Adipose tissue fibrosis in human cancer cachexia: The role of TGFβ pathway

Cancer cachexia is a multifactorial syndrome that dramatically decreases survival. Loss of white adipose tissue (WAT) is one of the key characteristics of cachexia. WAT wasting is paralleled by microarchitectural remodeling in cachectic cancer patients. Fibrosis results from uncontrolled ECM synthesis, a process in which, transforming growth factor-beta (TGFβ) plays a pivotal role.

Alves et al BMC Cancer (2017) 17:190 DOI 10.1186/s12885-017-3178-8 RESEARCH ARTICLE Open Access Adipose tissue fibrosis in human cancer cachexia: the role of TGFβ pathway Michele Joana Alves1* , Raquel Galvão Figuerêdo1, Flavia Figueiredo Azevedo6, Diego Alexandre Cavallaro1,5, Nelson Inácio Pinto Neto 5, Joanna Darck Carola Lima1, Emidio Matos-Neto1, Katrin Radloff1, Daniela Mendes Riccardi1, Rodolfo Gonzalez Camargo1, Paulo Sérgio Martins De Alcântara3, José Pinhata Otoch2,3, Miguel Luiz Batista Junior4 and Marília Seelaender1,2 Abstract Background: Cancer cachexia is a multifactorial syndrome that dramatically decreases survival Loss of white adipose tissue (WAT) is one of the key characteristics of cachexia WAT wasting is paralleled by microarchitectural remodeling in cachectic cancer patients Fibrosis results from uncontrolled ECM synthesis, a process in which, transforming growth factor-beta (TGFβ) plays a pivotal role So far, the mechanisms involved in adipose tissue (AT) re-arrangement, and the role of TGFβ in inducing AT remodeling in weight-losing cancer patients are poorly understood This study examined the modulation of ECM components mediated by TGFβ pathway in fibrotic AT obtained from cachectic gastrointestinal cancer patients Methods: After signing the informed consent form, patients were enrolled into the following groups: cancer cachexia (CC, n = 21), weight-stable cancer (WSC, n = 17), and control (n = 21) The total amount of collagen and elastic fibers in the subcutaneous AT was assessed by histological analysis and by immunohistochemistry TGFβ isoforms expression was analyzed by Multiplex assay and by immunohistochemistry Alpha-smooth muscle actin (αSMA), fibroblast-specific protein (FSP1), Smad3 and were quantified by qPCR and/or by immunohistochemistry Interleukin (IL) 2, IL5, IL8, IL13 and IL17 content, cytokines known to be associated with fibrosis, was measured by Multiplex assay Results: There was an accumulation of collagen and elastic fibers in the AT of CC, as compared with WSC and controls Collagens type I, III, VI, and fibronectin expression was enhanced in the tissue of CC, compared with both WSC and control The pronounced expression of αSMA in the surrounding of adipocytes, and the increased mRNA content for FSP1 (20-fold) indicate the presence of activated myofibroblasts; particularly in CC TGFβ1 and TGFβ3 levels were up-regulated by cachexia in AT, as well in the isolated adipocytes Smad3 and Smad4 labeling was found to be more evident in the fibrotic areas of CC adipose tissue Conclusions: Cancer cachexia promotes the development of AT fibrosis, in association with altered TGFβ signaling, compromising AT organization and function Keywords: Cancer cachexia, Fibrosis, Adipose tissue, Extracellular matrix, TGFβ * Correspondence: michelejalves@usp.br Cancer Metabolism Research Group, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil Full list of author information is available at the end of the article © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Alves et al BMC Cancer (2017) 17:190 Background Cancer cachexia is an irreversible syndrome in which involuntary weight loss occurs, due to skeletal muscle mass and adipose tissue wasting This condition is associated with poor prognosis, and decreased survival [1, 2] Cachexia is seldom diagnosed or treated, despite affecting around 80% of all cancer patients [3], and being the direct cause of 22–40% of cancer deaths [4, 5] The typical metabolic dysregulation acts in concert with the increase of inflammatory mediators [6, 7], as the syndrome develops in the manner of a chronic inflammatory state Profound wasting of WAT is frequently observed during cachexia, and recent evidence suggests that it precedes protein breakdown in the skeletal muscle or any decrease in food intake [8, 9] The WAT has been recognized as a highly dynamic organ [10–12] Therefore, changes in WAT may be proposed as early markers of the syndrome, thus presenting a potentially valuable tool for precocious diagnosis Previous studies [9, 13–16] have shown that the AT is deeply affected by local inflammation during cancer cachexia Several pro-inflammatory factors, such as TNFα, IL-1β and IL-6 are up-regulated by in the adipose tissue during cachexia, and we have shown before [14–16], that the subcutaneous depot presents a relevant contribution to systemic inflammation as these factors reach the circulation Each particular anatomical depot of adipose tissue exhibits specific arrangement and functions, along with diverse morphology and varying density of adipocytes, of pre-adipocytes, of fibroblasts, of endothelial cells and of resident macrophages [10, 17] The extracellular matrix (ECM) is a complex network essential for tissue architecture and cell functioning [10, 18] ECM acts as one of the most important reservoirs of growth factors, metalloproteinases, collagens and fibronectin Indeed, changes in ECM rapidly trigger signaling pathways controlling different cell functions, including development, migration, proliferation, apoptosis, and gene expression [19] Among the different extracellular matrix components, collagen VI is highly expressed in the adipose tissue, and is considered as the key form secreted by adipocytes The lack of collagen VI in col6 KO ob/ob mice results in the uninhibited expansion of individual adipocytes [20, 21] Studies with mice bearing the cachexia-inducing tumor MAC-16 show that adipocyte dimensions are reduced, the cell membranes disrupted, and that the tissue suffers fibrosis [9] Fibrosis results from persistent inflammation, in which activated mechanisms for repair response lead to excessive accumulation of extracellular matrix components [22, 23] TGFβ is one of the key cytokines taking part in wound-healing and tissue fibrosis [24] Binding of TGFβ to membrane receptors causes the assembly of a receptor complex that, in turn, phosphorylates SMAD Page of 12 proteins [25–27] Through the action of SMADs on target genes, the induction of differentiation of fibroblasts into myofibroblasts takes place [28] Myofibroblasts are contractile cells expressing alpha-smooth muscle actin (α-SMA), involved in the production of ECM proteins, such as collagen and fibronectin [29, 30] Myofibroblast activation, proliferation and survival are also mediated by other pro-inflammatory cytokines: TNF, IL13, IL1 and TGFβ [31–34] The microenvironment in fibrosis triggered by the TGFβ pathway often leads myofibroblasts to sustain uncontrolled deposition of extracellular matrix components, since these cells become resistant to the mechanisms of apoptosis [23] Recently, we demonstrated that cachectic patients with gastrointestinal cancer show morphological rearrangement in the subcutaneous AT, resulting in adipocyte size reduction, AT atrophy, formation of fibrotic areas and immune cell infiltration [35] In the current study, we hypothesized that the morphological changes in cancer cachexia concomitant with augmented expression of ECM elements occurs through enhanced signaling of the TGFβ pathway Methods Patient recruitment All subjects were selected between 2012 and 2015 at the ambulatory unit of the Surgical Medical Clinic at the University Hospital, after being scheduled for exploratory laparotomy or abdominal surgery (n = 212) The adopted exclusion criteria were: liver or kidney failure, AIDS, chronic inflammatory processes not related to cachexia, chemotherapy treatment (at the time), and chronic anti-inflammatory therapy All the procedures were performed according to the Declaration of Helsinki, and were approved by the Ethics Committee of Research Involving Human Subjects of the Institute of Biomedical Sciences/University of Sao Paulo (1082/CEP) and by the Human Ethics Committee of the University Hospital/University of Sao Paulo (CEP 752/07) The fully informed written consent signature was obtained from every patient after a detailed explanation of the study Following engagement in the study, patients were divided into three groups The control group (C) included weight stable patients subjected to surgical hernia removal Patients diagnosed previously with gastrointestinal cancer were divided in two groups: Weight-stable Cancer Group (WSC) and Cachectic Cancer Group (CC) The WSC group was comprised of patients with greater than 5% body weight loss in the previous months CC group included the patients with gastrointestinal cancer and cachexia, characterized according to the following criteria: weight loss greater than 5% in fewer than 12 months, fatigue, anorexia; and abnormal biochemical parameters (increased inflammatory markers: C-Reactive Alves et al BMC Cancer (2017) 17:190 Page of 12 Protein, anemia, and by low serum albumin) [1] The questionnaire EORTC QLQ-C30 was applied to assess quality-of-life of all patients and the obtained data indicated a reduced overall quality-of-life in the cachectic group A total of 153 patients were excluded from the study Forty patients refused to participate Further exclusion criteria were inconsistent data in questionnaires (n = 19) and BMI greater than 29.9 kg/m2 (n = 03) Patients of the control group showing inflammation (CRP > mg/L) (n = 27) and anemia (n = 01) were also excluded Some patients were excluded due to unconfirmed diagnosis after the final pathological analysis, as well as due to insufficient adipose tissue sample allowing analysis, or which could not be matched with a corresponding blood sample (n = 63) A total of 59 patients were recruited into the study Table shows the characteristics of the study groups Plasma and serum measurements Approximately 20 ml of blood were collected during admission at the hospital, and aliquots of both plasma and serum was stored at -80 °C for later measurements All analyses were performed in the automatic LABMAX 240® equipment from Labtest, using commercial standards for Albumin (LABTEST), C-Reactive Protein (LABTEST), and hemoglobin (LABTEST) Protein expression of cytokines (TGFβ1, TGFβ2, TGβ3, TNFα, IL6, IL2, IL5, IL8, IL13, IL17) employing Luminex® technology Approximately 100–200 mg of the subcutaneous AT from each sample were homogenized in 300 μL of ice-cold extraction protein buffer (10 mM Tris base, 0.01 mM EDTA, 0.1 mM Sodium Chloride and 1% Triton X-100) to which a protease inhibitor cocktail was added (1 tablet/50 ml extraction buffer) (Roche Diagnostics) Protein extraction from isolated adipocytes was performed in the same way as described above, and the adipocytes were isolated following the adapted protocol from Rodbell [36] The homogenate was then centrifuged at 18,000 g for 40 at °C and the fatty layer, discarded The supernatant was stored in aliquots at -80 °C Multi-species TGFβ plex Adipose tissue samples Subcutaneous adipose tissue was collected during the surgery procedure Fat pad slices were rapidly frozen in dry ice and maintained in 4% paraformaldehyde solution (w/v) for the histological analysis described below For protein and gene expression, analyses of the samples were maintained at -80 °C, prior to processing Table General and clinical characteristics of studied groups P value CONTROL WSC CC N 21 17 21 Male/Female (n) 16/5 9/8 13/8 Height (m) 1.65 ± 0.09 1.62 ± 0.09 1.63 ± 0.1 0.6735 Age (years) 54.10 ± 14.89 62.13 ± 11.78 63.00 ± 11.0 0.0642 Previous body mass (Kg) 71.43 ± 13.32 73.50 ± 13.28 73.78 ± 13.86 0.8431 Current body mass (Kg) 71.43 ± 13.32 69.28 ± 12.19 63.62 ± 12.90 0.1577 Clinical Parameters Δ Body mass (Kg) 0.00 [0.00; 0.00] 0.00 [−7.2; 0.0] −8.0 [−12.50;−7.0] P < 0.0001* BMI (Kg/m2) 25.82 ± 3.27 25.79 ± 4.62 23.68 ± 3.49 0.1558 C-Reactive Protein (mg/dL) 0.11 [0.0; 0.23] 0.1950 [0.0; 0.4] 1.170 [0.72; 1.3]