Identification of a novel 17,000-dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy

Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome which is characterized by hypercalcemia resulting from secretion by tumors of a circulating bone-resorbing factor. Evidence suggests that in many instances this factor is an adenylate cyclase-stimulating protein which shares features with, but is distinct from, parathyroid hormone (PTH). The current report describes the purification to homogeneity from a humoral hypercalcemia of malignancy-associated tumor of a novel, basic, highly potent PTH-like adenylate cyclase-stimulating protein. This factor differs from previously described PTH-like factors with respect to size, amino acid composition, and specific activity.     

Hypercalcemia Secondary to Gastrointestinal Stromal Tumors: Parathyroid Hormone-Related Protein Independent Mechanism?

ObjectiveHypercalcemia is a common paraneoplastic manifestation of many malignancies like breast, ovarian, and squamous-cell cancers of head and neck; however, there have been only a few case reports of hypercalcemia associated with gastrointestinal stromal tumors (GISTs). We report a case of GIST presenting with hypercalcemia without any osseous metastasis and provide a literature review regarding the mechanisms of hypercalcemia and therapeutic strategies.MethodsWe present a report of case and a review of the relevant literature.ResultsA 52-year-old woman with history of localized breast cancer in remission and a pelvic 13 × 12 cm GIST with peritoneal, liver, and lung metastases presented with hypercalcemia of 14.3 mg/dL (8.5-10.5 mg/dL). Parathyroid hormone-related protein (PTHrP) was undetectable, intact parathyroid hormone (PTH) was appropriately low at 1 pg/mL (10-65 pg/mL), and 1,25 dihydroxy vitamin D (1,25 OH₂ vit D) was elevated at 131 pg/mL (18-78 pg/mL) with normal renal function. Calcium responded transiently to tyrosine kinase inhibitor therapy and bisphosphonates but within a year, she expired due to tumor progression.ConclusionGIST is a rare cause of hypercalcemia. In addition to PTHrP expression, direct tumor production of 1,25(OH)₂ vit D or 1-α hydroxylase enzyme resulting in activation of 25-hydroxy vitamin D may be an alternative mechanism in GIST-related hypercalcemia. Therapy with tyrosine kinase inhibitors and bisphosphonates is recommended, though prognosis is poor. Further investigations are needed to characterize the etiology and management of hypercalcemia in these patients. (Endocr. Pract. 2013;19: e158-e162)     

Humoral hypercalcemia of malignancy: Some enigmas on the clinical features

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome mediated by tumor-derived parathyroid hormone-related peptide (PTHRP), which bears structural and functional similarities to PTH. Thus the clinical features of HHM are very similar to those of primary hyperparathyroidism (1° HPT), a prototype of humoral hypercalcemia caused by PTH. On the other hand, HHM syndrome differs from 1° HPT in several aspects, including serum 1,25(OH)₂D levels, acid-base balance, and bone remodeling process, the reason of which remains largely unknown. We approached these questions using a unique animal model of HHM, nude rats implanted with PTHRP-overproducing human carcinomas. In this review we will summarize the results and discuss the implications in understanding the disease mechanism.     

Endocrine regulation of calcium and phosphate in rat eye lens and its significance in cataract formation

Parathyroid hormone (PTH), calcitonin (CT)and calciferol (Vit. D3) operate synchronously to maintain a balance between calcium and phosphate levels in serum. An aberration of specific steps in the homeostatic process results in hypo/hyper phosphatemia. These aberrations may eventually lead to several diseased states. PTH and Vit. D3 induced hypercalcemia can, however, be significantly inhibited by calcitonin (CT). These findings have been correlated with the levels of calcium and phosphate obtained from human senile cataractous lenses of cortical and nuclear types. The comparison of the results indicate that amongst these three hormones PTH is most vulnerable in leading towards conditions for possible cataract formation in rat lens.     

Liposome-entrapped calcitonin and parathyroid hormone are orally effective in rats

We have demonstrated that liposomally entrapped calcitonin (CT) and parathyroid hormone (PTH) can be orally effective in regulating blood calcium. Liposomal CT produced hypocalcemia and liposomal PTH produced hypercalcemia upon oral administration to rats. Specific liposomal formulations were necessary to affect the appropriate decrease and increase in blood calcium concentration. Future extension of these studies may lead to clinically valuable forms of liposomal-peptide preparations.     

Decrease in calcitonin and parathyroid hormone mRNA levels and hormone secretion under long-term hypervitaminosis D3 in rats

In calcium homeostasis, vitamin D3 is a potent serum calcium-raising agent which in vivo regulates both calcitonin (CT) and parathyroid hormone (PTH) gene expression. Serum calcium is the major secretagogue for CT, a hormone product whose biosynthesis is the main biological activity of thyroid C-cells. Taking advantage of this regulatory mechanism, long-term vitamin D3-induced hypercalcemia has been extensively used as a model to produce hyperactivation, hyperplasia and even proliferative lesions of C-cells, supposedly to reduce the sustained high calcium serum concentrations. We have recently demonstrated that CT serum levels did not rise after long-term hypervitaminosis D3. Moreover, C-cells did not have a proliferative response, rather a decrease in CT-producing C-cell number was observed. In order to confirm the inhibitory effect of vitamin D3 on C-cells, Wistar rats were administered vitamin D3 chronically (25,000 IU/d) with or without calcium chloride (CaCl2). Under these long-term vitamin D3-hypercalcemic conditions, calcium, active metabolites of vitamin D3, CT and PTH serum concentrations were determined by RIA; CT and PTH mRNA levels were analysed by Northern blot and in situ hybridization; and, finally, the ultrastructure of calciotrophic hormone-producing cells was analysed by electron microscopy. Our results show, that, in rats, long term administration of vitamin D3 results in a decrease in hormone biosynthetic activities of both PTH and CT-producing cells, albeit at different magnitudes. Based upon these results, we conclude that hypervitaminosis D3-based methods do not stimulate C-cell activity and can not be used to induce proliferative lesions of calcitonin-producing cells.     

Nephrogenous cyclic adenosine monophosphate levels in normocalcemic cancer patients: its significance

We evaluated nephrogenous cyclic adenosine monophosphate ( NcAMP ) levels in 61 normocalcemic patients with documented cancer of various organs and cell types. NcAMP levels were elevated in 17 (28%) and decreased in 13 (21%) of the cancer patients. Both high and low NcAMP levels were seen within the various cancer groups. There was a significant correlation (r = 0.383, P less than 0.01) between NcAMP and serum parathyroid hormone (PTH) levels, suggesting that tumor-related factors affecting NcAMP , may be partially related to native PTH. Alternatively, these factors might be altering the effect of endogenous PTH on renal tubules. A significant negative correlation was also observed between NcAMP and tubular maximum for phosphate (r = -0.356, P less than 0.02) suggesting that either cAMP per se or factors affecting NcAMP alter phosphate excretion. Follow up serum calcium data was available on 48 of the 61 patients. Subsequent hypercalcemia developed independent of the initial nephrogenous cAMP levels. It therefore appears that NcAMP elevation and development of hypercalcemia are two separate paraneoplastic phenomena.     

Hypercalcemia in cancer.

Hypercalcemia may occur as a complication of haematological malignancies, in association with solid tumors with bone metastases, and with solid tumors in the absence of bone metastases. The latter syndrome, known as the humoral hypercalcemia of malignancy (HHM) shares many features with primary hyperparathyroidism. A parathyroid hormone-related protein (PTHrP) has been identified, isolated and cloned, which is most likely responsible for the calcium disturbances in HHM, PTHrP is a previously unrecognized hormone which has limited amino-terminal sequence homology with PTH and is the product of a separate gene. Tissue localization studies have identified PTHrP in squamous cell carcinomata, renal cortical carcinomata, in a proportion of breast cancers and in adult T-cell leukemia/lymphoma. In normal tissues, PTHrP has been immunohistochemically localized in keratinocytes, placenta and fetal parathyroid glands. In addition to its role in mediating hypercalcemia in cancer, PTHrP is likely to have an important endocrine role in the fetus, and perhaps a paracrine function in several organs.     

The Emerging Role of Denosumab in the Long-Term Management of Parathyroid Carcinoma-Related Refractory Hypercalcemia

Objective: The main cause of death in patients with parathyroid carcinoma is parathyroid hormone (PTH)-induced hypercalcemia. To date, the management of hypercalcemia has been based on the use of bisphosphonates and calcimimetic agents. In recent reports, the use of denosumab has shown encouraging results in cases of refractory hypercalcemia of malignancy. Our objective is to present a case of successful management of resistant hypercalcemia due to parathyroid carcinoma with denosumab, to review similar cases from the literature, and to propose denosumab's use in the clinical management of PTH-induced refractory hypercalcemiaMethods: Presentation of a case report and review of the literature for cases of parathyroid carcinoma–mediated hypercalcemia successfully treated with denosumab.Results: A 71-year-old man with metastatic parathyroid carcinoma was referred to our department for uncontrolled hypercalcemia, resistant to treatment with bisphosphonates and cinacalcet. Treatment with denosumab (120 mg per month) in addition to cinacalcet (180 mg per day) resulted in normalization of calcium levels and maintenance within the normal range for an observation period of 11 months. Review of the literature revealed 4 case reports and a letter to the editor, all of which reported the successful treatment of resistant hypercalcemia associated with parathyroid carcinoma.Conclusion: Based on the above findings of the effectiveness of denosumab in controlling refractory hypercalcemia, its safety in renal failure and the fact that denosumab may reduce PTH-induced bone loss, we endorse its use in the management of hypercalcemia in patients with parathyroid carcinoma and perhaps other conditions with PTH-induced hypercalcemia.Abbreviations: CT = computed tomography IV = intravenous PTH = parathyroid hormone     

Humoral hypercalcemia of malignancy

Studies of humoral hypercalcemia of malignancy (HHM) have provided evidence that tumors produce a protein that acts through the parathyroid (PTH) receptor but is immunologically distinct from PTH. We have recently purified and cloned a parathyroid hormone-related protein (PTHrP) implicated in HHM from a human lung cancer cell line (BEN). Full-length cDNA clones have been isolated and found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids. Eight of the first 13 amino-terminal residues are identical with human PTH, although antisera directed to the amino-terminus of PTHrP do not recognize PTH. The striking homology with PTH about the amino-terminal region is not maintained in the remainder of the molecule. PTHrP therefore represents a previously unrecognized hormone. A 34-amino acid synthetic peptide, PTHrP(1-34) was 2-4 times more potent than bovine or human PTH(1-34) in bioassays promoting the formation of cAMP and plasminogen activity in osteogenic sarcoma cells and activation of adenylate cyclase in chick kidney membranes. Like PTH, PTHrP peptides of less than 30 residues from the amino-terminus showed substantially reduced activity. PTHrP(1-34) was also more potent than hPTH(1-34) in stimulating cAMP and phosphate excretion and reducing calcium excretion in the isolated perfused rat kidney. Immunohistochemical localization of PTHrP was consistently demonstrated in squamous cell carcinomas. In normal tissues PTHrP has been immunohistochemically localized in keratinocytes and PTHrP-like activity has been extracted from ovine placenta and fetal ovine parathyroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction in animal numbers by long-term implantation of intravenous and intra-arterial catheters in thyroparathyroidectomized rats

The thyroparathyroidectomized (TPTx) rat has been extensively used to study parathyroid hormone (PTH)-mediated bone resorption by measuring systemic Ca2+ concentrations. Animals have been traditionally used acutely; that is, they are often infused immediately after surgery and are sacrificed after a single use. To perform multiple experiments using a single group of animals we developed a system of long-term implanted intravenous/arterial catheters. Using calcitonin (CT) as a positive control, we successfully completed 12 separate controlled subexperiments documenting significant reductions in PTH-induced hypercalcemia in rats of the CT group. We then successfully completed two separate TPTx subexperiments, using a 3 x 3 Latin square experimental design. In both subexperiments, CT significantly inhibited the increase of blood Ca2+ concentration resulting from continuous PTH infusion. Our results indicate that, by combining the long-term use of catheters with the Latin square design, we can successfully reduce the number of animals used, increase the number of compounds screened, and improve the quality of the data. Although results of this study confirmed the acceptability of multiple infusions in anti-resorptive studies, investigations into the applicability of this set up to other areas of study requiring infusions and frequent blood sample collections seem appropriate.     

A case of hypocalciuric hypercalcemia without family history.

Familial hypocalciuric hypercalcemia (FHH) is usually characterized by asymptomatic hypercalcemia, mild hypermagnesemia, and low urinary calcium excretion, and is occasionally associated with pulmonary fibrosis. It is inherited as an autosomal-dominant, and no sporadic case of hypocalciuric hypercalcemia has been heretofore reported. This report describes a patient with hypocalciuric hypercalcemia completely compatible with FHH but with no family history, suggesting that the most likely diagnosis is "nonfamilial" hypocalciuric hypercalcemia. We propose that the urinary excretion of calcium be examined in all patients with hypercalcemia, hypophosphatemia, and increased PTH before neck surgery, even if patients have no family history of hypercalcemia.     

Bioactivity of amino terminal fragments of parathyroid hormone and parathyroid hormone related peptide in rat kidney slices: no effect of dietary phosphate deprivation

Humoral hypercalcemia of malignancy has been associated with the production of a recently cloned peptide human parathyroid hormone related protein (hPTHRP). One of the markers of this disease is an increased urinary excretion of cyclic AMP. The postreceptor mechanism of action and physiological role of hPTHRP remain obscure. To study the activity of hPTHRP 1-34 compared to rat and human parathyroid hormone (PTH) 1-34 we incubated these peptides with rat kidney slices and measured the cyclic AMP generated in the supernatant. hPTHRP 1-34 was equipotent with human PTH 1-34 but both were 5 times less active than rat PTH 1-34. Previous studies have suggested that a low dietary phosphate intake results in renal resistance to the phosphaturic action of PTH perhaps mediated by reduced adenylate cyclase activation by PTH. To determine whether, during dietary phosphate restriction, hPTHRP 1-34 has actions different from hPTH 1-34 we studied their effects following dietary phosphate deprivation. Dietary phosphate restriction had no significant effect on the cyclic AMP generating activity of any of the peptides. We conclude that hPTHRP 1-34 may be operating through similar mechanisms as human PTH 1-34 and that the previously observed effects of dietary phosphate deprivation on PTH mediated cyclic AMP generation in a broken cell preparation do not occur in intact cell preparations.     

Gene for parathyroid hormone-like peptide is on rat chromosome 2

Parathyroid hormone-like peptide (PLP) is thought to be a mediator of hypercalcemia in both human and rodent malignancies. A rat PLP cDNA was used as a hybridization probe in Southern blot analysis of DNAs isolated from a panel of rat-mouse somatic cell hybrids. The single-copy gene for PLP was assigned to rat chromosome 2, whereas the rat parathyroid hormone (PTH) gene has previously been assigned to rat chromosome 1. Consequently, despite significant amino-terminal homology between PLP and PTH the genes encoding these peptides in the rat as well as human species have discrete chromosomal localizations.     

Paraneoplastic syndromes in urologic malignancy: the many faces of renal cell carcinoma

Renal cell carcinoma is unique among the genitourinary malignancies in that close to one third of affected patients show signs and symptoms of a paraneoplastic syndrome. The paraneoplastic syndromes associated with renal cell carcinoma range from those manifesting in constitutional symptoms (ie, fever, cachexia, and weight loss) to those that result in specific metabolic and biochemical abnormalities (ie, hypercalcemia, nonmetastatic hepatic dysfunction, amyloidosis, etc). The presence of a paraneoplastic syndrome in a patient with renal cell carcinoma is neither a marker of metastatic disease nor necessarily indicative of a poor prognosis. The importance of understanding the pathophysiology and biology behind the many paraneoplastic syndromes associated with renal cell carcinoma lies in the fact that the presence of these protean symptoms may be the initial presentation of either primary or recurrent disease. In this review, we will describe the proposed mechanisms of action of the many paraneoplastic syndromes associated with renal cell carcinoma as well as outline the clinical evaluation and treatment options currently available for these noteworthy disorders.     

Parathyroid hormone-related protein: biochemistry and molecular biology

This article critically reviews the current state of knowledge regarding the recently identified and cloned novel hormone parathyroid hormone-related protein (PTHrP). PTHrP is produced by tumors associated with the syndrome of humoral hypercalcemia of malignancy giving rise to the parathyroid hormone (PTH)-like symptoms characteristic of the syndrome. Areas that will be reviewed include identification, purification and cloning, localization, actions, and significance of PTHrP in cancers and normal physiology. The structure and regulation of the PTHrP gene that may be ancestrally related to the PTH gene will also be discussed. Studies in vivo and in vitro with synthetic and recombinant PTHrP sequences and antibodies developed against them have established that the PTH-like actions of PTHrP are mediated via the N-terminal sequences, which show some limited sequence homology with PTH. Evidence for PTH and non-PTH-like actions of PTHrP in normal physiology, which implicate a role for PTHrP in fetal and neonatal development, is also presented.     

Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1–108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an α-helical structure extending from residues 14–29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.The discovery of parathyroid hormone (PTH)6 -related protein (PTHrP) as the cause of hypercalcemia in many patients with cancer provided new insights into the pathogenesis of the skeletal complications of malignancy (1). It revealed PTHrP as a previously unrecognized hormone, related in evolution to the calcium-regulating PTH, but important in the pathogenesis of the humoral hypercalcemia of malignancy, a syndrome in which hypercalcemia occurs without evident bone metastases. Whereas PTH consists of 84 amino acids, human PTHrP has three alternative splice products of 139, 141, and 173 residues. Apart from 8 of the first 13 residues of PTH and PTHrP being identical, there is no significant identity between these peptides (2). PTHrP actively promotes bone resorption, doing so in a manner identical to that of PTH by acting upon the receptor (PTH1R) it shares with PTH. The PTH1R is located on cells of the osteoblast lineage, which program the formation and activation of osteoclasts, and on cells of the kidney tubule, through which both PTHrP and PTH promote cyclic AMP and phosphorus excretion but reduce calcium excretion. Other actions of PTHrP that reflect those of PTH include the ability to relax vascular and other smooth muscle. This response may reflect a physiological function of PTHrP rather than of PTH and is consistent with PTHrP production and local action on smooth muscles at various sites (3).The first 34 amino acids of each hormone contain the full biological activities of both PTH and of PTHrP to activate the PTH1R (4). The sequences of PTHrP and PTH between residues 14 and 34 are interesting in that, although they are not homologous, nevertheless they appear to be critical for binding of each to the seven transmembrane G protein-coupled receptor, PTH1R (4). Within the first 34 amino acids of PTH and PTHrP two functional regions have been revealed based on structural and cross-linking studies (5–8). These studies have indicated that the C-terminal half of the first 34 residues of each hormone comprises the high affinity binding domain, interacting with the N-terminal portion of the extracellular domain of the receptor. The N-terminal half of each hormone activates the receptor through contact points on the extracellular loops and juxtamembrane regions (9).Despite their equal ability to activate through the PTH1R, it was clear from the earliest work, even with antibodies against peptides within the first 14 residues of PTHrP, that highly specific antibodies could be generated that discriminate between PTH and PTHrP (10). Likewise, polyclonal antibodies against PTHrP-(1–34) that neutralized its effects completely in vitro in promotion of cyclic AMP production in response to PTHrP without any detectable neutralizing effect on PTH were used to prevent and to treat hypercalcemia in nude mice bearing xenografts of PTHrP-secreting human cancers (11, 12). Similar results were obtained with a neutralizing mouse monoclonal antibody against PTHrP (13). Subsequently, after the finding that breast cancer metastases to bone were enriched in PTHrP production (14), Guise and Mundy (15) used an experimental model in nude mice in which human breast cancer cells grow as lytic deposits in bone after intracardiac injection and showed that PTHrP production by the cancers contributed to the process of tumor establishment and growth in bone by promoting osteoclast formation and bone resorption. Furthermore, the tumor establishment and growth in bone could be prevented by treating the mice with a monoclonal antibody against PTHrP (16) or with a bisphosphonate (17) to inhibit bone resorption.The efficacy of anti-PTHrP antibodies in treating both humoral-mediated hypercalcemia in cancer and bone metastasis formation and growth in mouse models raises the prospect of humanized forms of these antibodies being used as therapeutic agents in these diseases in human subjects, and preclinical data have been obtained in support of that (18, 19). With that in mind, the present project was undertaken in which we have made use of a monoclonal antibody prepared against human PTHrP (residues 1–34), which neutralizes the actions of PTHrP through PTH1R without any action against PTH. The antibody has been complexed with recombinant human PTHrP (residues 1–108) to generate crystals that have been used to analyze the three-dimensional structure with the aim of discovering the structural basis of neutralization of PTHrP action by the antibody.     

Engineering a soluble parathyroid hormone GPCR mimetic

We designed and characterized a soluble mimic of the parathyroid hormone (PTH) receptor (PTH1R) that incorporates the N‐terminus and third extracellular loop of PTH1R, important for ligand binding. The engineered receptor (PTH1R‐NE3) was conceived to enable easy production and the use of standard biochemical and biophysical assays for the screening of competitive antagonists of PTH. We show that PTH1R‐NE3 is folded, thermodynamically stable and selectively binds PTH. We also demonstrate the utility of our mimic by identifying a small molecule that competes with PTH in our PTH1R‐NE3‐based fluorescence polarization assay. Antagonists to PTH1R, a transmembrane protein belonging to the class B G‐protein coupled receptor family, may provide new therapeutic options for calcium metabolism diseases like humoral hypercalcemia of malignancy. Proteins 2014; 82:1370–1375. © 2013 Wiley Periodicals, Inc.