Keith D. Kikawa

Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association

Docosahexaenoic acid (DHA), a 22:6 n-3 polyunsaturated fatty acid, is the longest and most highly unsaturated fatty acid found in most membranes and has been shown to inhibit cancer cell growth in part by modifying cell signaling. In the current study, alterations to epidermal growth factor receptor (EGFR) signaling upon DHA supplementation are examined in A549 lung adenocarcinoma, WiDr colon carcinoma and MDA-MB-231 breast carcinoma cell lines. Interestingly, EGFR phosphorylation, most notably at the tyrosine 1068 residue, is dramatically upregulated, and EGFR association with the Sos1 guanine nucleotide exchange factor is concomitantly increased upon DHA supplementation. However, guanosine triphosphate-bound Ras and phosphorylated extracellular signal-regulated kinase (Erk)1/2 are paradoxically downregulated in the same treatments. Previous reports have noted changes in membrane microdomains upon DHA supplementation, and our findings confirmed that EGFR, but not Ras, is excluded from caveolin-rich lipid raft fractions in DHA-treated cells, resulting in a decreased association of Ras with Sos1 and the subsequent downregulation of Erk signaling. Xenografts of the A549 cell line implanted in athymic mice fed a control high-fat diet or a diet high in DHA confirmed our in vitro data. These results demonstrate for the first time a functional consequence of decreased EGFR protein in lipid raft microdomains as a result of DHA treatment in three different cancer models. In addition, we report the ability of DHA to enhance the efficacy of EGFR inhibitors on anchorage-independent cell growth (soft agar), providing evidence for the potential development of enhanced combination therapies.

Docosahexaenoic acid attenuates breast cancer cell metabolism and the Warburg phenotype by targeting bioenergetic function

Docosahexaenoic acid (DHA; C22:6n−3) depresses mammary carcinoma proliferation and growth in cell culture and in animal models. The current study explored the role of interrupting bioenergetic pathways in BT‐474 and MDA‐MB‐231 breast cancer cell lines representing respiratory and glycolytic phenotypes, respectively and comparing the impacts of DHA with a non‐transformed cell line, MCF‐10A. Metabolic investigation revealed that DHA supplementation significantly diminished the bioenergetic profile of the malignant cell lines in a dose‐dependent manner. DHA enrichment also resulted in decreases in hypoxia‐inducible factor (HIF‐1α) total protein level and transcriptional activity in the malignant cell lines but not in the non‐transformed cell line. Downstream targets of HIF‐1α, including glucose transporter 1 (GLUT 1) and lactate dehydrogenase (LDH), were decreased by DHA treatment in the BT‐474 cell line, as well as decreases in LDH protein level in the MDA‐MB‐231 cell line. Glucose uptake, total glucose oxidation, glycolytic metabolism, and lactate production were significantly decreased in response to DHA supplementation; thereby enhancing metabolic injury and decreasing oxidative metabolism. The DHA‐induced metabolic changes led to a marked decrease of intracellular ATP levels by 50% in both cancer cell lines, which mediated phosphorylation of metabolic stress marker, AMPK, at Thr172. These findings show that DHA contributes to impaired cancer cell growth and survival by altering cancer cell metabolism, increasing metabolic stress and altering HIF‐1α‐associated metabolism, while not affecting non‐transformed MCF‐10A cells. This study provides rationale for enhancement of current cancer prevention models and current therapies by combining them with dietary sources, like DHA.

Induced Oxidative Stress and Cell Death in the A549 Lung Adenocarcinoma Cell Line by Ionizing Radiation Is Enhanced by Supplementation With Docosahexaenoic Acid

Both ionizing radiation and docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid (PUFA), have been shown to inhibit tumor cell growth at least in part by increasing oxidative stress. In this study, the effects of ionizing radiation, DHA, or a combination of the two on cell proliferation, anchorage-independent growth, apoptosis, and lipid peroxidation in A549 lung adenocarcinoma cells were examined. In this study, significant decreases in cell proliferation and colony formation were noted for ionizing radiation or DHA treatments, whereas a combination of the two showed significant reductions over either treatment alone. Conversely, lipid peroxidation and apoptotic cell death showed significant increases with ionizing radiation and DHA treatments, whereas cells receiving both treatments demonstrated further significant increases. Moreover, addition of vitamin E, an antioxidant, was able to completely reverse lipid peroxidation and cell death due to ionizing radiation and partially reverse these changes in DHA treatments. Finally, the preferential incorporation of DHA into lung and xenograft compared to liver tissue is demonstrated in an in vivo model. These findings confirm the potential of DHA supplementation to enhance the treatment of lung cancer using ionizing radiation by increasing oxidative stress and enhancing tumor cell death.

Key roles for GRB2-associated-binding protein 1, phosphatidylinositol-3-kinase, cyclooxygenase 2, prostaglandin E2 and transforming growth factor alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth

The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Docosahexaenoic Acid-mediated Inhibition of Heat Shock Protein 90-p23 Chaperone Complex and Downstream Client Proteins in Lung and Breast Cancer.

ABSTRACT The molecular chaperone, heat shock protein 90 (Hsp90), is a critical regulator for the proper folding and stabilization of several client proteins, and is a major contributor to carcinogenesis. Specific Hsp90 inhibitors have been designed to target the ATP-binding site in order to prevent Hsp90 chaperone maturation. The current study investigated the effects of docosahexaenoic acid (DHA; C22:6 n-3) on Hsp90 function and downstream client protein expression. In vitro analyses of BT-474 human breast carcinoma and A549 human lung adenocarcinoma cell lines revealed dose-dependent decreases in intracellular ATP levels by DHA treatment, resulting in a significant reduction of Hsp90 and p23 association in both cell lines. Attenuation of the Hsp90-p23 complex led to the inhibition of Hsp90 client proteins, epidermal growth factor receptor 2 (ErbB2), and hypoxia-inducible factor 1α (HIF-1α). Similar results were observed when employing 2-deoxyglucose (2-DG), confirming that DHA and 2-DG, both independently and combined, can disturb Hsp90 molecular chaperone function. In vivo A549 xenograft analysis also demonstrated decreased expression levels of Hsp90-p23 association and diminished protein levels of ErbB2 and HIF-1α in mice supplemented with dietary DHA. These data support a role for dietary intervention to improve cancer therapy in tumors overexpressing Hsp90 and its client proteins.

Abstract 483: Docosahexaenoic acid inhibits heat shock protein 90 complex and client proteins by reducing intracellular ATP levels in breast and lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA A major contributor to carcinogenesis is the molecular chaperone heat shock protein 90 (Hsp90). Hsp90 regulates the proper folding and stabilization of several client proteins, including human epidermal growth factor receptor 2 (ErbB2) and hypoxia-inducible factor 1α (HIF-1α). ErbB2 is a member of the epidermal growth factor receptor (EGFR) family of receptor-proteins, and is a clinical target for the treatment of metastatic cancer, while HIF-1α is often stabilized and overexpressed in several cancers. The ability for Hsp90 to become a mature complex and to properly fold client proteins is dependent on adenosine triphosphate (ATP) binding with the co-chaperone, p23. Specific Hsp90 inhibitors, like geldanamycin, target the Hsp90 ATP-binding site to prevent formation of the mature complex. Although supplementation with docosahexaenoic acid (DHA; C22:6 n-3) has not been shown to specifically target the Hsp90 ATP-binding site, the current study demonstrates a similar effect by DHA causing a reduction in available intracellular ATP levels. DHA is the longest, most unsaturated omega-3 polyunsaturated fatty acid (PUFA) existing in biological membranes and has been shown to possess anti-cancer activity in several cancer models. Studies from our laboratory have shown that DHA enrichment can reduce cancer cell metabolism and intracellular ATP levels in both breast and lung cancer models. In this study, a novel mechanism for the ability of DHA to inhibit the Hsp90 chaperone complex is defined. In vitro analyses of BT-474 human breast ductal carcinoma, as well as the A549 human lung adenocarcinoma cell lines were used to determine the impact of DHA-induced decreases of intracellular ATP levels. We found that the reduction in ATP levels by DHA treatment resulted in significant decreases in the association of Hsp90 and p23 in both cell lines. The decreased association of the Hsp90-p23 complex led to decreased levels of ErbB2 and HIF-1α client proteins, suggesting that DHA can modify Hsp90 chaperone function and attenuate client protein levels. Additionally, similar results were found when employing 2-deoxyglucose (2-DG), a glycolytic inhibitor, which confirms that DHA and 2-DG can disrupt Hsp90 molecular chaperone function by decreasing cellular ATP levels. Consistent with these observations, when using a dose of DHA that did not significantly reduce intracellular ATP levels, no change in the Hsp90-p23 chaperone complex or protein levels of ErbB2 and HIF-1α were seen. These results demonstrate a potential use for dietary intervention to improve cancer therapy in tumors that overexpress Hsp90 client proteins. Citation Format: Michael Mouradian, Irvin V. Ma, Erika D. Vicente, Keith D. Kikawa, Amy M. Chattin, Ronald S. Pardini. Docosahexaenoic acid inhibits heat shock protein 90 complex and client proteins by reducing intracellular ATP levels in breast and lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 483. doi:10.1158/1538-7445.AM2014-483

Abstract 1303: Key roles for Gab1, phosphatidylinositol-3-kinase, COX-2, prostaglandin E2 and TGF-alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth.

The omega-6 polyunsaturated fatty acid, linoleic acid (LA), is prevalent in Western diets and is known to enhance the tumorigenesis of mammary cancer models; with previous work demonstrating an upregulation of phosphatidylinositol-3-kinase (PI3K) signaling may play a key role. In this study, a mechanism for LA9s upregulation of cancer cell growth is defined. High levels of LA in animal diets or cell culture medium initiates a series of events beginning with increased cyclooxygenase (COX) activity, which leads to increased prostaglandin E2 (PGE2) production, that subsequently increases matrix metalloproteinase (MMP) levels and transforming growth factor alpha (TGF-α). TGF-α is a classic growth factor for the epidermal growth factor receptor 1 (EGFR), which plays a role in a large number of cancers. The Grb2-associated binding protein 1 (Gab1) is a scaffolding protein that can complex with EGFR to activate PI3K signaling. Recent studies in our laboratory reveal LA supplementation of the breast cancer cell line BT-474 and the lung adenocarcinoma cell line A549 greatly increases the association between Gab1 and EGFR, while at the same time dramatically decreasing Gab1 protein levels. These changes are concomitant with increases in activated Akt (pAkt), a downstream signaling component in the PI3K signaling pathway. Moreover, inhibitors of EGFR, PI3K and Gab1-specific siRNAs are capable of reversing LA-induced upregulation of pAkt, as well as observed increases in cell proliferation for these models. These data establish Gab1 as major target in LA-induced enhancement of tumorigenesis. Citation Format: Keith D. Kikawa, Clarissa R. Martins, Eric D. Johnson, Kristen Beck, Michael Mouradian, Ronald S. Pardini. Key roles for Gab1, phosphatidylinositol-3-kinase, COX-2, prostaglandin E2 and TGF-alpha in linoleic acid-induced upregulation of lung and breast cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2013-1303

Abstract 2918: Docosahexaenoic acid-induced oxidative stress increases cellular apoptosis through a c-MYC-dependent mechanism in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC MYC is a potent oncogene that transcriptionally regulates 15 percent of the genome and is found to be deregulated in approximately 70 percent of cancers (c-MYC), including breast cancer. Activation of c-MYC induces association between its required partners, MYC-associated factor X (MAX) or MAX dimerization protein (MAD), regulating pro-survival and pro-apoptotic pathways, respectively. c-MYC has proved to be a difficult molecular target of inhibition. Only recently have there been strategies providing a way to inhibit c-MYC-dependent tumors and its downstream targets. However, dietary factors like, long-chain fatty acids (LCFA), have been shown to change activity and expression levels of c-MYC in cell culture and animal models. LCFAs have also been reported to induce apoptosis through c-MYC. The present study evaluated whether changes in c-MYC expression or activity were responsible for induction of oxidative stress and apoptosis in response to docosahexaenoic acid (DHA, C22:6) supplementation in breast cancer. c-MYC mRNA and protein levels were determined in vitro in the breast cancer BT-474, MDA-MB-231, and the non-transformed MCF-10A cell lines. qPCR results showed increases in c-MYC mRNA expression upon PUFA treatment in both BT-474 and MDA-MB-231 cancer lines, but not in the MCF-10A non-transformed line. Protein analysis revealed no changes in total c-MYC expression, however total phosphorylation at Thr58 and Ser62 are significantly upregulated in a dose-response manner upon DHA supplementation in the BT-474 cell line. Remarkably, transcriptional activity of c-MYC is highly upregulated as well, suggesting that c-MYC activity increases in response to DHA treatment. Using dihydrodichlorofluorescein diacetate (H2-DCF-DA) assay, which measures intracellular reactive oxygen species (ROS) and oxidative stress, we found increases in response to DHA treatment in all cell lines. Finally, the increase in oxidative stress also resulted in an increase in apoptosis, which we suggest is through c-MYC activation. Further analysis of the c-MYC isoform status, association between MYC-MAX and MYC-MAD complexes, as well as inducers and inhibitors of c-MYC are currently underway to define specific targets where DHA is applying some of its anti-cancer effects. Ultimately, with a goal that this research will reveal dietary targets of DHA that could be used to enhance efficacy to current therapies. Citation Format: Michael Mouradian, Haviva Kobany, Christopher R. Douglas, Nam D. Hoang, Keith D. Kikawa, Ronald S. Pardini. Docosahexaenoic acid-induced oxidative stress increases cellular apoptosis through a c-MYC-dependent mechanism in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2918. doi:10.1158/1538-7445.AM2013-2918

Abstract 5430: Docosahexaenoic acid attenuates glycolytic metabolism and the Warburg phenotype by targeting hypoxia inducible factor 1 alpha in breast cancer.

Dietary factors have been estimated to account for approximately 30-40 percent of cancer incidence and research has shown that high fat diets rich in n-3 polyunsaturated fatty acids (PUFAs) are associated with lower cancer occurrences, including breast cancer. The glycolytic switch, known as the Warburg Effect, is observed in many malignant models, which results in a unique metabolic shift in energy metabolism where tumor cells increase anaerobic glycolysis even in the presence of normal oxygen levels. In our laboratory, we have shown that the n-3 PUFA, docosahexaenoic acid (DHA; C22:6), significantly decreases total cellular ATP and the transcriptional activity and protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), a major contributor to the Warburg phenotype. In this project, we set out to determine if DHA is responsible for attenuating the Warburg Effect through a HIF-1α-dependent mechanism. Alterations in glucose uptake and usage upon supplementation with DHA were examined in vitro in the breast cancer BT-474, MDA-MB-231, and non-transformed MCF-10A cell lines. Total glucose transporter 1 (GLUT 1) protein levels were determined in each cell line. No change was seen upon DHA supplementation in the MDA-MB-231 cell line, which may be due to overexpression of GLUT 1. However, GLUT 1 protein levels were significantly down in response to DHA treatment in the BT-474 cell line. We also observed significant decreases in glucose consumption through glycolysis and lactate production in the BT-474 and MDA-MB-231 cell lines upon DHA supplementation. However, no change was seen in the non-transformed cell line MCF-10A, suggesting that DHA is having anti-glucose-metabolism effects specifically on cancer cells, but not on non-transformed cells. Moreover, HIF-1α small interfering RNA (siRNA) experiments showed a similar trend in GLUT 1 expression and glycolytic capacity in the BT-474 and MDA-MB-231 cell lines, suggesting that DHA is exerting some of its anti-cancer effects through HIF-1α. Based on these findings, changes in glucose transporter expression and glucose usage likely contribute to the impaired growth and survival of malignant cell lines supplemented with DHA. Currently, therapies are being developed to target the Warburg Effect in the treatment of cancer and DHA could likely enhance such therapies. Citation Format: Michael Mouradian, Keith D. Kikawa, Ronald S. Pardini. Docosahexaenoic acid attenuates glycolytic metabolism and the Warburg phenotype by targeting hypoxia inducible factor 1 alpha in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2013-5430

Abstract 5440: Upregulation of prostaglandin E2 and TGF-alpha by linoleic acid enhances pro-oncogenic signaling in models of human lung and breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The omega-6 polyunsaturated fatty acid (PUFA), linoleic acid (LA), has been reported to have adverse affects in the growth and metastasis of a variety of cancer cell models. However, there has been some controversy over how much these models relate to human disease. In the current study, a novel cascade of actions behind the LA-induced upregulation of cell proliferation has been identified. It is well established that PUFAs serves as precursors for eicosanoids, and LA supplementation was found to significantly increase the levels of Prostaglandin E2 (PGE2) in a variety of lung and breast cancer cell lines. These increases in PGE2 are associated with elevations in the activity of Cyclooxygenase-2 (COX-2), increased levels of Tumor Growth Factor-alpha (TGF-alpha) and specific pro-proliferative signaling pathways. These findings were corroborated in animal xenografts derived from the same cell lines used for the in vitro studies, with serum levels of PGE2 showing significant increases in PGE2 in animals fed diets high in LA. Moreover, the omega-3 PUFA, docosahexaenoic acid (DHA), was found to have an opposing affect on PGE2 production by upregulating Thromboxane B2 (TXB2), a stable metabolite of Thromboxane A2 (TXA2), which inhibits COX-2 activity. These findings present specific mechanisms behind the upregulation of cancer cell growth due to LA supplementation that may contribute to a resolution in determining its role in human disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5440. doi:1538-7445.AM2012-5440