Rapalog resistance is associated with mesenchymal-type changes in Tsc2-null cells

Matthildi Valianou,Aristotelis Astrinidis,John J Bissler,Xiaolei Liu,Yiyang Lu,Erik Y Zhang,Peter Vogel,Natalia Filippidou,Jane J Yu,Daniel L Johnson

doi:10.1038/s41598-019-39418-5

Abstract

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.

Highlights

Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM) are tumor suppressor syndromes sharing the same primary genetic and biochemical features; inactivation of the tumor suppressors TSC1 or TSC2, and aberrant regulation of the PI3K/mechanistic target of rapamycin (mTOR) pathway
Inactivation of hamartin/tuberin, as in TSC and LAM, results in the hyperactivation of mTORC1. mTOR forms a second, distinct and partially rapamycin-insensitive multimeric complex consisting of mTOR, rictor, mLST8, DEPTOR, Protor1/2, and mSin1. mTORC2 is essential for the full activation of AKT, via direct phosphorylation at residue S473
The identification of mTORC1 hyperactivation as the main and most important biochemical event related to TSC and LAM pathogenesis[23,24], led to the first clinical trials and regulatory approval of the mTORC1 inhibitors sirolimus and everolimus (RAD001) for the management of brain, renal and pulmonary manifestations in TSC and LAM25–28

Summary

Introduction

Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM) are tumor suppressor syndromes sharing the same primary genetic and biochemical features; inactivation of the tumor suppressors TSC1 or TSC2, and aberrant regulation of the PI3K/mTOR pathway. Discontinuation of treatment leads to renal tumor re-growth and decline in pulmonary function even close to baseline values within a year after treatment cessation[25,32,33] Despite these drawbacks, rapalogs remain the only drugs for the treatment of renal, pulmonary, and brain lesions in TSC and LAM. We report the development and comprehensive characterization of the first tuberin-null rapamycin-resistant cell line Key features of these cells are the loss of epithelial markers, the acquisition of mesenchymal characteristics, the aberrant activation of signaling pathways in addition to PI3K/mTOR, and the enhanced tumorigenicity and metastatic potential

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Conclusion