Dataset Information

Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer

ABSTRACT: In phase I/II clinical trials, Z-endoxifen demonstrated substantial oral bioavailability and promising antitumor activity in endocrine-refractory estrogen-receptor positive breast cancer (ER+ BC) and other solid tumors, with plasma concentrations reportedly as high as 5 ï�M. Therefore, we explored the potential mechanisms of Z-endoxifen antitumor activity that extends beyond ERÎ± inhibition. In estrogen unstimulated aromatase-expressing ER+/human epidermal growth factor 2 receptor negative (HER2-) MCF7AC1 BC cells, Z-endoxifen at 5 ï�M, but not ERï�¡-targeting 0.01 and 0.1 ï�M concentrations inhibited growth and induced apoptosis, suggesting an ERÎ±-independent effect. Utilizing an unbiased mass spectrometry approach, we explored Z-endoxifen effects on other signaling pathways. Z-endoxifen at 5 ÂµM profoundly altered the phosphoproteome with minimal impact on total proteome. Computational analysis revealed Protein kinase C beta (PKCï�¢) and AKT1 as the prevalent upstream kinases for Z-endoxifen-downregulated protein phosphorylations. Notably, in ER+/HER2- BC models, Z-endoxifen at 5 ï�M attenuated AKTSer473 and AKT substrates in vitro in the presence of insulin and PKC agonist PMA and in vivo. Further, Z-endoxifen inhibited PKCï�¢1 kinase activity compared to other PKC isoforms in vitro and bound to PKCÎ²1. While PMA stimulated PKCï�¢1Ser661 phosphorylation correlated with AKTSer473 and AKT substrate phosphorylation, Z-endoxifen at 5 ï�M uniquely blocked these effects and induced PKCÎ²1 protein degradation. siRNA-mediated PKCï�¢1 knockdown attenuated AKTSer473 phosphorylation, suggesting PKCÎ²1-mediated suppression of AKT signaling by Z-endoxifen. Further, Z-endoxifen at 5 ï�M replicates the pan-AKT inhibitor MK-2206 effects on apoptosis. These findings implicate PKCÎ²1 as a novel Z-endoxifen substrate responsible for suppressing AKT signaling and inducing apoptosis in breast cancer.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD035007 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Endoxifen downregulates AKT phosphorylation through protein kinase C beta 1 inhibition in ERα+ breast cancer.

Jayaraman Swaathi S Wu Xinyan X Kalari Krishna R KR Tang Xiaojia X Kuffel Mary J MJ Bruinsma Elizabeth S ES Jalali Shahrzad S Peterson Kevin L KL Correia Cristina C Kudgus Rachel A RA Kaufmann Scott H SH Renuse Santosh S Ingle James N JN Reid Joel M JM Ames Matthew M MM Fields Alan P AP Schellenberg Matthew J MJ Hawse John R JR Pandey Akhilesh A Goetz Matthew P MP

NPJ breast cancer 20231219 1

Endoxifen, a secondary tamoxifen metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERα) binding at nanomolar concentrations. Phase I/II clinical trials identified clinical activity of Z-endoxifen (ENDX), in endocrine-refractory metastatic breast cancer as well as ERα+ solid tumors, raising the possibility that ENDX may have a second, ERα-independent, mechanism of action. An unbiased mass spectrometry approach revealed that ENDX concentrations achieved clinically with direc ...[more]

PMID: 38114522

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Project description:Intracellular bacterial pathogens inject a cocktail of effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. Salmonella enterica serovar Typhimurium (STm) uses two Type 3 secretion system (T3SS) to deliver a suite of effector proteins into host cells, however, knowledge of their host-targets remains sparse. To systematically map STm effector-host protein-protein interactions (PPIs) during active infection, we generated a library of 32 STm strains expressing affinity-tagged effector proteins from their endogenous chromosomal loci. Tagged-effector strains were then used to infect macrophages or epithelial cells, and PPIs were detected by Affinity-Purification Quantitative Mass-Spectrometry (AP-QMS) under both native and cross-linking conditions. We recovered 25 previously described effector-host PPIs, along with 421 novel interactions across the two cell lines tested, as well as several effector-effector PPIs. While effectors converged on specific host cellular processes, which varied across cell types, most had multiple host targets. Using reciprocal co-immunoprecipitations, we could validated 13 out of 22 new PPIs, demonstrating an accuracy of at least 59% for the AP-QMS approach. To illustrate the utility of this resource for discovering novel infection biology, we first showed that SseJ and SseL cooperate to control cholesterol transport at the Salmonella Containing Vacuole (SCV) via the Niemann-Pick C1 protein (NPC1). Second, we uncovered that PipB directly binds and recruits the organelle contact site protein PDZD8 to the SCV. Third, we elucidated a novel mechanism whereby the effector kinase SteC promotes host actin bundling, by directly interacting and phosphorylating formin-like proteins. Overall, we provide a systems-wide host-bacterial pathogen physical interactome resource, to our knowledge the first in an infection context, with broad implications for effector function and cooperation.

Dataset Information

Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer

Publications

Endoxifen downregulates AKT phosphorylation through protein kinase C beta 1 inhibition in ERα+ breast cancer.

Similar Datasets

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