The majority of breast cancer deaths result from the progression of this disease to a metastatic phenotype. Rac1 and Cdc42 are Rho family members that together with their downstream effectors, Wiskott-Aldrich Syndrome protein-family verprolin-homologous protein 2 (WAVE2) and Arp2/3, play an important role in cytoskeletal reorganization and the formation of membrane protrusions that promote cancer cell migration and invasion. γ-Tocotrienol, is a natural isoform within the vitamin E family of compounds that inhibits breast cancer cell growth and progression by suppressing various signaling pathways involved in mitogenic signaling and metastatic progression. Studies were conducted to examine the effects of γ-tocotrienol on Rac1/WAVE2 signaling dependent migration and invasion in highly metastatic mouse SA and human MDA-MB-231 mammary cancer cells. Exposure to γ-tocotrienol resulted in a dose-responsive decrease in Rac1/WAVE2 signaling as characterized by a suppression in the levels of Rac1/Cdc42, phospho-Rac1/Cdc42, WAVE2, Arp2, and Arp3 expression. Additional studies also demonstrated that similar treatment with γ-tocotrienol resulted in a significant reduction in tumor cell migration and invasion. Taken together, these findings indicate that γ-tocotrienol treatment effectively inhibits Rac1/WAVE2 signaling and reduces metastatic phenotypic expression in mammary cancer cells, suggesting that γ-tocotrienol may provide some benefit as a novel therapeutic approach in the treatment of metastatic breast cancer.Key words γ-tocotrienol; breast cancer; Rac1; Wiskott-Aldrich Syndrome protein-family verprolin-homologous protein 2 (WAVE2); migration; invasion Breast cancer is the most common form of cancer in women and is second only to lung cancer as a leading cause of cancer mortality in the United States.1,2) The vast majority of breast cancer deaths result from the development and progression of the metastatic form of this disease, that is characterized by cancer cells detaching from the primary tumor and spreading to distant sites in the body such as the bone, brain, surrounding lymph nodes, liver, and lungs.3-5) Breast cancer metastasis is a complex and multistep process that includes morphological changes, detachment from the basement membrane, increased mobility and invasion into surrounding tissues, intravasation, circulation, adhesion, extravasation, and growth at distant sites. 6) Since patient prognosis for metastatic breast cancer is very poor, there is great interest in developing antiinvasion therapies to improve patient treatment and survival. 7)An initial step in the metastasis process involves epithelialto-mesenchymal transition (EMT), where neoplastic epithelial cells lose their cellular polarity and acquire mesenchymal-like mobility that allows them to invade surrounding tissues. [8][9][10][11] This amoeboid-like movement requires the formation of cellular protrusions that result from the conversion of monomeric globular actin (G-actin) to polymerized filamentous actin (Factin). 12-15)The Rho family of sm...

Breast cancer is the most common cancer and the second leading cause of cancer death in American women. Rho GTPases play a crucial role in regulating different cellular functions, including cell proliferation, gene expression, cell morphology, actin polymerization, cell motility, metastasis and apoptosis. Wiskott-Aldrich syndrome protein family verprolin-homologous protein2 (WAVE2) is a member within the Wiskott-Aldrich syndrome protein (WASP) family displays a significant role in actin polymerization and cytoskeletal organization which, are essential for cellular migration and invasion. γ-Tocotrienol is a natural isoform within the vitamin E family that displays anticancer activity against a variety of cancer cell types. Pterostilbene is a natural dimethyl ether analog of resveratrol that has several beneficial effects such as anticancer activity. Studies were conducted to examine the effects of combined γ-tocotrienol and pterostilbene on Rac1/WAVE 2 signaling in mouse +SA and human MDA-MB231 breast cancer cells and cellular migration. Results show that combined treatment of γ-tocotrienol and pterostilbene caused a synergistic inhibition of both mouse +SA and human MDA-MB231 breast cancer cells growth, and corresponding reduction in Rac1/WAVE2 signaling as characterized by a significant inhibition in the levels of phospho-Rac1/cdc42, WAVE2, Arp2, and Arp3 expression. Additional studies indicated that this combination resulted in a significant inhibition in mammary cancer cell migration. In summary, these findings strongly suggest that combined treatment of γ-tocotrienol with pterostilbene may provide great benefit as a therapeutic approach in the treatment of metastatic breast cancer. Supported by a grant from the Louisiana Cancer Foundation. Citation Format: Ibrahim G. Algayadh, Paul William Sylvester. Synergistic anticancer effects of combined γ-tocotrienol and pterostilbene is associated with a suppression in Rac1/WAVE 2 signaling in highly malignant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1053. doi:10.1158/1538-7445.AM2017-1053

Previous studies showed that γ-tocotrienol, a natural vitamin E derivative is associated with decrease in the rate of glycolysis in human mammary tumor cells. Therefore, studies were conducted to investigate the effects of γ-tocotrienol on key regulatory pathways of glucose metabolism in MCF-7 and MDA-MB-231 breast cancer cells. Unregulated proliferative capacity of cancer cells places huge metabolic load on cancer cells, which ultimately promotes the dysregulation of normal metabolic pathways. This dysregulation of energy metabolism manifests as a characteristic phenomenon called as the “Warburg effect”. The Warburg effect is an over-reliance on aerobic glycolysis by cancer cells to meet energy demand of proliferation. Akt has been reported as key regulator of Warburg effect in several tumors and is a known promoter of glycolysis. Additionally, 5'-AMP activated protein kinase (AMPK) is a known metabolic sensor that is activated when energy balance is disrupted in cells. In addition to their role in regulating cellular energy metabolism, Akt and AMPK also share several downstream targets like FoxO3a, a transcription factor. In the present study, protein expression using western blot and immunocytochemistry revealed that in human breast cancer cell lines MCF-7 and MDA-MB-231, γ-tocotrienol induced dose-dependent inhibition of Akt. This was associated with a rescue of Akt-induced inhibition of FoxO3a. In addition, γ-tocotrienol also induced a decrease in aerobic glycolysis as evidenced by a decrease in consumption of glucose and a decrease in glycolytic enzyme expression. Furthermore, results showed that γ-tocotrienol treatment induced activation of AMPK in breast cancer cells. Immunoprecipitation analysis also revealed a possible interaction between AMPK and FoxO3a in γ-tocotrienol treated cells. To study the effect of γ-tocotrienol-induced activation of AMPK on its downstream targets, microarray analysis was done using RT2-Profiler PCR array. The microarray revealed that γ-tocotrienol-induced AMPK activation resulted in downregulation of several genes that are critical regulators of aerobic glycolysis. In summary, results show that anticancer effects of γ-tocotrienol treatment are associated with significant inhibition in pro-Warburg signaling such as Akt activation, and a corresponding increase in activity of AMPK in human breast cancer cells. In addition, γ-tocotrienol reversal of Warburg effect is directly related to a reduction in the rate of glycolysis in these breast cancer cells. Finally, γ-tocotrienol-induced activation of AMPK signaling was found to significantly alter gene expression of various downstream targets of AMPK. These findings highlight the potential of vitamin E derivatives in targeting the metabolism of breast cancer cells. This work was supported in part by funds provided by the Louisiana Cancer Foundation. Citation Format: Venkatesh Dronamraju, Ibrahim G. Algayadh, Paul William Sylvester. γ-Tocotrienol negative modulation of the Warburg effect is mediated by the AMPK activation in malignant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 376.