Chronic mTOR activation promotes cell survival in Merkel cell carcinoma
Introduction
Merkel cell carcinoma (MCC) is an aggressive skin cancer with a rising incidence (1500 cases per year in the US). Moreover, the incidence of MCC is approximately 11-fold greater in AIDS patients and 5-fold greater in organ transplant patients [9], [34]. In addition to a higher risk for other skin cancers, MCC patients also have an increased risk for hematologic malignancies [35]. In Australia between 1986 and 2001, the age-adapted incidence of MCC rose at an annual increase of 8%, compared to the 3% rise in cutaneous melanoma. Furthermore, 50% of patients are metastatic at presentation with a 5-year disease-associated mortality rate of 46%, far exceeding that of melanoma [3]. Nevertheless, there is no effective treatment so far [1], [4].
Despite this grim epidemiologic data, the cellular and molecular mechanisms dictating MCC pathogenesis and metastasis remain largely unknown. Studies have led to the discovery of Merkel cell carcinoma virus (MCV) [11]; however, the pathogenic role of MCV remains a captivating question. Intense investigations show no significant contribution of the p53, PTEN, MAPK pathways to MCC pathogenesis. PI3K/AKT and MAPK pathways, the two pathways that are most commonly dysregulated in human malignancies, are less frequently mutated in MCCs. Moreover, growth factor pathways are not constitutively activated in MCC. Interestingly, activation of the MAPK pathway leads to apoptosis in human MCC cells [17]. Most recently, studies have reported Akt hyper-phosphorylation in MCC regardless of the presence of MCV [15], [33]. In addition, up-regulation of the mammalian target of the rapamycin (mTOR) pathway has been found in MCCs that are positive for MCV through the interaction of eukaryotic translation initiation factor 4E-binding protein (4E-BP1) with MCV small T antigen [38].
mTOR is a critical mediator of the canonical pathway of the PI3K and MAPK pathways [24]. It resides in at least two functional multiprotein complexes, mTOR complex1 (mTORC1) and mTOR complex2 (mTORC2), which exhibit different subunit compositions and execute distinct cellular tasks. Tuberous sclerosis complex (TSC) is the key inhibitor of this pathway and functions as a guanosine triphosphatase-activating protein for the small guanine nucleotide-binding protein Rheb, which, in its GTP-bound form, is essential for the stimulation of mTORC1 activity. Disruption of this complex, through the loss of either TSC1 or TSC2 function results in constitutive activation of mTORC1 that is exemplified in the genetic disorder tuberous sclerosis. mTOR controls many cellular processes including apoptosis, autophagy, translation, energy metabolism, and inflammation. The mTORC1 pathway is frequently activated in many human cancers [6]. Clinically, rapalogs such as rapamycin has shown some therapeutic efficacy in a subset of tumors via inhibiting mTORC1 [45]. Moreover, mTOR kinase inhibitors have been shown to halt cell growth and survival in various cancers [18], [49]. However, the role of mTOR pathway in MCC carcinogenesis has not been extensively studied.
Autophagy refers to the highly regulated and evolutionally conserved process of turnover and maintenance of cellular component that is required for cellular homeostasis [25], [29]. The connections between autophagy malfunction and human diseases have been under recent intensive scrutiny [46]. Although autophagy has been assigned a largely cytoprotective role, it does accompany cell death, and its inhibition attenuates cellular demise in certain physiologic processes [2], [43]. In tumorigenesis, however, autophagy is a double-edged sword. From the perspective of cellular energetics, autophagy is likely a pro-survival mechanism in both early and late stages of cancer development. Conversely, autophagy may also have antagonistic roles in the oncogenic process, i.e., diminishing malignant transformation and facilitating tumor progression [14]. Several studies have documented increased autophagy in cancer cell death when anti-cancer agents were used [10], [13], [23]. More specifically, a recent study has shown that oncogenic RAS induces autophagy and autophagic cell death by up-regulation of the BH3-only protein NOXA [8]. Autophagic cell death has also been detected in cells lacking Bax and Bak, two pro-apoptotic factors, and can be induced by caspases-8 inhibition [50]. Therefore, defining the context-specific role for autophagy in cancer and determining the mechanisms involved will be important to guide autophagy-based therapeutic interventions. As autophagy is a downstream target of the mTOR pathway, we chose to examine its potential role in MCC pathogenesis.
To investigate whether mTOR signaling contributed to the development of MCC, we first evaluated mTOR pathway using formalin fixed paraffin embedded (FFPE) human MCC samples. Using tissue microarray (TMA) and immunohistochemistry, we found that mTOR pathway was activated as indicated by positive staining of phosphorylation-4E-BP1 (p-4E-BP1), phosphorylation-S6K (p-S6K), and phosphorylation-mTOR (p-mTOR) regardless of MCV status. Moreover, our data suggested alternative mechanisms of mTOR activation other than through MCV small antigen in MCCs. Furthermore, there was p62 accumulation in MCCs, indicative of impaired autophagy. To facilitate in vitro mechanistic studies, we then established two primary human MCC cell lines (MCC-2 and MCC-3) from two patients with lymph node metastases. We also demonstrated mTOR pathway up-regulation and decreased autophagy in both MCC cells. Moreover, we have shown that inhibition of mTOR pathway decreased cell proliferation and induced autophagy and cell death in MCC cells. Furthermore, cell death induced by mTOR inhibitors was independent of caspase activation and attenuated by an autophagy inhibitor. Thus, our study provides a new insight into the MCC pathogenesis and a rationale for potential new therapeutic targets.
Section snippets
Sample selection and tissue microarray
In accordance with institutional approvals for human study protocols, 65 archival formalin fixed and paraffin embedded (FFPE) MCC tissue blocks and 9 fresh MCC tissues were employed in this study. TMAs were prepared as previously described. For each case, a representative area from the tumor was carefully selected from a hematoxylin-eosin stained section of a MCC block. Core cylinders (0.6 mm) were punched from each FFPE tumor and deposited into a recipient paraffin block using the MTA-I manual
mTOR pathway is up-regulated in MCC tissue samples
Unlike other cancers, common tumor suppressor genes and oncogenes, such as p53, PTEN, Rb, Ras, B-RAF, c-kit, and β-catenin, are less frequently mutated in MCCs. Growth receptor pathways such as c-kit, VEGF, and PDGF are also not highly activated in MCC as in other skin cancers such as melanoma, indicating that proteins and/or pathways critical for MCC carcinogenesis are yet to be identified. Recently Shuda et al. described mTOR up-regulation in MCC with small T antigen [38]. Therefore, we
Discussion
In this study, by employing archival and fresh MCC tumors, as well as a primary human MCC cell line, we have first identified that mTOR activation, including activation of two downstream molecules 4E-BP1 and S6K and also mTOR per se, is common in MCCs. Second, we have shown that mTOR activation is independent of MCV and lead to impaired autophagy. Finally, augmentation of autophagy by mTOR inhibition potentiates cell death that is independent of caspase activation in MCC cells.
A recent study
Conflict of Interest
All authors have no financial disclosure.
Acknowledgements
The project described was supported by the Translational Research Institute (TRI), Grants UL1TR000039 and KL2TR000063 through the NIH National Center for Research Resources and the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
This study was also supported by funds from the Department of Dermatology and the Winthrop P. Rockefeller Cancer Institute, University of
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2018, Biomedicine and PharmacotherapyCitation Excerpt :mTOR acts as a sensor of cell nutrition status, stress, and growth factor signaling and plays an important role in autophagy [25]. Lin et al. showed that mTOR plays a potential role in Merkel cell carcinoma autophagy, as mTOR activation inhibits autophagy and promotes cell survival, whereas mTOR inhibition significantly induces autophagic cell death [26]. Wang et al. showed evidence that tetramethylpyrazine protects BMSCs from exposure to excess glucocorticoids by promoting autophagy through the AMPK/mTOR pathway, suggesting its potential as an effective agent for the prevention and treatment of glucocorticoid-induced osteoporosis [27].
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2016, NeuroscienceCitation Excerpt :Under lack of energy or stress, the mTOR signaling pathway is suppressed mainly through the mechanism of AMP-activated protein kinase (AMPK) activation (Inoki et al., 2012). In the research of Lin et al. (2014), mTOR activation in Merkel cell carcinoma was found to inhibit autophagy, whereas mTOR inhibition can induce autophagy. Wu et al. (2013) also found that the induction of autophagy simultaneously increases AMPK phosphorylation level, accompanied by phosphorylation of reducing S6 kinase p70 subtype (p70S6K).
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These authors contributed equally to this work.