Identification of novel pharmacological approaches to inhibit neurotensin receptor-1 mitogenic signaling in breast cancer cells.

摘要

G protein coupled receptors (GPCRs) represent the largest family of cell surface receptors and serve as the primary pharmacological targets for more than thirty percent of approved drugs on the market. In addition to targeting GPCRs to manage cardiovascular and central nervous system associated disease conditions, a growing body of literature suggests that many GPCRs are involved in cancer development, progression and metastasis.;Neurotensin receptor 1 (NTSR1) is a GPCR that has been recently identified as a mediator of tumorigenicity and metastasis. NTSR1, as well as its endogenous ligand, neurotensin (NTS), are coexpressed in several types of cancer including lung, pancreas, prostate, colon, head and neck as well as breast cancer cell lines and breast cancer tumor samples. We have previously reported that ceramide mimetics could inhibit breast cancer cell growth in vitro and in vivo. The direct effect of C6 ceramide on GPCR-mediated cancer progression has not been characterized. Thus, understanding the biochemical and biophysical regulation of NTSR1 by ceramide can help further define NTSR1 as a novel target in breast cancer. Our results show that nanoliposomal formulations of C6 ceramide inhibit NTSR1-mediated MDA-MB-231 breast cancer progression (mitogenesis, migration, and matrix metalloproteinase-9 activity). In addition, liposomal C6 ceramide inhibited NTSR1-mediated, but not phorbol 12-myristate 13-acetate-mediated, activation of the mitogen-activated protein kinase pathway. Mechanistically, nanoliposomal short-chain ceramide reduces NTSR1 interaction with Galphaq/11 subunits within structured membrane microdomains (SMDs), consistent with diminished NTS-induced translocation of NTSR1 into membrane microdomains.;We next hypothesized that agonist-induced receptor palmitoylation mediates dynamic receptor translocation and signaling within SMDs. We have identified that endogenously expressed NTSR-1 in MDA-MB-231 breast adenocarcinomas as well as exogenously expressed NTSR-1 in HEK293T cells that do not normally express NTSR-1 is palmitoylated at Cys381 and Cys383. Inhibition of NTSR-1 palmitoylation in MDAMB-231 cells as well as NTSR-1 expressing HEK293T cells diminished NTS-mediated ERK1/2 phosphorylation. Additionally, NTSR1 mutated at Cys381 and/or Cys383 to serine showed diminished ERK1/2 stimulation and reduced ability to protect HEK293T cells against apoptosis induced by serum starvation. Mechanistically, C381,383SNTSR-1 showed reduced ability to interact with Galphaq/11 and diminished localization to SMDs, where Galpha q/11 preferentially resides.;Based on these observations, we next hypothesized that ceramide effects on NTSR-1 localization and signaling could be in part mediated through interference with NTSR-1 palmitoylation/depalmitoylation cycle. Our results suggest that ceramide could either inhibit NTSR-1 palmitoylation through direct biochemical or biophysical mechanism(s) or through acceleration of NTSR-1 depalmitoylation.;Collectively, our findings suggest that liposomal short-chain C6 ceramide can be utilized to inhibit NTS-dependent breast cancer cell growth. Our data also establish palmitoylation as a novel pharmacological target to disrupt NTSR-1 mitogenic signaling in breast cancer.