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 fat. 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.

Extended Data Figure 1. Effects of LLC tumors 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 at day 4. Physical activity (a), food intake (b) CO₂ production (c), respiratory exchange ratio (RER) (d), and heat output (e) were monitored. Values are means ± SEM. Statistics by 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 tumor-induced adipose tissue browning and wasting

Wild-type (WT) and Prdm16-knockout (KO) mice inoculated with LLC cells were sacrificed at day 11 (n = 5 for the KO-LLC group and 6 for the other groups). Tissue weight (a) and tumor weight (b) were measured. Gene expression changes in iWAT (c), eWAT (d), iBAT (e) and gastrocnemius muscle (f) were determined by RT-qPCR. Values are means ± SEM. Statistics by two-tailed t-test. (*) refers to differences between the NTB and LLC groups. (#) refers to differences between the WT and KO groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005, ###P < 0.0005.

Extended Data Figure 3. Media conditioned by LLC sublones, EGF proteins and PTHrP/PTH (1-34) peptides stimulate Ucp1 and Dio2 expression in adipocytes

a, ParentalLLC (pLLC) cells or the subclones were cultured in serum-free medium for 24 hr. Primary adipocytes were treated for 24 hr with the LLC cell-conditioned media (n = 3). b, Primary adipocytes were treated with soluble EGF domains of EGF family proteins (100 ng/ml) for 24 hr (n = 3). c, Primary adipocytes were treated with AST-1306 (1 μM) and BTC, EREG or HBEGF (100 ng/ml) for 24 hr (n = 3). d, Primary brown adipocytes were treated with PTHrP or PTH peptides (100 ng/ml) for 2 hr (n = 3). mRNA levels were determined by RT-qPCR.Values are means ± SEM. Statistics by 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 PTHrP treatments of primary white and brown adipocytes

a, Primary white adipocytes were treated with PTHrP at various doses for 2hr (n = 3). b, Primary brown adipocytes were treated with PTHrP at 100 ng/ml for 2-24 hr or with Norepinephrine (NE; 100 nM) for 2 hr (n = 3). c, Primary brown adipocytes were treated with PTHrP at various doses for 2hr (n = 3). mRNA levels were measured by RT-qPCR. n = 3. Values are means ± SEM. Statistics by 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 signaling system

a-b, Primary adipocytes were treated with NE (100 nM), PTHrP or PTH (100 ng/ml each) for 24 hr and UCP1 protein levels were measured by western blotting in white (a) and brown (b) adipocytes. c, 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) and non-mitochondrial respiration (with rotenone) was determined using SeaHorse metabolic analyzer. Real-time triplicate readings (upper panel) and their averages (lower panel) were shown (n = 4). d-g, Primary adipocytes were serum-starved for 2 hr and pretreated with H89 (50 μM for white adipocytes and 30 μM for brown adipocytes) for 1 hr and then NE (100 nM), PTHrP or PTH (100 ng/ml each) were added for 30 min to analyze protein phosphorylation by western blotting in white (d) and brown adipocytes(e) or for 2hr to test gene expression by RT-qPCR (n = 3) in white (f) and brown adipocytes (g). Values are means ± SEM. Statistics by 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, Expression of Pth1r was tested in various tissues (n = 3). b, Plasma PTHrP levels were determined after subcutaneous injection of mice with 1mg PTHrP per kg body weight (n = 4). PTHrP (1-34) has a short half-life in the blood (less than 2 hours), 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 5 daily doses (d) of PTHrP (1 mg/kg body weight) and were sacrificed 2 hr 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 PTHrP (1 mg/kg body weight) and were sacrificed 4 hr later. More pronounced effects were observed at thermoneutrality, a condition under which basal thermogenic gene expression is diminished. Gene expression changes were measured by RT-qPCR in different fat depots. Vdr was identified as a robust mRNA target of PTHrP and included as a positive control. Values are means ± SEM. Statistics by two-tailed t-test. *P < 0.05, **P < 0.005, ***P < 0.0005.

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

a, Mice inoculated with LLC cells received IgG or anti-PTHrP (10 mg/kg body weight) every 3 days from day 6 to day 15 and were sacrificed at 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. Therefore, we performed another neutralization experiment (b) in which mice were treated similarly to a except that they received antibody injections at day 4 and 7 and were sacrificed at day 8. mRNA levels in iWAT were determined (n = 6 for each group). At this early stage of cachexia, weight loss is not evident. However, expression of thermogenic genes was increased in iWAT and the anti-PTHrP treatment blunted these changes. Values are means ± SEM. Statistics by two-tailed t-test. (*) compares NTB and IgG groups. (#) refers to differences between the IgG and anti-PTHrP groups. *P < 0.05, #P < 0.05.

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

Mice were placed into metabolic cages 4 days after inoculation with LLC cells. They received IgG or anti-PTHrP (10 mg/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), CO₂ production (c), respiratory exchange ratio (RER) (d) and heat output (e) were monitored. Values are means ± SEM. Statistics by two-tailed t-test. (*) compares the NTB and IgG groups. (#) refers to differences between the IgG and anti-PTHrP groups. *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

Mice described in Extended Data Fig. 8 were sacrificed at day 16 and their tissues were harvested for measurement of ex vivo respiration using a Clark electrode (n = 6 for the NTB group and 5 for the other groups). Values are means ± SEM. Statistics by two-tailed t-test. *P < 0.05 compared with the NTB group.

Extended Data Figure 10. LLC tumor-bearing mice do not display hypercalcemia and PTHrP treatment alone does not induce expression of muscle atrophy-related genes but exacerbates cachexia in the LLC tumor-bearing mice

a, H&E staining of representative cross sectional area of gastrocnemius muscles from the experiment described in Fig. 4b-g. b-c, Plasma calcium was measured in experiments described in Fig 4a and 4b-g. d-e, mRNA levels in 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 TNFα or PTHrP (100 ng/ml each) for 2hr to test gene expression by RT-qPCR or24 hr to measure myotube diameter (n = 3). Values are means ± SEM. Statistics by two-tailed t test. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with the control group. h-j, Mice inoculated with LLC cells were sacrificed after receiving daily injections of PTHrP (1 mg/kg body weight) between days 10-16 (n = 5 for the LLC-Vehicle group and 6 for the other groups). Carcass weight was measured by subtracting the tumor weight from the total weight (h). Fat tissues (i) and tumors (j) were dissected and weighed. Values are means ± SEM. Statistics by two-tailed t-test. (*) refers to differences compared to the NTB-Vehicle group. (#) refers to differences between the LLC groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005.

Figure 1. LLC tumors cause adipose tissue browning and cachexia

Mice inoculated with LLC cells were monitored up to 21 days (n = 6 for each group). NTB stands for non-tumor-bearing. 4 days after inoculation, a cohort of mice (n = 6 and 9 for the NTB and LLC groups respectively) was placed into metabolic cages to measure O₂ consumption (a). Carcass weight (calculated by subtracting tumor weight from the total weight) (b), weight of fat and muscle tissues (c) were measured. mRNA levels in iWAT (d), eWAT (e), iBAT (f) and gastrocnemius muscle (g) were determined by RT-qPCR. Values are means ± SEM. Statistics by two-tailed t-test. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with the NTB group.

Figure 2. LLC cell-conditioned medium stimulates thermogenic gene expression in fat cells

a-c,ParentalLLC cells or the subclones were cultured in serum-free medium for 24 hr. Primary adipocytes (n = 3 in a and b) were treated for 24 hr with the LLC cell-conditioned media (a and c) orthe filtered fractions (b). d, Gene expression heat map of the secreted factors identified in the microarray analysis. e, Primary adipocytes were treated with the indicated proteins (1 μg/ml) for 24 hr (n = 3). mRNA levels were determined by RT-qPCR. Ap2 and Pparg are controls for adipocyte differentiation.Values are means ± SEM. Statistics by two-tailed t-test. *P < 0.05, **P < 0.005, ***P < 0.0005 compared with the control group.

Figure 3. PTHrP is responsible for most of the LLC cell-derived browning activity

a-c, Primary adipocytes (n = 3 for each group) were treated with AST-1306 (1 μM) and LLC cell-conditioned medium for 24 hr (a) or with the indicated peptides (100 ng/ml) for 2 hr (b) or with PTHrP at 100 ng/ml for 2-24 hr and NE (100 nM) for 4 hr (c). d-e, Primary adipocytes (n = 3 for each group) were treated with IgG or anti-PTHrP (10 μg/ml) along with PTHrP (10 ng/ml) for 4 hr (d) or with LLC cell-conditioned medium for 24 hr (e). mRNA levels were measured by RT-qPCR. Values are means ± SEM. Statistics by two-tailed t-test. (*) refers to differences compared to the control or IgG groups. (#) refers to differences between the PTHrP groups or the LLC groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005, ###P < 0.0005.

Figure 4. Neutralization of PTHrP prevents tumor-induced adipose tissue browning

a, Plasma PTHrP levels in mice bearing LLC tumors up to 21 days (n = 6). b-g, Mice inoculated with LLC cells received IgG or anti-PTHrP (10 mg/kg body weight) every 3 days from day 6 to day 15 and were sacrificed at day 16 (n = 4, 5 and 6 for the NTB, IgG and anti-PTHrP groups respectively). Carcass weight (b), weight of tumors (c), fat and muscle tissues (d) and H&E staining of adipose tissues were shown (e). mRNA levels in eWAT (f) and iBAT (g) were measured by RT-qPCR. h, Mice were treated similarly to b-g except that they were placed into metabolic cages at day 4 to measure O₂ consumption (n = 6 for the NTB group and 5 for the other groups). Values are means ± SEM. Statistics by two-tailed t-test. (*) compares the NTB and IgG groups. (#) refers to differences between the IgG and anti-PTHrP groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005, ###P < 0.0005.

Figure 5. PTHrP is associated with wasting of lean body mass in cachectic mice and humans

a, mRNA levels in gastrocnemius muscle from the experiment described in Fig. 4b-g. b-e, Mice inoculated with LLC cells received IgG or anti-PTHrP (10 mg/kg body weight) every 3 days from day 6 to day 21-22 (n = 5 for the anti-PTHrP group and 6 for the other groups). Representative images of hindlimb and gastrocnemius muscles are shown (b). Fat and muscle tissues were weighed (c). Muscle function was analyzed by grip strength (d) and in situ contraction (e). f-h, Mice inoculated with LLC cells were sacrificed after receiving daily injections of PTHrP (1 mg/kg body weight) between days 10-16 (n = 5 for the LLC-Vehicle group and 6 for the other groups). Gastrocnemius muscle weight (f) and grip strength (g) were measured. mRNA levels in gastrocnemius muscle were determined by RT-qPCR (h). i-j, LBMand REE/LBM of the PTHrP(+) and PTHrP(−) patients were compared. k. A model for PTHrP action in cancer cachexia. Values are means ± SEM. Statistics by two-tailed t-test. (*) refers to differences compared to the NTB groups. (#) refers to differences between either the IgG and anti-PTHrP groups or the LLC-Vehicle and LLC-PTHrP groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005, ###P < 0.0005.