Sarah J. Roberts-Thomson

Calcium and cancer: targeting Ca2+ transport.

Ca2+ is a ubiquitous cellular signal. Altered expression of specific Ca2+ channels and pumps are characterizing features of some cancers. The ability of Ca2+ to regulate both cell death and proliferation, combined with the potential for pharmacological modulation, offers the opportunity for a set of new drug targets in cancer. However, the ubiquity of the Ca2+ signal is often mistakenly presumed to thwart the specific therapeutic targeting of proteins that transport Ca2+. This Review presents evidence to the contrary and addresses the question: which Ca2+ channels and pumps should be targeted?

Calcium channels and pumps in cancer: changes and consequences

Increases in intracellular free Ca2+ play a major role in many cellular processes. The deregulation of Ca2+ signaling is a feature of a variety of diseases, and modulators of Ca2+ signaling are used to treat conditions as diverse as hypertension to pain. The Ca2+ signal also plays a role in processes important in cancer, such as proliferation and migration. Many studies in cancer have identified alterations in the expression of proteins involved in the movement of Ca2+ across the plasma membrane and subcellular organelles. In some cases, these Ca2+ channels or pumps are potential therapeutic targets for specific cancer subtypes or correlate with prognosis.

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.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial–mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Peroxisome proliferator–activated receptor α in the human breast cancer cell lines MCF‐7 and MDA‐MB‐231

Peroxisome proliferator–activated receptor (PPAR) α is a ligand‐activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARα mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARα mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARα is expressed and dynamically regulated in human breast cancer MCF‐7 and MDA‐MB‐231 cells. Having established the presence of PPARα in both cell types, we then examined the consequence of PPARα activation, by its ligands Wy‐14,643 and clofibrate, on proliferation. With real‐time reverse transcriptase–polymerase chain reaction, we showed that PPARα mRNA was dynamically regulated in MDA‐MB‐231 cells and that PPARα activation significantly increased proliferation of the cell line. In contrast, PPARα expression in MCF‐7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA‐MB‐231 cells. However, PPARα ligand activation still significantly increased the proliferation of MCF‐7 cells. The promotion of proliferation in breast cancer cell lines following PPARα activation was in stark contrast to the effects of PPARγ‐activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARα in human breast cancer cell lines and showed for the first time that activation of PPARα in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland 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.

The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

BackgroundThe vanilloid receptor 1 (TRPV1) is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA) pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described.ResultsIn the present studies we investigated the hypothesis that the μ opioid receptor (MOP) agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+) responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine.Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1.ConclusionThe results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

Peroxisome proliferator-activated receptors in tumorigenesis: Targets of tumour promotion and treatment

The peroxisome proliferator‐activated receptors (PPAR) are ligand‐activated transcription factors. There are three genes that code for the PPAR isoforms: PPARα, PPARβ and PPARγ. In the present review, studies characterizing the various PPAR isoforms are discussed. Peroxisome proliferator‐activated receptor α has been implicated in the lipid‐lowering effects of the fibrate drugs. Peroxisome proliferator‐activated receptor γ has a clear role in adipocyte differentiation and is therapeutically targeted by the thiazolidinedione drugs for the treatment of type II diabetes. The physiological role of PPARβ is less well understood but, as described in the present review, recent studies have implicated it with a role in colon cancer. In the present review, particular attention is focused on the role of PPAR in the regulation of expression of proteins associated with cell cycle control and tumorigenesis.

Photochemical Studies On the Antiinflammatory Drug Diclofenac

Abstract— Irradiation with UVA light of the anti‐inflammatory drug diclofenac [2‐(2,6‐dichloroanilino) phenylacetic acid] in aqueous buffer or methanol solution leads to sequential loss of both chlorine substituents and ring closure to carbazole‐1‐acetic acid as the major product. Minor products result from substitution by the solvent. The photosensitizing properties of diclofenac and its major photoproduct were tested with singlet oxygen substrates and in the free radical polymerization of acrylamide. Although the major carbazole product is a weakly phototoxic agent, able to generate singlet oxygen more efficiently than diclofenac, the free radical photodechlorination process is postulated as the probable initiation step of in vivo photosensitivity responses.

Localization of plasma membrane and secretory calcium pumps in the mammary gland

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.