Donald L. Gill

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.

Being the largest the Ca²⁺ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca²⁺ signalling often involves both Ca²⁺ release via inositol 1, 4, 5-trisphosphate receptors (IP₃R) and store operated Ca²⁺ entries (SOCE) through Ca²⁺ release activated Ca²⁺ (CRAC) channels on plasma membrane (PM). IP₃Rs are functionally coupled with CRAC channels and other Ca²⁺ handling proteins. However, it still remains less well defined as to whether IP₃Rs could regulate ER-mediated Ca²⁺ signals independent of their Ca²⁺ releasing ability. To address this, we generated IP₃Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP₃Rs-TKO, IP₃Rs-DKO), and systemically examined ER Ca²⁺ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca²⁺ leakage and refilling, as well as SOCE were all significantly reduced in IP₃Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP₃R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP₃R3 is one crucial player in coordinating ER-mediated Ca²⁺ signalling.

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca²⁺ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca²⁺ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca²⁺ and Ca²⁺ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca²⁺ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca²⁺ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca²⁺ but is dispensable for the maintenance of long-term ER Ca²⁺ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1_1–491 and STIM1_1–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.