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. 2018 Sep 12;10(9):1289.
doi: 10.3390/nu10091289.

Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway

Yongzeng Ding  1 , Bhargava Mullapudi  2 , Carolina Torres  3 , Emman Mascariñas  4   5 , Georgina Mancinelli  6 , Andrew M Diaz  7 , Ronald McKinney  8 , Morgan Barron  9 , Michelle Schultz  10   11 , Michael Heiferman  12 , Mireille Wojtanek  13 , Kevin Adrian  14 , Brian DeCant  15   16 , Sambasiva Rao  17 , Michel Ouellette  18 , Ming-Sound Tsao  19 , David J Bentrem  20 , Paul J Grippo  21   22
Affiliations

Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway

Yongzeng Ding et al. Nutrients. .

Abstract

Pancreatic cancer remains a daunting foe despite a vast number of accumulating molecular analyses regarding the mutation and expression status of a variety of genes. Indeed, most pancreatic cancer cases uniformly present with a mutation in the KRAS allele leading to enhanced RAS activation. Yet our understanding of the many epigenetic/environmental factors contributing to disease incidence and progression is waning. Epidemiologic data suggest that diet may be a key factor in pancreatic cancer development and potentially a means of chemoprevention at earlier stages. While diets high in ω3 fatty acids are typically associated with tumor suppression, diets high in ω6 fatty acids have been linked to increased tumor development. Thus, to better understand the contribution of these polyunsaturated fatty acids to pancreatic carcinogenesis, we modeled early stage disease by targeting mutant KRAS to the exocrine pancreas and administered diets rich in these fatty acids to assess tumor formation and altered cell-signaling pathways. We discovered that, consistent with previous reports, the ω3-enriched diet led to reduced lesion penetrance via repression of proliferation associated with reduced phosphorylated AKT (pAKT), whereas the ω6-enriched diet accelerated tumor formation. These data provide a plausible mechanism underlying previously observed effects of fatty acids and suggest that administration of ω3 fatty acids can reduce the pro-survival, pro-growth functions of pAKT. Indeed, counseling subjects at risk to increase their intake of foods containing higher amounts of ω3 fatty acids could aid in the prevention of pancreatic cancer.

Keywords: chemoprevention; neoplastic disease; pancreatic cancer; polyunsaturated fatty acids; proliferation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ω3-enriched diets protect against, whereas ω6-enriched diets accelerate pancreatic tumorigenesis in vivo. EL-Kras (KRAS) mice with mutant KRAS expression restricted to the pancreas acinar compartment via a rat elastase promoter were employed as a model of early pancreatic tumorigenesis. Mice were then administered either a control diet, or a diet enriched in ω3 or ω6 fatty acids, respectively, for 7, 11, or 15 months. Mice were sacrificed, tissues were sectioned and stained with H&E, and lesion frequency/penetrance and cystic papillary neoplasm (CPN) size were quantified by two blinded investigators (C.T. and G. M). * p-value < 0.05: ** p-value < 0.01; *** p-value < 0.001. The asterisks above each column represent the significance compared to the control diet. The significance between both diet is indicated by connecting lines.
Figure 2
Figure 2
ω3-enriched diets prevent proliferation in vivo. Mice were injected with Bromo-deoxyuridine (BrdU) one hour prior to collection. Cell proliferation was assessed by immunohistochemistry to identify the incorporation of BrdU into the DNA of proliferating cells. Sections stained with BrdU antibody were scored as the number BrdU-positive nuclei per high power field by two independent researchers. * p-value < 0.05: ** p-value < 0.01; *** p-value < 0.001. The asterisks above each column represent the significance compared to the control diet. The significance between both diet is indicated by connecting lines.
Figure 3
Figure 3
ω3 fatty acids inhibit cell viability in vitro. (a) Human pancreatic ductal epithelial (HPDE) cells were administered increasing concentration of either ω3 fatty acids Docosahexaenoic Acid (DHA) and Eicosapentaenoic acid (EPA), or the ω6 fatty acid Linoleic acid (LA) in vitro. Proliferation was then assessed by 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay after 48 h, showing a similar reduction in proliferation in response to both ω3 fatty acids. (b–d) The experiment was next repeated in HPDE with stable expression of mutant KRASG12D (HPDE-KRAS), as well as human pancreatic nestin-expressing (HPNE) cells and HPNE cells with stable expression of mutant KRASG12D (HPNE-KRAS). * p-value < 0.05: ** p-value < 0.01; *** p-value < 0.001. The asterisk above each column represent the significance compared to the control diet. The significance between both diet is indicated by connecting lines.
Figure 4
Figure 4
ω3 fatty acids inhibit AKT phosphorylation in vitro. (a,b) HPDE cells were again administered fixed doses of 40 µM of either the ω3 fatty acids DHA and EPA, or the ω6 fatty acid LA in vitro. After 48 h, phosphorylated AKT (pAKT) expression was measured by Western blotting and densitometry normalizing expression to GAPDH. (c,d) The experiment was repeated in HPDE-KRAS cells. (* p < 0.05).
Figure 5
Figure 5
ω3 fatty acids prevent FOX3a and BAD phosphorylation in vitro and in vivo. (a,b) HPDE and HPDE-KRAS cells were incubated with fixed doses of 40 µM of the ω3 fatty acids DHA and EPA, or the ω6 fatty acid LA. Expression of the AKT-target pFOXO3a was assessed by Western blotting. (c) pFOXO3a expression was next evaluated in the KRASG12D mice given control, ω3-, or ω6-enriched diets for 11 months. (d–f) HPDE and HPDE-KRAS cells incubated with DHA, EPA, or LA and similarly evaluated for expression of the AKT-target pBAD. Additionally, pBAD expression was measured in KRAS mice fed control, ω3, or ω6 diets (for 11 months).
Figure 6
Figure 6
ω3 fatty acids promote apoptosis in vivo and in vitro. (a,b) Apoptosis was assessed in KRAS mice fed control, ω3, or ω6 diets (11 months) by immunohistochemistry for the surrogate marker cleaved-caspase 3. (c–f) Quantification of cell death (%) by apoptosis was performed in the HPDE, HPDE-KRAS, HPNE, and HPNE-KRAS cell lines incubated with the ω3 fatty acids DHA and EPA, or the ω6 fatty acid LA. Assays were performed in triplicate (* p < 0.05), and apoptosis measured via flow cytometry/Annexin–FITC assay. (* p < 0.05).
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