Extended Synaptotagmins 1 and 2 Are Required for Store-Operated Calcium Entry, Cell Migration and Viability in Breast Cancer Cells.

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1-STIM1 interaction at ER-PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells.

Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+-release-activated Ca2+ channels constituted by Orai family members, with predominance of calcium release-activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT-29 and Caco-2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT-29 and Caco-2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta-66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC.

Orai1α and Orai1ß support calcium entry and mammosphere formation in breast cancer stem cells.

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1ß, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1ß support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1ß in spheroid forming efficiency and self-renewal in breast cancer stem cells.

The Ca2+ Sensor STIM in Human Diseases.

The STIM family of proteins plays a crucial role in a plethora of cellular functions through the regulation of store-operated Ca2+ entry (SOCE) and, thus, intracellular calcium homeostasis. The two members of the mammalian STIM family, STIM1 and STIM2, are transmembrane proteins that act as Ca2+ sensors in the endoplasmic reticulum (ER) and, upon Ca2+ store discharge, interact with and activate the Orai/CRACs in the plasma membrane. Dysregulation of Ca2+ signaling leads to the pathogenesis of a variety of human diseases, including neurodegenerative disorders, cardiovascular diseases, cancer, and immune disorders. Therefore, understanding the mechanisms underlying Ca2+ signaling pathways is crucial for developing therapeutic strategies targeting these diseases. This review focuses on several rare conditions associated with STIM1 mutations that lead to either gain- or loss-of-function, characterized by myopathy, hematological and immunological disorders, among others, and due to abnormal activation of CRACs. In addition, we summarize the current evidence concerning STIM2 allele duplication and deletion associated with language, intellectual, and developmental delay, recurrent pulmonary infections, microcephaly, facial dimorphism, limb anomalies, hypogonadism, and congenital heart defects.

CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets.

BACKGROUND: Altered intracellular Ca2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. METHODS: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. RESULTS: Altered TG (thapsigargin)-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor) were found in neonatal platelets as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca2+-dependent inactivation, so reproducing the Ca2+-homeostasis pattern observed in neonatal platelets. CONCLUSIONS: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. s-STIM1 may avoid slow Ca2+-dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.

Correction: Sanchez-Collado et al. Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells. Cancers 2021, 14, 114.

In the original publication [...].

PKC-Mediated Orai1 Channel Phosphorylation Modulates Ca2+ Signaling in HeLa Cells.

The overexpression of the Orai1 channel inhibits SOCE when using the Ca2+ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.

Store-Operated Calcium Entry and Its Implications in Cancer Stem Cells.

Tumors are composed by a heterogeneous population of cells. Among them, a sub-population of cells, termed cancer stem cells, exhibit stemness features, such as self-renewal capabilities, disposition to differentiate to a more proliferative state, and chemotherapy resistance, processes that are all mediated by Ca2+. Ca2+ homeostasis is vital for several physiological processes, and alterations in the patterns of expressions of the proteins and molecules that modulate it have recently become a cancer hallmark. Store-operated Ca2+ entry is a major mechanism for Ca2+ entry from the extracellular medium in non-excitable cells that leads to increases in the cytosolic Ca2+ concentration required for several processes, including cancer stem cell properties. Here, we focus on the participation of STIM, Orai, and TRPC proteins, the store-operated Ca2+ entry key components, in cancer stem cell biology and tumorigenesis.

Store-Operated Calcium Entry in Breast Cancer Cells Is Insensitive to Orai1 and STIM1 N-Linked Glycosylation.

N-linked glycosylation is a post-translational modification that affects protein function, structure, and interaction with other proteins. The store-operated Ca2+ entry (SOCE) core proteins, Orai1 and STIM1, exhibit N-glycosylation consensus motifs. Abnormal SOCE has been associated to a number of disorders, including cancer, and alterations in Orai1 glycosylation have been related to cancer invasiveness and metastasis. Here we show that treatment of non-tumoral breast epithelial cells with tunicamycin attenuates SOCE. Meanwhile, tunicamycin was without effect on SOCE in luminal MCF7 and triple negative breast cancer (TNBC) MDA-MB-231 cells. Ca2+ imaging experiments revealed that expression of the glycosylation-deficient Orai1 mutant (Orai1N223A) did not alter SOCE in MCF10A, MCF7 and MDA-MB-231 cells. However, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) significantly attenuated SOCE in MCF10A cells but was without effect in SOCE in MCF7 and MDA-MB-231 cells. In non-tumoral cells impairment of STIM1 N-linked glycosylation attenuated thapsigargin (TG)-induced caspase-3 activation while in breast cancer cells, which exhibit a smaller caspase-3 activity in response to TG, expression of the non-glycosylable STIM1 mutant (STIM1N131/171Q) was without effect on TG-evoked caspase-3 activation. Summarizing, STIM1 N-linked glycosylation is essential for full SOCE activation in non-tumoral breast epithelial cells; by contrast, SOCE in breast cancer MCF7 and MDA-MB-231 cells is insensitive to Orai1 and STIM1 N-linked glycosylation, and this event might participate in the development of apoptosis resistance.

Role of Orai3 in the Pathophysiology of Cancer.

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca2+ currents, the store-operated current, Icrac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current Iarc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca2+ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca2+ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.

Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

The Orai1-AC8 Interplay: How Breast Cancer Cells Escape from Orai1 Channel Inactivation.

The interplay between the Ca2+-sensitive adenylyl cyclase 8 (AC8) and Orai1 channels plays an important role both in the activation of the cAMP/PKA signaling and the modulation of Orai1-dependent Ca2+ signals. AC8 interacts with a N-terminal region that is exclusive to the Orai1 long variant, Orai1α. The interaction between both proteins allows the Ca2+ that enters the cell through Orai1α to activate the generation of cAMP by AC8. Subsequent PKA activation results in Orai1α inactivation by phosphorylation at serine-34, thus shaping Orai1-mediated cellular functions. In breast cancer cells, AC8 plays a relevant role supporting a variety of cancer hallmarks, including proliferation and migration. Breast cancer cells overexpress AC8, which shifts the AC8-Orai1 stoichiometry in favor of the former and leads to the impairment of PKA-dependent Orai1α inactivation. This mechanism contributes to the enhanced SOCE observed in triple-negative breast cancer cells. This review summarizes the functional interaction between AC8 and Orai1α in normal and breast cancer cells and its relevance for different cancer features.

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.

Cross-Talk Between the Adenylyl Cyclase/cAMP Pathway and Ca2+ Homeostasis.

Cyclic AMP and Ca2+ are the first second or intracellular messengers identified, unveiling the cellular mechanisms activated by a plethora of extracellular signals, including hormones. Cyclic AMP generation is catalyzed by adenylyl cyclases (ACs), which convert ATP into cAMP and pyrophosphate. By the way, Ca2+, as energy, can neither be created nor be destroyed; Ca2+ can only be transported, from one compartment to another, or chelated by a variety of Ca2+-binding molecules. The fine regulation of cytosolic concentrations of cAMP and free Ca2+ is crucial in cell function and there is an intimate cross-talk between both messengers to fine-tune the cellular responses. Cancer is a multifactorial disease resulting from a combination of genetic and environmental factors. Frequent cases of cAMP and/or Ca2+ homeostasis remodeling have been described in cancer cells. In those tumoral cells, cAMP and Ca2+ signaling plays a crucial role in the development of hallmarks of cancer, including enhanced proliferation and migration, invasion, apoptosis resistance, or angiogenesis. This review summarizes the cross-talk between the ACs/cAMP and Ca2+ intracellular pathways with special attention to the functional and reciprocal regulation between Orai1 and AC8 in normal and cancer cells.

Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells.

Melatonin has been reported to induce effective reduction in growth and development in a variety of tumors, including breast cancer. In triple-negative breast cancer (TNBC) cells, melatonin attenuates a variety of cancer features, such as tumor growth and apoptosis resistance, through a number of still poorly characterized mechanisms. One biological process that is important for TNBC cells is store-operated Ca2+ entry (SOCE), which is modulated by TRPC6 expression and function. We wondered whether melatonin might intersect with this pathway as part of its anticancer activity. We show that melatonin, in the nanomolar range, significantly attenuates TNBC MDA-MB-231 cell viability, proliferation, and migration in a time- and concentration-dependent manner, without having any effect on nontumoral breast epithelial MCF10A cells. Pretreatment with different concentrations of melatonin significantly reduced SOCE in MDA-MB-231 cells without altering Ca2+ release from the intracellular stores. By contrast, SOCE in MCF10A cells was unaffected by melatonin. In the TNBC MDA-MB-468 cell line, melatonin not only attenuated viability, migration, and SOCE, but also reduced TRPC6 expression in a time- and concentration-dependent manner, without altering expression or function of the Ca2+ channel Orai1. The expression of exogenous TRPC6 overcame the effect of melatonin on SOCE and cell proliferation, and silencing or inhibition of TRPC6 impaired the inhibitory effect of melatonin on SOCE. These findings indicate that TRPC6 downregulation might be involved in melatonin's inhibitory effects on Ca2+ influx and the maintenance of cancer hallmarks and point toward a novel antitumoral mechanism of melatonin in TNBC cells.

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.

PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca2+ Entry Via STIM2 in MDA-MB-231 Cells.

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 effect 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.

Functional role of TRPC6 and STIM2 in cytosolic and endoplasmic reticulum Ca2+ content in resting estrogen receptor-positive breast cancer cells.

TRPC6 forms non-selective cation channels activated by a variety of stimuli that are involved in a wide number of cellular functions. In estrogen receptor-positive (ER+) breast cancer cells, the store-operated Ca2+ entry has been reported to be dependent on STIM1, STIM2 and Orai3, with TRPC6 playing a key role in the activation of store-operated Ca2+ entry as well as in proliferation, migration and viability of breast cancer cells. We have used a combination of biotinylation, Ca2+ imaging as well as protein knockdown and overexpression of a dominant-negative TRPC6 mutant (TRPC6dn) to show that TRPC6 and STIM2 are required for the maintenance of cytosolic and endoplasmic reticulum Ca2+ content under resting conditions in ER+ breast cancer MCF7 cells. These cells exhibit a greater plasma membrane expression of TRPC6 under resting conditions than non-tumoral breast epithelial cells. Attenuation of STIM2, TRPC6 and Orai3, alone or in combination, results in impairment of resting cytosolic and endoplasmic reticulum Ca2+ homeostasis. Similar results were observed when cells were transfected with expression plasmid for TRPC6dn. TRPC6 co-immunoprecipitates with STIM2 in resting MCF7 cells, a process that is impaired by rises in cytosolic Ca2+ concentration. Impairment of TRPC6 function leads to abnormal Ca2+ homeostasis and endoplasmic reticulum stress, thus, suggesting that TRPC6 might be a potential target for the development of anti-tumoral therapies.

TRPC6 channel and its implications in breast cancer: an overview.

TRPC6 channel is widely expressed in most human tissues and participates in a number of physiological processes. TRPC6 belongs to the DAG-activated subfamily of channels, but has also been postulated as a mediator in the store-operated calcium entry pathway. The recent characterization of TRPC6 crystal structure has granted a wonderful tool to finally dissect and understand TRPC6 physiological and biophysical properties. Growing evidences have demonstrated that the pattern of expression of TRPC6 proteins is upregulated in several pathophysiological conditions, including breast cancer. However, the real role of TRPC6 in breast cancer persists still unknown. Here we present the current state of the art concerning the function and significance of TRPC6 in this disease. Future investigations should be focus in the creation and identification of compounds that specifically target the channel to ameliorate TRPC6-related diseases.

ELA/APELA precursor cleaved by furin displays tumor suppressor function in renal cell carcinoma through mTORC1 activation.

Apelin is a well-established mediator of survival and mitogenic signaling through the apelin receptor (Aplnr) and has been implicated in various cancers; however, little is known regarding Elabela (ELA/APELA) signaling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here, we identified a function of mTORC1 signaling as an essential mediator of ELA that repressed kidney tumor cell growth, migration, and survival. Moreover, sunitinib and ELA showed a synergistic effect in repressing tumor growth and angiogenesis in mice. The use of site-directed mutagenesis and pharmacological experiments provided evidence that the alteration of the cleavage site of proELA by furin induced improved ELA antitumorigenic activity. Finally, a cohort of tumors and public data sets revealed that ELA was only repressed in the main human kidney cancer subtypes, namely clear cell, papillary, and chromophobe renal cell carcinoma. Aplnr was expressed by various kidney cells, whereas ELA was generally expressed by epithelial cells. Collectively, these results showed the tumor-suppressive role of mTORC1 signaling mediated by ELA and established the potential use of ELA or derivatives in kidney cancer treatment.