 (12756) and BAX (7480)…”

Linking genotypes with multiple phenotypes in single-cell CRISPR screens

“…Among those, CHEK1 is a checkpoint kinase that is essential for normal and cancer cells (Zhang and Hunter, 2014), GATA3 is a critical transcription factor with known oncogenic role (Mehra et al, 2005), and RAD51 has been reported as an oncogene with elevated expression in multiple cancer types including breast cancer (Maacke et al, 2000). CASP8 has multiple functions in different contexts (Stupack, 2013), with a possible essential role in breast cancer cell lines (De Blasio et al, 2016).…”