Julia M. Houthuijzen

Increased Plasma Levels of Chemoresistance-Inducing Fatty Acid 16:4(n-3) After Consumption of Fish and Fish Oil

IMPORTANCE Our research group previously identified specific endogenous platinum-induced fatty acids (PIFAs) that, in picomolar quantities, activate splenic macrophages leading to resistance to chemotherapy in mouse models. Fish oil was shown to contain the PIFA 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) and when administered to mice neutralized chemotherapy activity. OBJECTIVE Because patients with cancer frequently use fish oil supplements, we set out to determine exposure to 16:4(n-3) after intake of fish or fish oil. DESIGN, SETTING, AND PARTICIPANTS (1) In November 2011, 400 patients with cancer undergoing treatment at the University Medical Center Utrecht were surveyed to determine their use of fish oil supplements; 118 patients responded to the questionnaire (30%); (2) pharmacokinetic analysis of the 16:4(n-3) content of 6 fish oils and 4 fishes was carried out; (3) from April through November 2012, a healthy volunteer study was performed to determine 16:4(n-3) plasma levels after intake of 3 different brands of fish oil or 4 different fish species. Thirty healthy volunteers were randomly selected for the fish oil study; 20 were randomly selected for the fish study. These studies were supported by preclinical tumor experiments in mice to determine chemoresistance conducted between September 2011 and December 2012. MAIN OUTCOMES AND MEASURES (1) Rate of use of fish oil supplements among patients undergoing cancer treatment at our institution; (2) levels of 16:4(n-3) present in 3 brands of fish oil and 4 species of fish; and (3) plasma levels of 16:4(n-3) present in healthy volunteers after consuming fish oil or fish. RESULTS Eleven percent of respondents reported using omega-3 supplements. All fish oils tested contained relevant amounts of 16:4(n-3), from 0.2 to 5.7 µM. Mouse experiments showed that addition of 1 µL of fish oil to cisplatin was sufficient to induce chemoresistance, treatment having no impact on the growth rate of tumors compared with vehicle-treated controls (estimated tumor volume difference, 44.1 mm3; P > .99). When the recommended daily amount of 10 mL of fish oil was administered to healthy volunteers, rises in plasma 16:4(n-3) levels were observed, reaching up to 20 times the baseline levels. Herring and mackerel contained high levels of 16:4(n-3) in contrast to salmon and tuna. Consumption of fish with high levels of 16:4(n-3) also resulted in elevated plasma levels of 16:4(n-3). CONCLUSIONS AND RELEVANCE All tested fish oils and herring and mackerel fishes contained relevant levels of fatty acid 16:4(n-3), a fatty acid with chemotherapy-negating effects in preclinical models. After ingestion of these fish oils or fishes, 16:4(n-3) was rapidly taken up in the plasma of human volunteers. Until further data become available, fish oil and fish containing high levels of 16:4(n-3) may best be avoided on the days surrounding chemotherapy.

Lysophospholipids secreted by splenic macrophages induce chemotherapy resistance via interference with the DNA damage response

Host responses to systemic anti-cancer treatment play important roles in the development of anti-cancer drug resistance. Here we show that F4/80(+)/CD11b(low) splenocytes mediate the resistance to DNA-damaging chemotherapeutics induced by two platinum-induced fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) in xenograft mouse models. Splenectomy or depletion of splenic macrophages by liposomal clodronate protects against PIFA-induced chemoresistance. In addition, we find that 12-S-HHT, but not 16:4(n-3), functions via leukotriene B4 receptor 2 (BLT2). Genetic loss or chemical inhibition of BLT2 prevents 12-S-HHT-mediated resistance. Mass spectrometry analysis of conditioned medium derived from PIFA-stimulated splenic macrophages identifies several lysophosphatidylcholines as the resistance-inducing molecules. When comparing cisplatin and PIFA-treated tumours with cisplatin alone treated tumours we found overall less γH2AX, a measure for DNA damage. Taken together, we have identified an intricate network of lysophospholipid signalling by splenic macrophages that induces systemic chemoresistance in vivo via an altered DNA damage response.

For Better or Worse: FFAR1 and FFAR4 Signaling in Cancer and Diabetes

Increased energy intake can lead to obesity, which increases the risk for the development of diabetes and cancer. Free fatty acids regulate numerous cellular processes, like insulin secretion, inflammation, proliferation, and cell migration. Dysregulation of these cellular functions by increased lipid intake plays a significant role in the development of diseases like diabetes and cancer. Free fatty acid receptors 1 and 4 (FFAR1 and FFAR4) are two free fatty acid receptors under increasing investigation for their roles in diabetes and more recently also cancer. Both receptors bind medium- to long-chain, saturated and omega-3 unsaturated fatty acids. Increasing evidence shows that enhanced FFAR1 and FFAR4 signaling reduces diabetes symptoms but enhances tumor growth and migration of various cancer types like melanoma and prostate cancer. This review gives an overview of the role of FFAR1 and FFAR4 in diabetes and cancer and discusses their potential to function as targets for treatment.

Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum‐induced fatty acid 16: 4(n‐3) (hexadeca‐4,7,10,13‐tetraenoic acid) induces systemic resistance to a broad range of DNA‐damaging chemotherapeutics. We show that 16: 4(n‐3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16: 4(n‐3). Studies that used splenocytes from GPR120‐deficient mice have confirmed this conclusion. Activation of the 16: 4(n‐3)‐GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance‐inducing lipid mediator, lysophosphatidylcholine(24: 1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.—Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16: 4(n‐3) stimulates a GPR120‐induced signaling cascade in splenic macrophages to promote chemotherapy resistance. FASEB J. 31, 2195–2209 (2017). www.fasebj.org

Abstract 307: GPR120/FFAR4 activation by fatty acid 16:4(n-3) plays a key role in resistance to chemotherapy

Although chemotherapy is designed to eradicate tumor cells it also has a significant impact on normal tissues. These host-responses can have large effects on the efficacy of treatment and overall survival. Fatty acids are increasingly recognized to play important signaling roles. 12-S-HHT and 16:4(n-3) are two platinum-induced fatty acids (PIFAs) that induce systemic resistance to a broad range of DNA-damaging chemotherapeutics. PIFAs exert their chemoprotective effect on tumor cells via an indirect mechanism involving splenic F4/80 + /CD11b low macrophages. Here we identified GPR120 on mouse and human splenic macrophages as the relevant receptor for 16:4(n-3). GPR120 (FFAR4) is a free fatty acid receptor that binds medium- to long-chain fatty acids and omega-3 fatty acids and is involved in anti-inflammatory response and metabolic control. Although both GPR40 (FFAR1) and GPR120 can be activated by 16:4(n-3) in vitro, only inhibition or genetic loss of GPR120 was able to block 16:4(n-3)-mediated chemoresistance in vivo. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA 2 activity in splenic macrophages resulting in the production and secretion of resistance-inducing lysophosphatidylcholine 24:1 (LPC(24:1)). Taken together, we identified a novel function for GPR120. Activation by 16:4(n-3) leads to enhanced cPLA 2 activity and production of LPC(24:1) resulting in chemotherapy resistance. Citation Format: Julia Houthuijzen, Ilse Oosterom, Brian Hudson, Akira Hirasawa, Laura Daenen, Chelsea McLean, Steffen Hansen, Marijn van Jaarsveld, Daniel Peeper, sahar Jafari Sadatmand, Jeanine Roodhart, Chris van de Lest, Trond Ulven, Kenji Ishihara, Graeme Milligan, Emile Voest. GPR120/FFAR4 activation by fatty acid 16:4(n-3) plays a key role in resistance to chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 307.

Detection of endogenously circulating mesenchymal stem cells in human cancer patients: Mesenchymal stem cells in human cancer patients

Mesenchymal stem cells (MSCs) can play a vital role in tumor progression and anticancer therapy response, as demonstrated by various in vitro and in vivo model systems. Their ability to home to developing tumors and modulate the tumor microenvironment, by suppressing T‐cell responses and contributing to the tumor stroma, is suggested to have a significant impact on disease progression, metastasis formation, and therapy response. Most evidence, however, is derived from artificial models using exogenously administered MSCs. The contribution of endogenous MSCs to tumor progression is currently unclear. Furthermore, few studies have been conducted in humans. A prospective biomarker study was therefore undertaken in 40 human cancer patients and 10 healthy controls of similar age, aimed at (i) exploring and quantifying circulating MSC levels in healthy volunteers and patients with advanced malignancies, (ii) determining the variability of MSC levels between healthy volunteers and cancer patients with different histologic tumor types, and (iii) exploring biomarkers associated with MSC levels. Significantly increased levels of circulating MSC‐like cells were observed in cancer patients when compared to healthy individuals (1.72 fold difference, 95% CI 1.03–2.81%, p = 0.03). In addition, prior systemic therapy was associated with a significant increase in MSC‐like cells (1.73 fold difference, 95% CI 1.02–2.95, p = 0.04). These results indicate that the amount of endogenously circulating MSCs in humans is increased in response to cancer, and that systemic anticancer treatment can influence MSC levels. Further research is needed to determine whether MSCs have a predictive value.

Abstract 3771: Splenic macrophages induce chemotherapy resistance via DNA damage repair

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of resistance to chemotherapy is one of the most important obstacles to continued effective treatment of cancer in patients. We recently identified an important network that is responsible for reversible systemic resistance to chemotherapy. Mesenchymal stem cells (MSCs), activated by chemotherapy, secrete two specific polyunsaturated fatty acids that confer resistance to a broad spectrum of anti-cancer agents. These two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid (KHT) and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)), can independently induce chemotherapy resistance at picomolar concentrations. Here we show that these PIFAs do not induce resistance to the tumor cells directly but rather function systemically via F4/80+/CD11blow/Ly6G- splenocytes to enhance DNA damage repair within tumors. We found that the PIFAs only induce resistance against DNA damaging agents but not against non-DNA damaging chemotherapeutics. When comparing tumors from cisplatin and PIFAs treated mice with cisplatin alone treated mice we found overall less DNA damage and a quicker restoration of DNA damage. In BRCA1-/-/p53-/- mice lacking homologous recombination, PIFAs were unable induce chemotherapy resistance. Several lines of evidence identified F4/80+/CD11blow/Ly6G- splenocytes as key mediators within this mechanism of systemic chemoresistance. First, PIFAs were unable to induce resistance in splenectomized tumor-bearing mice. Second, administration of conditioned medium from splenocytes (sCM) activated by the PIFAs to splenectomized mice was able to re-introduce systemic chemotherapy resistance in various mouse models (mean tumor volume in mm3 8 days after treatment ± SEM control: 290.7 ± 33.4, cisplatin: 118.2 ± 8.9 and cisplatin + sCM: 261.1 ± 29.7). Third, analysis of the different cell types present in the spleen indicated that F4/80+/CD11blow expressing cells were able to induce systemic resistance in splenectomized mice. Finally, administration of liposomal clodronate to tumor-bearing mice treated with chemotherapy and PIFAs was able to inhibit induction of resistance. Given the fact that 12-S-HHT is a natural ligand of the leukotriene B4 receptor 2 (BLT2) we investigated if signaling via BLT2 is responsible for 12-S-HHT-induced chemoresistance. Both genetic loss of BLT2 or inhibition of BLT2 using LY255283 was able to prevent 12-S-HHT-mediated chemoresistance. Taken together, our findings provide a novel role for the spleen in inducing a systemic protection against chemotherapy via enhancing DNA damage repair. Citation Format: Julia M. Houthuijzen, Laura G.M, Daenen, Jeanine M.L. Roodhart, Klaas M. Govaert, Michelle E. Smith, Juergen Thomale, Sahar J. Sadatmand, Hilde Rosing, Fabian Kruse, Nico van Rooijen, Jos H. Beijnen, Piet Borst, Sven Rottenberg, Bodduluri Haribabu, Emile E. Voest. Splenic macrophages induce chemotherapy resistance via DNA damage repair. [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 3771. doi:10.1158/1538-7445.AM2014-3771

Abstract 893: The spleen as a key player in chemotherapy resistance.

The development of resistance to chemotherapy is one of the most important obstacles to continued effective treatment of cancer in patients. Chemotherapy resistance is generally believed to be mediated by tumor cell-intrinsic factors or by the tumor microenvironment. We recently identified an important network that is responsible for reversible systemic resistance to chemotherapy. Mesenchymal stem cells (MSCs), activated by chemotherapy, secrete two specific polyunsaturated fatty acids that confer resistance to a broad spectrum of anti-cancer agents. These two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid (KHT) and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)), can independently induce chemotherapy resistance at picomolar concentrations. Our findings clearly showed that these PIFAs do not directly cause chemotherapy resistance in the tumor cells. Here, we show that the intermediate compartment between PIFAs and chemoresistance are F4/80 positive cells residing in the spleen. Several lines of evidence identified these splenocytes as key mediators of systemic chemoresistance. First, PIFAs were unable to induce resistance in splenectomized tumor-bearing mice. Second, administration of conditioned medium from splenocytes (sCM) activated by the PIFAs to splenectomized mice was able to re-introduce systemic chemotherapy resistance in various mouse models (mean tumor volume in mm 3 8 days after treatment ± SEM control: 290.7 ± 33.4, cisplatin: 118.2 ± 8.9 and cisplatin + sCM: 261.1 ± 29.7). Third, analysis of the different cell types present in the spleen indicated that PIFA-activated F4/80 + /CD11b low expressing cells were able to induce systemic resistance in splenectomized mice. Finally, administration of liposomal clodronate to tumor-bearing mice treated with chemotherapy and PIFAs was able to inhibit induction of resistance. Thus, we propose a model where PIFAs activate F4/80 + /CD11b low plenocytes leading to the production and secretion of secondary factors that induce resistance to chemotherapy. In addition, mass spectrometry analysis of the sCM from F4/80 + /CD11b low splenocytes revealed that several lipid-like molecules were differentially present in the sCM of splenocytes cultured with 12-S-HHT versus splenocytes cultured with a vehicle control. Total lipids fractions extracted from spleen CM containing these lipid-like molecules were able to induce resistance in splenectomized mice. Taken together, our findings provide a novel role for the spleen in inducing a systemic protection against chemotherapy. Citation Format: Julia M. Houthuijzen, Laura G.M. Daenen, Jeanine M.L. Roodhart, Klaas M. Govaert, Nico van Rooijen, Chris van de Lest, Emile E. Voest. The spleen as a key player in chemotherapy resistance. [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 893. doi:10.1158/1538-7445.AM2013-893

Abstract 891: Platinum-induced fatty acids mediate resistance to DNA damaging agents via homologous recombination.

Background: We recently identified two fatty acids (PIFAs) that induced chemoresistance in mouse models. At picomolar concentrations, PIFAs 16:4(n-3) and KHT interfered with the antitumor activity of a broad spectrum of chemotherapeutics. Our aim was to unravel the mechanism through which PIFAs prevent chemotherapy-induced tumor cell death. Results: In several animal models, PIFAs were found to interfere with early effects of chemotherapeutics. Four hours after administration of cisplatin significant decreases in proliferation (BrdU) and increases in DNA damage (γH2AX-positive cells) were found in tumors. In contrast, combined therapy with cisplatin and PIFAs normalized proliferation and decreased DNA damage. In cisplatin-treated tumors 31±7% cells were γH2AX-positive as opposed to 20±5% in tumors treated with cisplatin and PIFAs (p Citation Format: Laura GM Daenen, Julia M. Houthuijzen, Fabian Kruse, Ariena Kersbergen, Jeanine M.L. Roodhart, Sven Rottenberg, Emile E. Voest. Platinum-induced fatty acids mediate resistance to DNA damaging agents via homologous recombination. [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 891. doi:10.1158/1538-7445.AM2013-891

Abstract 800: Formation of chemotherapy resistance-inducing fatty acids by mesenchymal stem cells depends on COX-1, thromboxane synthase and intracellular calcium

The development of resistance to chemotherapy is considered to be one of the most important obstacles to continued effective treatment of cancer in patients. We recently have identified an important mechanism that is responsible for systemic resistance to chemotherapy. Circulating mesenchymal stem cells (MSCs) activated by chemotherapy secrete specific polyunsaturated fatty acids that confer resistance to a broad spectrum of anti-cancer agents. These factors were identified to be two distinct platinum-induced polyunsaturated fatty acids (PIFAs) 12-S-keto-5,8,10-heptadecatrienoic acid (KHT) and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) that were able to induce chemotherapy resistance in picomolar concentrations. Interestingly, the production of these PIFAs is restricted to MSCs and mouse embryonic fibroblasts (MEFs), other stem cells like hematopoietic stem cells and cancer stem cells are not able to form the PIFAs after cisplatin stimulation. Research from our lab showed that the production of these PIFAs can be blocked by cyclooxygenase-1 (COX-1), thromboxane A synthase (TXAS) and intracellular calcium inhibitors, suggesting that these are important in the production of the PIFAs, and indicating that the PIFAs are formed in the pathways of arachidonic acid (AA) and eicosapentaenoic acid (EPA). Lipid peroxidation with subsequent release of oxygen radicals could be a trigger for the formation of PIFA. We therefore tested whether reactive oxygen species (ROS) were involved in the production of the PIFAs. Neither increasing ROS by hydrogen peroxide, nor scavenging of ROS by glutathione in the MSCs influenced the level of resistance to chemotherapy in mice. In addition, intracellular ROS levels in the MSCs treated with cisplatin for various intervals did not change as was determined by DCFDA fluorescence. Our findings provide a new paradigm on how MSCs can induce a systemic protection against chemotherapy through the secretion of two distinct fatty acids that are formed in response to platinum-containing chemotherapy. 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 800. doi:1538-7445.AM2012-800