Activation of the mTOR pathway in sporadic angiomyolipomas and other perivascular epithelioid cell neoplasms

Summary

Angiomyolipoma (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non–tuberous sclerosis complex (TSC), non–lymphangioleiomyomatosis-associated AMLs, and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14 of 15 cases of extrarenal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non–TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.

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 “mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cells” (PECs). Although the origin of these cells is unknown, PEComas include angiomyolipoma (AML), lymphangioleiomyomatosis (LAM), clear cell ‘sugar’ tumor of the lung, clear cell myomelanocytic tumor of the falciform ligament, and other “unusual clear cell tumors” 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.