Elsevier

Human Pathology

Volume 79, September 2018, Pages 199-207
Human Pathology

Original contribution
In situ analysis of mTORC1/2 and cellular metabolism–related proteins in human Lymphangioleiomyomatosis,☆☆

https://doi.org/10.1016/j.humpath.2018.05.018Get rights and content

Highlights

  • LAM is a rare lung disease characterized by mTOR hyperactivation.

  • In our study, high mTORC1 and -C2 activity was observed in the majority of LAM cases.

  • Bioenergetic pathways may also play an important role in the pathobiology of LAM.

Summary

Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease with features of a low-grade neoplasm. It is primarily caused by mutations in TSC1 or TSC2 genes. Sirolimus, an inhibitor of mTOR complex 1 (mTORC1), slows down disease progression in some, but not all patients. Hitherto, other potential therapeutic targets such as mTOR complex 2 (mTORC2) and various metabolic pathways have not been investigated in human LAM tissues. The aim of this study was to assess activities of mTORC1, mTORC2 and various metabolic pathways in human LAM tissues through analysis of protein expression. Immunohistochemical analysis of p-S6 (mTORC1 downstream protein), Rictor (mTORC2 scaffold protein) as well as GLUT1, GAPDH, ATPB, GLS, MCT1, ACSS2 and CPT1A (metabolic pathway markers) were performed on lung tissue from 11 patients with sporadic LAM. Immunoreactivity was assessed in LAM cells with bronchial smooth muscle cells as controls. Expression of p-S6, Rictor, GAPDH, GLS, MCT1, ACSS2 and CPT1A was significantly higher in LAM cells than in bronchial smooth muscle cells (P<.01). No significant differences were found between LAM cells and normal bronchial smooth muscle cells in GLUT1 and ATPB expression. The results are uniquely derived from human tissue and indicate that, in addition to mTORC1, mTORC2 may also play an important role in the pathobiology of LAM. Furthermore, glutaminolysis, acetate utilization and fatty acid β-oxidation appear to be the preferred bioenergetic pathways in LAM cells. mTORC2 and these preferred bioenergetic pathways appear worthy of further study as they may represent possible therapeutic targets in the treatment of LAM.

Introduction

Lymphangioleiomyomatosis (LAM) is a rare multisystem disorder with a strong female predilection, manifesting primarily as a progressive, diffuse cystic lung disease. Symptoms are generally due to obstructive lung disease; however, pleural complications such as pneumothorax and chylothorax may also occur. The lung cysts are related to proliferation of immature smooth muscle cells of perivascular phenotype (LAM cells) [1] and LAM is now considered as a low-grade neoplasm of the perivascular epithelioid cell tumor family [2]. The term sporadic LAM is used for patients without the tuberous sclerosis complex (TSC), while TSC-LAM refers to LAM that occurs in the setting of TSC. Both forms are primarily caused by TSC1 or TSC2 gene mutations [3]. These mutations lead to hyperactivation of the mammalian target of rapamycin (mTOR) and subsequently proliferation of LAM cells [4].

mTOR is a component of two multiprotein complexes: mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2) (Fig. 1). In addition to mTOR, mTORC1 contains scaffold protein Raptor and mTORC2 contains scaffold protein Rictor [5]. Through its participation in mTORC1 and mTORC2, mTOR integrates a variety of environmental signals and regulates cell growth and homeostasis [6]. Activation of mTORC1 leads to phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4EBP1), S6 kinase (S6K), and ribosomal S6 protein. Phospho-S6 (p-S6) facilitates protein translation, cell growth, and proliferation. mTORC1 also influences cellular metabolism (Fig. 1). Alternatively, mTORC2 is a mediator of actin cytoskeletal organization and promotes cell survival via phosphorylation of protein kinase B and serum- and glucocorticoid-induced protein kinase [5], [6].

mTOR also plays a central role in metabolic reprogramming of neoplastic cells with altered utilization of glucose, glutamine, and lipids [5], [6], [7], [8], [9]. Increased glucose uptake and overexpression of glucose transporter 1 (GLUT1) is well documented in most neoplasms [10], [11]. Regardless, certain neoplasms, including LAM, are undetectable by positron emission tomography using 2-deoxy-2-[18F]fluoro-D-glucose [12], [13], [14], suggesting that these neoplasms may use an alternative energy source such as glutamine or acetate instead of glucose.

Sirolimus is an mTORC1 inhibitor, which has been successfully utilized to attenuate disease progression in LAM patients [15], [16]. Unfortunately, lost lung function is not restored and disease progression resumes once treatment is discontinued [16]. In addition, certain disease subgroups, such as those that are post-menopausal, may have limited if any benefit from sirolimus. Lastly, the most effective dose and treatment duration are unknown, although presumed to be lifelong. Of course, some patients are unable to tolerate the treatment due to adverse events [17], [18]. It is therefore important to identify pathobiological targets that may respond to new or additional therapeutics to mitigate the proliferation of LAM cells.

The purpose of this study was to assess the significance of mTORC1, mTORC2 and various metabolic pathways (including glycolysis, oxidative phosphorylation, glutaminolysis, fatty acid β-oxidation, and acetate utilization) in the pathogenesis of LAM, using semiquantitative immunohistochemical methods on formalin-fixed paraffin-embedded human lung tissue.

Section snippets

Tissues

Our study was approved by the Mayo Clinic Institutional Review Board. Formalin-fixed paraffin-embedded lung tissue was available from the lung tissue registry for 11 patients with sporadic LAM. These patients underwent lung transplantation (7 patients) and diagnostic wedge biopsies (4 patients) at Mayo Clinic in Jacksonville, Florida, between January 1, 2004 and December 31, 2016. Diagnosis of LAM was based on the presence of characteristic clinical, radiologic, and histologic findings and was

Expression of LAM cell markers

Expression of LAM cell markers and hormone receptors are shown in Table 3. LAM cells were positive for HMB-45 in 9 of 11 cases (82%). All cases were positive for SMA and β-catenin.

Expression of mTOR-related proteins

In LAM cells, high p-S6 expression suggesting high mTORC1 activity was observed in 10 of 11 cases (91%), and high Rictor expression suggesting high mTORC2 activity was observed in 6 of 11 cases (55%) (Fig. 3). Low expression for both p-S6 and Rictor was observed in only 1 case. In contrast, no or low expression was

Discussion

Histologically, LAM is characterized by cystic spaces surrounded by bundles of proliferating LAM-type smooth muscle cells [20]. These LAM cells are typically immunoreactive for HMB-45, β-catenin, SMA, desmin, ER, and progesterone receptor [21], [22], [23], [24].

mTOR plays an important role in the regulation of protein translation, cell growth, proliferation, cytoskeletal organization, and cellular metabolism [5], [8], [9] (Fig. 1). In this study, expression of p-S6 (downstream target of mTORC1)

References (35)

  • A. Csibi et al.

    The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-dependent control of c-Myc translation

    Curr Biol

    (2014)
  • S.R. Johnson et al.

    European Respiratory Society guidelines for the diagnosis and management of lymphangioleiomyomatosis

    Eur Respir J

    (2010)
  • R.J. DeBerardinis et al.

    Fundamentals of cancer metabolism

    Sci Adv

    (2016)
  • D. Medvetz et al.

    Therapeutic targeting of cellular metabolism in cells with hyperactive mTORC1: a paradigm shift

    Mol Cancer Res

    (2015)
  • M. Shimobayashi et al.

    Making new contacts: the mTOR network in metabolism and signalling crosstalk

    Nat Rev Mol Cell Biol

    (2014)
  • M.B. Calvo et al.

    Potential role of sugar transporters in cancer and their relationship with anticancer therapy

    Int J Endocrinol

    (2010)
  • R.B. Hamanaka et al.

    Targeting glucose metabolism for cancer therapy

    J Exp Med

    (2012)
  • Cited by (16)

    View all citing articles on Scopus

    Disclosures/Conflict of Interest: none.

    ☆☆

    Funding/Support: This work was supported by the ÚNKP-17-3 New National Excellence Program of The Ministry of Human Capacities (I. K.), scientific grant of the Hungarian Respiratory Foundation (I. K.), Bolyai Fellowship of Hungarian Academy of Sciences (A. S.), and Semmelweis University Innovation Found STIA-KF-17 (A. S.).

    1

    These authors contributed equally to this work.

    View full text