Tang, Xiaojia

Abstract Cerebrovascular inflammation is prevalent in a majority of Alzheimer’s patients. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), circulating in the plasma have been shown to cause the inflammation of blood-brain barrier (BBB) endothelium lining the cerebral microvasculature. The BBB inflammation has been implicated in the increase of toxic Aβ accumulation within Alzheimer’s disease (AD) brain. TNF-alpha in the peripheral circulation can aggravate the accumulation of amyloid-beta (Aβ) peptides in Alzheimer’s disease brain. In the current study, we have shown that the exposure to TNF-alpha leads to an increase in Aβ42 accumulation in mice and BBB endothelial cells in vitro. Moreover, dynamic SPECT/CT imaging in wild-type (WT) mice infused with TNF-alpha increased the permeability and influx of Aβ42 into the mice brain. In addition, our results show that TNF-alpha modifies the expression of cofilin, actin, and dynamin, which are critical components for Aβ endocytosis by BBB endothelial cells. These results offer a mechanistic understanding of how TNF-alpha may promote Aβ accumulation at the BBB and the underlying interactions between inflammation and Aβ exposure that drives BBB dysfunction. Hence, a therapeutic intervention aimed at addressing cerebrovascular inflammation in Alzheimer’s disease may potentially reduce Aβ induced cerebrovascular toxicity in Alzheimer’s disease brain. Significance statement Increased levels of TNF-alpha circulating in the plasma are considered significant factors in the consequences of Aβ pathology in Alzheimer’s disease, where it can promote cerebrovascular inflammation and BBB dysfunction. However, the role of TNF-alpha, in exacerbating Aβ pathology by increasing Aβ accumulation at the BBB endothelial cells remains only partially understood. In this study, we demonstrated that TNF-alpha enhances Aβ42 accumulation in the BBB endothelium by altering the expression of the BBB endocytosis machinery, specifically cofilin, actin, and dynamin. These findings are anticipated to contribute to the development of therapeutic approaches aimed at addressing elevated cytokine levels in Alzheimer’s disease. Graphical Abstract

Abstract Background: In phase I/II clinical trials, the tamoxifen metabolite Z-endoxifen (ENDX) demonstrated substantial oral bioavailability and promising antitumor activity in endocrine-refractory estrogen-receptor positive breast cancer (ER+ BC) and other solid tumors, with ENDX plasma concentrations reaching as high as 5 µM, a concentration that far exceeds the ERα-targeting nanomolar concentrations. We previously identified protein kinase C beta 1 (PKCβ1), an oncogenic signaling kinase which regulates cell proliferation and tumorigenic transformation, as a novel ENDX receptor. ENDX-bound PKCβ1 (KD: 100 nM) and inhibited PKCβ1 kinase activity (IC50: 357 nM) in vitro, at concentrations achieved in phase I/II ENDX studies. Given the observations that ENDX induces antitumor activity in breast cancers resistant to endocrine therapies, including AIs, tamoxifen and fulvestrant, we postulated that ENDX may impact additional signaling pathways. Therefore, in this study we performed unbiased mass spectrometry to analyze the effects of ENDX on the phosphoproteome and total proteome. Methods: In estrogen unstimulated aromatase-expressing ER+/human epidermal growth factor 2 receptor negative (HER2-) MCF7AC1 breast cancer cells, the effects of ENDX on the phosphoproteome and total proteome were analyzed using ENDX concentrations achieved in tamoxifen treated patients (0.01 and 0.1 µM) and concentrations observed in the ENDX phase I/II trials (5 µM). NetWorKIN and RoKAI prediction tools were utilized to identify the upstream kinases of ENDX downregulated phosphoproteins. In estrogen unstimulated MCF7AC1 as well as in the ER+/HER2- T47D breast cancer cells, the ability of ENDX, tamoxifen, fulvestrant and PKC kinase inhibitor enzastaurin to block insulin or PKC agonist phorbol 12-myristate 13-acetate (PMA)-stimulated AKTSer473 phosphorylation was analyzed by immunoblot (IB) assay. Additionally, ENDX effects on AKTSer473 phosphorylation in the MCF7AC1 xenograft model in vivo was analyzed by IB assay. Further, the effects of PKCβ1 silencing in MCF7AC1 cells and the effects of AKT inhibitor MK-2206 treatment on growth and AKTSer473 phosphorylation in the MCF7AC1 as well as long-term estrogen deprived T47D (T47D-LTED) cell lines were analyzed by cell proliferation and IB assays. Results: In MCF7AC1 cells unstimulated with estrogen, ENDX at 5 µM but not at 0.01 and 0.1 µM concentrations inhibited growth, and induced apoptosis, suggesting an ERα-independent effect. Further, ENDX at 5 µM displayed three-fold greater effects in downregulating the phosphoproteome compared to the other concentrations, with minimal impact on the total proteome. Protein kinase C beta (PKCβ) and AKT1 were identified as the prevalent upstream kinases for ENDX downregulated protein phosphorylations. Notably, in estrogen unstimulated MCF7AC1 and T47D cells, ENDX at 5 µM attenuated phosphorylation of AKTSer473 and AKT substrates in vitro in the presence of insulin and in vivo. In MCF7AC1 cells, PMA-induced phosphorylation of PKCβ1 as well as AKTSer473 and AKT substrate phosphorylations were blocked by ENDX at 5 µM, with ENDX inducing PKCβ1 protein degradation both in the presence of insulin and PMA. Further, ENDX effects on growth and AKTSer473 phosphorylation were phenocopied by siRNA-mediated PKCβ1 knockdown as well as treatment with the pan-AKT inhibitor, MK-2206. Notably, the potent PKCβ1 kinase inhibitor, enzastaurin, had no effects either on PKCβ1 protein degradation nor on downstream AKT signaling. Conclusion: Taken together, these findings suggest that ENDX may exert anticancer activity via dual effects on blocking ERα as well as by targeting PKCβ1 for degradation, thus suppressing AKT signaling and induction of apoptosis. These preclinical findings will be studied in a planned neoadjuvant trial comparing ENDX with exemestane and ovarian function suppression (EVANGELINE) that will activate in the Fall of 2022. Citation Format: Swaathi Jayaraman, Xinyan Wu, Krishna R. Kalari, Xiaojia Tang, Mary Kuffel, Elizabeth S. Bruinsma, Santosh Renuse, James N. Ingle, Joel Reid, Matthew Schellenberg, John Hawse, Akhilesh Pandey, Matthew P. Goetz. Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-04.