PMCA2 regulates HER2 protein kinase localization and signaling and promotes HER2-mediated breast cancer

Abstract

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 ...

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

“…Initial experiments showed that PMCA2 silencing alone does not alter MDA-MB-231 cell viability. Since PMCA isoforms are known regulators of calcium-induced cell death [3,5,7], we assessed the consequence of PMCA2 silencing on ionomycin death responses. Our results indicated that PMCA2 silencing modestly promoted ionomycin MDA-MB-231 cell death at submaximal concentrations.…”

“…PMCA2 mRNA was higher in basal breast cancers compared to HER2, luminal A and luminal B subtypes [4]. Although a feature of some breast cancers, only a few studies have evaluated the functional significance of PMCA2 upregulation in the context of cell death in breast cancer; and detailed assessment of PMCA2 through overexpression or silencing techniques has been limited to luminal-like breast cancer cell lines that express at least one hormone receptor (estrogen or progesterone receptors) or those with HER2 amplification [3,5]. Heterologous PMCA2 expression in T47D cells attenuates global cytosolic free Ca 2+ levels ([Ca 2+ ]CYT) and protects T47D cells from calcium-induced cell death initiated with ionomycin [3].…”

“…Heterologous PMCA2 expression in T47D cells attenuates global cytosolic free Ca 2+ levels ([Ca 2+ ]CYT) and protects T47D cells from calcium-induced cell death initiated with ionomycin [3]. Assessment of PMCA2 silencing in the context of HER2 positive breast cancers has been examined in the SKBR3 and BT474 breast cancer cell lines [5], both of which are positive for HER2 amplification [6]. PMCA2 knockdown in SKBR3 promotes ionomycin cell death and downregulates HER2 signaling [5].…”

“…Assessment of PMCA2 silencing in the context of HER2 positive breast cancers has been examined in the SKBR3 and BT474 breast cancer cell lines [5], both of which are positive for HER2 amplification [6]. PMCA2 knockdown in SKBR3 promotes ionomycin cell death and downregulates HER2 signaling [5].…”

“…PMCA2 silencing-mediated effects on breast cancer cell death have focused on ionomycin [5], which can trigger cell death through sustained [Ca 2+ ]CYT increases of high magnitude [5,7,8].…”