In the lactating mammary gland, the plasma membrane calcium ATPase2 (PMCA2) transports milk calcium. Its expression is activated in breast cancers, where high tumor levels predict increased mortality. We find that PMCA2 expression correlates with HER2 levels in breast cancers and that PMCA2 interacts with HER2 in specific actin-rich membrane domains. Knocking down PMCA2 increases intracellular calcium, disrupts interactions between HER2 and HSP-90, inhibits HER2 signaling, and results in internalization and degradation of HER2. Manipulating PMCA2 levels regulates the growth of breast cancer cells, and knocking out PMCA2 inhibits the formation of tumors in mouse mammary tumor virus (MMTV)-Neu mice. These data reveal previously unappreciated molecular interactions regulating HER2 localization, membrane retention, and signaling, as well as the ability of HER2 to generate breast tumors, suggesting that interactions between PMCA2 and HER2 may represent therapeutic targets for breast cancer.calcium pumps | ErbB2 | receptor internalization | HSP-90 | epidermal growth factor receptor P lasma membrane calcium ATPases (PMCAs) are a family of ion pumps that transport calcium out of cells and maintain low resting intracellular calcium levels (1-3). PMCA2 (gene symbol Atp2b2) is highly expressed in the apical membrane of mammary epithelial cells only during lactation, where it has been shown to transport calcium into milk (4-6). After weaning, PMCA2 expression rapidly decreases, contributing to the initiation of programmed cell death and mammary gland involution (7,8). PMCA2 is also expressed in breast cancers (8-10), and high levels of tumor PMCA2 expression predict increased mortality in patients (8).Approximately 25-30% of invasive breast cancers overexpress human epidermal growth factor receptor 2 (HER2) as a result of amplification of the ERBB2 kinase gene (11-13), and overexpression of HER2 causes breast tumors in mouse mammary tumor virus (MMTV)-Neu transgenic mice (14). HER2 functions as a heterodimer with other ERBB family members, most commonly pairing with EGFR or human epidermal growth factor receptor 3 (HER3) in breast cancers (11, 13). For reasons that remain poorly understood, in contrast to other ERBB family members, which are internalized and degraded after stimulation, HER2 remains on the cell surface and continues to signal for prolonged periods (12,15).In this study, we describe a previously unrecognized function for PMCA2: supporting active HER2 signaling and HER2-mediated tumor formation. Our data suggest that PMCA2 interacts with HER2 within specific membrane domains and is required for HER2 expression, membrane retention, and signaling.Results PMCA2 and HER2 Are Coexpressed in Breast Cancers. PMCA2 levels correlate with HER2 in breast tumors (8). To further explore potential interactions between PMCA2 and HER2, we analyzed their expression in a previously reported tissue microarray consisting of 652 breast cancers with a median 9 y of clinical follow-up (8, 16). Patients with the highest quartiles of ...

Parathyroid hormone-related protein (PTHrP) contributes to the development and metastatic progression of breast cancer by promoting hypercalcemia, tumor growth and osteolytic bone metastases, but it is not known how PTHrP is upregulated in breast tumors. Here we report a central role in this process for the calcium-sensing receptor, CaSR, which enables cellular responses to changes in extracellular calcium, through studies of CaSR-PTHrP interactions in the MMTV-PymT transgenic mouse model of breast cancer and in human breast cancer cells. CaSR activation stimulated PTHrP production by breast cancer cells in vitro and in vivo. Tissue-specific disruption of the casr gene in mammary epithelial cells in MMTV-PymT mice reduced tumor PTHrP expression and inhibited tumor cell proliferation and tumor outgrowth. CaSR signaling promoted the proliferation of human breast cancer cell lines and tumor cells cultured from MMTV-PyMT mice. Further, CaSR activation inhibited cell death triggered by high extracellular concentrations of calcium. The actions of the CaSR appeared to be mediated by nuclear actions of PTHrP that decreased p27kip1 levels and prevented nuclear accumulation of the pro-apoptotic factor AIF. Taken together, our findings suggest that CaSR-PTHrP interactions might be a promising target for the development of therapeutic agents to limit tumor cell growth in bone metastases and in other microenvironments in which elevated calcium and/or PTHrP levels contribute to breast cancer progression.

ErbB2/HER2/Neu is a receptor tyrosine kinase that is overexpressed in 25–30% of human breast cancers, usually associated with amplification of the ERBB2 gene. HER2 has no recognized ligands and heterodimers between HER2 and EGFR (ErbB1/HER1) or HER2 and ErbB3/HER3 are important in breast cancer. Unlike other ErbB family members, HER2 is resistant to internalization and degradation, and remains at the cell surface to signal for prolonged periods after it is activated. Although the mechanisms underlying retention of HER2 at the cell surface are not fully understood, prior studies have shown that, in order to avoid internalization, HER2 must interact with the chaperone, HSP90, and the calcium pump, PMCA2, within specific plasma membrane domains that protrude from the cell surface. In this report, we demonstrate that HER2 signaling, itself, is important for the formation and maintenance of membrane protrusions, at least in part, by maintaining PMCA2 expression and preventing increased intracellular calcium concentrations. Partial genetic knockdown of HER2 expression or pharmacologic inhibition of HER2 signaling causes the depletion of membrane protrusions and disruption of the interactions between HER2 and HSP90. This is associated with the ubiquitination of HER2, its internalization with EGFR or HER3, and its degradation. These results suggest a model by which some threshold of HER2 signaling is required for the formation and/or maintenance of multi-protein signaling complexes that reinforce and prolong HER2/EGFR or HER2/HER3 signaling by inhibiting HER2 ubiquitination and internalization.

The calcium-sensing receptor (CaSR) is expressed in normal breast epithelial cells and in breast cancer cells. During lactation, activation of the CaSR in mammary epithelial cells increases calcium transport into milk and inhibits parathyroid hormone-related protein (PTHrP) secretion into milk and into the circulation. The ability to sense changes in extracellular calcium allows the lactating breast to actively participate in the regulation of systemic calcium and bone metabolism, and to coordinate calcium usage with calcium availability during milk production. Interestingly, as compared to normal breast cells, in breast cancer cells, the regulation of PTHrP secretion by the CaSR becomes rewired due to a switch in its G-protein usage such that activation of the CaSR increases instead of decreases PTHrP production. In normal cells the CaSR couples to Gαi to inhibit cAMP and PTHrP production, whereas in breast cancer cells, it couples to Gαs to stimulate cAMP and PTHrP production. Activation of the CaSR on breast cancer cells regulates breast cancer cell proliferation, death and migration, in part, by stimulating PTHrP production. In this article, we discuss the biology of the CaSR in the normal breast and in breast cancer, and review recent findings suggesting that the CaSR activates a nuclear pathway of PTHrP action that stimulates cellular proliferation and inhibits cell death, helping cancer cells adapt to elevated extracellular calcium levels. Understanding the diverse actions mediated by the CaSR may help us better understand lactation physiology, breast cancer progression and osteolytic bone metastases.

We examined whether the scaffolding protein sodium-hydrogen exchanger regulatory factor 1 (NHERF1) interacts with the calcium pump PMCA2 and the tyrosine kinase receptor ErbB2/HER2 in normal mammary epithelial cells and breast cancer cells. NHERF1 interacts with the PDZ-binding motif in PMCA2 in both normal and malignant breast cells. NHERF1 expression is increased in HER2-positive breast cancers and correlates with HER2-positive status in human ductal carcinoma (DCIS) lesions and invasive breast cancers as well as with increased mortality in patients. NHERF1 is part of a multiprotein complex that includes PMCA2, HSP90, and HER2 within specific actin-rich and lipid raft-rich membrane signaling domains. Knocking down NHERF1 reduces PMCA2 and HER2 expression, inhibits HER2 signaling, dissociates HER2 from HSP90, and causes the internalization, ubiquitination, and degradation of HER2. These results demonstrate that NHERF1 acts with PMCA2 to regulate HER2 signaling and membrane retention in breast cancers.

Edited by Alex TokerUnlike other ErbB family members, HER2 levels are maintained on the cell surface when the receptor is activated, allowing prolonged signaling and contributing to its transforming ability. Interactions between HER2, HSP90, PMCA2, and NHERF1 within specialized plasma membrane domains contribute to the membrane retention of HER2. We hypothesized that the scaffolding protein ezrin, which has been shown to interact with NHERF1, might also help stabilize the HER2-PMCA2-NHERF1 complex at the plasma membrane. Therefore, we examined ezrin expression and its relationship with HER2, NHERF1, and PMCA2 levels in murine and human breast cancers. We also used genetic knockdown and/or pharmacologic inhibition of ezrin, HSP90, NHERF1, PMCA2, and HER2 to examine the functional relationships between these factors and membrane retention of HER2. We found ezrin to be expressed at low levels at the apical surface of normal mammary epithelial cells, but its expression is up-regulated and correlates with HER2 expression in hyperplasia and tumors in murine mammary tumor virus-Neu mice, in human HER2-positive breast cancer cell lines, and in ductal carcinoma in situ and invasive breast cancers from human patients. In breast cancer cells, ezrin co-localizes and interacts with HER2, NHERF1, PMCA2, and HSP90 in specialized membrane domains, and inhibiting ezrin disrupts interactions between HER2, PMCA2, NHERF1, and HSP90, inhibiting HER2 signaling and causing PKC␣-mediated internalization and degradation of HER2. Inhibition of ezrin synergizes with lapatinib in a PKC␣-dependent fashion to inhibit proliferation and promote apoptosis in HER2-positive breast cancer cells. We conclude that ezrin stabilizes a multiprotein complex that maintains active HER2 at the cell surface. . 2 The abbreviations used are: EGFR, epidermal growth factor receptor; ERM, ezrin/radixin/moesin/merlin; MMTV, murine mammary tumor virus; DCIS, ductal carcinoma in situ; PH, pleckstrin homology; EGF, epidermal growth factor; PLA, proximity ligation assay; IP, immunoprecipitation; shRNA, short hairpin RNA; KD, knockdown; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; CA, constitutively active; BrdU, bromodeoxyuridine; DAPI, 4Ј,6-diamidino-2-phenylindole.

The root of Astragalus membranaceus B(UNGE) (AM) is a medicinal herb that has been capable of reducing the adverse effects of conventional chemotherapy. To investigate the effects of AM on cyclophosphamide (CP)-induced reproductive toxicity in mouse testes, 5-week-old male imprinting control region mice were divided into five groups; CP was treated on the first day of each week for 5 weeks (100 mg/kg, i.p.), and AM was treated for 5 days a week for 5 weeks. At the end of the treatment period, the testes were taken out, cleared of the adhering tissues, and weighed. Epididymis was taken out and used for sperm analysis. Testis samples were frozen for real-time quantitative PCR and Western blot analysis. AM treatment increased diminished relative testes weight, and sperm count and motility in mice treated with CP. CP treatment has detrimental effects on the expression of cAMP-responsive element modulator (CREM), a transcription factor that is highly expressed in male germ cells and is crucial to post-meiotic germ cell differentiation. AM restored CREM at both the mRNA and protein levels. AM has beneficial influences and appears able to ameliorate relative testes weight, sperm parameters, and CREM expression against CP-induced reproductive toxicity.

We hypothesized that adaptation to calcium supply demands of pregnancy and lactation do not require calcitriol. Adult Cyp27b1 null mice lack calcitriol and have hypocalcemia, hypophosphatemia, and rickets. We studied wild-type (WT) and null sister pairs raised on a calcium-, phosphorus-, and lactose-enriched "rescue" diet that prevents hypocalcemia and rickets. Bone mineral content (BMC) increased >30% in pregnant nulls, declined 30% during lactation, and increased 30% by 4 weeks post-weaning. WT showed less marked changes. Micro-CT revealed loss of trabecular bone and recovery in both genotypes. In lactating nulls, femoral cortical thickness declined >30%, whereas endocortical perimeter increased; both recovered to baseline after weaning; there were no such changes in WT. Histomorphometry revealed a profound increase in osteoid surface and thickness in lactating nulls, which recovered after weaning. By three-point bend test, nulls had a >50% decline in ultimate load to failure that recovered after weaning. Although nulls showed bone loss during lactation, their milk calcium content was 30% lower compared with WT. Serum parathyroid hormone (PTH) was markedly elevated in nulls at baseline, reduced substantially in pregnancy, but increased again during lactation and remained high post-weaning. In summary, pregnant Cyp27b1 nulls gained BMC with reduced secondary hyperparathyroidism, implying increased intestinal calcium delivery. Lactating nulls lost more bone mass and strength than WT, accompanied by increased osteoid, reduced milk calcium, and worsened secondary hyperparathyroidism. This implies suboptimal intestinal calcium absorption. Post-weaning, bone mass and strength recovered to baseline, whereas BMC exceeded baseline by 40%. In conclusion, calcitriol-independent mechanisms regulate intestinal calcium absorption and trabecular bone metabolism during pregnancy and post-weaning but not during lactation; calcitriol may protect cortical bone during lactation.