Abstract P1-09-12: mTORC1 is responsible for metastasis of ER+ breast cancer downstream of RON tyrosine kinase
Authors: Faham N , welm A 
Published: February 2018

Metastatic breast cancer is one of the major challenges of tumor biology, remaining the underlying cause of death in the vast majority of breast cancer patients. Despite advances in treatment strategies, metastatic breast cancer is still considered incurable and mostly resistant to the current treatment options. There is, therefore, a clear need for development of novel targeted therapeutic agents, which must be built on a foundation of a deeper understanding of the molecular mechanisms responsible for development of metastasis. It is well accepted that aberrant expression of the RON receptor tyrosine kinase in breast cancer contributes to development of metastasis, and is associated with poor patient prognosis. Elevated expression of RON has been reported in about 50% of primary breast cancer samples. However, in later metastatic stage, RON is overexpressed in 100% of breast cancer samples, pointing toward its important role in inducing metastasis.

RON is known to activate multiple signaling pathways, including MAPK, PI3K, Src, phospholipase C-γ and β-catenin. Although some of these pathways have been linked to specific biological outcomes in vitro, contributions of each signal transduction pathway to cancer progression and metastasis are still poorly defined. To address this critical question, we used a combination of inhibitory and mutational strategies. We engineered T47D cells to conditionally overexpress RON upon addition of doxycycline, enabling us to achieve titratable levels of RON. For inhibition of RON signaling, we used a highly selective RON inhibitor, ASLAN002. To obtain a broader knowledge about signaling networks downstream of RON, we used a high-throughput functional proteomics assay, Reverse Phase Protein Array (RPPA). RPPA analysis showed robust phosphorylation of ribosomal protein S6 (rpS6) upon RON activation, which could be reversed by RON inhibitor. We were then able to dissect the components of the pathway responsible for phosphorylation of rpS6 using inhibitors against different potential kinases. These analyses revealed that mTORC1 is the main kinase responsible for profound phosphorylation of rpS6 downstream of RON, which itself is fed dominantly by PI3K rather than MAPK. To validate the PI3K/mTORC1/p70S6K axis as the main feeder of rpS6, we took advantage of a mutational approach to modulate RON signaling to specific pathways. Biochemical analysis of various mutants in T47D cells showed that they signal differentially, with mutant A strongly signaling through PI3K/mTORC1. To assess the importance of PI3K/mTORC1 in RON-mediated metastasis, we conducted in vivo studies. Our results indicated that mutant A is the highest metastatic mutant, inducing metastasis in 100% of the mice studied. To examine whether mTORC1 is critical for induction of metastasis downstream of RON, we treated mice with mTORC1 inhibitor, everolimus, and monitored metastasis. Our data revealed that mTORC1 inhibition could dramatically shrink metastatic lesions, before resistance occurs.

We report a dissected analysis of the RON signaling pathway responsible for induction of metastasis in ER+ breast cancer. Our data strongly indicate that RON dominantly signals through PI3K/mTORC1/p70S6K/rpS6, and that this pathway is important in inducing metastasis.

Citation Format: Faham N, welm A. mTORC1 is responsible for metastasis of ER+ breast cancer downstream of RON tyrosine kinase [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-12.