Loss of Tuberous Sclerosis Complex-2 Function and Activation of Mammalian Target of Rapamycin Signaling in Endometrial Carcinoma

Abstract

Purpose: The involvement of phosphatase and tensin homologue deleted on chromosome ten (PTEN) in endometrial carcinoma has implicated phosphatidylinositol 3-kinase signaling and mammalian target of rapamycin (mTOR) activation in this disease. Understanding the extent of mTOR involvement and the mechanism responsible for activation is important, as mTOR inhibitors are currently being evaluated in clinical trials for endometrial carcinoma. Although tuberous sclerosis complex 2 (TSC2) is the ''gatekeeper'' for mTOR activation, little is known about defects in the TSC2 tumor suppressor or signaling pathways that regulate TSC2, such as LKB1/AMPactivated protein kinase, in the development of endometrial carcinoma. Experimental Design: We determined the frequency of mTOR activation in endometrial carcinoma (primary tumors and cell lines) and investigated PTEN, LKB1, and TSC2 defects as underlying cause(s) of mTOR activation, and determined the ability of rapamycin to reverse these signaling defects in endometrial carcinoma cells. Results: Activation of mTOR was a consistent feature in endometrial carcinomas and cell lines. In addition to PTEN, loss of TSC2 and LKB1 expression occurred in a significant fraction of primary tumors (13% and 21%, respectively). In tumors that retained TSC2 expression, phosphorylation of tuberin at S939 was observed with a high frequency, indicating that mTOR repression by TSC2 had been relieved via AKT phosphorylation of this tumor suppressor. In PTEN-null and LKB1-null endometrial carcinoma cell lines with functional inactivation of TSC2, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 were able to inhibit AKT and mTOR signaling and reverseTSC2 phosphorylation. In contrast, although rapamycin inhibited mTOR signaling, it did not relieve phosphorylation of TSC2 at S939. Conclusions: Inactivation ofTSC2 via loss of expression or phosphorylation occurred frequently in endometrial carcinoma to activate mTOR signaling. High-frequency mTOR activation supports mTOR as a rational therapeutic target for endometrial carcinoma. However, whereas rapamycin and its analogues may be efficacious at inhibiting mTOR activity, these drugs do not reverse the functional inactivation of TSC2 that occurs in these tumors.

Lkb1inactivation is sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy

“…In one study, western blotting showed a loss of LKB1 expression in 20% of primary tumors, which correlated with activation of the mTOR pathway (Lu et al, 2008). Similarly, we found that 20% of primary tumors showed immunohistochemical loss of LKB1 expression and, furthermore, that loss of the LKB1 protein significantly correlated with tumor stage, and hence, prognosis.…”

“…endometrial hyperplasias driven by Pten heterozygosity), rapamycin does slow the rate of disease progression, although most studies have not documented shrinkage of tumors of invasive cancers (Milam et al, 2007;Neshat et al, 2001). Interestingly, a recent phase II study of temsirolimus (an ester derivative of rapamycin) in endometrial cancer demonstrated a 26% objective response rate, similar to the 20% rate of endometrial tumors that express low to undetectable levels of the LKB1 protein (Contreras et al, 2008a;Lu et al, 2008). This leads us to speculate that LKB1-AMPK pathway status may be a better predictor of responses to rapalogs than the PI3K-AKT axis, particularly as PI3K…”

Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway

“…p-p70S6K is generally used in preference to p-mTOR to assess the tumour response to mTOR antagonists and to predict which tumours may be responsive to such agents (for reviews see Boulay et al (2004); MacKenzie and von Mehren (2007)). Indeed, phosphorylated mTOR has been generally found to be expressed in relatively low numbers of tumours, and several studies make no reference to tumour mTOR expression when assessing the effect of rapamycin and or its analogues (Jaeschke et al, 2002;Gao et al, 2003;Krishnan et al, 2006;El-Salem et al, 2007;Lu et al, 2008; Although the TMA was built with 50 chordomas, the total number described for each antibody represents the number that was possible to analyse. The scoring system employed was as follows: negative in the absence of immunoreactivity, 'low' when the immunoreactivity was unequivocal but less strong than the positive control and 'high' when the immunoreactivity was at least as strong as the positive control.…”

Targeting the mTOR/4E-BP Pathway in Endometrial Cancer

“…Studies of human endometrial tumors have shown that $20% are LKB1-negative, and as many as 65% have a functional deficit in the protein (25).…”

“…Rapalog studies in endometrial cancer have focused primarily on expression of mTOR pathway proteins (44,(52)(53)(54)(55)(56)(57)(58) and cellular responses to mTOR inhibitors (25,52,(59)(60)(61)(62). Several groups have also investigated the role of small-molecule mTOR kinase inhibitors in endometrial cancer cell lines (59,63) and the effects of mTOR inhibitors on mouse models of endometrial cancer (3,(64)(65)(66).…”