Original contributionActivation of the mTOR pathway in sporadic angiomyolipomas and other perivascular epithelioid cell neoplasmsā
Introduction
The World Health Organization recently recognized as a distinct entity a heterogeneous family of neoplasms with perivascular epithelioid cell differentiation known as PEComa [1]. These lesions are defined as āmesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cellsā (PECs). Although the origin of these cells is unknown, PEComas include angiomyolipoma (AML), lymphangioleiomyomatosis (LAM), clear cell āsugarā tumor of the lung, clear cell myomelanocytic tumor of the falciform ligament, and other āunusual clear cell tumorsā designated as PEComas not otherwise specified (NOS) [2]. Such tumors may be conceptualized as monotypic epithelioid or spindled nonrenal AML, akin to the monotypic tumors known to occur in tuberous sclerosis complex (TSC)-associated renal AML. The clinical course of these tumors is highly variable ranging from indolent benign lesions to lesions with an aggressive clinical course including distant metastases. Because of their relative scarcity, little is known about the underlying molecular mechanism. Currently, there is no effective medical therapy.
As suggested by its designation, PECs are characterized histologically by their epithelioid appearance and their physical relationship to blood vessels [1]. They display a distinct immunophenotype that includes the expression of melanocytic and smooth muscle markers (eg, gp100 and smooth muscle actin) but not epithelial antigens. Because the PECs have no normal anatomic counterpart, the origin of these tumors remains elusive.
Angiomyolipoma and LAM represent a subset of PEComas that are strongly associated with TSC [3]. The latter is an autosomal dominant disease characterized by the multisystem development of benign tumors stemming from underlying mutations of either the TSC1 or TSC2 tumor suppressor gene [4]. Angiomyolipoma of the kidney develops in more than 50% of patients with TSC and is a major source of morbidity secondary to hemorrhage and destruction of renal parenchyma. Recent studies suggest that up to one third of adult females with TSC also have manifestations of LAM based on radiologic characteristics of their lungs [5], [6]. Lymphangioleiomyomatosis occurs almost exclusively in females, although one case of LAM in a male patient has been reported [7]. The coexistence of AML and LAM in TSC suggests a common pathogenetic mechanism stemming from the disruption of the TSC1/2 pathway. However, it is not known if sporadic AMLs and other less common forms of PEComas have functional consequences of the loss of TSC1/2 activity.
A small number of studies that have examined the cytogenetic and molecular genetic features of PEComas have highlighted the clonal nature of these tumors but without consistent findings. In the context of the TSC genes, allelic loss of the TSC2 locus on 16p13 has been found repeatedly in sporadic AMLs and PEComas suggesting a potential causal link [8], [9], [10], [11]. However, many other recurrent chromosomal alterations including the loss of 1p, 17p, 18p, 19 as well as gain of 2q, 3q, 5, 12q, and X have been identified in a recent comparative genomic hybridization study of PEComas [12]. To better appreciate the functional significance of 16p LOH in these tumors, we used a biochemical and immunohistochemical (IHC) approach to assess the potential role of the TSC2 pathway.
Proteins encoded by the TSC1 and TSC2 genes (hamartin and tuberin, respectively) function as a complex to negatively regulate mTOR signaling in response to multiple environmental signals [13]. In the presence of growth factors, PI3K-mediated AKT activity promotes Rheb-dependent mTOR function to increase protein synthesis and, consequently, cell growth (Fig. 1A). This pathway is under the regulation of 3 tumor suppressor proteins, PTEN, TSC1, and TSC2. PTEN functions as a phosphatase to reduce intracellular lipid products (eg, PIP3) of PI3K, and in doing so, blocks the activation of AKT [14]. The TSC1/2 complex acts downstream of AKT to promote hydrolysis of Rheb-GTP via its GAP activity, which leads to the suppression of mTOR signaling. The mTOR serine/threonine kinase phosphorylates p70S6K and PHASI to promote ribosomal biogenesis and protein translation [15]. Recent studies identified other tumor suppressor proteins, LKB1 (Puetz-Jeghers syndrome) and NF1 (neurofibromatosis 1), that also regulate the mTOR pathway by modulating the activity of TSC1/2 [16], [17]. Thus, dyregulation of the TSC/mTOR pathway may be central to a diverse group of human tumors.
Although the loss of PTEN, TSC1, or TSC2 leads to mTOR activation during tumor development, there is a unique difference that distinguishes the molecular events in the respective tumors. When PTEN is absent, AKT becomes constitutively activated (ie, high phospho-AKT levels), whereas AKT phosphorylation is suppressed when either TSC1 or TSC2 is lost or mutated (Fig. 1B, C). The opposing effects on AKT activity in these 2 scenarios have been implicated in the observed differences in phenotypic behavior (ie, the benign nature of TSC-related tumors versus the frequent involvement of PTEN in human cancers) [18], [19]. The mechanism of AKT inhibition after disruption of TSC1/2 has been shown to be secondary to the negative feedback of p70S6K on the insulin receptor substrate 1, IRS1, leading to reduced insulin sensitivity [20], [21]. Therefore, the combined assessment of mTOR and AKT activities in tumors provides not only evidence for the possible involvement of the mTOR pathway but also clues to the underlying mechanisms. In this study, we show that sporadic forms of renal AML and nonrenal PEComas possess high levels of phospho-p70S6K and low levels of phospho-AKT consistent with the inactivation of TSC1/2. Indeed, our data indicate the loss of TSC2 but not PTEN expression in sporadic AMLs.
Section snippets
Tissue samples
Kidney AML samples (formalin-fixed, paraffin-embedded) were obtained from Elizabeth Henske, MD, Fox Chase Cancer Center (Philidelphia, PA), and from the University of Washington Medical Center (Seattle, WA). PEComa samples (formalin-fixed, paraffin-embedded) were obtained from Emory University (Atlanta, GA) and the University of Washington Medical Center (Seattle, WA). Fresh AML and kidney tissues were obtained from the University of Washington Medical Center, Seattle, WA. Samples were obtained
mTOR signaling is up-regulated in sporadic AMLs
The levels of p70S6K and AKT phosphorylation are commonly used to indicate their activities. In this study, we used phospho-specific antibodies in IHC analyses to dissect the mTOR pathway. Previously, we have shown that TSC-related AMLs exhibit evidence of mTOR activation [22], [23]. As an example, Fig. 2A and B illustrate the use of phospho-specific antibodies in a case of renal AML from a TSC patient with known TSC2 mutation. With the loss of TSC2 function, mTOR signaling in the tumor was
Discussion
The pathogenesis of AMLs has intrigued pathologists for many years. Genetic analyses of sporadic AMLs have demonstrated that these tumors are clonally derived, but the studies have not provided consistent results with respect to an underlying genetic mechanism [27], [28], [29]. Trisomy 7 has been independently reported in sporadic AMLs. Its significance remains uncertain given that nonneoplastic renal tissues often exhibit the same karyotypic change [30], [31], [32], [33]. The association
Acknowledgment
We thank Dr Elizabeth Henske (Fox Chase Cancer Center) for providing kidney AML samples for this study and Dr Elizabeth Barnes (University of Washington) for critical reading of the manuscript.
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2021, iScienceCitation Excerpt :More tdTomato+ NCCs engaging in self-renewal proliferative processes can account for large numbers of tumors in the same or varied organs which altogether index more aggressive tumorigenic phenotypes. Mutations in the Tsc1 and predominantly the Tsc2 gene loci give rise to constitutive mTOR kinase activity and unregulated cell growth and metabolism causing TS tumors (El-Hashemite et al., 2003; Giannikou et al., 2016; Inoki et al., 2003; Kenerson et al., 2007). However, in studies conducted by our group and others (Hartman et al., 2009; Higa et al., 2009; Lee et al., 2010; Li et al., 2014), several mTOR-independent pathogenic mechanisms for TS have been demonstrated in diseased tissues haploinsufficient at the Tsc2 locus.
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Supported in part by National Institutes of Health grant no. CA102662.