Analysis of the TCGA and GTEx databases using the GEPIA2 tool revealed differential OS depending on the level of STIM2 expression. Indeed, patients who express the most STIM2 have reduced survival and a predominantly monoblastic and monocytic cytological subtype. This high STIM2 expression as a negative marker was also observed in glioblastoma [50], but this contrasts with data obtained in other tumour types, such as colorectal cancers where high STIM2 expression led to suppression of growth [51] and cholangiocarcinoma where low of STIM2 expression was associated with a poor prognosis [52]. These discrepant effects of STIM2 depending on the cancer type reflect its heterogeneous pattern of expression and function in different cell systems. In this study, using a shRNA-mediated knockdown approach, we show that STIM2 is involved in genome integrity, cell cycle and apoptosis control of primary cells and leukemic monocytic cell lines. Intracellular Ca2+ is known to play a key role in the regulation of proliferation and the cell cycle in both normal and malignant cells [53]. SOCEs have been widely described as being linked to increased proliferation [54]. Studies have mainly focused on STIM1, ORAI1 and TRPC1, showing that a reduction in SOCE influx was correlated with a parallel decrease in cell proliferation [55]–[57]. A few studies on STIM2 have been carried out, with conflicting results on SOCE measurement and cell responses. In HUVECs and pulmonary artery smooth muscle cells, a decreased STIM2 expression led to a loss of proliferative capacity [58], [59]. We observed here a similar effect in both primary hematopoietic cells and leukemic cells. First, STIM2 KD induced apoptosis by the mitochondrial intrinsic pathway, with an increased ratio between pro-apoptotic and anti-apoptotic proteins. Second, it let to dysregulation of the cell cycle, characterized by G2/M phase arrest, with a decrease in cell cycle regulators controlling the G2/M transition. Indeed, in order to enter into mitosis from G2, cells must activate CDK1, which binds to cyclin B [60]. This activation depends on the phosphatase CDC25c, which removes a phosphate group from CDK1 [61]. The expression of CDK1, cyclin B1 and CDC25c were all drastically decreased following STIM2 KD.
We show here that both cell cycle blockage and the apoptotic response to STIM2 KD occurred through genomic stress (assessed by quantification of DSB) and p53 activation. The link between DNA breakage, H2AXγ phosphorylation and blockage in G2/M has been reported in other cell types, after low dose irradiation or exposure to toxins such as Benzo (a) pyrene [62]. p-H2AXγ allows recruitment at DNA double strands breakpoints of proteins involved in DNA repair, activates directly the p53 pathway [63] which induces p21 [63], inhibits the CDK1-cyclin B1 complex [48], [49] and represses CDC25c phosphatase [49], leading to cell blockage in G2/M to prevent defective mitosis. Of note, p53 is mutated in THP1 cells but is still expressed and functional, in agreement with other reports [64]–[66]. The central role of p53 in response to DNA stress mediated by STIM2 KD is highlighted by (i) the reversal of apoptosis and cell cycle blockage after cell exposure to the p53 inhibitor PFT-α and (ii) the absence of a phenotype in two cell lines defective for p53, HL60 and K562. Of note, it could be claimed that p-H2AXγ is a consequence and not a cause of p53 stabilization and of the subsequent activation of the apoptotic cascade since DNA ladder formation during apoptosis requires JNK-dependent phosphorylation of H2AXγ in cooperation with the caspase − 3/CAD pathway [67]. However, in leukemic and normal hematopoietic cells, PFT-α reversed cell cycle blockage and apoptosis but not p-H2AXγ induction, showing that genomic stress occurred upstream of p53 induction in STIM2 KD conditions. The cell response to p53 induction is either a reversible cell cycle blockage or apoptosis, depending on the balance between pro- and anti-apoptotic proteins that determines a “threshold” beyond which cells will die [68]–[70]. In OCI-AML3 and THP1 cells after STIM2 KD, this threshold is low as a consequence of decreased expression of MCL-1, a p53 target, as well as BCL2 and Bcl-XL, and increased expression of BAX and BAD. One can assume that this low “trigger” drives the massive apoptosis that we observed in response to the cell cycle blockage induced by p53 activation. Notably, the simultaneous decrease in BCL2 and MCL-1 levels induced by STIM2 KD is particularly interesting and relevant in therapeutics considering that high expression of MCL-1 is involved in the resistance of AML cells to targeted therapy, such as BCL2 Inhibitors [71].
To build a functional link between STIM2, Ca2+ response and DNA stress, we measured Ca2+ entry through SOCE after Thapsigargin exposure. One could assume that STIM2 KD would decrease the intracellular Ca2+ level in response to ER depletion. In contrast to another report [72], we observed increased Ca2+ entry in response to Thapsigargin after STIM2 KD. We first ruled out any compensatory mechanism by increased STIM1 expression since its level was stable after STIM2 KD (not shown). However, since STIM1 and STIM2 interact and since STIM1 activates ORAI more efficiently, we cannot rule out that STIM2 KD promotes the formation of STIM1 homomeric complexes capable of recruiting and stimulating SOC entry more efficiently [73]. Moreover, the effect of STIM2 modulation on SOCE is conflicting in the literature [13], [74], [75]. This may be due to the coexistence of different STIM2 isoforms, STIM2.1 and STIM2.2, which differ in their expression pattern and function on SOCE. Indeed, the largely expressed STIM2.2 isoform enhances SOCE whereas STIM2.1, which contains a new sequence within the CAD domain [76], [77], represses SOCE through abrogation of its interaction with ORAI. Despite the fact that all the shRNAs that we used targeted sequences outside the 8 residues specific to STIM2.1, the 2.2/2.1 STIM2 ratio was increased after shSTIM2 transduction, explaining the positive effect on SOCE. BAPTA, an intracellular Ca2+ chelator, was able to decrease STIM2 KD-mediated apoptosis in THP1 cells (Additional file 15 : Fig. S8), arguing for a link between STIM2 KD, deregulated Ca2+ signaling and cell death. Furthermore, the AUC results for Thapsigargin response showed that transfections do not appear to induced reticular calcium stress and therefore cannot explain any apoptotic effects. The basis of the obtained is therefore to be found at the level of the plasma membrane and calcium entry. However, the exact nature of this functional link between the altered SOCE response in monocytic cells and DNA stress is lacking.
In addition to these effects on the cell cycle and apoptosis, we pointed out a potential role of STIM2 in monocytic differentiation, as already described in naïve CD8+ T-cell maturation into cytotoxic terminal effector cells [78], [79].. STIM proteins are expressed in monocyte/macrophage function, as shown by KO mouse models. Sogkas et al. found that STIM1 was involved in phagocytosis whereas STIM2 was involved in cell migration and apoptosis, particularly in the production of pro-inflammatory cytokines [80], although these results were not confirmed by other teams [81]. In our study, we observed trends toward monocytic differentiation in AML expressing higher STIM2 levels and increased STIM2 expression during in vitro monocytic differentiation of CD34+ and leukemic cell lines. Moreover, STIM2 KD impaired CD14 expression whereas STIM2 overexpression increased its expression in THP1 cells exposed to low level of vitamin D. Once again, the functional link between SOCE deregulation after STIM2 KD and monocytic differentiation is lacking. However, such mechanism remains plausible considering the data in the literature [82], [83]. For example, differentiation of the myelomonocytic cell line U937 toward macrophage by dibutyrylcAMP was associated with upregulation of Calcium release-activated calcium channel (CRAC) activity. Thapsigargin-induced Ca2+ release from ER calcium was higher in differentiated U937 that their undifferentiated counterpart [82]. Macrophages and monocytes express ORAI 1, 2 and 3 as shown in transcriptomic studies, and CRAC is involved during macrophage activation and ROS production. ORAI1 may be the most abundant, while ORAI3 may induce a negative feedback in order to prevent cells from oxidative damages In THP1 cells, exposure to oxidized LDL increased ORAI-dependent Ca2+ intake, whereas Ca2+ chelation or ORAI1 inhibition decreased cell formation. Chemical inhibition of ORAI1 in apoE-/- mice drastically decreased atherosclerosis formation induced by a high cholesterol diet [84]. Taken together, these data, including ours, suggest a particular role of SOCE and Ca2+ signaling in monocytic/macrophage differentiation and function.