Wonnam Kim

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

Significance Unlike other ErbB receptors, human epidermal growth factor receptor 2 (HER2) does not generally become internalized after activation but, instead, remains on the cell surface to signal for prolonged periods. This property is thought to contribute to HER2’s ability to transform cells when overexpressed. The current study demonstrates that HER2’s resistance to endocytosis depends on the presence of the calcium pump, plasma membrane calcium ATPase2 (PMCA2), in specific membrane signaling domains in which intracellular calcium must be kept low to permit continued HER2 biochemical signaling. The dramatic reduction of mammary tumors in mouse mammary tumor virus (MMTV)-Neu mice in the absence of PMCA2 demonstrates its importance in supporting the development of breast tumors. Therefore, targeting interactions between PMCA2 and HER2 may offer therapeutic strategies for breast cancer. 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-Sensing Receptor Promotes Breast Cancer by Stimulating Intracrine Actions of Parathyroid Hormone–Related Protein

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 p27(kip1) levels and prevented nuclear accumulation of the proapoptotic factor apoptosis inducing factor. 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. Cancer Res; 76(18); 5348-60. ©2016 AACR.

The scaffolding protein NHERF1 regulates the stability and activity of the tyrosine kinase HER2.

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 in situ (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.

OPG Treatment Prevents Bone Loss During Lactation But Does Not Affect Milk Production or Maternal Calcium Metabolism

Lactation is associated with increased bone turnover and rapid bone loss, which liberates skeletal calcium used for milk production. Previous studies suggested that an increase in the skeletal expression of receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand (RANKL) coupled with a decrease in osteoprotegerin (OPG) levels likely triggered bone loss during lactation. In this study, we treated lactating mice with recombinant OPG to determine whether bone loss during lactation was dependent on RANKL signaling and whether resorption of the maternal skeleton was required to support milk production. OPG treatment lowered bone resorption rates and completely prevented bone loss during lactation but, surprisingly, did not decrease osteoclast numbers. In contrast, OPG was quite effective at lowering osteoblast numbers and inhibiting bone formation in lactating mice. Furthermore, treatment with OPG during lactation prevented the usual anabolic response associated with reversal of lactational bone loss after weaning. Preventing bone loss had no appreciable effect on milk production, milk calcium levels, or maternal calcium homeostasis when mice were on a standard diet. However, when dietary calcium was restricted, treatment with OPG caused maternal hypocalcemia, maternal death, and decreased milk production. These studies demonstrate that RANKL signaling is a requirement for bone loss during lactation, and suggest that osteoclast activity may be required to increase osteoblast numbers during lactation in preparation for the recovery of bone mass after weaning. These data also demonstrate that maternal bone loss is not absolutely required to supply calcium for milk production unless dietary calcium intake is inadequate.

HER2 signaling regulates HER2 localization and membrane retention

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.

Calcium-Sensing Receptor in Breast Physiology and Cancer

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.

Modernization of Traditional Oriental Medicine: New Dosage Forms and Medical Instruments

Traditional OrientalMedicine (TOM) has been developed for thousands of years; however recent developments in pharmaceutical technology promote modernization of TOM rapidly to expand accessibility, ease of administration, and costeffectiveness. Various forms of herbal medicine are improved for better efficiency and consumer preferences including soft extracts, granules, intranasal administrations, eye drop, drop pills, injection, and capsules. In-depth researches on safety and efficacy of these formulations are ongoing. Another trend on modernization of TOM is an integrated application with technology. Progress on technology has developed oriental medical instruments. For example, modern electrical engineering makes electroacupuncture, laser acupuncture, and laser moxibustion possible. Experimental and translational studies on safety and efficacy of these new methods are underway. Here we highlight some of the key ongoing challenges published in this special issue. S. Y. Suh and W. G. An used a modernized method to understand herbal medicine, such as herb-compound-target network and target-pathway network analysis, to study Bulsu-san commonly used for pregnant women in East Asia. In the results, the authors report that most compounds in Bulsu-san work together with multiple target genes in a synergetic way. C.-C. Yang et al. and M.L. Lin et al. used new treatment methods. C.-C. Yang et al. investigated vascular and autonomic impacts of combined acupuncture-far infrared radiation in improving peripheral circulation. M.-L. Lin et al. evaluated the effectiveness of laser acupuncture plus Chinese cupping in chronic nonspecific lower back pain treatment, which could further be a suitable option for lower back pain treatment in clinical settings. Y.-S. Lee et al. and T. Shu et al. integrated technology to increase understanding of TOM. Y.-S. Lee et al. used a data mining procedure, determined by the application of a term frequency-inverse document frequency weighting scheme to the cooccurrence table, to analyze relationships between emotions and the visceral system according to the principles of East Asian medicine. T. Shu et al. proposed an effective noninvasive computerized method based on facial images to quantitatively detect heart disease. In this special issue, we present 14 papers that address the issue about modernization of TOM, focusing on new approaches by herbal medicine and medical instruments.

Inhibitory effects of Aconiti Lateralis Radix Preparata on chronic intermittent cold-induced inflammation in the mouse hypothalamus

ETHNOPHARMACOLOGICAL RELEVANCE Aconiti Lateralis Radix Preparata (AR) is the most frequently used herb to generate heat and treat symptoms associated with coldness in Asia. AIMS OF THE STUDY The hypothalamus is one of the master regulators to maintain constant core body temperature. Chronic exposure to cold stress disturbs homeostatic regulation, gradually resulting in hypothalamic inflammation. This study investigate the effects of AR, on the chronic intermittent cold (CIC)-induced release of pro-inflammatory signaling molecules in the mouse hypothalamus. MATERIALS AND METHODS Aconiti Lateralis Radix Preparata extract (ARE) were solubilized in distilled water and diluted with saline before administration. Male ICR mice (7 weeks old, 30-32g) were divided randomly into 6 groups: (1) control, (2) cold stress, (3) ARE 30, (4) ARE 100, (5) ARE 300, and (6) ARE 1000mg/kg groups. Groups (2)-(6) were exposed to CIC stress once a day for 14 days. CIC stress was achieved by exposing the mice to 4°C and 60 ± 10% humidity for 120min once a day. Rectal temperature was measured after terminating cold stress. Cortisol levels were measured from serum. Hypothalamus tissue was used for western blot analysis, and IL-9, IL-13, PGE1, and PGE2 levels were assessed. RESULTS ARE treatment prevented the CIC-induced decrease in rectal temperature and increase in serum cortisol level. ARE-treated CIC-exposed mice demonstrated decrease in nuclear c-Fos levels dose-dependently compared to CIC-exposed mice. Nuclear NF-kB expression showed significant increase in CIC-exposed mice. ARE treatment significantly blunted the increase in nuclear NF-kB expression. CIC-exposed mice had significantly increased levels of both IL-9 and IL-13. Treatment with ARE suppressed the elevated IL-9 and IL-13 levels. Between control and CIC-exposed mice PGE1 levels showed no difference. However ARE (1000mg/kg)-treated CIC-exposed mice had a significant increase in PGE1 level compared to CIC-exposed mice. PGE2 levels were significantly higher in CIC-exposed mice compared to control mice. ARE treatment significantly attenuated the increase in PGE2 levels. CONCLUSIONS Our findings suggest CIC stress disturbs the anti-inflammatory effect of cortisol and maintenance of the body temperature. Thus AR contributes to suppress the activated proinflammatory factors, IL-9, IL-13, and PGE-2, and to increase the heat production.

An Insight into Ginsenoside Metabolite Compound K as a Potential Tool for Skin Disorder

Ginsenosides are the major bioactive natural compounds derived from Panax ginseng. Several studies report the pharmaceutical benefits of several ginsenosides, including antidementia, antitumor, and anti-inflammatory activity. Biotransformations by gut microbiome contribute to the biological function of these ginsenosides. After ingestion ginsenosides are hydrolyzed to Rg2, Rg3, compound K, and others by human gut flora. Compound K is considered the representative active metabolite after oral administration of ginseng or ginsenosides. Various studies report the diverse biological functions of compound K, such as antitumor, antidiabetic, antiallergic, and anti-inflammatory activity. Recent clinical trial and in vitro studies demonstrate the antiaging activities of ginsenosides in human skin. Ginsenosides have been considered as an important natural dermatological agent. In this review, we will cover the modern tools and techniques to understand biotransformation and delivery of compound K. Also the biological function of compound K on skin disorder and its potential dermatological application will be discussed.

Abstract P3-05-05: PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway

PTHrP affects mammary development and breast cancer. PTHrP is expressed in basal myoepithelial cells until late pregnancy, when it also becomes expressed in alveolar epithelial cells. During lactation, PTHrP is secreted into milk and into the circulation, and regulates systemic calcium and bone metabolism. Secretion of PTHrP by breast cancers stimulates bone resorption, promoting the skeletal metastases and/or development of hypercalcemia. Less is known about the contribution of PTHrP to development and/or progression of primary breast cancers. Recent GWAS reports have identified the PTHrP gene as a breast cancer susceptibility locus. Using data from the Cancer Genome Atlas (TCGA) Project and a Yale tissue microarray (YTMA49) we have found that increased breast tumor PTHrP expression predicts a more aggressive phenotype and increased mortality, signifying the clinical relevance of high tumor PTHrP levels. In an attempt to define how PTHrP affects breast cancer, we assessed the effects of PTHrP overexpression on the development of mammary tumors in mice. We developed a tetracyline-regulated, MMTV-driven transgenic model of PTHrP overexpression targeted to mammary epithelial cells (MMTV-rtTA;tetO-hPTHrP). Surprisingly, we found that PTHrP overexpression in luminal epithelial cells causes alveolar hyperplasia, secretory differentiation and milk production in virgin mice, associated with a lower number of luminal progenitor cells and basal stem cells upon FACS analysis. Additionally, Signal Transducer and Activator of Transcription 5 (Stat5) is highly activated in the setting of PTHrP-induced alveolar hyperplasisa. This activation is functionally relevant as evidenced by higher levels of β-Casein mRNA and protein, the transcription of which is regulated by activated Stat5 (pStat5). This effect of PTHrP was reversible upon withdrawal of doxycycline (Dox). Addition of Dox to primary mammary epithelial cell cultures from these mice recapitulated the findings. Knocking out PTHrP receptor (PTH1R) in these mice did not affect this phenotype, suggesting that PTHrP acts in a receptor-independent manner. Overexpression of PTHrP in MMTV-PyMT mice dramatically accelerated formation of mammary tumors reducing both latency of tumor formation and survival of the mice. It caused a higher rate of proliferation and a lower rate of apoptosis in vivo. All MMTV-rtTA;tetO-hPTHrP;MMTV-PyMT mice developed palpable tumors in all mammary glands within 3 weeks of age, became hypercalcemic and died before 4.5 weeks of age. These mice had higher levels of pStat5 and βCasein. Tumor cells cultured ex vivo and cell lines established from these tumors overexpressed PTHrP when stimulated with Dox. PTHrP overexpression, and not exogenous PTHrP, caused higher levels of proliferation, pStat5, and β-Casein. This PTHrP-induced activity of Stat5 was blocked by treatment of these cells with a pharmacological inhibitor of Jak2, a tyrosine kinase upstream of Stat5. Additionally, T47D cells transfected with PTHrP showed higher levels of pStat5, suggesting that PTHrP could activate Stat5 signaling regardless of the cell type. We show that PTHrP overexpression results in Stat5 activation and increased proliferation in breast cancer cells in a cell-autonomous fashion and independent of its receptor. Citation Format: Takyar FM, Boras-Granic K, Kim W, Dann P, Wysolmerski JJ. PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-05.