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Tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding neuropeptides via Lgr3/8 in the brain

Nature Cell Biology volume 23, pages 172–183 (2021)Cite this article

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Abstract

In patients with advanced-stage cancer, cancer-associated anorexia affects treatment success and patient survival. However, the underlying mechanism is poorly understood. Here, we show that Dilp8, a Drosophila homologue of mammalian insulin-like 3 peptide (INSL3), is secreted from tumour tissues and induces anorexia through the Lgr3 receptor in the brain. Activated Dilp8-Lgr3 signalling upregulated anorexigenic nucleobinding 1 (NUCB1) and downregulated orexigenic short neuropeptide F (sNPF) and NPF expression in the brain. In the cancer condition, the protein expression of Lgr3 and NUCB1 was significantly upregulated in neurons expressing sNPF and NPF. INSL3 levels were increased in tumour-implanted mice and INSL3-treated mouse hypothalamic cells showed Nucb2 upregulation and Npy downregulation. Food consumption was significantly reduced in intracerebrospinal INSL3-injected mice. In patients with pancreatic cancer, higher serum INSL3 levels increased anorexia. These results indicate that tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding hormones through the Lgr3/Lgr8 receptor in Drosophila and mammals.

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Fig. 1: Cancer anorexia precedes organ wasting in the Drosophila eye tumour model.
Fig. 2: Tumour-derived Dilp8 suppresses food intake through its receptor Lgr3.
Fig. 3: Dilp8-Lgr3 signalling regulates anorexigenic NUCB1 and orexigenic sNPF and NPF expression in the Drosophila brain.
Fig. 4: NUCB1- and Lgr3-expressing neurons are markedly increased and overlapped in the cancer condition.
Fig. 5: INSL3-LGR8 signalling regulates anorexigenic Nucb2 and orexigenic Npy expression in mouse hypothalamic cells.
Fig. 6: INSL3 regulates Nucb2 and Npy, and induces anorexia in the mouse tumour model.
Fig. 7: Serum INSL3 levels correlate with anorexia severity in patients with pancreatic cancer.

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Data availability

The RNA-seq data have been deposited in the GEO database under accession code GSE154404. Data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A.M. Gontijo, P. Leopold, M. Dominguez and P. Shen for providing reagents, D.-W. Kim and G. Wee for helping with experiments, O.-Y. Kwon for suggesting nesfatin in flies, and J.-S. Lee, K.-W. Choi, Y.-J. Kim and M. Subramanian for comments on the manuscript. Drosophila stocks were obtained from the Bloomington Stock Center (Bloomington) and Vienna Drosophila RNAi Center (VDRC). This work was supported by grants from the KRIBB Research Initiative Program, the National Research Council of Science & Technology (CRC-15-04-KIST), the National Research Foundation of Korea (2015R1A5A1009024, 2017K1A1A2013124, 2018R1A2A3075389, 2019R1A2C2004149 and 2019R1A2C2089484) and the Korean Health Technology R&D Project, Ministry of Health & Welfare, Korea (HI14C2640).

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Author notes

  1. These authors contributed equally: Eunbyul Yeom, Hyemi Shin, Wonbeak Yoo.

Authors and Affiliations

  1. Metabolism and Neurophysiology Research Group, Disease Target Structure Research Center, KRIBB, Daejeon, Korea

    Eunbyul Yeom, Seung Hyun Hong, Dae-Woo Kwon, Tae Hoon Ryu, Kyu-Sun Lee & Kweon Yu

  2. Tunneling Nanotube Research Center, Korea University, Seoul, Korea

    Eunbyul Yeom

  3. Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea

    Hyemi Shin & Jae Myoung Suh

  4. Environmental Disease Research Center, KRIBB, Daejeon, Korea

    Wonbeak Yoo

  5. Department of Convergence Medicine and Division of Hepato-Biliary and Pancreatic Surgery, Department of Surgery, University of Ulsan College of Medicine, Asan Institute for Life Sciences, AMIST, Asan Medical Center, Seoul, Korea

    Eunsung Jun

  6. Department of Medicinal Biotechnology, College of Health Sciences, Dong-A University, Busan, Korea

    Seokho Kim

  7. Department of Functional Genomics, UST, Daejeon, Korea

    Dae-Woo Kwon, Tae Hoon Ryu, Kyu-Sun Lee & Kweon Yu

  8. Division of Hepato-Biliary and Pancreatic Surgery, Department of Surgery, University of Ulsan College of Medicine, Asan Medical Center, Asan Biomedical Engineering Research Center, AMIST, Seoul, Korea

    Song Cheol Kim

  9. Convergence Research Center of Dementia, KIST, Seoul, Korea

    Kweon Yu

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Contributions

E.Y., K.-S.L. and K.Y. designed the research. S.C.K. provided key reagents. E.Y., H.S., W.Y., E.J., S.H.H., D.-W.K. and T.H.R. performed experiments. E.Y., S.K., J.M.S., S.C.K., K.-S.L. and K.Y. analysed the data. E.Y., K.-S.L. and K.Y. wrote the manuscript.

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Correspondence to Jae Myoung Suh, Song Cheol Kim, Kyu-Sun Lee or Kweon Yu.

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Extended data

Extended Data Fig. 1 Generation of the Drosophila cancer anorexia model.

a, Drosophila cancer model was established by overexpressing a wild-type (ykiWT) and a constitutively active form of yki (ykiS168A) using GMR-Gal4. Expression of the wild-type of yki (GMR > ykiWT) caused the mild rough eye phenotype, whereas the active form (GMR > ykiSA) showed dramatic protrusion of the eye. Scale bars are 200μm. b, The 10 day-old yki overexpression flies showed decreased food intake compared with that of the control in the blue dye feeding assay. n = 3 biologically independent experiments; 1.5 days *P = 0.022, 2 days **P = 0.003. c, UAS-ykiWT and UAS-ykiSA control flies showed normal feeding. n = 3 biologically independent experiments. d, The triglyceride levels of GMR > ykiSA was analyzed with female flies. n = 3 biologically independent experiments; day 10 *P = 0.038, day 15 *P = 0.035, day 20 *P = 0.012. e, Quantification of ovary size in Fig. 1c. n = 3 biologically independent experiments; day 10 ***P = 0.0006, day 15 **P = 0.0014, day 20 ***P < 0.001. f, The thoracic muscle of 15-day-old GMR > ykiSA showed abnormal mitochondrial structure and irregular packing of mitochondria between muscle fibers. n = 3 biologically independent experiments. Scale bar is 10μm. Data are presented as the mean ± s.e.m. Statistical significance was determined with two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001. Statistical source data.

Source data

Extended Data Fig. 2 RNA-seq analysis, anorexia in sev > Rasv12 flies, Lgr3Δ50 generation, Lgr3 expression in adult tissues, and eye phenotypes in GMR > ykiSA and the mutants of Dilp8 and Lgr3.

a, RNA-seq analysis showed the increased levels of three tumor-secreted factors (Dilp8, ImpL2, and Upd2) in GMR > ykiSA flies. b, sev > Rasv12 flies showed anorexia and this phenotype was effectively rescued by Dilp8-RNAi. n = 3 biologically independent experiments; sev > Rasv12 *P = 0.012, sev > Rasv12 Dilp8-RNAi *P = 0.013, sev > Rasv12 ImpL2-RNAi *P = 0.019. c, Dilp8 antibody test was performed by western blot and showed almost no expression in the Dilp8M100727 mutant. d, Dilp8 protein was increased in GMR > ykiSA fly heads and reduced in GMR > ykiSA, Dilp8-RNAi fly heads by western blot. e, The quantification of d using ImageJ program. n = 3 biologically independent experiments; GMR > ykiSA *P = 0.047, GMR > ykiSA Dilp8-RNAi *P = 0.045. f, The Lgr3Δ50 mutant was obtained using the CRISPR/Cas9-mediated mutagenesis. It consists of 50 base pair deletion in the intron region upstream of target sequences. g, Lgr3 mRNA expression in the Lgr3Δ50 mutant showed that the Lgr3Δ50 mutant is a strong hypomorphic allele. n = 3 biologically independent experiments, **P = 0.007. h, GMR > ykiSA,Dilp8MI00727 and GMR > ykiSA, Lgr3Δ50 flies respectively blunted or reversed the cancer anorexia phenotype observed in GMR > ykiSA flies. n = 4 biologically independent experiments; GMR > ykiSA *P = 0.010, GMR > ykiSADilp8M100727 *P = 0.012, GMR > ykiSALgr3Δ50 *P = 0.027. i, Dilp8 overexpression flies (GMR > Dilp8) showed reduced food intake. n = 6 biologically independent experiments, *P = 0.034. j, In GMR > ykiSA and GMR-Gal4 control flies, the Lgr3 mRNA expression level in the head is very high relative to other tissues. n = 3 biologically independent experiments; head *P = 0.017; thorax **P = 0.006, gut *P = 0.035. k, The eye phenotype in GMR > ykiSA is similar with those of GMR > ykiSA, Dilp8M100727 and GMR > ykiSA, Lgr3Δ50. Scale bar is 200μm. l, Lgr3-Gal4 expression marked by GFP. Lgr3 expression was not detectable in sNPF neurons. Scale bar is 100μm. m-n, sNPF (m) (*P = 0.018) and NPF (n) (*P = 0.048) mRNA levels were reduced by RNAi lines with Lgr3-Gal4. n = 3 biologically independent experiments. Data are presented as the mean ± s.e.m. Statistical significance was determined with two-tailed Student’s t-test; *P < 0.05, **P < 0.01. Statistical source data and unprocessed western blots.

Source data

Extended Data Fig. 3 Sequence homology of NUCB1 and NUCB2 in Drosophila and mammals.

The very similar signal peptide and cleavage site among mammalian NUCB2 and Drosophila NUCB1 suggest that the evolutionary conserved Nesfatin1 might be produced.

Extended Data Fig. 4 Intensity quantification of NUCB1 and Lgr3 antibodies immunostaining.

a, NUCB1 antibody specificity was tested by the NUCB1 inhibition using heat-shock Gal4 (Hs > NUCB1-RNAi). b, Staining intensity of neurons was measured with LSM 5 software program by drawing a dotted line as shown. c-e, The staining intensity of neuronal cells in Fig. 4 was quantified using LSM 5 software program and illustrated as intensity graphs. Control (n = 5), GMR > ykiSA (n = 10), GMR > ykiSA, Lgr3Δ50 (n = 5). f-g, A subset of sNPF (f) and NPF (g) expressing neurons in the cancer condition (GMR > ykiSA) were overlapped with NUCB1 expressing neurons. Scale bars are 100 μm. Statistical source data and unprocessed western blots.

Source data

Extended Data Fig. 5 NUCB1 protein expression in the cancer condition, ex vivo analysis, and Lgr3 expression by the Dilp8 treatment.

a, In western blot of adult heads with the NUCB1 antibody, NUCB1 protein level is increased in GMR > ykiSA, reduced in the Lgr3Δ50 mutant, and restored to the control level in GMR > ykiSA, Lgr3Δ50. b, The western blot bands of (a) were quantified using ImageJ program. n = 3 biologically independent experiments; GMR > ykiSA **P = 0.003, Lgr3Δ50 *P = 0.038, GMR > ykiSALgr3Δ50 ** P = 0.002. c, The schematic presentation of ex vivo culture assay in which the synthetic Dilp8 peptide was treated to the dissected adult brains in the culture media. d, Lgr3 mRNA level was increased after Dilp8 peptide treatment in the ex vivo culture. n = 3 biologically independent experiments; 0.5 hr *P = 0.020, 1 hr *P = 0.021. Data are presented as the mean ± s.e.m. Statistical significance was determined with two-tailed Student’s t-test; *P < 0.05, **P < 0.01. Statistical source data and unprocessed western blots.

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Extended Data Fig. 6 Conservation of INSL3/Lgr8 in mammals, and cachexia-anorexia phenotype of C26 or LLC implanted mice.

a. The phylogenetic comparative analysis of Drosophila Lgr3 and mammalian Lgr family shows that Lgr7/8 is a homologue of Lgr3. b, The phylogenetic comparative analysis of fly Dilp8 and mammalian INSL3 shows that they are classified as close peptides. c, Lgr8 protein is strongly expressed in the hypothalamic region and hippocampus of the mouse brain. d, In the RNA FISH, Lgr8 mRNA is strongly expressed in the hypothalamic region and hippocampus of the mouse brain. e, Lgr8 is expressed in the mouse hypothalamic N3 cells, but not in N39 cells. f-g, Body weight was changed in C26 tumor implanted mice (f) not in LLC tumor implanted mice (g). n = 7 mice for both groups. f, 2w + 2day *P = 0.027, 2w + 4day ***P = 0.0004, 2w + 6day **P = 0.0034. h, Tumor volume and weight changes were measured in C26 and LLC implanted mice. n = 6 mice for both groups. i-j, Muscle and fat weight of C26 tumor implanted mice at day 12 (i) and day 21 (j). Tibialis anterior (TA), gastrocnemius (GCM), inguinal white adipose tissue (iWAT), and epididymal white adipose tissue (eWAT). Control n = 3 mice, C26 day 12 n = 5 mice, C26 day 21 n = 4 mice. i, iWAT *P = 0.024, eWAT *P = 0.094. j, GCM *P = 0.038, iWAT **P = 0.001, eWAT *P = 0.013. k-l, Nucb2 (k) and Npy (l) mRNA levels were not changed in LLC implanted mice. n = 5 mice for both groups. m, INSL3 i.p. injected wild-type mice didn’t show reduction of food intake. n = 7 mice for both groups. n-o, Nucb2 (n) and Npy (o) mRNA levels were not changed in INSL3 i.p. injected wild-type mice hypothalamus. n = 3 mice for both groups. Data are presented as the mean ± s.e.m. Statistical significance was determined with two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001. Statistical source data and unprocessed western blots.

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Extended Data Fig. 7 Anorexia-associated body mass changes of human pancreatic cancer implanted mice.

a, Tumor volumes were increased in Panc1 and Capan1 implanted mice. n = 4 mice for three groups; 3 week *P = 0.049, 4 week *P = 0.049, 5 week *P = 0.033. b, Body mass were reduced in Panc1 and Capan1 implanted mice compared to the control mice. n = 4 mice for each group; 3 week Panc1 **P = 0.0014 and Capan1 *P = 0.049, 5 week Panc1 ***P < 0.0001 and Capan1 ***P < 0.0001. 6 week Panc1 ***P < 0.0001 and Capan1 ***P < 0.0001. c, Relaxin-INSL family mRNA expression levels in Capan1 cells. n = 3 biologically independent experiments; Rxn vs INSL3 **P = 0.003. Data are presented as the mean ± s.e.m. Statistical significance was determined with two-tailed Student’s t-test; *P < 0.05. Statistical source data.

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Supplementary Tables 1–3

Supplementary Table 1. Feeding regulatory neuropeptides in Drosophila were identified in the RNA sequencing analysis of GMR>ykiSA. Supplementary Table 2. Clinicopathological characteristics among patients with pancreatic cancer and other diseases. Supplementary Table 3. Clinicopathological characteristics of patients enrolled in food intake analysis.

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Yeom, E., Shin, H., Yoo, W. et al. Tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding neuropeptides via Lgr3/8 in the brain. Nat Cell Biol 23, 172–183 (2021). https://doi.org/10.1038/s41556-020-00628-z

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