Ping Xin

Summary: Invertebrates express one endoplasmic reticulum (ER)-resident Ca ²⁺ -sensing stromal-interaction molecule (Stim) and one Orai plasma membrane channel protein. Stim conveys store depletion to Orai, mediating the evolutionarily conserved Ca ²⁺ release-activated Ca ²⁺ (CRAC) current. The crucial role of their vertebrate homologues, STIM1 and Orai1 in mediating CRAC activity in mammals is well-established. However, mammals possess two STIM and three Orai isoforms and the choreography of their interactions under physiological receptor activation is unknown. We show that the five mammalian STIM1/2 and Orai1/2/3 isoforms have non-redundant functions. Yet, all five isoforms are always required together to ensure the graded diversity of mammalian Ca ²⁺ signaling events in response to the full spectrum of agonist strengths. Receptor-activated Ca ²⁺ signaling across the range of stimulus intensities requires functional interactions between not only STIM1/2 and Orai1/2/3, but also IP ₃ R, ensuring that receptor-mediated Ca ²⁺ release is precisely tailored to Ca ²⁺ entry and activation of nuclear factor of activated T-cells (NFAT). This is orchestrated by two interdependent and counterbalancing paradigms: the N-termini Ca ²⁺ -binding ER-luminal domains of unactivated STIM1/2 inhibit IP ₃ R-evoked Ca ²⁺ release. Gradual increase in agonist intensity leads to gradual STIM1/2 activation and relief of IP ₃ R inhibition. Concomitantly, the cytosolic C-termini of activated STIM1/2 differentially interact with Orai1/2/3 proteins as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai proteins and IP ₃ Rs at the ER-lumen and cytosol translate the strength of agonist stimulation to precise levels of Ca ²⁺ release, Ca ²⁺ entry and NFAT induction, ensuring the diversity and fidelity of complex mammalian Ca ²⁺ signaling. Highlights: All five STIM/Orai and IP ₃ R are always required together in mammalian Ca ²⁺ signalling Unactivated STIM1/2 inhibit IP ₃ R and activated STIM1/2 cooperatively activate Orai1/2/3 STIM1 contribution increases and that of STIM2 decreases as agonist intensifies Graded IP ₃ R disinhibition and Orai activation tailor receptor activity to NFAT induction

Summary: Despite the established role of mitochondria in tumorigenesis, the molecular mechanisms by which mitochondrial Ca ²⁺ (mtCa ²⁺ ) signaling regulates tumor growth and metastasis remain unknown. The crucial role of mtCa ²⁺ in tumorigenesis is highlighted by the altered expression of proteins mediating mtCa ²⁺ uptake and extrusion in cancer cells. Here, we demonstrate that expression of the mitochondrial Na ⁺ /Ca ²⁺ 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 mtCa ²⁺ 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, mtCa ²⁺ 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 mtCa ²⁺ levels is a novel therapeutic approach in metastatic colorectal cancer. Highlights: The 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 mtCa ²⁺ /ROS/HIF1α signaling axis. Graphical Significance: Mitochondrial Ca ²⁺ (mtCa ²⁺ ) homeostasis is essential for cellular metabolism and growth and plays a critical role in cancer progression. mtCa ²⁺ uptake is mediated by an inner membrane protein complex containing the mitochondrial Ca ²⁺ uniporter (MCU). mtCa ²⁺ uptake by the MCU is followed by a ∼100-fold slower mtCa ²⁺ extrusion mediated by the inner mitochondrial membrane ion transporter, the mitochondrial Na ⁺ /Ca ²⁺ exchanger NCLX. Because NCLX is a slower transporter than the MCU, it is a crucial rate-limiting factor of mtCa ²⁺ homeostasis that cannot easily be compensated by another Ca ²⁺ 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 mtCa ²⁺ 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 mtCa ²⁺ /mtROS/HIF1α signaling axis elicited through the loss of NCLX are a key adaptation and driver of metastatic colorectal cancer.