Helen M. Faddy

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?

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.

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.

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.

Implications of Dengue Outbreaks for Blood Supply, Australia

Dengue outbreaks have increased in size and frequency in Australia, and transfusion-transmitted dengue poses a risk to transfusion safety. Using whole blood samples collected during the large 2008–2009 dengue epidemic, we estimated the risk for a dengue-infectious blood donation as ≈1 in 7,146 (range 2,218–50,021).

Cytomegalovirus disease in immunocompetent adults.

Cytomegalovirus (CMV) is a highly prevalent and globally distributed virus. CMV infection in healthy adults is usually asymptomatic or causes a mild mononucleosis‐like syndrome. CMV disease causes significant morbidity and mortality in neonates and severely immunocompromised adults. CMV disease can present with a wide range of manifestations, with colitis being the most common. The incidence of severe CMV disease in immunocompetent adults appears to be greater than previously thought, which may be partly due to immune dysfunction related to comorbidities such as kidney disease or diabetes mellitus. CMV disease can mimic an array of alternative diagnoses and pose a significant diagnostic challenge, especially in immunocompetent adults, leading to delayed diagnosis, adverse health outcomes and unnecessary financial expense. Non‐invasive testing for CMV is widely available and can facilitate early diagnosis if used appropriately. Although limited, current evidence suggests that targeted antiviral therapy with ganciclovir or valganciclovir is appropriate for severe CMV disease in immunocompetent adults.

First reported case of transfusion-transmitted Ross River virus infection

PathWest Laboratory Medicine WA detected RRV IgM antibodies using an inhouse indirect immunofluorescence antibody (IFA) test, but no RRV antibodies were detected using an inhouse haemagglutination inhibition (HI) antibody test 10 days after blood donation. RRV IgM antibodies are detected by IFA testing within a few days of onset of illness and routinely persist for several weeks or, occasionally, months or years. IFA tests are less prone to false-positive results compared with enzyme immunoassays. The HI antibody test primarily detects IgG antibodies, which appear within several weeks but after the IgM response.

Hepatitis E virus and implications for blood supply safety, Australia.

To the Editor: Hepatitis E virus (HEV) is an emerging public health concern for industrialized countries (1). Although HEV infection has been associated with travel to countries where the virus is endemic, cases of autochthonous HEV are increasing (2). Detection of HEV RNA in blood donations in the United Kingdom, Germany, the Netherlands, Japan, and China and accumulating reports of HEV transmitted through blood transfusion highlight the potential risk this virus poses to blood supply safety (1–4). In Australia, where most HEV infections are associated with travel (5), an average of 25 HEV cases occurred each year during 1999–2013 (http://www9.health.gov.au/cda/source/rpt_3.cfm). HEV infection is nationally notifiable in Australia, but the presence of subclinical infections and the lack of recent seroprevalence studies have prevented the accurate estimation of HEV incidence and population exposure. Thus, we examined HEV seroprevalence in a cohort of Australian blood donors, assessed risk factors for exposure, and used the data to examine the effectiveness of current blood safety strategies for the management of HEV in Australia. Plasma samples (n = 3,237) were collected from donors during August–September 2013. Information on age, sex, state of residence, new/repeat donor status, and overseas travel disclosure was obtained. Details of any relevant blood donation deferral (malaria, diarrhea) applied on previous donation attempts were also collected. Application of a specific malaria deferral code is routine for donors disclosing travel to a malaria-endemic country, and a diarrhea deferral applies when a donor reports having had diarrhea (of viral or unknown cause) 1 week before any attempted donation. All samples were tested for HEV IgG by using the Wantai HEV-IgG ELISA (Beijing Wantai Biologic Pharmacy Enterprise Co., Ltd, Beijing, China). Positive samples were tested for HEV IgM by using the Wantai HEV-IgM ELISA and for HEV RNA by using a prototype transcription-mediated amplification assay (Hologic Inc., San Diego, CA, USA). Of 3,237 samples, 194 (5.99%) were positive for HEV IgG (95% CI 5.18–6.81). Compared with estimates from previous studies that used the Wantai ELISA (6–9), our estimate is comparable to those reported from Scotland (4.7%) and New Zealand (4.2%) but lower than those from the United States (18.8%) and southwestern France (52.5%). Considerable debate exists regarding the sensitivity and specificity of HEV detection methods (2,10); however, on the basis of studies in France and the United Kingdom (9,10), we believe that the measured seroprevalence in our study is accurate. Our findings showed an increased seroprevalence of HEV associated with previous malaria deferral, diarrhea deferral, and age (multivariate logistic regression) (Table), the latter of which is consistent with previous findings (9). IgG seropositivity was also higher (7.73%) in donors who had traveled to a malaria-endemic country. HEV is often endemic to malaria-endemic countries (http://wwwnc.cdc.gov/travel/yellowbook/2014); however, the HEV exposure status of travelers is unknown before departure, so the exact place of exposure cannot be determined. Furthermore, 3.37% of donors in our study had evidence of previous HEV exposure; these donors had not reported travel outside Australia, so they may have acquired HEV locally. Because subclinical HEV infection is possible, persons infected locally may not be identified by the current donor screening questionnaire and thus pose a potential risk to blood supply safety. Table HEV (IgG) seroprevalence, and risk factors for exposure, in Australian blood donors* Detection of HEV IgM in 4 (2.06%) of the 194 samples from IgG-positive donors indicates the donors had been recently exposed to HEV (95% CI 0.06–4.06). All 4 donors had traveled overseas; 3 reported travel to malaria-endemic countries. HEV RNA was not detected in any of the HEV IgG–positive samples. Although it is encouraging that HEV nucleic acid was not detected, the sample size is insufficient to accurately determine the true rate of HEV RNA carriage among donors in this study; a larger study is planned. Management strategies to safeguard the Australian blood supply against transfusion-transmitted HEV are based on donor selection guidelines. To identify donors with possible bacteremia/viremia, including HEV, blood donation staff members ask donors several medical, behavioral, and travel-based questions before donation. These include questions relating to general wellness, sex practices, gastric upset, diarrhea, abdominal pain, and vomiting within the previous week. In addition, for 4 months after a donor’s return from travel to a malaria-endemic country, donations are restricted to plasma for fractionation. Some protection against blood donations from HEV-infected persons may occur because HEV and malaria are co-endemic to many countries. Our findings showed a higher HEV seroprevalence among donors with prior malaria or diarrhea deferrals; thus, malaria- and diarrhea-related screening questions may reduce contributions from donors with travel-associated HEV infection. Our findings showed HEV exposure in travelers and nontravelers, suggesting the possibility of imported and locally acquired HEV in Australia. Prior HEV exposure was higher in donors who were temporarily excluded from donating blood on previous donation attempts, suggesting the current management strategy in Australia is partially effective in minimizing any risk of HEV transmission through blood transfusion. However, the presence of HEV IgG in donors who reported no overseas travel and/or no prior related deferrals, coupled with the knowledge that asymptomatic infection is possible, suggests that additional safety precautions may be warranted.

Inactivation of dengue, chikungunya, and Ross River viruses in platelet concentrates after treatment with ultraviolet C light

Arboviruses, including dengue (DENV 1‐4), chikungunya (CHIKV), and Ross River (RRV), are emerging viruses that are a risk for transfusion safety globally. An approach for managing this risk is pathogen inactivation, such as the THERAFLEX UV‐Platelets system. We investigated the ability of this system to inactivate the above mentioned arboviruses.

The effect of riboflavin and ultraviolet light on the infectivity of arboviruses

Arboviruses are an emerging threat to transfusion safety and rates of infection are likely to increase with the increased rainfall associated with climate change. Arboviral infections are common in Australia, where Ross River virus (RRV), Barmah Forest virus (BFV), and Murray Valley encephalitis virus (MVEV), among others, have the potential to cause disease in humans. The use of pathogen reduction technology (PRT) may be an alternative approach for blood services to manage the risk of arboviral transfusion transmission. In this study, the effectiveness of the Mirasol PRT (Terumo BCT) system at inactivating RRV, BFV, and MVEV in buffy coat (BC)‐derived platelets (PLTs) was investigated.