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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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Background Parathyroid hormone-related protein (PTHrP) is a major cause of humoral hypercalcemia of malignancy. PTHrP-induced hypercalcemia can also occur in benign disorders including pneumonia, SLE, HIV, sarcoidosis, and mammary hyperplasia. Treatment for PTHrP-induced hypercalcemia includes treating the underlying disorder and general management of hypercalcemia. Clinical Case A 70-year-old male with a chronic left thigh mass following trauma 12 years prior diagnosed as myositis ossificans, presented with worsening pain and swelling over the mass. Evaluation revealed serum calcium 18.6 mg/dl, PTH 10 ng/L (18-90), 25(OH)D 7 ng/ml (20-50), 1,25(OH)2D 15 pg/ml (20-79), PTHrP 9.3 pmol/L (≤ 4.2), negative SPEP, UPEP, serum light chains and immunofixation, and no bony lesions. MRI showed a large heterogenous mass centered in the left adductor muscle compatible with chronic hematoma with myositis ossificans. FDG PET/CT scan showed FDG avidity and calcification of the left thigh without any other site of uptake. Combination of IV hydration, denosumab and zoledronic acid lowered his calcium to 9.5 mg/dl, but levels increased again within 10 days. Repeated treatments with zoledronic acid initially led to modest lowering of calcium to 10.5 mg/dl, but he became refractory to treatment after 6 weeks, with calcium increasing to 12.3 mg/dl.Two biopsies of the mass were performed, including an open biopsy. The pathology showed nonpolarizable crystalline material with foreign giant cell reaction, without evidence for malignancy. It was concluded therefore that the patient's PTHrP-induced hypercalcemia was secondary to an inflammatory granulomatous condition. Since it had been reported that PTHrP-induced hypercalcemia in SLE and sarcoidosis responded to glucocorticoids, we initiated treatment with methylprednisolone 24 mg daily. After 7 days, the calcium declined from 12.3 to 10.5 mg/dl and PTHrP declined to 2.5 pmol/L. When the methylprednisolone dose was reduced to 12 mg daily, calcium increased to 11.4 mg/dl and PTHrP to 5 pmol/L. Increasing the methylprednisolone briefly back to 24 mg daily, and then 20 mg daily, lowered calcium to 9.0 mg/dl, increased PTH to 74 ng/L, and normalized PTHrP to 1.3 pmol/L. As long-term management with high-dose steroids was not a tenable option, a decision was made to resect the mass. The pathological diagnosis of the surgical mass was low-grade fibromyxoid sarcoma (LGFMS). Immunohistochemical studies identified PTHrP in surrounding vasculature, suggesting possible tumor-vessel interaction. Conclusion PTHrP-mediated hypercalcemia is rarely seen in sarcomas and treatments include coadministration of bisphosphonates, chemotherapy and surgery. We report here the first case of PTHrP-induced hypercalcemia from LGFMS with sustained lowering of calcium and PTHrP levels with glucocorticoid treatment. Further studies are needed to elucidate the mechanism of glucocorticoids in the treatment of PTHrP-induced hypercalcemia and could include lowering levels of inflammatory cytokines that stimulate PTHrP production from tumor cells, or by directly suppressing PTHrP gene expression. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning results in the distension of the alveolar structures by the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 hours of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GcAMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6 and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis. This is the result of increased TGFβ signaling and inhibition of cell cycle progression. Finally, we demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3, which also appears to be mediated by TGFβ signaling. In summary, these data suggest that intracellular calcium serves as an important biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.TeaserIncrease intracellular calcium leads to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

Human epidemiologic studies support a strong link between increased pancreatic adenocarcinoma (PDAC) risk and high fat diet (HFD) consumption, obesity, and overall energy imbalance. Given the rapid rise in worldwide obesity rates and the prevalence of western diets rich in fat, deciphering these mechanisms is not only a societal imperative but also represents a key untapped target to develop novel strategies for prevention and therapy. The translational relevance of diet research, however, has been limited by inconsistencies in fat source and consumption across human populations and mouse studies. Therefore, whether and how specific dietary fatty acids drive cancer development is poorly understood. To identify commonly consumed dietary fats capable of promoting pancreatic tumorigenesis, we fed a novel panel of 12 isocaloric HFDs – differing solely in fat source and representing the diversity of modern human fat consumption (as per statistics from the USDA) – to an oncogenic Kras-driven mouse model (KC: Pdx1-Cre; KrasLSL-G12D/+) that closely mimics the genetic and histologic features of human PDAC progression. Surprisingly, we found that diets rich in oleic acid (OA), a monounsaturated fat typically associated with good health, were strongly correlated with enhanced precancerous pancreatic intraepithelial neoplasia (PanIN) formation, arguing that OA enhances Kras-induced cellular transformation and early progression. High-oleic diets (HODs) and OA treatment of primary acinar cells induced loss of acinar cell identity and acquisition of ductal markers, consistent with a direct role for OA in acinar-to-ductal metaplasia (ADM), a prerequisite step in early PDAC development. Lipidomic analyses of plasma, liver, and muscle of mice fed HODs revealed greater circulating OA levels and increased tissue incorporation of OA into the acyl chains of phospholipids and sphingolipids, which make up the plasma membrane of cells and mediate intracellular and extracellular signaling. Furthermore, plasma and tissue OA levels more strongly correlated with tumor development, suggesting that direct OA pancreatic tissue incorporation could drive tumorigenesis. Indeed, molecular and biochemical analyses confirmed upregulation in the expression of de novo lipogenesis genes, alterations in lipid metabolism, and enhanced mTOR signaling in pancreata of mice fed HODs. Overall, these results directly link dietary OA to pancreatic lipid metabolism and transformation during PDAC development and highlight the complex pleiotropic effects of dietary fatty acids on health and disease: OA, while beneficial for heart health, may promote the development of certain cancers, such as PDAC. Uncovering the links between specific dietary fats and tumorigenesis are critical to enable precision nutritional guidance for the prevention and treatment of PDAC and potentially other obesity-associated cancers. Citation Format: Christian F. Ruiz, Rylee McDonnell, Jennifer Kaplan, Jasper de Jong, Christine Shugrue, Michael C. Rudolph, Fred S. Gorelick, John Wysolmerski, Matthew Rodeheffer, Mandar D. Muzumdar. Excess dietary oleic acid primes the pancreas for cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR015.

SUMMARY The lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target., Graphical Abstract, In brief Jeong et al. show that the formation of MAL2-mediated lipid raft-rich membrane protrusions is crucial for HER2 signaling in breast cancer cells. MAL2 is required for the formation of HER2/Ezrin/NHERF1/PMCA2 protein complexes. Formation of these protein complexes leads to a low calcium environment in the plasma membrane