Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.Ministerio de Ciencia e Innovación: PID2019-104084GB-C21Ministerio de Ciencia e Innovación: PID2019-104084GB-C22Consejería de Educación y Empleo, Junta de Extremadura: IB20007Consejería de Educación y Empleo, Junta de Extremadura: GR18061Consejería de Educación y Empleo, Junta de Extremadura: PD16072Consejería de Educación y Empleo, Junta de Extremadura: GR2100

Furin Prodomain ppFurin Enhances Ca2+ Entry Through Orai and TRPC6 Channels’ Activation in Breast Cancer Cells

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells’ malignant phenotype repression and reduction of their resistance to treatments.This research was funded by SIRIC-Brio, La Ligue Contre le Cancer and Region Nouvelle Aquitaine to A.M.K. and by PID2019-104084GB-C21 and PID2019-104084GB-C22/AEI/10.13039/501100011033, MICINN (Grant BFU2016-74932-C2) and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grants IB16046 and GR18061) to J.A.R. and T.S. J.J.L. is supported by a contract from Junta de Extremadura (TA18011). C.C. is supported by a contract from Junta de Extremadura—FEDER

Involvement of stanniocalcins in the deregulation of glycaemia in obese mice and type 2 diabetic patients

Las estanniocalcinas se expresan en el tejido del páncreas, y se sugirió una correlación directa entre la insulina circulante y las concentraciones de STC2 en el ser humano. Aquí, mostramos una correlación significativa entre STC1 y tanto la glucemia como la hemoglobina glicosilada entre los pacientes con DM2, mientras que los pacientes con DM2 que presentan los mayores valores de hemoglobina glicosilada exhibieron la menor expresión de STC2. Sin embargo, el tratamiento de los pacientes con fármacos antiglicémicos no modifica significativamente la expresión de ambas STC. Por otra parte, los ratones STC2-/- que mostraron sobrepeso neonatal y adulto presentaron además una glucemia desregulada cuando fueron alimentados con una dieta hipercalórica (pellet de cría, BP). Esta alteración es más evidente en las primeras etapas de la vida animal. La glucemia desregulada en estos ratones se confirmó mediante una prueba oral de glucosa. Además, los ratones STC2-/- presentan un aumento del tamaño del páncreas; así, el análisis histológico revela que los ratones WT responden a la dieta BP aumentando el tamaño de los islotes pancreáticos a través de la inducción de la división celular, y los ratones STC2-/- carecen de este mecanismo compensatorio. Contrariamente, los ratones alimentados con STC2-/- muestran un mayor número de islotes pero de tamaño similar a los alimentados con el pellet regular. El análisis histopatológico demuestra la alteración de la estructura de los tejidos y las infiltraciones de eritrocitos en los ratones STC2-/-, posiblemente debido al estrés evocado por la dieta BP. Por último, se observó una mayor inmunotinción de glucagón en el islote de los ratones STC2-/-, y el ensayo ELISA de glucagón confirmó el aumento del glucagón circulante. En resumen, presentamos pruebas del papel de los STC, principalmente el STC2, como posible marcador temprano durante el desarrollo de la diabetes mellitus.Stanniocalcins are expressed in the pancreas tissue, and it was suggested a direct correlation between circulating insulin and STC2 concentrations in human. Here, we show a significant correlation between STC1 and both glycaemia and glycosylated haemoglobin among DM2 patients, while DM2 patients who present the greatest glycosylated haemoglobin values exhibited the lowest STC2 expression. However, treatment of patients with antiglycaemic drugs does not significantly modify the expression of both STCs. On the other hand, STC2-/- mice that exhibited neonatal and adult overweight further presented deregulated glycaemia when they were feed with a hypercaloric diet (breeding pellet, BP). This alteration is more evident at the early stages of the animal life. Deregulated glycaemia in these mice was confirmed using glucose oral test. In addition, STC2-/- mice present enhanced pancreas size; thus, the histological analysis reveals that WT mice respond to BP diet by increasing the size of the pancreatic islets through inducing cell division, and STC2-/- mice lack this compensatory mechanism. Contrary, BP fed STC2-/- mice show enhanced number of islets but of similar size than those fed with regular pellet. Histopathological analysis demonstrates tissue structure disruption and erythrocytes infiltrations in STC2-/- mice, possibly due to the stress evoked by the BP diet. Finally, enhanced glucagon immunostaining was observed in the islet of STC2-/- mice, and the glucagon ELISA assay confirmed the increase in the circulating glucagon. Summarizing, we present evidence of the role of STCs, mainly STC2, as a possible early marker during development of diabetes mellitus.• Ministerio de Economía y Competitividad. Becas 2013‐45564C2‐1‐P, BFU‐2016‐74932‐C2‐1‐P • Programa Juan de la Cierva. Becas IJCI‐2015‐25665, JC‐2012‐ 2934 • Junta de Extremadura. Beca PRIIB16046peerReviewe

Remodelación de los componentes de la ECC en las células de cáncer de mama

Tesis doctoral con la Mención de "Doctor Internacional"Programa de Doctorado de Biomarcadores de Salud y Estados PatológicosEl cáncer de mama es la principal causa de muerte entre las mujeres de todo el mundo diagnosticadas de cáncer. Existe un consenso generalizado sobre los cambios asociados a la transición de célula sana a célula cancerígena. Especialmente, en las células de cáncer de mama, la regulación de la [Ca2+]c ejerce un papel fundamental a lo largo del desarrollo de esta patología. Son varios los mecanismos de homeostasis del Ca2+ que se han descrito fundamentales en la fisiopatogenia del cáncer. De entre ellos, el principal mecanismo de entrada de Ca2+ extracelular en células no excitables es la entrada capacitativa de Ca2+ (ECC), modulada por las familias de proteínas STIM, Orai y TRPC. Estudios, cada vez más numerosos, han demostrado que SOCE y sus componentes regulan una diversidad de procesos en el cáncer de mama. Se podría decir que STIM1 junto con Orai1 en muestras de tejido tumoral de pacientes son los más sobreexpresados en comparación con tejidos pre- o no cancerígenos. Orai3, por su parte, está más presente en los subtipos de cáncer de mama ER+. Además, otras isoformas de la familia TRP se han correlacionado con la progresión de cáncer de mama. Teniendo en cuenta estas consideraciones, las líneas de investigación expuestas en este trabajo se centraron en cómo se regulan varias líneas de cáncer de mama (TNBC y ER+) en la función de STIM1 a través de σ2R/TMEM97, la asociación de STIM2, Orai1 y TRPC1 con la progesterona, y el papel protector de Orai3 frente al ácido araquidónico.Breast cancer is the main cause of death between all women diagnosed of cancer around the world. The scientific community accepts the idea that specific changes in healthy cells lead to a neoplastic differentiation. Especially in breast cancer cells, the role of the [Ca2+]c and how it is regulated are crucial along the pathology. Nevertheless, many Ca2+ homeostatic pathways have been reported evolving cancer. Special mention deserves one of the major mechanisms for Ca2+ entry in non-excitable cells, capacitative Ca2+ entry (CCE), a mechanism for Ca2+ influx modulated by STIM, and conducted by Orai and TRPC proteins. At the moment, there is a body of evidence supporting the association between SOCE, and its components, and breast cancer especially related with the metabolism in these cells. In general, both STIM1 and Orai1 proteins are overexpressed in breast cancer samples as compared to non-tumoral breast samples. Orai3, for example, is highly expressed in the ER+ breast cancer subtype. In addition, a variety of TRP family isoforms have also been correlated with progression of breast cancer. In view of these considerations and its significant component, we have conducted experiments to ascertain the remodelling of CCE in breast cancer cells and we have described expression and function remodelling of STIM1 and, STIM2, Orai1 and TRPC1 in present of progesterone, as well as Orai3.• Ministerio de Economía y Competitividad. Proyectos BFU2013-45564-C2-1-P y BFU2016-74932-C2-1-P (I+D+i) • Junta de Extremadura y Fondos FEDER. Ayudas GR15029 y GR18061 • Junta de Extremadura. Beca-contrato para la formación predoctoral (PD16072), para Carlos Cantonero Chamorr

Furin prodomain ppfurin enhances ca2+ entry through orai and trpc6 channels’ activation in breast cancer cells

Furin, a proprotein convertase that belongs to a family of Ca2+-dependent serine peptidases, is involved in the maturation of a variety of proproteins, including growth factors, receptors and differentiation factors, adhesion molecules and proteases. Furin have been associated with tumorigenesis and tumor progression and metastasis; therefore, it has been hypothesized that Furin may constitute a new potential target for cancer therapy. In triple negative breast cancer cells, inhibition of Furin by the prodomain ppFurin results in enhancement of Ca2+ influx, which involves both the increase of store-operated calcium entry (SOCE) and the activation of constitutive Ca2+ entry. The latter involves the activation of Orai and TRPC6 channels, while the increase of SOCE observed in ppFurin-expressing cells is entirely dependent on Orai channels. As a result, ppFurin expression reduces triple negative breast cancer cell viability and ability to migrate and enhances their sensitization to hydrogen peroxide-induced apoptosis.The intracellular calcium concentration ([Ca2+]i ) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells’ malignant phenotype repression and reduction of their resistance to treatments.Junta de Extremadura-Fondo Europeo de Desarrollo Regional IB16046 y GR18061Junta de Extremadura TA18011SIRIC-Brio, La Ligue Contre le Cancer y Region Nouvelle Aquitania a A.M.K. y por PID2019-104084GB-C21 y PID2019-104084GB-C22 / AEI / 10.13039 / 501100011033

PGRMC1 inhibits progesterone-evoked proliferation and Ca2+ entry via STIM2 in MDA-MB-231 cells

Se ha demostrado que el componente 1 de la membrana del receptor de progesterona (PGRMC1) regula algunos características del cáncer. La progesterona (P4) provoca cambios en el calcio intracelular (Ca2+) en las líneas celulares de cáncer de mama triple negativo líneas celulares de cáncer de mama (MDA-MB-231, MDA-MB-468 y BT-20) y en otras líneas celulares de cáncer de mama como las células luminales MCF7. La expresión de PGRMC1 es elevada en las células MDA-MB-231 y MCF7 en comparación con la línea celular no tumoral MCF10A, y el silenciamiento de PGRMC1 aumenta la movilización de Ca2+ provocada por P4. En este trabajo hemos descubierto una nueva vía de movilización de Ca2+ dependiente de P4 en células MDA-MB-231 MDA-MB-231 y otras células de cáncer de mama triple negativo, así como en células MCF7, en las que interviene la molécula de interacción estromal 2 (STIM), la proteína 1 del canal de calcio activado por la liberación de calcio (Orai1) y el canal 1 del potencial receptor transitorio (TRP). La molécula de interacción estromal 1 (STIM1) no estaba implicada en esta nueva vía de Ca2+, como se demostró utilizando siRNA STIM1. El silenciamiento de PGRMC1 redujo el efecto negativo de P4 sobre la proliferación y muerte celular en células MDA-MB-231. En consonancia con esta última observación, la acumulación nuclear del Factor Nuclear de las Células T Activadas 1 (NFAT1) debida a la incubación de P4 durante 48 h aumentó las células transfectadas con el ARNsi de horquilla pequeña contra PGRMC1 (shPGRMC1). Estos resultados evidencian la existencia de una nueva vía de entrada de Ca2+ provocada por P4 que está regulada por PGRMC1.Progesterone receptor membrane component 1 (PGRMC1) has been shown to regulate some cancer hallmarks. Progesterone (P4) evokes intracellular calcium (Ca2+) changes in the triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and BT-20) and in other breast cancer cell lines like the luminal MCF7 cells. PGRMC1 expression is elevated in MDA-MB-231 and MCF7 cells as compared to non-tumoral MCF10A cell line, and PGRMC1 silencing enhances P4-evoked Ca2+ mobilization. Here, we found a new P4-dependent Ca2+ mobilization pathway in MDA-MB-231 cells and other triple-negative breast cancer cells, as well as in MCF7 cells that involved Stromal interaction molecule 2 (STIM2), Calcium release-activated calcium channel protein 1 (Orai1), and Transient Receptor Potential Channel 1 (TRPC1). Stromal interaction molecule 1 (STIM1) was not involved in this novel Ca2+ pathway, as evidenced by using siRNA STIM1. PGRMC1 silencing reduced the negative eect of P4 on cell proliferation and cell death in MDA-MB-231 cells. In line with the latter observation, Nuclear Factor of Activated T-Cells 1 (NFAT1) nuclear accumulation due to P4 incubation for 48 h was enhanced in cells transfected with the small hairpin siRNA against PGRMC1 (shPGRMC1). These results provide evidence for a novel P4-evoked Ca2+ entry pathway that is downregulated by PGRMC1.• Ministerio de Ciencia e Innovación. Proyecto BFU-2016-74932-C2-1-P • Junta de Extremadura y Fondos FEDER. Becas IB16046, IB18020 y GR18061 • Junta de Extremadura. Beca postdoctoral PD16072 para Carlos Cantonero ChamorropeerReviewe

Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

Arachidonic acid (AA) is a phospholipase A2 metabolite that has been reported to mediate a plethora of cellular mechanisms involved in healthy and pathological states such as platelet aggregation, lymphocyte activation, and tissue inflammation. AA has been described to activate Ca2+ entry through the arachidonate-regulated Ca2+-selective channels (ARC channels). Here, the analysis of the changes in the intracellular Ca2+ homeostasis revealed that, despite MDA-MB-231 cells expressing the ARC channel components Orai1, Orai3, and STIM1, AA does not evoke Ca2+ entry in these cells. We observed that AA evokes Ca2+ entry in MDA-MB-231 cells transiently expressing ARC channels. Nevertheless, MDA-MB-231 cell treatment with AA reduces cell proliferation and migration while inducing cell death through apoptosis. The latter mostly likely occurs via mitochondria membrane depolarization and the activation of caspases-3, -8, and -9. Altogether, our results indicate that AA exerts anti-tumoral effects on MDA-MB-231 cells, without having any effect on non-tumoral breast epithelial cells, by a mechanism that is independent on the activation of Ca2+ influx via ARC channels

NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

(1) Background: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) Methods and results: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) Conclusion: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation

STIM1 phosphorylation at Y316 modulates its interaction with SARAF and the activation of SOCE and ICRAC.

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca2+ entry (SOCE). Hence, identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE is of interest. By performing a computational analysis, we identified that the Y316 residue is susceptible to phosphorylation. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and the Ca2+ release-activated Ca2+ current (ICRAC). STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to in cells with wild-type (WT) YFP-tagged STIM1. Additionally, the Y316F mutation altered the pattern of interaction between STIM1 and SARAF under resting conditions and upon Ca2+ store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca2+-dependent inactivation (SCDI) as compared to STIM1 WT, an effect that was abolished by SARAF knockdown. Finally, in NG115-401L cells transfected with shRNA targeting SARAF, expression of STIM1 Y316F induced greater SOCE than STIM1 WT. Taken together, our results provide evidence supporting the idea that phosphorylation of STIM1 at Y316 plays a relevant functional role in the activation and modulation of SOCE

NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

ANTECEDENTES: Recientemente se ha publicado la estructura del receptor Sigma 2/TMEM97 (σ2RTMEM97). (2, 3) MÉTODOS Y RESULTADOS: Utilizamos enfoques genéticos y bioquímicos para identificar el mecanismo molecular aguas abajo de σ2R/TMEM97. El nuevo ligando fluorescente σ2R/TMEM97, NO1, redujo la proliferación y la supervivencia de las líneas celulares de cáncer de mama triple negativo (TNBC: líneas celulares MDA-MB-231 y MDA-MB-468), debido a la apoptosis inducida por NO1. También se demostró una mayor bioacumulación y una captación más rápida de NO1 en las células MDA-MB-231 en comparación con las líneas celulares MCF10A o MCF7. En consecuencia, la elevada expresión de σ2R/TMEM97 se confirmó mediante Western blotting. A diferencia de NO1, otros ligandos σ2R/TMEM97, como SM21 y PB28, potenciaron la proliferación y migración de las células MDA-MB-231. La entrada de calcio operada por almacenamiento (SOCE) es crucial para diferentes características del cáncer. Aquí demostramos que NO1, pero no otros ligandos σ2R/TMEM97, redujo la SOCE en células MDA-MB-231. Del mismo modo, el silenciamiento de TMEM97 en células MDA-MB-231 también afectó a SOCE. La administración de NO1 redujo la interacción STIM1-Orai1, probablemente al afectar al efecto regulador positivo de σ2R/TMEM97 sobre STIM1, ya que no pudimos detectar interacción con Orai1. (4) CONCLUSIONES: σ2R/TMEM97 es una proteína clave para la supervivencia de las células de cáncer de mama triple negativo mediante la promoción de SOCE; por lo tanto, NO1 puede convertirse en una buena herramienta farmacológica para evitar su proliferación.BACKGROUND: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) METHODS AND RESULTS: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) CONCLUSION: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation.• Ministerio de Industria, Economía y Competitividad. Proyectos BFU2013-45564C2-1-P y BFU2016-74932-C2-1-P • Junta de Extremadura y Fondos FEDER. Subvenciones GR18061 e IB16046 • Junta de Extremadura. Beca predoctoral PD16072, para Carlos Cantonero ChamorropeerReviewe