Source: UNIV OF THE DISTRICT OF COLUMBIA submitted to
NUTRIENT MODULATION OF P53-ATF3 SIGNALING IN BREAST CANCER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0217298
Grant No.
(N/A)
Project No.
DC-01KUMAR
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Feb 15, 2009
Project End Date
Feb 15, 2012
Grant Year
(N/A)
Project Director
Kumar, D.
Recipient Organization
UNIV OF THE DISTRICT OF COLUMBIA
4200 CONNECTICUT AVENUE N.W
WASHINGTON,DC 20008
Performing Department
BIOLOGY
Non Technical Summary
Vitamin E includes a family of micronutrients consisting of four tocopherols and four tocotrienols (α, β, γ and δ) both of which are present in various components of the human diet. Tocotrienols are minor constituents of vitamin E but possess several more powerful anti-cancer, cholesterol lowering, natriuretic (prevent hypertension and cardiovascular diseases caused by salt intake) and neuroprotective properties that are often lacking in tocopherols. Despite possessing these preventive/therapeutic advantages, tocotrienols have not been extensively studied compared to tocopherols. g-Tocotrienol (g-T3) induces apoptosis in a variety of cancer cell lines including ER+ and ER- breast cancer cells but, do not affect the proliferation of normal mammary cells. We identified ATF3, a transcription factor, as a novel target of g-T3 in breast cancer cells. We have shown that g -T3 induces apoptosis in caspase-3 deficient MCF-7 cells via caspase-7 dependent pathway. ATF3 interacts with p53 tumor suppressor protein and prevents its degradation by stabilizing p53 in the cells. P53 induces cell death in response to DNA damage by carcinogens thereby preventing cancer. We hypothesize that g -T3 stabilizes p53 via induction of ATF3 expression resulting in cellular apoptosis. To test this hypothesis we will examine whether (1) g-T3 induced apoptosis is ATF-3 dependent and (2) whether g-T3 stabilizes p53 via its interaction with ATF-3. The goal of this proposal is to investigate the molecular mechanism whereby g -T3 inhibits the growth of breast cancer cells. This study holds promise in identifying g-T3 as a chemo-preventative in breast cancer and/or its role in inhibiting the progression of breast cancer. POTENTIAL OUTCOMES: y-T3 is an understudied nutrient that has the ability to inhibit the progression of breast cancer cells. Importantly y-T3 inhibits cholesterol biosynthesis and can prevent cardiovascular disease and hypertension. Our proposal that y-T3 modulates p53-ATF3 cross talk may lead to broad (chemo-preventative and chemotherapeutic) applications for the inhibition of breast cancer cell proliferation and possibly cell transformation. Additionally, it might be useful as an adjuvant therapy along with standard chemotherapy and possibly radiotherapy regimens. If we validate the role of ATF-3 and p53 in y-T3 cell signaling, we will explore its role as a therapeutic agent using xenograft models. Our long term goal is to study the chemo-preventative and therapeutic effects of y-T3 in humans.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70240991040100%
Goals / Objectives
Vitamin E includes a family of lipophilic micronutrients consisting of four tocopherols and four tocotrienols (α, β, γ and δ) both of which are present in the human diet. Despite possessing several preventive/therapeutic advantages, tocotrienols are well understudied as compared to tocopherols. y-T3 induces apoptosis in a variety of cancer cell lines including breast cancer cells [45]. Tocotrienols do not affect the proliferation of normal mammary cells [46]. We have demonstrated that y-T3 induces apoptosis in caspase-3 deficient MCF-7 cells via caspase-7 dependent pathway. Using microarray we have identified for the first time that ATF3 is a novel target for y-T3 in MCF-7 breast cancer cells. ATF3 was the most upregulated (18.5 folds on Affymetrix HG-U133A2 Gene chip) gene in MCF-7 cells upon treatment with y-T3. ATF3 is a transcription factor that interacts with p53 tumor suppressor protein and prevents its degradation by inhibiting its ubiquitination [47]. Furthermore, this interaction is enhanced during genotoxic stress. We hypothesize that in p53 +/+ MCF-7 breast cancer cells, y-T3 induces apoptosis by inducing the expression of ATF3, which in turn stabilizes p53, resulting in cellular apoptosis. It has been shown that loss of ATF-3 impairs the p53-dependent cellular response to DNA damage and allows cellular transformation by oncogeneic ras in mouse embryonic fibroblasts (MEF) [45]. Also, ectopic expression of ATF-3 leads to apoptosis in cancer cells [48]. The goal of this project is to investigate the molecular mechanism whereby g-T3 inhibits the growth of breast cancer cells. Our long term goal is to study the chemo-preventative and therapeutic effects of y-T3 in humans. The specific aims of the proposal are: 1. Determine if y-T3 induces apoptosis in MCF-7 breast cancer cells is ATF3 dependent: We will examine if (a) ATF-3 promoter is transactivated by y-T3 using luciferase assay and (b) y-T3 induced apoptosis is ATF-3 dependent by studying the effects of (i) ATF-3 inhibition (using siRNA) and (ii) ATF-3 overexpression (using ATF-3 cDNA) on y-T3 induced apoptosis. 2. Determine whether y-T3 stabilizes p53 pathway via ATF3: We will examine (a) the post translational regulation of p53 by y-T3 using cyclohexamide chase experiment; (b) Interaction of ATF3 and p53 and determine whether ATF3 binding to p53 is enhanced upon y-T3 treatment and (c) effects of y-T3 treatment on p53 ubiquitination by MDM2 ligase.
Project Methods
Cell lines, Tocotrienols, Plasmids, transfections and treatment: We will use 3 breast cancer cell lines, MCF-7, T47D and ZR-75-1 for our experiments. Primarily we will use MCF-7 cells and any promising data will be validated in ZR-75-1 cells which are also ER+ and p53 +/+ cells. siRNA and transfections: siRNAs against ATF-3 will be obtained as validated stealth siRNA from Invitrogen. Cells will be transfected with ATF-3 siRNAs or the control scrambled siRNAs using RNAimax reagent (Invitrogen) according to the manufacturer's instructions. Cell viability and apoptosis assays: Cell survival following treatment with y-T3 will be determined by trypan blue dye exclusion and the colorimetric WST-1 cell viability/proliferation assay (Roche). Apoptosis will be measured using following assays: Apoptotic DNA content; Annexin V staining; Caspase-7 assay. ATF-3 promoter transactivation assay: To study ATF-3 promoter transactivation in response to y-T3, reporter assays will be performed using luciferase reporter vector driven by ATF-3 promoter. p53 stability in response bto y-T3 treatment: Stability of p53 protein response to y-T3 will be measured by cyclohedamide chase assay. Interaction between ATF-3 and p 53 proteins: We will study the interaction of ATF-3 and p53 in response to y-T3 in different cell lines by standard immunoprecipitations (IP). p53 ubiquitination assay: To examine the possible inhibition of p53 ubiquitination by ATF-3, we will transfect the cells with GFP-Ub, HA-p53 (from Addgene) and MDM2 plasmids. Statistical analysis: Most data-points will be analyzed using analysis of variance (ANOVA).

Progress 02/15/09 to 02/15/12

Outputs
OUTPUTS: Vitamin E consists of four tocopherols and four tocotrienols (alpha, beta, gamma and delta) both of which are present in various components of the human diet. Tocotrienols possess several powerful anti-cancer, cholesterol lowering, natriuretic and neuroprotective properties that are often lacking in tocopherols. Tocotrienols are well understudied as compared to tocopherols. Gamma Tocotrienols (gamma-T3) induces programmed cell death or apoptosis in a variety of cancer cell lines including breast cancer cells but not in normal cells. The molecular mechanisms involved in gamma-T3 action are not well understood. The goal of the proposal is to understand the molecular basis of the action of gamma-T3. The project made significant progress during the course of funding. Using microarray analysis and a range of cellular and molecular techniques, we demonstrated that gamma-T3 induces endoplasmic reticulum (ER) stress and activates multiple unfolded protein response (UPR) pathways. The results were disseminated in the form of research seminar sponsored by UDC AES at UDC and poster presentations at the National and International meetings. We published our results in the Journal of Nutritional Biochemistry that ranks sixth in the journals related to Nutrition and Dietetics. We further continued our studies using Tocomin, a commercially available mixture of naturally occurring tocotrienols (T3s) and tocopherol (Ts) extracted from palm oil/palm fruits. Our studies have demonstrated that both purified gamma-tocotrienol and Tocomin induce cell death and a self-eating process known as autophagy in breast cancer cells. A second manuscript was submitted to the PLOS one for publication demonstrating autophagic involvement of dietary tocotrienols in human breast cancer cells. The manuscript was returned for revision and we are currently performing additional experiments as suggested by reviewers. The manuscript will be submitted with revisions. In addition to the work covered in the two manuscripts, we also delineated further molecular pathways that are involved in cancer cell death and autophagy, Habib Kedir, an undergraduate student has demonstrated role of metabolic sensor AMPK in the autophagy induced by Tocotrienols. A manuscript on the role of AMPK is being prepared currently for submission. This proposal funded by the AES also leveraged laboratory development and other funding that my laboratory has received. Related to this proposal, we also investigated role of Vitamin E Succinate (VES), the most common form of Vitamin E in pancreatic cancer cells. Results were published last year in the Journal, Genes and Nutrition. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In this completed part of the study we demonstrate that gamma tocotrienols (gamma-T3) induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with gamma-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and Gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2 mediated oxidative stress response, TGF-beta signaling and Endoplasmic Reticulum (ER) stress response. Analysis of ER stress related proteins in MCF-7 and MDA-MB 231 cells treated with gamma-T3 demonstrated activation of PERK and pIRE1alpha pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most upregulated gene (16.8 folds) in response to gamma-T3. ATF3 knockdown using siRNA suggested essential role of ATF3 in gamma-T3 induced apoptosis. In summary, we demonstrate that gamma-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for gamma-T3 in breast cancer cells. As mentioned above, we demonstrated the modulation PERK and IRE1alpha dependent ER stress related pathways in MCF-7 and MDA-MB 231 cells when treated with gamma-T3. PERK phosphorylates eIF2alpha and attenuates translation machinery leading to ER stress. Remarkably, PERK signaling has also been shown to induce autophagy as a protective response to cellular insults, such as hypoxia and nutrient deprivation. Similarly, IRE1a has also been implicated in autophagic response. Continuing our studies, we included commercially available Tocomin as a source of naturally occurring dietary tocotrienols and studied its effects on inducing autophagy and apoptosis. We demonstrated that (a) gamma-T3 induces autophagy in MDA-MB 231 breast cancer cells by modulating the mTOR pathway and(b) Tocomin also induces cell death and autophagy in human breast cancer cells. Further, we demonstrated that 3-MA, a widely used autophagy inhibitor sensitizes MDA-MB 231 cells to Tocomin induced apoptosis. Further elucidation of molecular targets revealed that Tocotrienols modulate a metabolic sensor protein, AMPK and induces autophagy via modulation of Beclin-1. Related to this proposal, we also investigated role of Vitamin E Succinate (VES), the most common form of Vitamin E in pancreatic cancer cells and found that VES also induced cell death in pancreatic cancer cells by modulating an anti-apoptotic protein, survivin.

Publications

  • Patacsil, D., Tran, A.T., Cho, Y.S., Suy, S., Saenz, F., Malyukova, I., Ressom, H., Collins, S.P., Clarke, R., Kumar, D. Gamma-Tocotrienol induced Apoptosis is Associated with Unfolded Protein Response in Human Breast Cancer Cells. J. Nutr. Biochem., 2012 Jan;23(1):93-100
  • Patacsil D, Osayi S, Tran AT, Saenz F, Yimer L, Shajahan AN, Gokhale PC, Verma M, Clarke R, Chauhan SC, Kumar D. Vitamin E succinate inhibits survivin and induces apoptosis in pancreatic cancer cells. Genes Nutr. 2012 Jan;7(1):83-9.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Vitamin E consists of four tocopherols and four tocotrienols (alpha, beta, gamma and delta) both of which are present in various components of the human diet. Tocotrienols possess several powerful anti-cancer, cholesterol lowering, natriuretic and neuroprotective properties that are often lacking in tocopherols. Tocotrienols are well understudied as compared to tocopherols. Gamma Tocotrienols (gamma-T3) induces programmed cell death or apoptosis in a variety of cancer cell lines including breast cancer cells but not in normal cells. The molecular mechanisms involved in gamma-T3 action are not well understood. The goal of the proposal is to understand the molecular basis of the action of gamma-T3. The project made significant progress in the last year. Using microarray analysis and a range of cellular and molecular techniques, we demonstrated that gamma-T3 induces endoplasmic reticulum (ER) stress and activates multiple unfolded protein response (UPR) pathways. The results were disseminated in the form of research seminar sponsored by UDC AES at UDC and poster presentations at the National and International meetings. We published our results in the Journal of Nutritional Biochemistry that ranks sixth in the journals related to Nutrition and Dietetics. We have continued our studies using Tocomin, a commercially available mixture of naturally occurring tocotrienols (T3s) and tocopherol (Ts) extracted from palm oil/palm fruits. Our studies have demonstrated that both purified gamma-tocotrienol and Tocomin induce cell death and a self-eating process known as autophagy in breast cancer cells. A second manuscript is submitted for publication demonstrating autophagic involvement of tocotrienols in human breast cancer cells. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In the present study, we demonstrate that gamma tocotrienols (gamma-T3) induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with gamma-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and Gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2 mediated oxidative stress response, TGF-beta signaling and Endoplasmic Reticulum (ER) stress response. Analysis of ER stress related proteins in MCF-7 and MDA-MB 231 cells treated with gamma-T3 demonstrated activation of PERK and pIRE1alpha pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most upregulated gene (16.8 folds) in response to gamma-T3. ATF3 knockdown using siRNA suggested essential role of ATF3 in gamma-T3 induced apoptosis. In summary, we demonstrate that gamma-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for gamma-T3 in breast cancer cells. As mentioned above, we demonstrated the modulation PERK and IRE1alpha dependent ER stress related pathways in MCF-7 and MDA-MB 231 cells when treated with gamma-T3. PERK phosphorylates eIF2alpha and attenuates translation machinery leading to ER stress. Remarkably, PERK signaling has also been shown to induce autophagy as a protective response to cellular insults, such as hypoxia and nutrient deprivation. Similarly, IRE1a has also been implicated in autophagic response. Last year, continuing our studies, we included commercially available Tocomin as a source of naturally occurring dietary tocotrienols and studied its effects on inducing autophagy and apoptosis. We demonstrated that (a) gamma-T3 induces autophagy in MDA-MB 231 breast cancer cells by modulating the mTOR pathway and (b) Tocomin also induces cell death and autophagy in human breast cancer cells. Further, we demonstrated that 3-MA, a widely used autophagy inhibitor sensitizes MDA-MB 231 cells to Tocomin induced apoptosis.

Publications

  • Patacsil, D., Tran, A.T., Cho, Y.S., Suy, S., Saenz, F., Malyukova, I., Ressom, H., Collins, S.P., Clarke, R., Kumar, D. Gamma-Tocotrienol induced Apoptosis is Associated with Unfolded Protein Response in Human Breast Cancer Cells. J. Nutr. Biochem., 2012 Jan;23(1):93-100


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Gamma-Tocotrienol (γ-T3) is a member of the vitamin E family. Tocotrienols (T3) are powerful antioxidants and possess anti-cancer, neuroprotective and cholesterol lowering properties. T3s inhibit the growth of various cancer cell lines without affecting normal cells. Less is known about the exact mechanisms of action of T3s on cell death and other growth inhibitory pathways. The project made significant progress in the last year. We demonstrated using standard cell biological and protein biochemistry experiments that γ-T3 induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells. Gene expression microarray analysis also demonstrated that γ-T3 induces endoplasmic reticulum (ER) stress and activates multiple unfolded protein response (UPR) pathways. The results were disseminated in the form of a research seminar sponsored by UDC AES at UDC and poster presentations at a national meeting. We submitted a manuscript to publish the findings in refereed journal: Journal of Nutritional Biochemistry which has been accepted for publication. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In the present study we demonstrate that γ-T3 induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with γ-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and Gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2 mediated oxidative stress response, TGF-β signaling and Endoplasmic Reticulum (ER) stress response. Analysis of ER stress related proteins in MCF-7 and MDA-MB 231 cells treated with γ-T3 demonstrated activation of PERK and pIRE1α pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most upregulated gene (16.8 folds) in response to γ-T3. ATF3 knockdown using siRNA suggested essential role of ATF3 in γ-T3 induced apoptosis. In summary, we demonstrate that γ-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for γ-T3 in breast cancer cells.

Publications

  • No publications reported this period


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Vitamin E consists of four tocopherols and four tocotrienols (α, β, γ and δ) both of which are present in various components of the human diet. Tocotrienols possess several powerful anti-cancer, cholesterol lowering, natriuretic and neuroprotective properties that are often lacking in tocopherols. Tocotrienols are well understudied as compared to tocopherols. Gamma Tocotrienols (γ-T3) induces apoptosis in a variety of cancer cell lines including breast cancer but not in normal cells. The molecular mechanisms involved in γ-T3 action are not well understood. The goal of the proposal is to understand the molecular basis of the action of γ-T3. The project made significant progress in the last year. Using microarray analysis and a range of cellular and molecular techniques, we demonstrated that γ-T3 induces endoplasmic reticulum (ER) stress and activates multiple unfolded protein response (UPR) pathways. Specific results are discussed later. The results were disseminated in the form of research seminar sponsored by UDC AES at UDC and poster presentations at national meeting. We are currently preparing a manuscript to publish the findings in refereed journal. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This phase of the proposal generated significant data on the mechanisms of action of g-T3 on MCF-7 breast cancer cells. The microarray experiments performed generated gene expression data corresponding to modulation of multiple signaling pathways. Pathway analysis of the microarray data revealed the modulation of genes involved in ER stress response and unfolded protein response (UPR) signaling. Expression of various genes was validated using Western Blot in MCF-7 cells. The data suggested that g-T3 induces ER stress mediated apoptosis in MCF-7 cells. g-T3 induces apoptosis in both MCF-7 (ER+; p53 wild type) and MDA-MB 231 breast cancer cells (ER-, PR-, HER2- and p53 mutant). We asked if ER stress induction is a common mechanism of action for gT3 and whether g-T3 induces ER stress in both MCF-7 and MDA-MB 231 cells. ER stress is mediated by 3 interconnected pathways. Further we asked, which ER stress pathway(s) is involved in g-T3 induced apoptosis. Our recent results revealed that g-T3 indeed induces ER stress response in both cell lines. ATf3 is involved in ER stress response and was induced significantly (18.5 folds) in MCF-7 cells treated with gT3. We asked, whether ATF3 is essential for g-T3 induced apoptosis. Using siRNA to ATF3 we demonstrated that ATF3 induction is essential for gT3 induced apoptosis in MCF-7 cells. We are currently in process of preparing a manuscript for the publication of these data. The proposal employed a range of cellular and molecular biological techniques and a range of data analysis packages. Involvement of students allowed student learning and motivating them into research. The students also presented the data at 2 different conferences. One manuscript is under preparation.

Publications

  • No publications reported this period