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.

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH-and salt-sensitive manner (K D ‫؍‬ 101 ؎ 31 nM in 10 mM HEPES, 150 mM NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/ SPRY/B30.2 domain. The potential significance of BTN1A1/ XOR interactions in the mammary gland and other tissues is discussed. Members of the butyrophilin (BTN)3 gene family are attracting increasing attention because they may play multifunctional roles in diverse physiologies, including lactation (1, 2), selection and regulation of T-cells in the immune system (3-6), and modulation of autoimmune disease (7-9). BTN proteins have the canonical structures of cell surface receptors, which, after an N-terminal signal sequence, generally comprise two exoplasmic Ig folds (10, 11), a membrane anchor and a cytoplasmic domain consisting of a stem region, a PRY/SPRY/B30.2 domain (12, 13), and a cytoplasmic tail at the C terminus (14).The eponymous BTN1A1 protein has been linked to the secretion of milk lipid droplets because it is highly expressed in the mammary epithelium during lactation and is incorporated into the surface membrane coat surrounding cytoplasmic lipid droplets (the milk fat globule membrane (MFGM)) as they bud into milk from the apical surface (15). Furthermore, ablation of the Btn1a1 gene disrupts lipid secretion, causing the accumulation of large pools of triacylglycerol in the cytoplasm of Btn1a1 null mice (1). In a different context, dietary exposure to BTN1A1 in dairy products has been associated with modulation of the autoimmune disease multiple sclerosis because of structural similarities between the IgI fold of BTN1A1 (16) and the IgV fold of myelin oligodendrocyte glycoprotein (MOG) (17) an antigen on the myelin nerve sheath that is...

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.

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts, either with itself, or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced-green-fluorescent-protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5–0.7%, (w/w) of the total protein, i.e., over fifty-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets, mark it as a potential mobile signaling molecule in milk.

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.

Summary Enhanced stemness in colorectal cancer has been reported and it contributes to aggressive progression, but the underlying mechanisms remain unclear. Here we report a Wnt ligand, Dickkopf-2 (DKK2) is essential for developing colorectal cancer stemness. Genetic depletion of DKK2 in intestinal epithelial or stem cells reduced tumorigenesis and expression of the stem cell marker genes including LGR5 in a model of colitis-associated cancer. Sequential mutations in APC , KRAS , TP53 , and SMAD4 genes in colonic organoids revealed a significant increase of DKK2 expression by APC knockout and further increased by additional KRAS and TP53 mutations. Moreover, DKK2 activates proto-oncogene tyrosine-protein kinse Src followed by increased LGR5 expressing cells in colorectal cancer through degradation of HNF4α1 protein. These findings suggest that DKK2 is required for colonic epithelial cells to enhance LGR5 expression during the progression of colorectal cancer.