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Cancer Therapy: Preclinical

Parathyroid -Related Protein Induces Cachectic Syndromes without Directly Modulating the Expression of Hypothalamic Feeding-Regulating Hirofumi Hashimoto,1Yumiko Azuma,2 Makoto Kawasaki,1Hiroaki Fujihara,1Etsuro Onuma,2 Hisafumi Yamada-Okabe,2 Yo h Ta ku w a , 3 Etsuro Ogata,4 and Yoichi Ueta1

Abstract Purpose: Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hyper- calcemia of malignancy (HHM) and concurrent anorexia and wasting. Because changes in the expression of hypothalamic feeding-regulating peptides can directly affect appetites and thereby can cause anorexia and wasting, we addressed whether the cachectic syndromes induced by PTHrP rely on the action of hypothalamic feeding-regulating peptides. Experimental Design: Rats were inoculated with a LC-6 human cancer xenograft that secreted PTHrP, and the mRNA levels of the hypothalamic feeding-regulating genes and serum levels were examined before and after the development of HHM by in situ hybridization histochemistry and ELISA, respectively. Some rats were given the anti-PTHrP antibody. Results and Conclusion: The mRNA levels for the orexigenic peptides, such as the agouti- related protein and the Y in the arcuate nucleus (Arc), were significantly increased after the development of HHM, whereas the mRNA levels for the anorexigenic peptides, such as the in theArc, the cocaine and amphetamine-regulated transcript in theArc, and the corticotropin-releasing factor in the paraventricular nucleus, were significantly decreased after the development of HHM. Plasma leptin levels were also reduced in cachectic rats, and the administration of anti-PTHrP antibody to the cachectic rats not only improved the cachectic symptoms but also restored the mRNA levels of these orexigenic and anorexigenic peptides, except for . Thus, PTHrP induces HHM and concurrent cachectic syndromes by mecha- nisms other than directly modulating the leptin or hypothalamic feeding-regulated peptides.

The severity of cachexia from such illnesses as cancer, end-stage studies led to the hypotheses that body weight is regulated by a renal disease, and HIV may be the primary determining factor feedback loop in which peripheral signals report nutritional in both the quality of life and the eventual mortality (1, 2). information to an integrating center in the of the Anorexia-cachexia syndrome is observed in a large number of and that are essential molecules within the patients in the advanced stages of cancer (3, 4). It is a complex hypothalamus (9–13). disease characterized by several metabolic and behavioral It is known that appetite is under chemical control and that abnormalities, including early satiety, weakness, general fa- feeding behavior is regulated by various hypothalamic peptides, tigue, depression, and weight loss. Although the etiology of the orexigenic peptides, such as agouti-related protein (AgRP), cachexia is not well defined, several hypotheses have been (NPY), and (hypocretins), and anorex- explored for cytokines, , neuropeptides, neurotrans- igenic peptides, such as cocaine- and amphetamine-regulated mitters, tumor-derived factors, and others (1, 5–8). Previous transcript (CART), proopiomelanocortin (POMC), corticotro- pin-releasing factor (CRF), a-melanocyte-stimulating hormone, and leptin (14, 15). Among these peptides, functions of leptin have been well studied in mice and rats lacking the functional Authors’Affiliations: 1Department of Physiology, School of Medicine, University ob 2 leptin or its receptor. Leptin is the product of the gene, and of Occupational and Environmental Health, Kitakyushu, Japan; Kamakura Research ob Laboratories, Chugai Pharmaceutical Co. Ltd., Kanagawa, Japan; 3Department of fasting suppresses in adipocytes (16, 17), Physiology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; leading to a decrease in the plasma concentration of leptin and 4Cancer Institute Hospital,Tokyo, Japan (18). Leptin treatment impairs fasting-induced neuroendocrine Received 6/20/06; revised 10/10/06; accepted 10/16/06. responses in the gonadal-adrenal- axis (19), and leptin The costs of publication of this article were defrayed in part by the payment of page ob ob/ob advertisement deficiency due to mutation of the gene ( mouse)and charges. This article must therefore be hereby marked in accordance db/db with 18 U.S.C. Section 1734 solely to indicate this fact. leptin resistance due to mutation of the ( Note: H. Hashimoto andY.Azuma contributed equally to this work. mouse and fa/fa rat)cause severe obesity (16, 20, 21). Requests for reprints: Yoichi Ueta, Department of Physiology, School of Furthermore, NPY mRNA levels in the arcuate nucleus (Arc) Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, of ob/ob mouse, db/db mouse, and fa/fa rat were significantly Yahatanishi-ku, Kitakyushu 807-8555, Japan. Phone: 81-93-691-7420; Fax: 81- 93-692-1711; E-mail: [email protected]. increased in comparison with controls (22, 23). F 2007 American Association for Cancer Research. Previously, we showed that animals carrying tumors secreting doi:10.1158/1078-0432.CCR-06-1487 parathyroid hormone-related protein (PTHrP)exhibited

Clin Cancer Res 2007;13(1) January 1, 2007 292 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Hypothalamic Neuropeptides in Cachectic Rats humoral hypercalcemia of malignancy (HHM), reduced hybridization procedures have been described in detail previously (29). food intake, and body weight loss, and administration of the In brief, hybridization was done at 37jC overnight in a 45 AL buffer   humanized anti-PTHrP antibody raised against the NH - solution consisting of 50% formamide and 4 SSC (1 SSC = 150 2 A terminal 34 amino acids of the human PTHrP (PTHrP ) mmol/L NaCl, 15 mmol/L sodium citrate), which contained 500 g/mL 1-34 sheared salmon sperm DNA (Sigma, St. Louis, MO), 250 Ag/mL baker’s improved these symptoms (24, 25). Because the anti-PTHrP yeast total RNA (Roche Molecular Biochemicals GmbH, Mannheim, antibody augmented the food intake in cachectic animals, Germany), 1 Denhardt’s solution, and 10% dextran sulfate with we speculated that PTHrP secreted from tumor induced HHM molecular weight 500,000 (Sigma). The hybridization was done and concurrent anorexia by affecting the gene expression of under a Nescofilm coverslip (Bando Chemical IMD Ltd., Osaka, hypothalamic feeding-regulating peptides and leptin. Japan). [35S]3¶-end-labeled deoxyoligonucleotides complementary to In the present study, we examined whether orexigenic and transcripts coding for AgRP (5¶-CGACGCGGAGAACGAGACTCGC- anorexigenic peptides are involved in the PTHrP-induced GGTTCTGTGGATCTAGCACCTCTGCC-3¶), NPY (5¶-GGAGTAGTATCT- anorexia and cachexia and found that PTHrP induced anorexia GGCCATGTCCTCTGCTGGCGCGTC-3¶),CART(5¶-TGGGGAC- ¶ ¶ and cachexia without directly up-regulating the anorexigenic TTGGCCGTACTTCTTCTCATAGATCGGAATGC-3 ),POMC(5- peptides or down-regulating the orexigenic peptides in the CTTCTTGCCCAGCGGCTTGCCCCAGCAGAAGTGCTCCATGGAC- TAGGA-3¶),CRF(5¶-CAGTTTCCTGTTGCTGTGAGCTTGCTGAGC- hypothalamus. TAACTGCTCTGCCCTGGC-3¶), thyrotropin-releasing hormone (TRH; 5¶-GTCTTTTTCCTCCTCCTCCCTTTTGCCTGGATGCTGCGCTTTTGT- Materials and Methods GAT-3¶),orexin(5¶-TTCGTAGAGACGGCAGGAACACGTCTTCTGGC- GACA-3¶), and melanin-concentrating hormone (MCH; 5¶-CCAAC- Cells and animals. Human lung cancer cell line LC-6-JCK originat- AGGGTCGGTAGACTCGTCCCAGCAT-3¶)were used as specific probes. ing from human large cell lung cancer was purchased from the Central Institute for Experimental Animals (Kawasaki, Japan). The cells were maintained in vivo in nude mice (BALB/cAnN Crj-nu/nu), and small pieces of tumor tissues (f10 mm3)were s.c. implanted into 5-week-old male F344/N Jcl-rnu nude rats. The rats displayed hypercalcemia and weight loss between 42 and 60 days after implantation of the tumor, and those with blood ionized (iCa)levels higher than 1.8 mmol/L and at least 0.5 mmol/L higher than the nontumor-bearing rats were used as HHM rats (24, 25–27). Day 0 represents the day when the tumors were implanted. Nude mice and nude rats purchased from Charles River Japan, Inc. (Yokohama, Japan)and Clea Japan, Inc. (Tokyo, Japan), respectively, were kept in sterilized cages. Drugs. The humanized anti-PTHrP antibody raised against the

NH2-terminal 34 amino acids of the human PTHrP (PTHrP1-34; ref. 25) was dissolved in saline. Rats were given 3 mg/kg of the anti-PTHrP antibody i.v. once weekly. Experimental procedures. The body weight and iCa in nontumor- bearing rats and HHM rats were measured 28, 38, 46, 52, 56, 60, and 63 days after implantation of the tumor. Blood was collected from the tail vein, and the concentration of iCa was measured by the electrode method using a Ca2+/pH analyzer (Bayer 634, Bayer Diagnostics, Sudbury, United Kingdom). Fifty-two days after implan- tation of the tumor, nude rats bearing the LC-6-JCK xenograft were divided into three groups: the group for decapitation for in situ hybridization histochemistry, the group given with vehicle, and the group given with anti-PTHrP antibody (n = 6 per group). Animals used for in situ hybridization histochemistry were decapi- tated on day 53 (nontumor-bearing rats and HHM rats)and on day 63 (nontumor-bearing rats, HHM rats, and rats given with anti-PTHrP antibody)after implantation of tumor ( n = 6 per group). were rapidly removed, placed on powdered dry ice, and stored at À80jC until use. Trunk blood was collected and serum concentrations of leptin were measured using an ELISA kit (YK051 Rat Leptin-HS, Yanaihara Institute, Inc., Shizuoka, Japan). Animals used in this experiment were treated in accordance with the ethical guidelines of animal care, handling, and termination of the Chugai Pharmaceutical (Kanagawa, Japan). In situ hybridization histochemistry. In situ hybridization histo- chemistry was done on frozen 12-Am-thick coronal brain sections cut on a cryostat at À20jC, thawed, and mounted onto gelatin/chrome alum-coated slides. Brain tissue was stored at À80jC before cutting. The locations of the paraventricular nucleus (PVN), Arc, and lateral Fig. 1. Changes in body weight (A)andiCa(B) in the nontumor-bearing rats hypothalamic area (LH)were determined according to coordinates (Normal), HHM rats (HHM), and rats injected with anti-PTHrP antibody (HHM + Ab). Points, mean (n = 6); bars, SE. *, P < 0.05, compared with nontumor-bearing given by the atlas of Paxinos and Watson (28). Ten sets of two sections rats; **, P < 0.01, compared with nontumor-bearing rats; #, P < 0.05, compared containing the PVN and four sections containing the Arc and LH were with rats given with anti-PTHrP antibody; ##, P < 0.01, compared with rats given used from each rat to measure the density of autoradiography. In situ with anti-PTHrP antibody.

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Fig. 2. Expression of mRNA forAgRP (A, B, C,andG)andNPY(D, E, F,andH) in the Arc of the nontumor-bearing rats (Normal), HHM rats (HHM), and rats injected with anti-PTHrP antibody (HHM + Ab) 53and 63d after implantation of tumor. Representative autoradiographs of sections hybridized to a 35S-labeled oligodeoxynucleotide probe complementary to mRNA forAgRP (A-C)andNPY(D-F)intheArc63d after transplantation of tumor showing signal intensity from high (white)to low (black). A and D, sections from a nontumor-bearing rat. B and E, sections from a HHM rat. C and F, sections from a HHM rat given with anti-PTHrP antibody. Bar,1mm. Columns, mean (n = 6); bars, SE. **, P < 0.01, compared with nontumor- bearing rats; ##, P < 0.01, compared with rats given with anti-PTHrP antibody.

The specificity of the probes has been described previously, except for Results CART and MCH (23, 29–32). The specificity of the probes for CART and MCH was established from the nucleotide database at the National Expression of hypothalamic peptides, orexigenic peptides, and Center for Biotechnology Information.5 Total counts of 6  105 cpm/ anorexigenic peptide mRNAs in nontumor-bearing rats, HHM slide for AgRP, NPY, CRF, POMC, CART, and TRH and 4  105 cpm/ slide for orexin and MCH transcripts were used. Hybridized sections rats, and rats given with anti-PTHrP antibody. Consistent containing the PVN, the Arc, and the LH were exposed to autoradio- with previous results (24, 26, 27), body weight of HHM rats graphy film (Hyperfilm, Amersham, Buckinghamshire, United King- significantly decreased in comparison with nontumor-bearing dom)for 4 days for orexin and 7 days for AgRP, NPY, CRF, POMC, rats after day 30 (30 days after tumor implantation)but CART, TRH, and MCH. The autoradiographic images were quantified significantly increased after HHM rats were given the anti- using an MCID imaging analyzer (Imaging Research, Inc., St. PTHrP antibody (Fig. 1A). In addition, the level of iCa in HHM Catherines, Ontario, Canada). The images were captured by a charge- rats became significantly higher than that of nontumor-bearing  coupled device camera (Dage-MTI, Inc., Michigan City, IN)at 40 rats after day 30 but it was decreased after HHM rats received magnification. The mean absorbance of the autoradiographs was the anti-PTHrP antibody (Fig. 1B). Single administration of measured by comparing with simultaneously exposed 14C microscale 3 mg/kg anti-PTHrP antibody to HHM rats restored blood samples (Amersham). 14C was used as the standard for quantification of the absorbance of autoradiographs for in situ hybridization calcium for at least 2 weeks. In addition, these rats continued to histochemistry. The standard curve was fitted by the absorbance of gain weight and showed increased food uptake for at least the 14C microscale on the same film. 2 weeks compared with vehicle control (26). Furthermore, Statistical analysis. All data are given as mean F SE calculated from repeated weekly administration of the anti-PTHrP antibody the results of the in situ hybridization histochemistry. Each group nearly completely restored blood calcium and body weight for within an experiment was compared with the control group. The data at least 6 weeks (25). were analyzed using a one-way fractional ANOVA followed by a To address whether feeding-regulated neuropeptides are Bonferroni correction for multiple comparisons. The changes in body involved in the wasting in this animal model, we first examined weight, iCa, and plasma leptine were also statistically analyzed using the expression of the orexigenic peptide genes in HHM rats and one-way ANOVA followed by a Bonferroni correction for multiple comparisons. Statistical significance was defined as P < 0.05. compared it with that in the control nontumor-bearing rats at the time body weights of the rats bearing LC-6 began to decrease (day 53)and after they reached a plateau (day 63).The mRNA levels of the genes for orexigenic peptides, AgRP and 5 http://www.ncbi.nlm.nih.gov/. NPY, in the Arc were significantly higher in HHM rats than in

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Fig. 3. Expression of mRNA for CART (A, B, C,andG)andPOMC(D, E, F,andH) in the Arc of the nontumor-bearing rats (Normal), HHM rats (HHM), and rats injected with anti-PTHrP antibody (HHM + Ab) 53and 63d after implantation of tumor. Representative autoradiographs of sections hybridized to a 35S-labeled oligodeoxynucleotide probe complementary to mRNA for CART (A-C)andPOMC(D-F)in the Arc 63d after transplantation of tumor showing signal intensity from high (white) to low (black). A and D, sections from a nontumor-bearing rat. B and E, sections from a HHM rat. C and F, sections from a HHM rat given with anti-PTHrP antibody. Bar,1mm. Columns, mean (n = 6); bars, SE. **, P < 0.01, compared with nontumor-bearing rats; ##, P < 0.01,compared with rats given with anti-PTHrP antibody.

Fig. 4. Expression of mRNA for CRF (A, B, C,andG)andTRH(D, E, F,andH)inthe PVN of nontumor-bearing rats (Normal), HHM rats (HHM), and rats injected with anti-PTHrP antibody (HHM + Ab) 53and 63d after implantation of tumor. Representative autoradiographs of sections hybridized to a 35S-labeled oligodeoxynucleotide probe complementary to mRNA for CRF (A-C) and TRH (D-F)inthePVN63dafter transplantation of tumor showing signal intensity from high (white)tolow(black). A and D, sections from a nontumor-bearing rat. B and E, sections from a HHM rat. C and F, sections from a HHM rat given with anti-PTHrP antibody. Bar, 1mm. Columns, mean (n =6); bars, SE. **, P < 0.01,compared with nontumor- bearing rats; ##, P < 0.01, compared with rats given with anti-PTHrP antibody.

www.aacrjournals.org 295 Clin Cancer Res 2007;13(1) January 1, 2007 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical nontumor-bearing rats on days 53 and 63 (Fig. 2). Administra- anti-PTHrP antibody to HHM rats greatly increased the plasma tion of the anti-PTHrP antibody to HHM rats showed reduced leptin level, but it was still lower than that of the nontumor- levels of the AgRP and NPY mRNA compared with levels in the bearing rats. control rats (Fig. 2). Next, we examined the expression of the anorexigenic genes in HHM rats. The mRNA levels of the genes Discussion for the anorexigenic peptides, CART and POMC in the Arc and CRF in the PVN, in HHM rats were all lower than those in PTHrP has been isolated as a principal factor responsible for nontumor-bearing rats on days 53 and 63 (Figs. 3 and 4A, B, C, HHM (34). As previous studies have shown, rats carrying the and G). Administration of the anti-PTHrP antibody to the HHM LC-6 xenograft display HHM and concurrent anorexia, and rats significantly increased the levels of CART, PONC, and CRF administration of anti-PTHrP antibody restored blood calcium, mRNA, but their levels were still lower than those in the food intake, and body weight in these rats (24, 26, 27). nontumor-bearing rats even after HHM rats received the anti- Capparelli et al. (35)reported that s.c. administration of osteo- PTHrP antibody (Figs. 3 and 4A, B, C, and G). We also protegerin improved hypercalcemia in mice bearing the Colon- examined the expression of the TRH gene in the PVN and found 26 tumor, which produced and secreted PTHrP into plasma and that the level of TRH mRNA was more or less the same between induced hypercalcemia; however, body weight loss and high HHM and nontumor-bearing rats (Fig. 4D, E, F, and H), and the levels of PTHrP in plasma remained. The results suggest that administration of the anti-PTHrP antibody had no effect. the anorexia and wasting in these mice did not solely depend The mRNA level of the orexin gene in the LH of HHM rats on hypercalcemia and that tumor-secreted PTHrP might be was similar to that of the nontumor-bearing rats on day 53 but directly involved. became lower than nontumor-bearing rats by day 63 and was In the present study, we focused on the hypothalamic not increased by the administration of the anti-PTHrP antibody function in HHM rats treated or untreated with anti-PTHrP (Fig. 5). There was no significant difference in the mRNA level antibody because the hypothalamus is well known to be an of the MCH gene in the LH of HHM rats given the anti-PTHrP integrative site in the regulation of feeding (36). The mRNA antibody and nontumor-bearing rats (Fig. 5). levels of orexigenic peptides, such as AgRP and NPY, in HHM Expression of the NPY gene has been reported to be affected rats were significantly higher than those seen in nontumor- by leptin (33). Therefore, we also examined the plasma leptin bearing rats, whereas the mRNA levels of anorexigenic peptides, levels in HHM rats. Concentration in plasma of leptin in such as CART, POMC, and CRF, in HHM rats were lower than HHM rats was significantly lower than that in nontumor- those seen in nontumor-bearing rats. Administration of anti- bearing rats on days 53 and 63 (Fig. 6). Administration of the PTHrP antibody to HHM rats restored these mRNA levels. The

Fig. 5. Expression of mRNA for orexin (A, B, C,andG)andMCH(D, E, F,andH) in the LH of nontumor-bearing rats (Normal), HHM rats (HHM), and rats injected with anti-PTHrP antibody (HHM + Ab) 53and 63d after implantation of tumor. Representative autoradiographs of sections hybridized to a 35S-labeled oligodeoxynucleotide probe complementary to mRNA for orexin (A-C)andMCH(D-F)intheLH63d after transplantation of tumor showing signal intensity from high (white)to low (black). A and D, sections from a nontumor-bearing rat. B and E, sections from a HHM rat. C and F, sections from a HHM rat given with anti-PTHrP antibody. Bar,1mm. Columns, mean (n =6); bars, SE. **, P < 0.01, compared with nontumor-bearing rats.

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G361 human melanoma xenograft model (37)and in lung cancer patients who developed cachexia and chemotherapy- induced anorexia (38). In the present study, the plasma leptin level also declined in the HHM rats and anti-PTHrP antibody restored it. Many appetite-regulating peptides are under the control of leptin (14, 15), and leptin activates neuroendocrine cells in the hypothalamus, particularly in the Arc, which contains both orexigenic and anorexigenic peptides, such as AgRP, NPY, CART, and POMC (33, 39, 40). Therefore, it seems that the adipose tissues and hypothalamus where leptin is produced and acts, respectively, are not the primary target sites of PTHrP to induce anorexia and cachexia. On the other hand, the mRNA levels of orexins and MCH, which are expressed in the neurons of the LH and enhance feeding (41, 42), were not markedly altered in HHM rats, and anti-PTHrP antibody had little effect. Thus, PTHrP may affect factors and mechanisms that function between hypothalamic feeding-regulating peptides and central nervous system pathways downstream of these peptides. However, we cannot rule out the possibility that PTHrP directly activates neurons in the hippocampus because PTHrP induced a marked elevation in the concentration of cytosolic free calcium in a hippocampal single neuron (43)and the calcium- sensing receptor is localized in the brain, especially in the subfornical organ, olfactory bulbs, and hippocampus in mice Fig. 6. Plasma level of leptin in nontumor-bearing rats (Normal), HHM rats and rats (44, 45). (HHM), and rats injected with anti-PTHrP antibody (HHM + Ab)53and63dafter In conclusion, we showed that increased plasma levels of implantation of tumor. Columns, mean (n = 4-6); bars, SE. **, P < 0.01, compared PTHrP cause HHM and concurrent anorexia and cachexia with nontumor-bearing rats; ##, P < 0.01, compared with rats given with anti-PTHrP antibody. without up-regulating plasma leptin levels and anorexigenic peptide expression and down-regulating orexigenic peptide expression. Thus, modulation of leptin or the hypothalamic results suggest that the HHM rats were eager to eat but, in fact, feeding-regulating peptides is not the primary action of PTHrP could not, and restoration by the administration of anti-PTHrP that induces anorexia and cachexia, and other mechanisms are antibody was the consequence of the improvement of their involved in the PTHrP-induced anorexia and cachexia. feeding. Although it has been generally believed that anorexia is accompanied by the up-regulation of anorexigenic peptides Acknowledgments and/or down-regulation of orexigenic peptides, the hypotha- lamic signals clearly tended to stimulate feeding in HHM rats. We thank Y. Asao and N. Aoyama for their technical assistance and F. Ford for Elevated plasma levels of have also been reported in the editing the article.

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