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

Abstract

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.

Figure 1

The TSC/mTOR signaling pathway. A) PI3K-dependent activation of AKT leads to the inhibition of TSC2 activity resulting in an increase in Rheb-GTP level and downstream activation of mTOR, which in turn phosphorylates p70S6K and PHASI to promote protein synthesis. LKB1 phosphorylates AMPK to enhance TSC1/2 inhibition of mTOR. Acting down-stream of Ras, ERK and RSK phosphorylates TSC2 to repress its GAP activity. In a negative feedback loop, p70S6K phosphorylates IRS1 to reduce insulin sensitivity and AKT activity. Tumor suppressor genes that inhibit mTOR activity are in bold. B, C) Simplified versions of the pathway to highlight the functional consequences of the loss of PTEN (B) and TSC1/2 (C) are shown to illustrate the unique difference in AKT activity of the two scenarios. Arrows indicate relative change in activities.

Figure 2

Immunophenotype of a TSC2-related AML. The loss of TSC2 function results in A) up-regulation of mTOR as illustrated by increased levels of phospho-p70S6K(Thr389) expression in the tumor (T) compared to adjacent normal kidney (N), and B) down-regulation of AKT as shown by the lack of phospho-AKT(Ser473) immuoreactivity in the tumor (T). C) In contrast, prostate carcinoma, which frequently undergo PTEN loss, expressed high levels of phospho-AKT(Ser473) compared with adjacent atrophic gland (N). Magnification 400x.

Figure 3

Immunohistochemical analysis of the mTOR pathway in sporadic AMLs. Examples of two sporadic, non-TSC AMLs analyzed by immunohistochemistry for A) phospho-p70S6K, B) phospho-S6, and C) phospho-AKT. T, tumor; N, normal kidney. Magnification: for A and B, top panel: 100x; bottom panel: 400x; for C, 200x.

Figure 3

Immunohistochemical analysis of the mTOR pathway in sporadic AMLs. Examples of two sporadic, non-TSC AMLs analyzed by immunohistochemistry for A) phospho-p70S6K, B) phospho-S6, and C) phospho-AKT. T, tumor; N, normal kidney. Magnification: for A and B, top panel: 100x; bottom panel: 400x; for C, 200x.

Figure 3

Immunohistochemical analysis of the mTOR pathway in sporadic AMLs. Examples of two sporadic, non-TSC AMLs analyzed by immunohistochemistry for A) phospho-p70S6K, B) phospho-S6, and C) phospho-AKT. T, tumor; N, normal kidney. Magnification: for A and B, top panel: 100x; bottom panel: 400x; for C, 200x.

Figure 4

Biochemical analysis of the mTOR pathway in sporadic AMLs. Four independent, non-TSC AMLs were analyzed by Western blotting using antibodies indicated. Panel A highlights the targets of mTOR: p70S6K, ribosomal S6, and PHASI, as well as AKT. Panel B illustrates the up-stream negative regulators of mTOR: TSC1, TSC2, PTEN, LKB1, AMPK, and ERK. Each sample is represented by normal kidney (NK), and tumor (AML). Actin serves as loading control.

Figure 4

Biochemical analysis of the mTOR pathway in sporadic AMLs. Four independent, non-TSC AMLs were analyzed by Western blotting using antibodies indicated. Panel A highlights the targets of mTOR: p70S6K, ribosomal S6, and PHASI, as well as AKT. Panel B illustrates the up-stream negative regulators of mTOR: TSC1, TSC2, PTEN, LKB1, AMPK, and ERK. Each sample is represented by normal kidney (NK), and tumor (AML). Actin serves as loading control.

Figure 5

Immunohistochemical analysis of p70S6K in PEComas. Two examples of PEComas immunostained for phospho-p70S6K. Magnification: 100x (top) and 400x (bottom).