American Association for Cancer Research
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supplemental figures from Calcium-Sensing Receptor Promotes Breast Cancer by Stimulating Intracrine Actions of Parathyroid Hormone–Related Protein

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posted on 2023-03-30, 23:48 authored by Wonnam Kim, Farzin M. Takyar, Karena Swan, Jaekwang Jeong, Joshua VanHouten, Catherine Sullivan, Pamela Dann, Herbert Yu, Nathalie Fiaschi-Taesch, Wenhan Chang, John Wysolmerski

Supplemental Figure 1. A&B) Whole mount preparations of mammary glands from 9-week-old virgin CaSR; Supplemental Figure 2. A) Representative immunoblot showing total cdk2 levels in BT474 cells {plus minus} NPS2143 or control, CaSRKD, and PTHrPKD BT474 cells at different calcium concentrations; Supplemental Figure 3. A) Representative TUNEL staining in control MDA-MB-231.1833 cells {plus minus} NPS2143, and CaSRKD and PTHrPKD MDA-MB-231.1833 cells at different calcium concentrations; Supplemental Figure 4. A) Representative immunoblot for AIF in cytoplasmic or nuclear extracts from cells cultured from control MMTV-PyMT tumors {plus minus} NPS2143 and exposed to 5mM extracellular calcium; Supplemental Figure 5. BrdU incorporation in CaSRKD (A) or PTHrPKD (B) MDA-MB-231.1833 cells {plus minus} 10nM or 100nM PTHrP.



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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 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.

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