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The ubiquitous store-operated Ca2+ entry pathway mediated by plasma membrane Ca2+ release-activated Ca2+ (CRAC) channels regulates a wide variety of physiological functions. While it is clearly established that the ORAI1 protein is essential for native mammalian CRAC channels, the contribution of ORAI2 and ORAI3 have remained nebulous. The crystal structure of the sole Orai isoform in drosophila (dOrai) revealed a hexameric assembly, suggesting that mammalian CRAC channels are hexamers of ORAI. Nevertheless, the relative contribution of each isoform of the mammalian ORAI trio to the stoichiometry of native CRAC channels remains elusive. The recent generation of ORAI isoform single, double and triple knockout cell lines and tissue-specific knockout mice has shed light on how native ORAI isoform heteromerization fine tunes CRAC-mediated Ca2+ signaling.

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.

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., eLife digest 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.

SummaryDespite the established role of mitochondria in tumorigenesis, the molecular mechanisms by which mitochondrial Ca2+ (mtCa2+) signaling regulates tumor growth and metastasis remain unknown. The crucial role of mtCa2+ in tumorigenesis is highlighted by the altered expression of proteins mediating mtCa2+ uptake and extrusion in cancer cells. Here, we demonstrate that expression of the mitochondrial Na+/Ca2+ exchanger NCLX (SLC8B1) is decreased in colorectal tumors and is associated with advanced-stage disease in patients. We reveal that downregulation of NCLX leads to mtCa2+ overload, mitochondrial depolarization, mitophagy, and reduced tumor size. Concomitantly, NCLX downregulation drives metastatic spread, chemoresistance, the expression of epithelial-to-mesenchymal transition (EMT), hypoxia, and stem cell pathways. Mechanistically, mtCa2+ overload leads to an increase in mitochondrial reactive oxygen species (mtROS) which activates HIF1α signaling supporting the metastatic behavior of tumor cells lacking NCLX. Our results reveal that loss of NCLX expression is a novel driver of metastatic progression, indicating that control of mtCa2+ levels is a novel therapeutic approach in metastatic colorectal cancer.HighlightsThe expression of NCLX is decreased in colorectal tumors and is associated with advanced-stage disease in patients.NCLX plays a dichotomous role in colorectal tumor growth and metastasis.NCLX downregulation causes mitophagy and reduced colorectal cancer tumor growth.NCLX downregulation induces stemness, chemoresistance and metastasis through mtCa2+/ROS/HIF1α signaling axis.Graphical AbstractSignificanceMitochondrial Ca2+ (mtCa2+) homeostasis is essential for cellular metabolism and growth and plays a critical role in cancer progression. mtCa2+ uptake is mediated by an inner membrane protein complex containing the mitochondrial Ca2+ uniporter (MCU). mtCa2+ uptake by the MCU is followed by a ∼100-fold slower mtCa2+ extrusion mediated by the inner mitochondrial membrane ion transporter, the mitochondrial Na+/Ca2+ exchanger NCLX. Because NCLX is a slower transporter than the MCU, it is a crucial rate-limiting factor of mtCa2+ homeostasis that cannot easily be compensated by another Ca2+ transport mechanism. This represents the first study investigating the role of NCLX in tumorigenesis and metastasis. We demonstrate for the first time that colorectal cancers exhibit loss of NCLX expression and that this is associated with advanced-stage disease. Intriguingly, decreased NCLX function has a dichotomous role in colorectal cancer. Thus, we reveal that NCLX loss leads to reduced primary tumor growth and overall tumor burden in vivo. Yet, the consequential increases in mtCa2+ elicit pro-survival, hypoxic and gene transcription pathways that enhance metastatic progression. This dichotomy is a well-established feature of chemoresistant and recurrent tumor cells including cancer stem cells. Moreover, the downstream changes elicited by NCLX loss are reminiscent of mesenchymal colorectal cancer subtypes that display poor patient survival. Our data indicate that the demonstrated changes to the mtCa2+/mtROS/HIF1α signaling axis elicited through the loss of NCLX are a key adaptation and driver of metastatic colorectal cancer.

Individuals with diverticula or outpouchings of the colonic mucosa and submucosa through the colonic wall have diverticulosis, which is usually asymptomatic. In 10-25% of individuals, the diverticula become inflamed, resulting in diverticulitis. Very little is known about the pathophysiology or gene regulatory pathways involved in the development of diverticulitis. To identify these pathways, we deep sequenced RNAs isolated from full-thickness sections of sigmoid colon from diverticulitis patients and control individuals. Specifically for diverticulitis cases, we analyzed tissue adjacent to areas affected by chronic disease. Since the tissue was collected during elective sigmoid resection, the disease was in a quiescent state. A comparison of differentially expressed genes found that gene ontology (GO) pathways associated with the immune response were upregulated in diverticulitis patients compared with nondiverticulosis controls. Next, weighted gene coexpression network analysis was performed to identify the interaction among coexpressed genes. This analysis revealed RASAL3, SASH3, PTPRC, and INPP5D as hub genes within the brown module eigengene, which highly correlated ( r = 0.67, P = 0.0004) with diverticulitis. Additionally, we identified elevated expression of downstream interacting genes. In summary, transcripts associated with the immune response were upregulated in adjacent tissue from the sigmoid colons of chronic, recurrent diverticulitis patients. Further elucidating the genetic or epigenetic mechanisms associated with these alterations can help identify those at risk for chronic disease and may assist in clinical decision management. NEW & NOTEWORTHY By using an unbiased approach to analyze transcripts expressed in unaffected colonic tissues adjacent to those affected by chronic diverticulitis, our study implicates that a defect in the immune response may be involved in the development of the disease. This finding expands on the current data that suggest the pathophysiology of diverticulitis is mediated by dietary, age, and obesity-related factors. Further characterizing the immunologic differences in diverticulitis may better inform clinical decision-making.

Summary Stromal-interaction molecules (STIM1/2) sense endoplasmic reticulum (ER) Ca2+ depletion and activate Orai channels. However, the choreography of interactions between native STIM/Orai proteins under physiological agonist stimulation is unknown. We show that the five STIM1/2 and Orai1/2/3 proteins are non-redundant and function together to ensure the graded diversity of mammalian Ca2+ signaling. Physiological Ca2+ signaling requires functional interactions between STIM1/2, Orai1/2/3, and IP3Rs, ensuring that receptor-mediated Ca2+ release is tailored to Ca2+ entry and nuclear factor of activated T cells (NFAT) activation. The N-terminal Ca2+-binding ER-luminal domains of unactivated STIM1/2 inhibit IP3R-evoked Ca2+ release. A gradual increase in agonist intensity and STIM1/2 activation relieves IP3R inhibition. Concomitantly, activated STIM1/2 C termini differentially interact with Orai1/2/3 as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai and IP3Rs translate the strength of agonist stimulation to precise levels of Ca2+ signaling and NFAT induction, ensuring the fidelity of complex mammalian Ca2+ signaling.