Gain-of-function mutations in the calcium-sensing receptor are associated with a familial syndrome of hypocalcemia with hypercalciuria that needs to be distinguished from hypoparathyroidism.

Background Ferroptosis is a novel mode of non-apoptotic cell death induced by build-up of toxic lipid peroxides (lipid-ROS) in an iron dependent manner. Cancer-associated fibroblasts (CAFs) support tumor progression and drug resistance by secreting various bioactive substances, including exosomes. Yet, the role of CAFs in regulating lipid metabolism as well as ferroptosis of cancer cells is still unexplored and remains enigmatic. Methods Ferroptosis-related genes in gastric cancer (GC) were screened by using mass spectrum; exosomes were isolated by ultra-centrifugation and CAF secreted miRNAs were determined by RT-qPCR. Erastin was used to induce ferroptosis, and ferroptosis levels were evaluated by measuring lipid-ROS, cell viability and mitochondrial membrane potential. Results Here, we provide clinical evidence to show that arachidonate lipoxygenase 15 (ALOX15) is closely related with lipid-ROS production in gastric cancer, and that exosome-miR-522 serves as a potential inhibitor of ALOX15. By using primary stromal cells and cancer cells, we prove that exosome-miR-522 is mainly derived from CAFs in tumor microenvironment. Moreover, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was found to mediate miR-522 packing into exosomes, and ubiquitin-specific protease 7 (USP7) stabilizes hnRNPA1 through de-ubiquitination. Importantly, cisplatin and paclitaxel promote miR-522 secretion from CAFs by activating USP7/hnRNPA1 axis, leading to ALOX15 suppression and decreased lipid-ROS accumulation in cancer cells, and ultimately result in decreased chemo-sensitivity. Conclusions The present study demonstrates that CAFs secrete exosomal miR-522 to inhibit ferroptosis in cancer cells by targeting ALOX15 and blocking lipid-ROS accumulation. The intercellular pathway, comprising USP7, hnRNPA1, exo-miR-522 and ALOX15, reveals new mechanism of acquired chemo-resistance in GC. Graphical abstract

Nearly 30 mutations have been identified to date in the coding region of the extracellular calcium-sensing receptor (CaR) that are associated with inherited human hypo-and hypercalcemic disorders. To understand the mechanisms by which the mutations alter the function of the receptor may help to discern the structurefunction relationships in terms of ligand-binding and G protein coupling. In the present studies, we transiently expressed eight known CaR mutations in HEK293 cells. The effects of the mutations on extracellular calciumand gadolinium-elicited increases in the cytosolic calcium concentration were then examined. Seven inactivating mutations, which cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, show a reduced functional activity of the receptor because they may 1) reduce its affinity for agonists; 2) prevent conversion of the receptor from a putatively immature, high mannose form into the fully glycosylated and biologically active form of the CaR, in addition to lowering its affinity for agonists; or 3) fail to couple the receptor to and/or activate its respective G protein(s). Conversely, one activating mutation, which causes a form of autosomal dominant hypocalcemia, appears to increase the affinity of the receptor for its agonists.The recently cloned extracellular Ca 2ϩ (Ca 2ϩ o )-sensing receptor (CaR) 1 (1) has provided key insights into the pathogenesis of inherited human hypo-and hypercalcemic disorders (2, 3). The receptor, BoPCaR (bovine parathyroid Ca 2ϩ -sensing receptor) was first isolated from bovine parathyroid using expression cloning in Xenopus laevis oocytes and shows pharmacological properties nearly identical to those of the native receptor in its responses to extracellular divalent cations (i.e. Ca o ), and polyamines (e.g. neomycin) (1). Subsequently, cDNAs encoding the human homolog of the same receptor have been cloned from human parathyroid (4) and kidney (5), using a homology-based strategy. The human and bovine receptors share a high degree of homology at the amino acid level (93% identity). Stimulation of the CaR by agonists activates phospholipase C, with resultant increases in inositol phosphates and the cytosolic calcium concentration (Ca 2ϩ

The metastatic organotropism has been one of the cancer's greatest mysteries since the ‘seed and soil' hypothesis. Although the role of EGFR in cancer cells is well studied, the effects of secreted EGFR transported by exosomes are less understood. Here we show that EGFR in exosomes secreted from gastric cancer cells can be delivered into the liver and is integrated on the plasma membrane of liver stromal cells. The translocated EGFR is proved to effectively activate hepatocyte growth factor (HGF) by suppressing miR-26a/b expression. Moreover, the upregulated paracrine HGF, which binds the c-MET receptor on the migrated cancer cells, provides fertile ‘soil' for the ‘seed', facilitating the landing and proliferation of metastatic cancer cells. Thus, we propose that EGFR-containing exosomes derived from cancer cells could favour the development of a liver-like microenvironment promoting liver-specific metastasis.

Malignant tumors, including colorectal cancer (CRC), usually rely on ATP generation through aerobic glycolysis for both rapid growth and chemotherapy resistance. The M2 isoform of pyruvate kinase (PKM2) has a key role in catalyzing glycolysis, and PKM2 expression varies even within a single tumor. In this study, we confirmed that expression of PKM2 is heterogeneous in CRC cells, namely high in oxaliplatin‐resistant cells but relatively low in sensitive cells, and found that chemoresistant cells had enhanced glycolysis and ATP production. In addition, we report a PKM2‐dependent mechanism through which chemosensitive cells may gradually transform into chemoresistant cells. The circular RNA hsa_circ_0005963 (termed ciRS‐122 in this study), which was determined to be a sponge for the PKM2‐targeting miR‐122, was positively correlated with chemoresistance. In vitro and in vivo studies showed that exosomes from oxaliplatin‐resistant cells delivered ciRS‐122 to sensitive cells, thereby promoting glycolysis and drug resistance through miR‐122 sponging and PKM2 upregulation. Moreover, si‐ciRS‐122 transported by exosomes could suppress glycolysis and reverse resistance to oxaliplatin by regulating the ciRS‐122–miR‐122–PKM2 pathway in vivo. Exosomes derived from chemoresistant CRC cells could transfer ciRS‐122 across cells and promote glycolysis to reduce drug susceptibility in chemosensitive cells. This intercellular signal delivery suggests a potential novel therapeutic target and establishes a foundation for future clinical applications in drug‐resistant CRC.

The extracellular calcium (Ca 2؉ o )-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface crosslinking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.

Hepatocellular carcinoma (HCC), the major form of liver cancer, has shown increasing incidence and poor prognosis. Adipose tissue is known to function in energy storage and metabolism regulation by the secretion of adipokines. Circular RNAs (circRNAs), a novel type of noncoding RNA, have recently been recognized as key factors in tumor development, but the role of exosome circRNAs derived from adipose tissues has not been defined yet. Here, adipose-secreted circRNAs were found to regulate deubiquitination in HCC, thus facilitating cell growth. It was observed that exosome circ-deubiquitination (circ-DB) is upregulated in HCC patients with higher body fat ratios. Moreover, in vitro and in vivo studies showed that exo-circ-DB promotes HCC growth and reduces DNA damage via the suppression of miR-34a and the activation of deubiquitination-related USP7. Finally, the results showed that the effects of adipose exosomes on HCC cells can be reversed by knockdown of circ-DB. These results indicate that exosome circRNAs secreted from adipocytes promote tumor growth and reduce DNA damage by suppressing miR-34a and activating the USP7/Cyclin A2 signaling pathway.

Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium (Ca2+o)-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating Ca2+o or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high Ca2+o-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both Gq and G(i). High Ca2+o increased serine phosphorylation of the 85-kDa cytosolic phospholipase A2 (cPLA2) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-Ca2+o)-induced ERK1/2 activation and reduced cPLA2 phosphorylation in both cell types, documenting MAPK's role in cPLA2 activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through Gq/11-mediated activation of PI-PLC, as well as through G(i)- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in cPLA2 activation.