Effect of different simvastatin doses on cellular proliferation, migration and actin cytoskeleton
The IC50 value of simvastatin for the Hs578 cell line was determined at 2.092µM ± 0.0665 (Fig. 1). In subsequent experiments, concentrations of simvastatin that corresponds to 85% (IC15), 70% (IC30) and 50% (IC50) of the cellular viability of Hs578T cells were used, which are respectively 0.5µM, 1µΜ and 2µΜ.
Interestingly, treatment of simvastatin exhibited a dose differential effect on the wound healing capacity of Hs578T cells. In fact, simvastatin at IC15 and IC30 seems to promote cell migration by almost 20%, but at a higher level of cytotoxicity (IC50, 2µM) wound healing was decreased to about 70% of the control (Fig. 1).
Moreover, simvastatin at IC30 and IC50, but not at IC15, promoted actin cytoskeleton rearrangement on Hs578T cells (Fig. 2). Untreated and treated with simvastatin at IC15 Hs578T cells appeared with characteristic stellate shape and elongated bodies, exhibiting many protrusions (Fig. 2A-2B). Interestingly, simvastatin at IC30 (Fig. 2C) and IC50 (Fig. 2D) promoted actin cytoskeleton rearrangement on Hs578T cells, where cells appeared with a more rounded shape and significantly reduced protrusions, perhaps pointing to a decrease in the number of stress fibers.
Differential effect of simvastatin doses on EMT- and stemness- related molecules
Due to the differential effect of simvastatin on cellular migration and actin cytoskeleton organization on Hs578T cells, EMT related markers were analyzed at IC15, IC30 and IC50 values of simvastatin. Hs578T cells treated with simvastatin at IC15 exhibited an EMT promoting gene signature characterized by elevated mRNA levels of the stemness markers, KLF4 and CD44 (Fig. 3), as well as of the majority of the investigating EMT markers including SNAI1, SNAI2, ZEB1 and VIM (Fig. 3). The elevation ranged from 20% (CD44) up to more than 100% (SNAl2). On the contrary, simvastatin at IC50 diminished the cellular migration of Hs578T cells, which is accompanied by actin cytoskeleton reorganization and reduced mRNA levels of KLF4 and TWIST to about 80% of the control (Fig. 3), indicating a less aggressive phenotype.
Statins can modulate epigenetics alterations, such as methylation, acetylation and the expression of noncoding RNAs , such as miRNAs . Using bioinformatics tools and literature searching, four miRNAs specifically important for TNBC and presenting validated EMT-related targets with a distinct role in breast cancer were chosen. Our results show that simvastatin reduces the expression of the investigated miRNAs in a manner independently of the used dose and the function of the miRNA. Specifically, simvastatin at all used doses corresponding to IC15, IC30 and IC50 decreased by 30–40% the expression of the oncomiR miR-155 and the tumor suppressor miR-34a, miR-200b and miR-340 (Fig. 3).
Our data indicate that simvastatin at IC15 induces cellular migration on Hs578T TNBC cells without structural alterations on actin cytoskeleton, stimulating an EMT-related and stemness gene signature. Simvastatin at IC50 attenuated the migration of Hs578T cells followed by actin reorganization and reduction of some EMT and stemness markers. Interestingly, Hs578T treated with simvastatin at IC30 exhibited a mixed effect of the alterations observed at IC15 and IC50.
Simvastatin has been shown to inhibit the geranylgeranylation of RhoA which is important to preserve the undifferentiated status of cells, thus simvastatin can stimulate differentiation of BCSCs. Moreover, blocking of RhoA can inhibit ROCK2 activity. ROCK2 can modulate actin cytoskeleton reorganization and regulate mechanosensory stimulus that are involved to transcriptional regulation of genes related to proliferation and differentiation status . Additionally, RhoA can influence Cdc42 and Rac proteins modulating stress fibers and cellular protrusions, while simvastatin can also inhibit the prenylation of Rac1 and Cdc42 . Overall, simvastatin seems to regulate crucial mechanical pathways related to the aggressive characteristics of tumor cells, influencing the transcription of EMT- and stemness-associated genes . To continue, simvastatin effect on apoptosis is mainly and markedly observed in doses near and above the IC50 value on MDA-MB-231 TNBC cells . Recent studies, using different concentrations of statins exhibits that higher concentrations of 90% of EC50 had a major and more clear effect on attenuating cellular migration and colony formation of ERα + MCF-7 and TNBC MDA-MB-231 cells, compared to lower statins concentrations of 10% of EC50 which couldn’t affect those cellular functions in 2D cultures. Moreover, statins concentrations of 90% of EC50 a small effect on inhibiting the size of 3D spheroids of MDA-MB-231 mixed with fibroblasts, while increased the size of MCF-7/fibroblasts spheroids. These data suggests that different concentrations of statins can orchestrate different mechanisms of action which are also depending on the tumor microenvironment (TME) , as well as the treatment duration .
Our data indicate that simvastatin at all used concentrations attenuated the examined miRNAs independently of their role in BC progression. Nevertheless, simvastatin at concentrations below IC50 induced the expression of miR-140-5p in a dose-dependent manner via a mechanism that involves induction of oxidative stress and the TF NRF1, which binds to the promoter of the pre-miR-140, leading to cell death . To note, miRNAs can also regulate the actin-myosin cytoskeleton dynamics , indicating that statins, which are crucial regulators of actin cytoskeleton may exert their pleiotropic effects via multiple not-yet elucidated mechanisms.