Humoral Hypercalcemia Of Malignancy Syndrome Research Articles

Parathyroid Hormone-Related Protein (PTHrP): A Key Regulator of Life/Death Decisions by Tumor Cells with Potential Clinical Applications

Parathyroid hormone-related protein (PTHrP), classically regarded as the mediator of the humoral hypercalcemia of malignancy syndrome, is a polyhormone that undergoes proteolytic processing into smaller bioactive forms. These bioactive forms comprise an N-terminal-as well as midregion-and C-terminal peptides, which have been shown to regulate various biological events, such as survival, proliferation and differentiation, in diverse cell model systems, both normal and pathological. A number of experimental data have demonstrated that PTHrP is also able to modulate tumor-relevant phenotypic expressions, thereby playing a role in early and advanced tumorigenesis, and in the response to treatment. In particular, interest has mainly been focused on the effects of PTHrP on cell proliferation/apoptosis, migration and invasion, which are the main roles involved in cancer development in vivo. The objective of this review is to discuss collectively the literature data on the molecular and biochemical basis of the mechanisms underlying the different, and sometimes opposite, effects exerted by PTHrP on various neoplastic cytotypes, with some final comments on both present and potential utilization of PTHrP as a target for anti-cancer therapy.

Effect of parathyroid hormone related protein, and dihydrotestosterone on proliferation and ornithine decarboxylase mRNA in human prostate cancer cell lines.

Parathyroid hormone related protein (PTHrP) has been identified as the major hormone responsible for the syndrome of humoral hypercalcemia of malignancy (HHM). Recent studies have shown that a large number of prostate tumors demonstrate the presence of PTHrP despite the fact that prostate cancer is rarely associated with the HHM syndrome. Other studies have indicated that PTHrP behaves as an early response gene, which stimulates ornithine decarboxylase (ODC) enzyme activity, an enzyme, involved in the biosynthesis of polyamines. It is therefore possible that PTHrP regulates prostate tumor cell proliferation via ODC gene expression. In the present study, we evaluated the effects of PTHrP and/or dihydrotestosterone (DHT) treatment on DNA synthesis by thymidine incorporation in androgen-dependent (LnCaP) and androgen-independent (PC3) human prostate adenocarcinoma cell lines. In addition, we utilized Northern blot analysis to investigate the effect of PTHrP [1-34] alone or in combination with DHT on ODC mRNA. PTHrP [1-34] treatment resulted in an increase in thymidine uptake in PC3 cells by 50%, whereas no such increase was seen in LnCaP cells. However, in the LnCaP cells, in the presence of DHT, PTHrP stimulated DNA synthesis to a level greater than that seen with DHT alone. DHT (10 nM) treatment resulted in an induction of PTHrP as well as ODC mRNAs in the androgen-dependent (LnCaP) but not in androgen-independent (PC3) cell line. PTHrP [1-34] treatment resulted in induction of ODC mRNA in the LnCaP cells. Addition of DHT resulted in a further increase in the ODC mRNA expression. These data suggest that PTHrP may play a role in prostate cancer cell proliferation and the increased ODC gene expression may be one possible mechanisms responsible for this phenomenon.

Role of parathyroid hormone-related peptide and Indian hedgehog in skeletal development.

Parathyroid hormone-related peptide (PTHrP), which frequently causes the humoral hypercalcemia of malignancy syndrome, is an autocrine/paracrine regulator of chondrocyte proliferation and differentiation that acts through the PTH/PTHrP receptor (PTH1R). PTHrP is generated in response to Indian hedgehog (Ihh), which mediates its actions through the membrane receptor patched, but interacts also with hedgehog-interacting protein (Hip). Mice lacking PTHrP show accelerated chondrocyte differentiation, and thus premature ossification of those bones that are formed through an endochondral process, and similar but more-severe abnormalities are observed in PTH1R-ablated animals. The mirror image of these skeletal findings, i.e., a severe delay in chondrocyte differentiation and endochondral ossification, is observed in transgenic mice that overexpress PTHrP under the control of the alpha1(II) procollagen promoter. Severe abnormalities in chondrocyte proliferation and differentiation are also observed in two genetic disorders in humans that are most likely caused by mutations in the PTH1R. Heterozygous PTH1R mutations that lead to constitutively activity were identified in Jansen metaphyseal chondrodysplasia, and homozygous or compound heterozygous mutations that lead to less-active or completely inactive receptors were identified in patients with Blomstrand lethal chondrodysplasia. Based on the growth plate abnormalities observed in these human disorders and in mice with abnormal expression of either PTHrP or the PTH1R, it appears plausible that impaired expression of PTHrP and/or its receptor contributes to the growth abnormalities in children with end-stage renal disease. In fact, mild-to-moderate renal failure leads in animals to a reduction in PTH1R expression in growth plates and impaired growth, but it remains uncertain whether this contributes to altered chondrocyte growth and differentiation.

Role of interleukin-6 in uncoupling of bone in vivo in a human squamous carcinoma coproducing parathyroid hormone-related peptide and interleukin-6.

OCC tumor has been established from a human squamous carcinoma associated with humoral hypercalcemia of malignancy (HHM) and shown to overproduce parathyroid hormone-related peptide (PTHrP) and cause aggressive hypercalcemia when implanted into nude rats. In the present study, we have demonstrated by reverse transcription-polymerase chain reaction and Northern blot analysis that OCC tumor also overexpressed interleukin 6 (IL-6) mRNA and that tumor-bearing animals exhibited a marked increase in plasma IL-6 as well as PTHrP concentrations. When a monoclonal antibody against human IL-6 was injected to block the activities of tumor-derived IL-6, bone loss in tumor-bearing animals was significantly prevented. Quantitative bone histomorphometric analysis revealed that treatment with anti-IL-6 antibody caused a substantial decrease in both osteoclast number and eroded surface (as parameters of bone resorption) and also a significant increase in the mineral apposition rate, but little effect on the osteoblastic surface. These results provide in vivo evidence suggesting that in tumors coproducing IL-6 and PTHrP, IL-6 is involved not only in the acceleration of osteoclastic bone resorption but also, at least in part, in the suppression of osteoblastic functions in HHM syndrome.

Neoplastic Transformation of Normal Rat Embryo Fibroblasts by a Mutated p53 and an Activated ras Oncogene Induces Parathyroid Hormone-related Peptide Gene Expression and Causes Hypercalcemia in Nude Mice

Parathyroid hormone-related peptide (PTHRP) is a 141-amino acid protein identified in various carcinomas associated with humoral hypercalcemia of malignancy (HHM). Although the causal role of PTHRP in HHM syndrome has been established, the molecular and cellular mechanism by which PTHRP gene is overexpressed in certain malignancies remains unknown. We have demonstrated in the present study that PTHRP secretion was markedly induced concomitantly with the formation of transformed foci after normal rat embryo fibroblasts (REFs) were co-transfected with an activated ras (ras) and a mutated form of p53 (p53-mt) genes. In either ras- or p53-mt-transfected (nontransformed) cells, only modest or barely detectable secretion of PTHRP was observed, respectively. Northern blot analysis revealed that PTHRP mRNA was markedly induced in fully transformed cells 11 days after transfection with both ras and p53-mt genes. Inhibition of RNA synthesis with actinomycin D resulted in almost complete disappearance of PTHRP mRNA at 2-3 h, suggesting a transcriptional mechanism. Transient transfection experiments revealed that PTHRP promoter activity was induced in ras + p53-mt transfectants. REFs transformed by ras and p53-mt genes and thereby induced to secrete PTHRP in vitro produced aggressively growing tumors associated with HHM syndrome when injected into nude mice. These results suggest that activation of PTHRP gene is closely related to malignant transformation of normal mammalian cells and that ras and p53 may be important regulators of PTHRP gene transcription. The transfection-focus formation system of REFs should provide an excellent model to study the molecular and cellular mechanism underlying concomitant overexpression of PTHRP gene with carcinogenesis.

Hypercalcemia of malignancy: the central role of parathyroid hormone-related protein.

Hypercalcemia is the most common metabolic complication of cancer. Malignancy-associated hypercalcemia (MAHC) can be divided into two syndromes, humoral hypercalcemia of malignancy (HHM) and local osteolytic hypercalcemia (LOH), based on whether a circulating hormone or local paracrine factors mediate accelerated bone resorption. Over the past decade, studies have shown that parathyroid hormone-related protein is the cause of the HHM syndrome, and recent data suggest that this protein may also play a paracrine role in some patients with local osteolytic hypercalcemia. Study of the regulation of parathyroid hormone-related protein gene expression is beginning to shed some light on the molecular mechanisms responsible for this common clinical problem.

In situ localization of parathyroid hormone-like protein and mRNA in intraepithelial neoplasia and invasive carcinoma of the uterine cervix

Parathyroid-like protein (PLP), or parathyroid hormone-related peptide, is a well-recognized mediator of paraneoplastic hypercalcemia (humoral hypercalcemia of malignancy syndrome). In this study we examined the expression of PLP by 40 invasive squamous cell carcinomas (SCCs) of the cervix and selected carcinomas of nonsquamous histology. Using a polyclonal antibody to human PLP, 93% of SCCs, including two tumors from patients with humoral hypercalcemia of malignancy syndrome, showed moderate to strong cytoplasmic immunoperoxidase staining for PLP. The strongest staining often was observed in areas of invasion associated with stromal desmoplasia. The small number of weak or negatively stained SCCs were all poorly differentiated tumors. Although native uninvolved squamous epithelium showed weak to moderate staining of the superficial layers, there was variable or full-thickness immunostaining in areas of dysplasia. Normal endocervical glands and stroma as well as cervical adenocarcinomas and neuroedocrine carcinomas were negative. In situ hybridization studies showed abundant PLP mRNA within SCC in patients with hypercalcemia. However, PLP mRNA was of relatively low abundance in tumors of normocalcemic patients. Ultrastructural studies showed cytoplasmic, membrane-bound, granular inclusions in tumor cells from the hypercalcemic patients. Our data suggest that increased PLP gene transcription contributes to the increased production of PLP and the pathogenesis of humoral hypercalcemia of malignancy syndrome.

Parathyroid Hormone Related Peptide (1-34) and 1,25-Dihydroxyvitamin D, Have No Additive Effects on Bone Tartrate Resistant Acid Phosphatase Activity in Fetal Rat Calvaria Cultures

Parathyroid hormone related peptide (PTHrP) and 1,25-dihydroxyvitamin D are known to be resorptive agents which could contribute to the development of hypercalcemia in humoral hypercalcemia of malignancy (HHM) syndrome in Walker 256 tumor bearing rats. In order to clarify some aspects about the relative contribution of these factors to bone resorption, we have determined the effects produced by PTHrP (1-34) and/or 1,25(OH)2D3 on tartrate resistant acid phosphatase (TRAP), a biochemical marker of bone resorption, in fetal rat calvaria cultures. At the same time, bovine parathyroid hormone (PTH) (1-34) was used as a control in the experiments in order to compare its effects with those produced by the other two agonists. In the present work, 10(-7) M PTH (1-34), 10(-7) M PTHrP (1-34) and 10(-8) M or 10(-10) M 1,25(OH)2D3 produced a significant increase in TRAP activity, when these agonists were added to the calvaria culture. Surprisingly, and in spite of the different ways of action of PTH, PTHrP and 1,25(OH)2D3, their actions are not additive in our experiment. The results of the present work suggest that any of the two implicated factors PTHrP or 1,25(OH)2D3 could be individually responsible for the high rate of bone resorption that takes place in HHM syndrome in Walker 256 carcinosarcoma bearing rats, although other different agents, like TGF, could also be implicated.

Osseous and intestinal compartments in the humoral hypercalcemia of malignancy associated to Walker 256 tumor in rats.

The objective of the present work has been to study some aspects of bone and intestinal compartments in rats with Walker 256 carcinosarcoma, an experimental model of humoral hypercalcemia of malignancy (HHM). The results have been compared to those obtained in control animals and, also, to those obtained in Yoshida sarcoma-bearing rats, which were used as tumoral controls without hypercalcemia. Urinary hydroxyproline/creatinine ratio (OHProl/creat) is increased, in both Walker 256 and Yoshida tumor-bearing animals, showing the nonspecifity of this bone marker. However, serum tartrate-resistant acid phosphatase (TRAP) levels are increased in Walker 256 tumor-bearing animals, but they are normal in Yoshida tumor-bearing animals, indicating that TRAP is a better index of bone resorption than OHProl/creat in the HHM syndrome. The decrease of bone calcium content in Walker 256 tumor-bearing rats, not shown by Yoshida-bearing rats, also reflects an increased bone resorption due to HHM. Serum and bone osteocalcin levels are similar in control, Walker 256 and Yoshida tumor-bearing rats, but we observed a decrease in serum alkaline phosphatase levels in Walker 256 and Yoshida tumor-bearing animals, which could also be a nonspecific tumor effect, due to the presence of the neoplasia. Our results support the convenience of the employment of a nonhypercalcemic tumor group as control in the HHM study, in addition to the healthy controls. We have also observed higher 1,25-dihydroxyvitamin D serum levels in Walker 256 tumor-bearing rats than in control and Yoshida tumor-bearing rats. On the other hand, we have found normal levels in the fractional rate of intestinal calcium absorption in Walker-256 tumor-bearing rats, in spite of their high calcium levels, and a significant decrease of this parameter in Yoshida sarcoma-bearing animals. These results support the concomitant contribution of intestinal compartment to hypercalcemia, in the experimental model of HHM studied.

Parathyroid hormone-related protein stimulates prostaglandin E2 release from human osteoblast-like cells: modulating effect of peptide length.

Parathyroid hormone-related protein (PTHrP) is a potent bone-resorbing protein that frequently mediates the humoral hypercalcemia of malignancy syndrome. Since prostaglandins may mediate the bone-resorptive action of certain hormones, we examined the effect of PTHrP on prostaglandin E2 (PGE2) secretion by human osteoblast-like cells. There was low-level basal secretion of PGE2 by Saos-2 cells (8.1 +/- 0.6 pg/ml). Using four different preparations of PTHrP, it was observed that with increasing peptide length, from 36 to 141 amino acids, a significant increase in efficacy for PGE2 release was seen in these cells. All forms of PTHrP were agonists for PGE2 release, with effects seen at concentrations as low as 10(-12) M in 48 h conditioned media. The amino terminus of the molecule appeared critical for this effect since the truncated derivative PTHrP-(7-34) did not induce significant PGE2 secretion. However, the influence of peptide length could not be explained by differential activation of adenylate cyclase since [Tyr36]PTHrP-(1-36)amide was equipotent to the longest peptide preparation, PTHrP-(1-141), in stimulating cyclic AMP accumulation in the Saos-2 cells. In contrast, PTHrP-(1-141) was significantly more effective than [Tyr35]PTHrP-(1-36)-amide in inducing a rise in cytosolic calcium. Further, this effect was noted at concentrations lower than those that caused significant cyclic AMP accumulation in the Saos-2 cells. PTHrP-(1-141) induced the release of PGE2 from primary human bone cell cultures to levels entirely comparable to those seen in the Saos-2 cells. PTHrP-(1-141) also induced PGE2 release by cultured fetal rat long bones at 72 h. We conclude that the carboxy-terminal region of PTHrP has important effects on cellular signal transduction pathways and on the release of a potent bone-active cytokine, PGE2.

Effect of parathyroid hormone-like peptides on 25-hydroxyvitamin D-1 alpha-hydroxylase activity in rodents

The role of vitamin D metabolism in the humoral hypercalcemia of malignancy syndrome (HHM) is unclear. We studied in vivo and in vitro effects of synthetic parathyroid hormone-like peptides (PTH-LPs) on rodent renal 25-OHD-1 alpha-hydroxylase activity. Infusion of mice with PTH-LP-(1-36) at 10 pmol/h for 12 and 24 h showed significant (429 +/- 139% and 937 +/- 413%, respectively) stimulation of control enzyme activity. Infusion for 36 h demonstrated diminution of activity to levels nearer to the unstimulated state (228 +/- 36% of control). In that maximal activity was observed after 24 h of infusion, we examined 1 alpha-hydroxylase activity after variable dosages of PTH-LP-(1-36) at this time point. Animals infused with PTH-LP-(1-36) at dosages of 2.5, 10, and 30 pmol/h for 24 h demonstrated 1 alpha-hydroxylase activities of 0.71 +/- 0.12, 4.74 +/- 2.09, and 9.91 +/- 1.01 ng.mg protein-1.20 min-1 (means +/- SD), respectively, all significantly greater than control activity (0.51 +/- 0.20 ng.mg protein-1.20 min-1). PTH-LP-(1-36) and PTH-LP-(1-74) were comparable in potency to bovine (b)PTH-(1-34) in stimulating 1 alpha-hydroxylase. Direct in vitro incubation of PTH-LP-(1-36) with renal slices resulted in stimulation of 1 alpha-hydroxylase activity up to 200% of control levels, comparable to that seen with equimolar concentrations of bPTH-(1-34).(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid Hormone-Like Proteins: Biochemical Responses and Receptor Interactions*

I. Introduction HUMORAL hypercalcemia of malignancy (HHM) was first described almost 50 yr ago (1) and is the most common paraneoplastic syndrome. It appears to result in most instances from the production by tumors of a novel class of protein hormones which share amino acid sequence homology with PTH. These PTH-like proteins (PTHLPs)1, purified and amino-terminally sequenced from human HHM-associated tumors, appear to mimic certain of the renal and skeletal actions of PTH, thereby leading to the clinical and biochemical features of the HHM syndrome. The structure, location, and transcriptional pattern of the PTHLP gene have been elucidated and the full complementary DNA-predicted amino acid sequences of three PTHLP translation products have been revealed (2–7). Further, PTHLPs and their mRNA transcripts have been identified in a variety of normal, nonmalignant tissues (8, 9), and may have important regulatory or functional roles in these tissues. The renal tubule and skeletal osteoblast are regarded as t...

A hypercalcemic nude rat model that completely mimics human syndrome of humoral hypercalcemia of malignancy.

Tumors causing humoral hypercalcemia of malignancy (HHM) were implanted to athymic nude rats. In one of these rat models transplanted with uterine cancer (UCC), a complete reproduction of human HHM syndrome was achieved: hypercalcemia, hypophosphatemia with increased urinary phosphate and cyclic AMP excretion, and suppressed serum 1,25-dihydroxy-vitamin D (1,25(OH)2D) level. In another hypercalcemic nude rat model implanted with oral cavity cancer (OCC), all the features were similar except for markedly elevated serum 1,25(OH)2D. Hypercalcemia disappeared by surgical removal of the tumors in both models, confirming the humoral mechanisms for causing these features. Furthermore, in UCC tumor-bearing rats, hypophosphatemia, increased renal phosphate excretion, and reduced serum 1,25(OH)2D concentration were already present when these rats were only marginally hypercalcemic. These results raise the possibility that the changes in renal tubular phosphate handling and vitamin D metabolism in HHM are not secondary to hypercalcemia but are due to direct effects of the humoral factor(s) that cause this syndrome. Extracts of both tumors exhibited stimulation of cyclic AMP production in osteoblastlike cells, UMR 106, which could be almost completely inhibited by parathyroid hormone (PTH) antagonist, human PTH(3-34). By comparing the nature and characteristics of humoral factor(s) from UCC and OCC models, mechanisms responsible for causing these abnormalities can be explored. Thus, these nude rat models can be useful for elucidating the underlying mechanism of the development of HHM.