Loss of STIM2 in colorectal cancer drives growth and metastasis through metabolic reprogramming and PERK-ATF4 endoplasmic reticulum stress pathway.

The endoplasmic reticulum (ER) stores large amounts of calcium (Ca2+), and the controlled release of ER Ca2+ regulates a myriad of cellular functions. Although altered ER Ca2+ homeostasis is known to induce ER stress, the mechanisms by which ER Ca2+ imbalance activate ER stress pathways are poorly understood. Stromal-interacting molecules STIM1 and STIM2 are two structurally homologous ER-resident Ca2+ sensors that synergistically regulate Ca2+ influx into the cytosol through Orai Ca2+ channels for subsequent signaling to transcription and ER Ca2+ refilling. Here, we demonstrate that reduced STIM2, but not STIM1, in colorectal cancer (CRC) is associated with poor patient prognosis. Loss of STIM2 causes SERCA2-dependent increase in ER Ca2+, increased protein translation and transcriptional and metabolic rewiring supporting increased tumor size, invasion, and metastasis. Mechanistically, STIM2 loss activates cMyc and the PERK/ATF4 branch of ER stress in an Orai-independent manner. Therefore, STIM2 and PERK/ATF4 could be exploited for prognosis or in targeted therapies to inhibit CRC tumor growth and metastasis.

Orai3 and Orai1 mediate CRAC channel function and metabolic reprogramming in B cells.

The essential role of store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels in T cells is well established. In contrast, the contribution of individual Orai isoforms to SOCE and their downstream signaling functions in B cells are poorly understood. Here, we demonstrate changes in the expression of Orai isoforms in response to B cell activation. We show that both Orai3 and Orai1 mediate native CRAC channels in B cells. The combined loss of Orai1 and Orai3, but not Orai3 alone, impairs SOCE, proliferation and survival, nuclear factor of activated T cells (NFAT) activation, mitochondrial respiration, glycolysis, and the metabolic reprogramming of primary B cells in response to antigenic stimulation. Nevertheless, the combined deletion of Orai1 and Orai3 in B cells did not compromise humoral immunity to influenza A virus infection in mice, suggesting that other in vivo co-stimulatory signals can overcome the requirement of BCR-mediated CRAC channel function in B cells. Our results shed important new light on the physiological roles of Orai1 and Orai3 proteins in SOCE and the effector functions of B lymphocytes.

The airway smooth muscle sodium/calcium exchanger NCLX is critical for airway remodeling and hyperresponsiveness in asthma.

The structural changes of airway smooth muscle (ASM) that characterize airway remodeling (AR) are crucial to the pathogenesis of asthma. During AR, ASM cells dedifferentiate from a quiescent to a proliferative, migratory, and secretory phenotype. Calcium (Ca2+) is a ubiquitous second messenger that regulates many cellular processes, including proliferation, migration, contraction, and metabolism. Furthermore, mitochondria have emerged as major Ca2+ signaling organelles that buffer Ca2+ through uptake by the mitochondrial Ca2+ uniporter and extrude it through the Na+/Ca2+ exchanger (NCLX/Slc8b1). Here, we show using mitochondrial Ca2+-sensitive dyes that NCLX only partially contributes to mitochondrial Ca2+ extrusion in ASM cells. Yet, NCLX is necessary for ASM cell proliferation and migration. Through cellular imaging, RNA-Seq, and biochemical assays, we demonstrate that NCLX regulates these processes by preventing mitochondrial Ca2+ overload and supporting store-operated Ca2+ entry, activation of Ca2+/calmodulin-dependent kinase II, and transcriptional and metabolic reprogramming. Using small animal respiratory mechanic measurements and immunohistochemistry, we show that smooth muscle-specific NCLX KO mice are protected against AR, fibrosis, and hyperresponsiveness in an experimental model of asthma. Our findings support NCLX as a potential therapeutic target in the treatment of asthma.

STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells.

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma.

Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

The Mitochondrial Ca2+ uniporter is a central regulator of interorganellar Ca2+ transfer and NFAT activation.

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release-activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.

Antipsychotic drugs elicit cytotoxicity in glioblastoma multiforme in a calcium-dependent, non-D2 receptor-dependent, manner.

Dopamine D2 -like receptor antagonists have been suggested as being potential anticancer therapeutics with specific utility for central nervous system cancers due to their ability to cross the blood-brain barrier. Despite a plethora of data reporting anticancer effects for D2 R antagonists in cell or animal studies, the ligand concentrations or doses required to achieve such effects greatly exceed the levels known to cause high degrees of occupancy of the D2 receptor. To resolve this conundrum, we interrogated a panel of glioblastoma multiforme (GBM) cell lines using D2 antagonists of varying chemotype. We studied the cytotoxic effects of these compounds, and also ascertained the expression of D2 receptors (D2 R) on these cells. Although several chemotypes of D2 R antagonists, including phenothiazines and phenylbutylpiperidines, were effective against GBM cell line cultures, the highly selective antagonist remoxipride had no anticancer activity at biologically relevant concentrations. Moreover the D2 R antagonist-induced cytotoxicity in monolayer cultures was independent of whether the cells expressed D2 R. Instead, cytotoxicity was associated with a rapid, high-magnitude calcium flux into the cytoplasm and mitochondria, which then induced depolarization and apoptosis. Blocking this flux protected the GBM cell lines U87MG, U251MG, and A172. Together, these data suggest that the cytotoxicity of these D2 R antagonists involves calcium signaling mechanisms, not D2 R antagonism. Repurposing of existing drugs should focus on the former, not latter, mechanism.

Dichotomous role of the human mitochondrial Na+/Ca2+/Li+ exchanger NCLX in colorectal cancer growth and metastasis.

Despite the established role of mitochondria in cancer, the mechanisms by which mitochondrial Ca2+ (mtCa2+) regulates tumorigenesis remain incompletely understood. The crucial role of mtCa2+ in tumorigenesis is highlighted by altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer. Here, we demonstrate decreased expression of the mitochondrial Na+/Ca2+/Li+ exchanger NCLX (SLC8B1) in human colorectal tumors and its association with advanced-stage disease in patients. Downregulation of NCLX causes mtCa2+ overload, mitochondrial depolarization, decreased expression of cell-cycle genes and reduced tumor size in xenograft and spontaneous colorectal cancer mouse models. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, and expression of epithelial-to-mesenchymal, hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to increased mitochondrial reactive oxygen species, which activate HIF1α signaling supporting metastasis of NCLX-null tumor cells. Thus, loss of NCLX is a novel driver of metastasis, indicating that regulation of mtCa2+ is a novel therapeutic approach in metastatic colorectal cancer.

Colorectal cancer is the second largest cause of cancer deaths worldwide. Even in cases where the cancer is diagnosed and treated early, cells can sometimes survive treatment and spread to other organs. Once the cancer has spread, the survival rate is less than 15%. Mitochondria are compartments in the cell that produce energy, and they play an important role in supporting the rapid growth of cancer cells. The levels of calcium ions in mitochondria control how they produce energy, a process that is altered in cancer cells. To better understand how calcium ions influence colorectal cancer growth, Pathak, Gueguinou et al. studied a protein called NCLX, which controls calcium levels by pumping them out of the mitochondria. Two mouse strains that were used to study what happens if NCLX is missing. The first strain was genetically modified to disable the gene for NCLX and then exposed to carcinogens. The second strain was injected with colorectal cancer cells from a human tumor that were lacking NCLX. In both strains, the tumors that formed were smaller than in mice with NCLX. However, the human cancer cells in the second model were more likely to spread to other organs. This is likely because the build-up of calcium ions in the mitochondria of mice lacking NCLX led to an increase in the production of hypoxia-inducible factor-1a, a protein that is a common driver of cancer spread. Pathak, Gueguinou et al. demonstrated how NCLX can affect colorectal cancer progression. It suggests that it may have opposing effects during early and late-stage colorectal cancer, encouraging tumor growth but also decreasing the spread to other organs. Further research could help refine treatments at different stages of the disease.

Distinct pharmacological profiles of ORAI1, ORAI2, and ORAI3 channels.

The ubiquitous Ca2+ release-activated Ca2+ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd3+ (5µM), 2-APB (5 µM and 50 µM), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.

L-type Ca2+ channel blockers promote vascular remodeling through activation of STIM proteins.

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

The native ORAI channel trio underlies the diversity of Ca2+ signaling events.

The essential role of ORAI1 channels in receptor-evoked Ca2+ signaling is well understood, yet little is known about the physiological activation of the ORAI channel trio natively expressed in all cells. The roles of ORAI2 and ORAI3 have remained obscure. We show that ORAI2 and ORAI3 channels play a critical role in mediating the regenerative Ca2+ oscillations induced by physiological receptor activation, yet ORAI1 is dispensable in generation of oscillations. We reveal that ORAI2 and ORAI3 channels multimerize with ORAI1 to expand the range of sensitivity of receptor-activated Ca2+ signals, reflecting their enhanced basal STIM1-binding and heightened Ca2+-dependent inactivation. This broadened bandwidth of Ca2+ influx is translated by cells into differential activation of NFAT1 and NFAT4 isoforms. Our results uncover a long-sought role for ORAI2 and ORAI3, revealing an intricate control mechanism whereby heteromerization of ORAI channels mediates graded Ca2+ signals that extend the agonist-sensitivity to fine-tune transcriptional control.