The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic.

The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth; and dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2 diabetes, and cancer. We previously reported that a combination of grape polyphenols, (resveratrol, quercetin and catechin: RQC) at equimolar concentrations, inhibits breast cancer (BC) growth and metastasis in nude mice. The current objective is to investigate a role for RQC in BC therapy via inhibition of the Akt/AMPK/mTOR pathway. We found that in metastatic BC cells, RQC at 5uM each inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR. To determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling, metastatic BC cells were treated with RQC individually and in combination, at various concentrations. The activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors p70S6 kinase (p70S6K) and 4E binding protein (4EBP1) were determined by Western blot. Results show that at 蠅5uM, individual compounds downregulated Akt activity to a level similar to combined RQC at 5uM each, indicating a single mechanism of action. Quercetin at 15uM was the most efficient compound at both AMPK activation and mTOR inhibition. Therefore, regulation of cancer cell metabolism by quercetin has the potential to block BC cell growth and migration, and thus, metastatic progression. In conclusion, quercetin is a viable candidate for future development as an anti BC therapeutic. Grant Funding Source: Supported by US Army DoD/BCRP W81XWH‐07‐1‐0330 to SD, U54 CA096297 to UPRM‐MSC, MBRS‐RISE

The lymphocyte activation gene 3 (LAG‐3) protein was first identified as a cell surface molecule selectively expressed in activated human T lymphocytes, Natural Killer cells and T‐cell‐dependent activated B cells. The LAG‐3 is a transmembrane protein homolog of CD4 protein. LAG‐3 binds Major Histocompatibility Complex (MHC) class II molecules with much higher affinity than CD4 and acts as an inhibitor of antigen‐driven T cell expansion. This protein can also be detected in a soluble form (sLAG‐3), and in fact, it is now known that can be cleaved from the cell surface. In the soluble form, sLag‐3 induces MHC class II mediated signaling in dendritic cells, leading to their activation and maturation as well as production of cytokines. Also high levels of sLAG‐3 protein in the sera of breast cancer patients tend to correlate with good prognosis of the disease. This correlation suggests that sLAG‐3 may well be an important effectors molecule regulating the immune response to tumor antigens. We plan to study the structure of the extracellular part of the protein and the interaction with MHCII. Here we reported the insertion of the different variants of the LAG‐3 gene into the pENTR vector, and the transfection in mammalian cells. In addition, we demonstrated the expression of the LAG‐3 protein. This information will give us a better understanding of the function of sLAG‐3 and why this protein is a good adjuvant for vaccines.