Adipose tissue browning in cancer-associated cachexia can be attenuated by inhibition of exosome generation

https://doi.org/10.1016/j.bbrc.2018.09.139Get rights and content

Highlights

  • Exosomes produced by Lewis lung carcinoma (LLC) cells can cause lipolysis in 3T3-L1 adipocytes, as well as in white adipose tissue in mice, and are one of the factors that contribute to cancer-induced cachexia (CAC).

  • The neutral sphingomyelinase inhibitor, GW4869 not only inhibits exosome generation and release by tumor cells, but also alleviates exosome-induced lipolysis in 3T3-L1 adipocytes.

  • Inhibition of exosome generation and release with GW4869 can prevent the onset of CAC in mice bearing LLC tumors.

Abstract

Cancer-associated cachexia (CAC) is a disorder characterized by unintended weight loss due to skeletal muscle wasting and adipose tissue loss. Although muscle atrophy in this condition has been well studied, the mechanisms underlying adipose tissue loss, which include browning, have not been investigated in detail. In this respect, though recent studies have shown that exosomes from cancer cells can promote lipolysis, the link between exosomes from cancer cells and CAC has not been clearly established. In this study, we investigate if exosomes from Lewis lung carcinoma (LLC) cells can induce lipolysis in vitro (in 3T3-L1 adipocytes) and in vivo (in LLC tumor-bearing mice). We find that exosomes from LLC cells do induce lipolysis in 3T3-L1 adipocytes and that the white adipose tissues of mice with LLC tumors show clear signs of lipolysis. We also find that this lipolysis can be inhibited using the neutral sphingomyelinase inhibitor GW4869. Our results indicate that GW4869 not only inhibits exosome generation and release from LLC cells, but can also inhibit lipolysis induced by LLC-derived exosomes in 3T3-L1 adipocytes. Furthermore, we also demonstrate that LLC tumor-bearing mice treated with GW4869 did not develop CAC. In summary, our results suggest that inhibiting exosome generation and release can inhibit lipolysis and adipose tissue browning, and may be useful as a novel strategy for treating CAC.

Introduction

Cancer-associated cachexia (CAC) is a disorder characterized by unintended weight loss due to skeletal muscle wasting and adipose tissue loss [1]. The condition often occurs in patients with pancreatic, lung, and colon cancers during advanced stages. Furthermore, CAC is also known to occur in people who have a poor quality of life, or have been exposed to radiotherapy and chemotherapy; CAC is one of the most common causes of death in patients with advanced-stage cancers [2,3]. As of now, no treatment options are available to prevent or reverse cachexia [1,4]. A better understanding of the mechanisms underlying CAC is therefore needed to devise effective therapies to treat this condition.

White adipose tissue (WAT), which is responsible for storing energy, is converted to brown adipose tissue (BAT), which is generally responsible for dissipating energy. However, brown fat cells also emerge in white adipose tissue, we called this process ‘browning’. One of the hallmark characteristics of CAC is the ‘browning’ of WAT. In this process, the ‘browning’ of WAT is known to be mediated by the uncoupling protein 1 (UCP1), which is abundant in brown adipose tissue and uncouples oxidative phosphorylation from ATP synthesis in the inner mitochondrial membrane to generate heat [5]. Since adipose tissue ‘browning’ contributes to lipolysis and adipose tissue loss in CAC, we hypothesize that the inhibition of this process may aid in alleviating CAC [6,7].

The molecular mechanisms influencing CAC are complex, and several tumor-associated factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and parathyroid hormone-related protein are known to contribute to CAC [4,7,8]. However, Recent studies indicate that exosomes (produced by many different cell types containing proteins, nucleic acids, and lipids) can mediate communication within tumor microenvironment, and that tumor cell-derived exosomes can contribute to cachexia [9]. Exosomes containing heat-shock proteins (HSPs) such as HSP70 and HSP90 produced by Lewis lung carcinoma (LLC) cells have been found to induce CAC-like muscle wasting [10]; similarly, exosomes from K7M2 cells can mediate muscle atrophy through notch signaling in CAC in mice [11]. We therefore hypothesize that CAC may be alleviated by inhibiting exosome release by tumor cells. We use the neutral sphingomyelinase inhibitor, GW4869, which is known to block exosome generation and release from RAW264.7 macrophages, HEK293 cells, and lung epithelial cells [[12], [13], [14]], to investigate the effects of inhibiting exosome generation on lipolysis in cultured adipocytes in vitro and on WATs in vivo in LLC tumor-bearing mice.

Section snippets

Cell culture and differentiation

LLC and 3T3-L1 cell lines were obtained from ATCC (American type culture collection). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin.

To differentiate 3T3-L1 cells into adipocytes, cells were treated with 0.5 mM isobutylmethylxanthine, 1 mM dexamethasone, 5 mg/mL insulin, and 5 mM troglitazone (all from Sigma, St. Louis, MO, USA). Two days after differentiation, cells were

LLC-derived conditioned medium (LCM) induces lipolysis in 3T3-L1 adipocytes

Since numerous studies have shown that LLC can induce cachexia in mice, we investigated the effects of LLC cell-derived factors on lipolysis in 3T3-L1 adipocytes. Western blotting and densitometric analyses indicated that adipocytes treated with LLC-derived conditioned medium (LCM) have higher levels of UCP1 and phospho-hormone sensitive lipase (P-HSL, a markers of activated lipolysis) than those in the control group (CN) (Fig. 1A and B). Furthermore, adipocytes treated with LCM released higher

Discussion

Our study establishes that exosomes from LLC cells can cause lipolysis in vitro and in vivo, and also explores the effects of blocking exosome release from LLC cells under in vitro and in vivo conditions. We find that GW4869 can effectively inhibit exosome release in LLC cells, and that it inhibits the development of CAC in mice. Our work shows that GW4869 attenuates LLC cell-induced lipolysis in 3T3-L1 adipocytes in vitro, and adipose tissue browning in LLC tumor-bearing mice. These results

Declaration of interest statement

The authors report no conflicts of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (NSFC) Grant [81773282, 81672528, and 81773282] and Hubei Provincial Health Project Grant WJ2015MB020.

References (24)

  • J.M. Argiles et al.

    Cancer cachexia: understanding the molecular basis

    Nat. Rev. Canc.

    (2014)
  • M. Xu et al.

    Deficiency in hormone-sensitive lipase accelerates the development of pancreatic cancer in conditional KrasG12D mice

    BMC Canc.

    (2018)
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