Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia

Abstract

Cachexia is a wasting disorder of adipose and skeletal muscle tissues that leads to profound weight loss and frailty. About half of all cancer patients suffer from cachexia, which impairs quality of life, limits cancer therapy and decreases survival. One key characteristic of cachexia is higher resting energy expenditure levels than in healthy individuals, which has been linked to greater thermogenesis by brown fat1,2,3,4,5,6. How tumours induce brown fat activity is unknown. Here, using a Lewis lung carcinoma model of cancer cachexia, we show that tumour-derived parathyroid-hormone-related protein (PTHrP) has an important role in wasting, through driving the expression of genes involved in thermogenesis in adipose tissues. Neutralization of PTHrP in tumour-bearing mice blocked adipose tissue browning and the loss of muscle mass and strength. Our results demonstrate that PTHrP mediates energy wasting in fat tissues and contributes to the broader aspects of cancer cachexia. Thus, neutralization of PTHrP might hold promise for ameliorating cancer cachexia and improving patient survival.

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Figure 1: LLC tumours cause adipose tissue browning and cachexia.
Figure 2: LLC-cell-conditioned medium stimulates thermogenic gene expression in fat cells.
Figure 3: PTHrP is responsible for most of the LLC-cell-derived browning activity.
Figure 4: Neutralization of PTHrP prevents tumour-induced adipose tissue browning.
Figure 5: PTHrP is associated with wasting of the LBM in cachectic mice and humans.

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Gene Expression Omnibus

Data deposits

The microarray data set has been deposited in the Gene Expression Omnibus (GEO) database under the accession GSE57797.

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Acknowledgements

We thank M. Mourtzakis and C. Prado for their help with the human study. S. Kir is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation (DRG-2153-13), and J.P.W. is supported by a postdoctoral fellowship from the American Cancer Society (PF-13-385-01-TBE). This work was supported by National Institutes of Health grant DK31405 to B.M.S.

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Authors

Contributions

S. Kir and B.M.S. conceived and designed the experiments. S. Kir, J.P.W., S. Kleiner., L.K. and P.C. performed the experiments. S. Kir, J.P.W. and V.E.B. analysed the data. S. Kir and B.M.S. wrote the manuscript.

Corresponding author

Correspondence to Bruce M. Spiegelman.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Effects of LLC tumours on whole-body metabolism, food intake and physical activity.

Mice inoculated with LLC cells (n = 6 and 9 for the NTB and LLC groups, respectively) were placed into metabolic cages on day 4. Physical activity (a), food intake (b), carbon dioxide production (c), respiratory exchange ratio (RER) (d) and heat output (e) were monitored. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

Extended Data Figure 2 Fat-specific Prdm16-deficient mice are resistant to LLC-tumour-induced adipose tissue browning and wasting.

Wild-type (WT) and Prdm16-knockout (KO) mice inoculated with LLC cells were killed on day 11 (n = 5 for the KO-LLC group and 6 for the other groups). Tissue weight (a) and tumour weight (b) were measured. Gene expression changes in iWAT (c), eWAT (d), iBAT (e) and the gastrocnemius muscle (f) were determined by qRT–PCR. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, ** and *** denote differences between the NTB group and the LLC group. #, ## and ### denote differences between the WT group and the KO group. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; #, P < 0.05; ##, P < 0.005; ###, P < 0.0005.

Extended Data Figure 3 LLC-subclone- conditioned medium, EGF proteins, and PTHrP(1–34) and PTH(1–34) peptides stimulate Ucp1 and Dio2 expression in adipocytes.

a, Parent LLC (pLLC) cells or subclones were cultured in serum-free medium for 24 h. Primary adipocytes were treated for 24 h with the LLC-cell-conditioned media (n = 3). b, Primary adipocytes were treated with 100 ng ml−1 soluble EGF domains from the indicated EGF family proteins for 24 h (n = 3). c, Primary adipocytes were treated with 1 μM AST-1306 and 100 ng ml−1 BTC, EREG or HBEGF for 24 h (n = 3). d, Primary brown adipocytes were treated with 100 ng ml−1 PTHrP or PTH peptides for 2 h (n = 3). The mRNA levels were determined by qRT–PCR. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared with the control group.

Extended Data Figure 4 Time-course and dose-response of PTHrP treatment effects on primary white and brown adipocytes.

a, Primary white adipocytes were treated with PTHrP at various doses for 2 h (n = 3). b, Primary brown adipocytes were treated with 100 ng ml−1 PTHrP for 2–24 h or with 100 nM noradrenaline (NE) for 2 h (n = 3). c, Primary brown adipocytes were treated with PTHrP at various doses for 2 h (n = 3). The mRNA levels were measured by qRT–PCR. n = 3. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared with the control group.

Extended Data Figure 5 PTHrP stimulates cellular respiration and acts via the PKA signalling pathway.

a, b, Primary adipocytes were treated with 100 nM NE, 100 ng ml−1 PTHrP or 100 ng ml−1 PTH for 24 h, and UCP1 protein levels were measured by western blotting in white (a) and brown (b) adipocytes. c, The oxygen consumption rate (OCR) of primary white adipocytes, including basal respiration, uncoupled respiration (by blocking ATP synthase with oligomycin), maximal respiration (by stimulating uncoupling with FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone)) and non-mitochondrial respiration (with rotenone), was determined using an XF24 Extracellular Flux Analyzer. Real-time triplicate readings (upper panel) and their averages (lower panel) are shown (n = 4). dg, Primary adipocytes were serum-starved for 2 h and pretreated with H89 (50 μM for white adipocytes and 30 μM for brown adipocytes) for 1 h, and then 100 nM NE, 100 ng ml−1 PTHrP or 100 ng ml−1 PTH was added for 30 min to analyse protein phosphorylation by western blotting in white (d) and brown (e) adipocytes or for 2 h to assess gene expression by qRT–PCR (n = 3) in white (f) and brown (g) adipocytes. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared with the control group.

Extended Data Figure 6 PTHrP induces thermogenic gene expression patterns in vivo.

a, The expression of Pth1r (PTH and PTHrP receptor) was assessed in various tissues (n = 3). b, Plasma PTHrP levels were determined after subcutaneous injection of mice with 1 mg PTHrP per kg body weight (n = 4). PTHrP(1–34) has a short half-life in the blood (less than 2 h); however, its tissue retention might be different. c, d, Mice (n = 5 for the control group and 7 for the PTHrP group) received a single dose (c) or five daily doses (d) of 1 mg PTHrP per kg body weight and were killed 2 h after the final dose. e, Mice (n = 5 for the control group and 7 for the PTHrP group) were transferred to thermoneutrality (30 °C), and after two days of acclimation they received a single dose of 1 mg PTHrP per kg body weight and were killed 4 h later. More pronounced effects were observed at thermoneutrality, a condition under which basal thermogenic gene expression is diminished. Gene expression changes were measured by qRT–PCR in different fat depots. Vdr was identified as a robust mRNA target of PTHrP and was included as a positive control. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

Extended Data Figure 7 Neutralization of PTHrP prevents tumour-induced adipose tissue browning.

a, Mice inoculated with LLC cells received 10 mg kg−1 IgG or anti-PTHrP antibody every 3 days from day 6 to day 15 and were killed on day 16 (n = 4, 5 and 6 for the NTB, IgG and anti-PTHrP groups, respectively). At this time point, we did not observe any increase in the thermogenic gene profile in iWAT. b, Therefore, we performed another neutralization experiment in which the mice were treated similarly to a except that they received antibody injections on days 4 and 7 and were killed on day 8. The mRNA levels in iWAT were determined (n = 6 for each group). At this early stage of cachexia, weight loss was not evident. However, the expression of thermogenic genes was increased in iWAT, and the anti-PTHrP treatment blunted these changes. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. * denotes the difference between the NTB group and the IgG group. # denotes the difference between the IgG group and the anti-PTHrP group. *, P < 0.05; #P < 0.05.

Extended Data Figure 8 Effects of PTHrP neutralization on whole-body metabolism, food intake and physical activity of cachectic mice.

Mice were placed into metabolic cages 4 days after inoculation with LLC cells. They received 10 mg IgG or anti-PTHrP per kg body weight every 3 days from day 6 to day 15 (n = 6 for the NTB group and 5 for the other groups). Food intake (a), physical activity (b), carbon dioxide production (c), respiratory exchange ratio (RER) (d) and heat output (e) were monitored. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, ** and *** denote the difference between the NTB group and the IgG group. #, ## and ### denote the difference between the IgG group and anti-PTHrP group. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; #, P < 0.05; ##, P < 0.005; ###, P < 0.0005.

Extended Data Figure 9 Effects of PTHrP neutralization on ex vivo adipose and skeletal muscle tissue respiration.

The mice described in Extended Data Fig. 8 were killed on day 16, and their tissues were harvested for the measurement of ex vivo respiration using a Clark electrode (n = 6 for the NTB group and 5 for the other groups). The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *P < 0.05 compared with the NTB group.

Extended Data Figure 10 LLC-tumour-bearing mice did not display hypercalcaemia, and PTHrP treatment alone did not induce expression of muscle-atrophy-associated genes but exacerbated cachexia in the LLC-tumour-bearing mice.

a, Haematoxylin and eosin staining of a representative cross-sectional area of the gastrocnemius muscles from the experiment described in Fig. 4b–g. b, c, The plasma calcium level was measured in the experiments described in Fig. 4a–g. d, e, The mRNA levels in the quadriceps muscles from the experiment described in Extended Data Fig. 6c, d were measured (n = 5 for the control group and 7 for the PTHrP group). f, g, Primary myotubes were treated with 100 ng ml−1 tumour-necrosis factor-α (TNF-α) or PTHrP for 2 h to test gene expression by qRT–PCR or for 24 h to measure myotube diameter (n = 3). The values are mean ± s.e.m. Statistical analysis was conducted by two-tailed t-test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared with the control group. hj, Mice inoculated with LLC cells were killed after receiving daily injections of 1 mg PTHrP per kg body weight between days 10 and 16 (n = 5 for the LLC-Vehicle group and 6 for the other groups). The carcass weight was measured by subtracting the tumour weight from the total weight (h). The fat tissues (i) and tumours (j) were dissected and weighed. The values are mean ± s.e.m. Statistical analysis was conducted using the two-tailed t-test. *, ** and *** denote differences from the NTB-Vehicle group. # and ## denote differences between the LLC-Vehicle group and the LLC-PTHrP group. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; #, P < 0.05; ##, P < 0.005.

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This file contains all metabolic and clinical data for the cancer patients included in the study. (PDF 86 kb)

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Kir, S., White, J., Kleiner, S. et al. Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia. Nature 513, 100–104 (2014). https://doi.org/10.1038/nature13528

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