Desma Grice

Store-Independent Activation of Orai1 by SPCA2 in Mammary Tumors

Ca(2+) is an essential and ubiquitous second messenger. Changes in cytosolic Ca(2+) trigger events critical for tumorigenesis, such as cellular motility, proliferation, and apoptosis. We show that an isoform of Secretory Pathway Ca(2+)-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuates basal Ca(2+) levels and tumorigenicity. Contrary to its conventional role in Golgi Ca(2+) sequestration, expression of SPCA2 increased Ca(2+) influx by a mechanism dependent on the store-operated Ca(2+) channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca(2+) stores or STIM1 and STIM2 sensors and uncoupled from Ca(2+)-ATPase activity of SPCA2. Binding of the SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca(2+) influx. Our findings reveal a signaling pathway in which the Orai1-SPCA2 complex elicits constitutive store-independent Ca(2+) signaling that promotes tumorigenesis.

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced store-operated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1high/STIM2low tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women. Mol Cancer Ther; 10(3); 448–60. ©2011 AACR.

ORAI-mediated calcium entry: Mechanism and roles, diseases and pharmacology

ORAI1 is a protein located on the plasma membrane that acts as a calcium channel. Calcium enters via ORAI1 as a mechanism to refill the sarcoplasmic/endoplasmic reticulum calcium stores, the depletion of which can be detected by the sensor protein STIM1. Isoforms of these proteins ORAI2, ORAI3 and STIM2 also have roles in cellular calcium homeostasis but are less well characterized. This pathway of filling the calcium stores is termed store-operated calcium entry and while the pathway itself was proposed in 1986, the identity of the key molecular components was only discovered in 2005 and 2006. The characterization of the ORAI and STIM proteins has provided clearer information on some calcium-regulated pathways that are important in processes from gene transcription to immune cell function. Recent studies have also suggested the importance of the components of ORAI-mediated calcium entry in some diseases or processes significant in disease including the migration of breast cancer cells and thrombus formation. This review will provide a brief overview of ORAI-mediated calcium entry, its role in physiological and pathophysiological processes, as well as current and potential pharmacological modulators of the components of this important cellular calcium entry pathway.

Golgi calcium pump secretory pathway calcium ATPase 1 (SPCA1) is a key regulator of insulin-like growth factor receptor (IGF1R) processing in the basal-like breast cancer cell line MDA-MB-231.

Calcium signaling is a key regulator of pathways important in tumor progression, such as proliferation and apoptosis. Most studies assessing altered calcium homeostasis in cancer cells have focused on alterations mediated through changes in cytoplasmic free calcium levels. Here, we show that basal-like breast cancers are characterized by an alteration in the secretory pathway calcium ATPase 1 (SPCA1), a calcium pump localized to the Golgi. Inhibition of SPCA1 in MDA-MB-231 cells produced pronounced changes in cell proliferation and morphology in three-dimensional culture, without alterations in sensitivity to endoplasmic reticulum stress induction or changes in global calcium signaling. Instead, the effects of SPCA1 inhibition in MDA-MB-231 cells reside in altered regulation of calcium-dependent enzymes located in the secretory pathway, such as proprotein convertases. Inhibition of SPCA1 produced a pronounced alteration in the processing of insulin-like growth factor receptor (IGF1R), with significantly reduced levels of functional IGF1Rβ and accumulation of the inactive trans-Golgi network pro-IGF1R form. These studies identify for the first time a calcium transporter associated with the basal-like breast cancer subtype. The pronounced effects of SPCA1 inhibition on the processing of IGF1R in MDA-MB-231 cells independent of alterations in global calcium signaling also demonstrate that some calcium transporters can regulate the processing of proteins important in tumor progression without major alterations in cytosolic calcium signaling. Inhibitors of SPCA1 may offer an alternative strategy to direct inhibitors of IGF1R and attenuate the processing of other proprotein convertase substrates important in basal breast cancers.

Non-Stimulated, Agonist-Stimulated and Store-Operated Ca2+ Influx in MDA-MB-468 Breast Cancer Cells and the Effect of EGF-Induced EMT on Calcium Entry

In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca2+ reserves, molecular components of the store-operated Ca2+ entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca2+ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca2+ influx. The potential roles for specific Ca2+ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca2+ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca2+ influx in a manner dependent on Ca2+ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca2+ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (time to peak [Ca2+]CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca2+ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca2+ influx in non-stimulated cells.

UV-Induced DNA Damage Promotes Resistance to the Biotrophic Pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

Arabidopsis thaliana Y-family DNA polymerase η catalyses translesion synthesis and interacts functionally with PCNA2

SUMMARY Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Poleta, although AtPoleta exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPoleta in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPoleta were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPoleta can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPoleta in UV-irradiated cells to PCNA2 and PCNA- and ubiquitin-binding motifs in AtPoleta.

The Golgi Associated Calcium Pumps in Human Breast Cancer.

In breast cancer there is evidence of alterations in Golgi-dependent functions, some of which are regulated by calcium. The Golgi apparatus is an intracellular calcium store; calcium stored in the Golgi has a role in cell signalling and cell proliferation. A decrease in Golgi calcium concentration can limit some Golgi-dependent processes and is associated with an increase in endoplasmic reticulum stress. Previous studies have shown that Golgi associated calcium pumps sequester calcium into the Golgi apparatus. We are investigating Golgi associated calcium pumps in the context of breast cancer. We queried microarray databases of clinical breast samples and breast cancer cell lines. Golgi associated calcium pumps were differentially expressed between breast cancer subtypes (P<0.05). To reduce Golgi calcium, we inhibited expression of one Golgi associated calcium pump isoform using Dharmacon siRNA in MDA-MB-231 breast cancer cells. Inhibition (~96%) was confirmed with real time RT-PCR. Knockdown was present at 24, 72, 96 and 120 hours (P<0.05). Using this model we are assessing the effect of siRNA-mediated inhibition on cell proliferation, lipid production and sensitivity to endoplasmic reticulum stress. Many of these assays have required specific optimization of siRNA treatment conditions to enable transfection at low seeding densities. Collectively our results suggest that some breast cancers are associated with a remodelling of Golgi calcium transport via alterations in the transcription of Golgi associated calcium pumps