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Progression of benign tumors to invasive, metastatic cancer is accompanied by the epithelial-to-mesenchymal transition (EMT), characterized by loss of the cell-adhesion protein E-cadherin. Although silencing mutations and transcriptional repression of the E-cadherin gene have been widely studied, not much is known about posttranslational regulation of E-cadherin in tumors. We show that E-cadherin is tightly coexpressed with the secretory pathway Ca2+-ATPase isoform 2, SPCA2 (ATP2C2), in breast tumors. Loss of SPCA2 impairs surface expression of E-cadherin and elicits mesenchymal gene expression through disruption of cell adhesion in tumorspheres and downstream Hippo-YAP signaling. Conversely, ectopic expression of SPCA2 in triple-negative breast cancer elevates baseline Ca2+ and YAP phosphorylation, enhances posttranslational expression of E-cadherin, and suppresses mesenchymal gene expression. Thus, loss of SPCA2 phenocopies loss of E-cadherin in the Hippo signaling pathway and EMT–MET transitions, consistent with a functional role for SPCA2 in E-cadherin biogenesis. Furthermore, we show that SPCA2 suppresses invasive phenotypes, including cell migration in vitro and tumor metastasis in vivo. Based on these findings, we propose that SPCA2 functions as a key regulator of EMT and may be a potential therapeutic target for treatment of metastatic cancer.Implications:Posttranslational control of E-cadherin and the Hippo pathway by calcium signaling regulates EMT in breast cancer cells.

Supplementary Movie 2

Supplementary Movie 1

Background: Voltage-gated sodium (Na(V)) channels help regulate electrical activity of the plasma membrane. Mutations in associated subunits can result in pathological outcomes. Here we examined the interaction of Na(V) channels with cardiac arrhythmia-linked mutations in SCN2B and SCN4B, two genes that encode auxiliary β-subunits. Materials and Methods: To investigate changes in SCN2B(R137H) and SCN4B(I80T) function, we combined three-dimensional X-ray crystallography with electrophysiological measurements on Na(V)1.5, the dominant subtype in the heart. Results:SCN4B(I80T) alters channel activity, whereas SCN2B(R137H) does not have an apparent effect. Structurally, the SCN4B(I80T) perturbation alters hydrophobic packing of the subunit with major structural changes and causes a thermal destabilization of the folding. In contrast, SCN2B(R137H) leads to structural changes but overall protein stability is unaffected. Conclusion:SCN4B(I80T) data suggest a functionally important region in the interaction between Na(V)1.5 and β4 that, when disrupted, could lead to channel dysfunction. A lack of apparent functional effects of SCN2B(R137H) on Na(V)1.5 suggests an alternative working mechanism, possibly through other Na(V) channel subtypes present in heart tissue. Indeed, mapping the structural variations of SCN2B(R137H) onto neuronal Na(V) channel structures suggests altered interaction patterns.

How activation leads to gating Voltage-gated sodium (Na v ) channels are key players in electrical signaling. Central to their function is fast inactivation, and mutants that impede this cause conditions such as epilepsy and pain syndromes. The channels have four voltage-sensing domains (VSDs), with VSD4 playing an important role in fast inactivation. Clairfeuille et al. determined the structures of a chimera in which VSD4 of the cockroach channel Na v PaS is replaced with VSD4 from human Na v 1.7, both in the apo state and bound to a scorpion toxin that impedes fast activation (see the Perspective by Chowdhury and Chanda). The toxin traps VSD4 in a deactivated state. Comparison with the apo structure shows how interactions between VSD4 and the carboxyl-terminal region change as VSD4 activates and suggests how this would lead to fast inactivation. Science , this issue p. eaav8573 ; see also p. 1278

Fast inactivation of voltage-gated sodium (Na

Progression of benign tumors to invasive, metastatic cancer is accompanied by the epithelial-to-mesenchymal transition (EMT), characterized by loss of the cell-adhesion protein E-cadherin. Although silencing mutations and transcriptional repression of the E-cadherin gene have been widely studied, not much is known about posttranslational regulation of E-cadherin in tumors. We show that E-cadherin is tightly coexpressed with the secretory pathway Ca2+-ATPase isoform 2, SPCA2 (ATP2C2), in breast tumors. Loss of SPCA2 impairs surface expression of E-cadherin and elicits mesenchymal gene expression through disruption of cell adhesion in tumorspheres and downstream Hippo-YAP signaling. Conversely, ectopic expression of SPCA2 in triple-negative breast cancer elevates baseline Ca2+ and YAP phosphorylation, enhances posttranslational expression of E-cadherin, and suppresses mesenchymal gene expression. Thus, loss of SPCA2 phenocopies loss of E-cadherin in the Hippo signaling pathway and EMT–MET transitions, consistent with a functional role for SPCA2 in E-cadherin biogenesis. Furthermore, we show that SPCA2 suppresses invasive phenotypes, including cell migration in vitro and tumor metastasis in vivo. Based on these findings, we propose that SPCA2 functions as a key regulator of EMT and may be a potential therapeutic target for treatment of metastatic cancer. Implications: Posttranslational control of E-cadherin and the Hippo pathway by calcium signaling regulates EMT in breast cancer cells.

Progression of benign tumors to invasive, metastatic cancer requires loss of the celladhesion protein E-cadherin. Although intensive efforts have focused on gene repression and silencing mutations, much less is known about posttranslational control of E-cadherin expression in cancer. SPCA2 is a secretory pathway Ca2+-ATPase that is down-regulated in metastatic breast cancer. We show that SPCA2 is tightly co-expressed with epithelial signature genes and required for E-cadherin biogenesis and cell surface expression. Unexpectedly, this function is uncoupled from Ca2+ pumping and mediated by binding to E-cadherin. Loss of SPCA2 is sufficient to disrupt cell-cell adhesion in tumorspheres and elicit mesenchymal gene expression through Hippo-YAP signaling. These findings point to a causal link between low SPCA2 levels and the epithelial-mesenchymal transition required for breast cancer metastasis.

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A 260/A 280 between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A 260/A 280 ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A 260/A 280 measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.

Epidermal growth factor receptor (EGFR) signalling is a potent driver of glioblastoma, a malignant and lethal form of brain cancer. Disappointingly, inhibitors targeting receptor tyrosine kinase activity are not clinically effective and EGFR persists on the plasma membrane to maintain tumour growth and invasiveness. Here we show that endolysosomal pH is critical for receptor sorting and turnover. By functioning as a leak pathway for protons, the Na/H exchanger NHE9 limits luminal acidification to circumvent EGFR turnover and prolong downstream signalling pathways that drive tumour growth and migration. In glioblastoma, NHE9 expression is associated with stem/progenitor characteristics, radiochemoresistance, poor prognosis and invasive growth in vitro and in vivo. Silencing or inhibition of NHE9 in brain tumour-initiating cells attenuates tumoursphere formation and improves efficacy of EGFR inhibitor. Thus, NHE9 mediates inside-out control of oncogenic signalling and is a highly druggable target for pan-specific receptor clearance in cancer therapy., This work was supported by grants from the National Institutes of Health NS070024 (A.Q.H.), and DK054214 and GM62142 (R.R.). K.C.K. was supported by an American Heart Association postdoctoral fellowship (11POST7380034); J.P. received support from the Johns Hopkins PREP program; H.P. was a Fulbright Fellow supported by the International Fulbright Science and Technology Award; and A.H. was a Porter fellow of the American Physiological Society.

Secretory Pathway Ca2+-ATPases are P-type ATPases comprising 2 isoforms, SPCA1 and SPCA2 that localize to the Golgi and to vesicular compartments. They sequester Ca2+ into stores for protein processing, modification and sorting, and regulate basal cytosolic levels. The two isoforms play distinct roles in breast cancer: SPCA2 expression is greatly amplified in luminal subtypes but has a lower expression in basal subtypes, with opposite expression patterns for SPCA1. Previously, we showed that SPCA2, but not SPCA1, interacts with the Ca2+ channel Orai1 to initiate store-independent Ca2+ entry, which promotes tumorigenesis. We sought to understand the roles of SPCA1 and SPCA2 in breast cancer and how they correlate with the different subtypes. We present analysis of patient survival and microarray data for both SPCA1 and SPCA2 expression, and evaluate isoform-specific roles in two different breast cancer cell lines, which represent the luminal (MCF7) and the basal (MDA-MB-231) subtypes. Using knockdown and overexpression approaches, we have correlated Ca2+ dynamics with cell proliferation, tumorsphere formation, and plasma membrane trafficking of a cell adhesion protein critical for tumorigenesis, E-cadherin. From these studies, we found that SPCA2 and not SPCA1 promotes oncogenesis by regulating E-cadherin in luminal mammary tumors and that SPCA2 down regulation may be critical for metastasis in both subtypes.

Na+(K+)/H+ Exchangers (NHEs) are a large family of transport proteins that conduct electroneutral countertransport of cations with protons across lipid bilayers. A newly discovered Na+/H+ Exchanger...

Secretory Pathway Ca2+-ATPases (SPCA) are P-type pumps that localize to the Golgi and post-Golgi vesicular compartments into which they sequester Ca2+ and Mn2+ ions required for protein processing, glycosylation and sorting. There are two isoforms in human that play distinct roles in breast cancer, a leading cause of death in women worldwide. Previously, we showed that SPCA2, but not SPCA1, interacts with the Ca2+ channel Orai1 to initiate store-independent Ca2+ entry, which promotes tumorigenesis. We sought to understand the roles of SPCA1 and SPCA2 in breast cancer and how they correlate with the different subtypes and with epithelial-mesenchymal transition (EMT). SPCA2 expression is greatly amplified in luminal subtypes but has a lower expression in basal subtypes, with opposite expression patterns for SPCA1. We present analysis of patient survival and microarray data for both SPCA1 and SPCA2 expression, and evaluate isoform-specific roles in two different breast cancer cell lines, which represent epithelial (MCF7) and post-EMT (MDA-MB-231) subtypes. Using knockdown and overexpression approaches, we correlate isoform expression with cell proliferation, tumorsphere formation, and plasma membrane trafficking of a cell adhesion protein critical for tumorigenesis, E-cadherin. From these studies, we conclude that SPCA2 promotes oncogenesis by regulating E-cadherin and that SPCA2 down regulation may be critical for metastasis.

Breast cancer is one of the most common forms of cancer in women, causing over 40,000 deaths per year. The major concern with breast cancer is its tendency to metastasize, preferentially, to the brain, bone and other areas of high physiological calcium. Calcium is a vital component of the breast, as mammary tissue has the ability to sequester and compartmentalize such calcium. During lactation, mammary cells are required to concentrate the 2mM blood concentration of milk into the 40-80mM concentration of calcium found in milk. This action requires a highly coordinated group of calcium channels and pumps to seize the calcium into intracellular storage compartments. We have shown in previous studies that SPCA2 is highly upregulated during lactation and tumorigenesis. Additional studies from others have shown that TRP channels are also upregulated in lactation and tumorigenesis. Here we show that this induction is due to the interaction of these two molecular components of calcium homeostasis in breast tissue.